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Department of Neurobiology and Physiology (M.I.S., D.J.L., T.K.W.), Northwestern University, Evanston, Illinois 60208; Department of Medicine (T.K.W.), Northwestern Medical School, Chicago, Illinois 60611; and Department of Human Genetics (H.S., S.A.C.), University of Michigan, Ann Arbor, Michigan 48109
Address all correspondence and requests for reprints to: Teresa K. Woodruff, Ph.D., Department of Neurobiology and Physiology, Northwestern University, 2205 Tech Drive, Evanston, Illinois 60208. E-mail: tkw{at}northwestern.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The activin signaling pathway is conserved in a number of cells, but FSHß expression is restricted to gonadotropes. A pituitary-specific transcription factor necessary for activin-dependent induction of the FSHß promoter has been identified that permits FSHß expression in nongonadotrope cells. Pitx2 is a member of Pitx subfamily of bicoid-related homeodomain factors that is required for pituitary development and is present in the adult pituitary. This factor was transfected into LßT2 cells, where it caused up-regulation of basal and activin-mediated FSHß promoter activity. Furthermore, cotransfection of Pitx2c with Smad3 in kidney-derived TSA cells resulted in activin-regulated FSHß response, suggesting its important role in tissue-restricted regulation of FSHß by activin. A Pitx2c binding site was identified within the proximal promoter, and elimination of this region also resulted in a loss of activin-regulated FSHß promoter activity. Taken together, these studies suggest that the regulation of FSHß is dependent on activin-mediated signaling factors in concert with pituitary-derived nuclear regulatory proteins.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ß, encoded by separate genes. The glycoprotein - subunit (GSU) is common to all members of the pituitary glycoprotein family, whereas the ß-subunit is unique to each member. It is the ß-subunit that gives biological specificity to each hormone and constitutes a possible target for molecular regulation of these proteins (3, 4). Production and secretion of the gonadotropins is highly regulated by a variety of positive (GnRH, activin, steroids) and negative (inhibin, follistatin, and steroids) factors. The pulse frequency of GnRH is partially responsible for the differential regulation of FSH and LH. Slow frequency (5) and low amplitude (6) GnRH pulses increase FSHß mRNA, whereas fast pulses stimulate LHß synthesis. Inhibin, activin, and follistatin are proteins that specifically regulate FSH. Inhibin and activin are structurally related members of the TGFß superfamily of ligands. At the level of the pituitary, activin stimulates (7) and inhibin inhibits the release of FSH (8, 9). Follistatin is a known activin binding protein and inhibitor of activin action (10).
As activin is a FSH-specific modulator, many studies have investigated the tissue-specific effect of this ligand. In primary pituitary cell cultures, activin causes a dose-dependent increase in FSHß mRNA levels and FSH secretion, whereas the levels of LHß and GSU remain unchanged (3). These studies suggest that the regulatory action of activin on FSH secretion may be accounted for by transcriptional stimulation of FSHß mRNA within gonadotrope cells. A number of studies support a hypothesis that activin, rather than GnRH, may be the primary regulator of FSH. Specifically, activin stimulates FSH secretion in rat primary pituitary cells that have been desensitized to GnRH (11). Furthermore, GnRH stimulation of FSHß mRNA in a pituitary perfusion system is activin-dependent because addition of follistatin attenuates the increase in FSHß mRNA expression (12).
These in vitro data are supported by a number of in vivo studies showing increased levels of plasma FSH in female and male rats injected with recombinant human activin A (activin; Refs. 13 and 14). Activin also stimulates FSH secretion in female rats treated with a GnRH antagonist, which suggests a role for activin in FSH stimulation that is independent of a GnRH mechanism (15). Previous studies also suggest that activin acts on the FSHß gene at the level of transcription because the transcriptional inhibitor actinomycin-D prevents an activin-stimulated increase in FSHß primary transcript levels (4).
As a member of the TGFß superfamily of proteins, activin signals through a heteromeric receptor complex and intracellular signaling molecules known as Smad proteins. The activin signal transduction pathway is initiated by binding of activin to its transmembrane type II serine/threonine kinase receptor, ActRII (16) or ActRIIB (17). The ligand-bound type II receptor associates with and phosphorylates the type I receptor, the activin receptor-like kinase, ALK4 (ActRIB; Ref. 18). The activated type I receptor, in turn, phosphorylates cytoplasmic coactivating factor Smads. The phosphorylation of activin-specific, receptor-restricted Smads, Smad2 and Smad3 (19), allows their association with a common mediator Smad, Smad4, and subsequent translocation of the Smad complex into the nucleus. Once in the nucleus, the Smad complex binds specific DNA sequences, known as Smad-binding elements (SBE), which consist of CAGA- or GTCT-like sequences (20, 21, 22, 23). The presence of a SBE within the regulatory region of a gene, however, is not sufficient for activin regulation. This short, 4-bp sequence can be found on average once every 256 bp in the genome, at least once in every promoter. In addition, the affinity of Smad molecules for the SBE is very low (24). Because this element is bound equally well by Smads activated by all TGFß ligands, signal specificity is presumed to be achieved by the interaction of Smads with tissue- and cell-specific DNA-binding partners. In the nucleus, the activin-specific Smad complex can interact with DNA-binding adapters, such as forkhead activin signal transducer (FAST) proteins (25, 26, 27). Subsequently, this complex can recruit coactivators such as cAMP response element binding protein-binding protein and its homolog p300 (28) or corepressors such as TGIF (29), and thus positively or negatively regulates the transcription of different target genes. Alternatively, Smad molecules can associate with known transcription factors, such as TFE3 (30), and modulate activity of existing transcriptional complexes (for reviews, see Refs. 31, 32, 33, 34).
Although a great deal is known regarding activin signaling in a variety of cells, little is known about the specific signal transduction pathway leading to FSH regulation by activin. As the FSHß-subunit is a likely target of activin-Smad signaling, we investigated the role of activin-specific Smads (Smad2, Smad3, and Smad4) in FSH regulation using transient transfection studies. It has been shown previously that Smad2 and Smad3 have distinct transcriptional properties. Smad2, in cooperation with forkhead domain protein (FAST2), is required for induction of the mouse goosecoid (gsc) promoter, whereas Smad3 suppresses activation of this promoter (27). Here, we demonstrate that the pituitary gonadotrope also uses these two coactivators differentially to mediate activin-stimulated FSHß synthesis.
Although activin affects the expression of many genes, activin-regulated FSH expression is exclusive to pituitary gonadotropes. This selectivity suggests that the cellular specificity of activin signal is achieved by transcriptional coregulators acting in concert with Smad proteins. Therefore, we also attempted to identify the pituitary-specific transcription factors necessary for activin-dependent stimulation of the FSHß promoter. To find such a protein, a candidate gene approach was used to identify transcription factors expressed in the pituitary that may play a role in FSH regulation. In particular, Pitx2, a member of the Pitx subfamily of bicoid-related homeodomain factors, was selected as an appropriate transcriptional cofactor for investigation. Three isoforms of Pitx2 are generated by alternative promoter usage and alternative splicing. These transcription factors contain identical homeodomain and C-terminal sequences but different N termini (35, 36). Pitx2 is essential for the development of the pituitary (for reviews, see Refs. 37, 38, 39) because Pitx2 knockout mice (-/-) exhibit an arrest of pituitary growth and differentiation (35). Unfortunately, the Pitx2 (-/-) phenotype is embryonic lethal and does not provide a good in vivo model of Pitx2 transcriptional properties. The Pitx2 hypomorph mice (neo/neo), on the other hand, live until postnatal d 1 and exhibit significant effects of reduced Pitx2 function (36). Interestingly, these mice have no markers of differentiated gonadotrope cells, including LHß, FSHß, and GnRH receptors, as compared with only a slight deficit in the somatotrope and thyrotrope markers, GH and TSHß-subunit, respectively. These findings suggest that gonadotrope differentiation and function are especially sensitive to a decreased dosage of Pitx2.
In addition to pituitary development, Pitx2 plays an important role in left-right axis formation. Pitx2c specifically functions as a mediator of nodal signaling in left lateral plate mesoderm. It has also been found that activin A can induce Pitx2c mRNA, confirming the role of activin in left-right asymmetry and indicating a possible role of Pitx2c as a downstream comodulator of activin signaling (40). In addition, Pitx2 is expressed in the adult pituitary (35), where it is involved in transcriptional regulation of pituitary-specific genes (41). Here, we demonstrate that Pitx2c acts as a pituitary-specific transcriptional coactivator involved in FSH regulation by activin. Importantly, we show that Pitx2c, in conjunction with Smad3, transactivates the FSHß promoter in both pituitary and nonpituitary cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Previously published studies have demonstrated that LßT2 cells express FSHß mRNA and secrete FSH upon activin stimulation in a dose- and time-dependent manner (43, 44). Similarly, we have shown by Northern analysis that the FSHß-subunit and other known activin responsive genes are regulated in LßT2 cells (data not shown). The regulatory action of activin on FSH secretion may be due to transcriptional stimulation of FSHß mRNA (3, 4). To directly investigate the effect of activin on transcriptional control of FSHß-subunit, we used the rat FSHß promoter in transfection studies. LßT2 cells were transiently transfected with 338 bp of the 5'-flanking region of rat FSHß gene fused to the luciferase reporter gene (-338rFSHß-Luc). Cells were subsequently treated with activin in the presence or absence of follistatin. Activin was able to stimulate activity of the -338rFSHß-Luc promoter construct up to 4-fold over basal levels, and cotreatment with follistatin prevented this stimulatory effect (Fig. 1A
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). As Smad4 is a common mediator of Smad action, the effects of cotransfection of Smad2/Smad4 and Smad3/Smad4 on transcription were investigated. As expected, there was a synergistic effect of Smad3 and Smad4 on FSHß promoter activity when cells were treated with activin. Altogether, the combination of Smad3, Smad4, and activin resulted in 74-fold stimulation of the reporter gene as compared with only 9-fold and 40-fold stimulation by Smad3 or Smad3 in the presence of activin, respectively. Conversely, neither Smad2 nor Smad2/Smad4 was able to stimulate the FSHß promoter in LßT2 cells. Furthermore, overexpression of increasing amounts of Smad3 transactivated the FSHß promoter in a dose-dependent manner, whereas Smad2 had no effect on the promoter activity at either concentration (Fig. 1C).
The Proximal Rat FSHß Promoter Contains a Functional SBE
Activated receptor-restricted-Smad/common mediator-Smad complexes can bind specific DNA sequences, known as SBEs, which consist of CAGA- or GTCT-like sequences (20, 21, 22, 23). We visually inspected the sequence of the rat -338 FSHß promoter for these motifs. Several possible SBEs were identified (Fig. 2A) and examined by transient transfections of FSHß promoter deletion constructs. Unlike the remarkable activation of the -338 promoter by activin, a construct restricted to -106 bp of the proximal promoter was not stimulated by activin, Smad3, or Smad3/4 (Fig. 2B).
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), only the probe that included a CAGA followed by a 7-bp linker and an inverted SBE palindrome (-281 to -253) was able to bind Smad proteins as measured by a change in protein-DNA mobility when antibodies directed against Smad proteins were incubated with the complexes (Fig. 3A). It is apparent that multiple proteins can bind this DNA fragment based on the number of complexes detected on the nondenaturing gels. The identity of the proteins present in these intermediate complexes is not known but may represent different stoichiometric assemblies of Smad3, Smad4, and other factors binding to the DNA. The protein-DNA complex denoted a(i) was ligand-specific, because it appears only in the nuclear protein extracts from cells treated with activin. The protein-DNA complex a(ii) was eliminated after addition of Smad3 antisera, possibly due to antibody interference with the DNA-interacting domain of Smad3. A new complex was revealed after addition of Smad4 antisera (*) representing a true supershift of a DNA-Smad4 interaction. A heavy band, denoted b, was decreased, and a new DNA-protein complex, c, formed upon addition of antisera directed against Smad2, Smad3, or Smad4.
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). Although no activin-specific band was recognized, the Smad3 interaction was again identified by inhibition of the DNA-protein complex (band a) when Smad3 antibody was preincubated with the nuclear extract. There was little or no effect of the Smad2 antibody on the protein-DNA complex, whereas Smad4 specific binding was observed with the same region (Fig. 3B). As seen in Fig. 3A, a higher molecular weight band was observed upon addition of Smad4 antibody, representing a complex of Smad4 protein, antibody, and DNA (*). To identify the specific sequence required for the Smad4-DNA interaction and further investigate the nature of the DNA-protein interactions, several oligonucleotides containing mutations within the inverted palindromic SBE sequence were designed and used in EMSA experiments (Fig. 3C). The Smad4 supershift (*) seen in Fig. 3, A and B, was again observed when Smad4 antisera was incubated with the wild-type DNA-protein lysate complex. As before, there was a loss of complex b and appearance of complex c upon addition of antibodies. Protein binding to mutated oligonucleotides 1, 2, and 3 was still apparent; however, Smad4 antisera failed to cause a shift in any protein-DNA complex, suggesting that the interaction of the DNA with Smad4 was disrupted. Oligonucleotide mutant 3 had a more complex interaction with endogenous proteins, disrupting band a as well as the ability of Smad4 antisera to create a slower migrating band. Mutations 4 and 5, on the other hand, did not disrupt the Smad4 supershift band (*). Thus, mutations 1, 2, and 3, which interrupted the core SBE, caused a loss of Smad4 supershift, whereas mutants 4 and 5, which lie outside the CAGA motif, did not interrupt Smad4 interaction.
Pituitary-Specific Factors Are Necessary for FSHß Regulation
To examine whether the -338rFSHß promoter was responsive in other activin-sensitive cell lines, this construct was transfected into the TSA monkey kidney epithelial cell line (45). To confirm that these cells contain a functional activin signaling pathway, a well known activin/TGFß responsive reporter gene construct, the plasminogen activator inhibitor-1 promoter ligated upstream of the luciferase reporter gene (p3TP-Lux), was used in transient transfection experiments (22, 46, 47). Cells were transfected with p3TP-Lux and treated with activin for 24 h (Fig. 4A). Activin stimulated luciferase activity 4-fold over basal levels. Cotransfection of both Smad2 and Smad3 expression vectors resulted in ligand-independent stimulation of the promoter, indicating involvement of these molecules in activin signaling in TSA cells. The rat FSHß promoter, however, was not active in this cell line under any of the conditions described above (Fig. 4B). These data suggest that although an intact activin signaling pathway exists in TSA cells, pituitary-specific transcription factors exist that are necessary for activin-dependent stimulation of the FSHß promoter and are not expressed in TSA cells.
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GSU, LHß, and FSHß (48). We investigated whether another member of this family, Pitx2, can transactivate the FSHß promoter in LßT2 cells. Pitx2 is expressed in adult pituitary gonadotrope cells (35) and in LßT2 cells (data not shown), making it a good candidate for a pituitary-specific factor involved in FSH regulation. Cells were transiently transfected with -338rFSHß-Luc reporter and Pitx2 isoforms (Fig. 5A), then treated with activin for 24 h. Cotransfection of any of the Pitx2 isoforms led to a significant up-regulation of the -338rFSHß promoter activity. In each case, activin treatment resulted in an additional increase of luciferase expression (Fig. 5B). Pitx2c, however, was found to be the most potent activator of the FSHß promoter. Cotransfection of this factor resulted in 9-fold stimulation of reporter gene activity, and cotreatment with activin induced the promoter an additional 40%. Overexpression of increasing amounts of Pitx2c transactivated the FSHß promoter in a dose-dependent manner (Fig. 5C), and specificity of this induction was confirmed by inability of Pitx2c to transactivate p3TP-Lux promoter in both LßT2 and TSA cell lines (data not shown).
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). Unlike in LßT2 cells, activin treatment of TSA cells caused only a slight (1.4-fold) additional increase of luciferase activity. As Smad3 is necessary for activin-mediated induction of FSHß-Luc expression in LßT2 cells, we examined whether Smad3 was limiting. Cotransfection of Smad3 and Pitx2c with the rat FSHß promoter into both LßT2 and TSA cell lines resulted in greatly enhanced responsiveness of the FSHß promoter to activin. In pituitary-derived cells, activin stimulation of the promoter reached 11-fold over basal in the presence of Pitx2c alone, whereas cotransfection of Smad3 and Pitx2c augmented this induction to 23-fold over basal (Fig. 6A). There was also a significant additive effect of both of these factors on FSHß promoter in kidney-derived cells (Fig. 6B). Activin was more potent when both Smad3 and Pitx2c were coexpressed. Under these conditions, activin induced luciferase activity an additional 2-fold over Pitx2c and Smad3.
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). Although DNA binding sites have been well characterized for Pitx1 and other bicoid-related homeoproteins (48), no consensus DNA binding element for Pitx2 has been found to date. To investigate whether Pitx2c directly binds the FSHß promoter in the -230 to -185 promoter interval, whole cell extracts from untransfected TSA cells or cells transfected with HA-tagged Pitx2c were made. Expression of Pitx2c was confirmed by Western blot analysis using antibody against HA tag (data not shown) and DNA-protein interactions identified by EMSA. Interaction between Pitx2c and DNA (shift) was detected with double-stranded probe spanning a -230 to -199 region of the promoter, and these interactions were effectively competed with 100-fold molar excess of the unlabeled probe (Fig. 7B). This result is consistent with our transfection data and, taken together, suggest that Pitx2c can directly bind this region of the rat FSHß promoter to modulate activin-stimulated FSHß transcription.
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) is necessary for transcriptional activation of the promoter, the site was deleted and used in transient transfection studies in LßT2 cells. The -338rFSHß(SBEdel)-Luc reporter construct was cotransfected with the indicated Smad and Pitx2c expression vectors. Elimination of the palindromic Smad4 binding site resulted in a loss of basal activation, stimulation by both Smad3 and Smad3/4 (Fig. 8A), as well as activin-mediated (Fig. 8B) FSHß promoter activity. In addition, deletion of this site caused a loss of promoter stimulation by Pitx2c (Fig. 8A), suggesting that these elements are involved in Pitx2c-mediated induction of the FSHß promoter.
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), suggesting complex interactions between these two factors. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The main obstacle in prior investigations of activin-regulated gene expression in pituitary gonadotrope cells was the lack of a pituitary cell line that contained all of the necessary components to permit the study of FSH synthesis and secretion. However, a recently derived gonadotrope cell line, LßT2, has proven to be a very useful tool in studying the molecular mechanisms underlying expression of FSHß (51). As reported previously (43, 44), these cells express FSHß mRNA and secrete FSH upon activin stimulation. Thus, the LßT2 cell line represents a cell model that responds to activin with specific gene activities predicted by in vivo analysis of the ligand.
To permit the cell type-restricted response that characterizes the activin signal in the pituitary, gonadotrope-specific transcription factors must be present in the model cell line. For example, activin inhibits the production of GH by somatotrope cells, while stimulating FSHß in gonadotrope cells. Evidence of cell type selectivity for expression of the FSHß gene is the relatively low basal expression of the FSHß promoter in most cell lines, including the pituitary-derived T3 line. When the ovine FSHß promoter is transfected into LßT2 cells, both basal and activin-stimulated transcription is significantly enhanced (42, 44). Here, we have shown that the rat FSHß-subunit promoter is also directly responsive to activin in LßT2 cells. The 338 bp of 5'-flanking region of the rat FSHß gene (-338rFSHß) has been found to be the most activin-responsive domain of this promoter, and a construct containing this region was stimulated by the ligand up to 4-fold over basal. These results indicate that the activin signaling pathway leading to FSHß stimulation is intact and functional in the LßT2 cell line. Based on the well characterized signal transduction pathway for activin, our initial studies were directed at the role of the canonical activin-stimulated signaling factors, the Smads, in FSHß transcription stimulation.
The TGFß superfamily of ligands binds ligand-specific receptors, which phosphorylate transcriptional modulators known as Smads (for reviews, see Refs. 31, 32, 33, 34). To ask whether the FSHß promoter is regulated by Smad proteins, we examined activin signaling in LßT2 cells via the Smad pathway. Although both Smad2 and Smad3 are known to be TGFß/activin-specific factors (47, 52, 53, 54), these proteins have different roles in the transcriptional activation of the rat FSHß promoter. Overexpression of Smad3 resulted in 8.5-fold induction of the promoter, and this stimulation was further (40-fold over basal) augmented by activin treatment. Interestingly, there was no effect of Smad2 on the promoter despite the fact that this molecule was able to transactivate p3TP-Lux promoter in TSA cells. Smad4, a common mediator of Smad signaling (55, 56, 57), binds DNA directly and is required for formation of stable transcriptional complexes (58). In our system, Smad4 had no effect on the promoter, but it elicited a synergistic activity when cotransfected with Smad3, and not with Smad2. Activin treatment caused additional, up to 74-fold, stimulation of the reporter gene activity. Taken together, our data suggest that there is indeed a functional difference between Smad2 and Smad3 transcriptional properties and that Smad3 and Smad4 are necessary for activin regulation of FSHß.
Functional differences between Smad2 and Smad3 have been described previously. It has been found that the most N-terminal portion of Smad3 is critical for its function and that this structural difference may account for its transcriptional properties as compared with Smad2 (59). Although Smad2 is necessary for TGFß/activin-mediated stimulation of the mouse gsc promoter, Smad3 inhibits this activation (27). In the present study, however, we found little effect of Smad2 on Smad3-mediated activation of the FSHß promoter (data not shown), suggesting that the mechanism of different regulation of FSHß by these factors is distinct from that of gsc.
Structurally, the main difference between Smad2 and Smad3 is the presence of two extra protein loops in the MH1 domain of Smad2. The first loop consists of 10 amino acid residues in positions 21–30. The second loop covers 30 amino acids spanning residues 79–108. It is likely that these loop structures result in the differential ability of the two transcription factors to interact with tissue-restricted coregulators of gene function, thus providing an important level of control to the ubiquitous TGFß superfamily of ligands. A naturally occurring Smad2 variant has been identified that lacks the third exon, and this protein may function more like a Smad3 molecule in some cells (60). In addition to future studies that will address the regulation of Smads during the normal reproductive cycle, it will be of interest to determine whether this truncated Smad2 protein is expressed in the pituitary gonadotrope and what role it might play in the modulation of FSHß in response to activin. It will be interesting to study binding of this truncated Smad2 form to DNA in light of our findings that the ability of wild-type Smad2 and Smad3 to form complexes with the same region of the FSHß promoter is distinct.
Transient transfections using further deletions of the -338rFSHß 5' end resulted in a loss of activin and Smad-dependent promoter activity (i.e. -106rFSHß) and narrowed the activin responsive region of the promoter to the interval between -338 and -106 bp. Additional transfection studies, however, using a heterologous promoter construct containing two copies of this region resulted in a lack of Smad3-stimulated luciferase activity, indicating that there are a series of complex interactions that extend beyond the simple CAGA site (data not shown). This result is consistent with the current Smad literature, which indicates that although the CAGA DNA sequence will bind Smads, it is not sufficient to confer Smad responsiveness.
By visual inspection, a number of CAGA sites were identified in the -338 to -106 region of the rat FSHß promoter, each of which could be capable of binding Smad proteins. However, the only site that was found to bind Smads was an inverted palindrome 5'GTCTAGAC3'. We have shown that Smad4 is present within transcriptional complexes that interact with the DNA fragment containing this sequence. This is indicated by the appearance of the supershift band (*) upon addition of Smad4 antibody (Fig. 3, A–C). However, there was no change in this banding pattern in the presence of both Smad2 and Smad4 antibodies, suggesting that Smad2 is not a part of these complexes. Conversely, a disruption of Smad4 supershift band was observed upon addition of Smad3 antisera. This is possibly due to interference of this antibody with binding of the protein complex to DNA, and indicates the presence of Smad3 protein within this complex. Functional analysis of this SBE indicates that Smad3 and Smad4 act directly through this site, because the removal of this sequence resulted in a loss of both basal and activin-stimulated, as well as Smad3 and Smad3/4-mediated, FSHß promoter activity. Along with the binding studies and heterologous promoter experiments, our data suggest that the inverted palindrome 5'GTCTAGAC3' sequence (-281 to -253) is necessary but not sufficient for transcriptional activation of the rat FSHß promoter.
The observation that not all CAGA intervals are created equal is a common finding in the Smad literature. It is clear that not all genes are Smad-responsive, yet, if the CAGA motif were all that was necessary to confer binding and activation, then it follows that all genes should be TGFß or activin responsive. Therefore, the finding that the FSHß promoter has multiple CAGA sites but not all of them bind Smads is not surprising. Collectively, our data demonstrate that FSHß promoter is complex and contains as yet unidentified cell and tissue-specific transcription elements (for reviews, see Refs. 61 and 62). Remarkably, different and complex banding patterns were revealed when only slight changes in the sequence of oligonucleotides used in EMSA experiments were made (Fig. 3, compare panels A and B). This indicates that other unidentified factors are involved in DNA interactions that may be needed for cooperativity with Smads to regulate FSHß transcription. The effect of activin and the Smad proteins on FSHß transcription was specific to pituitary gonadotrope cells, as the promoter was not responsive in a kidney-derived cell line (TSA). Despite the fact that TSA cells contain a functional activin signaling pathway, the FSHß promoter was not induced by activin, Smad molecules, or a combination of both. This result suggests that pituitary-specific transcription factors exist that are necessary for activin-dependent stimulation of the FSHß promoter. Thus, the specificity of the activin signal in LßT2 cells is achieved by Smad interaction with pituitary-specific DNA-binding proteins. FAST was the first Smad nuclear target identified and was found to be necessary for transcription of Xenopus homeobox gene Mix.2 (FAST1; Ref. 25) and mouse gsc gene (FAST2; Ref. 27). As FAST is expressed only during gastrulation, the cooperation of this protein with Smad complexes provides for a specific regulation of activin target genes at this stage of development. FAST assists in the recruitment and assembly of transcriptional coactivators (28) or corepressors (29) with Smads within a specific cell at a specific time. Smad molecules can also associate with known transcription factors, such as TFE3 (30) or Jun/Fos (63), thus modulating the activity of existing transcriptional complexes and other signaling pathways. For example, activity of promoters containing activator protein-1 (AP-1) sites has been shown to be highly stimulated by TGFß and activin, suggesting a cooperation between Smad molecules and AP-1 binding proteins in the regulation of these genes (63, 64, 65). Moreover, only the activated forms of Jun family transcription factors can interact with Smads, and, consistently, in the absence of active Jun and Fos, the Smads only weakly activate AP-1-containing promoters (63, 64). This suggests that TGFß and activin regulation of specific target genes through AP-1 sites may be limited to cells containing functional Jun and Fos transcription factors. Similarly, we postulate that gonadotrope-specific elements are involved in activin-mediated regulation of FSH. Here, we have investigated Pitx2 as a candidate factor that is expressed in the adult pituitary (35) and has been shown to be essential for development of all five pituitary cell lineages (for reviews, see Refs. 37, 38, 39).
Very little is known regarding signaling pathways that lead to Pitx2 activation and cooperation of this factor with other proteins. In contrast, the transcriptional properties of Pitx1, another member of Pitx family of transcription factors, have been studied extensively. Pitx1 has been shown to regulate a variety of pituitary-specific promoters, including gonadotropin subunits GSU, LHß, and FSHß. In addition, activation of the LHß-subunit promoter is significantly augmented by synergism of Pitx1 with SF-1 and Egr-1 transcription factors (48, 66, 67). Similarly, although to a lesser extent, Pitx2a and Pitx2b isoforms can synergize with SF-1 and Egr-1 to transactivate the LHß promoter (41). Because LßT2 cells express Pitx2 mRNA, we investigated whether this transcription factor can transactivate the FSHß promoter in this cell line. Indeed, all three isoforms of Pitx2 caused an up-regulation of the -338rFSHß promoter activity. We found, however, that addition of Pitx2c resulted in the highest stimulation of the rat FSHß promoter. Although activin treatment augmented stimulatory effects of all Pitx2 isoforms on the FSHß promoter, we chose to concentrate our further studies on Pitx2c as the most potent activator of the FSHß promoter.
We hypothesize that expression of Pitx2c, as a pituitary-specific factor that mediates regulation of FSH by activin, should result in ectopic expression of FSH. Indeed, overexpression of Pitx2c in the TSA kidney epithelial cell line resulted in 20-fold induction of the rat FSHß promoter. Importantly, transfection of both Pitx2c and Smad3 permitted activin stimulation of reporter gene activity. Clearly, Pitx2c rendered the FSHß promoter responsive to activin in TSA cells and, accordingly, Pitx2c enhanced responsiveness of the FSHß promoter to activin in LßT2 cells when cotransfected with Smad3. These results suggest that there is a cooperation between Smad3 and Pitx2c that leads to activin-mediated regulation of the FSHß promoter, and they further imply that Pitx2 is a tissue-restricted transcription factor necessary for activin-mediated FSH regulation in the gonadotrope. We have also shown that the elimination of the Smad-binding site affected transcriptional activation of the promoter by Pitx2c, suggesting the existence of a Smad-Pitx interaction within the context of the -338rFSHß promoter. Based on these results, we speculate that these two factors physically interact. It will be interesting to further investigate the nature of the Smad-Pitx association and delineate precisely how this family of factors interact and interrelate to other proteins that bind within the FSHß proximal promoter. Additional experiments are underway to identify other transcription factors that may influence differential Smad binding to this region. Additionally, it is predicted that Smad2 does not transactivate FSHß because it is unable to bind or interact with Pitx2. This hypothesis will be tested by examining the different protein-protein binding sites in Smad2, Smad3, and Pitx2c. Furthermore, because Pitx2 was found to be important for expression of gonadotrope-specific transcription factors such as GATA2, Egr-1, and SF-1 (36), additional studies are evaluating the involvement of these factors in FSHß regulation and their cooperation with Pitx2 or Smad3. Finally, it is clear that Pitx2c binds directly to DNA, specifically, to a region of the promoter including -230 to -199 interval. A recent report indicates that the Pitx2 molecules form homo- and heterodimers (68). Future studies will be conducted to clarify the stoichiometry of Pitx2 and Smad binding to the FSHß promoter.
Consistent with the results of our progressive deletion studies, we have found that Pitx2c can directly bind a -230 to -199 region of the FSHß promoter to modulate activin-stimulated FSHß transcription. We have shown that, at least partially, this factor acts directly through this site, because deletion of this DNA sequence significantly blunted Pitx2c-dependent transcription of the promoter. DNA binding to a -230/-199 region, however, was not strictly required for this activation, suggesting that Pitx2c can modulate FSHß transcription either through a different DNA-binding site or a DNA-independent mechanism. Recently, two Pitx1 binding sites were identified within the proximal rat FSHß promoter (69). It is possible that Pitx2 can act through these sites, or through other, as yet unidentified elements. Pitx1 has also been shown to modulate activity of the LHß promoter through a DNA-independent interaction with SF-1 (70). Potentially, Pitx2 can affect transcription of the FSHß gene through a similar mechanism and protein-protein interaction with Smad3 or other factors. This Pitx2c-Smad3 cooperation in the context of the rat FSHß promoter is supported by the fact that the removal of the Pitx2 binding element affected Smad3-mediated activation of FSHß.
In summary, our data show that LßT2 cells contain a functional activin signaling pathway and that Smad3 and Smad4 are necessary for stimulation of the rat FSHß promoter by this ligand. Furthermore, we have established that there is a functional difference between Smad3 and Smad2 biochemical and transcriptional properties with respect to FSHß gene regulation, supporting previous observations in other systems (27). We have found that the region from -270 to -248 of the rat FSHß promoter binds Smad3 and Smad4, and that an inverted palindromic sequence within this region is necessary for basal, activin, Smad-, and Pitx2-dependent activation. We also identified Pitx2c as a candidate pituitary transcriptional cofactor that is involved in FSHß regulation through a possible cooperation with Smad proteins. This factor not only transactivates the FSHß promoter, but also potentiates the effects of activin and Smads on FSHß transcription. We have shown that Pitx2c binds the region of the promoter that encompasses the sequence from -230 to -199, and that this element is also necessary for full transcriptional activation. It is evident that the identification of transcriptional cofactors involved in the regulation of FSHß promoter activity is essential for a greater understanding of the molecular mechanisms underlying the regulation of FSHß by activin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of the Rat FSHß Promoter Deletion Plasmids
Reporter constructs containing different deletions of the rat FSHß promoter genes fused to the luciferase reporter gene in the pXP2
2 vector were developed by PCR using the previously described plasmid as a template (71). All sense primers included a BamHI restriction site engineered at the 5' terminus, and antisense primers had a KpnI restriction site engineered at the 3' end. All amplified products were ligated upstream of the luciferase reporter gene into a promoterless pXP22 vector (72). Sequences of all constructs were confirmed by cycle sequencing on a PTC-100 Thermocycler (MJ Research, Inc., Waltham, MA). Smad expression vectors and p3TP-Lux reporter plasmid were provided by the laboratory of Dr. J. Massague. Pitx2 expression vectors were described previously (36).
Cell Culture and Transient Transfections
The pituitary gonadotrope cell line LßT2 (73) was carried on plates coated with matrigel (BD Biosciences, Bedford, MA) in F12:DMEM supplemented with 5% fetal bovine serum, 0.45% glucose, and 1% antibiotic. The monkey embryonic kidney cell line TSA (45) was carried in DMEM, 10% fetal bovine serum, and 1% antibiotic. Both cell lines were carried in a humidified atmosphere (37 C) of 5% CO2 and passaged as necessary. All experimental treatments were done in phenol-free serum-free (LßT2) or serum-free (TSA) media to avoid the potential steroid effect. For transfections, cells were plated 1 d before transfection in 24-well plates and transfected with 250 ng of the reporter DNA and 25 ng of the expression vector per well using LipoFectamine Plus (Invitrogen, Carlsbad, CA). Empty vectors of both reporter and expression plasmids were used to balance DNA where necessary. Cells were treated with control media, activin (30 ng/ml), or activin and follistatin (30 ng/ml) for 24 h. Cells were lysed in GME buffer [25 mM glycylglycine (pH 7.8), 15 mM MgSO4, 4 mM EGTA, 1 mM dithiothreitol, and 1% Triton X-100], and lysates were added to assay buffer (GME buffer, 16.5 mM KPO4, 2.2 mM ATP, and 1.1 mM dithiothreitol). Luciferase activity was measured for 30 sec using an AutoLumat (Berthold Technologies Co., Oak Ridge, TN). We attempted to use internal controls for all transfection experiments by dual luciferase and ß-galactosidase assays. Unfortunately, cotransfection of both renilla-luciferase and ß-galactosidase expression vectors caused a significant decrease in activin response. LßT2 cells were grown on a matrigel matrix, which interfered with normalization of luciferase activity to protein content. The data shown here reflect the actual RLUs and are representative of the mean and SEM of at least three separate transfection experiments.
EMSA
Oligonucleotides representing the sense strands of the rat FSHß promoter were labeled with [
-32P]dATP using T4 kinase polymerase (Promega Corp., Madison, WI), purified with ProbeQuant G-50 micro column (Amersham Pharmacia Biotech, Piscataway, NJ) and annealed with their respective antisense strands. Labeled nucleotides (
50,000–100,000 cpm) were incubated with LßT2 nuclear extract (5 µg) or TSA whole cell extract (20 µg) in a final reaction volume of 30 µl containing 40 mM HEPES (pH 7.5), 100 mM KCl, 10 mM MgCl2, 12% glycerol, and 500 ng poly(dI-dC). After incubation for 15 min at room temperature, the samples were loaded onto a 5% polyacrylamide gel and electrophoresed at room temperature for 3 h at 160 V in a buffer containing 25 mM Tris-HCl (pH 7.8), 192 mM glycine, and 1 mM EDTA. After electrophoresis, the gels were dried and exposed to Kodak BioMax MR-1 film (Fisher Scientific, Pittsburgh, PA). For competition experiment, proteins and buffer were preincubated with unlabeled oligonucleotides, at 100-fold excess molar concentrations, for 15 min at room temperature before addition of labeled DNA. A similar preincubation was included with addition of antisera against Smad2, Smad3, and Smad4 (courtesy of Dr. Peter ten Dijke, The Netherlands Cancer Institute) and 12CA5 HA antibody (courtesy of Dr. Robert A. Lamb, Northwestern University).
Statistical Analysis
All values are expressed as the mean ± SEM. Student’s t test was used to evaluate differences between the control samples and activin-treated samples or between samples transfected with vector alone and samples transfected with different Smad and Pitx expression constructs. A P value less than 0.05 was considered statistically significant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AP-1, Activator protein-1; FAST, forkhead activin signal transducer; gsc, goosecoid; GSU, glycoprotein -subunit; RLUs, relative light units; SBE, Smad-binding element.
Received for publication February 22, 2002. Accepted for publication December 12, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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