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Ontogeny and Reproduction Research Unit, Centre Hospitalier de l’Université Laval (CHUL) Research Center and Centre de Recherche en Biologie de la Reproduction (CRBR), Department of Obstetrics and Gynecology, Université Laval, Ste-Foy, Québec, Canada G1V 4G2
Address all correspondence and requests for reprints to: Dr. Robert S. Viger, Ontogeny and Reproduction Research Unit, T1-49, Centre Hospitalier de l’Université Laval (CHUL) Research Center, 2705 Laurier Boulevard, Ste-Foy, Québec, Canada G1V 4G2. E-mail: robert.viger{at}crchul.ulaval.ca.
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Introduction |
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GATA factors are also expressed in a variety of other tissues, such as the gut, pituitary, brain, adrenals, and gonads, where they control many important physiological processes through the regulation of numerous downstream target genes (10, 11, 12, 13). As the adrenals and gonads represent the main steroid-producing organs, this suggests that GATA factors might play an essential role in steroidogenesis. Indeed, GATA-6 is abundantly expressed in the fetal, neonatal, and adult adrenal cortex (14). Within the gonads, both GATA-4 and GATA-6 are expressed in the ovarian steroidogenic compartment (15, 16), whereas in the testis, GATA-4 is present in fetal and adult Leydig cells (13, 17, 18, 19). Proposed steroidogenic target genes for GATA factors include those that code for the steroidogenic factor 1 (SF-1) transcription factor as well as three key enzymes/proteins involved in steroid hormone biosynthesis: 17ß-hydroxysteroid dehydrogenase type 1, aromatase, and steroidogenic acute regulatory protein (StAR) (20, 21, 22, 23, 24).
Steroidogenesis is tightly regulated by a complex regulatory cross-talk involving pituitary trophic hormones and the newly synthesized steroid hormones themselves. Temporally, the increase in steroid hormone production can be divided into two sequential steps: first, the acute effects that occur within minutes and result in increased mobilization and delivery of cholesterol precursors to the inner mitochondrial membrane, and second, the chronic effects leading to increased transcription of genes encoding essential components of steroidogenesis (reviewed in Ref. 23). The acute induction of steroidogenesis is mediated by StAR (25). Indeed, the requirement of StAR for intracellular cholesterol trafficking was strengthened by the identification of StAR mutations in patients with congenital lipoid adrenal hyperplasia, a lethal condition characterized by a dramatic reduction of steroidogenesis, accumulation of cholesterol, and lipid deposits in steroidogenic tissues (23). The absolute requirement for StAR was further corroborated by gene inactivation experiments in which StAR-/- mice have a phenotype similar to that of human congenital lipoid adrenal hyperplasia (26, 27).
Trophic hormone-induced steroidogenesis is predominantly mediated through the cAMP-dependent intracellular signaling pathway leading to protein kinase A (PKA) activation and is associated with a rapid increase in StAR transcription (28, 29). Although the StAR promoter lacks typical cAMP-responsive elements (CRE), a recent study has identified three CRE half-sites in the proximal StAR promoter, thus revealing a potential role for cAMP response element modulator (CREM) in StAR transcription (30). The region responsible for cAMP induction is located within the first 254 bp upstream of the transcription start site (31). Interestingly, the mechanism of cAMP-induced StAR transcription differs among species. The nuclear receptor SF-1 appears to be involved in both basal and cAMP-stimulated activity of the human StAR promoter (32, 33). In contrast, SF-1 is not involved in acute induction of the mouse StAR promoter (21, 22, 31), suggesting that other transcription factors are involved. These include members of the CCAAT-enhancer binding proteins such as C/EBPß. The onset of C/EBPß expression occurs just before StAR gene transcription, and its expression is increased about 4-fold in both testicular Leydig and ovarian granulosa-lutein cells upon stimulation with gonadotropins and cAMP analogs, supporting a role for this factor in StAR transcription (21, 34, 35). Another factor recently shown to contribute to StAR promoter activity is GATA-4. Indeed, GATA-4 was shown to be a potent activator of the mouse StAR promoter (20), and conversely, mutation of the -65-bp StAR GATA element leads to a decrease in basal and cAMP-induced StAR promoter activity in both MA-10 Leydig tumor cells and granulosa cells (21, 22).
Although SF-1, C/EBPß, and GATA-4 are clearly key regulators of StAR promoter activity, how these factors are integrated as downstream effectors of the trophic hormone stimulatory signal ultimately leading to increased StAR gene transcription remains unknown. An important feature of the initial period of cAMP-induced StAR promoter activity is that it does not require de novo protein synthesis (31), suggesting that all essential transcription factors are present and that posttranslational modifications of the transcription factors might be involved. In the present study we provide the first evidence that in MA-10 cells, GATA-4 is phosphorylated by PKA in response to cAMP stimulation. Using heterologous CV-1 cells to reconstitute the molecular mechanism involved in cAMP-induced StAR promoter activity, we show that transcriptional cooperation between GATA-4 and C/EBPß is PKA dependent.
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Materials and Methods |
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N2, aa 255–440; N3, aa 302–440; internal, aa 201–255 and 302–440; N1C1, aa 201–332; N1C2, aa 201–261) have been described previously (20, 36, 37). The remaining GATA-4 deletion constructs (N1, aa 201–440; N2C1, aa 255–332; C1, aa 1–332) were obtained by PCR using the full-length GATA-4 vector as template and the following pairs of primers: N1 (forward primer, 5'-GATCTAGAAAGCCTCAGCGCCGGCTGTCT-3'; reverse primer, 5'-ATGGATCCTTACGCGGTGATTATGTCCCC-3'), N2C1 (forward primer, 5'-GCTCTAGAAAGCCTCAGCGCCGGCTGTCT-3'; reverse primer, 5'-AGTGGATCCTTAACCTGCTGGTGTCTTAGATTTATT-3'), and C1 (forward primer, 5'-GATCTAGATACCAAAGCCTGGCTATGGCC-3'; the same reverse primer as for N2C1). A GATA-4 deletion construct containing a Cys to Ala substitution at aa 294 (N1 C294A) was first obtained by transferring a HindIII/BamHI fragment from N1C1 C294A (described in Ref. 37) into pBluescript SK (Stratagene). The resulting plasmid was digested with NcoI/BamHI (the NcoI site is a naturally occurring site immediately downstream of the second zinc finger), and the backbone vector was purified and ligated to a fragment containing the entire C-terminal domain, obtained via a partial NcoI and a total BamHI digest of the full-length GATA-4 cDNA, to produce N1 C294A. All GATA-4 deletion constructs were subcloned into the XbaI/BamHI sites of a pcDNA3 expression vector (Invitrogen, Carlsbad, CA) modified to contain an ATG sequence in-frame with an added XbaI site. The validity of all of our constructs was verified by sequencing. Expression plasmids for SF-1 (38), Friend of GATA 2 (FOG-2) (39), PKA catalytic 
-subunit (40), protein kinase inhibitor (PKI) (41), and C/EBP isoforms (42) were provided by Drs. Keith Parker, Eric Olson, Marc Montminy, Richard Maurer, and Steven McKnight, respectively.
Cell culture and transfections
African green monkey kidney CV-1 cells were grown in DMEM supplemented with 10% newborn calf serum at 37 C and 5% CO2. The MA-10 mouse Leydig tumor cell line was provided by Mario Ascoli (43). MA-10 cells were grown in Waymouth’s medium containing 15% horse serum at 37 C under 5% CO2. Transfections of CV-1 cells were performed in 24-well plates using the calcium phosphate precipitation method. Detailed transfection conditions have been described previously (20). Data reported represent the average of at least three experiments, each performed in duplicate.
Nuclear extracts and Western blots
Nuclear extracts were prepared by the procedure outlined by Schreiber et al. (44). In Western analyses, 20-µg aliquots of nuclear extracts from MA-10 cells were separated by SDS-PAGE and then electrotransferred to Hybond polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech, Baie-D’Urfé, Canada). Immunodetection of GATA-4 was achieved using commercially available GATA-4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antiserum and a Vectastain-ABC-Amp Western blot detection kit (Vector Laboratories, Inc., Burlingame, CA). For immunodetection of a Ser/Thr-phosphorylated GATA-4 protein, the GATA-4 protein was first purified by immunoprecipitation using a GATA-4-specific antiserum (Santa Cruz Biotechnology, Inc.), and the immunoprecipitate was separated by SDS-PAGE and then electrotransferred to a Hybond PVDF membrane. The detection of Ser/Thr-phosphorylated GATA-4 protein was then achieved using a phospho-(Ser/Thr) PKA substrate antiserum (Cell Signaling Technology, Beverly, MA) and a Vectastain-ABC-Amp Western blot detection kit.
Protein-protein interaction assays
In vivo interactions between GATA-4 and C/EBPß were assessed by coimmunoprecipitation experiments using 100 µg nuclear extracts from MA-10 Leydig cells and 3 µl C/EBPß antiserum or rabbit IgG (used as control) in 500 µl binding buffer [150 mM NaCl, 50 mM Tris-Cl (pH 7.5), 0.3% Igepal (Sigma-Aldrich, Oakville, Canada), 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonylfluoride, and 10 mM ZnCl2] for 2 h at 4 C with agitation, and then for an additional 3 h in the presence of 20 µl protein G-Sepharose beads (Amersham Pharmacia Biotech). Bound immunocomplexes were washed four times with binding buffer, resuspended in 30 µl 1x Laemmli buffer, and subjected to SDS-PAGE. Proteins were electrotransferred to Hybond PVDF membrane and subjected to immunoblotting using a 1:1000 dilution of GATA-4 antiserum. Immune complexes were revealed with a 1:4000 dilution of biotinylated antigoat antibody using the Vectastain-ABC-Amp Western blot detection kit.
In vitro interactions were analyzed using 35S-labeled full-length C/EBPß and either full-length or deleted/mutated GATA-4 proteins. All proteins were obtained using the TnT in vitro transcription/translation kit (Promega Corp., Madison, WI). Proteins were incubated in 500 µl binding buffer supplemented with 0.25% BSA and 1 µl C/EBPß antiserum for 2 h at 4 C with agitation, and then for an additional 3 h in the presence of 20 µl protein G-Sepharose beads. Bound immunocomplexes were washed three times in binding buffer supplemented with BSA and once in binding buffer without BSA, resuspended in 30 µl 1x Laemmli buffer, and subjected to SDS-PAGE. Proteins were finally electrotransferred to Hybond PVDF membrane and visualized by autoradiography.
In vitro kinase assay
In vitro translated GATA-4 protein was synthesized and purified by immunoprecipitation as described above. Immunoprecipitated GATA-4 was then subjected to an in vitro kinase reaction using commercially available PKA catalytic subunit (New England Biolabs, Inc., Mississauga, Canada) according to the manufacturer’s recommendations in the presence of 0.5 µl [
-32P]ATP for 30 min at 30 C. Reactions were terminated by adding 1x Laemmli loading buffer and heating for 5 min at 95 C. After SDS-PAGE, labeled proteins were electrotransferred to a Hybond PVDF membrane and visualized by autoradiography.
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). Interestingly, cotransfection of PKA led to a significant enhancement of the ability of GATA-4 to trans-activate the StAR promoter (an average of 
40-fold compared with 20-fold in the absence of PKA). Similar experiments performed with PKC did not result in an enhancement of GATA-4 transcriptional activity (data not shown). In vitro kinase assays confirmed that GATA-4 can be directly phosphorylated by PKA (Fig. 1B). The in vivo phosphorylation status of GATA-4 was next investigated in MA-10 cells using a phospho-(Ser/Thr) PKA substrate antibody that specifically recognizes serine or threonine residues that have been phosphorylated by PKA. As shown in Fig. 1C, endogenous GATA-4 protein is constitutively phosphorylated in unstimulated MA-10 cells (vehicle lane). After cAMP stimulation, however, phosphorylated GATA-4 protein levels were significantly increased by about 5-fold (cAMP lane). This could be specifically attributed to increased phosphorylation, as total GATA-4 protein levels remained unchanged upon hormonal stimulation (Fig. 1D). In CV-1 cells the enhancement of GATA-4 transcriptional activity is indeed PKA dependent, as this effect could be blocked by PKI, a PKA-specific inhibitor (Fig. 1E). The fact that PKI alone had no effect on GATA-4-mediated trans-activation in CV-1 cells suggests that GATA-4 is poorly phosphorylated in the absence of PKA in these cells.
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, FOG-2 repressed GATA-4-stimulated StAR promoter activity regardless of the presence of PKA, suggesting that phosphorylation of GATA-4 does not hinder its interaction with FOG-2.
Cooperation between GATA-4 and C/EBPß on the StAR promoter requires PKA
Although PKA stimulates GATA-4 transcriptional activity, it cannot be the sole determinant of hormone-induced StAR gene expression because PKA and GATA-4 are present in many tissues and cell types where StAR is not. As GATA-4 is known to contribute to cell-specific gene expression through synergistic interactions with other transcription factors (2), the potential for cooperativity among GATA-4, SF-1, and C/EBPß on the StAR promoter was studied by transfecting different combinations of the three factors in CV-1 cells with or without PKA. As shown in Fig. 2A, addition of PKA did not lead to any transcriptional enhancement between SF-1 and C/EBPß or between GATA-4 and SF-1. Surprisingly, cotransfection of GATA-4 and C/EBPß in the presence of PKA led to a synergistic activation (up to 75-fold) of the StAR promoter. This level of activation was not seen in the absence of PKA (20-fold, which corresponds to the activation by GATA-4 alone). This synergy could also be repressed by FOG-2, indicating that the presence of C/EBPß does not impair recruitment of this cofactor (Fig. 2B). Interestingly, PKA-dependent transcriptional cooperation between GATA-4 and C/EBPß on the StAR promoter was also observed with another GATA family member, GATA-6 (Fig. 3A), as well as between GATA-4 and two other C/EBP proteins, C/EBP and C/EBP
(Fig. 3B). The fact that synergy is not limited to specific members of the GATA and C/EBP family of factors suggests that this mechanism might contribute to StAR gene expression in steroidogenic tissues where GATA and C/EBP factors are coexpressed. Data from the literature have revealed the importance of the multiple regulatory elements that hinge on an intact GATA element for conveying cAMP responsiveness of the StAR promoter (21, 22). We therefore tested whether the PKA-dependent synergy in CV-1 cells between GATA-4 and C/EBPß on the StAR promoter was also dependent on an intact GATA element. As shown in Fig. 4, a point mutation that inactivates the GATA element (GATA into GGTA) completely abolished both GATA-4-mediated activation and PKA-dependent synergy with C/EBPß. These data are thus consistent with previous studies that demonstrated the requirement of a GATA regulatory element for StAR promoter activity in steroidogenic cells (21, 22).
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, GATA-4 and C/EBPß were coimmunoprecipitated from MA-10 cells, which confirms that the two proteins interact in vivo. Similar results were obtained using nuclear extracts from cAMP-treated MA-10 cells (data not shown). To determine whether this interaction was direct, in vitro coimmunoprecipitation experiments were used (Fig. 5B). A mixture of in vitro translated-35S-labeled GATA-4 and C/EBPß proteins were immunoprecipitated using a C/EBPß-specific antiserum. Under these conditions, both proteins were clearly coimmunoprecipitated (Fig. 5B, lane 4). The coimmunoprecipitation was specific, as the C/EBPß antiserum could immunoprecipitate its own protein (Fig. 5B, lane 3), but not GATA-4 or a luciferase control protein (Fig. 5B, lanes 1 and 2). Thus, the transcriptional cooperation between GATA-4 and C/EBPß on the StAR promoter is probably the result of a direct protein-protein interaction between the two factors.
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). Again, a mixture of in vitro translated, 35S-labeled GATA-4 and C/EBPß proteins was immunoprecipitated using a C/EBPß-specific antiserum. The relative sizes of the 35S-labeled GATA-4 proteins before immunoprecipitation with the C/EBPß antiserum are indicated in the left panel of Fig. 5C. In addition to the full-length GATA-4 protein (wild type; aa 1–440), C/EBPß also interacted with truncated GATA-4 proteins deleted of the entire N-terminal region up to and including the first zinc finger (N2, aa 255–440). However, C/EBPß was not able to interact with a further N-terminal deletion that removed both zinc fingers (N3, aa 302–440), indicating that the second zinc finger is sufficient for an interaction with C/EBPß. Moreover, an internal deletion (internal), which removes the second zinc finger but retains the first zinc finger, did not impair interaction with C/EBPß. Thus, the portion of the GATA-4 protein that interacts with C/EBPß is localized to either zinc finger of its DBD. Consistent with this finding, a small GATA-4 protein that consists solely of its two zinc fingers (N1C1, aa 201–332) still interacted with C/EBPß.
PKA-dependent cooperation between C/EBPß and GATA-4 requires GATA-4 activation and DBD
To map the domains of the GATA-4 protein required for PKA-dependent synergy with C/EBPß on the StAR promoter, a series of truncated GATA-4 proteins was used (Fig. 6). The GATA-4 protein contains two independent activation domains (ADs) that flank its DBD. Although GATA-4 proteins deleted of either the N-terminal (N1) or the C-terminal (C1) AD were weaker activators than the full-length GATA-4 protein (wild type), their transcriptional enhancement by PKA or cooperation with C/EBPß was unaffected. However, removal of both ADs (N1C1 and N2C1) resulted in a loss of StAR promoter activation, transcriptional enhancement by PKA, and cooperation with C/EBPß. Consistent with the requirement of the GATA-4 DBD for an interaction with C/EBPß (Fig. 5), GATA-4 proteins that were either deleted (internal, N1C2) or point mutated (N1 C294A) in the second zinc finger were unable to transcriptionally cooperate with C/EBPß in the presence of PKA. Thus, the GATA-4 DBD and at least one of its ADs are required for PKA-dependent cooperation with C/EBPß.
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Discussion |
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A unique PKA-dependent transcriptional cooperation involving GATA factors
The functional specificity of GATA proteins is achieved by synergistic or antagonistic interactions with other cell- restricted transcription factors, thus creating a unique combinatorial code of factors necessary for proper spatio- temporal gene expression. To date, all synergistic interactions involving GATA factors have been shown to elicit an immediate effect on target gene expression without the need for additional regulatory steps. An example directly related to the present work is the previously described synergy between C/EBPß and GATA-1 on the eosinophil granule major basic protein promoter, which occurs in the absence of any stimulatory signal (53). Our data, however, revealed that in the absence of stimulatory signal, synergy between C/EBPß and GATA-4 does not occur on the mouse StAR promoter. Rather, transcriptional cooperation between C/EBPß and GATA-4 on the StAR promoter requires PKA, which presumably phosphorylates GATA-4. Indeed, we have shown that GATA-4 is phosphorylated in response to cAMP stimulation in MA-10 cells. The GATA-4 domain required for the physical interaction with C/EBPß was mapped to the DNA-binding region. This is not surprising given that the zinc finger region of GATA-4 and other GATA factors has been reported to be crucial for protein-protein interactions with different transcription factors (2). Taken together, this constitutes the first example of a transcriptional cooperation involving a GATA factor that is dependent on PKA. Thus, as depicted in Fig. 7, this represents a novel and potentially important mechanism for regulating StAR gene expression in response to hormonal stimuli.
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Two independent studies have suggested that C/EBP and GATA regulatory elements are essential for maximal cAMP responsiveness of the mouse StAR promoter (21, 22). However, the molecular mechanism through which the C/EBP and GATA proteins contribute to cAMP-induced StAR transcription has not yet been defined. Although GATA-4 contributes to cAMP-induced StAR promoter activity in MA-10 cells, we and others have shown that GATA-4 protein levels are nonetheless unaffected by cAMP treatment (Fig. 1D and Ref. 22). This suggests that a posttranslational modification of GATA-4 might be involved in conveying cAMP responsiveness of the StAR promoter. Indeed, we showed that GATA-4 is phosphorylated by PKA in MA-10 cells upon cAMP stimulation, and in CV-1 cells, PKA enhances GATA-4 transcriptional activity on the StAR promoter. Interestingly, phosphorylation by the MAPKs, ERK1/2 and p38, has been recently reported to enhance GATA-4 transcriptional activity in the heart (62, 63, 64). Thus, phosphorylation appears to be an important mechanism for regulating GATA-4 activity in different tissues. Several mechanisms, none of which is exclusive, can explain the enhancement of GATA-4-mediated transcriptional activity in response to PKA. First, phosphorylation may increase GATA-4 DNA binding affinity, as previously reported in the heart (62, 63, 65, 66, 67, 68). Similarly, phosphorylation of GATA-1 has been shown to increase its DNA binding affinity in erythroid cells (69). Second, in vivo cooperation of GATA-4 with other transcription factors might be influenced by phosphorylation. This may be the case for GATA-4 and C/EBPß, because in CV-1 cells, transcriptional cooperation between the two factors was dependent on the presence of PKA. Interestingly, in MA-10 cells we could detect a GATA-4-C/EBPß interaction with or without cAMP stimulation, which at first glance would suggest that this interaction does not depend on the phosphorylation status of the individual factors. However, we cannot formally exclude the possibility that phosphorylation contributes to this interaction, because GATA-4 is constitutively phosphorylated in these cells. Third, transcription factor phosphorylation has also been shown to regulate coactivator recruitment. This has been extensively studied for the CREB transcription factor as well as for nuclear receptors (61, 70, 71). As GATA-4 has been shown to interact with p300/CBP (72), phosphorylation of GATA-4 might also regulate coactivator recruitment. Finally, phosphorylation could prevent or disrupt an interaction between GATA-4 and a corepressor. It is unlikely that this possibility involves a disruption of FOG-2 recruitment, because the PKA-dependent enhancement of GATA-4 transcriptional activity or cooperation with C/EBPß was still repressed by the FOG-2 cofactor. Interestingly, antagonism between FOG-1 and C/EBPß has been shown to regulate eosinophil lineage commitment of multipotent hemopoietic progenitors (73). It is therefore tempting to speculate that the opposing effects of FOG-2 and C/EBPß on GATA-4 transcriptional activity might contribute to Leydig cell differentiation in addition to fine-tuning the regulation of StAR promoter activity.
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Acknowledgments |
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-subunit expression plasmid), and Mario Ascoli (MA-10 cell line) for generously providing material used in this study. |
Footnotes |
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Abbreviations: aa, Amino acids; AD, activation domain; C/EBP, CCAAT/enhancer-binding protein; CRE, cAMP-responsive element; CREB, CRE-binding protein; DBD, DNA-binding domain; FOG, Friend of GATA; PKA, protein kinase A; PKI, protein kinase inhibitor; PVDF, polyvinylidene difluoride; SF-1, steroidogenic factor 1; StAR, steroidogenic acute regulatory protein.
Received April 17, 2002.
Accepted for publication June 17, 2002.
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- and ß-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression. Mol Endocrinol 6:2114–21221-adrenergic agonist-responsive transcription of the endothelin-1 gene in cardiac myocytes. J Biol Chem 275:13721–13726This article has been cited by other articles:
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Y. Jo and D. M. Stocco Regulation of Steroidogenesis and Steroidogenic Acute Regulatory Protein in R2C Cells by DAX-1 (Dosage-Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, Critical Region on the X Chromosome, Gene-1) Endocrinology, December 1, 2004; 145(12): 5629 - 5637. [Abstract] [Full Text] [PDF]
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J. K. Divine, L. J. Staloch, H. Haveri, C. M. Jacobsen, D. B. Wilson, M. Heikinheimo, and T. C. Simon GATA-4, GATA-5, and GATA-6 activate the rat liver fatty acid binding protein gene in concert with HNF-1{alpha} Am J Physiol Gastrointest Liver Physiol, November 1, 2004; 287(5): G1086 - G1099. [Abstract] [Full Text] [PDF]
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N. A. Rahman, S. Kiiveri, A. Rivero-Muller, J. Levallet, S. Vierre, J. Kero, D. B. Wilson, M. Heikinheimo, and I. Huhtaniemi Adrenocortical Tumorigenesis in Transgenic Mice Expressing the Inhibin {alpha}-Subunit Promoter/Simian Virus 40 T-Antigen Transgene: Relationship between Ectopic Expression of Luteinizing Hormone Receptor and Transcription Factor GATA-4 Mol. Endocrinol., October 1, 2004; 18(10): 2553 - 2569. [Abstract] [Full Text] [PDF]
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 R. S. Viger, H. Taniguchi, N. M. Robert, and J. J. Tremblay The 25th Volume: Role of the GATA Family of Transcription Factors in Andrology J Androl, July 1, 2004; 25(4): 441 - 452. [Full Text] [PDF]
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H. A. LaVoie, D. Singh, and Y. Y. Hui Concerted Regulation of the Porcine Steroidogenic Acute Regulatory Protein Gene Promoter Activity by Follicle-Stimulating Hormone and Insulin-Like Growth Factor I in Granulosa Cells Involves GATA-4 and CCAAT/Enhancer Binding Protein {beta} Endocrinology, July 1, 2004; 145(7): 3122 - 3134. [Abstract] [Full Text] [PDF]
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H. Hiroi, L. K. Christenson, L. Chang, M. D. Sammel, S. L. Berger, and J. F. Strauss III Temporal and Spatial Changes in Transcription Factor Binding and Histone Modifications at the Steroidogenic Acute Regulatory Protein (StAR) Locus Associated with StAR Transcription Mol. Endocrinol., April 1, 2004; 18(4): 791 - 806. [Abstract] [Full Text] [PDF]
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P. Val, C. Aigueperse, B. Ragazzon, G. Veyssiere, A.-M. Lefrancois-Martinez, and A. Martinez Adrenocorticotropin/3',5'-Cyclic AMP-Mediated Transcription of the Scavenger akr1-b7 Gene in Adrenocortical Cells Is Dependent on Three Functionally Distinct Steroidogenic Factor-1-Responsive Elements Endocrinology, February 1, 2004; 145(2): 508 - 518. [Abstract] [Full Text] [PDF]
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H. A. LaVoie The Role of GATA in Mammalian Reproduction Experimental Biology and Medicine, December 1, 2003; 228(11): 1282 - 1290. [Abstract] [Full Text] [PDF]
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P. Jimenez, K. Saner, B. Mayhew, and W. E. Rainey GATA-6 Is Expressed in the Human Adrenal and Regulates Transcription of Genes Required for Adrenal Androgen Biosynthesis Endocrinology, October 1, 2003; 144(10): 4285 - 4288. [Abstract] [Full Text] [PDF]
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J. J. Tremblay and R. S. Viger Transcription Factor GATA-4 Is Activated by Phosphorylation of Serine 261 via the cAMP/Protein Kinase A Signaling Pathway in Gonadal Cells J. Biol. Chem., June 6, 2003; 278(24): 22128 - 22135. [Abstract] [Full Text] [PDF]
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) or expression vectors for SF-1 (10 ng/well;
), GATA-4 (50 ng/well; 
) or C/EBPß (50 ng/well; 
) in the absence (-) or presence of increasing doses (50, 100, and 250 ng/well) of an expression vector encoding the PKA catalytic 
). B, Unprogrammed rabbit reticulocytes (control lane) or in vitro translated GATA-4 protein (GATA-4 lane) were purified by immunoprecipitation with a GATA-4-specific antiserum, and the immunoprecipitates were then used in an in vitro PKA phosphorylation assay in the presence of [
) or presence (
