Insights Into the Roles of GATA Factors in Mammalian Testis Development and the Control of Fetal Testis Gene Expression.

Defining how genes get turned on and off in a correct spatiotemporal manner is integral to our understanding of the development, differentiation, and function of different cell types in both health and disease. Testis development and subsequent male sex differentiation of the XY fetus are well-orchestrated processes that require an intricate network of cell-cell communication and hormonal signals that must be properly interpreted at the genomic level. Transcription factors are at the forefront for translating these signals into a coordinated genomic response. The GATA family of transcriptional regulators were first described as essential regulators of hematopoietic cell differentiation and heart morphogenesis but are now known to impact the development and function of a multitude of tissues and cell types. The mammalian testis is no exception where GATA factors play essential roles in directing the expression of genes crucial not only for testis differentiation but also testis function in the developing male fetus and later in adulthood. This minireview provides an overview of the current state of knowledge of GATA factors in the male gonad with a particular emphasis on their mechanisms of action in the control of testis development, gene expression in the fetal testis, testicular disease, and XY sex differentiation in humans.

Identification of novel genes and pathways regulated by the orphan nuclear receptor COUP-TFII in mouse MA-10 Leydig cells†.

In males, Leydig cells are the main producers of testosterone and insulin-like 3 (INSL3), two hormones essential for sex differentiation and reproductive functions. Chicken ovalbumin upstream promoter-transcription factors I (COUP-TFI/NR2F1) and COUP-TFII (NR2F2) belong to the steroid/thyroid hormone nuclear receptor superfamily of transcription factors. In the testis, COUP-TFII is expressed and plays a role in the differentiation of cells committed to give rise to fully functional steroidogenic adult Leydig cells. Steroid production has also been shown to be diminished in COUP-TFII-depleted Leydig cells, indicating an important functional role in steroidogenesis. Until now, only a handful of target genes have been identified for COUP-TFII in Leydig cells. To provide new information into the mechanism of action of COUP-TFII in Leydig cells, we performed microarray analyses of COUP-TFII-depleted MA-10 Leydig cells. We identified 262 differentially expressed genes in COUP-TFII-depleted MA-10 cells. Many of the differentially expressed genes are known to be involved in lipid biosynthesis, lipid metabolism, male gonad development, and steroidogenesis. We validated the microarray data for a subset of the modulated genes by RT-qPCR. Downregulated genes included hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1), cytochrome P450, family 11, subfamily a, polypeptide 1 (Cyp11a1), prolactin receptor (Prlr), nuclear receptor subfamily 0, group B, member 2 (Shp/Nr0b2), ferredoxin 1 (Fdx1), scavenger receptor class B, member 1 (Scarb1), inhibin alpha (Inha), and glutathione S-transferase, alpha 3 (Gsta3). Finally, analysis of the Gsta3 and Inha gene promoters showed that at least two of the downregulated genes are potentially new direct targets for COUP-TFII. These data provide new evidence that further strengthens the important nature of COUP-TFII in steroidogenesis, androgen homeostasis, cellular defense, and differentiation in mouse Leydig cells.

Notch signaling represses GATA4-induced expression of genes involved in steroid biosynthesis.

Notch2 and Notch3 and genes of the Notch signaling network are dynamically expressed in developing follicles, where they are essential for granulosa cell proliferation and meiotic maturation. Notch receptors, ligands, and downstream effector genes are also expressed in testicular Leydig cells, predicting a potential role in regulating steroidogenesis. In this study, we sought to determine if Notch signaling in small follicles regulates the proliferation response of granulosa cells to FSH and represses the up-regulation steroidogenic gene expression that occurs in response to FSH as the follicle grows. Inhibition of Notch signaling in small preantral follicles led to the up-regulation of the expression of genes in the steroid biosynthetic pathway. Similarly, progesterone secretion by MA-10 Leydig cells was significantly inhibited by constitutively active Notch. Together, these data indicated that Notch signaling inhibits steroidogenesis. GATA4 has been shown to be a positive regulator of steroidogenic genes, including STAR protein, P450 aromatase, and 3B-hydroxysteroid dehydrogenase. We observed that Notch downstream effectors HEY1, HEY2, and HEYL are able to differentially regulate these GATA4-dependent promoters. These data are supported by the presence of HEY/HES binding sites in these promoters. These studies indicate that Notch signaling has a role in the complex regulation of the steroidogenic pathway.

GATA4 knockdown in MA-10 Leydig cells identifies multiple target genes in the steroidogenic pathway.

GATA4 is an essential transcription factor required for the initiation of genital ridge formation, for normal testicular and ovarian differentiation at the time of sex determination, and for male and female fertility in adulthood. In spite of its crucial roles, the genes and/or gene networks that are ultimately regulated by GATA4 in gonadal tissues remain to be fully understood. This is particularly true for the steroidogenic lineages such as Leydig cells of the testis where many in vitro (promoter) studies have provided good circumstantial evidence that GATA4 is a key regulator of Leydig cell gene expression and steroidogenesis, but formal proof is still lacking. We therefore performed a microarray screening analysis of MA-10 Leydig cells in which Gata4 expression was knocked down using an siRNA strategy. Analysis identified several GATA4-regulated pathways including cholesterol synthesis, cholesterol transport, and especially steroidogenesis. A decrease in GATA4 protein was associated with decreased expression of steroidogenic genes previously suspected to be GATA4 targets such as Cyp11a1 and Star. Gata4 knockdown also led to an important decrease in other novel steroidogenic targets including Srd5a1, Gsta3, Hsd3b1, and Hsd3b6, as well as genes known to participate in cholesterol metabolism such as Scarb1, Ldlr, Soat1, Scap, and Cyp51. Consistent with the decreased expression of these genes, a reduction in GATA4 protein compromised the ability of MA-10 cells to produce steroids both basally and under hormone stimulation. These data therefore provide strong evidence that GATA4 is an essential transcription factor that sits atop of the Leydig cell steroidogenic program.

Functional cooperation between GATA factors and cJUN on the star promoter in MA-10 Leydig cells.

Steroid hormone biosynthesis requires the steroidogenic acute regulatory protein (STAR). STAR is part of a protein complex that transports cholesterol through the mitochondrial membrane where steroidogenesis begins. Several transcription factors participate to direct the proper spatiotemporal and hormonal regulation of the Star gene in Leydig cells. Mechanistically, this is believed to involve the functional interplay between many of these factors. Here we report a novel transcriptional cooperation between GATA factors and cJUN on the mouse Star and human STAR promoters in MA-10 Leydig cells. This cooperation was observed with different GATA members (GATA1, 4, and 6), whereas only cJUN could cooperate with GATA factors. GATA/cJUN transcriptional cooperation on the Star promoter is mediated via closely juxtaposed GATA and AP-1 binding motifs. Mutation of all functional GATA and cJUN elements abolished GATA/cJUN cooperation, which is in agreement with previous data reporting a direct interaction between GATA4 and cJUN in a heterologous system. These data add valuable new insights that further define the molecular mechanisms that govern Star transcription in steroidogenic cells of the testis.

Sertoli cell behaviors in developing testis cords and postnatal seminiferous tubules of the mouse.

Sertoli cells are the primary structural component of the fetal testis cords and postnatal seminiferous tubules. Live imaging technologies facilitate the visualization of cell morphologies and behaviors through developmental processes. A transgenic mouse line was generated using a fragment of the rat Gata4 gene to direct the expression of a dual-color fluorescent protein reporter in fetal and adult Sertoli cells. The reporter encoded a red fluorescent protein, monomeric Cherry (mCherry), fused to histone 2B and enhanced green fluorescent protein (EGFP) fused to a glycosylphosphatidylinositol sequence, with a self-cleaving 2A polypeptide separating the two fusion proteins. After translation, the red and green fluorescent proteins translocated to the nucleus and plasma membrane, respectively, of Sertoli cells. Transgene expression in testes was first detected by fluorescent microscopy around Embryonic Day 12.0. Sertoli cell division and migration were visualized during testis cord formation in organ culture. Initially, the Sertoli cells had mesenchyme-like morphologies and behaviors, but later, the cells migrated to the periphery of the testis cords to become epithelialized. In postnatal seminiferous tubules, Sertoli nuclei were evenly spaced when viewed from the external surface of tubules, and Sertoli cytoplasm and membranes were associated with germ cells basally in a rosette pattern. This mouse line was bred to previously described transgenic mouse lines expressing EGFP in Sertoli cytoplasm or a nuclear cyan fluorescent protein (Cerulean) and mCherry in plasma membranes of germ cells. This revealed the physical relationship between Sertoli and germ cells in developing testis cords and provided a novel perspective on Sertoli cell development.

The effect of human GATA4 gene mutations on the activity of target gonadal promoters.

GATA transcription factors are crucial regulators of cell-specific gene expression in many tissues including the gonads. Although clinical cases of reproductive dysfunction have yet to be formally linked to GATA gene mutations, they have begun to be reported in other systems. Heterozygous GATA4 mutations have been associated with cases of congenital heart defects. Little is known, however, about the effect of these mutations on gonadal gene transcription. Since individuals carrying these mutations do not appear to suffer from gross reproductive defects, we hypothesized that this might be due to the differential transcriptional properties of the mutant proteins on heart versus gonadal target genes. Five mutations (S52F, E215D, G295S, V266M, and E359X) were recreated in the rat GATA4 protein. Several parameters were used to analyze the transcriptional properties of the mutants: activation of known gonadal target promoters (Star, Cyp19a1, and Inha), DNA binding, and interaction with GATA4 transcriptional partners. Three mutations (S52F, G295S, and E359X) reduced GATA4 transcriptional activity on the different gonadal promoters. With the exception of the G295S mutant, which showed a significant loss of DNA-binding affinity, the decrease in activity of the other GATA4 mutants was not associated with a change in DNA binding. All GATA4 mutants retained their ability to interact and cooperate with their major gonadal partners (NR5A1 and NR5A2) thereby compensating in part for the loss in intrinsic GATA4 transcriptional activity. Thus, unlike the heart, where the GATA4 mutations have deleterious effects, our data suggest that they would have a lesser impact on gonadal gene transcription and function.

A GATA4/WT1 cooperation regulates transcription of genes required for mammalian sex determination and differentiation.

BACKGROUND: In mammals, sex determination is genetically controlled. The SRY gene, located on Y chromosome, functions as the dominant genetic switch for testis development. The SRY gene is specifically expressed in a subpopulation of somatic cells (pre-Sertoli cells) of the developing urogenital ridge for a brief period during gonadal differentiation. Despite this tight spatiotemporal expression pattern, the molecular mechanisms that regulate SRY transcription remain poorly understood. Sry expression has been shown to be markedly reduced in transgenic mice harboring a mutant GATA4 protein (a member of the GATA family of transcription factors) disrupted in its ability to interact with its transcriptional partner FOG2, suggesting that GATA4 is involved in SRY gene transcription. RESULTS: Although our results show that GATA4 directly targets the pig SRY promoter, we did not observe similar action on the mouse and human SRY promoters. In the mouse, Wilms' tumor 1 (WT1) is an important regulator of both Sry and Müllerian inhibiting substance (Amh/Mis) expression and in humans, WT1 mutations are associated with abnormalities of sex differentiation. GATA4 transcriptionally cooperated with WT1 on the mouse, pig, and human SRY promoters. Maximal GATA4/WT1 synergism was dependent on WT1 but not GATA4 binding to their consensus regulatory elements in the SRY promoter and required both the zinc finger and C-terminal regions of the GATA4 protein. Although both isoforms of WT1 synergized with GATA4, synergism was stronger with the +KTS rather than the -KTS isoform. WT1/GATA4 synergism was also observed on the AMH promoter. In contrast to SRY, WT1/GATA4 action on the mouse Amh promoter was specific for the -KTS isoform and required both WT1 and GATA4 binding. CONCLUSION: Our data therefore provide new insights into the molecular mechanisms that contribute to the tissue-specific expression of the SRY and AMH genes in both normal development and certain syndromes of abnormal sex differentiation.

Role of the GATA family of transcription factors in endocrine development, function, and disease.

The WGATAR motif is a common nucleotide sequence found in the transcriptional regulatory regions of numerous genes. In vertebrates, these motifs are bound by one of six factors (GATA1 to GATA6) that constitute the GATA family of transcriptional regulatory proteins. Although originally considered for their roles in hematopoietic cells and the heart, GATA factors are now known to be expressed in a wide variety of tissues where they act as critical regulators of cell-specific gene expression. This includes multiple endocrine organs such as the pituitary, pancreas, adrenals, and especially the gonads. Insights into the functional roles played by GATA factors in adult organ systems have been hampered by the early embryonic lethality associated with the different Gata-null mice. This is now being overcome with the generation of tissue-specific knockout models and other knockdown strategies. These approaches, together with the increasing number of human GATA-related pathologies have greatly broadened the scope of GATA-dependent genes and, importantly, have shown that GATA action is not necessarily limited to early development. This has been particularly evident in endocrine organs where GATA factors appear to contribute to the transcription of multiple hormone-encoding genes. This review provides an overview of the GATA family of transcription factors as they relate to endocrine function and disease.

LRH-1/NR5A2 cooperates with GATA factors to regulate inhibin alpha-subunit promoter activity.

Inhibin alpha is the common subunit of the dimeric inhibin proteins known for their role in suppressing pituitary FSH secretion. In this study, we have examined the role of GATA factors and the nuclear receptor, LRH-1/NR5A2, in the regulation of inhibin alpha-subunit promoter activity. The inhibin alpha promoter contains two GATA-binding motifs that can be activated by GATA4 or GATA6. The GATA-dependence of the promoter was demonstrated by downregulating GATA expression in MA-10 cells using siRNA technology. We next examined whether GATA factors could cooperate with LRH-1, a factor recently proposed to be an important regulator of inhibin alpha-subunit transcription. Both GATA4 and GATA6 strongly synergized with LRH-1. Consistent with the cAMP-dependence of the inhibin alpha-subunit promoter, GATA/LRH-1 synergism was markedly enhanced by PKA and the co-activator protein CBP. Thus, our results identify LRH-1 as a new transcriptional partner for GATA factors in the regulation of inhibin alpha-subunit gene expression.

Protein kinase A-dependent synergism between GATA factors and the nuclear receptor, liver receptor homolog-1, regulates human aromatase (CYP19) PII promoter activity in breast cancer cells.

Cancers, including that of the breast, are the result of multiple contributing factors including aberrant gene expression. Indeed, the CYP19 gene encoding P450 aromatase, the key enzyme for estrogen biosynthesis, is up-regulated in breast tumors predominantly via the cAMP-responsive gonad-type PII promoter, ultimately leading to increased intratumoral estrogen production and tumor growth. Thus, identifying the molecular factors involved in aromatase PII promoter regulation is essential for our understanding and treatment of the disease. Because we have previously shown activity of the murine aromatase PII promoter to be markedly up-regulated by GATA factors with respect to the gonads, we hypothesized that GATA factors are also key determinants of human PII promoter-driven aromatase transcription in breast tumors. We now show that GATA3 and GATA4 are indeed expressed in several breast cancer cells lines. Consistent with the cAMP dependence of the PII promoter, activation elicited by GATA3 or GATA4 alone and the striking synergism between GATA3 or GATA4 and the nuclear receptor liver receptor homolog (LRH)-1 was intimately linked to forskolin treatment or overexpression of protein kinase A (PKA) catalytic subunit. PKA-mediated phosphorylation increases the interaction between GATA3 and LRH-1 and the requirement for PKA in aromatase PII promoter stimulation involves at least three specific amino acid residues: GATA3 Ser308, GATA4 Ser261, and LRH-1 Ser469. Finally, we show that the human LRH-1 promoter is itself a target for GATA factors. Thus, taken together, our results suggest that GATA factors likely contribute to aberrant aromatase expression in breast tumors through two distinct, yet complementary mechanisms.

Characterization of a novel rat epididymal cell line to study epididymal function.

The epididymis is an androgen-dependent organ that allows spermatozoa to become fully functional as they pass through this tissue. The specialized functions of the epididymis are mediated by interactions between epididymal epithelial cells and between epididymal cells and spermatozoa. Although the critical role of the epididymis in sperm maturation is well established, the mechanisms regulating cell-cell interactions remain poorly understood because of the lack of appropriate cell line models. We now report the characterization of a novel rat caput epididymal cell line (RCE) that was immortalized by transfecting primary cultures of rat epididymal cells with the simian virus 40 large T antigen. At the electron microscope level, the cell line was composed of epithelial principal cells with characteristics of in vivo cells; principal cells had well-developed Golgi apparatus, abundant endoplasmic reticulum cisternae, and few endosomes. RCE cells expressed the mRNAs coding for the androgen receptor, estrogen receptor alpha, and 4-ene-steroid-5-alpha-reductase types 1 and 2 as well as epididymal-specific markers Crisp-1 and epididymal retinoic acid binding protein. Epididymal retinoic acid binding protein expression was significantly induced with dihydrotestosterone, although this effect was not blocked by flutamide, suggesting that RCE cells are not androgen responsive. Neighboring cells formed tight and gap junctions characteristic of epididymal cells in vivo and expressed tight (occludin and claudin-1, -3, and -4) and gap junctional proteins (connexin-26, -30.3, -32, and -43). The RCE cell line displays many characteristics of epithelial principal cells, thus providing a model for studying epididymal cell functions.

Transcription factor GATA-4 is activated by phosphorylation of serine 261 via the cAMP/protein kinase a signaling pathway in gonadal cells.

Gonadal gene expression is regulated by pituitary hormones acting through the cAMP/protein kinase A (PKA) signal transduction pathway. The downstream molecular effectors of these signals, however, have yet to be fully understood. We have recently shown that cAMP stimulation of gonadal cells leads to phosphorylation of the transcription factor GATA-4, a key regulator of gonadal gene expression, thus suggesting that this factor might be a novel target for the cAMP/PKA signaling pathway. We now show that the rapid phosphorylation of GATA-4 induced by cAMP in vivo can be blocked by a PKA-specific inhibitor but not by mitogen-activated protein kinase inhibitors, indicating that GATA-4 is predominantly phosphorylated by PKA in response to cAMP in gonadal cells. In addition, using in vitro kinase assays, we show that PKA phosphorylation of GATA-4 occurs predominantly on an evolutionarily conserved serine residue located at position 261. Phosphorylation of GATA-4 Ser261 by PKA enhances its transcriptional activity on different gonadal promoters, an effect that was markedly reduced with a S261A mutant. Moreover, the S261A mutant blunted cAMP-induced promoter activity in gonadal cells. Finally, PKA-dependent phosphorylation of GATA-4 also led to enhanced recruitment of the CREB-binding protein coactivator. This recruitment and transcriptional cooperation were dramatically impaired with the S261A mutant. Thus, our results identify GATA-4 as a novel downstream effector of cAMP/PKA signaling in gonadal cells, where phosphorylation of Ser261 and recruitment of CREB-binding protein likely represent a key mechanism for conveying the cAMP responsiveness of gonadal genes that lack classical cAMP regulatory elements.

Protein kinase A-dependent cooperation between GATA and CCAAT/enhancer-binding protein transcription factors regulates steroidogenic acute regulatory protein promoter activity.

Steroidogenic acute regulatory protein (StAR) is an essential cholesterol transporter in steroidogenic tissues. Hormone-induced StAR expression is regulated through the cAMP-dependent pathway involving activation of protein kinase A (PKA). The StAR promoter contains several conserved DNA regulatory elements. These include binding sites for steroidogenic factor 1, CCAAT/enhancer-binding protein (C/EBP), and GATA transcription factors. Although these elements are important for StAR promoter activity, how the various transcription factors that bind these elements cooperate to confer cAMP responsiveness remains poorly understood. As induction of StAR transcription by cAMP in steroidogenic MA-10 cells does not require de novo protein synthesis, this suggests that all essential transcription factors are present and that posttranslational modifications of the factors are involved. We now report that GATA-4 is phosphorylated in MA-10 cells in response to cAMP and in heterologous CV-1 cells, GATA-4 transcriptional activity is stimulated by PKA. Moreover, we show that GATA-4 and C/EBPbeta directly interact in vitro and in vivo and synergistically activate the StAR promoter in CV-1 cells exclusively in the presence of PKA. As PKA-dependent synergy was also observed with other GATA and C/EBP family members, this transcriptional cooperation may contribute to hormone-stimulated StAR expression in all steroidogenic tissues.