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Endocrinology Vol. 142, No. 2 751-759
Copyright © 2001 by The Endocrine Society

ARTICLES

Requirement of Ras-Dependent Pathways for Activation of the Transforming Growth Factor ß3 Promoter by Estradiol1

Desheng Lu and Vincent Giguère2

Molecular Oncology Group (D.L., V.G.), McGill University Health Center, Montréal, Québec, Canada H3A 1A1; and Departments of Biochemistry, Medicine and Oncology (V.G.), McGill University, Montréal, Québec, H3G 1Y6 Canada

Address all correspondence and requests for reprints to: Dr. Vincent Giguère, Molecular Oncology Group, McGill University Health Centre, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1. E-mail: vgiguere{at}dir.molonc.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been previously observed that the transforming growth factor ß3 (TGFß3) gene can be activated by both estradiol (E2) and selective estrogen receptor modulators (SERMs) in vivo but that only SERMs have a potent stimulatory effect on the TGFß3 promoter in cultured cells. We demonstrate in this report that E2 can act also as a potent inducer of the TGFß3 promoter via a novel and specific estrogen receptor (ER){alpha}-mediated mechanism. Our results show that treatment with epidermal growth factor or transfection of a constitutively active Ras mutant allows E2 to induce the TGFß3 promoter via ER{alpha} in cotransfected HeLa and osteosarcoma MG63 cells. Both protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors can block the combined stimulatory effect of E2 and epidermal growth factor/Ras. However, E2 induction of the TGFß3 promoter was found to be unaffected by mutation of ER{alpha} serine 118, a well-characterized target of MAPK. Progressive deletion analysis of the ER{alpha} amino-terminal region delineated three separate domains modulating the E2/activated Ras response, revealing a complex functional organization of the ER{alpha} A/B domain required for regulation of the TGFß3 promoter. In addition, PKC and MAPK inhibitors had no effect on the induction of TGFß3 promoter activity by the SERM EM-652. These results indicate that induction of the TGFß3 promoter by the E2/ER{alpha} complex requires the concomitant activation of PKC and MAPK signaling and provide a novel framework for the design of more effective estrogen-based therapeutic strategies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS REGULATE A wide variety of physiological processes, such as maturation of reproductive tissues, bone homeostasis, and protection of the cardiovascular system (1). The estrogen signal is known to be mediated by two related proteins, estrogen receptors (ERs) {alpha} and ß (2, 3, 4, 5, 6). In the classical activation pathway, upon estrogen binding, the transformed receptors form dimers and bind to estrogen response elements (EREs) within their target genes (7). This complex then interacts with coactivator proteins and the basal transcription machinery, leading to the transcriptional activation of estrogen-responsive genes (8, 9). Estrogens also possess the ability to regulate some non-ERE-containing genes. The transcriptional activation of these genes requires interaction of ERs with heterologous transcription factors such as AP-1, SP-1, and other unidentified proteins (10, 11, 12, 13).

It has been shown that nonsteroidal agents, including dopamine, growth factors, cAMP, and phorbol esters, could activate nuclear receptors (reviewed in 14). Stimulation of cellular activity by growth factors, such as epidermal growth factor (EGF) and IGF-1, results in both estrogen-independent and ligand-dependent ER{alpha} activation. The activation of ER{alpha} by EGF and IGF-1 involves phosphorylation through the Ras-mitogen-activated protein kinase (Ras-MAPK) signaling of a serine residue at position 118 located in the amino-terminal activation function (AF-1) domain of ER{alpha} (15, 16, 17, 18). Similarly, the activation of ERß can be regulated by the Ras-MAPK pathway: phosphorylation of specific serine residues within the AF-1 domain after EGF stimulation leads to the recruitment of the steroid receptor coactivator-1 (SRC-1) (6, 19). In addition to MAPK, the cyclin-dependent kinases, pp90rsk1, src family tyrosine kinases, and protein kinase A (PKA) have all been shown to phosphorylate ER{alpha}, which affects its transcriptional activity in ERE-containing genes (20, 21, 22, 23, 24). However, a role of nonsteroidal agents in the activation of non-ERE-containing genes remains to be demonstrated.

Antiestrogens have been developed to antagonize estrogen cancer-promoting effects in reproductive tissues. Some antiestrogens, such as raloxifene (RAL) and tamoxifen, have been demonstrated to have tissue-selective activity (25). Whereas these compounds display antiestrogen action in the breast, they exert estrogenic action, at various degrees, in the uterus and mimic the beneficial functions of estrogens in bone and cardiovascular tissues. Because these synthetic molecules can act as both agonist and antagonist, depending on their site of action and target gene, they are currently referred to as selective ER modulators (SERMs). Although crystallographic studies have shown that binding of estradiol (E2) and SERMs can lead the ERs to adopt distinct structures (26, 27, 28), exactly how SERMs exert potent antiestrogenic activity in one tissue while mimicking estrogen action in another remains elusive. Among SERM-inducible genes recently identified, the transforming growth factor ß3 (TGFß3) gene displays some specific characteristics (29, 30, 31). The TGFß3 promoter does not contain a classical ERE (the DNA-binding domain of ER{alpha} is dispensable for its activation by SERMs); and whereas the TGFß3 gene was found to be activated by E2 and SERMs in vivo in the bone of ovariectomized rats, only SERMs potently activate the TGFß3 promoter in cultured cells. The molecular mechanisms underlying the response of ER to its natural ligand on the TGFß3 promoter in vivo, but not in cultured cells, remain to be resolved. In this study, we report that activation of the growth factor/Ras signaling pathway confers the ability to E2 to induce transcription from the TGFß3 promoter and that both PKC and MAPK activities are required to obtain this effect. These results reveal a novel estrogen activation pathway and suggest that SERMs may stimulate the activity of ER{alpha} on the TGFß3 promoter by mimicking the action of this pathway on the ER{alpha}/TGFß3 promoter-binding protein transcriptional complex.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and plasmids
Human ER{alpha} was a gift from Pierre Chambon (Institut National de la Santé et de la Recherche Médicale, Illkirch, France). ER{alpha} was cloned into the EcoRI site of pCMX vector (32). All deletion mutants of ER{alpha} were constructed by amplifying the appropriate fragments by PCR, using Pfu polymerase (Stratagene, San Diego, CA), and subcloning them into the expression vector pCMX. All mutants were confirmed by sequencing with the T7 sequencing kit from Pharmacia Biotech (Piscataway, NJ). The pCMXhERß plasmid was described previously (19). The TGFß3Luc reporter plasmid was a gift from Dr. Claude Labrie, the Laboratory of Molecular Endocrinology, Centre Hospitalier de l’Université Laval (CHUL) Research Center (Québec City, Canada). H-RasS17N and H-RasV12 expression plasmids were kindly provided by Dr. Morag Park (McGill University, Montréal, Québec, Canada). The pCMXßgal has been previously described (19).

E2, tumor promoting agent (TPA), and H89 were obtained from Sigma (St. Louis, MO). EM-652 and RAL were synthesized in the medicinal chemistry division of the Laboratory of Molecular Endocrinology, CHUL Research Center. Tamoxifen (OHT) was kindly provided by Dr. Salin-Drouin (Besins-Iscovesco, Paris, France). EGF and GF109203x were obtained from Roche Diagnostics (Laval, Canada). PD98059 was purchased from New England Biolabs, Inc. (Beverly, MA).

Cell culture and transfection
HeLa and MG63 cells were maintained in DMEM supplemented with 10% FCS and 100 µg/ml penicillin and 100 µg/ml streptomycin. Cells were grown in phenol red-free DMEM (Life Technologies, Inc., Gaithersburg, MD) with 10% charcoal dextran-treated FCS for 24 h before transfection. Cells were maintained in a humidified atmosphere at 37 C and 5% CO2 and plated in 12-well plates for transfection. At about 50% confluence, cells were transfected by the calcium phosphate-DNA precipitation method, as previously described (33), typically with 1.0 µg TGFß3Luc reporter, 0.5 µg pCMXßgal, 0.25 µg ER{alpha} or ß expression plasmid, and carrier DNA pBluescript KSII for a total 5 µg per well. After 16 h, the cells were washed and given fresh medium that contained 2.5% charcoal dextran-treated FCS with 10 nM E2 or EM-652, unless otherwise stated, for 20–24 h. If necessary, EGF and kinase inhibitors (PD98059, H89, and GF109203x) were added immediately after transfection. For TPA treatment, cells were treated with TPA (100 ng/ml) for 4–6 h after transfection. For luciferase assay, cells were lysed in potassium phosphate buffer containing 1% Triton X-100, and light emission was detected in the presence of luciferin using a microtiter plate luminometer (Dynex Technologies, Chantilly, VA). Luciferase values were normalized for variations in transfection efficiency, using the ß-galactosidase internal control, and expressed as relative luciferase units (RLU). The values for luciferase activity presented in this study represent means of a minimum of three independent transfections performed in duplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E2 stimulates the TGFß3 promoter in the presence of EGF
To assess the molecular mechanisms underlying E2 and SERMs action on the TGFß3 promoter, we first employed HeLa cells cotransfected with the pTGFß3Luc reporter and human ER{alpha} expression plasmids. The pTGFß3Luc construct contains the human TGFß3 promoter linked to the luciferase reporter gene. As shown in Fig. 1AGo, three SERMs [including RAL, OHT, and EM-652, a novel nonsteroidal compound (34, 35)] increased transcriptional activity of the TGFß3 promoter 4- to 6-fold above the basal levels. In contrast, E2 treatment had little effect. A similar activation pattern was observed in human osteosarcoma MG63 cells (Fig. 1BGo). Because EM-652 displayed the most potent effect on the TGFß3 promoter at low concentrations (Fig. 1Go), EM-652 was chosen as a reference for SERM activity in subsequent experiments. The lack of significant induction by E2 but potent activation by SERMs, in these systems is consistent with previous reports (29, 30).



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  		Figure 1. E2 stimulates the TGFß3 promoter, in the presence of EGF. A, SERMs activate the TGFß3 promoter through ER{alpha}. HeLa cells were transfected with 1.0 µg TGFß3Luc reporter plasmid and 0.25 µg of the ER{alpha} expression plasmid pCMXhER{alpha}. Cells were treated with increasing concentrations of E2, EM-652, RAL, and tamoxifen (OHT). B, Same as A, except that human MG63 osteosarcoma cells were used for transfection. C, EGF influences E2 induction of the TGFß3 promoter. HeLa cells were transfected with 1.0 µg TGFß3Luc reporter and pCMXhER{alpha} expression plasmid. After transfection, cells were grown in the presence or absence of 10 nM E2 or EM-652 and treated with 20 ng/ml EGF.

 
Based on the prior observations that growth factors can modulate ERs activity in a promoter- and cell-type dependent manner, we next considered the possibility that growth factor stimulation may be required to generate an E2 response in this system. As seen in Fig. 1CGo, treatment with 20 ng/ml EGF increased the transcriptional activity of the TGFß3 promoter in HeLa cells by more than 2-fold in the presence of E2 but had no effect on basal activity and EM-652 response. These results suggest that growth factor signaling may play a physiological role in the induction of the TGFß3 promoter by E2.

Ras modulates E2 activation of the TGFß3 promoter specifically through ER{alpha}
In a cell type- and promoter-specific manner, EGF has been shown to activate ER{alpha} through the Ras-MAPK signaling cascade (17, 18). We therefore examined a possible role for Ras signaling in the E2 activation of the TGFß3 promoter by ER{alpha}. We first used dominant negative Ras (H-RasS17N) and dominant active Ras (H-RasV12) constructs in transactivation studies performed in HeLa cells. As shown in Fig. 2AGo, the presence of the dominant negative H-RasS17N effectively inhibited the permissive action of EGF on the E2 induction of the TGFß3 promoter. In contrast, H-RasS17N had no effect on the EM-652 response. E2-induced activation of TGFß3 promoter activity was also enhanced by amounts of H-RasV12 expression plasmid (Fig. 2BGo). Because previous studies have demonstrated that phosphorylation of serine 118 of ER{alpha} is critical for activation through the Ras-MAPK signaling cascade (17, 18), we thus explored the role of serine 118 in E2-induced activation of the TGFß3 promoter using the ER{alpha}S118A mutant in transfection experiments. In the presence of ER{alpha}S118A, H-RasV12 enhanced E2-induced activation of the TGFß3 promoter to the same extent as with the wild-type receptor (Fig. 2CGo). Parallel experiments were also performed in MG63 cells. These experiments demonstrate that E2-induced activation of TGFß3 promoter activity was also enhanced by the presence of H-RasV12 in human osteosarcoma cells (Fig. 3AGo), and that these cells seem to be more sensitive to the action of Ras (Fig. 3BGo) because less H-RasV12 expression plasmid was required to obtain a maximal E2 response. These results also show that serine 118 is not required for E2-induced TGFß3 promoter activation (Fig. 3CGo). Taken together, these results clearly indicate that Ras signaling modulates E2-induced activation of the TGFß3 promoter but that ER{alpha} may not be the direct target of MAPK.



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  		Figure 2. Ras regulates E2 induction of the TGFß3 promoter in HeLa cells. A, H-RasS17N inhibits E2 induction of the TGFß3 promoter in the presence of EGF. The TGFß3Luc reporter plasmid was transfected into HeLa cells with pCMXhER{alpha}, in the presence or absence of H-RasS17N expression plasmid. After transfection, cells were treated with or without 10 nM E2 or EM-652, in the presence or absence of 20 ng/ml EGF. B, H-RasV12 enhances E2 induction of the TGFß3 promoter. Increasing concentrations of H-RasV12 expression vector were transfected, together with the TGFß3Luc reporter plasmid and pCMXhER{alpha}. After transfection, cells were treated with or without 10 nM E2. C, Serine 118 in ER{alpha} is not required for E2 induction mediated by H-RasV12. The TGFß3Luc reporter plasmid was transfected, together with wild-type ER{alpha} or ER{alpha}S118A expression vector, in the presence or absence of the H-RasV12 expression plasmid. HeLa cells were then incubated with 10 nM E2.

 


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  		Figure 3. Ras regulates E2 induction of the TGFß3 promoter in MG63 cells. A, H-RasV12 promotes E2 induction of the TGFß3 promoter. The TGFß3Luc reporter plasmid was transfected into MG63 cells with pCMXhER{alpha}, in the presence or absence of H-RasV12 expression plasmid. After transfection, cells were treated with or without 10 nM E2 or EM-652. B, H-RasV12 enhances E2 induction of the TGFß3 promoter. Increasing concentrations of H-RasV12 expression vector were transfected, together with the TGFß3Luc reporter plasmid and pCMXhER{alpha}. After transfection, cells were treated with or without 10 nM E2. C, Serine 118 in ER{alpha} is not required for E2 induction mediated by H-RasV12. The TGFß3Luc reporter plasmid was transfected, together with wild-type ER{alpha} or ER{alpha}S118A expression vector, in the presence or absence of the H-RasV12 expression plasmid. MG63 cells were then incubated with 10 nM E2.

 
Because previous studies had found that the activity of ER{alpha} and ß can be cell type-, promoter-, and ligand-dependent, we next explored whether ERß could also activate the TGFß3 promoter in this system. As shown in Fig. 4Go, A and B, ERß caused a 2- to 3-fold induction of TGFß3 promoter activity in the presence of EM-652 or RAL in HeLa and MG63 cells, whereas E2 had no effect. However, unlike ER{alpha}, cotransfection of ERß into HeLa or MG63 cells did not result in E2-inducted activation of the TGFß3 promoter (Fig. 4Go, C and D). These results suggest that the activation of the TGFß3 promoter by E2 is receptor-specific.



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  		Figure 4. Role of ERß in activation of the TGFß3 promoter. A, Dose-dependent effect of E2 and SERMs on the TGFß3 promoter in HeLa cells. HeLa cells were transfected with 1 µg TGFß3Luc reporter and 0.25 µg of the ERß expression plasmid pCMXhERß. Cells were treated with increasing concentrations of E2, EM-652, and RAL. B, Dose-dependent effect of E2 and SERMs on the TGFß3 promoter in MG63 cells. Transfection conditions were the same as in A, except MG63 cells were used. C, Effect of cotransfection of ERß and H-RasV12 on E2-induced activation of the TGFß3 promoter in HeLa cells. HeLa cells were transfected with 1 µg TGFß3Luc reporter and 0.25 µg pCMXhERß with or without H-RasV12. After transfection, cells were grown with 10 nM E2 or EM-652. D, Effect of cotransfection of ERß and H-RasV12 on E2-induced activation of the TGFß3 promoter in MG63 cells. Transfection conditions were the same as in A, except MG63 cells were used.

 
Amino-terminal deletion mutants reveal the presence of multiple functional domains required for the E2 response and its modulation by Ras
Given the observation that serine 118 is not involved in modulating the effect of Ras on E2 induction of the TGFß3 promoter, we next undertook the mapping of the domains within ER{alpha} responsible for this effect. Various N-terminal truncated mutants of ER{alpha} were transfected into HeLa cells with the TGFß3Luc reporter gene, in the presence or absence of activated H-RasV12. The ability of H-RasV12 to facilitate the E2 activation of the TGFß3 promoter was greatly attenuated when amino acids 1–26 were deleted, and it was completely abolished after removal of the first 67 amino acids (Fig. 5Go). These results delineated the region encompassing the first N-terminal 67-amino acid of ER{alpha} as essential for Ras modulation of the E2 response on the TGFß3 promoter. Surprisingly, further deletion of the N-terminal region to amino acid 107 allowed E2 induction of the TGFß3 promoter in the absence of Ras, suggesting the presence of a strong inhibitory domain within amino acids 68–107 (Fig. 5Go). As expected, the presence of H-RasV12 exerted no positive influence on the activity of this mutant. Further deletion of the N-terminal region to amino acid 156 almost completely abolished E2 response, indicating an essential requirement for this domain in this process. Complete deletion of the N-terminal region (CDEF{alpha} mutant) generated a similar response. These experiments reveal the presence of at least three distinct functional domains within the ER{alpha} A/B region that modulate E2 response of the TGFß3 promoter: a Ras regulatory domain (1–67), an inhibitory domain (68–107), and a transactivation domain required for E2 induction (107–155).



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  		Figure 5. Multiple ER{alpha} amino-terminal regulatory domains are involved in E2/Ras induction of the TGFß3 promoter. The TGFß3Luc reporter plasmid (1 µg) was transfected into HeLa cells with expression vectors carrying wild-type ER{alpha} and various N-terminal deletion mutants (0.25 µg) with or without H-RasV12 expression plasmid. After transfection, cells were treated with 10 nM E2. A schematic representation of various ER{alpha} deletion mutants is displayed on the left of the graph.

 
MAPK and PKC inhibitors block E2 activation of the TGFß3 promoter
To identify possible downstream effectors of EGF/Ras signaling in these cell systems, PKA, PKC, and MEK inhibitors (H89, GF109203x, and PD98059, respectively) were next tested for their ability to interfere with E2-induced TGFß3 promoter activity in both HeLa (Fig. 6AGo) and MG63 cells (Fig. 6BGo). The PKA inhibitor H89 had no effect on E2- or EM-652-induced activation of the TGFß3 promoter. In sharp contrast, both the specific MEK inhibitor PD98059 and PKC inhibitor GF109203x blocked the E2 but not the EM-652 response. Together, these data suggest that EGF-Ras signaling facilitates E2-induced activation of the TGFß3 promoter through both MEK- and PKC-dependent pathways.



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  		Figure 6. MAPK and PKC inhibitors block E2 activation of the TGFß3 promoter. A, HeLa cells were transfected with 1.0 µg TGFß3Luc reporter and 0.25 µg ER{alpha} expression plasmid with or without 0.3 µg H-RasV12 expression vector. Cells were then treated with 10 nM E2 or EM652 in the presence or absence of 100 nM H89, 50 µM PD98059, and 5 µM GF109203x. B, Transfection conditions were the same as in A, except MG63 cells were treated as indicated.

 
To assess whether PKC signaling alone is sufficient to confer E2 induction of the TGFß3 promoter, cells were concomitantly treated with TPA, E2, and EM-652. As seen in Fig. 7Go, treatment with TPA did not lead to an E2 induction of TGFß3 promoter activity. This result suggests that direct activation of PKC by TPA is not sufficient to promote E2 induction of the TGFß3 promoter.



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  		Figure 7. PKC signaling is not sufficient for E2 induction of the TGFß3 promoter. A, HeLa cells were transfected with 1 µg TGFß3Luc reporter and 0.25 µg pCMXhER{alpha} plasmid. Sixteen hours after transfection, cells pretreated, for 4–6 h, with 100 ng/ml TPA. Cells were subsequently grown with 10 nM E2 or EM-652. B, Transfection conditions were the same as in A, except MG63 cells were used.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
E2 reduces bone resorption and has been used to both prevent and treat osteoporosis (36). It is assumed that E2 mediates bone maintenance by influencing the expression of local factors that play important roles in bone homeostasis. TGFß3, a crucial factor in bone remodeling, has been identified as a target gene for estrogen-mediated bone maintenance (29). Though both natural estrogens and SERMs have been shown to activate TGFß3 gene expression in vivo, it remained unclear why only certain SERMs and (to a lesser extent) estrogen metabolites, and not E2, had a potent effect on the TGFß3 promoter in cultured cells (29, 30). In this study, we explored the possible mechanisms underlying E2-dependent induction of the TGFß3 promoter by ER{alpha} and ERß. Our results demonstrate that activation of growth factor/Ras signaling cascades involving both the PKC and MAPK pathways allows E2 to induce the activity of the TGFß3 promoter specifically via ER{alpha} in cultured HeLa and MG63 osteosarcoma cells.

Our observation that Ras-mediated PKC and MAPK signaling is crucial for E2 induction of the TGFß3 promoter raises several interesting issues that remain to be investigated. It is not presently known whether there is a direct requirement for PKC action on one of the transcription factors/coactivators involved in this process, because PKC has been previously shown to activate the MAPK pathway through phosphorylation of Raf (37). However, several putative PKC target sites, one within the A/B domain (serine 46) and the others within the ligand-binding domain (serines 432, 518 and 527, and threonines 465 and 553), are present in ER{alpha}. Experiments are in progress to identify whether any of these residues are directly involved in E2-induced activation of the TGFß3 promoter. With respect to the MAPK pathway, our experiments have shown that the effect of activated Ras is not modulated through serine 118 of ER{alpha}, a result which is in agreement with our deletion analysis of ER{alpha}, which positioned the Ras regulatory domain within the first 68 amino acid residues of the protein (discussed below). Furthermore, the absence of MAPK phosphorylation sites within the Ras regulatory domain suggests that MAPK acts on another component of the TGFß3 promoter activation complex. In the event that direct phosphorylation on ER{alpha} is found not to be involved in regulating the E2-induced transactivation of the TGFß3 promoter, identification of the associated factor(s) or coactivator(s) implicated in this system would be of great interest. The implication of an intermediary factor has also been invoked recently to explain the observation that PKA decreases the association of the progesterone receptor with the corepressor N-CoR and SMRT, in the absence of direct phosphorylation of the receptor by PKA (38). Finally, the identification of which PKC isoform(s) is implicated in E2 induction of the TGFß3 promoter after Ras activation is crucial to our full understanding of how PKCs target the ER{alpha} pathway. We are therefore currently engaged in the process of determining which PKC isoform(s) mediates E2-induced activation of the TGFß3 promoter.

Using progressive N-terminal deletions, we identified three domains within the ER{alpha} A/B region that modulate E2-dependent transcription of the TGFß3 promoter (Fig. 5Go). The first one is located at the N-terminal end of the protein, encompassing amino acids 1–68. This domain is required for the permissive effect of EGF and activated Ras on E2-stimulated transcription of the TGFß3 promoter. The second domain, comprised of amino acids 68 and 107, inhibits the E2 response, because its deletion leads to a better induction by E2 (compared with the inactive ER{alpha}{Delta}1–67 mutant). Finally, we have shown that amino acids 107–156 are required for the E2 response. Interestingly, previous studies have shown that amino acids 102–149 of ER{alpha} comprise an activating domain that synergizes with AF-2 in response to E2 at ERE-containing promoters in HeLa cells (39). These results suggest that the Ras-regulatory domain (1–68) exerts its modulating function through relieving the action of the inhibitory domain (68–107) on the E2-activation domain (107–156). We hypothesize that the folded inhibitory domain covers the E2-regulatory domain and prevents its interactions with associated factor(s) necessary for recognition of the TGFß3 promoter (see below) and/or coactivator protein(s). Upon stimulation of the Ras pathway by growth factors, MAPK and PKC signaling is activated. The direct or indirect action of PKC and MAPK on the Ras regulatory domain (1–68) likely results in a conformational change in the A/B domain, relieving the action of the inhibitory domain and leading to the exposure of the E2 activation domain. The exposed E2 activation domain then cooperates with the essential AF-2, ultimately initiating gene transcription. Our description of a complex functional organization of the ER{alpha} A/B domain, required for E2 regulation of the TGFß3 promoter, is in agreement with previous investigations of this domain and its role in the promoter- and cell-specific transcriptional activity of ER{alpha} induced by E2 and the SERM tamoxifen (39, 40). It is now clear, from these studies, that intramolecular communication between multiple regions within the ER{alpha} A/B domain are required to support transcriptional activation by E2 and SERMs of specific subsets of target genes.

The results described in this report suggest a model for TGFß3 promoter activation by E2 and SERMs (Fig. 8Go). In this model, the action of E2 requires the dual activation of the Ras and PKC pathways by one or more growth factors produced locally in the bone. This is demonstrated by the inhibition of the E2 response in the presence of EGF through expression of a dominant negative interfering mutant of Ras (RasS17N), as well as by treatments with the MEK inhibitor PD98059 and the PKC inhibitor GF109203X. The combined activities of the PKC and MAPK pathways is likely to result in the posttranslational modification of either ER{alpha}, the TGFß3 promoter binding protein (referred to here as TPBP), presumably required for recognition of the previously defined RAL response element (30, 31), and/or coactivators, such as SRC-1, recently shown to be phosphorylated through the MAPK pathway (41). These events would lead to the formation of transcriptionally competent E2-dependent complex. On the other hand, SERMs such as EM-652, RAL, and tamoxifen may induce a conformational change in ER{alpha} that mimics the combined effects of E2 binding and PKC/MAPK action, therefore bypassing the need for Ras signaling, as demonstrated by the insensitivity of EM-652 induction of the TGFß3 promoter to the action of the various inhibitors used in this study.



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  		Figure 8. Proposed mechanism that mediates E2 activation of the TGFß3 promoter. Growth factors such as EGF have the ability to stimulate both the Ras/Raf/Mek/Erk and PKC pathways, either directly or through cross-talk between Ras and PKC or PKC and Raf. Because the PKC inhibitor GF109203x blocks the effect of activated RasV12, it is likely that PKC functions downstream of Ras. The exact target(s) of the Ras/Raf/Mek/Erk and PKC pathways remains to be delineated, but it is likely to involved ER{alpha} as well as coactivator (CoA) and TPBP. The possible effect of the phosphorylation of one or more of these effectors is to increase their affinity for each other, resulting in the formation of a competent TGFß3 promoter activation complex. SERMs may bypass the need for covalent modification of these proteins by changing the conformation of ER{alpha} so that it increases its affinity for CoA and TPBP. The amino-terminal domain is marked as N, the DNA-binding domain as DBD, and the ligand-binding domain as LBD.

 
In conclusion, this work identifies a new ER{alpha} activation pathway and presents further insight into the molecular mechanisms that help explain the tissue-selective activity of SERMs. More importantly, our findings provide the basic framework for the design of more effective therapeutic strategies for the treatment of diseases such as osteoporosis.


   Footnotes
 
1 This work was supported by the Canadian Institutes of Health Research and the National Cancer Institute of Canada through the Canadian Breast Cancer Research Initiative program. Back

2 Supported by a Senior Scientist Award of the Canadian Institutes of Health Research. Back

Received August 11, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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