0163-769X/96/$03.00/0
Endocrine Reviews
Copyright © 1996 by The Endocrine Society
Vol. 17, No. 3
Printed in U.SA.
Role of the Cytoskeleton in Adrenocortical Cells*
MARC FEUILLOLEY AND HUBERT VAUDRY
European Institute for Peptide Research, Laboratory of Cellular and Molecular Neuroendocrinology,
INSERM U 413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France
I. Introduction
I. Introduction
II. Role of Microtubules
A. Microtubules in adrenocortical cells
1. Organization
2. Specific microtubule-associated proteins (MAPs)
B. Involvement of microtubules in spontaneous adrenal steroidogenesis
C. Role of microtubules in the steroidogenic response
of adrenocortical cells to corticotropic factors
1. Microtubules and the plasma membrane: involvement of microtubules in signal transduction
2. Microtubules, mitochondria, and cholesterol
transport
3. Interaction of microtubules with the Golgi complex and cytoplasmic vesicles
D. Microtubules and steroidogenesis in adrenocortical
rumor cells
III. Role of Microfilaments
A. The microfilament network in adrenocortical cells
1. Structure of the network
2. Microfilament-associated proteins in adrenocortical cells
B. Interaction of microfilaments with the plasma
membrane
C. Microfilaments and mitochondria: role in cholesterol and intermediate steroid transport
D. Microfilaments and steroidogenesis in adrenocortical tumor cells
IV. Role of Intermediate Filaments
A. Expression of intermediate filament proteins in adrenocortical cells
B. Intermediate filaments and adrenal steroidogenesis
1. Role in normal adrenocortical cells
2. Role in adrenocortical tumor cells
V. Cytoskeleton and Cell Contacts: Role in Adrenal Steroidogenesis
T
VI. Cytoskeleton and the Nuclear Matrix: Control of
Genomic Expression
A. Microtubules in adrenocortical cells
HE cytoskeleton is formed by three types of high molecular weight polymers, namely microtubules, microfilaments, and intermediate filaments. Microtubules and microfilaments are homogeneous and ubiquitous structures (1),
whereas intermediate filaments represent a heterogeneous
family of fibers whose expression depends upon the level of
differentiation of the cells (2-4). All three types of cytoskeletal elements are intimately associated with cytoplasmic organelles, particularly mitochondria (5-7), lysosomes (8, 9),
and secretory vesicles (10,11). Microtubules, microfilaments,
and intermediate filaments also interact with the nucleus
(12-14), the plasma membrane (15,16), and, indirectly, with
the extracellular matrix (17-19). There is now ample evidence
that cytoskeletal fibers play a pivotal role in cell motility
(20-23) and various forms of intracellular movements including axonal transport (24, 25), chromosome migration
(26-28), and pigment dispersion (29, 30). Cytoskeletal components are also involved in the process of capping and
receptor-mediated endocytosis (31-33). In exocrine (34-36)
and endocrine cells (10, 37-39), which export their secretory
products by exocytosis, microtubules and microfilaments are
implicated in both the traffic of secretory granules in the
cytoplasm and the fusion of the vesicles with the plasma
membrane. Although the presence of vesicles has been observed in steroidogenic cells (40-42), it is generally accepted
that steroids are released immediately after synthesis by
diffusion through the plasma membrane (43). However, during the last decade, a number of studies have demonstrated
that pharmacological agents that induce either disruption or
stabilization of cytoskeletal fibers strongly affect steroid hormone secretion (44-46). The present review examines the
evidence for the involvement of the different components of
the cytoskeleton in the activity of normal and tumoral cells.
II. Role of Microtubules
1. Organization. Microtubules are dynamic structures formed
by the polymerization of heterodimers of a- and j3-tubulin
(47, 48). Adrenocortical cells contain a dense network of
microtubules (49) that surrounds the nucleus (50, 51) and
radiates from the centrosome to the submembrane area (52,
53) (Fig. 1). In bovine adrenocortical cells, tubulin represents
1-3% of total cytoplasmic proteins (54,55). Tubulin extracted
from the adrenal cortex is similar to brain tubulin with regard
to isoelectric pH (pHi) (49) and affinity for colchicine (55).
VII. Conclusion
Address reprint requests to: Marc Feuilloley, Ph.D., European Institute for Peptide Research (IFRMP n°23), Laboratory of Cellular and
Molecular Neuroendocrinology, INSERM U 413, UA CNRS, University
of Rouen, 76821 Mont-Saint-Aignan, France.
*This work was supported by grants from INSERM (U 413), the
Direction des Recherches et Etudes Techniques (No. 92-099), and the
Conseil Regional de Haute-Normandie.
269
FEUILLOLEY AND VAUDRY
270
COLCHICINE
NOCOOAZOLE
Vol. 17, No. 3
absent, and only traces of the tau protein have been detected
(66). The major MAP expressed in these cells is a 190-kDa
polypeptide, which exhibits only very limited sequence similarity with MAPI and MAP2 (67). Proteins related to this
190-kDa MAP have been identified in other cell types, notably in the Y-l murine adrenal tumor cell line (68). Since this
protein appears to be an ubiquitous member of a group of
nonneuronal MAPs, it has been designated as MAP-U. In
vitro, the stabilizing effect of MAP-U on microtubules and the
size of the bridges formed by this MAP are similar to those
of MAP2 and tau (69). However, while various MAPs identified in neurons bind to microfilaments (70), MAP-U expressed in adrenocortical cells is totally devoid of affinity for
actin fibers (69). The stabilizing activity of MAPs on microtubules is regulated by phosphorylation (71, 72), and the
protein kinases involved in the phosphorylation of MAPs are
associated with the cytoskeleton (71). A cAMP-dependent
protein kinase bound to the cytoskeleton has been identified
only in adrenocortical tumor cells (73). Concurrently, a Ca2+calmodulin-dependent protein kinase, tightly linked to the
cytoskeleton, has been characterized in normal adrenal fasciculata cells (74). This latter protein kinase has several substrates including /3-tubulin itself and calcineurin (74), a Ca2+calmodulin-dependent phosphatase that also appears to be
associated with the cytoskeleton in adrenocortical cells (75).
B. Involvement of microtubules in spontaneous adrenal
steroidogenesis
The role of microtubules in adrenal steroidogenesis has
been investigated by using antimicrotubular agents such as
colchicine (76) and vinblastine (77,78). This kind of approach
inevitably raises the question of the possible nonspecific effects of the drugs used to disrupt cytoskeleton elements. For
FIG. 1. Schematic representation of the structure of tubulin dimers
instance,
it has been shown that, in adrenocortical cells, mil(A), microtubules (B), and their relations with cellular organelles (C).
limolar
concentrations
of colchicine inhibit cAMP phosA, Each subunit of the heterodimer of a- and /3-tubulin can bind a
phodiesterases (79). Vinblastine exhibits a low affinity for
molecule of GTP as well as different types of depolymerizing agents
such as vinblastine, colchicine, or nocodazole. B, Microtubules are
acidic molecules (77, 78), but these nonspecific ionic interformed by the association of tubulin dimers. C, Microtubules radiate
actions occur at concentrations of the drug that are 100 to
from the centrosome (CT), encircle the nucleus (N), cross the Golgi
1000 times higher than those necessary to induce disruption
area (G), and extend to the plasma membrane (PM). Microtubules are
of the microtubular network (53, 62, 80). Nonspecific effects
linked through MAPs with lysosomes (L), cytoplasmic vesicles, mitochondria (M), and other cytoskeletal elements such as intermediate
may also originate from the close association that exists befilaments (IF).
tween microtubules and intermediate filaments (81). For instance, microtubule depolymerization has been shown to
induce collapse of intermediate filaments in fibroblasts (82).
However, different isoforms of tubulin are found in epitheHowever, in human adrenocortical cells, complete depolylial cells (56, 57), suggesting that various forms of tubulin
merization of microtubules does not significantly affect the
may also be present in adrenocortical cells. In addition to the
organization of the intermediate filament network (62).
tissue-specific expression of tubulins, species differences in
In vivo studies conducted in rat have shown that vincristhe structure of tubulin have also been reported. For instance,
tine and colchicine induce a dose-related increase in plasma
a special type of a-tubulin, which has a low sensitivity to
corticosterone (83, 84) and aldosterone (85), whereas vindepolymerizing agents (58, 59), has been identified in poikiblastine
causes a rapid decrease of corticosterone levels (86).
lotherms (60). Nevertheless, in both frog and human adreIn
spite
of their opposite effects on plasma corticosteroid
nocortical cells, vinblastine induces total disruption of the
levels, colchicine and vinblastine both induce an increase of
microtubular network (61, 62) (Fig. 2).
the concentration of glucocorticoids in the adrenal cortex (84,
2. Specific microtubule-associated proteins (MAPs). MAPs are
86). Inaba and Kamata (84) suggested that part of the effects
essential for the stability and function of microtubules (63).
of colchicine and vinblastine in vivo could be ascribed to
MAPs were originally identified in neurons and have been
indirect effects of the drugs on ACTH release by pituitary
classified in three groups, i.e. MAPI, MAP2, and tau (64, 65). cells. In fact, colchicine inhibits ACTH secretion (87), and it
has been demonstrated that microtubules are required for
In bovine adrenocortical cells, MAPI and MAP2 are totally
June, 1996
CYTOSKELETON IN ADRENAL CELLS
FIG. 2. Confocal laser scanning microscope photomicrographs of frog and human adrenocortical cells stained with
tubulin antiserum. In control frog (A)
and human (B) cells, a dense network of
microtubules is seen radiating from the
centrosome to the submembrane area.
A 5-h incubation with vinblastine (10~5
M) causes total disruption of the microtubular network, and the appearance of
intensely immunoreactive crystals of
tubulin (arrows) in the cytoplasm of frog
(C) and human (D) adrenocortical cells.
N, Nucleus (x 1000).
271
io)
% **•
anterograde transport of secretory vesicles in corticotrophs
(10). Therefore, the effect of microtubule-disrupting agents
cannot be ascribed to their action on pituitary corticotrophs.
However, it should be noticed that the activity of adrenocortical cells is under multifactorial control (88-91). In addition to the conventional humoral factors, ACTH and angiotensin II, other agents, either produced locally by
chromaffin cells of the adrenal medulla or released by fibers
innervating the adrenal cortex, can modulate corticosteroid
secretion (88-91). Since microtubules are involved in the
migration of the secretory vesicles containing neurotransmitters and regulatory peptides (92-94), the in vivo effect of
antimicrotubular agents on corticosteroid secretion may be
accounted for, at least in part, by the action of the drugs on
the release of paracrine or neuronal factors.
In vitro, the basal secretion of corticosteroids in rat adrenocortical cells is not modified by antimicrotubular agents
such as colchicine, vinblastine, and podophyllotoxine (84,
95). Similarly, colchicine, vinblastine, and nocodazole are
devoid of action on the spontaneous release of corticosterone
or cortisol in birds (80) and humans (62). The frog adrenal
gland has also been widely used to investigate the possible
effects of antimicrotubular agents on corticosteroid secretion
(53, 61, 96, 97). In this particular model, the adrenal gland is
composed of a single population of adrenocortical cells that
produce both corticosterone and aldosterone and thus are
considered to be homologous to mammalian granulosa cells
(98, 99). In vitro studies using perifused frog adrenal tissue
confirmed that colchicine and vinblastine have no effect on
the spontaneous secretion of corticosteroids (53,61). Analysis
of the steroids released by adrenocortical cells has demonstrated that antimicrotubular agents do not modify the biosynthetic pathways of corticosteroids (53). In agreement with
these observations, Rainey et al. (100) have shown that taxol,
which induces the polymerization of nonfunctional microtubules (101, 102), does not affect the basal level of corticosterone and cortisol secretion in bovine adrenocortical cells.
A stimulatory action of antimicrotubular agents on the spontaneous production of corticosteroids has been only reported
in special circumstances, i.e. after in vivo pretreatment of the
animals with colchicine (85) or after addition of FCS and
cholesterol to the incubation medium (103). In this latter
study, an effect of antimicrotubular agents was observed
only after long-term treatment (24-40 h) or in the presence
of very high doses of colchicine (>10~4 M) (103) which may
have caused nonselective effects (79).
While microtubules do not play a significant role in the
spontaneous secretion of corticosteroids, antimicrotubular
agents markedly affect the conversion of pregnenolone into
progesterone in rat (104, 105) and chicken (45) granulosa
cells. Formation of progesterone, which is catalyzed by the
enzyme 3/3-hydroxysteroid dehydrogenase/ A5-A4 isomerase
272
FEUILLOLEY AND VAUDRY
(3/3-HSD), is an obligatory step in the biosynthetic pathway
of all steroid hormones, including adrenal steroids, androgens, and estrogens. However, multiple 3/3-HSD isoenzymes, exhibiting tissue-specific distribution, have been
identified (106). In addition, 3/3-HSD has been localized in
different subcellular compartments, i.e. the endoplasmic reticulum and the mitochondria (107), suggesting that microtubules may play distinct functions in steroid biosynthesis in
the various steroidogenic cell types.
C. Role of microtubules in the steroidogenic response of
adrenocortical cells to corticotropic factors
1. Micmtubules and the plasma membrane: involvement of microtubules in signal transduction. Interaction of microtubules
with plasma membrane proteins has been described in various cell types. Specifically, in neuronal cells and in sex
steroid-producing cells, tubulin binds to various membraneassociated proteins, including fodrin (108, 109), calmodulindependent protein kinase (110), GTP-binding regulatory proteins of the adenylyl cyclase complex (111, 112), opioid
receptors (113), cholinergic nicotinic receptors (114), a-aminobutyric acidA receptors (115), glycine receptors (116), and
lectine-binding sites (32). In adrenocortical cells, administration of ACTH or (Bu)2cAMP provokes a rearrangement of
microtubules into a radial pattern (50, 51) and markedly
alters the overall morphology of the cells (117-121), suggesting that microtubules are involved in the response to corticotropic factors.
In frog adrenocortical cells, vinblastine reduces by 50% the
corticotropic response to ACTH (61) and serotonin (53), two
factors that act through the adenylyl cyclase pathway (122).
In contrast, vinblastine does not affect the response to
(Bu)2cAMP and to forskolin or fluoride ions, which activate
the catalytic and regulatory subunits, respectively, of adenylyl cyclase. These observations may suggest that microtubules are either involved in the binding of ACTH to its
receptor, or more probably, in the coupling of the receptor
with the Gs regulatory protein of adenylyl cyclase (61). In
support of this latter hypothesis, it has been shown that
binding of tubulin to G proteins is required for anchoring
these proteins in the plasma membrane (111). In addition,
recent studies have demonstrated that disruption of microtubules blocks the GTPase activity of G proteins (123). The
involvement of microtubules in the coupling of the ACTH
receptor to the Gs protein should be directly tested by ADP
ribosylation.
A number of studies have also been performed to investigate the possible implication of microtubules in the mechanism of action of corticotropic factors whose receptors are
not coupled to the adenylyl cyclase pathway. In amphibians,
the stimulatory effect of angiotensin II is mediated through
activation of the arachidonic acid cascade (99). In vitro studies
have shown that vinblastine, at a dose of 10~5 M, which
causes complete depolymerization of microtubules (Fig. 2),
does not impair the steroidogenic action of angiotensin II (53,
96) or prostaglandins (96). At the same dose, vinblastine does
not block the response of adrenocortical cells to acetylcholine
and endothelin (53,97), which act through the phospholipase
C and phospholipase A2 pathways (124, 125). It appears
Vol. 17, No. 3
therefore that, in the frog adrenal gland, microtubules are
only involved in the response to corticotropic factors whose
receptors are coupled to adenylyl cyclase (Fig. 3).
A dense network of microtubules is present in the cytoplasm and in the submembrane area of duck adrenocortical
cells (121, 126). However, in vitro studies indicate that col- _
chicine and vinblastine do not affect the morphological characteristics and the steroidogenic actions of ACTH in duck
adrenal cells (80), suggesting that, in the bird, microtubules
do not play a significant role in ACTH-evoked corticosteroid
biosynthesis and release.
In rat adrenocortical cells, the role of microtubules is still
being debated. In vivo studies have shown that colchicine and r
vinblastine cause accumulation of corticosterone and aldosterone in the adrenal cortex (43, 85, 127). In vitro, microtubule-disrupting agents do not modify the intracellular con- .
centration of corticosteroids (84) but induce reversible
inhibition of ACTH-stimulated steroid secretion (84, 95,128,
129). In fact, the inhibitory effect of colchicine and vinblastine ,
in rat adrenocortical cells can be accounted for by both inhibition of coupling between the ACTH receptor and the Gs
protein (129) and blockage of the translocation of cholesterol
into the mitochondria (130). However, the inhibitory effect of
dbcAMP
FIG. 3. Schematic representation of the ACTH receptor complex illustrating the possible interaction with microtubules in adrenocortical cells. Ac, Adenylate cyclase; dbcAMP, (Bu)2cAMP; F~, fluoride
ions; FK, forskolin; R, receptor protein; aB, a-subunit of the GTPbinding regulatory protein of adenylyl cyclase; /3, j3-subunit of the
GTP-binding regulatory protein of adenylyl cyclase; 7, 7-subunit of
the GTP-binding regulatory protein of adenylyl cyclase; ju,T, microtubule.
June, 1996
CYTOSKELETON IN ADRENAL CELLS
antimicrotubular agents is weak and requires high doses of
the drugs. For instance, millimolar concentrations of vinblastine only induce partial inhibition of ACTH-evoked corticosteroid release in rat zona glomerulosa cells (130).
In bovine adrenocortical cells, the organization of the microtubular network and the morphology of the cells are
markedly affected by ACTH (50,51). Angiotensin II does not
give rise to similar morphological changes (51). The role of
microtubules in the transduction of the corticotropic message
of ACTH and angiotensin II in bovine adrenocortical cells has
not yet been investigated in detail.
The role of microtubules has been studied in normal human adrenocortical cells only recently (62). Vinblastine reduces by 50% the corticotropic response of human adrenal
slices to ACTH. Conversely, vinblastine does not affect the
response to (Bu)2cAMP (62), suggesting that, in humans as
in amphibians, microtubules are involved in the transduction
pathway at some step before cAMP formation.
273
vinblastine affects the cytoplasmic distribution of lysosomes
(153). Consistent with these data, it has been shown that
lysosomes, in very much the same way as coated vesicles, are
associated with the microtubular network (8,154) and transported by a mechanism triggered by MAPs (155-157). Therefore, it appears that, in adrenocortical cells, microtubules are
involved in the catabolism of LDL. Studies performed in
luteal and tumor Leydig cells indicate that microtubules are
also necessary for cholesterol uptake from LDL in other types
of steroidogenic cells (138, 158).
3. Interaction of microtubules with the Golgi complex and cyto-
plasmic vesicles. Microtubules are associated with the Golgi
membrane by proteins located in their minus end region
(159), and the microtubular network appears to be required
for maintaining the structure of the Golgi apparatus (160). In
adrenocortical cells, as in other cell types, microtubule bundles pass through the Golgi complex (52). Ultrastructural
studies have shown that ACTH and angiotensin II induce the
formation
of electron-dense granules in the vicinity of the
2. Microtubules, mitochondria, and cholesterol transport. InterGolgi apparatus of adrenocortical cells (86, 127, 161). The
action of microtubules with mitochondria has been docusignificance of these granules remains enigmatic inasmuch as
mented in various types of steroid-secreting glands includthe release of steroid hormone does not occur through a
ing the testis (131,132), the ovary (133), and the adrenal (52).
classic exocytotic mechanism, and thus should not depend
Steroid biosynthesis requires the transfer of cholesterol from
on
the microtubular network. Immunohistochemical studies
cytoplasmic stores of free and esterified cholesterol to the
suggest
that the granules appearing after exposure of adrenal
inner mitochondrial membrane where cholesterol is concells to corticotropic factors are lysosomal vesicles involved
verted to pregnenolone (134,135). In resting conditions, adin lipoprotein degradation (41).
renocortical cells use primarily the cytoplasmic stores of
cholesteryl esters as a source of cholesterol (136, 137),
whereas Leydig cells employ essentially free cholesterol
D. Microtubules and steroidogenesis in adrenocortical
from the plasma membrane (138). In rat adrenocortical cells,
tumor cells
colchicine and vinblastine inhibit the formation of the comThe role of the microtubular network in adrenocortical
plex between cholesterol and cytochrome P450scc (128, 130,
tumor cells has been investigated in detail in the Y-l cell line
139), suggesting the involvement of microtubules in the
(162-166). The steroidogenic pathway of Y-l cells is truntranslocation of cholesterol from the cytoplasm to the mitocated and leads only to the formation of 20a-dihydroprochondria. Similarly, in granulosa cells, microtubules faciligesterone and ll/3-hydroxy-20a-dihydroprogesterone as fitate the transfer of cholesterol from lipid droplets to the
nal
steroid products (167). The number and composition of
mitochondria (104, 105). Conversely, in Leydig cells, which
the proteins associated with the plasma membrane in Y-l
use a different store of cholesterol, depolymerization of micells are noticeably different from those found in normal
crotubules produces an increase in androgen biosynthesis
adrenocortical cells (168). For instance, Y-l cells express LDL
(131, 132).
receptors similar to those identified in normal cells (167,168)
During acute stimulation, adrenal cells require more chobut exhibit unique angiotensin II receptors negatively coulesterol than can be produced by neosynthesis from acetylcoenzyme A (140-142). Therefore, biosynthesis of steroids by pled to adenylyl cyclase (169). The spectrin-like protein characterized in the plasma membrane of Y-l cells only shares
adrenal cell depends on the supply of exogenous cholesterol
limited homology with that identified in normal cells (170).
provided by high-density (HDL) or low-density (LDL) liIn contrast to normal adrenocortical cells, Y-l cells contain a
poproteins (137). The uptake of cholesterol from HDL is
protein kinase C (171) that is loosely associated with the
mediated by a mechanism which appears to be independent
plasma
membrane but tightly bound to the cytoskeleton
of microtubules and microfilaments (143, 144). In contrast,
(172).
the release of cholesterol from LDL occurs after receptorIn Y-l cells, microtubules are scarce (100) and tubulin is
mediated endocytosis (145-147), a mechanism that requires
mainly associated with granules scattered in the cytoplasm
an intact microtubular network (148,149). Contacts between
(173). Exposure of the cells to ACTH induces retraction of the
coated vesicles and microtubules are frequently observed in
plasma membrane, disappearance of tubulin granules, and
adrenocortical cells (52). Vesicles containing LDL exhibit a
formation of a dense network of microtubules (40,173,174).
dense lattice of clathrin on their cytoplasmic side (150,151),
ACTH exerts similar effects in other adrenal tumor cell lines
and clathrin is tightly associated with a- and j3-tubulin (42).
of murine origin (175). In Y-l cells, the corticotropic effects
Biochemical and histological studies also suggest that MAPs
of ACTH (162,163) and (Bu)2cAMP (164) are not modified by
are involved in the binding of microtubules to the clathrin
antimicrotubular agents, suggesting that microtubules are
lattice (152). Cholesterol is released from LDL after fusion of
not involved in the steroidogenic response to ACTH. Colthe vesicles with lysosomes (137). In rat adrenocortical cells,
274
chicine and vinblastine are also devoid of effect on the basal
formation of cAMP in Y-l cells (162, 165), whereas these
drugs induce a marked increase of the spontaneous secretion
of steroids (162, 165, 166). In murine adrenal tumor cells,
steroidogenesis requires the mobilization of the cellular
stores of free (176) and esterified cholesterol (177). The tubulin granules that have been detected in the cytoplasm of
Y-l cells are associated with cholesterol and cholesteryl esters (173). Since colchicine causes disruption of the association of tubulin with cholesterol in Y-l cells (178), it appears
that the stimulation of steroid secretion induced by antimicrotubular agents is due to the release of cholesterol from the
aggregates formed with tubulin (173). In agreement with
these data, the microtubule-stabilizing agent taxol blocks
ACTH-induced synthesis of corticosteroids in Y-l cells (44,
100,179). Taxol, which increases the level of polymerization
of tubulin (180, 181), might stimulate the formation of the
complex between cholesterol and tubulin and therefore
might decrease the level of free cholesterol available for
steroid biosynthesis. Although a nonspecific effect of taxol
cannot be excluded, the response to taxol is not mediated by
inhibition of protein synthesis (100) or cholesterol transport
(44, 179). In other tumor cell lines, such as the human adrenocortical cell line SW-13, colchicine causes morphological
changes identical to those observed in Y-l cells (182).
Taken together, these data show that microtubules are not
FIG. 4. Confocal laser scanning microscope photomicrographs of frog and human adrenocortical cells stained with
an actin antiserum. In control frog (A)
and human (B) cells, microfilaments
are abundant in the submembrane area
and in the perinuclear region. A 2-h incubation with cytochalasin B (5 X 10~5
M) causes total disappearance of microfilaments and the formation of immunoreactive aggregates randomly distributed in the cytoplasm of frog (C) and
human (D) adrenocortical cells. In addition, cytochalasin B induces a retraction of the cytoplasm, which conferes to
the cells a stellate aspect. N, Nucleus
(X1000).
Vol. 17, No. 3
FEUILLOLEY AND VAUDRY
necessary for the basal secretion of corticosteroids. In normal
cells, microtubules are required for the coupling of the secretory responses to ACTH, LDL endocytosis and degradation, and translocation of cholesterol to the mitochondria.
Conversely, in adrenocortical tumor cells, microtubules do
not play a significant role in ACTH-evoked corticosteroid
secretion and in cholesterol transport.
III. Role of Microfilaments
A. The microfilament network in adrenocortical cells
1. Structure of the network. Microfilaments are homopolymers
of actin arranged in a two-stranded helix made by the association of filaments resulting from the linear and noncovalent polymerization of actin (183, 184). The molecular
weight, immunological characteristics, and affinity for various proteins and drugs of actin extracted from rat adrenocortical cells are identical to those of actin purified from other _,
nonmuscle cells (185). In rat adrenal cells, actin represents
more than 7% of total proteins (185) and has been identified
as the major protein in the peripheral zone of the cytoplasm
(186). In various vertebrate species, including frog (187),
duck (80,126), rat (188), bovine (51,189), and human (62), a
dense network of microfilaments is lining the internal surface
of the plasma membrane of adrenocortical cells (Fig. 4). Mi-
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i
June, 1996
CYTOSKELETON IN ADRENAL CELLS
crofilaments are also present in the cytoplasm and are found
in close contact with the nuclear envelope (51,121,126,190).
In adrenocortical cells from intact tissue, microfilaments
form a diffuse network of individual fibers (80,126,190). In
cultured adrenocortical cells, the microfilament network is
barely detectable, and microfilaments frequently form thick
bundles of parallel fibers designated as stress fibers (62,121,
187, 191, 192).
275
been tested by using chaetoglobosin C (an inactive cytochalasin analog), which had no effect on corticosteroid secretion
(187).
In frog adrenocortical cells, cytochalasin B inhibits the
steroidogenic response to ACTH, vasoactive intestinal
peptide, and serotonin (53, 190), three hormones whose
receptors are coupled to the adenylyl cyclase system (122,
208). The stimulatory effect of (Bu)2cAMP on corticosterone and aldosterone secretion is also blocked by cytocha2. Microfilament-associated proteins in adrenocortical cells. The
lasin B (190), suggesting that, in amphibians, microfilastructure of the microfilament network is mainly regulated
ments are involved in a step beyond production of cAMP.
by specific associated proteins (20). Several microfilamentIn the rat, cytochalasin B induces a weak inhibition of the
associated proteins, including a-actinin, fodrin, myosin, procorticotropic action of ACTH in vivo (128, 139). However,
filin, and tropomyosin, appear to be ubiquitous (193). In
these data should be interpreted with a degree of caution
particular, myosin has been identified in mouse tumor adsince cytochalasin B interferes with the secretion of varirenocortical cells (172). Concurrently, other microfilamentous peptidic hormones (39, 209) and neurotransmitters
associated proteins, such as vinculin, have been detected in
(94) involved in the control of corticosteroid secretion. In
human granulosa cells (194). In addition, adrenal cells exvitro
studies have shown that cytochalasins reduce ACTHpress specific microfilament-associated proteins. For inevoked
cAMP production in rat zona glomerulosa cells in
stance, spectrin-related proteins have been characterized in
primary
culture (129). In these cells, the Gs protein of
normal and tumoral adrenocortical cells (74, 170). In these
adenylyl
cyclase is tightly associated with F actin and
cells, binding of calmodulin to actin and spectrin-related
transferred
to tubulin during ACTH stimulation (129). In
proteins stimulates a cytoskeleton-associated kinase that
freshly
dispersed
adrenocortical cells, cytochalasin B also
phosphorylates actin and the a-subunit of spectrin (74). In
causes
partial
inhibition
of the response to ACTH (128,
contrast, it has been demonstrated that gelsolin and ad130),
but
the
action
of
cytochalasin
B depends on the
severin, two calcium-regulated microfilament-disrupting
composition of the culture medium (130). At a dose of 10~5
proteins that play pivotal roles in exocytosis in chromaffin
M, cytochalasin B does not impair the stimulatory effect of
cells (195, 196), are not present in adrenocortical cells (197,
ACTH
on rat zona glomerulosa explants (210) while cy198).
tochalasin B markedly reduces the response of frog adrenal explants to ACTH (190), suggesting that, in the rat, the
B. Interaction of microfilaments with the plasma membrane association of actin with the Gs protein of the ACTH receptor complex is not a limiting process in the steroidoIn rat adrenocortical cells, ACTH causes a marked degenic response of adrenal cells to ACTH. In human adrecrease of the total amount of actin (199) but induces a sinocortical
cells, cytochalasin B inhibits ACTH-evoked
multaneous increase of the local concentration of actin in the
cortisol
secretion
by acting at a step located beyond the
submembrane area (188). Polymerization of microfilaments
coupling
of
the
receptor
to adenylyl cyclase (62). These
participates in the formation of microvilli that appear in the
observations
indicate
that
microfilaments are mainly inplasma membrane of ACTH-stimulated adrenocortical cells
volved
in
events
occurring
downstream of the binding of
(188, 200). These microvilli, which augment the exchange
ACTH
to
its
receptor
and
cAMP
formation.
surface of the cells, may facilitate endocytosis of LDL (201)
In contrast to ACTH, angiotensin II does not affect the
and diffusion of newly synthesized steroids (188, 200). Restructure of the microfilament network (51). In frog adorganization of actin filaments at the periphery of the cell
renocortical cells, cytochalasin B blocks the stimulatory
after stimulation of steroidogenesis has also been observed
effect
of angiotensin II (53,190) and endothelin (97), whose
in granulosa cells (202).
receptors
are both coupled to phospholipases. Analysis of
Cytochalasins, which cause depolymerization of microinositol
phosphates
and phospholipids formed in adrenal
filaments (203,204), have been widely used to investigate the
cells
has
shown
that
cytochalasin B blocks the hydrolysis
role of these cytoskeletal elements in adrenocortical cells. It
of polyphosphoinositides and the formation of lysophosis significant to note that glucocorticoids stabilize microfilaphatidylinositides induced by angiotensin II (187). These
ments and can reverse the depolymerizing effect of cytocharesults suggest that microfilaments are required for the
lasins (205). Nonspecific effects of cytochalasins cannot be
coupling of the angiotensin II receptor to phospholipase C
ruled out. In particular, cytochalasin B has been shown to
and phospholipase-A2 (187). In support of this notion, it
block glucose uptake in intestinal epithelial cells (206) and in
has
been shown that, in neutrophils, microfilaments conLeydig cells (207) and to increase the intracellular concentrol the dissociation of the GTP-binding regulatory protein
tration of free hexoses in adrenocortical cells (187). It should
associated with phospholipase C (211). Direct association
be noticed, however, that suppression of glucose from the
of
actin filaments with the a-subunit of Gq, the regulatory
incubation medium has no effect on corticosteroid secretion
protein
involved in phospholipase C activation, has been
from frog adrenal explants (190), suggesting that the inhibdemonstrated
in mammary cells (212). In addition, studies
itory effect of cytochalasin B on spontaneous corticosteroid
performed
in
rat
kidney cells indicate that microfilaments
secretion cannot be accounted for by blockage of glucose
are
required
for
endocytosis
of the angiotensin II-receptor
transport. The specificity of action of cytochalasin B has also
276
FEUILLOLEY AND VAUDRY
complex (213), which is a key step in phospholipase C
activation (214).
Vol. 17, No. 3
D. Microftlaments and steroidogenesis in adrenocortical
tumor cells
In the Y-l murine adrenal tumor cell line, microfilaments
and stress fibers are prominent within cell extensions in
C. Microftlaments and mitochondria: role in cholesterol andcontact with the substratum (40, 220). Actin cables are also
intermediate steroid transport
observed randomly distributed in the cytoplasm (220, 221).
In response to various stimulating agents including ACTH,
In adrenocortical cells, mitochondria and lipid vesicles are
vasoactive intestinal peptide, adenosine, cAMP, and cholera
intimately associated with microfilaments and stress fibers
toxin, Y-l cells exhibit a rapid rounding of the cytoplasm (40,
(126, 188). These interactions are so tight that substantial
222, 223) associated with a marked decrease of the number
amounts of actin have been detected in the external memof microfilaments and stress fibers at the cell periphery (40).
brane of mitochondria (215). Disruption of microfilaments
Exposure of Y-l cells to ACTH causes the splitting of stress 1
induces the aggregation of mitochondria and lipid vesicles
fibers into individual microfilaments, while the remaining
around the nucleus (191). Although ACTH markedly moddismantled bundles disperse randomly in the cytoplasm
ifies the distribution of the microfilament network, ACTH
(224).
does not affect the concentration of actin associated with
Changes in the extracellular concentration of calcium
mitochondrial or vesicular membranes (188). The limiting
markedly affects the distribution of microfilaments and the
step in corticosteroid biosynthesis is the translocation of chomorphological response of Y-l cells to ACTH (221). This
lesterol from the cytoplasmic compartment to cytochrome
observation is consistent with the identification, in Y-l cells,
P450scc in the internal membrane of mitochondria (134, 216). of calcium-binding proteins that regulate the functions of
Studies performed on rat adrenal mitochondria have demmicrofilaments. Calmodulin, which in Y-l cells binds to sevonstrated that microfilament-disrupting agents, such as cyeral cytoskeletal proteins including actin, myosin, and spectochalasin B, markedly reduce the association of cholesterol
trin (74), appears to be required for the coupling of the ACTH
to cytochrome P450scc, suggesting that microfilaments are
receptor to the regulatory protein of adenylyl cyclase (225).
Identification of a calmodulin-dependent protein kinase,
involved in the translocation of cholesterol to the mitochondria (139). Similarly, microfilaments appear to be involved in which phosphorylates the a-subunit of spectrin, suggests
that the association of microfilaments with the plasma memthe transport of cholesterol to the mitochondria in frog adbrane itself is controlled by calcium-binding proteins (74). A
renal cells (217). In frog adrenocortical explants, cytochalasin
calcium- and phospholipid-dependent protein kinase, which
B blocks the formation of lljS-hydroxysteroids, suggesting
phosphorylates the light chains of myosin, is also involved
that microfilaments may also be required for the transport of
in the organization of the microfilament network in Y-l cells
11-desoxycorticosterone from the endoplasmic reticulum to
(172). Several other enzymes, including a cAMP-dependent
the mitochondria (217). Consistent with these observations,
protein
kinase (73) and a calmodulin-regulated phosphatase
it has been shown that microfilaments control the translo(75),
control
the degree of polymerization of microfilaments
cation of cholesterol and precursor steroids to the internal
and
their
association
with the plasma membrane in Y-l cells.
membrane of the mitochondria in other types of steroid
Y-l
cells
to cytochalasin B induces a retraction
Exposure
of
hormone-producing cells, i.e. granulosa cells (45, 218) and
of
the
cytoplasm,
similar
to that observed in normal adreLeydig cells (219).
nocortical
cells
(220).
The
organized
network of stress fibers
In spite of the ubiquitous function played by microfilais
replaced
by
a
filamentous
felt,
particularly
in the area of
ments in cholesterol transport, the effect of microfilamentcontact of the cells with the substratum (220). In addition,
disrupting agents on spontaneous secretion of corticostecytochalasin B causes the formation of vacuoles (220) and, as
roids markedly differ from one species to the other.
observed by video recording, a total arrest of the movement
Cytochalasin B totally inhibits the spontaneous secretion of
of all particles in the cytoplasm (175). These effects of cy- '
corticosterone and aldosterone in the frog (53,190), induces
tochalasin B are totally reversible (175, 220, 224) and have
a transient and weak decrease of the spontaneous secretion
been reported also in the human adrenocortical cell line
of corticosteroids in the rat (210), and has no effect on the
SW-13 (182). In Y-l cells, cytochalasin B inhibits the release *
basal production of cortisol in the human (62). It should be
of 20a-dihydroprogesterone and Ilj3-hydroxy-20a-dihydronoticed that all these studies were conducted in serum-free
progesterone, the two major steroids produced in this cell
medium, using the same dose of cytochalasin B. An imporline (175), and blocks the steroidogenic response to ACTH
tant aspect to consider is the origin of cholesterol employed
and (Bu)2cAMP (220, 226, 227). The effect of cytochalasin B
by adrenocortical cells from each species for steroid biosynon ACTH- and (Bu)2cAMP-evoked steroid secretion cannot
thesis. For instance, in human adrenal cells, LDL are the
be ascribed to inhibition of nucleic acid or protein synthesis
predominant source of cholesterol whereas, in the rat adresince it has also been observed in enucleated cells (228). In
nal, a large proportion of cholesterol originates from HDL
fact, in Y-l cells as in normal cells, cytochalasin B blocks the
(137). As mentioned previously, cytoskeletal elements are
accumulation of cholesterol in the mitochondria, suggesting
only required for cholesterol uptake from LDL; thus, it can
that microfilaments are involved in the translocation of chobe assumed that, depending on the source of cholesterol
lesterol from the cytoplasm to the mitochondria (229-231). In
support of this notion, DNase 1, which binds to monomeric
employed by the cell, the role of the microfilament network
G actin, inhibits both the polymerization of microfilaments
on corticosteroid biosynthesis may vary.
June, 1996
CYTOSKELETON IN ADRENAL CELLS
and the transport of cholesterol to the mitochondria (232).
Inhibition of pregnenolone biosynthesis has also been observed in rat adrenal carcinoma cells treated with cytochalasin B (233).
Mattson and Kowal (220) have reported that, in Y-l cells,
low concentrations of cytochalasin B, which are not sufficient
to cause visible depolymerization of microfilaments, stimu• late steroid secretion. The intensity of the stimulatory effect
of cytochalasin B depends on the concentration of FCS in the
culture medium (231). In Y-l cells, cytochalasin B stimulates
the uptake of HDL and cholesteryl esters (234, 235) and,
consequently, may increase the steroidogenic activity of the
cells (231). In medium containing fetal serum, botulinum C2
toxin, whose substrate is Rho proteins and actin (236), mimics
the effects of cytochalasin B, including depolymerization of
, microfilaments, retraction of the cytoplasm, and stimulation
of steroid secretion (237). Although the specificity of action
of botulinum C2 toxin on microfilaments has not been demonstrated, its effects are consistent with those of microfilament-disrupting agents on the uptake of steroid precursors
** in Y-l cells, inasmuch as the C2 toxin increases the concentration of cholesterol in the mitochondria without modifying
cAMP level (237). Concurrently, it has been demonstrated
that, in fibroblasts, lysophospholipids contained in the fetal
serum affect the organization of the microfilament network
(238, 239). Therefore, it is conceivable that, in Y-l cells, lysophospholipids may be involved in the effect of microfilament-disrupting agents on cholesterol uptake.
Taken together, these data suggest that, in spite of their
dependence upon the composition of the culture medium,
microfilaments are required for the transport of cholesterol
and / or intermediate metabolites to the mitochondria both in
normal cells and adrenocortical tumor cells. Microfilaments
' may also control LDL endocytosis, the turnover of polyphosphoinositides, and the coupling of the ACTH receptor
1
to adenylyl cyclase. The involvement of microfilaments in the
mechanism of action of corticotropic factors has not been
described in adrenocortical tumor cells.
IV. Role of Intermediate Filaments
A. Expression of intermediatefilamentproteins in
adrenocortical cells
Intermediate filaments form a heterogenous class of fibers,
which have been divided into six distinct classes, i.e. acidic
and basic cytokeratin filaments (class I and II), vimentin,
desmin, peripherin, and glial fibrillary acidic protein fibers
(class III), neurofilaments and a-internexin fibers (class IV),
nuclear lamins (class V), and nestinfilaments(class VI) (240).
Intermediate filament proteins are expressed in a tissuespecific manner (2). For instance, cytokeratin intermediate
filaments are only present in epithelial cells (2, 240). Consistent with the "cell migration" theory establishing that the
adrenal cortex derives from the mesodermal epithelium
(241), cytokeratin has been identified in adrenocortical cells
(242, 243). At least two types of cytokeratins, namely cytokeratin 8 and 18, are expressed in human adrenocortical cells
(244). Cytokeratin fibers observed in human adrenal cells
exhibit the typical organization of intermediate filaments
277
present in all epithelial cells of vertebrates: they form a cagelike structure around the nucleus and a dense network in the
cytoplasm (62) (Fig. 5). Vimentin intermediate filaments have
also been visualized in 10% of human adrenocortical cells
(244) and in bovine fasciculata cells (245). In contrast, neurofilaments, which are found in adrenal chromaffin cells
(243), have never been detected in the adrenal cortex (243,
244).
During carcinomal transformation of human adrenocortical cells, a progressive decrease of the expression of keratin
occurs, associated with an induction of the synthesis of vimentin (244, 246). Therefore, coexpression of keratin and
vimentin is frequently noted in human adrenal carcinoma
cells (243, 244, 247). In highly differentiated adrenocortical
tumor cells, such as SW-13 human cells, vimentin is the
unique form of cytoplasmic intermediate filaments (248).
Subclones of SW-13 cells appear to be totally devoid of intermediate filaments (248, 249). Vimentin intermediate filaments have also been visualized in Y-l cells (245, 250).
Desmin intermediate filaments are present in some clones of
Y-l cells, but keratinfibershave not been detected in this cell
line (178).
B. Intermediatefilamentsand adrenal steroidogenesis
1. Role in normal adrenocortical cells. Morphological studies
have shown that, in bovine adrenal fasciculata cells, vimentin
intermediate filaments are in close contact with mitochondria (245) and lipid vesicles (251), suggesting that intermediate filaments may serve in the transport of lipid droplets
to the mitochondria (252). However, it has not been formally
established that cytokeratin filaments are bound to the mitochondrial membrane (253). Treatment of human adrenocortical cells with the intermediatefilament-disruptingagent
/3-/3'-iminodipropionitrile (IDPN) (254) causes disorganization of the network of cytokeratin fibers (62). IDPN selectively affects intermediate filaments (254, 255) and does not
modify the organization of microtubules and microfilaments
in adrenocortical cells (62). In spite of its action on keratin
intermediate filaments, IDPN has no effect on the spontaneous secretion of steroids in human (62), rat (256), chicken
(256), and frog (257) adrenal cells, suggesting that intermediate filaments are not involved in corticosteroid biosynthesis in normal cells.
Keratin intermediate filaments are associated with the
plasma membrane (14, 81, 258) and appear to be involved in
the process of receptor-mediated endocytosis (259, 260). In
rat and chicken adrenocortical cells, IDPN inhibits the steroidogenic effect of ACTH (256). However, the inhibitory
action of IDPN has only been observed with high concentrations of the drug (>50 HIM), which are known to induce
nonspecific effects (261). Millimolar doses of IDPN, which
are sufficient to disorganize the intermediate filament network (62), do not modify ACTH-evoked corticosteroid secretion in human (62) and frog adrenocortical cells (257).
Intermediate filament proteins can be directly associated
with membrane phospholipids (262). Cytokeratin (263), vimentin (264, 265), and presumably other intermediate filament proteins (266) exhibit high affinity for phosphatidylinositol-4,5-bisphosphate, a membrane component that
278
FEUILLOLEY AND VAUDRY
Vol. 17, No. 3
FIG. 5. Confocal laser scanning microscope photomicrographs of frog and human adrenocortical cells stained with a
keratin antiserum. In control frog (A)
and human (B) cells, keratin intermediate filaments form a cage around the
nucleus and a dense network in the cytoplasm. A 6-h incubation with IDPN
(10~3 M) causes partial disorganization
of intermediate filaments in frog (C)
and human (D) adrenocortical cells.
However, many keratin fibers remain
intact, particularly in amphibian cells.
N, Nucleus (x 1000).
plays a major role in the transduction of the corticotropic
message of angiotensin II (267, 268). In frog adrenocortical
cells, IDPN inhibits the response to angiotensin II (257), suggesting the involvement of intermediate filaments in the
mechanism of action of angiotensin II. In support of this
suggestion, IDPN does not modify the stimulatory effect of
prostaglandins (269) that act as second messengers of angiotensin II (99, 270). In addition, studies performed in bovine fasciculata cells have shown that calmodulin, which acts
with calcium as an intracellular messenger of angiotensin II,
can phosphorylate vimentin intermediate filaments (189),
suggesting that phosphorylation of these cytoskeletal fibers
is implicated in transduction of the messages of certain corticotropic factors. It has been shown recently that IDPN does
not affect the response of frog adrenocortical cells to endothelin (97), although ET-1 receptors are also coupled to phospholipase activity and prostaglandin synthesis (125). Therefore, it appears that, in normal adrenocortical cells,
intermediate filaments play a specific role in response to
certain corticotropic factors whose receptors are coupled to
phospholipases such as angiotensin II.
250, 251). In these cells, IDPN does not modify the spontaneous secretion of 20a-dihydroprogesterone (261), whereas
acrylamide, which is also thought to specifically disrupt intermediate filaments (271, 272), increases the spontaneous
secretion of steroids (273). In the human adrenal tumor cell
line SW-13, vimentin filaments are required for the transport
and esterification of cholesterol (274), suggesting a possible
role of intermediate filaments in steroid biosynthesis. Further
studies with other drugs and cell types are thus needed to
determine the function of intermediate filaments in spontaneous steroidogenesis in adrenocortical tumor cells.
Exposure of Y-l cells to ACTH induces rounding up of the
cells and disappearance of desmin intermediate filaments
(178). Although IDPN and acrylamide have opposite effects
on the spontaneous secretion of corticosteroids, these two
intermediate filament inhibitors exert a stimulatory effect on
ACTH-evoked steroid secretion in Y-l cells (261, 273). The
action of acrylamide is not mediated by an increase of cAMP
but involves a step of ACTH action upstream of pregnenolone formation (273). Inasmuch as IDPN does not modify the steroidogenic response of Y-l cells to (Bu)2cAMP and
2. Role in adrenocortical tumor cells. In Y-l cells, the distribution cholera toxin, it appears that, in adrenocortical tumor cells,
intermediate filaments are probably involved in a step of the
of vimentin intermediate filaments is similar to that of keratin
transduction of the corticotropic message of ACTH occurring
fibers in normal cells (245, 250, 251). Close contacts between
after cAMP formation and before pregnenolone biosynthesis.
vimentin fibers, mitochondria, lipid vesicles, and other cytoskeletal elements have been visualized in Y-l cells (245,
Therefore, it appears that, in normal adrenocortical cells,
June, 1996
CYTOSKELETON IN ADRENAL CELLS
279
F/B
NORMAL ADRENOCORTICAL
CELL
FIG. 6. Schematic representation of a normal adrenocortical cell illustrating the possible involvement of microtubules, microfilaments, and
intermediate filaments in the process of corticosteroid secretion. A, Aldosterone; AA, arachidonic acid; AC, adenylyl cyclase; B, corticosterone;
Ca/CM-PK, calcium/calmodulin-dependent protein kinase; Ch, cholesterol; Ch.E, cholesteryl ester; CT, centrosome; DG, diacylglycerol; DOC,
11-deoxycorticosterone; F, cortisol; G, GTP-binding regulatory protein of adenylyl cyclase; Gs, stimulatory GTP-binding regulatory protein of
adenylyl cyclase; I, inositol; IP, inositol phosphate; IP 2 , inositol bisphosphate; IP 3 , inositol trisphosphate; L, lysosome; LysoPI, lyso-phosphatidylinositol; N, nucleus; P, progesterone; PG, prostaglandins; PI, phosphatidylinositol; PIP, phosphatidylinositol monophosphate; PIP 2 ,
phosphatidylinositol bisphosphate; PKA, protein kinase A; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; P4508CC,
cytochrome P450scc; P450 n p, cytochrome P45011/3; R, membrane receptor; S, compound S (11-desoxycortisol); A5P, pregnenolone; 17OH-P,
17-hydroxyprogesterone.
intermediate filaments play a specific role in the response to
certain corticotropic factors whose receptors are coupled to
phospholipases such as angiotensin II. In contrast, intermediate filaments are not necessary for the spontaneous and the
ACTH-evoked secretion of corticosteroids. The role of intermediate filaments in adrenocortical tumor cells is not clear
since some clones, perfectly viable, are virtually devoid of
intermediate filaments. Studies performed in Y-l cells suggest the involvement of intermediate filaments in the mechanism of action of ACTH.
V. Cytoskeleton and Cell Contacts: Role in Adrenal
Steroidogenesis
The plasma membrane of adrenocortical cells exhibits specialized structures that are involved in the formation of con-
tacts between neighboring cells and with the extracellular
matrix. In the duck adrenal cortex, gap junctions and intermediate (or adherens) junctions are the most common forms
of cell contacts (126). In the rat adrenal cortex, a high density
of gap junctions is present in the zona glomerulosa and
reticularis, whereas zona fasciculata cells are mainly connected by septate-like junctions and desmosomes (200, 275).
Gap junctions and desmosomes are also the two major types
of specialized structures detected in the membrane of adrenocortical tumor cells (182, 276). Tight junctions (zonulae
occludentes) are rarely observed in the adrenal cortex of
vertebrates (126, 275).
Identification of cell-cell adherens junctions in the adrenal
cortex strongly suggests that molecules related to cadherins,
a class of transmembrane glycoproteins typical of adherens
junctions (277), are expressed in adrenocortical cells. The
FEUILLOLEY AND VAUDRY
280
Vol. 17, No. 3
11R-2Q>diOH-P
20«diOH-P
Y-1 ADRENOCORTICAL TUMOR CELL
FIG. 7. Schematic representation of a Y-1 adrenocortical tumor cell illustrating the possible involvement of micro tubules, microfilaments, and
intermediate filaments in the process of corticosteroid secretion. Gi; Inhibitory GTP-binding regulatory protein of adenylyl cyclase; 11/320adiOH-P, Ilj3-hydroxy-20a-dihydro-progesterone; 20adiOH-P, 20a-dihydroprogesterone. See legend to Fig. 6 for other abbreviations.
observation that, in granulosa cells, cadherins are involved
in the response to FSH (278) suggests that adherens junctions
may regulate the activity of steroid-secreting cells. In support
of this notion, it has been demonstrated that cadherins are
linked to microfilaments (279) that play pivotal roles in steroid biosynthesis in adrenocortical cells (see Section III). Although most of the constitutive proteins of desmosomes
(desmoplakin, desmoglein, desmocollin, and plakophilin)
are attached to intermediate filaments (280), desmosomes
only appear as physical links between cells, without major
functions in stabilization of the intermediate filament network (281) or in cell growth and division (280). Direct contacts between gap junctions and cytoskeletal fibers have
never been described (282), but gap junctions appear to play
an important role in the response of bovine and human
adrenal cells to ACTH (283). In human adrenocortical cells,
depolymerization of microtubules and microfilaments does
not prevent the association of gap junctions to the plasma
membrane (182).
The extracellular matrix is a complex network of fibrous
proteins. In the rat adrenal gland, collagen, elastin, and
contractile fibers, generated by fibroblasts and myoid cells
originating from the capsule (284), form a dense plexus of
fibrils surrounding the capillaries and delimiting the three
cortical zones (285). Adrenocortical cells also produce extracellular matrix proteins, such as tenascin, which has
been identified recently in the human adrenal cortex (286).
A complex matrix, similar to that produced by fibroblasts,
is required for the growth and the steroidogenic activity
of bovine adrenocortical cells in vitro (287). The fact that
microfilament-disrupting agents exert different effects on
rat zona glomerulosa explants (210) and freshly dispersed
rat zona glomerulosa cells (130) suggests that contacts
between microfilaments and the cell adhesion molecules
are necessary for the steroidogenic activity of adrenocortical cells.
It thus appears that, through their association with plasma
membrane structures involved in cell contacts, microfilaments and intermediate filaments may modulate the steroidogenic activity of adrenocortical cells in response to their
extracellular environment.
June, 1996
CYTOSKELETON IN ADRENAL CELLS
VI. Cytoskeleton and the Nuclear Matrix: Control of
Genomic Expression
The nuclear matrix designates elements of the nucleus
including the peripheral nuclear lamina and the internal
fibrogranular material (19). The nuclear lamina is a network
of intermediate filaments bordering the inner surface of the
nuclear membrane (288). Lamin fibers bind to DNA (289,
290), polynucleosomes (291), and chromosomes (292), and
thus these fibers serve as links between cytoplasmic intermediate filaments and nucleic acid.
Actin and actin-binding proteins have been detected
within the nucleus in different types of cells, including
epithelial cells (293, 294). In adrenocortical cells, microfilaments have been visualized in close contact with the
nuclear membrane (62, 187), suggesting that actin filaments interact with the nuclear matrix. Consistent with
this notion, it has been demonstrated that cytochalasin B
and D inhibit DNA synthesis in normal and adrenocortical
tumor cells (295). This effect of cytochalasins, which is not
due to inhibition of nucleotide transport, suggests that
actin and/or microfilaments may play a role in the mechanism of replication (295). Corticotropic factors, such as
ACTH, induce reorganization of the structure of the nucleus in adrenal cells (296) and a rapid stimulation of the
synthesis of mRNAs encoding for j3-actin, before the expression of the genes encoding for the enzymes involved
in steroid biosynthesis (297). Other studies performed in
granulosa cells have shown that microfilaments are involved in the process of differentiation (298). Since it has
been demonstrated that certain classes of translationally
active mRNAs and ribosomes are associated with the microfilament network (299, 300), it is conceivable that microfilaments may be involved in the regulation of transcription in adrenocortical cells.
Studies performed in other cell types have shown that
cytoplasmic intermediate filaments, including keratin and
vimentin fibers (two classes of fibers expressed in normal
and tumoral adrenocortical cells), are connected to the
nuclear lamina (14) and have high affinity for nucleic acids
and DNA (301, 302). The functional significance of these
contacts is unknown, although several lines of evidence
suggest that intermediate filaments exert gene-regulatory
functions (303). Isolation of subclones of the human adrenal cell line SW13, devoid of cytoplasmic intermediate
filaments (249), indicates that the absence of these cytoskeletal elements does not impair cell growth and division. In addition, injection of vimentin in these adrenocortical tumor cells causes rapid polymerization of
intermediate filaments in the cytoplasm without any noticeable influence on the organization of the nuclear membrane (304).
Altogether, these studies show that microfilaments probably exert regulatory functions both at the transcriptional
and translational levels. Intermediate filaments are closely
associated with nuclear structures but do not apparently play
a significant role in the regulation of gene expression in
adrenocortical cells.
281
VII. Conclusion
During the last 10 yr, many studies have demonstrated
that the elements of the cytoskeleton play major functions in
adrenocortical cells. However, the role of the cytoskeleton in
corticosteroid biosynthesis differs markedly among vertebrate species as well as between normal and tumoral cells. In
normal adrenocortical cells, microtubules are required for
transduction of the corticotropic message of ACTH and cholesterol transport, whereas microfilaments control the metabolism of polyphosphoinositides as well as the translocation of cholesterol and intermediate steroids from the
cytoplasm to the mitochondria. Concurrently, intermediate
filaments are involved in the stimulus-response coupling of
certain corticotropic factors such as angiotensin II (Fig. 6). In
the Y-l adrenocortical tumor cells, the role of microtubules
appears to be limited to cholesterol storage, while microfilaments are required for the transport of cholesterol to the
mitochondria. Finally, in tumor cells that express intermediate filaments, these fibers are probably involved in the
mechanism of action of ACTH (Fig. 7).
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