0163-769X/00/$03.00/0
Endocrine Reviews 21(3): 313–345
Copyright © 2000 by The Endocrine Society
Printed in U.S.A.
Leukemia-Inhibitory Factor—Neuroimmune Modulator
of Endocrine Function*
C. J. AUERNHAMMER†
AND
S. MELMED
Academic Affairs, Cedars-Sinai Research Institute, University of California Los Angeles School of
Medicine, Los Angeles, California 90048
ABSTRACT
Leukemia-inhibitory factor (LIF) is a pleiotropic cytokine expressed by multiple tissue types. The LIF receptor shares a common
gp130 receptor subunit with the IL-6 cytokine superfamily. LIF signaling is mediated mainly by JAK-STAT (janus-kinase-signal transducer and activator of transcription) pathways and is abrogated by
the SOCS (suppressor-of cytokine signaling) and PIAS (protein inhibitors of activated STAT) proteins. In addition to classic hematopoietic and neuronal actions, LIF plays a critical role in several
endocrine functions including the utero-placental unit, the hypo-
thalamo-pituitary-adrenal axis, bone cell metabolism, energy homeostasis, and hormonally responsive tumors. This paper reviews
recent advances in our understanding of molecular mechanisms regulating LIF expression and action and also provides a systemic overview of LIF-mediated endocrine regulation. Local and systemic LIF
serve to integrate multiple developmental and functional cell signals,
culminating in maintaining appropriate hormonal and metabolic homeostasis. LIF thus functions as a critical molecular interface between the neuroimmune and endocrine systems. (Endocrine Reviews
21: 313–345, 2000)
I. Introduction
V. LIF—Hematopoietic and Neuropoietic Cytokine
A. Hematopoietic system
B. Nervous system
VI. LIF and Endocrine Systems
A. Utero-placental unit—blastocyst implantation
B. Hypothalamo-pituitary-adrenal axis
C. Bone metabolism
D. Energy metabolism
E. Endocrine-responsive tumors
VII. Integrative Section—The Neuroimmune-Endocrine Interface
II. LIF—Gene Structure and Regulation
A. LIF gene and primary structure
B. LIF expression
C. LIF protein tertiary structure
III. LIF Receptor—Gene Structure and Regulation
A. The cytokine receptor superfamily
B. LIFR gene and structure
C. Membrane-bound LIFR
D. Soluble LIFR
E. gp130 Gene and structure
F. IL-6 cytokine family and the gp130 receptor subunit
G. LIFR-gp130 complex
H. The LIFR-gp130 complex signals OSM, CNTF, and
CT-1
I. IL-6:IL-6R and IL-11:IL11R complex
J. LIF binding and LIFR expression
IV. LIF Signaling
A. Jak-STAT pathway
B. Jaks
C. STATs
D. Cytoplasmic receptor domains
E. Negative feedback regulators of Jak-STAT signaling
F. SHP-2
G. SOCS proteins
H. PIAS
I. Mitogen-activated protein kinase (MAPK)
J. Others
I. Introduction
A
LTHOUGH classic endocrinology involved the study
of secreted hormones impacting a distant target tissue, it has become increasingly clear that local regulatory
molecules play a critical role in endocrine function. Thus,
growth factors and cytokines that act in a paracrine or autocrine fashion have been shown to regulate hormone secretion, hormone action, and metabolic homeostasis. One
such cytokine molecule, LIF, has recently been shown to
exert striking control of endocrine systems and, as such, has
been elucidated as a key component of endocrine control.
Leukemia inhibitory factor (LIF) is a polyfunctional cytokine of the interleukin-6 (IL-6) cytokine family, sharing the
common gp130 receptor subunit together with IL-6, interleukin-11 (IL-11), oncostatin (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin (CT-1). The leukemia inhibitory factor receptor (LIFR) is a class I cytokine receptor,
belonging to the hematopoietic cytokine receptor superfamily. In addition to classical hematopoietic effects, LIF affects
various endocrine tissues and cell types, including proliferation of primordial germ cells, maintenance of pluripotent
embryonal stem cells, endometrial decidualization and blastocyst implantation, hypothalamus-pituitary-adrenal (HPA)
Address reprint requests to: Dr. Shlomo Melmed, Academic Affairs,
Cedars-Sinai Medical Center, 8700 Beverly Boulevard, 2015, Los Angeles, California, 90048 USA. E-mail: [email protected].
* This work was supported in part by a scholarship of the Deutsche
Forschungsgemeinschaft (Au 139/1–1) and by NIH grant DK 50238.
† Current address: Department of Internal Medicine II, Klinikum
Grosshadern, Ludwig-Maximilians-University, Munich 81366, Germany.
313
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AUERNHAMMER AND MELMED
axis activation and pituitary development, osteoblast and
osteoclast function, adipocyte lipid and energy homeostasis,
and auto/paracrine growth regulation of endocrine-responsive neoplasms.
The topic of LIF in the endocrine system was reviewed in
this journal in 1991 by Kurzrock et al. (1), and several recent
reviews have summarized general aspects of LIF action (2–5).
In the past few years, however, significant new knowledge
has been gained on both LIF signaling as well as immuneendocrine functions of LIF. Recent studies have highlighted
the LIF-induced Jak-STAT (janus kinase-signal transducer
and activator of transcription) signaling cascade, and its negative feedback-regulation by suppressor-of cytokine-signaling proteins (SOCS) and protein inhibitors of activated STAT
(PIAS). A number of recent animal and human studies have
indicated an important immune-endocrine role for LIF in
blastocyst implantation and early pregnancy. Recent studies
in infertile women suggest a potential link for LIF in unexplained failure of implantation in humans. An important
functional role for LIF as a neuroimmune-endocrine modulator in the hypothalamo-pituitary-adrenal axis and in pituitary development has recently been demonstrated. These
findings have strong pathophysiological implications on the
role of LIF in the HPA axis response to various afferent
stimuli including stress and inflammation. There is also increasing evidence favoring a significant role for LIF in bone
development and metabolism, energy metabolism, and as an
auto/paracrine growth factor in endocrine-responsive tumors, including breast cancer.
The recent unraveling of the LIF-induced Jak-STAT signaling and SOCS-mediated autoregulatory feedback, as well
as the immune-endocrine function of LIF in blastocyst implantation and infertility and the neuroimmune-endocrine
modulation of HPA axis activity, link LIF to currently important and topical areas of endocrine research. Taking into
account the recent enhanced understanding of this ubiquitous cytokine and its various functions, this review therefore
focuses on the LIF signaling cascade and its immuno-endocrine functions.
II. LIF—Gene Structure and Regulation
LIF was originally characterized and cloned as a differentiation factor for the murine leukemic M1 cell line. Because
of its multifunctional actions, LIF has been independently
characterized by various groups and named with different
synonyms (including differentiation-stimulating factor, differentiation-inducing factor, differentiation-inhibitory factor, differentiation-retarding factor, human interleukin for
DA cells, melanoma-derived lipoprotein lipase inhibitor, osteoclast-activating factor, cholinergic neuronal differentiation factor, hepatocyte-stimulating factor III), later proven to
be authentic LIF (for review see Refs. 1, 5, and 6).
A. LIF gene and primary structure
To date, the murine (GenBank Accession X06381, M63419
J05435, X12810 m60289, S73374) (7–12), human (GenBank
Accession M63420 J05436, X13967) (12–18), porcine (19),
ovine (19), bovine (GenBank Accession D50337, U63311) (20,
Vol. 21, No. 3
21), rat (22), and mink (GenBank Accession AF048827) (23)
genes for LIF have been cloned.
Southern blot analysis with human and murine probes of
the LIF coding region yield a unique hybridization pattern
(12), indicating a single gene locus. The murine LIF gene is
located on chromosome 11A1 (24, 25), while the human LIF
gene is located on chromosome 22q12.1–12.2 (15, 16, 26 –28).
In the murine and human genome, the LIF gene is in close
proximity to the OSM gene (15, 16, 24 –29).
The length of the murine and human LIF gene is approximately 6.0 kb and 6.3 kb, respectively (12). Northern blot
analysis with a specific murine LIF probe detects a single
approximately 4.2-kb transcript (12) and a similar transcript
size is found for human LIF. The human and murine LIF gene
consist of 3 exons and 2 introns (12). Exon 1 encodes the first
6 amino acids of the hydrophobic leader, exon 2 encodes the
rest of the hydrophobic leader and the first 53 amino acids
of the mature protein, while exon 3 encodes the C-terminal
137 amino acids and an extremely long 3⬘-untranslated region spanning approximately 3.2 kb (12). The human and
murine LIF genes show a high homology of 78 –94% in their
coding regions (12, 19), while the noncoding regions are
much less conserved. At the amino acid level, human and
murine LIF show 79% homology (19). Murine glycosylated
LIF is a 38- to 67-kDa protein (5, 6), which can be deglycosylated to an approximately 20-kDa protein consisting of 180
amino acids (aa), without losing its biological activity (5, 6).
In contrast, N-linked glycosylation of rat LIF at various sites
has been shown to differently alter LIF bioactivity in a cellspecific manner (30, 31).
The minimal LIF promoter extends from ⫺103 to ⫹1 (31),
and this region is totally conserved between murine, human,
ovine, and porcine LIF genes, except for a single insertion
(19). The major transcription start site has been located 60 – 64
bp upstream of the translation initiation codon (12). Further
functional elements in the 5⬘-region of murine LIF are negative regulatory elements between ⫺360 and ⫺249 (31) and
in a GC-rich hypomethylated region between the respective
first exons of diffusible and matrix-associated LIF (11, 32).
Distal positive enhancers, which can overcome the negative
elements, are located in the murine LIF gene at ⫺860 to ⫺661
(33) and at ⫺3,200 to ⫺1,200 nucleotides (11). The presence
of negative regulatory elements might explain the very low
constitutive expression of LIF in most tissues, which, however, can be induced by several cytokines and mitogens.
In addition to the originally described form of murine LIF,
designated diffusible LIF (LIF-D), an alternative form,
termed matrix-associated LIF (LIF-M), has been described
(11, 34). Initially, matrix-associated LIF-M has been considered a murine entity, as it was not found in the human
genome (11, 19, 34). However, recent studies demonstrate the
existence of human and porcine LIF-M (35, 36). In addition,
a truncated LIF version (LIF-T) was found in the murine,
human, and porcine genome (35, 36). These findings indicate
a complex and conserved organization of the mammalian LIF
gene (35, 36). Expression of LIF-D, LIF-M, and LIF-T transcripts differs in a cell-specific manner (35, 36). LIF-M and
LIF-T arise by alternate promoter usage and splicing of different exons 1 to exon 2 and 3 (11, 34, 35). The respective DNA
sequences encoding exon 1 of LIF-M and LIF-T are located
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LIF — MODULATOR OF ENDOCRINE FUNCTION
within the first intron of LIF-D (11, 19, 34 –36). LIF-T lacks an
in-frame initiation codon in exon 1, and a truncated approximately 17-kDa LIF protein is translated from a transcript,
initiated by an in-frame initiation codon in exon 2 (35, 36).
LIF-T is expressed intracellularly, but no significant amounts
are secreted (35, 36). This is due to protein translation initiated downstream of the secretion signaling sequence (35, 36).
Exon 1 of murine LIF-M harbors an in-frame initiation codon,
and the resulting N-terminal protein region directs LIF protein secretion to the extracellular matrix (11, 19, 34). Human
LIF-M—similar to LIF-T transcripts—lacks an in-frame initiation codon in exon 1 (36) but also does not utilize the
in-frame initiation codon in exon 2 (36). As significant
amounts of a secreted 20-kDa LIF protein are translated from
human LIF-M transcripts (36), an atypical mode of translation initiation using a non-AUG codon has been suggested
(36).
B. LIF expression
LIF is expressed and secreted in a variety of tissues and cell
types (for review see Ref. 6). Basal LIF tissue expression is
usually low and often not detectable by Northern blot analysis (6, 37). LIF gene expression can be induced by several
proinflammatory agents, e.g., lipopolysaccharide (6, 37, 38),
IL-1 (6, 39 – 48, 49), IL-17 (48), and tumor necrosis factor-␣
(TNF-␣) (6, 39 – 42, 45, 47, 49), or inhibited by antiinflammatory agents, e.g., glucocorticoids (40, 44, 50, 51), IL-4 (43, 46,
48), and IL-13 (48), respectively.
C. LIF protein tertiary structure
LIF is a long-chain four-␣-helix bundle cytokine (5, 52– 60).
The four-␣-helix bundle cytokines are subdivided into shortchain and long-chain cytokines, as their helices comprise
approximately 15 or 25 residues, respectively (52–54). Crystal
structures have been determined for the long-chain four-␣helix bundle cytokines LIF (57), IL-6 (61), CNTF (62), GH (63),
granulocyte-colony stimulating factor (G-CSF) (64), and leptin (65). Although exhibiting only a low degree of homology
in their primary structures, they show a high homology in
their tertiary structures and in their functional receptor
epitopes (66). The tertiary structure of LIF, from the N to the
C terminus, consists of helices A, B, C, and D, linked by two
long loops AB and CD, as well as the short loop BC (5, 55– 60).
Three functional binding sites, interacting with the LIFR and
gp130 receptor subunit, respectively, have been characterized (67– 69) (Fig. 1A).
III. LIF Receptor—Gene Structure and Regulation
A. The cytokine receptor superfamily
The class I cytokine receptor superfamily (5, 54, 70) is
characterized by structural and sequence similarities in
their extracellular regions containing cytokine-binding
domains (CBD), a single transmembrane domain, and an
intracellular domain of variable length, lacking endogenous kinase activity. Each CBD spans approximately 200
residues, consisting of two fibronectin-type-III (FNT-III)
modules with four conserved cysteine residues in the N-
315
terminal and a Trp-Ser-X-Trp-Ser (WSXWS) motif in the
C-terminal domain (5, 54, 70). In addition, further FNT-III
and Ig-like domains can be identified in the extracellular
region (5, 54, 70). Subfamilies of the class I cytokine receptor superfamily are characterized by either homodimerization of their specific receptor subunit (GH,
PRL, Epo, G-CSF, TPO), or by sharing a common ␤-chain
(IL-3, IL-5, GM-CSF), ␥-chain (IL-2, IL-4, IL-7, IL-9, IL-13,
IL-15), or gp130 receptor subunit (LIF, OSM, IL-6, IL-11,
CNTF, CT-1), respectively (71). The LIFR—also referred to
as low-affinity LIF receptor, LIFR␣, LIFR␤, or gp190 —and
the common gp130 receptor subunit both belong to the
class I cytokine receptor superfamily (72–75).
B. LIFR gene and structure
The human (GenBank Accession X61615) (72), murine
(GenBank Accession D26177, S73496, S73495, S81861,
X99778, X99779) (72, 76 –78) and rat (GenBank Accession
D86345) (79) gene for LIFR␣ have been cloned. The LIFR
gene is located on human chromosome 5p12–13 and murine chromosome 15 within a cluster of cytokine receptor
genes, including IL-7, PRL, and GH receptor (80), suggesting ancestral emergence from multiple gene duplications. The human LIFR gene spans more than 70 kb and
contains 20 exons (81). Alternative promoter usage of the
human LIFR␣ gene yields a placental tissue-specific promoter (GenBank Accession U78104) with a novel placenta
specific enhancer element, as well as an alternative promoter active in nonplacental tissues (GenBank Accession
AF018079) (82, 83).
C. Membrane-bound LIFR
Human LIFR (GenBank Accession X61615), is an approximately 110-kDa protein that is glycosylated to about 190 kDa
at multiple potential N-linked glycosylation sites (72). Northern probe analysis using a probe specific for human LIFR␣
exhibits placental mRNA transcripts of approximately 6.0 kb
and 4.5 kb, as well as a minor transcript of about 5.0 kb (72).
Human LIFR␣ preprotein is 1097 aa, encompassing a signal
sequence of 44 aa, an extracellular domain of 789 aa, a transmembrane domain of 26 aa, and a cytoplasmic domain of 238
aa. Human and murine LIFR␣ share 76% amino acid sequence homology in their extracellular domain (72, 79). The
extracellular region of the LIFR consists of two CBDs separated by an Ig-like domain, and three membrane-proximal
FBT III modules (69, 72) (Fig. 1B).
D. Soluble LIFR
Murine LIFR exists in both a membrane-bound and a soluble form, the latter lacking the transmembrane and cytoplasmic domains. In Northern blot analysis for soluble or
membrane-bound murine LIFR, transcripts yield different
sizes of approximately 3 kb or about 5 kb and 10 kb, respectively (76 –78). Both murine receptor forms are derived from
a single gene locus by alternative splicing. The cDNA of
membrane-bound murine LIFR␣ (Gen Bank Accession
D26177 and S81861) is derived by alternative splicing, skip-
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AUERNHAMMER AND MELMED
Vol. 21, No. 3
FIG. 1. A, The three-dimensional structure of murine LIF [reproduced with permission from D. K. Smith and H. R. Treutlein: Protein Sci
7:886 – 896, 1998 (69)]. MOLSCRIPT diagram of the crystal structure of murine LIF (57) showing binding site regions I to III (68, 107). LIF
is a long-chain four-␣-helix bundle cytokine (5, 52– 60). The tertiary structure of LIF, from the N- to the C terminus, consists of helices A, B,
C, and D, linked by two long loops, AB and CD, as well as the short loop BC (5, 55– 60). LIF possesses three binding regions on distinct epitopes
(66 – 69, 109). Site I binds to the membrane-proximal cytokine binding domain (CBD) of LIFR, while site II binds to the CBD of gp130 (69). Site
III binds also to the LIFR. The membrane-distal CBD as well as the Ig-like domain of the LIFR are currently discussed as putative binding
sites (115, 116). B, Schematic diagram of the LIFR-gp130 complex and its molecular signaling pathways [adapted from T. Hirano (2), T. Taga
and T. Kishimoto (3), C. J. Auernhammer and S. Melmed (201), and A. B. Vojtek and C. J. Der (225)]. Heterodimerization of the LIFR-gp130
complex by LIF activates Jak kinase activity (150 –153), followed by phosphorylation of gp130 and the LIFR (150, 151, 154, 155). Phosphorylated
tyrosine residues on LIFR and gp130 provide specific docking sites for the SH2 domains of STAT proteins (149, 160 –162), causing receptor
association and subsequent phosphorylation of STATs (128, 150, 151, 153–155, 159 –171). The pattern of Jak/STAT protein activation by LIF
is cell type specific (150). Distinct functions of box 1, box 2, and box 3, as well as specific tyrosine residues in the cytoplasmic LIFR and gp130
domains, are discussed in Section IV. Negative regulators of Jak-STAT signaling, e.g., SOCS and PIAS, interfere at specific sites in the signaling
cascade. In addition to activating the Jak-STAT cascade, LIF also stimulates the Ras-MAPK pathway (2–5, 144, 145).
ping an exon that is specific for the soluble LIFR form, and
contains a translation termination codon (77). A B2 repetitive
sequence, contained within the 3⬘-untranslated region of soluble LIFR cDNA (GenBank Accession X99778), may cause
polyadenylation and regulate expression of soluble LIFR (77,
78). Murine serum levels of soluble LIF-R are highest during
pregnancy (84), while a profound increase of soluble LIFR
mRNA has been demonstrated in liver during gestation
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LIF — MODULATOR OF ENDOCRINE FUNCTION
317
TABLE 1. IL-6 cytokine family: overlapping biological effects
Cytokine-mediated effect
IL-6
IL-11
LIF
OSM
CNTF
CT-1
Maintenance of ES cell pluripotentiality
Macrophage differentiation in M1 cells
Growth promotion of myeloma cells
Promotion of thrombopoiesis
Induction of hepatic acute phase proteins
Induction of ACTH secretion in vivo
Induction of ACTH secretion in vitro
Neural differentiation
Induction of bone loss/osteoclast formation
Induction of cardiac hypertrophy in vitro
⫺/⫹
⫹
⫹
⫹
⫹
⫹
⫺/⫹
⫹
⫹
⫺/⫹
⫺
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺/⫹
⫹
nd
⫹
⫹
nd
⫹
nd
⫹/⫺
⫹
⫹
nd
nd
⫹
⫹
nd
⫹
nd
⫹
[Reproduced with permission from C. J. Auernhammer and S. Melmed: In Molecular Regulation, Humana Press, 1999 (201); adapted from
T. Taga and T. Kishimoto (3) and K. Nakashima and T. Taga (4).]
(days 8 –19), peaking at day 12 with an approximately 20-fold
increase (77). Alternative promoter usage for transmembrane
and soluble LIFR has been suggested, as a 5⬘-untranslated
exon 1 is expressed in most tissues, while an alternative exon
1a is restricted to liver and, during gestation, is profoundly
increased in the liver and uterus (85). Murine soluble LIFR
inhibits LIF action in vitro and in vivo (86, 87), thus acting as
an important antiinflammatory modulator of LIF action. Recently, a human soluble LIFR has also been described (81, 88)
and shown to act as an antagonist (88).
Soluble receptors, lacking their respective transmembrane
and cytoplasmic domains by alternative splicing events,
have also been identified for several other IL-6 family cytokines, including sIL-6R (89 –91), sIL-11R (92–94), and
sCNTFR (95, 96). These soluble receptor isoforms possess
agonistic activity with their respective ligand in cell lines
expressing gp130, but lacking the membrane-bound specific
R␣ subunit (89, 93–96). On the other hand, antagonistic
effects have also been observed in other models (91, 94),
indicating a complex modulation of cytokine bioactivity by
soluble cytokine receptors.
E. gp130 Gene and structure
Human (GenBank Accession M57230) and murine (Genbank Accession M83336, X62646) gp130 cDNAs have been
cloned (73, 74). Human gp130 is an approximately 100-kDa
protein in its deglycosylated state, but exhibits 14 potential
N-linked glycosylation sites (73). Northern probe analysis
with a specific probe for the entire coding region of human
gp130 reveals ubiquitous gp130 mRNA expression with a
single transcript of approximately 7.0 kb (73). The human
gp130 gene is located on chromosome 5q11 (97, 98). Human
gp130 preprotein is 918 aa, comprising a 22-aa signal sequence, a 597-aa extracellular domain, a 22-aa transmembrane domain, and a 277-aa cytoplasmic domain (73). The
extracellular gp130 domain consists of a N-terminal Ig-like
domain, a CBD, and three membrane-proximal FNT-III modules (73), thus exhibiting structural similarity with the LIFR␣
(Fig. 1B). The cytoplasmic domain of LIFR and gp130 both
contain three homologous and functionally important motifs, termed box1, box2, and box3 (3, 99). The crystal structure
of the CBD of human gp130 has recently been resolved (75).
Soluble forms of gp130 with a molecular mass of 90 to 110
kDa exist in human serum (100) and have been suggested to
arise by proteolytic cleavage, rather than alternative splicing
(101). Soluble gp130 has also been demonstrated to act as an
antagonist of IL-6 (91, 100, 102) and LIF (101, 102) signaling,
respectively.
F. IL-6 cytokine family and the gp130 receptor subunit
The IL-6 cytokine family is characterized by their receptors
sharing the common gp130 receptor subunit (2– 4) and consists of LIF, IL-6, IL-11, OSM, CNTF, and CT-1. Ligand binding of LIF, OSM, CNTF, or CT-1 causes heterodimerization
of gp130 with LIFR, and a third cytokine-specific receptor
subunit in the case of CNTF and CT-1 (2– 4). In contrast,
ligand binding of IL-6 or IL-11 to their specific receptor
subunits does not involve LIFR and has been suggested to
involve gp130 homodimerization (2– 4), although other models will be discussed below. Due to the shared receptor subunit and signaling cascade of all IL-6 cytokine family members, several of these cytokines show partially overlapping or
redundant hematological effects (2– 4) (Fig. 2 and Table 1).
Recently, two new members of the IL-6 cytokine family,
named cardiotrophin-like cytokine (103) and neurotrophin1/B-cell-stimulating factor-3 (104), have been reported. Initial studies demonstrated cardiotrophin-like cytokine (103)
to involve tyrosine phosphorylation of gp130 and STAT1,
and neurotrophin-1/B-cell-stimulating factor-3 (104) to involve tyrosine phosphorylation of gp130, LIFR, and STAT3
in their respective signaling cascades.
G. LIFR-gp130 complex
The LIFR forms a heterodimer with gp130, enabling LIF
signal transduction (5, 69, 105–108). While LIF binds at relatively low affinity [dissociation constant (Kd) ⬃ 1 ⫻ 10⫺9 m]
to its specific LIFR, subsequent association with gp130 forms
a high affinity (Kd ⬃ 0.1 ⫻ 10⫺10 m) complex (72, 105).
LIF possesses three binding regions on distinct epitopes,
similar to CNTF, IL-6, and IL-11 (66 – 69, 109). Mutagenesis
analysis of each cytokine revealed site I to bind with the
specific cytokine receptor, while site II binds to gp130 (66 – 69,
109). Binding site III has various functions, as it binds to the
LIFR in the case of LIF (67– 69), but allows contact with a
second gp130 molecule in the case of IL-6 and IL-11 (67, 68,
109 –113). In an electrostatic analysis model, derived from the
crystal structure (57, 58) and mutagenesis studies of LIF (67,
68, 114), LIF binds to the membrane-proximal CBD of LIFR
(site I) and to the CBD of gp130 (site II) (69) (Fig. 1B). While
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AUERNHAMMER AND MELMED
Vol. 21, No. 3
FIG. 2. The IL-6 cytokine family and
their receptors. A, IL-6 cytokine family
receptor complexes sharing LIFR and
gp130 [reproduced with permission
from C. J. Auernhammer and S. Melmed: In Molecular Regulation, Humana
Press, 1999 (201); adapted from T.
Hirano (2)].
some data suggest that LIF binding site III binds to the
membrane-distal CBD of the LIFR (69), others have found the
Ig-like domain of the murine LIFR to be essential for highaffinity LIF binding (115, 116). Based on LIFR mutagenesis
studies, two distinct LIF binding sites in the membrane-distal
CBD and Ig-like domain have also been proposed, while the
interacting membrane-proximal CBD was suggested to be
important for protein conformation (116). Phe156 and
Lys159, located in site III at the N-terminal end of the D helix,
are important residues for binding to the LIFR and are conserved in LIF, OSM, CNTF, and CT-1 (68), all of which bind
to the LIFR.
H. The LIFR-gp130 complex signals OSM, CNTF, and CT-1
In addition to LIF signaling, the LIFR and gp130 heterodimer is also required for signal transduction of OSM,
CNTF, and CT-1 (2– 4). Signaling of OSM is achieved by
either a heterodimer of the common LIFR and gp130 (OSM
receptor type I) or the specific OSMR and gp130 (OSM receptor type II), respectively (117–122). While human OSM
activates OSM type I receptors to a similar extent as does LIF
(120), murine (m)OSM exhibits a 30- to 100-fold lower binding and no activation of the OSM receptor type I (120 –122).
In contrast, mOSM specifically activates only the OSM receptor type II (117, 120 –122). CNTF signaling is mediated by
a tripartite complex of CNTFR, LIFR, and gp130 (123–126).
CT-1 also requires the LIFR and gp130 for signaling (127–129)
and has been suggested to form a tripartite receptor complex
similar to CNTF, including LIFR, gp130, and a glycosylated
80-kDa protein (128).
I. IL-6:IL-6R and IL-11:IL-11R complex
In contrast to LIFR-mediated signaling of LIF, OSM,
CNTF, and CT-1, the LIFR is not involved in signaling of IL-6
and IL-11. Both IL-6-IL-6R (130 –136) and IL-11-IL-11R (137–
139) require gp130 for complex formation, recognize two
distinct binding motifs on gp130, and compete for binding to
gp130 (111, 112). In different models (2– 4, 61, 111, 112, 130 –
136), IL-6 binds to its specific IL-6R subunit, causing either
a hexameric complex consisting of IL-6:IL-6R:gp130 in 2:2:2
formation, or homodimerization of gp130 in a tetrameric
complex. A similar model of gp130 homodimerization has
been proposed for IL-11 (2– 4, 109, 111, 112); however, monomeric gp130 further enables a pentameric complex that
consists of two of each IL-11 and IL-11Ra (139).
J. LIF binding and LIFR expression
Low- and high-affinity binding sites for LIF have been
described in several cell types (72, 140, 141). A low number
of approximately 150 – 400 high-affinity binding sites with a
Kd of 10 - 200 ⫻ 10⫺12 m is found on most cells responsive
to LIF. Furthermore, approximately 1,000 – 6,000 low-affinity
binding sites with a Kd of 1– 4 ⫻ 10⫺9 m are present on many
cell types. While LIFRa constitutes the low-affinity binding
site, association of the LIF-LIFR complex with gp130 results
in its conversion to a high-affinity binding site (72, 105).
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LIF — MODULATOR OF ENDOCRINE FUNCTION
A recent study demonstrated the mannose-6-phosphate/
insulin-like growth factor II receptor (Man-6-P/IGFII-R) to
be a nanomolar affinity receptor for glycosylated, but not for
deglycosylated, human LIF (142, 143). Several human cell
lines exhibiting no detectable binding of nonglycosylated
human LIF, revealed 3,000 to 40,000 binding sites for glycosylated human LIF, due to the Man-6-P/IGFII-R (142). Therefore, low-affinity binding of glycosylated human LIF seems
to be not only mediated by the low-affinity receptor LIFR, but
also to a large extent by the Man-6-P/IGFII-R. Binding of LIF
to the Man-6-P/IGFII-R caused no downstream functional
effects, but mediated a rapid internalization and degradation
of LIF (143). Therefore, the Man-6-P/IGFII-R might regulate
LIF bioavailability (143).
IV. LIF Signaling
A. Jak-STAT pathway
All IL-6 cytokine family receptors lack intrinsic kinase
activity. Ligand binding causes conformational changes and
hetero- or homodimerization of their respective receptor subunits, followed by activation of cytoplasmic Janus kinases
(Jaks), tyrosine phosphorylation of the receptor and signal
transducers and activators of transcription (STATs), and further downstream events. The Jak-STAT signaling cascade is
a common signaling pathway, shared by several type I and
type II cytokine receptors and has been extensively discussed
in several recent reviews (2–5, 71, 144 –148).
B. Jaks
Jak 1 and Jak 2 associate with the cytoplasmic receptor
subunits gp130 and LIFR␤ in the absence of ligand, but are
autophosphorylated and activated only after ligand binding
and heterodimerization of the LIFR-gp130 complex (149)
(Fig. 1B). Heterodimerization of the LIFR-gp130 complex by
LIF activates Jak1 (150 –153), Jak2 (151–153), and Tyk2 (151)
kinase activity, followed by phosphorylation of gp130 and
the LIFR (150, 151, 154, 155). Targeted disruption of the Jak1
gene abrogates gp130-mediated signaling (156), while targeted disruption of the Jak2 gene does not abolish LIF or IL-6
responsiveness (157, 158). Similarly, in vitro transient overexpression of a dominant negative Jak1 mutant almost completely abrogated LIF responsiveness, while a dominant negative Jak2 mutant attenuated LIF signaling only by
approximately 30% (159). All these data suggest an essential
role of Jak1 for LIF signaling.
C. STATs
Phosphorylated tyrosine residues on LIFR and gp130 provide specific docking sites for the SH2-domains of STAT
proteins (149, 160 –162), causing receptor association and
subsequent phosphorylation of STAT1 (128, 150, 159 –164)
STAT3 (128, 150, 151, 153–155, 163–169), or STAT5a (170,
171), respectively. The pattern of Jak/STAT protein activation by LIF is cell type specific (150). STAT1(⫺/⫺) embryonic
stem cells derived from mice with targeted disruption of the
STAT1 gene are no longer responsive to interferons (IFNs),
but still respond to LIF (172). However, overexpression of
319
STAT3 dominant negative mutants (173, 174) or lowering of
activated STAT3 levels (175) inhibits LIF-induced maintenance of pluripotent embryonic stem cells. Similarly, overexpression of STAT3 dominant negative mutants also inhibits LIF-induced differentiation of leukemic M1 cells (176),
POMC, and SOCS-3 expression of corticotroph AtT-20 cells
(177, 178), as well as c-fos and atrial natriuretic factor expression in cardiocytes (150). These results demonstrate a
compelling role of STAT-3 for LIF-signaling in several cell
types (Fig. 1B).
D. Cytoplasmic receptor domains
Using chimeric receptor models, homodimers of gp130 as
well as LIFR␤ were shown to be sufficient for STAT3 tyrosine
phosphorylation (179). Carboxy-terminal truncation of the
cytoplasmic gp130 or LIFR␤ domain, respectively, revealed
that the membrane-proximal box 1 and box 2 regions are not
sufficient for STAT3 phosphorylation (161, 152, 179). In contrast, the 74 membrane-proximal aa of the LIFR␤ are sufficient for binding of Jak1 and Jak2 (149). Further mutagenesis
analysis demonstrated a consensus sequence YXXQ, located
on several membrane-distal locations in the cytoplasmic domains of gp130 and LIFR␤, respectively, which is required
for STAT3 association with the receptor and subsequent
phosphorylation (Fig. 1B). Thus, tyrosine phosphorylation of
the YXXQ motif provides a binding motif for the highly
specific SH2-domain of STAT3 (160, 162, 179). Binding of
STATs to the cytoplasmic receptor subunit causes a closer
steric association with Jak kinases, which may result in STAT
tyrosine phosphorylation. Phosphorylation of C-terminal tyrosine sites in STAT3 (Tyr 705) and in STAT 1 (Tyr 701) causes
the SH2 domains to enable homo- or heterodimerization of
STAT-3-STAT3, STAT1-STAT3, or STAT-1-STAT1, respectively (146, 147). Crystal structure analysis of STAT3 and
STAT1 homodimers demonstrates that the SH2 domain of a
STAT monomer binds to the C-terminal phosphotyrosine of
the other, thus enabling homodimerization (180, 181). The
dimerized STAT complexes are translocated to the nucleus,
and their DNA-binding domain (aa 400 –500) binds to specific DNA STAT-binding elements (SBE), causing transcriptional activation (146, 147, 182) (Fig. 1B). In addition to primary tyrosine phosphorylation, IL-6 cytokine family
members also cause secondary serine phosphorylation of
STAT3 and STAT1 (147, 169, 183–187). Secondary serine
phosphorylation of STATs has been controversially shown to
either enhance DNA binding of STAT3-STAT3 complexes
(183) or to have no effect (147, 169, 186, 187). However,
despite not directly effecting DNA binding of STAT3 complexes, serine phosphorylation of STAT3 seems to be required for full transactivation of STAT-responsive genes
(147, 169, 186, 187).
E. Negative feedback regulators of Jak-STAT signaling
Negative feedback regulators of gp-130-mediated activation of Jak-STAT signaling include the tyrosine phosphatase
SHP-2, as well as members of the newly described SOCS and
PIAS protein families. Those negative feedback regulators
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negatively interfere with the LIF-induced Jak-STAT signaling cascade at different levels (Fig. 1B).
spectively, and inhibit their binding to specific DNA sequences.
F. SHP-2
I. Mitogen-activated protein kinase (MAPK)
The SH2-containing protein tyrosine phosphatase-2
(SHP-2) is a cytosolic protein involved in regulation of tyrosine kinase-mediated signaling pathways (for review see
Refs. 188 –190). LIF stimulates tyrosine phosphorylation of
SHP-2 (191) by a Jak-1-dependent pathway (192). After LIF
stimulation, SHP-2 associates with the cytoplasmic gp130
receptor subunit (191). A membrane-proximal tyrosine phosphorylation site in the cytoplasmic domain of the gp130
receptor (Y118) is essential for tyrosine phosphorylation of
SHP-2 (179, 193–195). Overexpression of dominant negative
SHP-2 variants (194, 196, 197) or a mutated gp130 subunit
lacking the cytoplasmic binding site for SHP-2 (194) significantly enhanced CNTF- or LIF-induced effects in different
cell models. Therefore, a negative feedback regulation of
SHP-2 on gp130-mediated STAT activation has been suggested.
In addition to activating the Jak-STAT cascade, several IL-6
cytokine family members also stimulate the Ras-MAPK pathway (2–5, 144, 145) (Fig. 1B). In the Shc/Grb2/SOS/Ras/
Raf/Mek/Erk signaling cascade, serine/threonine kinases
Erk1/2 themselves activate numerous nuclear transcription
factors, as well as cytosolic and cytoskeletal targets (for review see Refs. 144, 145, and 225).
LIF has been demonstrated to stimulate Shc (152), Ras (152,
226), Raf-1 (227), MAPKK (228), as well as Erk1 and Erk2
activity (141, 150, 152, 165, 168, 173, 227–229). The ability of
LIF to induce MAPK tyrosine phosphorylation and activity
is cell-type specific (141, 152, 168, 177, 227–229) and is probably essentially required for distinct LIF activities, while it is
not required for others (152, 161, 177). SHP-2 has been shown
to be essential for LIFR/gp130-mediated activation of MAPK
(161, 192, 229, 230), despite its inhibitory function on gp130mediated STAT activation (194, 196, 197). As discussed
above, LIF-induced SHP2 activation requires specific cytoplasmic tyrosine residues on gp130 (Y118) and LIFR (Y115)
(161, 179, 193–195). Deletion of these essential tyrosine residues or coexpression of a dominant-negative SHP2 mutant
blocks subsequent MAPK activity (161, 229). These data suggest LIFR/gp130-stimulated MAPK activity to be mediated
through activation of SHP-2 (Fig. 1B). Phosphatidylinositol
(PI) 3-kinase also seems to be an essential mediator of LIFand IL-6-induced MAPK activation (230, 231), as the PI-3kinase inhibitor wortmannin inhibits LIF- and IL-6-induced
activation of MAPK activity, while STAT3 phosphorylation
was mostly unaffected.
Bidirectional interactions of the Jak-STAT and the RasMAPK pathway are suggested by several lines of evidence.
The LIFR itself is a target of LIF-induced MAPK activity and
is phosphorylated on Ser-1044 in its cytoplasmic domain
(229). Whether secondary serine phosphorylation of STAT3
(183, 184) depends on MAPK activity is controversial (167,
169, 185, 232). The biological significance of secondary serine
phosphorylation of STATs is also still controversial and
might differ among cell types (169, 183–185, 233). Recently,
activation of Erk1/2 has been demonstrated to inhibit Jak1
and Jak2 kinase activity, while serine phosphorylation of
STAT3 did not play an essential role (233). Based on these
data, a close interaction of the Jak-STAT and Ras-MAPK
pathways is now apparent. Further studies are needed to
understand these interactions.
G. SOCS proteins
SOCS proteins are a new family of proteins termed suppressors of cytokine signaling (SOCS), STAT-induced STAT
inhibitors (SSI), cytokine-inducible SH2 containing protein
(CIS), and Jak-binding protein (JAB). Several current reviews
have summarized the fast growing knowledge on this protein family (198 –201). SOCS-1 and/or SOCS-3 can inhibit the
signaling cascade of several Jak-STAT-dependent cytokines,
including the gp130 sharing cytokines LIF (202–207), IL-6
(202, 208 –210), OSM (202), and CNTF (211), as well as GH
(212), PRL (213), leptin (214, 215), IL-4 (216, 217), and IFNs
(218, 219). Overexpression of SOCS-3 inhibits LIF-induced
phosphorylation of gp130 and STAT3, as well as STAT3mediated downstream events (206). Recent studies revealed
SOCS-1 to inhibit Jak2 activity by binding to the catalytic JH1
domain of Jak2 (204, 207–210). A similar mechanism of JakSTAT inhibition has also been suggested for SOCS-3 (209,
215, 220), while others suggested a slightly different mechanism with no direct inhibition of Jak kinase activity (221).
SOCS protein expression is stimulated by multiple cytokines
in a tissue- and cell type-specific manner (198 –202). As both
SOCS-1 (203) and SOCS-3 (178) gene expression have been
demonstrated to be STAT-3 dependent, there exists a negative autoregulatory feedback of SOCS-1 and SOCS-3 on their
own gene expression. In addition, a recent study also found
STAT-independent induction of SOCS-3 gene expression by
IL-10 (222), while we observed induction of SOCS-3 by IL-1␤,
which was not mediated by the ⫺72 to ⫺64 STAT-RE in the
SOCS-3 promoter (our unpublished results).
H. PIAS
Another family of negative regulators of STAT signaling,
termed PIAS, has recently been described (223, 224). In contrast to SOCS proteins inhibiting Jak activity and subsequent
STAT phosphorylation and activation, PIAS1 and PIAS3 interact directly with both activated STAT-1 and STAT3, re-
J. Others
1. Insulin receptor substrate (IRS). IRS proteins are adaptor
proteins with multiple tyrosine phosphorylation sites, serving as docking sites for SH2-domains of various proteins. IRS
proteins are involved in signaling of insulin and various
cytokines (for review see Refs. 145 and 234). In 3T3-F442A
fibroblasts, LIF stimulates tyrosyl phosphorylation of IRS-1
(235) and IRS-2 (236), respectively. Phosphorylated IRS-1 or
IRS-2 associates with various proteins, including p-85 reg-
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LIF — MODULATOR OF ENDOCRINE FUNCTION
ulatory subunit of phosphatidylinositol 3⬘-kinase, Grb-2, or
protein tyrosine phosphatase SHP-2, respectively (145, 234).
As these molecules have been ascribed to involvement in LIF
signaling, IRS proteins likely play a modulatory function in
the LIF signaling cascade and therefore merit further investigation.
2. Tyrosine kinases. In addition to the Jak-STAT signaling
cascade, several other cytoplasmic tyrosine kinases are activated by IL-6, including Btk (237), Tec (237), Fes (238),
p59Fyn (239), p56/59Hck (239), and p56Lyn (239). Because of
largely overlapping actions in the IL-6 cytokine family, there
might also be a potential role for some of these kinases in the
LIF signaling pathway. So far, to the best of our knowledge,
only LIF-induced activation of Hck has been reported (152,
240). Therefore, further investigation should elucidate the
potential involvement of these cytoplasmic kinases in LIF
signaling.
V. LIF—Hematopoietic and Neuropoietic Cytokine
A. Hematopoietic system
As this review is focused on the endocrine actions of LIF,
the hematopoietic effects of LIF are only briefly discussed
herein, and the topic is extensively reviewed elsewhere (4, 5,
241).
LIF was first cloned in 1987 and characterized by its ability
to stimulate differentiation of the murine myeloblastic leukemia cell line M1 (7). Thereafter, numerous studies used M1
cells as an in vitro model for studying LIF binding on LIFR/
gp130 complex, intracellular mechanisms of LIF action (171,
176), and regulation of the Jak STAT-induced signaling cascade by SOCS proteins. Studies overexpressing SCL (242,
243), flt3 ligand (244), or Wilm’s tumor suppressor gene (245)
in M1 cells have partially enlightened the downstream
events of LIF-induced M1 cell differentiation.
Due to the close similarities of the gp130-related IL-6 cytokine family, several of these cytokines show partially overlapping or redundant hematological effects (2– 4) (Table 1).
Animal models have confirmed important involvement of
gp130-related cytokines on the hematopoietic system. Targeted disruption of gp130 causes death of homozygous murine embryos (gp130 ⫺/⫺) between 12.5 days postcoitum
(pc) and term, because of cardiac hypoplasia and hematological disorders with greatly reduced fetal liver pluripotential and committed hematopoietic progenitor cells (246).
Postnatally induced inactivation of gp130 resulted in a less
pronounced decrease in hematopoietic progenitor cells,
while in the peripheral blood, reduced platelet counts was
the most striking finding (247). Homozygous LIF knockout
mice (LIF ⫺/⫺) have reduced numbers of pluripotent hematopoietic stem cells in spleen and bone marrow and impaired thymic maturation (248), indicating an important role
of LIF in hematopoietic stem cell survival/proliferation.
Strikingly, homozygous LIFR knockouts (LIFR ⫺/⫺) did not
reveal major hematological abnormalities and showed normal colony formation of pluripotent hematopoietic progenitor cells (249). Taken together, these animal models demonstrate that gp130-related cytokines, including LIF, are
essential for hematopoiesis.
321
Although devoid of intrinsic proliferative action, LIF acts
as a hematopoietic growth factor that synergistically costimulates hematopoietic progenitor cell proliferation (241,
250, 251). Another indirect mechanism of LIF action on hematopoietic stem cells is mediated by LIF-induced upregulation of stromal bone marrow-derived cytokines (252).
LIF stimulates megakaryocyte proliferation and platelet production (241, 253–257) and specifically induces proliferation
of IL-3-stimulated murine and human megakaryocytes in
vitro (241, 253). In vivo experiments in mice (241, 254, 255) and
primates (256, 257) demonstrate that daily LIF administration for 1–2 weeks causes an approximately 2-fold increase
in circulating platelet levels. Recent studies also suggest costimulatory effects of LIF on murine and human erythroid
(258, 259) and macrophage (260, 261) progenitor cells.
In addition to IL-6, other members of the IL-6 cytokine
family, including LIF, also play an important role in stimulating survival and proliferation of multiple myeloma cells.
This topic has been recently reviewed (262, 263). LIF stimulates myeloma cell growth, probably acting as a paracrine
growth factor (107, 264).
B. Nervous system
LIF has been termed a cytokine at the interface between
neurobiology and immunology (265); in addition to its effects
on the hematopoietic system, various neuropoietic effects
(265, 266), e.g., switching of sympathetic neuronal phenotype
and rescue and differentiation of sensory and motor neurons
as well as glia cells, have been demonstrated. This manuscript will only briefly discuss recent knockout animal studies, elucidating the physiological roles of LIF, LIFR, and
gp130 in the nervous system. For extensive information on
LIF- and LIFR-mediated effects in the nervous system, we
recommend the comprehensive review provided by Murphy
et al. (266).
LIF stimulates cholinergic differentiation of sympathetic
neurons in vitro (267) and in vivo (268). However, studies on
mice deficient in LIF or CNTF demonstrated that neither LIF
nor CNTF is essential for cholinergic differentiation of sweat
gland sympathetic neurons (269). On the other hand, in vitro
blockade of LIFR in neuron/gland cocultures inhibited cholinergic differentiation activity (270). These data demonstrate
that cholinergic differentiation of sympathetic neurons requires LIFR activation, while LIF and CNTF can act as stimuli. However, as LIF- and/or CNTF-knockout mice show
intact cholinergic differentiation of sympathetic neurons,
other cytokines acting through the LIFR can probably compensate for their deficiency.
Gene knockout studies have demonstrated differentiation
of astrocytes and expression of the astrocyte marker glial
fibrillary acidic protein (GFAP) to be mediated by LIFR (249,
271, 272) and gp130 (273, 274). Furthermore, stimulation of
astrocyte differentiation is dependent on the Jak-STAT pathway (271, 275), as shown by mutation analysis of chimeric
cytoplasmic gp130 or LIFR components and overexpression
of STAT3 dominant negative mutants (271). In LIF knockout
animals, decreased numbers of GFAP-positive cells are
found in the hippocampus (273, 276). These data indicate LIF
to be essential for differentiation of astrocytes in certain brain
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AUERNHAMMER AND MELMED
areas. However, other cytokines acting through the LIFRgp130 receptor complex might compensate for most neurotrophic LIF actions.
VI. LIF and Endocrine Systems
A. Utero-placental unit— blastocyst implantation
LIF is an important cytokine in early pregnancy because of
its essential function in uterine blastocyst implantation. The
critical role of LIF in blastocyst implantation is derived from
in vivo and in vitro animal studies, as well as results of human
studies, including several recent reports, that indicate a link
of altered LIF expression in the utero-placental unit to unexplained fertility with failure of implantation. Several recent
reviews on this topic provide complementary information
(277–282).
1. Knockout models and implantation. Striking evidence for the
essential role of LIF in embryonic implantation was provided
by Stewart et al. (283), who showed that female LIF knockout
LIF⫺/LIF⫺ mice are infertile, due to a defect in endometrial
decidualization and embryonic implantation. Blastocyst
transfer from female LIF⫺/LIF⫺ mice to pseudopregnant
wild-type (wt) LIF⫹/LIF⫹ mice resulted in implantation and
successful pregnancies. Treatment of LIF⫺/LIF⫺mice with
recombinant LIF also enabled successful implantation. Thus,
in the mouse, LIF plays an obligatory role in embryonic
implantation. The requirement of LIF for successful murine
implantation seems to be similar for other mammals, as passive immunization of ewes and cows against LIF results in
a reduced pregnancy rate (284). Due to multiple systemic
abnormalities, both the LIFR⫺/LIFR⫺ (249) and gp130⫺/
gp130⫺ /(246) knockout models are not viable beyond term,
and therefore implantation in female knockout animals cannot be studied in these models.
Recently, the essential role of IL-11, another member of the
IL-6 cytokine family, has been demonstrated for successful
decidualization. Robb et al. (285) demonstrated female IL11Ra⫺/IL-11Ra⫺ knockout mice to be infertile due to defective decidualization. This lesion appeared isolated, as the
female IL-11Ra⫺/IL-11Ra⫺ mice exhibited otherwise normal
estrous cycle, oozyte fertilization, and blastocyst development. Male IL-11Ra⫺/IL-11Ra⫺ mice are normally fertile. As
members of the IL-6 cytokine family exhibit partially overlapping and redundant functions, these findings resemble
the results in LIF⫺/LIF⫺ knockout mice (283). However,
RNAse protection analysis showed a distinct temporal pattern of decidual cytokine expression, indicating a cascade of
different events. While decidual LIF expression peaked on
days 2.5 to 3.5 pc and rapidly declined thereafter, IL-11
expression peaked at days 5.5 to 7.5 pc (285). Thus, despite
partially overlapping functions in promoting uterine decidualization and blastocyst implantation, LIF and IL-11 appear
to act in a specific temporal cascade in the uterus.
2. Uterine LIF expression. Uterine LIF expression in adult
virgin mice is barely detectable during diestrous, proestrous,
and metestrous II (286), but peaks during estrous and
metestrous I, corresponding to late endometrial proliferative
and early secretory phases, including ovulation (286). During
Vol. 21, No. 3
early pregnancy, uterine LIF expression at day 0.5 pc is
similar to that found during estrous and metestrous I. At day
1.5 and 2.5 pc, uterine LIF expression declines, but peaks at
day 3.5 pc shortly before implantation of the blastocyst. After
implantation at day 4.5 pc, uterine LIF expression then declines again, becoming nearly undetectable (286). Thus, peak
endometrial LIF expression occurs early, preceding or coinciding with the time of blastocyst implantation, in the mouse
(286 –288), rat (289), rabbit (290), pig (291), mink (23), and
western spotted skunk (292). In contrast, in sheep endometrial LIF expression is relatively constant throughout the
estrous cycle and early pregnancy (293), and in pseudopregnant mice (287) and rabbits (290) the pattern of uterine LIF
expression is similar to that observed in pregnant animals.
These data indicate endometrial LIF expression to be under
maternal control, independent of stimuli from the conceptus.
In humans, LIF mRNA and protein are maximally expressed in endometrial samples derived from normal cycling
women in the mid and late secretory phase (45, 294 –298).
Glandular and luminal epithelial cells account for the majority of endometrial LIF mRNA and protein (45, 294, 297–
299). Immunohistochemical studies show luminal and glandular epithelial LIF staining to be cycle dependent, peaking
during the mid and late secretory phase (294, 297–299). In
contrast, during the proliferative phase, luminal and glandular epithelial LIF mRNA and protein were not at all (294,
298) or only faintly (297, 299) expressed. Results of immunohistochemical studies of LIF expression in stromal cells are
controversial. Stromal cells consist mostly of fibroblasts and
leukocytes (300). While some studies report modest LIF expression in endometrial stromal cells only during the proliferative (299) or secretory phase (294), respectively, others
found stromal LIF constantly expressed throughout the menstrual cycle (292). Thus, human endometrial luminal and
glandular cells are the major contributor of endometrial LIF
mRNA and protein expression and show a cycle-dependent
peak of LIF expression in the mid and late secretory phase.
This is also the timepoint when blastocyst implantation occurs and suggests an important role for LIF in human implantation, as has been demonstrated in the LIF knockout
mouse (283).
In vitro, a permanent human epithelial endometrial cell
line has been shown to produce small amounts of LIF (291).
Explant cultures from human endometrial glandular epithelial cells exhibit significantly higher LIF mRNA expression
and secretion than do stromal cells (45, 296, 301, 302). In vitro
LIF expression by stromal cells was also induced by IL-1␣,
TNF␣, platelet-derived growth factor (PDGF), epidermal
growth factor (EGF), and transforming growth factor-␤
(TGF␤), while interferon-␥ inhibits LIF expression in these
cells (45). Similarly, LIF secretion from first-term decidual
cells is stimulated by IL-1, TNF␣, and TGF␤ (303).
3. LIF effects on decidual cell cultures. During pregnancy, decidual cells as well as cytotrophoblasts express LIF mRNA
and protein. Decidual culture explants from pregnant
women show significant LIF production and secretion (304,
305), which correlates with the pregnancy duration (304).
High levels of LIF are encountered during the first trimester
and at term, but lower LIF secretion occurs during the second
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LIF — MODULATOR OF ENDOCRINE FUNCTION
trimester (304). LIF secretion by decidual explants derived
from women with early ectopic pregnancy between days 35
to 76, all showed high levels of LIF secretion, irrespective of
the pregnancy term (305). A stimulatory effect of estradiol on
LIF secretion was observed in this primary culture model
(305).
Serum levels of LIF are lower in pregnant women in comparison to nonpregnant women (306), while serum levels of
soluble LIF-R increase severalfold during pregnancy in mice
(84) and humans (306). Although the significance of these
findings is not yet understood, locally produced uterine LIF
seems to act in an autocrine/paracrine fashion rather then
systemically. The soluble LIF-R, however, might act as a
negative regulator (84, 306), modulating local as well as systemic LIF actions.
4. Hormonal regulation of uterine LIF expression. Hormonal
regulation of endometrial LIF expression is not fully understood. Data on possible effects of estrogen or progesterone on
endometrial LIF expression are contradictory. Comparison
of in vivo studies in different species is complicated by variation of implantation type, modus, and hormonal regulation
(281, 307). In vitro, primary endometrial cell cultures are
derived from different sources, including different cell subtype enrichments, primary cultures started at different stages
of the ovulatory cycle, or early pregnancy, respectively.
Implantation in mice is dependent on estrogens, and endometrial LIF protein expression in ovariectomized mice is
up-regulated by estrogen, while progesterone has no stimulatory effect (307). Mixed monolayers derived from whole
murine uteri at day 3 pc exhibited expression of diffusible LIF
in an RNAse protection assay (308). However, expression of
diffusible LIF was not altered by estrogen, progesterone, or
a combination of estrogen plus progesterone treatment (308).
In contrast to mice, rabbits are not dependent on maternal
estrogens for implantation, and endometrial LIF protein expression is up-regulated by progesterone, while estrogen has
no effect (307). In ovariectomized ewes, both estrogen and
progesterone had an inhibitory effect on endometrial LIF
expression (293).
Although LIF expression in human glandular endometrial
epithelial cells is highest during the progesterone-driven secretory phase (45, 294 –298), data on the effects of estradiol
and progesterone on LIF expression are incongruent, which
in part might be explained by tissue- and phase-specific
effects. Reporter gene activity of a human LIF promoter
luciferase construct was stimulated 3.5- to 7-fold by medroxyprogesterone acetate in uterine tumor SKUT-1B cells,
cotransfected with progesterone receptor A or B, respectively
(309). Treatment of fertile women with 200 mg of the progesterone antagonist, mifepristone, on day LH⫹2 resulted in
a decreased LIF expression in glandular endometrial epithelial cells on day LH⫹6, while the steroid had no effect on
luminal epithelial or stromal cells (310). In contrast, in a
primate model, treatment of rhesus monkeys with 2 mg/kg
mifepristone on day LH⫹2 had no effect on endometrial LIF
expression on day LH⫹6 (311). In vitro, estradiol, progesterone, and medroxyprogesterone acetate have been reported to
lack an effect on endometrial stromal cells (45). These data
would explain why stromal LIF expression is not cycle de-
323
pendent (294, 297, 299). LIF expression in epithelial endometrium explant cultures slightly decreases during incubation with estradiol and progesterone (302). ␤hCG did not
show an in vitro effect on LIF expression of mixed endometrial cells derived from women undergoing oocyte retrieval
for in vitro fertilization (IVF) (312).
5. Uterine LIFR and gp130 expression. Similar to uterine LIF
expression during early pregnancy, expression of LIFR and
gp130 in the endometrium is up-regulated during early pregnancy (288, 313, 314). LIF binding and gp130 immunoreactivity peak on days 3 and 4 of mouse pregnancy (288) and
days 5 and 6 of rabbit pregnancy (313), while blastocyst
implantation takes place on day 3.5 pc in the mouse and day
7 pc in the rabbit, respectively. Using in situ hybridization,
murine LIFR and gp130 mRNA expression were detected in
decidual tissue, with highest expression evident in decidua
directly surrounding the embryo (314). By day 8.5 pc, LIFR
expression decreased and was only detectable in a small area
near the placenta, while gp130 mRNA increased in the whole
decidua beyond day 8.5 pc. Northern blot analysis revealed
a 3.0- and 10.0-kb decidual LIFR transcript, compatible with
the soluble and membrane-bound form of the murine LIFR
(314). From preimplantation to day 8.5 pc, LIFR and gp130
mRNA was also expressed in uterine endometrial glands
(314). Several possible functions of LIFR and gp130 in the
murine decidua have been proposed (314), including the
notion that LIF may act directly, regulating decidual growth
and maturation.
Using Northern blot analysis of total RNA, human LIFR
could not be detected in endometrial samples derived during
the proliferative or secretory phase (298, 315), while gp130
was low but constitutively expressed, peaking during the
secretory phase (315). Low LIFR mRNA expression was
found in first trimester decidua, while chorionic villi of the
first trimester exhibited high expression of LIFR mRNA
(315). Using in situ hybridization, human LIFR was found to
be expressed in the luminal epithelium of the endometrium,
but not in glandular epithelium or stromal cells (298). gp130
Was detected in luminal as well as glandular epithelium
(298).
6. LIF in follicular fluid. LIF is present in human follicular fluid
from women undergoing IVF and embryo transfer (316 –
318). LIF levels in follicular fluid were significantly higher in
preovulatory follicles derived from women after ␤hCG treatment, as compared with immature follicles derived from
women before ␤hCG treatment (316, 317). Cultured ovarian
stromal cells (316) as well as granulosa-lutein cells (316, 317)
exhibit low constitutive LIF expression. Furthermore, cultured granulosa cells derived from mature follicles exhibit
increased LIF production after ␤hCG stimulation, while
granulosa cells from immature follicles do not respond to
␤hCG (317). Thus, LIF might be involved in ovulation and/or
final oocyte development. However, no correlation between
LIF levels in follicular fluid and IVF outcome could be established (318), and LIF⫺/LIF⫺ mice exhibit normal blastocyst formation rates before implantation (283). Therefore, LIF
does not appear to be essential in this process, or its loss
might be compensated by other factors.
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7. Embryonic LIF, LIFR, and gp130 expression. By RT-PCR, LIF
mRNA is first detectable in the murine embryo at the morula
stage, while LIFR and gp130 mRNA are first detectable at the
blastocyst stage (314). In situ hybridization reveals a distinct
localization of LIF expression in the trophoectoderm, but not
in the inner cell mass, while LIFR and gp130 are primarily
localized in the inner cell mass (314). This expression pattern
is highly suggestive of a paracrine regulation of the inner cell
mass by trophoectodermal-derived LIF (314).
In vitro, cultures of mouse embryos for 7 days in LIFsupplemented medium resulted in an approximately 40 –
50% increased inner cell mass and trophoblastic area, in
comparison to embryos cultured in nonsupplemented medium (308). In vitro, supplementing culture media with LIF
increases the percentage of murine eight-cell embryos to
develop beyond the hatched blastocyst stage, to hatch or
exhibit trophoblast outgrowth in vitro (308).
Human morula- and blastocyst-stage embryos also express gp130 mRNA and LIFR mRNA (295, 319, 320). Furthermore, gp130 (321) and LIFR (322) are expressed on cytotrophoblasts.
LIF had no effect on cell proliferation or expression of
integrins ␣1, ␣5, or ␤1 by cultured human trophoblast cells
(322). However, LIF affects cytotrophoblast ␤hCG and fibronectin production in a phase-specific manner, although
the available data are discordant. LIF increases ␤hCG production of cytotrophoblasts derived from first trimester placentas (304, 322), while it decreases ␤hCG production of
cytotrophoblasts purified from placentas of term pregnancies (304, 323). In contrast, another study reported a decrease
of ␤hCG production in cytotrophoblasts derived from first
trimester (324). LIF also inhibits forskolin- and cAMPinduced ␤hCG production by the human choriocarcinoma
cell lines BeWo (313) and JEG-3 (304). LIF also increases
fibronectin expression by cytotrophoblasts purified from
placentas of term pregnancies (323), while no effect was
observed in cytotrophoblasts derived from first trimester
placentas (324).
8. LIF and embryonic stem cells. LIF is essential for inhibition
of pluripotent embryonic stem cell differentiation (152, 161,
173–175, 197, 308, 325–330) and promotion of primordial
germ cell growth by inhibiting apoptosis (331–336). These
features attribute LIF an essential factor for in vitro maintenance and growth of pluripotential ES cells and PG cells. For
inhibition of pluripotent embryonic stem cell differentiation
by LIF, STAT3 activation is essential, while SHP2 and MAP
kinase activation are not required (330). Interestingly, recent
data demonstrate that some pluripotent ES cells may be LIF
independent (337, 338). This LIF-independent alternative
pathway of pluripotent ES cell maintenance provides a possible explanation why gp130 (246) and LIFR (249) could be
successfully targeted to generate knockout mice. Also, the
primordial germ cell compartment was not affected in LIFR
knockout mice (249), indicating the presence of an alternative
developmental pathway. LIF action on ES and PG cells is
important for in vitro research, as well as laboratories interested in IVF or gene targeting procedures.
9. Uterine LIF and immune cells. The action of LIF as an “interplayer” between the immune and endocrine systems has
Vol. 21, No. 3
recently been reinforced by a study demonstrating LIF production by decidual T cell leukocytes (322, 339) producing
also the TH2 cytokine subset (IL-4, IL-10) (339). Progesterone
stimulates T cell LIF production via IL-4 (339). Production of
LIF, IL-4, and IL-10 was significantly reduced in decidual
CD4⫹ cell clones derived from women with unexplained
recurrent abortions in comparison to control fertile women,
while no differences were observed in the respective peripheral blood T cells of both groups (339). Similarly, another
study reported that while decidual CD56⫹CD16⫺ NK cells
and CD3⫹ T cells expressed LIF, no LIF expression was
evident in the same peripheral blood leukocyte subsets (340).
These results indicate an important microenvironmental role
of decidual CD4⫹ cells as mediators of paracrine LIF secretion. This hypothesis is also strengthened by a recent animal
study (341) reporting that intravenous administration of
CD4⫹ thymocytes in pseudopregnant mice increased uterine LIF mRNA expression and successful implantation rate
after blastocyst transfer.
10. LIF and failure of implantation. Although the role of LIF in
human embryonic implantation is not totally resolved, recent
studies have linked some cases of unexplained infertility to
an altered pattern of endometrial LIF expression (302, 339,
342, 343) or to LIF gene mutations (344), possibly causing
decreased availability or biological activity of LIF in the
uterus. In two studies, endometrial explant cultures derived
from women with unexplained infertility and multiple implantation failures demonstrated reduced LIF expression in
the secretory phase to approximately 30 – 40% of LIF secretion by control fertile women (342, 343). Another study examining LIF content in uterine flushings showed that on day
LH⫹10, intrauterine LIF was lower in samples obtained from
women with unexplained infertility than from control fertile
women (302). As mentioned above, production of LIF, IL-4,
and IL-10 was reduced in decidual CD4⫹ cell clones derived
from women with unexplained recurrent abortions in comparison to control fertile women (339). In a study examining
nulligravid infertile women (n ⫽ 74) and fertile controls (n ⫽
75), heterozygous point mutations in the coding-region of the
LIF gene were found in three infertile women, while only one
point mutation/polymorphism in the non-coding region
was observed in the fertile women (344). During the proliferative phase (cycle day 10), endometrial LIF concentrations
correlate negatively with sonographic endometrial thickness
(345), suggesting a role for LIF not only in the secretory
phase, but also in the proliferative phase of the cycle. These
data indicate that the low amounts of endometrial LIF expressed during the proliferative phase are physiological,
while higher LIF concentrations appear associated with disturbed endometrial proliferation (345).
11. LIF in ectopic pregnancy. A recent study showed elevated
LIF expression in human fallopian tubes, located ipsi- and
contralateral to ectopic pregnancies (346). Cultured human
fallopian tube epithelial cells showed a high constitutive LIF
expression and secretion, while LIF expression in stromal
cells was significantly induced by the inflammatory cytokines IL-1␣ and TNF␣ (346). Similarly, in cultured bovine
oviduct cells, LIF expression is stimulated by TNF␣ (347).
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LIF — MODULATOR OF ENDOCRINE FUNCTION
These findings might provide a link between salpingitis and
ectopic implantation, caused by higher tubal LIF production,
thus providing a more favorable milieu for ectopic implantation.
12. Summary. In summary, LIF has been demonstrated in
several knockout models to be essential for successful murine
implantation. In humans, available data also indicate an important auto-/paracrine role for LIF in implantation. Endometrial LIF expression is regulated during the menstrual
cycle, peaking in the postovulatory/secretory phase. Some
women with previously unexplained infertility show reduced endometrial LIF expression. Further studies are required to examine the incidence and role of disturbed LIF
expression in unexplained infertility. The underlying pathophysiology of disturbed endometrial LIF expression or LIF
action require further characterization and should provide
new therapeutic strategies in the treatment of previously
unexplained infertility. Auto/paracrine LIF actions on the
preimplantation embryo suggest that there might also be a
therapeutic use for LIF in IVF procedures.
B. Hypothalamo-pituitary-adrenal axis
Much insight has recently been gained regarding the
neuro-immuno-endocrine interface involving different cytokines and pituitary function. Cytokines are secreted as auto/
paracrine factors from pituitary cells and are involved in
pituitary development, cell proliferation, and tumor formation, as well as modulation of hormone secretion. Several
recent reviews address various aspects of this topic (348 –
353). As the auto/paracrine role of LIF in pituitary function
and development has recently received increasing attention,
a comprehensive review of our current understanding of LIF
in the pituitary follows.
1. LIF in corticotroph function.
a. LIF and LIF-R expression. LIF was first described in bovine
pituitary folliculostellate cells by Ferrara et al. (354). LIF
mRNA expression has also been detected in murine corticotroph AtT-20 cells (47, 355), and pituitary cells derived
from mouse (38), rat (50, 356), and sheep (357). LIF is expressed in human fetal pituitary as early as 14 weeks of
gestation (355), while LIFR and gp130 are expressed in human fetal pituitaries at weeks 18 and 31 of gestation, as well
as in adult pituitary tissue (358) (Fig. 3). Immunohistochemistry and ligand immunostaining of human fetal pituitary
cells exhibited LIF and LIF binding sites (LIFR) on one third
of ACTH-positive cells and approximately 20% of GHpositive cells, respectively (355). Ten to 15% of cells that
costained with TSH, PRL, gonadotropins, or ␣-subunit, as
well as cells exhibiting no hormone costaining, were positive
for LIF or LIFR, respectively (355). LIF immunostaining was
also detected in all adult human pituitaries examined and in
pituitary somatotroph and corticotroph adenomas (355). In
contrast, in sheep pituitary, LIF immunostaining was found
predominantly in LH- and TSH-positive cells (357). Immunoelectron microscopy of human fetal pituitary cells (20
weeks gestation) showed immunostaining with LIF antiserum in the membrane-proximal cytoplasmic region of se-
325
cretory granules (355). When cultured, FACS-sorted LIFbinding human fetal pituicytes showed abundant ACTH
secretion, indicating enrichment of corticotroph cells in this
population (355).
In several tissues, LIF, LIFR, and gp130 mRNA expression
is stimulated by various inflammatory stimuli (37– 48, 359),
while glucocorticoids negatively regulate LIF gene expression (44, 50, 51).
In murine hypothalamus and pituitary, LIF gene expression is up-regulated by lipopolysaccharide (38) or IL-1␤ (47),
respectively. Using RT-PCR, both diffusible LIF and matrixassociated LIF and LIFR were shown to be increased 30 and
60 min after systemic lipopolysaccharide (LPS) administration (50 ␮g ip) in murine hypothalamus and pituitary (38).
Northern blot analysis also demonstrated a severalfold increase of pituitary LIF mRNA 60 min after systemic administration of IL-1␤ (100 ng ip) in C57BL/6 mice (47). In vitro,
incubation of corticotroph AtT-20 cells with IL-1␤ (0.1–10.0
ng/ml) stimulates LIF mRNA expression 5- to 10-fold (47).
This effect can be blocked by coincubation with human IL-1
receptor antagonist (100 ng/ml) or neutralizing mIL-1␤ antibody (47). While TNF␣ (20 ng/ml) exhibits only a modest
stimulatory effect on corticotroph LIF expression in vitro,
coincubation of IL-1␤ plus TNF␣ resulted in synergistic induction of LIF expression in comparison to IL-1␤ alone (47).
b. LIF-induced POMC and ACTH. In vitro, LIF stimulates
ACTH secretion in murine corticotroph AtT-20 cells (163,
206, 355, 360), primary murine (361), rat (356), and ovine (357)
pituitary cultures, as well as in fetal human pituitary cells
derived from week 16 –31 gestation (355, 358). Murine corticotroph AtT-20 cells exhibit a 2- to 4-fold increase of ACTH
secretion during incubation with 1 nm LIF for 24 h (163, 206,
355, 360). While CRH (10 or 20 nm) alone exerts a 3- to 7-fold
stimulation of ACTH secretion, coincubation of CRH with
LIF results in a further synergistic 2- to 3-fold increase of
ACTH secretion in comparison to CRH alone (163, 355).
Similarly, stably transfected AtT-20 cells overexpressing LIF
exhibit a positive correlation between secretion of LIF and
ACTH in conditioned medium and demonstrate enhanced
sensitivity to CRH stimulation, with an increased ACTH
production within 8 h (360). In human fetal pituitary cells,
incubation with 1 nm LIF for 24 h caused a 29% induction of
ACTH secretion. CRH (10 nm) alone induced a 3- to 4-fold
increase of ACTH secretion, while coincubation of CRH plus
LIF resulted in synergistic 5- to 6-fold elevated ACTH levels
(358). Shorter in vitro incubation with LIF for 6 h had no effect
on ACTH levels (358). LIF-induced ACTH secretion in
AtT-20 cells is inhibited by coincubation with LIFR or gp130
antiserum (163). Coincubation with gp130 antiserum also
decreases LIF-induced ACTH secretion in human fetal pituitary cells (358). These data demonstrate a specific action
of LIF mediated through its high affinity LIFR-gp130 complex. Coincubation with specific antisera directed against LIF
(355), LIFR (163), or gp130 (358) also decreased basal ACTH
secretion rates, demonstrating an auto/paracrine stimulation of ACTH secretion by corticotroph-derived LIF. Dexamethasone down-regulates LIF expression in cultured rat
anterior pituitary tissue (50) and inhibits basal and LIFinduced ACTH secretion (163). Part of the suppressive effect
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Vol. 21, No. 3
FIG. 3. A, Expression of human LIF receptor (LIFR) was detected in adult and fetal pituitaries, as early as 18 weeks of gestation (358) by
RT-PCR. B, Human LIF mRNA expression was detected in fetal pituitaries, as early as 16 weeks of gestation (355). A specific band of 400 bp
was protected by RNase protection assay. C, LIF-induced tyrosine phosphorylation of Jak2 and STAT3 in murine corticotroph AtT-20 cells (177).
Cell lysates were precipitated with Jak2 or STAT3 antibody, respectively. Immunoprecipitates were separated followed by immunoblotting with
a specific antityrosine antibody, and with Jak2 or STAT3 antibody, respectively. D, In vivo administration of LIF in C57BL6 mice (12 ␮g
LIF/mouse ip) stimulated pituitary POMC gene expression and plasma ACTH levels (362). E, Negative regulation of LIF-induced POMC
promoter activity, POMC gene expression, and ACTH secretion by SOCS-3 overexpression in murine corticotroph AtT-20 cells (206). After LIF
stimulation, SOCS-3 overexpressing AtT-20 cells (S) showed significantly attenuated POMC promoter activity, POMC gene expression, and
ACTH secretion in comparison to mock-transfected AtT-20 cells (M). F, LIF-induced SOCS-3 mRNA expression and autoregulation of pituitary
corticotroph SOCS-3 gene expression by a STAT-dependent mechanism (178). In mock-transfected AtT-20 cells (M) LIF (10 ng/ml) rapidly
induced exogenous SOCS-3 mRNA expression. In contrast, LIF-induced expression of endogenous SOCS-3 mRNA was abrogated in AtT-20 cells
overexpressing SOCS-3 (S).
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LIF — MODULATOR OF ENDOCRINE FUNCTION
of dexamethasone on basal ACTH secretion might occur
indirectly by suppression of auto-/paracrine LIF. In favor of
this hypothesis, stable overexpression of LIF in AtT-20 cells
blunted dexamethasone suppression of CRH-induced ACTH
secretion (360).
In vivo, systemic LIF administration rapidly induces
ACTH secretion in mice (362) and nonhuman primates (363).
In C57BL/6 mice, ip injection of 12 ␮g recombinant murine
LIF resulted in an approximately 4-fold increase of ACTH
and corticosterone levels at 60 min (362) (Fig. 3). In chronically catheterized fetal rhesus monkeys (Macaque mulatta),
systemic intracarotid administration of recombinant human
LIF (100 ␮g/kg) was followed by a 12-fold increase of plasma
ACTH levels after 60 min (363). While CRH alone (10 ␮g/kg)
induced ACTH secretion only 4.8-fold, coadministration of
LIF (50 ␮g/kg) and CRH (10 ␮g/kg) synergistically stimulated ACTH levels 23-fold in comparison to controls (363).
Stimulation of pituitary ACTH secretion is a characteristic
of several gp130-mediated cytokines. In addition to the potent action of LIF, direct stimulation of ACTH secretion in
vitro has been demonstrated for OSM (355, 358), IL-11 (364),
and IL-6 (355). In mice in vivo, coadministration of IL-1 with
each respective member of the IL-6 cytokine family (LIF,
OSM, CNTF, IL-11, IL-6, CT-1, NNT-1/BSF-3) induced corticosterone secretion significantly more than IL-1 alone (104,
365).
In addition to ACTH secretion, LIF also stimulates POMC
gene expression (163, 177, 206, 355, 364, 366). Incubation of
AtT-20 cells with LIF for up to 48 h stimulates POMC mRNA
expression about 2-fold (163, 177, 206, 355, 364, 366). In vivo,
pituitary POMC mRNA is stimulated in mice 1 and 3 h after
systemic LIF administration (362). In AtT-20 cells transfected
with a ⫺706/⫹64 rat POMC promoter-luciferase construct,
luc activity was stimulated 2- to 4-fold by LIF (1 nm) alone
(163, 177, 206, 364, 366) and up to 7-fold by CRH (10 nm) or
(Bu)2cAMP (5 mm) (163, 206, 364, 366). A striking synergism
of luc activity was seen during coincubation of CRH and LIF
(163, 366). In primary human fetal pituitary cell cultures, a
⫺879/⫹6 human POMC promoter-luciferase construct was
induced 7-fold by LIF (1 nm), 3- to 4-fold by CRH (10 nm), and
with potent synergism (22-fold) by CRH plus LIF (358).
The signaling mechanism for LIF, inducing POMC promoter activity, gene expression, and ACTH secretion, has
been extensively studied (163, 164, 177, 178, 206, 355, 358,
366).
Immunoneutralization studies show dependence of corticotroph LIF signaling on LIFR and gp130, indicating specific LIF signaling through the high-affinity LIFR-gp130 complex (163, 164, 355, 358). In the corticotroph cell, LIF rapidly
induces phosphorylation of gp130 (206), STAT3 (163, 164,
206), STAT1␣ (163, 164), STAT1␤ (163, 164), and a novel
STAT1␣-related protein p115 (164). LIF-induced POMC gene
expression and ACTH secretion have recently been shown to
be STAT3 dependent in AtT-20 cells (177). Stable transfection
of AtT-20 cells with dominant negative STAT-3 mutants,
including mutation of a carboxy-terminal tyrosine phosphorylation site Tyr705 to Phe705 (STAT-3F) or alanine substitutions at positions (E434 and E435) important for DNA binding (STAT-3D), inhibits LIF-induced POMC gene expression
and ACTH secretion (177). The suppressor of cytokine sig-
327
naling SOCS-3, harboring an essential STAT1/STAT3 binding element in its promoter region (178), is potently stimulated by LIF in AtT-20 cells (178, 206). Overexpression of
SOCS-3, known to inhibit the Jak-STAT signaling cascade,
blocks LIF-induced gp130 and STAT3 phosphorylation in
AtT-20 cells, subsequently inhibiting LIF-stimulated POMC
promoter activity, gene expression, and ACTH secretion
(206). All these observations demonstrate pituitary corticotroph LIF signaling through the Jak-STAT signaling cascade,
while alternative LIF signaling pathways, e.g., MAPK pathway, do not seem to be important in the corticotroph cell
(177).
LIF stimulates promoter activity of a ⫺706/⫹64 rat POMC
promoter-luciferase construct, alone and in striking synergism with CRH (163, 366). CRH stimulates POMC expression
through a cAMP-dependent pathway with activation of protein kinase A, associated with increased intracellular cAMP
levels, CREB phosphorylation, and a transient increase of
c-fos mRNA (366). Although LIF stimulates POMC expression synergistically with CRH, incubation of AtT-20 cells
with LIF does not alter cAMP levels, CREB phosphorylation,
or c-fos mRNA expression (366), indicating a c-fos-independent pathway for LIF action on CRH-induced POMC transcription. A ⫺173/⫺160 element within the rat POMC promoter was shown, at least in part, to mediate the LIF-CRH
synergy on POMC transcription (366). The nuclear binding
factors involved have been characterized to be serine-phosphorylated proteins (366). In contrast, STAT3 and STAT1 are
apparently not involved in the observed LIF-CRH synergy
on element ⫺173/⫺160, as no supershift was observed in
EMSA with respective STAT3, STAT1␣, or antiphosphotyrosine antibodies to this DNA motif (366). Although LIF
affects POMC expression and ACTH secretion in a STAT-3
dependent manner (177), the specific binding element in the
POMC promoter responsible for this STAT-mediated activation has not yet been defined.
c. LIF-stress response of the HPA axis. As discussed above, in
response to various inflammatory stimuli, LIF and LIFR expression is up-regulated in different tissues. In the circulation, increased serum levels of LIF have been found in septic
patients (361, 367–370). LIF serum levels are also increased
(range, 0.55 to 1.45 ng/ml) in a variety of other acute and
chronic inflammatory conditions (361). Systemic LIF appears
to play an essential role in sepsis, as indicated by observations that serum LIF levels positively correlate with lethality
in sepsis (367, 369, 371). Furthermore, when given a lethal
dose of LPS, preadministration of LIF significantly improved
survival in the mouse (372, 373). Increasing observations
suggest an important role for LIF in the neuro-immunoendocrine interface, modulating the pituitary ACTH response to various stimuli, including inflammatory stimuli
and different stressors (38, 47, 362, 374).
LPS injection results in a concordant increase of plasma LIF
and ACTH levels in mice (361). This systemic source of LIF
might contribute to HPA axis activation in inflammatory
states, as systemic LIF injection stimulates the HPA axis in
vivo (362, 363). In addition, local expression of murine LIF
and LIFR in the hypothalamus and pituitary is up-regulated
by systemic LPS administration in vivo (38). However, in
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Vol. 21, No. 3
FIG. 4. Model of pituitary development in LIF transgenic mice [with permission from H. Yano et al.: Mol Endocrinol 12:1708 –1720, 1998 (379).
© The Endocrine Society.] A, In mice overexpressing a pituitary-directed tandem construct EGFP/LIF (LIF-transgenic mice) (12 weeks),
pituitary LHX3 and Pit-1 expression was lower in comparison to mice overexpressing EGFP alone (controls). Pituitary POMC expression was
higher in LIF-transgenic mice in comparison to wt controls. B, In comparison to wt animals, LIF-transgenic mice exhibit significantly higher
serum corticosterone levels and show no sufficient suppression by dexamethasone. C, In the pituitary, LIF overexpression blocks the stream
of Lhx3 and subsequent Pit-1-dependent cell lineages. Directly or indirectly, LIF stimulates the corticotroph lineage and also diverts pituitary
progenitor cells to nasal epithelial cells. Solid arrows depict normal pituitary ontogeny.
June, 2000
LIF — MODULATOR OF ENDOCRINE FUNCTION
329
FIG. 4. Continued
vitro, no stimulation of LIF secretion was observed in primary cultured murine pituitary cells during 24 h of LPS
incubation (361). This result might be explained by the in vitro
system used, possibly lacking other pituitary-derived cells,
which might express the essential receptor components for
LPS signaling, e.g., CD14 and Toll receptor 4 (375, 376).
IL-1 is an important inflammatory cytokine, thought to
activate the HPA axis predominantly through stimulation of
hypothalamic CRH and subsequent ACTH secretion (348,
349). IL-1␤ stimulates pituitary LIF expression in vivo and in
vitro (47). IL-1␤-stimulated ACTH secretion from AtT-20 cells
is partially inhibited by coincubation with LIF antiserum
(47). After systemic IL-1␤ administration in vivo (100 ng ip),
LIF⫺ /LIF⫺ knockout mice exhibit reduced ACTH and corticosterone levels in comparison with B6D2F1 wt mice (47).
Thus, LIF modulates IL-1␤-induced activation of the HPA
axis.
The model of LIF⫺/LIF⫺ knockout mice has yielded interesting insights into the integrative function of LIF in pituitary function. Baseline plasma ACTH and corticosterone
levels are not significantly different (362) or are decreased
(47, 374) in LIF⫺/LIF⫺ knockout in comparison to wt LIF⫹/
LIF⫹ B6D2F1 mice. As LIF⫺ /LIF⫺ mice show a diminished
ACTH response to various stressors (47, 362, 374), stress
caused during blood collection, handling of animals, anesthesia, or retroorbital sinus puncture might be responsible for
the different baseline ACTH results observed. A small study
showed that LIF⫺/LIF⫺ mice (n ⫽ 4 –5) exhibited 27% lower
baseline ACTH levels and 62% lower “stressed” ACTH levels
after 36 h fasting (374). Infusion of LIF⫺/LIF⫺ mice with
recombinant LIF for 3 days (1.2 ␮g/day) stimulated ACTH
and corticosterone levels by 70% and 54%, respectively (374).
In a larger study, no significantly altered baseline ACTH and
corticosterone levels were found in LIF⫺/LIF⫺ mice (n ⫽ 7–9)
(362). After short immobilization stress for 15 min LIF⫺/LIF⫺
mice exhibited modestly decreased ACTH secretion in comparison with LIF⫹/LIF⫹ mice. In contrast, after prolonged
immobilization stress for 30 or 45 min, ACTH responses in
LIF⫺/LIF⫺ mice were not different from baseline controls,
while LIF⫹/LIF⫹ mice demonstrated robustly enhanced
ACTH levels (362). Basal and poststress pituitary POMC
mRNA content was significantly decreased in LIF⫺/LIF⫺
mice in comparison with LIF⫹/LIF⫹ mice (362). Although
LIF⫺/LIF⫺ mice mounted an attenuated ACTH response,
especially to prolonged stressors, serum corticosterone levels
after immobilization stress were unaltered in comparison to
wt animals (362). This phenomenon can be explained by very
small amounts of ACTH being sufficient for a maximal adrenal response.
2. LIF and pituitary development. In murine corticotroph
AtT-20 cells, LIF inhibits cell cycle progression from G1 into
S phase and proliferation, while enhancing ACTH secretion
(377). Thus, LIF acts as a differentiation factor in murine
AtT-20 cells, causing a phenotypic switch from proliferative
to synthetic (377).
Two transgenic mouse models with pituitary-directed LIF
expression have demonstrated LIF to be an important neuroimmuno-endocrine modulator of pituitary development
(378, 379). Transgenic mice expressing pituitary-directed LIF
driven by the rat GH promoter (378) showed striking dwarfism, with undetectable serum GH levels and IGF-I levels
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AUERNHAMMER AND MELMED
diminished to 30% of wt controls. In the pituitary, the number of GH and PRL cells was decreased, while the number of
ACTH cells was increased 2.2-fold. The anterior pituitary
contained cystic cavities, lined by cuboidal, ciliated epithelial
cells, focally immunopositive for cytokeratin and S-100 protein and immunonegative for adenohypophyseal hormones.
Human Rathke’s cysts also exhibit LIF immunoreactivity in
cyst-lining cells. Thus, LIF overexpression might perturb
differentiation of Rathke’s pouch, an invagination of oral
ectoderm and source of common progenitor cells, believed to
differentiate into distinct hormone-secreting cell lines (378).
During murine pituitary ontogeny, GH expression emerges
at a relatively late stage [embryonic day 16 –17 (E16 –17)],
while ␣GSU is the earliest hormone-specific transcript (E9.5),
followed by POMC (E12), TSH (E12–13), and LH and FSH
(E15). To examine the impact of LIF on earlier pituitary
development, a second transgenic model with pituitarydirected LIF driven by the ␣GSU promoter was established
(379). Phenotypically, these transgenic mice were dwarfs
with extremely low IGF-I serum levels. Infertility of both
sexes was due to central hypogonadotropism. In comparison
to wt mice, the transgenic mice exhibited Cushingoid features including truncal obesity and thin skin, elevated basal
corticosterone levels, and incomplete suppression of corticosterone levels by dexamethasone (379). The LIF transgenic
pituitary was hypoplastic due to a dramatic decrease of somatotroph, lactotroph, and gonadotroph cells, as well as a
variably diminished number of thyrotroph cells. ACTHimmunopositive cells were increased in absolute numbers
and accounted for approximately 65% of anterior pituitary
cells in the LIF-transgenic pituitaries, in comparison with
about 13% in wt pituitaries (379). On E14.5, the transcription
factors Lhx3 and Pit-1 were also decreased in transgenic
pituitaries (379). As a hypothetical model, suppression of
LHX3 expression in the fetal pituitary by LIF might direct
differentiation of progenitor cells away from Lhx3-dependent cell lineages (gonadotroph, thyrotroph, somatotroph,
and lactotroph) toward the corticotroph lineage and ciliated
epithelial cells (379) (Fig. 4).
3. Summary. In summary, LIF stimulates pituitary corticotroph function in vivo and in vitro. LIF is a modulator of HPA
axis function, as knockout mice demonstrate an attenuated
response of the HPA axis to various inflammatory and stress
stimuli. Deficiency of LIF might be compensated by partially
overlapping functions of other gp130-sharing cytokines. Using LIFR⫺/⫺ and gp130⫺/⫺ knockout animals, the impact of
IL-6-related cytokine signaling in the pituitary should be
further evaluated. Pituitary-targeted overexpression of LIF
directs fetal pituitary differentiation toward the corticotroph
cell lineage and prevents somatotroph and lactotroph lineage
differentiation. Thus, early pituitary LIF expression appears
to regulate both spatial and temporal pituitary development,
independently of transcription factor function. Thus, LIF behaves as a soluble differentiation factor in fetal pituitary
development. Further studies should focus on the effects of
other gp130-sharing cytokines on pituitary function and development and characterization of overlapping or specific
properties.
Vol. 21, No. 3
C. Bone metabolism
Several cytokines play a role in proliferation and function
of osteoblasts and osteoclasts, thus affecting physiological
bone formation and remodeling, as well as pathophysiological states, e.g., osteoporosis. Several current reviews (380 –
385) discuss the growing evidence supporting the role of
gp130-sharing cytokines, especially IL-6 and IL-11, in osteoclast differentiation and bone resorption. IL-6 and IL-11 are
secreted by osteoblast-like cells and act in a paracrine fashion
on osteoclasts. IL-6 expression in osteoblast-like cells is repressed by estrogens (385) and androgens (386), and stimulated by T3 (387–390). Loss of estrogens also increases IL-6R
and gp130 expression in osteoblasts (388). Thus, lowering sex
steroids or hyperthyroidism might be associated with increased bone resorption and increased risk of osteoporosis
due to increased osteoblast IL-6 expression, subsequently
stimulating osteoclast differentiation and proliferation.
In addition, other cytokines of the IL-6 family, namely LIF
and OSM, exert effects on bone resorption and formation.
Although the topic of “LIF and bone metabolism” has been
extensively reviewed several years ago (391, 392), recent
studies have provided new insights for specific LIF function
in bone metabolism. Similar to other cytokine functions,
these effects of members of the IL-6 cytokine family on bone
metabolism appear to partially overlap.
1. LIF expression on osteoblasts and osteoclasts. LIF expression
has been reported in various osteoblastic cell lines, including
murine MC3T3-E1 (393–395), rat UMR 106 – 06 (396), rat
UMR 201 (396), in primary cell cultures derived from newborn rat long bones (396) or fetal rat calvarias (397), and
finally in U-OS and SaoS-2 human osteosarcoma cell lines
(398), as well as in benign and malignant human primary
bone tumors (399). As demonstrated by Northern blot analysis, LIF expression in murine MC3T3-E1 cells is rapidly and
transiently stimulated by IL-1␣, IL-1␤, TNF-␣, or LPS (395).
Similarly, LIF expression in rat UMR 201 cells is stimulated
by TNF␣ (396). By Northern blot analysis, no stimulation of
LIF mRNA expression in MC3T3-E1 cells was observed after
incubation with PTH, 1␣,25(OH)2D3, or LIF itself (395). However, using more sensitive semiquantitative RT-PCR analysis, a rapid and transient stimulation of LIF and IL-6 mRNA
was detected in MC3T3-E1 cells incubated with PTH (394).
Stimulation of LIF and IL-6 expression by PTH in osteoblasts
is an immediate-early gene response induced by cAMP signal transduction (400). Also in vivo, injection of PTH into the
subcutaneous space overlying mouse parietal bones rapidly
and transiently induces parietal expression of LIF and IL-6
transcripts (401), suggesting that LIF and IL-6 may be mediators of initial PTH effects in vivo.
2. LIFR expression on osteoblasts and osteoclasts. Using ligand
autoradiography, specific LIF binding was found in osteoblasts, but not in multinucleated osteoclasts derived from
newborn rat long bones (396). Scatchard analysis of the osteoblast-like rat osteogenic sarcoma cell line UMR 106 – 06
revealed 300 LIF binding sites per cell with a Kd of 60 pm
(396). A similar number and affinity for LIF receptors was
found on preosteoblastic rat calvaria (RCT-1) cells (402). By
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LIF — MODULATOR OF ENDOCRINE FUNCTION
RT-PCR, expression of gp130, LIFR, and IL-6R has been demonstrated in primary calvarial cultures isolated from 21-dayold rat fetuses (397).
Murine osteoblastic MC3T3-E1 cells exhibit approximately
1,100 LIF binding sites per cell with a Kd of 161 pm (394).
Immunoprecipitation verified expression of LIFR and an alternative gp130 form in MC3T3-E1 cells (153, 390). LIFinduced signal transduction in osteoblastic MC3T3-E1 cells
involves tyrosine phosphorylation of Jak1 and, to a lesser
extent, Jak2 (153), gp130 (403), and LIFR (153, 403), as well as
predominantly STAT1 and, to a lesser extent, STAT3 (153).
LIF stimulates expression of gp130 in MC3T3-E1 cells (390).
In contrast, human osteoblast-like MG-63 osteosarcoma
cells do not express LIFRs (403, 404). LIFR antibodies failed
to precipitate a specific protein (403), and LIF does not phosphorylate gp130 (403), form STAT1 and STAT3 complexes
(404), or activate MAPK (404) in these cells. However, despite
the lack of LIF activity in MG-63 cells, OSM stimulates gp130
tyrosine phosphorylation (403), STAT1, and STAT3 (404), as
well as Erk1 and Erk2 (376), probably acting through a specific OSMR type II (403).
Both murine MC3T3-E1 and human MG-63 cells are modestly induced by IL-6 plus sIL-6R and IL-11, respectively
(403). Thus, various members of the IL-6 cytokine family can
affect osteoblastic MC3T3-E1 or MG-63 cells. Depending on
the cell type-specific expression of LIFR or OSMR type II, LIF
and OSM appear to exhibit overlapping effects mediated by
shared LIFR signaling, or selective effects of OSM, signaling
through the specific OSMR type II.
3. LIF effect on bone resorption and bone formation. LIF was first
reported to stimulate bone resorption by Abe et al. (405),
when LIF purified from conditioned medium of mitogenactivated spleen cells exhibited osteoclast-activating factor
activity. In vitro, LIF induced bone resorption in neonatal
mouse calvaria cell cultures and increased osteoclast numbers (406). But, in the same culture system, LIF also induced
3
H-thymidine incorporation in AP-positive stained osteoblastic cells and in the osteoprogenitor region of cultured
hemicalvaria of 6-day-old mice (406, 407). In fetal mouse
calvarial cultures, a combination of LIF plus IL-1 induced
bone resorption, while each cytokine by itself was inactive
(395). In vivo, local injection of LIF (0.5 ␮g/day) over a hemicalvarial region for 5 days accelerated bone turnover (408).
LIF increased bone resorption as evidenced by increased
osteoblast numbers (3-fold), osteoclast surface (5-fold), and
eroded bone surface (10-fold) (408). However, net bone formation was also increased, as osteoblast numbers, osteoblast
surface, osteoid area, and overall bone thickness doubled
(408). Systemic overexpression of LIF in syngeneic DBA/2
mice engrafted with hemopoietic FDC-P1 cells transfected
with LIF cDNA resulted in increased osteoclastic resorption,
as well as increased numbers of osteoblasts and a large increase in net bone formation and bone mass (409). Thus
available data suggest a stimulatory effect of LIF on bone
resorption by inducing osteoclast proliferation and differentiation (395, 405, 406, 410), while few studies report an
inhibitory effect of LIF on bone resorption (411, 412). Another
study reported LIF to increase osteoclastic activity in cul-
331
tured cells derived from a giant cell tumor of bone, while
osteoclast numbers did not change significantly (413).
Although most available data suggest a stimulatory effect
of IL-6 family cytokines on bone resorption by inducing
osteoclast proliferation and differentiation (380 –385, 395,
405, 406, 410), osteoclasts are not dependent on gp130 signaling, as osteoclasts are present in gp130-deficient mice
(414). LIFR⫺/⫺ knockout mice exhibit bone development
abnormalities with an approximately two thirds reduction in
bone mass, 6-fold increase in osteoclast numbers, and 7-fold
increase in osteoclast surfaces, while bone formation is only
nonsignificantly reduced by 30% (249). In view of the indirect
osteoclast stimulation by LIF (395, 405, 406, 410), these findings (249) are unexpected. Other indirect effects of LIF causing a balance between osteoclast activation and inhibition
might contribute to this finding. Similarly, other cytokines
using the LIFR for signaling might exert negative direct or
indirect effects on osteoclast proliferation and function.
Bone remodeling is a dynamic process of osteoclastmediated bone resorption and subsequent bone formation by
osteoblasts. A mechanism for osteoblasts modulating proliferation and differentiation of osteoclast progenitor cells is
by secretion of cytokines, which stimulate osteoclasts in a
paracrine fashion (380 –385). Stimulation of osteoblasts with
IL-1, TNF␣, or PTH induces expression of IL-6 (380 –385),
IL-11 (415, 416), and LIF (38, 397, 401), respectively. A direct
paracrine effect of osteoblast-derived LIF on osteoclasts is
unlikely, as LIFRs have not been demonstrated on osteoclasts, but only on osteoblasts (396). However, LIF stimulates IL-6 expression from fetal rat calvaria cell cultures (397)
and neonatal mouse osteoblasts (417). Thus, mitogenic LIF
effects on osteoclasts could be indirect, mediated by osteoblast-derived IL-6. This theory of LIF acting indirectly via
osteoblasts on osteoclasts is also supported by the observation that LIF stimulates bone resorption only in cultures
containing both osteoclasts and osteoblasts, while highly
purified osteoclast cultures were not stimulated by LIF (392).
In addition, as LIF stimulates collagenase-3 expression in
osteoblasts, the increase in collagenase might be responsible,
in part, for the stimulation of osteolytic activity by LIF (418).
On the other hand, although osteoclast-like cells have been
considered to lack LIFR expression (396), osteoclast-like cells
from a human giant cell tumor of the bone have been found
to express LIF and LIFR (419). In these multinucleated giant
cells, LIF directly stimulated proliferation, although decreasing their ability of resorption (419). Therefore, at least in this
tumor cell model, direct autocrine/paracrine LIF effects in
osteoclast-like cells seem to exist.
Conflicting effects of LIF on differentiation and proliferation of osteoblast-like cells have been reported (402, 420).
Osteoblast differentiation from murine embryonic fibroblasts is not stimulated by LIF, OSM, or CNTF, while IL-6
plus sIL-6R, or IL-11 promote differentiation of AP-positive
cells (421). Accordingly, murine embryonic fibroblasts express gp130, but not the IL-6R(gp80) or the LIFR (gp190)
(421). LIF was observed to either inhibit DNA synthesis in
MC3T3-E1 cells (153, 420) or to have no effect (422), while
OSM inhibited 3H-thymidine incorporation in MG-63 cells
(404). The prodifferentiation and antiapoptotic effects of
OSM in MG-63 cells may be mediated by the cyclin-depen-
332
AUERNHAMMER AND MELMED
dent kinase inhibitor p21 (423). Similarly, DNA synthesis in
calvarial osteoblast cultures derived from newborn mice
(153) and bone nodule formation in primary cultures of rat
calvarial cells derived from 21-day old fetuses (397) were
inhibited by LIF. In contrast, high concentrations of LIF (10
to 1,000 ng/ml) increased DNA synthesis and 3H-thymidine
incorporation of AP-positive cells in human trabecular bone
cultures (424). LIF (100 ng/ml) and OSM (100 ng/ml) stimulated 3H-thymidine incorporation 2.5- to 3.0-fold in primary
cultures of parietal bone-derived osteoblastic cultures isolated from 22-day old rat fetuses (417). LIF also stimulated in
vitro proliferation of stromal progenitors with osteogenic
potential from isolated bone marrow (424). In a murine heterotopic calcification model, LIF and OSM lowered the Ca/P
ratio and mineral density of newly induced ossifications
(425). Therefore, in addition to an increase of net bone formation, LIF also seems to alter the mineral phase quality of
the newly formed bone.
4. Summary. In summary, LIF modulates bone formation by
direct or indirect paracrine effects on osteoblasts and osteoclasts, respectively. Several cytokines, including IL-1,
TNF␣, LPS, and PTH stimulate LIF secretion from osteoblastic cells. LIF is believed to act indirectly on osteoclasts by
stimulating stromal/osteoblastic expression of other cytokines, which mediate LIF-induced osteoclast proliferation
and bone resorption (Fig. 5). In addition to indirect stimulation of bone resorption, LIF also increases new bone formation, resulting in increased net bone mass. The observed
contradictory results of LIF on osteoblasts and osteoclasts
might be explained by several factors. First, different experimental conditions, e.g., cell isolation procedures, might per
se influence cell subsets and their specific responsiveness.
Second, as cell preparations for primary culture are isolated
from animals of different ages, osteoblasts at different stages
of development might respond differently to LIF. Future
studies should focus on experiments evaluating the specific
and essential role of LIF and mechanisms for direct or indirect osteoclast stimulation.
FIG. 5. Model of LIF effects on osteoblasts and osteoclasts. Factors as IL-1,
TNF␣, LPS, and PTH stimulate LIF
and IL-6 secretion from ostoblasts. IL-6
is a direct activator of osteoclast proliferation and function. LIF effects on osteoclasts are indirect by enhancing osteoblast cytokine secretion, e.g., IL-6.
Thus, LIF indirectly activates osteoclast function and bone resorption.
Vol. 21, No. 3
D. Energy metabolism
Cytokines play an important role in anorexia, weight loss,
wasting, and cachexia associated with chronic illness including cancer and infections. Current evidence points to a complex and partially overlapping action of several cytokines,
including IL-1, TNF␣, IL-6, LIF, IFN␥, and others in inducing
weight loss (426 – 428).
Based on studies from Mori et al. (429, 430) and others (257,
431– 434), LIF is thought to play a role in the cancer cachexia
syndrome by inhibition of adipocyte lipoprotein lipase (LPL)
activity. Similarly, IL-1, TNF␣, IL-6, and IFN␥ decrease LPL
activity (for review see Ref. 433). In rats, systemic LIF administration increases hepatic triglyceride secretion by stimulating both lipolysis and de novo fatty acid synthesis (435).
Serum levels of LIF are increased in most patients with
cancer and lymphomas (361, 399, 436). Nude mice bearing
tumors of the SEKI human melanoma cell line develop cachexia and loose 25– 40% of total body weight (429, 432).
Resection of the tumor results in normalization of body
weight and abrogation of the cachexia (429). Mori et al. purified a “lipoprotein lipase-inhibitor” derived from conditioned media of SEKI human melanoma cells, and subsequently found it to be identical with LIF (429, 430). Injection
of six different human carcinoma cell lines in nude mice
resulted in cachexia and substantial weight loss in each case
(431, 432). However, while SEKI (melanoma), NAGAI (neuroepithelioma), and OCC-1C (oral cavity carcinoma) cells
were associated with high levels of LIF expression and secretion, MKN-1 (gastric carcinoma), LS180 (colon carcinoma), or LX-1 (lung carcinoma) cell lines do not express LIF,
IL-6, or IL-11 (432). Accordingly, conditioned medium derived from SEKI, NAGAI, or OCC-1C cultures inhibits LPL
activity by 80 –90%, while conditioned medium derived from
MKN-1, LS180, or LX-1 cultures causes only a 20 –30% decrease of LPL activity (432). These data indicate that some
tumor cells cause cachexia by production of LIF and related
cytokines and subsequent inhibition of LPL activity. However, other tumors may cause similar cancer cachexia syn-
June, 2000
LIF — MODULATOR OF ENDOCRINE FUNCTION
dromes by other, LIF-independent mechanisms (432). LIF
signaling in 3T3-L1 adipocytes involves tyrosine phosphorylation of STAT 3 and STAT1 (155, 167). LPL activity in
3T3-L1 cells is decreased by LIF, mostly by inhibition of LPL
transcription (433). Although showing similar half-maximal
doses for inhibition of LPL activity in 3T3-L1 cells, LIF decreased LL activity less potently than TNF␣ (433). Additional
proof supporting a role for LIF in energy homeostasis derives
from the observation that mice engrafted with cells producing high systemic LIF levels developed a fatal syndrome
accompanied by weight loss of 20 –25%, associated with loss
of all subcutaneous and abdominal fat and generalized organ
atrophy within 12–70 days (409). Similarly, injection of recombinant human LIF to nonhuman primates (80 ␮g/kg/
day for 14 consecutive days) results in weight loss of 10% and
a reduction in subcutaneous fatty tissue (257).
The cytokine-induced decrease in adipocyte LPL expression and decreased LPL activity is a possible mechanism for
LIF causing cachexia. However, anorexia, which often accompanies infectious diseases or cancer, cannot be explained
by this mechanism. In a recent study by Sarraf et al. (427),
LPS, TNF␣, IL-1, and to a somewhat lesser degree LIF and
IL-6, were found to stimulate murine leptin mRNA expression in fat and serum leptin levels. Thus, the anorectic effect
of inflammatory cytokines might be mediated, at least in
part, by leptin, and in cancer-associated anorexia, cytokinemediated regulation of adipocyte leptin might be involved.
However, direct expression of leptin could not be detected by
RT-PCR in several human cancer cell lines (432).
1. Summary. In summary, LIF induces weight loss and cachexia in vivo. Two possible mechanisms might mediate this
LIF-induced weight loss including LIF-induced decrease in
adipocyte LPL activity, and LIF-induced increase of serum
leptin levels. However, these effects are not specific LIF actions. Similar effects can also be caused by other cytokines,
e.g., IL-1 and TNF-␣. As many tumor patients show elevated
LIF serum levels and various tumor cell lines secrete LIF, LIF
is one potential cachectic factor in these patients.
E. Endocrine-responsive tumors
In 90% of cancer patients (n ⫽ 75) LIF was detectable in the
serum with a median value of 0.4 ⫾ 0.1ng/ml (361). No
differences in LIF levels were observed in patients with different carcinoma types (361). Significantly increased LIF serum levels were also found in Hodgkin’s and Non-Hodgkin
lymphomas in comparison to controls (436). In vitro, human
carcinoma cell lines derived from pancreas (n ⫽ 8), lung (n ⫽
7), stomach (n ⫽ 5), colon (n ⫽ 2), breast (n ⫽ 2), melanocytes
(n ⫽ 2), liver (n ⫽ 1), and gall bladder (n ⫽ 1), all exhibited
LIF mRNA expression detected by Northern blot analysis
(437, 438). gp130 And LIFR were also detected by RT-PCR in
virtually all examined cancer cell lines (438). Thus, LIF, LIFR,
and gp130 seem to be constitutively expressed in most cancer
cells, indicating the possibility of auto/paracrine stimulation
of cancer cells by LIF. Secretion of either LIF or IL-6 from the
murine sarcoma cell line 4JK is induced by IL-1 and TNF␣
severalfold (439). Culture of RAW 264 macrophages with
conditioned medium from 4JK cells activates TNF␣ secretion
333
about 10-fold, while coculture of RAW and 4JK cells results
in increased LIF and IL-6 production (439). These results
suggest an interaction of tumor-infiltrating monocytes/macrophages and sarcoma cells, to enhance tumor cell LIF secretion. Both induction of cell proliferation as well as growth
inhibition and apoptosis can be stimulated by LIF (438),
indicating cell type-specific signal transduction in different
cancer cells.
LIFR and gp130 are expressed in many human breast
carcinoma cell lines (440), as well as human breast cancer
specimens (440, 441) and human mammary epithelial cells
derived from normal breast tissue (441, 442). LIFR and gp130
were detected by RT-PCR in human breast cell lines, including 184 and 184B5 cells derived from nonmalignant breast
epithelial cells; MCF-7M, T-47D, MDA-MB-134, and MDAMB-361cells derived from estrogen receptor-positive breast
cancer cells; and ASK-BR-3, BT-20, BT-549, and MDA-MB231 derived from estrogen receptor-negative breast cancer
cells, respectively (440). Similarly, IL-11R was also expressed,
while transcripts for the IL-6R and CNTFR were not detected
in the examined cell lines (440). Ligand binding of 125I-LIF to
MCF-7M cells and Scatchard plot analysis revealed a single
class of high-affinity (Kd 1.5 ⫻ 10⫺11 m to 27.0 ⫻ 10⫺11 m)
binding sites (60 – 430 receptors per cell) (440, 443).
In most studies, LIF has been reported to stimulate proliferation of estrogen receptor-positive MCF-7M cells (438,
442– 444) and T-47D cells (443, 444). MCF-7M cells (443, 444)
and T-47D cells (444 – 446) do not express LIF, indicating a
paracrine role of LIF, possibly derived from adjacent nontumorous cells. LIF also stimulated proliferation of the estrogen receptor-negative breast tumor cells SK-BR3 (442, 443)
and BT20 (442). However, LIF showed no effect on proliferation of estrogen receptor-negative breast tumor cells BT549 and MDA-MB-231 (440), as well as normal human breast
cells, including primary human mammary epithelial cells,
HBL 1000 and HS578BST (442, 443). Despite being nonresponsive to LIF (444), estrogen receptor-negative MDA-MB231 cells express and secrete LIF (444, 446). A 666-nucleotide
human LIF promoter luciferase construct, cotransfected together with progesterone receptors into MDA-MB-231 cells,
showed stimulated luc activity after stimulation with
medroxy-progesterone acetate (446). LIF treatment of primary human breast cancer cells, derived from 6 patients,
resulted in a 12–110% increase in colony formation (443). In
contrast to these data indicating a stimulatory effect of LIF on
proliferation of different breast cancer cells (438, 442– 444),
one study observed an inhibitory effect of LIF on proliferation of MCF-7M cells (440). This discrepancy might be due
to different batches of MCF-7M cells exhibiting different
states of differentiation, as well as different incubation time
periods.
OSM inhibits proliferation of MCF-7M, SK-BR3, BT-549,
and MDA-MB231 cells, as well as normal human mammary
epithelial cells (440, 442). These opposing effects of LIF and
OSM on cell proliferation may be explained by differing
signal transduction of LIF and OSM through either the LIFR
(shared by LIF and OSM, OSMR type I) or the OSMR (specific
for OSM, OSMR type II), respectively. OSMR are more abundantly expressed than LIFR in normal human mammary
epithelial cells and in most breast cancer cell lines (440, 442).
334
AUERNHAMMER AND MELMED
RT-PCR of mRNA derived from 50 human breast cancer
specimens revealed expression of transcripts for gp130, LIFR,
IL-11R, IL-6R, and CNTFR in virtually all samples (440). A
small study reported detection of LIF, IL-6, IL-11, and OSM
transcripts by RT-PCR in breast tumor samples (445). In
another study, immunohistochemistry of 50 human breast
cancer specimens revealed staining for LIF and LIFR in 80%
of the tumors, as well as most adjacent normal breast epithelium (441). In normal breast epithelium, immunostaining
for LIF and LIFR did not differ in samples derived from
premenopausal and postmenopausal women (441). In breast
cancer, LIF and LIFR coexpression correlated significantly
with diploidy and a low S-phase fraction (441).
1. Summary. In summary, LIF plays an important, yet incompletely understood, role in breast cancer proliferation.
First, LIF is expressed by various cancer cell lines and in
primary tumors (441, 444 – 446). LIFR and gp130 are expressed in most breast cancer cells (440 – 442), and the data
suggest a paracrine stimulatory effect of LIF on breast cancer
cell proliferation. Second, as LIF indirectly stimulates osteoclast proliferation and bone resorption, LIF derived from
breast cancer cells could stimulate osteoclasts in the process
of bone metastasis. The mouse mammary tumor cell line
MMT060562 secretes LIF and supports osteoclast formation
in a mouse bone marrow coculture system (447). In addition,
the osteoclast-activating fraction of the conditioned medium
derived from MMT060562 cells could be inactivated by LIF
antibody (447).
Therefore, the role of LIF in breast cancer might be of
clinical relevance and merits further evaluation. Targeted
overexpression of inhibitors of cytokine signaling (SOCS proteins) in breast carcinoma cell lines might provide insight in
the effect of different gp130 sharing cytokines stimulating
these cells and also prove be a potential therapeutic tool to
disrupt auto-/paracrine stimulation of tumor growth by LIF
and other IL-6 cytokine family members.
VII. Integrative Section—The NeuroimmuneEndocrine Interface
There is now compelling evidence that cytokine functions are not restricted to the hemopoietic and immune
system. Their multiple metabolic cellular and tissue regulatory functions place the cytokines clearly in the realm
of endocrine signaling molecules. Their actions are evident
both in terms of classic endocrine secretions, as well as in
terms of paracrine and autocrine actions. As neuroimmune
neuroendocrine modulators, cytokines serve as critical
transducers for the interface between peripheral and
central stress and inflammatory signals, and the hypothalamic-pituitary unit. Cytokines also serve to regulate important metabolic functions including fuel and bone metabolism, reproductive function, and immune responses.
LIF, in this regard, appears to play a critical role in neuroimmune transduction of central and immune signals to the
adrenal axis. Furthermore, LIF regulation of cell growth,
reproductive function, bone metabolism, and energy homeostasis all point to a pivotal role for gp-130-mediated
signaling in endocrine control. Further understanding of
Vol. 21, No. 3
mechanisms regulating LIF expression and LIFR signaling
will thus elucidate multiple endocrine-regulatory processes.
Clearly, the regulatory biology of cytokine synthesis and
action impacts on protean physiological and pathological
processes. Perhaps as a reflection of their ubiquitous yet
critical actions, cytokines and their respective signaling molecules share significant structural and functional overlap and
redundancy.
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