[CANCER RESEARCH 64, 6444 – 6452, September 15, 2004]
Transcriptional Regulation of Signal Regulatory Protein ␣1 Inhibitory Receptors by
Epidermal Growth Factor Receptor Signaling
Gurpreet S. Kapoor,1 Dmitri Kapitonov,1 and Donald M. O’Rourke1,2
Departments of 1Neurosurgery and 2Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
ABSTRACT
Signal regulatory protein (SIRP) ␣1 is a membrane glycoprotein and a
member of the SIRP receptor family. These transmembrane receptors
have been shown to exert negative effects on signal transduction by
receptor tyrosine kinases via immunoreceptor tyrosine-based inhibitory
motifs in the carboxyl domain. Previous work has demonstrated that
SIRPs negatively regulate many signaling pathways leading to reduction
in tumor migration, survival, and cell transformation. Thus, modulation
of SIRP expression levels or activity could be of great significance in the
field of cancer therapy. The aim of the present study was to determine the
factors that regulate levels of SIRP␣1 in human glioblastoma cells that
frequently overexpress the epidermal growth factor receptor (EGFR)
because SIRPs have been shown to negatively regulate EGFR signaling.
Northern blot analysis and immunoprecipitation assays showed variable
expression levels of endogenous SIRP␣ transcripts in nine well-characterized glioblastoma cell lines. We examined SIRP␣1 regulation in
U87MG and U373MG cells in comparison with clonal derivatives that
express a truncated form of erbB2, which negatively regulates EGFR
signaling by inducing the formation of nonfunctional heterodimeric complexes. Mutant erbB2-expressing cells contained more SIRP␣1 mRNA
when compared with the parental cells in presence or absence of serum.
Similarly, immunoprecipitation assays showed increased SIRP␣1 protein
levels in erbB-inhibited cells when compared with parental cells. Messenger RNA stability assays revealed that the increased mRNA levels in
EGFR-inhibited cells were due to an induction of transcription. Consistent
with this finding, expression of the erbB2 mutant receptor up-regulated
SIRP␣1 promoter activity in all cell lines tested. Interestingly, pharmacological inhibition of the kinase activities of EGFR, erbB2, and src and
activation of mitogen-activated protein kinase, but not phosphatidylinositol 3ⴕ-kinase, significantly up-regulated SIRP␣1 promoter activity. Based
on these observations, we hypothesize that down-modulation of EGFR
signaling leads to transcriptional up-regulation of the inhibitory SIRP␣1
gene. These data may be important in the application of erbB-inhibitory
strategies and for design of therapies for the treatment of glial tumors and
other epithelial malignancies.
INTRODUCTION
Signal regulatory proteins (SIRPs) were described as transmembrane
phosphoproteins that coprecipitated with SH2 domain-containing protein
tyrosine phosphatases (1, 2). SIRP receptors were first identified as SH2
domain-containing protein tyrosine phosphatase substrate (SHPS)-1 (1)
and brain immunoglobulin-like molecules with a tyrosine-based activation motif (BIT; ref. 3). The SIRP family currently contains more than 15
members that vary with respect to subtle amino acid differences in their
extracellular domains. The family was further divided into two types, ␣
and ␤, which differ by the presence (SIRP␣) or absence (SIRP␤) of
an intracytoplasmic domain (2). The SIRP␣ subtype has an apparent
Received 1/26/04; revised 6/23/04; accepted 7/14/04.
Grant support: National Institutes of Health grant R01 CA-90586 and grants from
The Department of Veterans Affairs (Merit Review Program) and The Brain Tumor
Society (D. O’Rourke).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Donald M. O’Rourke, 502 Stemmler Hall, Department of
Neurosurgery, University of Pennsylvania School of Medicine, 36th and Hamilton Walk,
Philadelphia, PA 19104. Phone: 215-898-2871; Fax: 215-898-9217; E-mail: orourked@
mail.med.upenn.edu.
©2004 American Association for Cancer Research.
molecular weight of between 85,000 and 95,000 in humans and rats and
100,000 to 120,000 in the mouse and is heavily glycosylated.
Several groups have discovered various SIRP␣-like molecules,
which are designated as SHPS-1, p84, BIT, MFR, MyD-1, and
SIRP␣1 (1, 2, 4 –7). The intracytoplasmic domain of SIRP␣ proteins
contains a four tyrosine-based regulatory motif called the immunoreceptor tyrosine-based inhibitory motif, which, on phosphorylation,
recruits the SH2 domain-containing protein tyrosine phosphatases
SHP-1 and SHP-2. This physical association has been shown to
induce negative regulatory effects on cell proliferation induced by
growth factor stimulation of receptor tyrosine kinases and by viral
oncogene products (1–3, 8). On the other hand, SIRP␤ contains no
intracytoplasmic domain and associates with other adapter molecules
to modulate signaling (9). Two such adapter molecules, DAP-12 (also
known as KARAP; refs. 10 –12) and DAP-10 (also called KAP-10;
refs. 13 and 14), have been shown to interact with SIRP␤. In contrast
to SIRP␣, SIRP␤ molecules possess immunoreceptor tyrosine-based
activating motifs, which confer a positive regulatory effect (10, 15,
16).
SIRP␣1 proteins are differentially expressed in a variety of tissues
but are abundant in myeloid cells, neurons, and brain (2, 17–20).
Various studies have linked SIRP␣1 receptors to different biological
processes, most notably cell growth and motility. SIRP␣1 proteins
were reported to interact with CD47 on cerebral epithelium to facilitate monocyte migration across the brain, which is considered to be
a critical event in various neuroinflammatory disorders, such as multiple sclerosis or human immunodeficiency virus-associated dementia
(21). A study using mice expressing a mutant SHPS-1 [SHPS-1cyto(⫺/⫺)] lacking most of the cytoplasmic region indicated that
SHPS-1 (SIRP␣1) contributes to the survival of circulating platelets
by down-regulating the macrophage phagocytic response (22). Overexpression studies of SHPS-1 in fibroblasts have shown that SHPS-1
plays a crucial role in integrin-mediated cytoskeletal reorganization,
cell motility, and adhesion-induced activation of Rho and in the
negative regulation of growth factor-induced activation of mitogenactivated protein kinases [MAPKs (23)]. SIRP␣1/SHPS-1 receptors
have also been shown to positively or negatively regulate MAPK
pathways after stimulation of many different receptors, including
epidermal growth factor receptor (EGFR) and the insulin receptor. A
previous study has shown that overexpression of SHPS-1 resulted in
increased SHPS-1/SHP-2 complex formation, which potentiated the
Ras/Raf/MAPK pathway in response to insulin (24). On the contrary,
SIRP␣1 overexpression in NIH3T3 cells inhibited DNA synthesis and
MAPK phosphorylation after epidermal growth factor (EGF) or insulin stimulation (2). Previously, we reported that SIRP␣1 associates
with SHP-2 to negatively regulate EGFR-mediated phosphatidylinositol 3⬘-kinase (PI3K) signaling and resulted in reduced transformation,
reduced cell migration, and cell spreading and enhanced apoptosis
after DNA damage in human glioblastoma cells (25). Recently, we
demonstrated that an association between the SHP-2 phosphatase and
Gab1 adapter protein is critical for EGFR-mediated positive signaling
(26) and that SHP-2 plays a positive role in regulating the response of
astrocytes and fibroblasts to growth factors (27). Therefore, modulation of SIRP␣1 activity and expression levels may play an important
role in regulating EGFR-mediated Gab1/SHP-2 association and cell
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TRANSCRIPTIONAL REGULATION OF SIRP␣1 BY EGFR SIGNALING
signaling. However, mechanisms that regulate SIRP␣1 expression and
function have not been well characterized.
In this study, we have evaluated the transcriptional regulation of
SIRP␣1 in human cancer cells to understand SIRP expression and
function. We have chosen human glioblastoma cells due to the central
role played by EGFR in glial tumorigenesis (28 –31), the coupling
between EGFR and SIRP, and the high levels of endogenous SIRP
expression in the mammalian brain. We used nine well-characterized
human glioblastoma cell lines to determine factors responsible for
transcriptional regulation of SIRP␣1 levels. Northern blot and immunoprecipitation analyses in U87MG and U373MG cells in comparison
with erbB-inhibited clonal derivative U87MG/T691 and U373MG/
T691 cells showed that phenotypic inhibition and serum starvation
resulted in increased SIRP␣1 expression. These results suggest that
down-regulated EGFR signaling cooperated with serum starvation to
up-regulate SIRP␣1 expression. To further evaluate mechanisms regulating inhibitory SIRP␣1 receptors in glioblastoma cells, we used a
luciferase reporter construct carrying an isolated ⬃2.0-kb fragment of
the 5⬘-untranslated region (UTR) from the SIRP␣1 gene for promoter
studies. A luciferase reporter plasmid containing this SIRP␣1 promoter region showed increased luciferase activity when cotransfected
with a plasmid carrying a truncated erbB2 receptor, which interferes
with EGFR signaling in different cell lines. Furthermore, pharmacological inhibition of tyrosine kinase activity of EGFR and erbB2
increased SIRP␣1 promoter activity. Interestingly, pharmacological
inhibition of p42/44 MAPK and src kinase showed statistically significant up-regulation of promoter activity as compared with control
cells. Transcriptional up-regulation of SIRP␣1 expression is therefore
coupled to down-modulation of EGFR signaling. Collectively, our
data suggest that mechanisms up-regulating inhibitory SIRP␣1 receptor expression and/or function could be used as an adjunct to existing
therapies for erbB-driven tumors including glioblastomas and other
epithelial malignancies.
Northern Blot Analysis. Total RNA was isolated using TRIzol, according
to the manufacturer’s directions. Briefly, 10 to 20 ␮g of total cellular RNA
were fractionated on 1% formaldehyde agarose gel, transferred to Magnacharge nylon membrane by capillary blotting, and fixed by baking at 80°C
under vacuum. Labeling of radioactive probes was performed using
[␣-32P]dCTP and a Prime-It kit (Stratagene, La Jolla, CA) according to the
manufacturer’s instructions. Hybridization was carried out at 65°C, after which
membranes were washed to a stringency of 1⫻ SSC, 0.1% SDS at 65°C.
Autoradiography was carried out at ⫺80°C. A 2.2-kb SIRP␣1 cDNA fragment
excised with NheI and PstI from pIRES hygro2 plasmid was used to make
radioactive probe for hybridization. To verify equal loading, all gels were
stained with ethidium bromide. Furthermore, SIRP␣1 mRNA levels were
normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
mRNA levels, which remained relatively constant under different treatment
conditions.
Cell Lysis, Immunoprecipitation, and Immunoblotting. Cells were
scraped off in lysis buffer containing 50 mmol/L Tris (pH 7.5), 150 mmol/L
NaCl, 2 mmol/L EGTA, 0.1% Triton X-100, 1 mmol/L phenylmethylsulfonyl
fluoride, 100 ␮g/mL aprotinin and leupeptin, and 1 mmol/L sodium orthovanadate. After a 30-minute incubation on ice, soluble fractions were collected, and
protein concentrations were determined using a Dc Protein Assay Kit (BioRad, Hercules, CA). Approximately 400 –500 ␮g of soluble fraction were
incubated with anti-SIRP␣1-antibody (2 ␮g) for 2 hours at 4°C. Immune
complexes were collected with protein A-Sepharose CL-4B for 1 hour; washed
three times with wash buffer containing 50 mmol/L Tris (pH 7.5), 133 mmol/L
NaCl, 2 mmol/L EGTA, and 0.1% Triton X-100; and boiled for 5 minutes in
1⫻ SDS-PAGE sample buffer [250 mmol/L Tris (pH 6.8), 10% SDS, 10%
␤-mercaptoethanol, and 40% glycerol]. Protein samples were resolved by
SDS-PAGE, transferred to nitrocellulose, and incubated with anti-SIRP␣1
antibody (1:3,000) for 1 hour, followed by incubation with horseradish peroxidaselinked anti-IgG secondary antibody (1:3,000; Amersham Pharmacia Biotech).
Immunoreactive proteins were detected by enhanced chemiluminescence as described by the supplier.
Isolation of the 5ⴕ-Flanking Region Fragment of SIRP␣1. Based on the
published sequence of SIRP␣1/PTPNS1/SHPS-1 (GenBank accession number
NM_080792, NM_004648), the 5⬘-UTR of SIRP␣1 was used to BLAST
search the expressed sequence tag database for the transcripts containing the
longest 5⬘-UTR. A clone (BG421441) that contained an additional 2,000
MATERIALS AND METHODS
nucleotides at the 5⬘-end was used to search the human genome for the
Reagents. Cycloheximide, puromycin, and actinomycin were purchased sequence upstream of the SIRP␣1 5⬘-UTR. A Homo sapiens PAC clone
from Calbiochem (San Diego, CA). EGF was purchased from Becton Dick- RP4-539M6 from chromosome 22 was retrieved (locus AC004832) and anainson (Franklin Lakes, NJ). Anti-SIRP␣1 antibody was kindly provided by Dr. lyzed for putative transcription start sites. Using the Promoter Prediction tool
Gibbes R. Johnson (Food and Drug Administration, Bethesda, MD). Anti-␤- of the Berkeley Drosophila Genome Project, a putative transcription start site
actin monoclonal antibody (clone AC-15) was purchased from Sigma (St. was predicted to be located 50 bp upstream of the BG421441 5⬘-UTR with a
Louis, MO). TRIzol and all tissue culture supplies were obtained from Invitro- score of 0.99. Translation is predicted to originate at position ⫹261.
gen (Carlsbad, CA). Diethyl pyrocarbonate and protein A-Sepharose CL-4B
Plasmid Construct. Based on the sequence of the RP4-539M9 PAC clone
were purchased from Sigma. Magnacharge nylon membrane was purchased from chromosome 22 (AC004832 locus), two oligonucleotide primers, a plus
from Osmonics Inc. (Westborough, MA). [␣-32P]dCTP (300 Ci/mmol) was primer (5⬘-CTTACGCGTAACTCATGGGCATTAAGATCAATTACTTGGobtained from NEN Life Science Products, Inc. (Boston, MA), and the enCCAGGTGAGG-3⬘, ⫺1908/⫺1868) and a minus primer (5⬘-CAAAGATCTThanced chemiluminescence detection kit was obtained from Amersham PharTGCGCAAACTTGTTTTTCTGAGGTCAGCGCTGCGAGC-3⬘, ⫹176/⫹136),
macia Biotech (Buckinghamshire, United Kingdom). A light chemilumineswere designed to amplify the UTR of the SIRP␣1 gene using normal human
cence reporter gene assay system for the detection of luciferase activity was
genomic DNA (Promega) as a template. The 2084-bp fragment was confirmed
purchased from Promega (Madison, WI).
by both plus and minus primers by DNA sequencing. Plasmid pGL3-2084
Cell Lines and Culture Conditions. Human glioblastoma cell lines
SIRP␣1 was generated by fusing this fragment with pGL3-Basic vector at the
U87MG, U373MG, U343, U251, U118, LN18, LN229, SF767, and T98G
MluI
and BglII sites.
were maintained in Dulbecco’s modified Eagle’s medium (Cellgro, Herndon,
Transient Transfections. Transfections were performed using the FuGene
VA), with 10% fetal bovine serum (Hyclone, Ogdon, UT) at 37°C in 95%
air/5% CO2. U87MG/T691 and U373MG/T691, clonal derivatives of U87MG reagent (Roche Molecular Biochemicals) according to the 5manufacturer’s
and U373MG, respectively, were supplemented with 0.4 mg/mL G418 (In- instructions. Briefly, cells were seeded at a density of 1 ⫻ 10 cells/well in a
vitrogen) to maintain stable expression of truncated ErbB2/Neu receptor 12-well plate 1 day in advance. Transfections were performed in duplicate
(T691stop) with large cytoplasmic deletion, which include the tyrosine kinase using 6 ␮L of FuGene and 1 ␮g of the reporter plasmid carrying the SIRP␣1
domain of p185/neu (31). Another derivative, U87MG/SIRP␣1, which stably promoter. The pSV2-␤-galactosidase vector (0.4 ␮g; Promega) was used to
expresses the SIRP␣1 protein, was supplemented with 40 ng/mL hygromycin control transfection efficiency. Forty-eight hours after transfection, cells were
harvested by removing the media, washing twice with PBS, and directly
(Roche Molecular Biochemicals, Indianapolis, IN; ref. 25).
Messenger RNA Stability Assay. To measure the half-life of the SIRP␣1 adding 100 ␮L of lysis buffer per well. Of this lysate, 10 ␮L were used for
message, we incubated U373MG and U373MG/T691 cells with 10 ␮g/mL luciferase determination, and 50 ␮L were used for ␤-galactosidase determinaactinomycin D, and samples were harvested for total RNA at the designated tion. These determinations were performed using the luciferase kit and the
intervals thereafter.
␤-galactosidase enzyme assay system (Promega).
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TRANSCRIPTIONAL REGULATION OF SIRP␣1 BY EGFR SIGNALING
RESULTS
Human Glioblastoma Cells Express SIRP␣1. SIRP␣1 molecules
are expressed at different levels in a variety of tissues (2). Two SIRP␣
transcripts of 3.9 and 2.5 kb have been detected by Northern hybridization in heart, brain, placenta, lung, liver, skeletal muscle, kidney,
and pancreas, with higher expression in brain, liver, and heart (2).
Similarly, other workers have reported transcripts of 4.4 and 2.4 kb in
different tissues, suggesting the occurrence of alternative splice forms
(19, 32). In the present study, we investigated the presence of SIRP␣1
transcripts in RNAs from nine well-characterized human glioblastoma
cell lines by Northern blotting using a 32P-labeled, 2.2-kb SIRP␣1
cDNA fragment. We also used U87MG glioblastoma cells ectopically
expressing SIRP␣1 protein (U87MG/SIRP␣1) as a positive control.
We observed one prominent mRNA transcript of 3.9 kb and three
additional transcripts of 6.0, 2.4, and 2.3 kb in SIRP␣1-expressing
U87MG cells (Fig. 1A). There was variable expression of SIRP␣1
Fig. 1. Differential expression of SIRP␣1 in human glioblastoma cell lines. A. Nine
glioblastoma cell lines were plated on 25-cm2 flasks at a density of 1 ⫻ 106 cells/flask at
day 0 and refed with fresh medium at day 2. At day 4, total RNA was extracted from each
cell line and subjected to Northern blot analysis using 32P-labeled, 2.0-kb SIRP␣1 cDNA
insert. Ethidium bromide staining of the gel confirmed equal loading by visual inspection.
The nylon membrane was stripped and then probed with 32P-labeled GAPDH probe as a
loading control (see Materials and Methods). B. Cells were seeded at density of 2 ⫻ 106
cells/10-mm dish at day 0. After 24 hours of serum starvation after day 3, cells were lysed,
and lysates were subjected to immunoprecipitation with anti-SIRP␣1 antibody followed
by SDS-PAGE and immunoblotting with anti-SIRP␣1 antibody. IP, immunoprecipitation;
IB, immunoblotting.
transcripts in glioblastoma cells lines with greatest SIRP␣1 abundance
in U87MG, U373MG, U343, and LN229 cells (Fig. 1A).
We then assessed the expression of SIRP␣1 protein by immunoprecipitating SIRP␣ polypeptides with an anti-SIRP␣1 antibody in
glioblastoma cell lysates, followed by immunoblotting with a polyclonal anti-SIRP␣1 antibody. The immunoblot results showed a prominent single Mr 90,000 band in U87MG/SIRP␣1 cells (Fig. 1B). The
expression of SIRP␣1 in different cell lines was variable, and the
highest levels of SIRP␣1 protein were found in U373 and LN229
cells. Collectively, our results indicate that the majority of glioblastoma cell lines express SIRP␣1 with multiple transcripts that may
represent various alternative splice forms of the SIRP␣1 gene.
ErbB Inhibition and Serum Starvation Cooperate to UpRegulate SIRP␣1 Expression in Human Glioblastoma Cells. EGF
treatment induces phosphorylation of the SIRP␣1/SHPS-1 receptor
(25, 33–35), suggesting that EGFR signaling may modulate SIRP␣1
expression and/or activation. In our previous work, we showed that
overexpression of a truncated erbB2 mutant receptor (T691stop) reduced cell transformation and conferred increased susceptibility to
apoptosis in U87MG glioblastoma cells by inhibiting EGFR/erbB
signaling (30, 31). Similarly, we also showed that overexpression of
SIRP␣1 in U87MG glioblastoma cells reduced cell transformation
and motility, and enhanced DNA damage-induced apoptosis (25).
These studies suggested a possible link between EGFR signaling and
SIRP␣ expression and/or function. However, there are no reports
demonstrating that SIRP␣ expression is regulated by EGFR function
or kinase activation. To evaluate the relationship between EGFR
signaling and SIRP␣ expression, we performed Northern blot analysis
using a 32P-labeled, 2.2-kb SIRP␣1 cDNA on total RNAs from
U87MG and U373MG glioblastoma cells in the presence or absence
of serum. Parental cells were compared with their clonal derivatives
expressing either the constitutively active EGFRvIII oncoprotein (also
termed ⌬EGFR) or the truncated erbB2 mutant (T691stop), all of
which have well-characterized phenotypes with distinct transforming
efficiencies (30, 31). ⌬EGFR (or EGFRvIII) lacks a portion of the
extracellular domain, is constitutively phosphorylated, and confers a
more malignant phenotype (30, 36). T691stop is a truncated form of
erbB2 with a large cytoplasmic deletion, which includes the tyrosine
kinase domain (p185neu) and the entire COOH terminus. This mutant
receptor inhibits cell transformation and confers increased susceptibility to apoptosis by inducing the formation of nonfunctional heterodimeric complexes (30, 31). Interestingly, U87MG/T691 and
U373MG/T691 cell clones showed increased SIRP␣1 mRNA expression when compared with their parental controls (Figs. 2A and 3A).
However, U87MG.⌬EGFR cells showed no change in SIRP␣1
mRNA expression as compared with parental U87MG cells (Fig. 3A).
These studies suggest that down-modulation of EGFR signaling leads
to up-regulation of SIRP␣1 expression. Moreover, on serum starvation, all cells showed up-regulation of SIRP␣1 expression, suggesting
that low serum might be an additional stimulus that increases SIRP␣1
mRNA synthesis. We then evaluated SIRP␣1 protein expression
under similar conditions by immunoprecipitation assay using an antiSIRP␣1 antibody and Western blot analysis. Consistent with the
mRNA expression results, we observed increased SIRP␣1 protein
levels in EGFR-inhibited clones and with serum starvation of cells
(Figs. 2B and 3B).
To further evaluate our findings, U373MG/T691 cells were treated
with EGF at different concentrations and for various times, and the
effect on SIRP␣1 receptor expression was determined by Northern
blotting. EGF treatment reduced SIRP␣1 receptor expression in a
dose-dependent manner without substantially affecting expression of
the housekeeping GAPDH gene (Fig. 4A and B). The most effective
concentration of EGF for down-regulating SIRP␣1 expression was 50
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TRANSCRIPTIONAL REGULATION OF SIRP␣1 BY EGFR SIGNALING
half-lives for both mRNA species were nearly identical in both cell
lines (3.9-kb species, ⬃5 hours; 6.0-kb species, ⬃2 hours), indicating
that SIRP␣1 mRNA stability does not account for the differences in
SIRP␣1 levels in the two isogenic cell lines. This observation suggested that there might be a difference in transcriptional regulation of
the SIRP␣1 gene between the two cell lines distinguished by the level
of endogenous EGFR activation.
To address transcriptional regulation of SIRP␣1, we identified a
2084-bp region upstream of the SIRP␣1 5⬘-UTR on chromosome 22
and cloned this fragment into the pGL3 basic reporter plasmid carrying a luciferase gene (pGL3-SIRPprom-Luc) as described in Materials
and Methods (Fig. 6A). The pGL3-SIRP-Luc reporter was cotransfected with either empty vector or the T691 inhibitory receptor into
different glioblastoma cells to determine the effect of EGFR inhibition
on SIRP␣1 promoter activity. We observed a 2- to 3.5-fold increase in
SIRP␣1 promoter activity in all cells transfected with the T691 EGFR
Fig. 2. EGFR/erbB inhibition and serum starvation up-regulate SIRP␣1 expression in
human glioblastoma cells. A. U373MG and U373MG/T691 cells were seeded in 25-cm2
flasks at a density of 1 ⫻ 106 cells/flask in duplicates. On day 3, medium in one flask from
each cell line was replaced with serum-free medium and incubated for another 24 hours.
At day 4, total RNA was extracted from each cell line and subjected to Northern blot
analysis using 32P-labeled, 2.0-kb SIRP␣1 cDNA insert. Ethidium bromide staining of the
gel confirmed equal loading by visual inspection. Nylon membrane was stripped and then
probed with 32P-labeled GAPDH probe as a loading control (see Materials and Methods).
B. Cells (5 ⫻ 105) were seeded in 6-well plates in duplicates at day 0. After 24 hours of
serum starvation after day 3, cells were lysed, and lysates were separated on SDS-PAGE,
transferred onto nitrocellulose membrane, and immunoblotted with anti-SIRP␣1 antibody.
Membrane was stripped and blotted with anti-␤-actin monoclonal antibody to determine
the amount protein sample loaded in each well.
ng/mL, and saturation occurred after 100 ng/mL (Fig. 4A). A significant decrease in SIRP␣1 receptor expression was apparent at 6 hours,
and the plateau was reached after 12 hours (Fig. 4B). These observations suggest that ligand-induced activation of EGFR/erbB2 complexes regulates expression of SIRP␣1 inhibitory receptor.
Up-Regulation of SIRP␣1 Messenger RNA Involves Induction
of Gene Transcription. Our results indicate that overexpression of
the T691 mutant erbB receptor up-regulated SIRP␣1 mRNA expression. To evaluate whether increase in SIRP␣1 expression is regulated
at the level of transcription or on RNA stability, we inhibited the
transcription of newly synthesized mRNA with actinomycin D (10
␮g/mL) at different time intervals in both U373MG parental and
EGFR-inhibited U373MG/T691 cells (Fig. 5A). We then plotted the
relative SIRP␣1 mRNA levels (Fig. 5B) and calculated the half-lives
of the 3.9- and 6.0-kb mRNA species in both cell lines. The calculated
Fig. 3. Down-modulation of EGFR signaling and serum starvation up-regulates
SIRP␣1 expression in glioblastoma cells. A. U87MG, U87MG/T691, and
U87MG.⌬EGFR cells were seeded in 25-cm2 flasks at a density of 1 ⫻ 106 cells/flask in
duplicates. On day 3, medium in one flask from each cell line was replaced with
serum-free medium and incubated for another 24 hours. At day 4, total RNA was extracted
from each cell line and subjected to Northern blot analysis using 32P-labeled, 2.0-kb
SIRP␣1 cDNA insert. Ethidium bromide staining of the gel confirmed equal loading by
visual inspection. The nylon membrane was stripped and then probed with 32P-labeled
GAPDH probe to normalize SIRP␣1 mRNA levels (see Materials and Methods). B.
U87MG, U87MG/T691, and U87MG.⌬EGFR cells were seeded in 10-mm dishes in
duplicates at day 0. After 24 hours of serum starvation after day 3, cells were lysed, and
lysates were subjected to immunoprecipitation with anti-SIRP␣1 antibody followed by
SDS-PAGE and immunoblotting with anti-SIRP␣1 antibody. These figures are the representative of three independently reproducible experiments.
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TRANSCRIPTIONAL REGULATION OF SIRP␣1 BY EGFR SIGNALING
Fig. 4. EGFR activation negatively regulates SIRP␣1 expression. A. U87MG/T691 cells were seeded at a density of 1 ⫻ 106 cells/flask on day 0. On day 2, the media were replaced
by fresh serum-free medium. After 24 hours, cells were treated with the indicated concentrations of EGF (ng/mL) for another 6 hours. RNA was extracted and subjected to Northern
blot analysis using 32P-labeled, 2.0-kb SIRP␣1 cDNA insert. RNA gel stained with ethidium bromide before blotting is shown to demonstrate equal loading. SIRP␣1 mRNA levels
were normalized with levels of GAPDH mRNA. B. U87MG/T691 cells were seeded at a density of 1 ⫻ 106 cells/flask on day 0. Cells were incubated in fresh serum-free medium.
After 24 hours, cells were treated with EGF (100 ng/mL) at the indicated intervals of time. RNA was extracted and subjected to Northern blot analysis using 32P-labeled, 2.0-kb SIRP␣1
cDNA insert. RNA gel stained with ethidium bromide before blotting is shown to demonstrate equal loading. SIRP␣1 mRNA levels were normalized with levels of GAPDH mRNA.
inhibitory receptor when compared with empty vector controls (Fig.
6B). To further support this observation, we treated U87MG parental
cells with the EGFR kinase inhibitor AG1478 and the erbB2 kinase
inhibitor AG825 to evaluate the effects on SIRP␣1 promoter activity.
As expected, we observed enhanced promoter activity with both the
inhibitors, suggesting that differences in the SIRP␣1 mRNA levels
between two isogenic glioblastoma cell lines distinguished only by
levels of functional activation of the erbB kinases could only be
attributed to differences in activation of the SIRP␣1 promoter (Fig. 6C).
Inhibition of MAPK and src Kinase but not PI3K Leads to
Increased SIRP␣1 Promoter Activity. Having shown that inhibition of the EGFR and erbB2 kinase activation leads to increased
SIRP␣1 promoter activity, we examined whether pathways downstream of the EGFR activation loop played any role in SIRP␣1
promoter activation. U87MG cells were transiently transfected with
the pGL3-SIRPprom-Luc reporter, serum-starved, and then treated
with various pharmacological inhibitors. We observed that treatment
of U87MG cells with the p42/44MAPK inhibitors PD98059 and
U0126 up-regulated SIRP␣1 promoter activity (Fig. 7). Interestingly,
treatment with PP1, a src kinase inhibitor, also increased SIRP␣1
promoter activity. Our results support a previous study showing that
overexpression of v-src suppressed SHPS1 (rodent homologue of
SIRP) mRNA expression via the Ras-MAPK pathway to promote
transformation in fibroblast cells (37) and suggest that src kinase and
MAPK pathways may play an important role in regulating SIRP
expression at the level of transcription. However, LY294002 and
wortmannin, pharmacological inhibitors of the PI3K/Akt pathway, did
not affect SIRP␣1 promoter activity (Fig. 7). This observation suggests that transcriptional activation of the SIRP␣1 gene may be
regulated by EGFR/erbB2 and src kinases via extracellular signalregulated kinase (ERK) 1/2.
Up-Regulated SIRP␣ Expression Is Dependent on New Protein
Synthesis. To examine whether the induction of SIRP␣ expression
required new protein synthesis, phenotypically inhibited U87MG cells
expressing the ErbB2 (T691stop) mutant were incubated with different concentrations of cycloheximide and puromycin in the presence or
absence of EGF (100 ng/mL). Inhibition of translation reduced mRNA
levels of the 3.9-kb SIRP␣1 transcript, indicating that additional
protein synthesis is required to stimulate SIRP␣1 expression on inhibition of the EGFR signaling module (Fig. 8A and B). However, there
was an increase in the levels of 6.0-kb transcript on cycloheximide
and puromycin treatment in the presence or absence of EGF, suggesting that the 6.0-kb transcript could be a precursor mRNA and that
ligand-mediated EGFR activation may cooperate with inhibition of
translation to block generation of mature SIRP␣1 mRNAs, to downregulate SIRP␣1 expression (Fig. 4A and B).
To further support our observations, U87MG and U87MG/T691
cells were transiently transfected with pGL3-SIRPprom-Luc reporter,
serum-starved, and treated with the indicated concentrations of cycloheximide. As expected, we observed increased promoter activity in
EGFR-inhibited cells as compared with parental cells. Interestingly,
cycloheximide-treated cells showed reduced promoter activity as
compared with untreated cells, indicating that synthesis of new transcription factors is required for activation and expression of the
SIRP␣1 gene (Fig. 8C).
DISCUSSION
Previous studies have shown that SIRP␣1 is tyrosine-phosphorylated and activated by a variety of ligands, including EGF, plateletderived growth factor, insulin, neurotrophins, lysophosphatidic acid,
adhesion to fibronectin, growth hormone, colony-stimulating factor,
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for both biology and therapy. However, there are no reports regarding
the specific factors regulating the expression of SIRP␣1 inhibitory
proteins.
In this study, we provide the first evidence, to our knowledge, of
transcriptional regulation of SIRP␣1. Variable amounts of SIRP␣1
mRNA and protein in different glioblastoma cell lines suggest that
expression of SIRP␣1 may be under some regulatory control mediated
by EGFR kinase function. It has been established both in vitro and in
vivo systems that EGFR interacts and forms active heterodimeric
complexes with neu/c-erbB2 (p185neu) in response to EGF and neu
differentiation factor (heregulin) to potentiate cell signaling and transformation (41– 46). In accordance with previous reports, we demonstrated that erbB/EGFR inhibition due to nonfunctional EGFR/erbB2
complexes resulted in inhibition of glioblastoma transformation and
increased susceptibility to apoptosis (30, 31). Furthermore, we have
also shown that overexpression of SIRP␣1 in glioblastoma cells led to
reduced EGFR-mediated transformation, motility, and spreading and
Fig. 5. SIRP␣1 mRNA decay in U373MG and U373MG/T691 cells with actinomycin
D. A. Cells were seeded at a density of 1 ⫻ 106 cells/flask on day 0. On day 2, the media
were replaced by fresh serum-free medium containing actinomycin D (10 ␮g/mL).
Samples were obtained at regular intervals as indicated after the addition of actinomycin
D. RNA was extracted and subjected to Northern blot analysis using 32P-labeled, 2.0-kb
SIRP␣1 cDNA insert. RNA gel stained with ethidium bromide before blotting is shown
to demonstrate equal loading. SIRP␣1 mRNA levels were normalized with levels of
GAPDH mRNA. B. Bands on the gel from A were quantified by Scion Image ␤ Release
3b software and then plotted using the Microsoft Excel program. Curves were fitted using
the assumption of a first-order exponential decay. X axis, hours after addition of actinomycin D. Y axis, SIRP␣1 mRNA levels. ‚ and Œ, U373MG cells; 䡺 and f, U373MG/
T691 cells. ‚ and 䡺, 3.9-kb band; Œ and f, 6.0-kb band. The half-lives of the 3.9-kb
SIRP␣1 message in U373MG cells and U373MG/T691 cells were calculated to be 5.0
hours (r2 ⫽ 0.99) and 5.4 hours (r2 ⫽ 0.98), respectively. The half-lives of the 6.0-kb
SIRP␣1 message in U373MG cells and U373MG/T691 cells were calculated to be 1.8
hours (r2 ⫽ 0.88) and 2.0 hours (r2 ⫽ 0.86), respectively.
and serum (1–3, 8, 17, 24, 25, 33, 35, 38 – 40). Tyrosine phosphorylation of SIRP␣1 leads to recruitment of either of the two SH2
domain-containing protein tyrosine phosphatases, SHP-1 and SHP-2
(2, 17, 19). Binding of these protein tyrosine phosphatases to SIRP
stimulates the catalytic activity of these phosphatases, which in turn
exerts a negative regulatory effect on cell signaling. Previously, we
reported (25) that in glioblastoma cells, EGF stimulation led to an
association between tyrosine-phosphorylated SIRP␣1 and SHP-2, resulting in reduced transformation, cell migration, and cell spreading
and enhanced apoptosis after DNA damage. These observations suggest that modulation of SIRP␣1 levels or function may be important
Fig. 6. EGFR/erbB inhibition up-regulates SIRP␣1 promoter activity in glioblastoma
cells. A, schematic of pGL3–2.0kb SIRP␣1 promoter. After sequence confirmation, the
2.0-kb fragment was directionally cloned (Mlu1-BglII) in pGL3-basic vector to generate
pGL3-SIRPprom-Luc reporter construct. B. A pGL3–2.0kb SIRP␣1 promoter-luciferase
reporter construct was cotransfected into U87MG, U118MG, U251MG, U343MG, and
LN229 cells with vectors expressing either mutant erbB2 or pSV-␤-galactosidase. Fortyeight hours after transfection, cells were serum-starved for 24 hours. C. U87MG cells were
cotransfected with pGL3–2.0kb SIRP␣1 promoter-luciferase reporter construct and vector
expressing pSV-␤-galactosidase. Cells were serum-starved for 24 hours and treated with
AG1478 (10 ␮mol/L) or AG825 (20 ␮mol/L) for another 24 hours. Cell lysates were made
and assayed for luciferase activity according to the manufacturer’s instructions (Promega).
All activities were normalized by ␤-galactosidase activity from three different experiments. The results are reported as the mean ⫾ SD of fold induction, considering 1.0 as the
relative luciferase activity of the cells transfected with corresponding empty vector.
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Fig. 7. Inhibition of MAPK and src kinase but not PI3K up-regulates SIRP␣1 promoter
activity. U87MG cells were cotransfected with pGL3–2.0kb SIRP␣1 promoter-luciferase
reporter construct and vector expressing pSV-␤-galactosidase. Cells were serum-starved
for 24 hours and treated with LY294002 (25 ␮mol/L), wortmannin (100 nM), PD98059
(25 ␮mol/L), U0126 (10 ␮mol/L), and PP1 (20 ␮mol/L) for another 24 hours. Cell lysates
were made and assayed for luciferase activity according to the manufacturer’s instructions
(Promega). All activities were normalized by ␤-galactosidase activity from three different
experiments. The results are reported as the mean ⫾ SD of fold induction, considering 1.0
as the relative luciferase activity of the cells transfected with corresponding empty vector.
resulted in increased susceptibility to apoptosis (25). These observations strongly indicate a link between EGFR/erbB2 signaling and
SIRP␣1 expression and function in glioblastoma cells. The present
observations using Northern blot analysis and immunoprecipitation
assays showed elevated SIRP␣1 levels in cells expressing an erbB2
mutant receptor in comparison with parental cells. Based on these
results, we report that modulation of EGFR receptor assembly and
subsequent kinase activation at the cell surface modulate SIRP␣1
gene expression. SIRP␣1 may then participate in exerting negative
regulatory effects on cell signaling cascades in erbB-driven human
cancer cells, including glioblastoma cells.
We further extended our work to examine whether up-regulation of
SIRP␣1 by EGFR blockade is controlled at the level of transcription.
We observed that blockade of mRNA synthesis by actinomycin D in
parental and mutant ErbB2-expressing cells did not affect the half-life
of SIRP␣1 mRNAs in both the cell phenotypes, indicating that upregulation of SIRP␣1 levels was not due to alteration in mRNA
transcript stability. This suggested that down-modulation of EGFR
signaling might up-regulate SIRP␣1 expression at the level of transcription. To more clearly define this observation, we cloned a
2084-bp fragment representing the 5⬘-UTR of SIRP␣1 transcripts into
a luciferase reporter vector. A significant increase in promoter activity
was observed when different cell lines were cotransfected with the
T691 erbB2 inhibitory receptor and pGL3-SIRP-Luc reporter vector.
This is in agreement with our Northern blot results that showed the
ability of the erbB2 mutant to induce SIRP␣1 mRNA expression.
Furthermore, pharmacological inhibition of EGFR and erbB2 tyrosine
kinase activity increased SIRP␣1 promoter activity, supporting our
hypothesis that down-modulation of erbB kinase activation and signaling leads to enhanced SIRP␣1 gene expression, which results in
reduced transformation and increased apoptosis (25). Interestingly,
pharmacological inhibition of src kinases and ERK1/2, but not PI3K/
Akt, significantly up-regulated SIRP␣1 promoter activity, suggesting
that expression of SIRP␣1 is regulated by EGFR and src kinases via
the MAPK pathway.
We also investigated the effects of cycloheximide and puromycin,
two well-known protein synthesis inhibitors, on SIRP␣1 mRNA ex-
pression. Surprisingly, inhibition of protein translation reduced the
3.9-kb mRNA but increased 6.0-kb mRNA levels in the presence or
absence of EGF. One possible explanation is that the 6.0-kb mRNA
may be an unspliced pre-mRNA. Thus, blocking of translation would
block the synthesis of components of spliceosome required to splice
6.0-kb mRNA. Furthermore, there are two hypotheses that may explain our observations. One study showed that mRNA degradation is
coupled to translation. Thus, blocking of protein synthesis prolongs
mRNA half-life (47). Another hypothesis is that degradation is dependent on a labile protein whose synthesis is blocked by the translational inhibitor (48). Additionally, some translational inhibitors,
such as cycloheximide, cause ribosomes to “freeze” on the mRNA,
potentially shielding it from degradation by cytoplasmic RNases
(49 –52). Our results demonstrating EGF-mediated down-regulation
of SIRP␣1 expression (Fig. 4) and a substantial increase in the 6.0-kb
mRNA by cycloheximide or puromycin in the presence of EGF (Fig.
8) suggest that EGFR activation might regulate the protein synthesis
machinery to inhibit splicing and/or degradation of 6.0-kb SIRP␣1
mRNA. On the other hand, cycloheximide treatment down-modulated
SIRP␣1 promoter activity, which correlates with the cyclohexaimideinduced decrease in the 3.9-kb mRNA transcript. This observation
clearly suggest that SIRP␣1 up-regulation in EGFR-inhibited cells
requires de novo protein synthesis and the synthesis of new intermediatory molecules that are essential for the activation of transcriptional
machinery.
Serum starvation is a routinely used technique for pushing cells into
a quiescent state to study the mechanism for a particular pathway or
the mode of action of a specific enzyme under optimal conditions. It
is generally believed that serum-starved cells are arrested in G1 or G0
phase, with most of the genes functioning at their basal expression
level (53–55). However, there are some reports showing that serum
starvation leads to up-regulation of certain genes. A study in breast
carcinoma, bladder carcinoma, and osteosarcoma cell lines and in
SV40-immortalized keratinocytes showed that serum starvation led to
an increase in human alternative reading frame (ARF) mRNA levels,
suggesting that ARF responds not only to oncogenic hyperproliferative signals but also to suboptimal growth conditions (56). The ARF
protein is encoded by the INK4a locus as an alternative INK4a
transcript (57–59). ARF has been shown to have tumor suppressor
activity (60) and to induce G1 cell cycle arrest in a p53-dependent
manner (60 – 62). Similarly, a study in human colon carcinoma cells
demonstrated that serum starvation up-regulated vascular endothelial
growth factor mRNA expression and promoter activity via ERK1/2
activation (63). Interestingly, it was reported that in NIH3T3 and
MCF-7 breast carcinoma cells, serum starvation up-regulated protein
kinase C␩, which associates with the cyclin E/cyclin-dependent kinase 2 complex and exerts a negative regulatory effect on cell cycle
progression (64). A study using human T-cell lymphotrophic virusinfected T-cell lines N1186 [interleukin (IL)-2 dependent] and
N1186-94 (IL-2-independent) showed that N1186 cells became arrested in G1 after serum and IL-2 deprivation and this resulted in
elevated levels of p27Kip1 bound to the cyclin E/cyclin-dependent
kinase 2 complex. However, N1186-94 cells failed to arrest in G1 on
serum deprivation, suggesting that serum starvation cooperates with
down-modulation of IL-2 receptor signaling to produce antiproliferative effects (65). In the present study, we observed that serum starvation led to up-regulation of SIRP␣1 levels in both parental and
EGFR-inhibited T691-expressing cells, which is in agreement with
the previously discussed studies. Collectively, the present observations and our previous reports on the effect of EGFR and SIRP␣1 on
transformation, motility, and susceptibility to apoptosis (25, 30, 31)
suggest that an erbB-inhibited phenotype and serum starvation cooperate to induce SIRP␣1 gene transcription, which might result in
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TRANSCRIPTIONAL REGULATION OF SIRP␣1 BY EGFR SIGNALING
Fig. 8. Up-regulation of SIRP␣1 expression by EGFR/erbB inhibition requires de novo protein synthesis. A and B. U373MG/T691 cells were seeded in 25-cm2 flasks at a density
of 1 ⫻ 106 cells/flask at day 0. On day 3, cells were changed to serum-free conditions and incubated with the indicated concentrations of cycloheximide and puromycin in the presence
or absence of EGF (100 ng/mL) for 12 hours. RNA was extracted and subjected to Northern blot analysis using 32P-labeled, 2.0-kb SIRP␣1 cDNA insert. RNA gel stained with ethidium
bromide before blotting is shown to demonstrate equal loading. SIRP␣1 mRNA levels were normalized with levels of GAPDH mRNA. These blots are representative of two
independently reproducible experiments. C. U87MG and U87MG/T691 cells were cotransfected with pGL3–2.0kb SIRP␣1 promoter-luciferase reporter construct and vector expressing
pSV-␤-galactosidase. Cells were serum-starved for 24 hours and treated with the indicated concentrations of cycloheximide for another 24 hours. Cell lysates were made and assayed
for luciferase activity according to the manufacturer’s instructions (Promega). All activities were normalized by ␤-galactosidase activity from three different experiments. The results
are reported as the mean ⫾ SD of fold induction, considering 1.0 as the relative luciferase activity of the untreated parental cells.
activation of certain cell cycle-regulatory proteins that exert the negative effects on cell signaling reported previously (25). Present studies
are under way to define mechanisms of SIRP␣1 inhibitory functions.
Future studies will aim to identify various transcriptional factors
required for SIRP␣1 gene expression and to delineate putative cisregulatory element-binding sites on the SIRP␣1 promoter to gain
understanding of the regulatory mechanisms required for the upregulation of SIRP␣1 gene expression. Defining the coupling between
EGFR down-modulation and SIRP␣1 expression and functional activation may be important for the development of future therapies for
human cancers, in particular central nervous system and lymphoidrelated malignancies.
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Transcriptional Regulation of Signal Regulatory Protein α1
Inhibitory Receptors by Epidermal Growth Factor Receptor
Signaling
Gurpreet S. Kapoor, Dmitri Kapitonov and Donald M. O'Rourke
Cancer Res 2004;64:6444-6452.
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