Francis Bergeron

The nuclear receptors SF1 and LRH1 are expressed in endometrial cancer cells and regulate steroidogenic gene transcription by cooperating with AP-1 factors

Excessive exposure to estradiol represents the main risk factor for endometrial cancer. The abnormally high estradiol levels in the endometrium of women with endometrial cancer are most likely due to overproduction by the tumour itself. Endometrial cancer cells express the genes encoding the steroidogenic enzymes involved in estradiol synthesis. Here we used RT-PCR and Western blot to show that the nuclear receptors SF1 and LRH1, two well-known regulators of steroidogenic gene expression in gonadal and adrenal cells, are also expressed in endometrial cancer cell lines. By transient transfections, we found that SF1 and LRH1, but not the related nuclear receptor NUR77, can activate the promoters of three human steroidogenic genes: STAR, HSD3B2, and CYP19A1 PII. Similarly, forskolin but not PMA, could activate all three promoters. In addition, we found that both SF1 and LRH1 can transcriptionally cooperate with the AP-1 family members c-JUN and c-FOS, known to be associated with enhanced proliferation of endometrial carcinoma cells, to further enhance activation of the STAR, HSD3B2, and CYP19A1 PII promoters. All together, our data provide novel insights into the mechanisms of steroidogenic gene expression in endometrial cancer cells and thus in the regulation of estradiol biosynthesis by tumour cells.

A cell-autonomous molecular cascade initiated by AMP-activated protein kinase represses steroidogenesis.

ABSTRACT Steroid hormones regulate essential physiological processes, and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by luteinizing hormone (LH) via its receptor leading to increased cyclic AMP (cAMP) production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Steroidogenesis then passively decreases with the degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP-to-AMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis, including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutively steroidogenic R2C cells. We have also determined that maximum AMPK activation following stimulation of steroidogenesis in MA-10 Leydig cells occurs when steroid hormone production has reached a plateau. Our data identify AMPK as a molecular rheostat that actively represses steroid hormone biosynthesis to preserve cellular energy homeostasis and prevent excess steroid production.

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.

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.

Novel Targets for the Transcription Factors MEF2 in MA-10 Leydig Cells

ABSTRACT Testosterone production by Leydig cells is a tightly regulated process requiring synchronized expression of several steroidogenic genes by numerous transcription factors. Myocyte enhancer factor 2 (MEF2) are transcription factors recently identified in somatic cells of the male gonad. In other tissues, MEF2 factors are essential regulators of organogenesis and cell differentiation. So far in the testis, MEF2 factors were found to regulate Leydig cell steroidogenesis by controlling Nr4a1 and Star gene expression. To expand our understanding of the role of MEF2 in Leydig cells, we performed microarray analyses of MEF2-depleted MA-10 Leydig cells, and the results were analyzed using Partek and Ingenuity Pathway Analysis software. Several genes were differentially expressed in MEF2-depleted Leydig cells, and 16 were validated by quantitative RT-PCR. A large number of these genes are known to be involved in fertility, gonad morphology, and steroidogenesis. These include Ahr, Bmal1, Cyp1b1, Hsd3b1, Hsd17b7, Map2k1, Nr0b2, Pde8a, Por, Smad4, Star, and Tsc22d3, which were all downregulated in the absence of MEF2. In silico analyses revealed the presence of MEF2-binding sites within the first 2 kb upstream of the transcription start site of the Por, Bmal1, and Nr0b2 promoters, suggesting direct regulation by MEF2. Using transient transfections in MA-10 Leydig cells, small interfering RNA knockdown, and a MEF2-Engrailed dominant negative, we found that MEF2 activates the Por, Bmal1, and Nr0b2 promoters and that this requires an intact MEF2 element. Our results identify novel target genes for MEF2 and define MEF2 as an important regulator of Leydig cell function and male reproduction.

GATA4 Autoregulates Its Own Expression in Mouse Gonadal Cells via Its Distal 1b Promoter

ABSTRACT Transcription factor GATA4 is required for the development and function of the mammalian gonads. We first reported that the GATA4 gene in both human and rodents is expressed as two major alternative transcripts that differ solely in their first untranslated exon (exon 1a vs. exon 1b). We had also showed by quantitative PCR that in mouse tissues, both Gata4 exon 1a- and 1b-containing transcripts are present in all sites that are normally positive for GATA4 protein. In adult tissues, exon 1a-containing transcripts generally predominate. A notable exception, however, is the testis where the Gata4 exon 1a and 1b transcripts exhibit a similar level of expression. We now confirm by in situ hybridization analysis that each transcript is also strongly expressed during gonad differentiation in both sexes in the rat. To gain further insights into how Gata4 gene expression is controlled, we characterized the mouse Gata4 promoter sequence located upstream of exon 1b. In vitro studies revealed that the Gata4 1b promoter is less active than the 1a promoter in several gonadal cell lines tested. Whereas we have previously shown that endogenous Gata4 transcription driven by the 1a promoter is dependent on a proximally located Ebox motif, we now show using complementary in vitro and in vivo approaches that Gata4 promoter 1b-directed expression is regulated by GATA4 itself. Thus, Gata4 transcription in the gonads and other tissues is ensured by distinct promoters that are regulated differentially and independently.

The Transcription Factor MEF2 Is a Novel Regulator of Gsta Gene Class in Mouse MA-10 Leydig Cells.

Testosterone is essential for spermatogenesis and the development of male sexual characteristics. However, steroidogenesis produces a significant amount of reactive oxygen species (ROS), which can disrupt testosterone production. The myocyte enhancer factor 2 (MEF2) is an important regulator of organogenesis and cell differentiation in various tissues. In the testis, MEF2 is present in Sertoli and Leydig cells throughout fetal and adult life. MEF2-deficient MA-10 Leydig cells exhibit a significant decrease in steroidogenesis concomitant with a reduction in glutathione S-transferase (GST) activity and in the expression of the 4 Gsta members (GST) that encode ROS inactivating enzymes. Here, we report a novel role for MEF2 in ROS detoxification by directly regulating Gsta expression in Leydig cells. Endogenous Gsta1-4 mRNA levels were decreased in MEF2-deficient MA-10 Leydig cells. Conversely, overexpression of MEF2 increased endogenous Gsta1 levels. MEF2 recruitment to the proximal Gsta1 promoter and direct binding on the -506-bp MEF2 element were confirmed by chromatin immunoprecipitation and DNA precipitation assays. In MA-10 Leydig cells, MEF2 activates the Gsta1 promoter and cooperates with Ca(2+)/calmodulin-dependent kinases I to further enhance Gsta1 promoter activity. These effects were lost when the -506-bp MEF2 element was mutated or when a MEF2-Engrailed dominant negative protein was used. Similar results were obtained on the Gsta2, Gsta3, and Gsta4 promoters, suggesting a global role for MEF2 factors in the regulation of all 4 Gsta genes. Altogether, our results identify a novel role for MEF2 in the expression of genes involved in ROS detoxification, a process essential for adequate testosterone production in Leydig cells.