1Pulmonary and Critical Care Division, Department of Medicine, Tupper Research Institute, New England Medical Center, Boston, Massachusetts 02111; 2Department of Carcinogenesis, University of Texas, M. D. Anderson Cancer Center, Science Park Research Division, Smithville, Texas 78957; and 3Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111
Submitted 16 October 2002 ; accepted in final form 9 April 2003
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ABSTRACT |
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tuberous sclerosis complex 2 gene; tuberin; cell growth; mitogen-activated protein kinase
The genomic and nongenomic effects of estrogen are mediated by estrogen receptors (ER) (19). Unlike other growth factors, estrogen diffuses through the cell membrane, binds to ER, and localizes to the nucleus to activate gene transcription (19). However, not all the effects of ER are mediated genomically. Accumulating evidence indicates that some of the effects of ER are mediated through the early nongenomic activation of signaling intermediates within the cytosol including members of the ras-raf-MEK-1-mitogen-activated protein kinase (MAPK) pathway (34). The ras-raf-MEK-1 pathway is frequently activated during cell growth (7, 8). This results in the downstream activation of extracellular signal-regulated kinase-1 and -2 (ERK-1p44mapk and ERK-2p42mapk), members of the family of MAPKs (7, 8). ERK-1 and ERK-2 play a vital role in the downstream activation of several nuclear transcription factors that regulate genes that are involved in governing the cell cycle. It has been demonstrated that cross talk exists between the MAPK pathway and steroid hormone signaling (31, 36). MAPK is frequently activated by ER and, conversely, activated MAPK mediates translocation of ER to the nucleus (23, 31, 36). Understanding the genomic and nongenomic effects of ER, including the regulation of the MAPK pathway, is key to understanding the mechanism that underlies the diverse effects of estrogen.
The factors that predict mitogenesis in response to estrogen are likely very complex. For example, it has been demonstrated during development that specific gene expression profiles in VSMCs are associated with a growth response (55). In response to estrogen, the exact nature of such genes and the cellular processes that they alter are unknown. It has been shown that expression of the tumor suppressor gene tuberous sclerosis complex 2 (TSC2), which encodes the protein tuberin, can regulate ER-mediated gene transcription (30). Loss of function of the TSC2 gene results in a well-defined clinical syndrome, tuberous sclerosis complex (TSC), that is associated with the development of multiple benign tumors (1, 17), some of which express increased amounts of ER (3, 26, 29, 39). Loss of function of TSC2 has also been implicated in the pathogenesis of pulmonary LAM, a disease with a female preponderance that is characterized by smooth muscle cell proliferation and is also associated with increased ER expression (2, 6, 48, 50). The Eker rat, which is heterozygous for TSC2, spontaneously develops tumors of mesenchymal cell origin including tumors of the reproductive tract (24, 58). ELT-3 cells are Eker rat uterine leiomyoma-derived smooth muscle cells that are null for the TSC2 gene and grow in response to estrogen (20). In contrast, VSMCs highly express TSC2 and are growth inhibited by estrogen (10, 11, 14, 33, 53). In this study we used TSC2-expressing vascular cells and TSC2-null ELT-3 cells to compare and contrast the mechanisms that regulate cellular growth in response to estrogen. We describe the coordinate regulation by estrogen of platelet-derived growth factor (PDGF) and MAPK to regulate cell growth. The growth-inhibiting effect of estrogen is associated with the downregulation of PDGF and MAPK in TSC2-expressing cells. In contrast, upregulation of PDGF and MAPK was essential for estrogen-mediated growth of TSC2-null cells. These data suggest that TSC2 gene expression may be associated with the cellular growth response to estrogen.
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EXPERIMENTAL PROCEDURES |
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Inhibitors. Inhibitors used in these studies included the MEK-1 inhibitor PD-98059 (Calbiochem, La Jolla, CA); suramin, which inhibits growth factor-receptor interactions (Sigma); the specific estrogen receptor inhibitor ICI-182780 (Tocris, Ellisville, MO); and PDGF receptor (PDGFR) inhibitors 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (tyrphostin AG 17) and 6,7-dimethoxy-2-phenylquinoxaline (AG 1296), which block PDGFR kinase and signaling from the activated PDGFR (25, 28) (Calbiochem). Length of treatment and doses are indicated in RESULTS.
PDGF neutralizing antibody. For neutralizing antibody experiments, cells were treated with E2. Polyclonal anti-human PDGF-BB neutralizing antibody (R&D Systems) was added to culture medium at 8 h of E2 treatment. Cell extracts were prepared 12 h after the addition of neutralizing antibody. Nonspecific immunoglobulin was used as a control and added in the same manner to culture medium. The neutralizing antibody dose (ND) used was 20 times the ND50 recommended by the manufacturer.
Preparation of cell extracts. Preparation of whole cell extracts was described previously (13). Membrane extracts for PDGFR and epidermal growth factor receptor (EGFR) immunoblotting experiments were prepared in a similar manner with EB buffer (10 mM Tris, pH 7.4, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.1% BSA) plus inhibitors, 50 mM NaF, 1 mM PMSF, 2 mM Na3VO4, and 20 µg/ml aprotinin.
Immunoprecipitation and kinase assays. MAPK activity was measured
as previously described (13).
Immunoprecipitation experiments were performed by binding 5 µg of
PDGFR- polyclonal antibody (R&D Systems), EGFR antibody (Cell
Signaling Technology), or phosphotyrosine (Santa Cruz Biotechnology, Santa
Cruz, CA) with 1 mg of protein extracts prepared as described
(13). Samples were rocked at
4°C overnight. Protein A Sepharose beads (Repligen, Cambridge, MA) were
then added and rocked at 4°C for a further 2 h. The precipitated immune
complexes were washed, and the reaction was terminated by the addition of
2x Laemmli sample buffer. Samples were boiled and resolved by 7%
SDS-PAGE. In all experiments, membranes were subsequently stripped and
reblotted with appropriate antibody to ensure actual immunoprecipitation of
the protein of interest.
Immunoblot analysis. Cell extracts were electrophoresed on
SDS-PAGE gels. Gel proteins were electrophoretically transferred to a
polyvinylidene difluoride (PVDF) membrane (wet transfer). The blot was blocked
with 5% nonfat dry milk or 1% BSA at room temperature for 1 h. Blots were
incubated with primary antibodies to ERK-1, phosphotyrosine, ER-,
tuberin (C-20), actin (Santa Cruz Biotechnology) PDGF, PDGFR-
(R&D
Systems), phospho-ERK, phospho-EGFR, and EGFR (Cell Signaling Technology) at
4°C overnight, washed, and incubated for 1 h with appropriate secondary
horseradish peroxidase (HRP)-conjugated antibody (Cell Signaling Technology).
The chemiluminescent signal was detected with enhanced chemiluminescence (ECL)
(Cell Signaling Technology). Immunoblots for PDGF-BB included a positive
control for accurate identification of the BB isomer of PDGF. All experiments
were performed in triplicate. Densitometric semiquantitative analysis was
performed by Image-Quant software (Molecular Dynamics, Sunnyvale, CA). Data
were expressed as fold activation relative to control. Results are expressed
as means ± SD. Statistical analysis by a nonparametric test
(Mann-Whitney U-test) was performed with SPSS software. Significance
was assumed at a P value of <0.05.
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RESULTS |
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The growth response to E2 (01,000 nM) was next examined. Growth of VECs was unaffected by E2 (Fig. 1B), and, as previously shown by others, VSMCs were growth inhibited by E2 (Refs. 10, 14, 53; Fig. 1C). In contrast, a significant dose-dependent growth response to E2 was observed in ELT-3 cells (Fig. 1D). The dose of E2 used for the remaining experiments was 10 nM, a dose that induced significant and consistent inhibition of VSMC growth (mean ± SD: 21 ± 12.2%) and promotion of ELT-3 cell growth (2.05 ± 0.53%). In ELT-3 cells, cell proliferation or apoptosis was not directly measured. However, cell morphology and trypan blue exclusion dye testing were similar between control and treatment groups (results not shown), suggesting that the growth effects of estrogen were not mediated through decreased apoptosis.
PDGF participates in E2-mediated growth of
TSC2-null cells. The induction of autocrine factors by E2 has
been implicated in hormonally regulated cell growth
(27,
61). To investigate potential
growth factors that might be involved in E2-mediated cell growth,
TSC2-null ELT-3 cells were treated with the peptide growth factors
PDGF-BB (0.110 ng/ml), aFGF and bFGF (510 ng/ml), and
TGF-1 (25 ng/ml) and growth was examined with Coulter counting.
Figure 2A demonstrates
significant ELT-3 cell growth in response to PDGF-BB and both forms of FGF.
Stimulation of cell growth was not observed in response to TGF-
1. To
examine the possibility of synergy between E2 and the mitogens
identified from this experiment, ELT-3 cell growth was assessed after
treatment with E2 together with either PDGF-BB or bFGF.
E2 combined with PDGF-BB resulted in cell counts that were
moderately synergistic, an effect not observed with bFGF
(Fig. 2B). To further
explore this synergy, lower doses of bFGF and PDGF-BB were used together with
E2 treatment. Again, moderate synergy was observed for PDGF-BB plus
E2 but no additional stimulation was noted for bFGF plus
E2 (results not shown). These results suggested that E2
and PDGF-BB are synergistic in promoting TSC2-null ELT-3 cell
growth.
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To investigate the hypothesis that PDGF participates in E2-induced ELT-3 cell growth, the proliferative effect of E2 was assessed in the presence and absence of the PDGFR inhibitor AG 17 (tyrphostin), which inhibits the phosphorylation of the PDGFR (28). Complete inhibition of E2-stimulated ELT-3 cell growth was achieved with 10 µM AG 17 without significantly affecting baseline control cell growth (Fig. 3A). A similar significant inhibition of E2-induced growth was observed with a second PDGFR inhibitor, AG 1296 (20 µM) (Ref. 25; results not shown). Successful inhibition of PDGF-BB-induced ELT-3 cell growth, which served as a positive control, was also seen in response to AG 17 (Fig. 3B). Thus these results suggest that PDGF plays an integral part in mediating TSC2-null ELT-3 cell growth in response to E2.
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E2 differentially regulates an autocrine
pathway involving PDGF-BB in TSC2-expressing cells compared with TSC2-null
cells. The preceding experiments indicated that PDGF was involved in
mediating a positive growth response to E2 in TSC2-null
ELT-3 cells. To investigate the participation of PDGF in
E2-regulated cell growth, VECs, VSMCs, and ELT-3 cells were treated
with E2 (048 h) and cell lysates were subjected to
immunoblot analysis for PDGFR- and PDGF-BB protein
(Fig. 4). In VECs, PDGFR-
and PDGF-BB protein remained unaltered by E2. In VSMCs,
downregulation of PDGFR-
and PDGF-BB protein was observed. In contrast,
time-dependent induction of PDGFR-
protein was observed in
TSC2-null ELT-3 cells beginning at 2 h, with maximum sustained
induction by 2448 h (1.89 ± 0.31-fold by densitometry). A more
pronounced induction of PDGF-BB protein was also observed in ELT-3 cells (2.56
± 0.89-fold by densitometry), which temporally paralleled PDGFR-
induction by E2. To investigate whether this pathway was activated
by E2 in an autocrine fashion, PDGFR-
was immunoprecipitated
from E2-treated lysates and subjected to immunoblotting for
phosphotyrosine (Fig.
5A). Again, TSC2-expressing VECs and VSMCs were
compared with TSC2-null ELT-3 smooth muscle cells. No phosphorylation
of PDGFR-
was observed in TSC2-expressing VECs or VSMCs despite
adequate amounts of PDGFR-
in immunoprecipitated samples
(Fig. 5A). In
contrast, substantial phosphorylation of PDGFR-
by E2 was
observed in TSC2-null ELT-3 cells
(Fig. 5A).
Furthermore, inhibition by the ER antagonist ICI-182780 (100
nM)ofE2-induced PDGFR-
phosphorylation in TSC2-null
ELT-3 cells was also observed, confirming that this response was ER mediated
(Fig. 5A).
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Previous authors have shown that the response to estrogen can be mediated
through EGFR (9,
12,
27,
46). Furthermore, it has also
been shown that AG 17 can also inhibit the phosphorylation of EGFR
(42) and can inhibit growth by
disrupting mitochondria (4).
Thus AG 17 is not truly specific as an inhibitor of PDGFR activation. The
effect of estrogen on the activation of EGFR and the effect of AG 17 on
phosphorylation of both PDGFR and EGFR were next examined. ELT-3 cells were
treated with estrogen in the presence and absence of AG 17. Cell lysates were
subjected to immunoprecipitation with a polyclonal antibody for
phosphotyrosine followed by immunoblotting for PDGFR- and EGFR
(Fig. 5B). Both
estrogen- and PDGF-BB-induced phosphorylation of PDGFR-
were
significantly inhibited by AG 17. In contrast, estrogen did not activate EGFR
and AG 17 did not significantly inhibit EGF-induced phosphorylation of EGFR.
To ensure that phosphorylation of EGFR was not occurring at an earlier time
point, estrogen-treated lysates were subjected to Western immunoblot with an
antibody for phospho-EGFR (Fig.
5C). No significant phosphorylation of EGFR was observed
over the time period examined.
E2-induced cell growth and ERK activation in TSC2-null cells are MEK-1 dependent. The ras-raf-MEK-1-ERK pathway is frequently activated during cell growth (7, 8), and E2 has been shown to result in the immediate activation of the ERK pathway (36, 54). To investigate the potential mechanisms used by E2 to regulate cell growth, TSC2-expressing VECs and VSMCs and TSC2-null ELT-3 smooth muscle cells were compared for ERK activation in response to E2. Cell extracts were subjected to Western immunoblot for phospho-ERK (048 h). In TSC2-expressing VSMCs and VECs the activation of ERK-1 and ERK-2 by E2 was immediate, occurring at 15 min and slowly declining to baseline levels by 24 h (Fig. 6A). Densitometry of phospho-ERK-2 revealed a fourfold activation of ERK in VECs and a twofold activation of ERK in VSMCs at 15 min, returning to baseline levels by 24 h (Fig. 6A). In contrast, the pattern of E2-induced ERK-1 and ERK-2 activation in TSC2-null ELT-3 cells was biphasic (Fig. 6B). Immunoblotting did not reveal any increase in the amount of ERK protein in E2-treated samples, and immunoprecipitation kinase assay also confirmed this pattern of ERK activation in response to E2 in ELT-3 smooth muscle cells. This suggests that ERK activation occurred via phosphorylation rather than induction of ERK protein. Densitometry, performed on Western immunoblots of phospho-ERK, revealed a twofold activation of ERK-2 at 15 min, returning to baseline levels by 12 h (Fig. 6B). A second wave of ERK activation began at 48 h, with maximum activation by 2448 h. The biphasic pattern of ERK activation by E2 in ELT-3 cells was not observed in TSC2-expressing VSMCs or VECs.
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To investigate the role of E2-induced ERK activation in TSC2-null ELT-3 cell growth, cells were treated with E2 in the presence and absence of PD-98059, a MEK-1 inhibitor, and cell growth was assessed. Figure 7A demonstrates that E2-stimulated growth was significantly inhibited by PD-98059, indicating that E2-induced growth of ELT-3 cells is MEK-1 dependent.
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E2-induced late activation of ERK in TSC2-null cells is dependent on MEK-1 and autocrine induction of PDGF-BB. In ELT-3 cells, the delayed second wave of ERK-1 and -2 activation and the known induction of PDGF by E2, which temporally paralleled E2-induced activation of ERK, suggested that an autocrine factor, possibly PDGF, mediated this later phase of ERK phosphorylation. To test this hypothesis, cultured ELT-3 cells were treated with E2 in the presence and absence of PD-98059 (10 µM), a MEK-1 inhibitor, suramin (300 µM), an inhibitor of growth factor-receptor interactions, and AG 17 (10 µM), a PDGFR inhibitor, and PDGF-neutralizing antibody (20 µg/ml). The inhibitors and neutralizing antibody were added 8 h after E2 treatment, at a time when PDGF induction by E2 was previously observed, and lysates were prepared 12 h later. Samples were subjected to immunoblot analysis with phospho-ERK-1/2 antibody. Significant inhibition of E2-induced ERK-1/2 activation was observed for PD-98059, suramin, and AG 17 (Fig. 7B). More specifically, significant inhibition of E2-induced ERK-1/2 activation was also observed for PDGF-neutralizing antibody (Fig. 7C). Early activation (15 min) of ERK by E2 was also inhibited by pretreating ELT-3 cells with PD-98059 1 h before E2 treatment (results not shown). These results demonstrated that late-phase ERK-1/2 activation by E2 in ELT-3 smooth muscle cells is MEK-1 dependent and mediated by PDGF-BB.
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DISCUSSION |
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This study shows that the growth response to estrogen is mediated, not by the immediate activation of the ERK pathway or by direct genomic transactivation of cell cycle genes, but rather by the induction of a PDGF autocrine loop that results in a more sustained pattern ERK activity. Conversely, the growth inhibitory response to estrogen is associated with the downregulation of these proteins. Studies have shown that PDGF is induced by estrogen (18, 47). This study now demonstrates that the autocrine production of PDGF can be upregulated and downregulated by estrogen to switch on or switch off cellular growth. Furthermore, Lange et al. (27) showed that potentiation of EGF-induced cell growth by the steroid hormone progesterone involves the enhanced activation of all members of the MAPK families, ERK, JNK, and p38, as well as the cell cycle proteins, cyclin D1, cyclin E, and p21WAF1. This suggests that the autocrine production of polypeptide growth factors by steroid hormones is designed to augment or potentiate specific signaling pathways within the cell to promote cell growth.
In the present study we demonstrate that the divergent growth response to
estrogen is associated with the altered expression of ER- and TSC2.
TSC2-expressing cells contained comparatively low levels of ER-
expression and were growth inhibited by estrogen. TSC2-null cells
highly expressed ER-
and exhibited a significant growth response to
estrogen. Thus loss of TSC2 expression may be associated with
enhanced expression of ER-
as a mechanism to promote a growth response
to estrogen. In support of this theory, increased ER expression has been
observed in conditions associated with functional loss of TSC2 such
as LAM, AML, and hepatic hemangioma
(3,
26,
29,
37,
39). Furthermore, estrogen has
been implicated as a growth-promoting hormone in the pathogenesis of both LAM
and AML, which are associated with aberrant expression of TSC2 and
characterized smooth muscle cell proliferation
(51).
The regulation of cellular growth by estrogen is likely determined by the expression of a gene or set of genes that are cell or tissue specific. One weakness of this study is that the effects of estrogen were not tested in comparable TSC2-expressing and TSC2-null cell lines. Furthermore, the cells tested were not derived form the same tissue source. However, our data and those of others do suggest that TSC2 may be one of the genes that promote or inhibit the growth response to estrogen. Other groups have observed that TSC2-null ELT-3 cells, derived from uterine leiomyomas in the Eker rat, grow in response to estrogen (21). In contrast, mature myometrium from this model, which possesses one wild-type allele for TSC2, does not grow in response to estrogen (5). Furthermore, in Hep G2 cells, Lou et al. (30) demonstrated reduced ER-mediated gene transcription by the expression of TSC2. Interestingly, neither the PDGF-B chain promoter or PDGFR promoter contain a classic estrogen response element (ERE) site (National Center for Biotechnology Information GenBank accession no. NM0026009). However, the promoters for these genes do contain consensus binding sites for transcription factors, AP-1 and SP-1, which are known to directly activate transcription in response to the estrogen-ER complex (52, 56). It could be speculated that expression of TSC2 regulates ER-mediated transcription of PDGF and PDGFR via transcription factors such as AP-1 and SP-1. Collectively, these data support the hypothesis that TSC2 gene expression regulates the growth response to estrogen.
We observe that the altered production of PDGF and its receptor was associated with opposing functions for estrogen. TSC2 expression has the potential to regulate the described autocrine pathway at multiple levels. In this study we showed that a major component of estrogen-regulated cellular growth of TSC2-null cells involved the MAPK pathway. It is widely believed that the rapid activation of MAPK after estrogen treatment is a signaling effect of membrane-associated ER (mER) (12, 44, 45). The function of the early-immediate activation of ERK was not fully examined in this study. Although early activation of ERK was not different in TSC2-expressing and TSC2-null smooth muscle cells, the second phase of estrogen-induced ERK activation was very apparent in TSC2-expressing ELT-3 smooth muscle cells. This second wave was mediated by the autocrine induction of PDGF-BB. It has been demonstrated that tuberin, the protein product of the TSC2 gene, possesses Rap1GAP activity (57). Rap1GAP GTPases share homology with the ras family of signal molecules, and B-raf is activated by Rap1 (40, 41). When microinjected into cells, Rap1 can induce DNA synthesis and dysregulation of the ERK pathway, which has been implicated as a mechanism through which Rap1 can alter cell growth (59, 60). Furthermore, a prosite computer search (ExPASy; http://www.expasy.ch/) of the tuberin sequence reveals a MAPK phosphorylation site. Thus TSC2 has the potential to play a vital role in modulating at least the pattern of MAPK/ERK activation to alter estrogen-regulated cell growth. Recently, much attention has been paid to the trans-activation of membrane receptors such as EGFR by mER (12, 45). Trans-activation usually occurs as an early membrane-associated event that affects downstream signaling events including the activation of MAPK. TSC2 localizes to the cell membrane, and it could be speculated that loss of function of TSC2 potentially regulates receptor function at the cell membrane to alter downstream signaling events (38). Finally, TSC2 has been shown to antagonize insulin- and growth factor-mediated cell growth by antagonizing the phosphatidylinositol 3-kinase pathway distal of Akt (16, 32, 43). Thus it is reasonable to speculate the converse, i.e., that loss of function of TSC2 is associated with augmentation of growth factor-regulated pathways to promote cell growth, a hypothesis that is supported by the observations in this study.
In conclusion, we have described an ER-regulated autocrine loop involving PDGF in TSC2-null smooth muscle cells that is not present in cells that express TSC2. Estrogen initiates a coordinated cellular response to ensure continued cell growth via amplification of the MAPK pathway. Understanding of the complex integration of signaling pathways between steroid and peptide growth factors may bear relevance to estrogen-related diseases that are characterized by aberrant cell proliferation.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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