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Ontogeny and Reproduction Research Unit, Centre Hospitalier de l’Université Laval (CHUL) Research Centre and Centre for Research in Biology of 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 Centre, 2705 Laurier Boulevard, Ste-Foy, Québec, Canada G1V 4G2. E-mail: robert.viger{at}crchul.ulaval.ca.
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and genes involved in steroidogenesis like P450 aromatase (Cyp 19) and steroidogenic acute regulatory protein. GATA factors also contribute to cell-specific gonadal gene expression through cooperative interactions with other transcription factors such as the orphan nuclear receptor steroidogenic factor-1. GATA transcriptional activity is also modulated by two multitype zinc finger proteins called the Friend of GATA (FOG) proteins, which were cloned as GATA-specific cofactors. The FOG proteins (FOG-1 and FOG-2) can act as either enhancers or repressors of GATA transcriptional activity, depending on the cell and promoter context. We now report that the FOG proteins are coexpressed with GATA factors in testicular cells in which they differentially repress the promoter activities of several GATA-dependent target genes. These findings implicate the FOG proteins in the regulation of GATA-dependent gene transcription in the gonads. |
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GATA factors are also emerging as crucial regulators of gonadal function. GATA-like DNA-binding proteins are found in the gonads of several species including worms, flies, snakes, birds, rodents, pigs, and humans (15, 16, 17, 18, 19, 20, 21, 22, 23). Four GATA factors are expressed in the mammalian gonads: GATA-1 (21, 22, 24), GATA-2 (25), GATA-4 (16, 17, 21, 23), and GATA-6 (16, 17). GATA-1 is predominantly found in postnatal Sertoli cells of the testis (21, 22). In the mouse and pig, GATA-4 is abundantly expressed from the onset of gonadal development and is later found in multiple cell lineages including testicular Sertoli and Leydig cells and granulosa cells of the ovary (17, 21, 23). GATA-6 mRNA is present in granulosa cells, corpora lutea, and Sertoli cells. Interestingly, GATA-2 has been recently reported to be expressed in germ cells of the ovary during a discrete period of early fetal development (25). Thus, GATA factors are likely key regulators of gene expression in the gonads. Indeed, several GATA-dependent gonadal promoters have now been identified. These include the Müllerian inhibiting substance (MIS), inhibin , steroidogenic acute regulatory protein (StAR), and aromatase (Cyp 19) PII promoters (17, 26, 27, 28, 29). Moreover, GATA factors contribute to cell-specific gonadal gene expression through cooperative interactions with other transcription factors such as steroidogenic factor (SF)-1 on the MIS, inhibin , and aromatase promoters (28, 30, 31).
All of the vertebrate GATA proteins contain a conserved DNA-binding domain composed of two multifunctional zinc fingers. The C-terminal zinc finger is required for DNA binding to the core GATA motif (32, 33), whereas both fingers are involved in protein-protein interactions with other cell-restricted factors (1, 34). Importantly, these interactions modulate the transcriptional activities of GATA factors, thereby contributing to their functional specificities. Recently a new family of large multitype zinc finger proteins termed Friend of GATA (FOG), FOG-1 and FOG-2, were identified through their ability to interact with the N-terminal zinc fingers of the GATA factors (35, 36, 37, 38, 39). Like GATA-1, FOG-1 is highly expressed in developing hematopoietic cells. Similarly, FOG-2 is coexpressed with GATA-4 in the heart, brain, and gonads. Mouse knockout studies have revealed that FOG proteins, like their GATA counterparts, have crucial developmental functions in vivo. The lack of FOG-1 leads to a block in erythroid and megakaryocytic differentiation (40), whereas FOG-2-/- mouse embryos die because of early defects in heart morphogenesis and coronary vascular development (41, 42).
Although the FOG proteins do not appear to bind DNA alone, they can act as either enhancers or repressors of GATA transcriptional activity depending on the cell and promoter context being studied (35, 36, 37, 38, 39, 43, 44). In this regard, it has been suggested that the FOG proteins act as bridging molecules that link GATA proteins with other factors involved in either activation or repression. Although the FOG proteins have been reported to be expressed in the gonads (36, 37, 38, 39, 45), their role in modulating GATA-dependent transcription in these endocrine tissues has not yet been addressed. In the present study, we have performed an exhaustive analysis of the transcriptional properties of FOG-1 and FOG-2 on multiple GATA-dependent gonadal promoters. We show that FOG proteins are coexpressed with GATA factors and act as repressors of GATA-mediated transcription. The magnitude of repression, however, is dependent on specific GATA/FOG combinations.
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Materials and Methods |
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promoter constructs and the synthetic 2XGATA-MIS and 3X(SF-1:GATA)-MIS luciferase reporters have been described previously (28, 30). A description of expression vectors for full-length GATA-1, GATA-4, and GATA-6 have also been reported elsewhere (28). A mutated GATA-4 protein containing a glutamic acid to lysine substitution at amino acid 215 (GATA-4 E215K) was obtained by site-directed mutagenesis using the QuikChange XL mutagenesis kit (Stratagene, La Jolla, CA) and the following pair of oligonucleotide primers (in which the mutation is italicized) on the wild-type GATA-4 cDNA: forward, 5'-CTTCTCAGAAGGCAGAAAGTGTGTCAACTGCGG-3', and reverse, 5'-CCGCAGTTGACACACTTTCTGCCTTCTGAGAAG-3'. Expression plasmids for SF-1, FOG-1, and FOG-2 were kindly provided by Drs. Keith Parker (University of Texas Southwestern Medical Center, Dallas, TX), Stuart Orkin (Harvard Medical School, Boston, MA), and Eric Olson (University of Texas Southwestern Medical Center), respectively.
Cell culture and transfections
African green monkey kidney CV-1 cells were grown in DMEM supplemented with 10% newborn calf serum. The MA-10 mouse Leydig tumor cell line was generously provided by Dr. Mario Ascoli (University of Iowa, Iowa City, IA) (46). MA-10 cells were grown in Waymouth’s media containing 15% horse serum at 37 C under 5% CO2. MSC-1 Sertoli and 293 embryonal kidney cells were maintained in DMEM containing 10% fetal bovine serum. The TM3 Leydig cell line was cultured in a 1:1 mixture of Ham’s F12 and DMEM containing 5% horse serum and 2.5% fetal bovine serum. Transfections of CV-1 and MSC-1 cells were done in 24-well plates using the calcium phosphate precipitation method as described previously (28). Data reported represent the average of at least three experiments, each done in duplicate.
EMSA and Western blot
EMSAs were done using 0.5 µl in vitro translated (TnT kit, Promega Corp., Madison, WI) wild-type or mutated GATA-4 proteins that were prepared according to the manufacturer’s instructions. EMSAs were performed as described elsewhere (21), except that 100 ng dI-dC was used when working with in vitro translated proteins. For the Western blot, recombinant GATA-4 proteins were obtained by transfecting 293 cells with expression vectors encoding the respective wild-type and mutated GATA-4 proteins. Nuclear extracts were prepared 48 h following transfection by the procedure outlined by Schreiber et al. (47). EMSAs were performed using a 32P-labeled double-stranded oligonucleotide corresponding to the consensus GATA element in the proximal SF-1 promoter (sense oligo: 5'-CCCATAAAGATAGGGATATT-3', antisense oligo: 5'-AATTATCCCTATCTTTATGGG-3'). Binding reactions and electrophoresis conditions were as previously described (21). A double-stranded mutant oligonucleotide (sense oligo: 5'-CCCATAAAGGTAGGGATATT-3', antisense oligo: 5'-AATATCCCTACCTTTATGGG-3') was used to confirm the specificity of GATA binding. In the Western blot analysis, 20-µg aliquots of 293 cell nuclear extracts containing either the GATA-4 wild-type or mutated (GATA-4 E215K) proteins were separated by SDS-polyacrylamide electrophoresis and then transferred to Hybond polyvinylidene difluoride membranes (Amersham Biosciences, Baie-D’Urfé, Canada). Immunodetection of the GATA proteins was achieved using an antiserum directed against the C-terminal region of the GATA-4 protein (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and a commercially available Vectastain-ABC-Amp Western blot detection kit (Vector Laboratories, Burlingame, CA).
RNA isolation and RT-PCR
Total cellular RNA was prepared from neonate rat primary Sertoli cell cultures and the TM3, MSC-1, and MA-10 cell lines by the single-step acid guanidinium thiocyanate-phenol-chloroform method (48). Primary rat Sertoli cell cultures were prepared as previously described (31). A sample of total RNA prepared from adult (90-d-old) rat Leydig cells (ALCs) was kindly provided by Dr. Matthew Hardy (The Population Council, New York, NY). The preparation of purified ALC (>95% purity based on 3ß-hydroxysteroid dehydrogenase staining) by centrifugal elutriation and Percoll density gradient sedimentation has been well documented by Shan and Hardy (49). Different first-strand cDNAs were then synthesized from the RNA samples using AMV reverse transcriptase (Amersham Biosciences). The cDNAs were used as templates in the PCR using VENT DNA polymerase (New England Biolabs, Inc., Mississauga, Canada) and oligonucleotide primers specific for GATA-1 (forward primer: CTAAGCTTATGGATTTTCCTGGTCTAGGGG-3', reverse primer: 5'-CTGGATCCGTACCTTCAAGAACTGAGTGG-3'), GATA-4 (forward primer: 5'-CTTCTAGACAACCCAATCTCGATATG-3', reverse primer: 5'-CAGGATCCAAGTCCGAGCAGGAATTG-3'), GATA-6 (forward primer: 5'-CGGGATCCCGGAGGAAATGTACCAGAC-3', reverse primer: 5'-CGGGATCCCGGAATTCCTAGGGGAAGCGTGCAGAG-3'), FOG-1 (forward primer: 5'-AACCGGCTACAGCAGGGTGCAGG-3', reverse primer: 5'-CCCTCGAGAAGTGTCAAGGGTCCTGGTGGTG-3'), and FOG-2 (forward primer: 5'-CCCTCGAGGGTGACTGCTTTCTTTAGTAACTC-3', reverse primer: 5'-ATGTGCCTACCTGAGCAGGAACA-3').
In situ hybridization and immunohistochemistry
A 361-bp GATA-6 cDNA probe corresponding to the N-terminal portion of the coding region of the rat GATA-6 cDNA (nucleotide positions -8 to +353 relative to the ATG) was obtained by PCR (forward primer: 5'-CGGGATCCCGGAGGAAATGTACCAGAC-3', reverse primer: 5'-CGGGATCCCGGAATTCCTAGGGGAAGCGTGCAGAG-3') and cloned into pcDNA3 (Invitrogen, Carlsbad, CA). Similarly, fragments of the mouse FOG-1 (nucleotides 2068–2504) and FOG-2 (nucleotides 1802–2315) cDNAs were amplified by PCR and cloned into pcDNA3: FOG-1 (forward primer: 5'-CGGGATCCCGGCGCAGCGGGAACACCCAC-3', reverse primer: 5'-CGGAATTCCGCAGTAGTAGCGCTTGTGC-3') and FOG-2 (forward primer: 5'-CGGGATCCCGGCTGTGAGTCCTAACACTG-3', reverse primer: 5'-CCCTCGAGGGCTACATCAAGAAATCTCTG-3'). Sense and antisense digoxigenin-labeled riboprobes for GATA-6, FOG-1, and FOG-2 were subsequently obtained by linearizing the plasmids followed by in vitro transcription using T7 or Sp6 RNA polymerase (Amersham Biosciences) in the presence of digoxigenin-UTP (Roche Diagnostics, Laval, Canada). The digoxigenin-labeled riboprobes were then used in in situ hybridization experiments on paraformaldehyde-fixed, paraffin-embedded tissue sections. In brief, testis sections were initially washed in PBS, fixed in 4% paraformaldehyde in PBS, embedded in paraffin, and cut into 4-µm sections. Sections were dewaxed in xylene, rehydrated in graded alcohols (95%, 70%, and 50%) and diethylpyrocarbonate-treated water, digested by proteinase K for 15 min, refixed in 4% paraformaldehyde, treated with 0.25% acetic anhydride in 100 mM triethanolamine (pH 8.0) for 10 min, and finally washed several times in PBS. The sections were then prehybridized in hybridization solution (0.3 M NaCl; 10 mM Tris-HCl, pH7.5; 1 mM EDTA; 1x Denhardt’s; 5% dextran sulfate; 0.02% sodium dodecyl sulfate; 50% formamide; and 250 µg/ml tRNA) at 42 C and finally hybridized in 30 µl of the same solution containing 7.5 µg digoxigenin-labeled GATA-6 antisense or sense riboprobe for 12–16 h at 42 C.
On the following day, the sections were successively washed in 2x, 1x, and 0.2x sodium chloride/sodium citrate buffer and then incubated with an alkaline phosphatase-conjugated antidigoxigenin antiserum (Roche) for 2 h at room temperature. Nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine (NBT/BCIP) were used as substrates for the alkaline phosphatase reaction. Sections were counterstained with 5% neutral red and mounted in Permount (Fisher Scientific, Montréal, Quebéc, Canada). FOG-1 and FOG-2 proteins in the testis were detected by immunohistochemistry using commercially available antisera (Santa Cruz Biotechnology, Inc.). Normal goat serum was used as a negative control. Immunodetection of GATA-4 was done using a Vectastain-ABC Elite kit (Vector Laboratories) as previously described (21). Diaminobenzidine was used as substrate for the peroxidase reaction, and the sections were counterstained with hematoxylin.
Statistical analysis
Statistical analyses were done by one-way ANOVA, followed by Tukey’s honestly significant difference tests to detect differences between groups. The analyses were done with the aid of the SPSS, Inc. software package (SPSS, Inc., Chicago, IL). P < 0.05 was considered significant.
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). In the testis, GATA factors are predominantly expressed in Sertoli and Leydig cells (17, 21, 22, 23). Consistent with this, FOG-1 and FOG-2 were found to be abundantly expressed in MSC-1 Sertoli cells and both the TM3 and MA-10 Leydig cell lines. Primary Sertoli cells prepared from neonate rats contained high levels of FOG-2 and low levels of FOG-1. Purified ALCs specifically expressed GATA-4 and low levels of FOG-1 and FOG-2. The MA-10 Leydig tumor cell line contained significant levels of all the testis-expressed GATA factors as well as the two FOG cofactors. Thus, like cardiac and hematopoietic cells, FOG-1 and FOG-2 are also coexpressed with GATA factors in testicular cells.
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, A–D). Abundant GATA-6 mRNA transcripts were present in the testicular cords of the early fetal testis (Fig. 2A) and in the seminiferous tubules of the neonate and prepubertal testis (Fig. 2, B and C). No signal was observed with the sense probe (Fig. 2D). The nonradioactive technique allowed to unambiguously determine that GATA-6 is indeed expressed in Sertoli cells. In the neonate testis, the absence of GATA-6 mRNA from the cytoplasm of germ cells was particularly evident (Fig. 2B). Thus, multiple GATA factors (GATA-1, -4, and -6) are expressed in Sertoli cells of the testis. Because FOG-1 and FOG-2 are often coexpressed with GATA factors, we expected the two cofactors to be strongly expressed in Sertoli cells. Indeed, in situ hybridization and immunohistochemistry experiments revealed the presence of FOG-1 and FOG-2 mRNA and protein in Sertoli cells (Fig. 3).
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). The FOG proteins behaved as repressors on all promoters tested. The degree of repression, however, varied as a function of the FOG/GATA pair studied. Transactivation induced by GATA-1 was the most sensitive to repression by both FOG-1 and FOG-2. Significant repression could be observed starting at the weakest FOG dose, and at the strongest dose, promoter activity was potently repressed (on average, >100-fold), well below unstimulated levels. Similarly, cotransfection of equivalent amounts of either FOG-1 or FOG-2 completely repressed GATA-4-mediated promoter activity. Interestingly, FOG-1 was a weak repressor of GATA-6-mediated transactivation. With respect to GATA-6, significant repression by FOG-1 could be achieved only at the highest FOG dose and only on the MIS promoter and the 2x GATA reporter. In contrast, FOG-2 was a more efficient repressor of GATA-6-stimulated promoter activity in which repression levels were comparable with those observed on GATA-4-mediated transactivation. In all transfections, the inhibition of GATA-dependent promoter activity was not due to a generalized repression of transcription because the FOG proteins alone (gray bars in Fig. 4) had little effect on the promoters tested.
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). Similar to their ability to repress GATA-mediated transactivation, FOG-1 and FOG-2 were also efficient repressors of GATA/SF-1 synergism on three different gonadal target promoters. In all cases, the highest FOG doses not only abrogated GATA/SF-1 synergism but also repressed promoter activities well below those involving GATA alone. The FOG proteins were most potent at repressing GATA/SF-1 synergisms involving GATA-1 and GATA-4. Although FOG-2 efficiently repressed GATA-6/SF-1 synergism, once again FOG-1 was a comparatively weaker repressor especially on the aromatase promoter (Fig. 5, stippled bars in the right panel).
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. Because the mutation does not affect formation of the zinc fingers, the mutant protein bound to DNA as efficiently as the wild-type GATA-4 protein (Fig. 6A). Western blot data also confirmed that the mutant and wild-type GATA-4 proteins were expressed at similar levels (Fig. 6B). Overexpression of the mutated GATA-4 protein in CV-1 cells transactivated two highly responsive GATA-dependent reporters to the same extent as the wild-type protein. However, because the mutated protein is unable to interact with FOG proteins, its transcriptional activity, unlike the wild-type GATA-4 protein, was unaffected by overexpression of either FOG-1 or FOG-2 (compare Fig. 6C with Fig. 4
). The ability of the mutated GATA-4 protein was then tested for its ability to synergize with SF-1 on different gonadal promoters (Fig. 7). Like its wild-type counterpart (Fig. 5), the mutated GATA-4 protein strongly synergized with SF-1 (Fig. 7). However, this synergism was unaffected by overexpression of either FOG-1 or FOG-2, indicating that a GATA/FOG interaction is essential for repression to occur (Fig. 7).
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). When overexpressed in MSC-1 cells, both FOG-1 and FOG-2 significantly repressed the activities of three different gonadal promoters (Fig. 8, upper panel). This effect was dependent on the presence of intact GATA regulatory elements in the target promoters because deletion or mutation of these sites completely abrogated FOG repression (Fig. 8, lower panel). Taken together, these results indicate that in gonadal cells, the FOG proteins repress GATA-dependent target genes by interacting with GATA factors bound to their respective regulatory elements.
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) or endogenous (Fig. 8) GATA factors. Moreover, this is the first report showing that the degree of FOG repression is dependent on specific GATA/FOG combinations.
The FOG proteins differentially repress GATA activity on target gonadal promoters
In vitro studies of hematopoietic- and cardiac-specific promoters have shown that the FOG-1 and FOG-2 can function as either enhancers or repressors of GATA-mediated transcription (35, 36, 37, 38, 39, 43, 44). Our present study, however, suggests that the FOG proteins act as repressors on multiple gonadal promoters. FOG repression appears to be an evolutionarily conserved mechanism because the FOG-related U-shaped protein also represses the transcriptional activity of the GATA protein Pannier in Drosophila (52). The repressive nature of FOG-like proteins is due in part to their association with the potent transcriptional corepressor, C-terminal-binding protein-2 (35, 44, 53). Recently, a non-C-terminal-binding protein-2-dependent repression domain has also been identified in the N-terminal regions of the FOG proteins (44). Thus, FOG repression is likely an important mechanism for modulating the expression of GATA-dependent genes in the gonads of several species.
The FOG proteins interact with GATA actors via their first and sixth zinc fingers (43, 44). The corresponding contact residues in the GATA N-terminal zinc fingers are found in all vertebrate GATA proteins. As such, the FOG proteins are capable of interacting with all GATA factors. At the transcriptional level, we therefore expected the FOG proteins to behave similarly at repressing gonadal promoter activity. Surprisingly, the extent of FOG-mediated repression was dependent on specific FOG/GATA combinations. For example, at similar doses, FOG-1 was a strong repressor of GATA-1 and GATA-4 but only weakly repressed GATA-6-mediated transcription. Although all GATA factors share a similar FOG interaction domain, subtle differences in FOG/GATA interaction affinities likely account for the observed differences in FOG repression. The fact that significant repression of GATA-6-mediated transcription could be achieved only at the highest FOG-1 dose supports this notion. The apparent selectivity of FOG proteins for certain GATA family members could be an important mechanism for fine-tuning the expression of multiple GATA-dependent genes. For example, in Sertoli cells that coexpress multiple GATA factors, limiting amounts of FOG-1 may be sufficient for repressing GATA-1 and GATA-4 target genes but not those under the control of GATA-6.
A role for FOG proteins in gonad-specific gene expression
Although FOG-1 and FOG-2 transcripts can be detected in adult Leydig cells (Fig. 1), their low level of expression suggests that they serve to fine-tune the expression of GATA-4 dependent genes in these cells. In contrast, the abundance of FOG-1 and FOG-2 in Sertoli cells suggests that these two proteins likely play a key role in down-regulating GATA-dependent expression of some Sertoli cell genes. The best studied GATA target gene in Sertoli cells is MIS. MIS secretion by fetal Sertoli cells is essential for normal male sex differentiation because it induces regression of the Müllerian ducts in the developing male embryo. Several transcription factors have been proposed to be involved in the spatiotemporal expression of the MIS gene such as Sry HMG box-related gene 9 (Sox9), SF-1, and GATA-4 (54). Although the sexually dimorphic expression of MIS is largely controlled by Sox9 (55), how MIS levels are specifically down-regulated in postnatal Sertoli cells remains unclear. We have previously reported that GATA-4 regulates MIS transcription by directly binding the MIS promoter and by synergistic interaction with SF-1 (30, 31). GATA-6 may also contribute to the regulation of MIS transcription because it is also expressed in fetal and neonate Sertoli cells (Fig. 2). Because these GATA-dependent activities (both GATA-4 and GATA-6) are repressed by FOG-1 and FOG-2 (Figs. 4 and 5), FOG repression of GATA activity in postnatal Sertoli cells likely contributes to the down-regulation of MIS expression. Indeed, we have previously shown that overexpression of FOG-2 in primary Sertoli cell cultures markedly decreases MIS promoter activity (31).
Interestingly, the FOG-dependent repression of MIS transcription can be related to the repressive role played by the orphan nuclear receptor Dax-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome). In contrast to the FOG proteins, Dax-1 mediates its effect through a direct interaction with SF-1 (56, 57). The recent demonstration that FOG-2 could specifically interact with the orphan nuclear receptors chicken ovalbumin upstream promoter-transcription factor 2 and 3 (58) raised the possibility that the FOG inhibition of GATA/SF-1 synergism could be mediated directly through an interaction with another orphan nuclear receptor, SF-1. However, the fact that FOG-2 did not repress GATA/SF-1 synergism involving a GATA-4 protein (GATA-4 E215K) mutated in its ability to interact with the FOG proteins (Fig. 6) confirmed that a GATA/FOG interaction is indeed essential for repression to occur.
In summary, the FOG proteins are coexpressed with GATA transcription factors in testicular cells and are repressors of GATA-mediated transactivation and GATA/SF-1 synergism on a number of gonadal target genes. Thus, our results implicate the FOG proteins as important modulators of GATA-dependent gene transcription in multiple cell types of the testis. Given the crucial in vivo roles played by FOG-1 and FOG-2 in the hematopoietic and cardiac systems, the FOG proteins are likely to have similar functional roles in the gonads.
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Acknowledgments |
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Footnotes |
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Abbreviations: ALC, Adult Leydig cell; FOG, Friend of GATA; MIS, Müllerian inhibiting substance; NBT/BCIP, nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine; SF-1, steroidogenic factor 1; Sox9, Sry HMG box-related gene 9; StAR, steroidogenic acute regulatory protein.
Received March 7, 2002.
Accepted for publication June 25, 2002.
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J. J. Tremblay and R. S. Viger A Mutated Form of Steroidogenic Factor 1 (SF-1 G35E) That Causes Sex Reversal in Humans Fails to Synergize with Transcription Factor GATA-4 J. Biol. Chem., October 24, 2003; 278(43): 42637 - 42642. [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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 I. Ketola, J. Toppari, T. Vaskivuo, R. Herva, J. S. Tapanainen, and M. Heikinheimo Transcription Factor GATA-6, Cell Proliferation, Apoptosis, and Apoptosis-Related Proteins Bcl-2 and Bax in Human Fetal Testis J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1858 - 1865. [Abstract] [Full Text] [PDF]
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J. J. Tremblay, F. Hamel, and R. S. Viger Protein Kinase A-Dependent Cooperation between GATA and CCAAT/Enhancer-Binding Protein Transcription Factors Regulates Steroidogenic Acute Regulatory Protein Promoter Activity Endocrinology, October 1, 2002; 143(10): 3935 - 3945. [Abstract] [Full Text] [PDF]
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