Classical cadherins in the testis: how are they regulated?

Cadherins (CDH) are crucial intercellular adhesion molecules, contributing to morphogenesis and creating tissue barriers by regulating cells' movement, clustering and differentiation. In the testis, classical cadherins such as CDH1, CDH2 and CDH3 are critical to gonadogenesis by promoting the migration and the subsequent clustering of primordial germ cells with somatic cells. While CDH2 is present in both Sertoli and germ cells in rodents, CDH1 is primarily detected in undifferentiated spermatogonia. As for CDH3, its expression is mainly found in germ and pre-Sertoli cells in developing gonads until the establishment of the blood-testis barrier (BTB). This barrier is made of Sertoli cells forming intercellular junctional complexes. The restructuring of the BTB allows the movement of early spermatocytes toward the apical compartment as they differentiate during a process called spermatogenesis. CDH2 is among many junctional proteins participating in this process and is regulated by several pathways. While cytokines promote the disassembly of the BTB by enhancing junctional protein endocytosis for degradation, testosterone facilitates the assembly of the BTB by increasing the recycling of endocytosed junctional proteins. Mitogen-activated protein kinases (MAPKs) are also mediators of the BTB kinetics in many chemically induced damages in the testis. In addition to regulating Sertoli cell functions, follicle stimulating hormone can also regulate the expression of CDH2. In this review, we discuss the current knowledge on regulatory mechanisms of cadherin localisation and expression in the testis.

Regulation of Cdh2 by the AP-1 family transcription factor Junb in TM4 Sertoli cells.

Cadherins are transmembrane proteins that mediate cell-to-cell adhesion and various cellular processes. In Sertoli cells of the testis, Cdh2 contributes to the development of the testis and the formation of the blood-testis barrier, being essential for germ cells' protection. Analyses of chromatin accessibility and epigenetic marks in adult mouse testis have shown that the region from -800 to +900 bp respective to Cdh2 transcription start site (TSS) is likely the active regulatory region of this gene. In addition, the JASPAR 2022 matrix has predicted an AP-1 binding element at about -600 bp. Transcription factors of the activator protein 1 (AP-1) family have been implicated in the regulation of the expression of genes encoding cell-to-cell interaction proteins such as Gja1, Nectin2 and Cdh3. To test the potential regulation of Cdh2 by members of the AP-1 family, siRNAs were transfected into TM4 Sertoli cells. The knockdown of Junb led to a decrease in Cdh2 expression. ChIP-qPCR and luciferase reporter assays with site-directed mutagenesis confirmed the recruitment of Junb to several AP-1 regulatory elements in the proximal region of the Cdh2 promoter in TM4 cells. Further investigation with luciferase reporter assays showed that other AP-1 members can also activate the Cdh2 promoter albeit to a lesser extent than Junb. Taken together, these data suggest that in TM4 Sertoli cells, Junb is responsible for the regulation of Cdh2 expression which requires its recruitment to the proximal region of the Cdh2 promoter.

Involvement of calmodulin-dependent protein kinase I in the regulation of the expression of connexin 43 in MA-10 tumor Leydig cells.

Connexin 43 (Cx43, also known as Gja1) is the most abundant testicular gap junction protein. It has a crucial role in the support of spermatogenesis by Sertoli cells in the seminiferous tubules as well as in androgen synthesis by Leydig cells. The multifunctional family of Ca2+/calmodulin-dependent protein kinases (CaMK) is composed of CaMK I, II, and IV and each can serve as a mediator of nuclear Ca2+ signals. These kinases can control gene expression by phosphorylation of key regulatory sites on transcription factors. Among these, AP-1 members cFos and cJun are interesting candidates that seem to cooperate with CaMKs to regulate Cx43 expression in Leydig cells. In this study, the Cx43 promoter region important for CaMK-dependent activation is characterized using co-transfection of plasmid reporter-constructs with different plasmids coding for CaMKs and/or AP-1 members in MA-10 Leydig cells. Here we report that the activation of Cx43 expression by cFos and cJun is increased by CaMKI. Furthermore, results from chromatin immunoprecipitation suggest that the recruitment of AP-1 family members to the proximal region of the Cx43 promoter may involve another uncharacterized AP-1 DNA regulatory element and/or protein-protein interactions with other partners. Thus, our data provide new insights into the molecular regulatory mechanisms that control mouse Cx43 transcription in testicular Leydig cells.

Basic Leucine Zipper Transcription Factors as Important Regulators of Leydig Cells' Functions.

Transcription factors members of the basic leucine zipper (bZIP) class play important roles in the regulation of genes and functions in testicular Leydig cells. Many of these factors, such as cAMP responsive element binding protein 1 (CREB1) and CCAAT enhancer binding protein beta (CEBPB), are regulated by the cAMP/protein kinase A (PKA) pathway, the main signaling pathway activated following the activation of the luteinizing hormone/choriogonadotropin membrane receptor LHCGR by the - hormone LH. Others, such as X-box binding protein 1 (XBP1) and members of the cAMP responsive element binding protein 3 (CREB3)-like superfamily, are implicated in the endoplasmic reticulum stress by regulating the unfolded protein response. In this review, the influences of bZIP transcription factors, including CREB1, CEBPB and activator protein 1 (AP-1) family members, on the regulation of genes important for cell proliferation, steroidogenesis and Leydig cell communication will be covered. In addition, unresolved questions regarding the mechanisms of actions of bZIP members in gene regulation will be identified.

Transcriptomic analysis of MA-10 tumor Leydig cells treated with adipose derived hormones adiponectin and resistin.

Obesity contributes to a decrease in testosterone production in men. Indeed, adipose tissue produces several hormones, including adiponectin and resistin, and these may influence the activity of signaling pathways responsible for regulating the expression of genes related to steroidogenesis. In this study, we wanted to identify which genes are directly regulated by these hormones using the MA-10 tumor Leydig cell model. To do this, we treated these cells with adiponectin or resistin, followed by RNA extraction and RNA-Seq transcriptome analysis. Interestingly, genes upregulated by the globular form of adiponectin (gACRP30) were associated to steroid hormones biosynthesis, whereas resistin had no effect on the transcriptome of MA-10 Leydig cells. Moreover, the expression of the Star gene, encoding the steroidogenic acute regulatory protein, was increased in response to treatments with 0.5 mM 8Br-cAMP. Such stimulation was further increased by adiponectin, resulting in increased progesterone production. However, resistin had no effect on steroid production from MA-10 tumor Leydig cells under the treatment conditions investigated. Thus, our data suggest that a direct regulation of steroidogenic genes' expressions in Leydig cells by adipose derived hormones involves cooperation between the cAMP/PKA pathway and adiponectin, but not resistin, to activate Star expression and improve progesterone synthesis.

The AP-1 family of transcription factors are important regulators of gene expression within Leydig cells.

PURPOSE: Members of the AP-1 family of transcription factors are immediate early genes being modulated by different extracellular signals. The aim of this review is to highlight the important roles of AP-1 members in transcriptional regulation of genes important for testicular Leydig cell function and male testosterone production. METHODS: A search of the relevant literature was performed in Google Scholar and NCBI Pubmed for AP-1 members and Leydig cells. Additional information was accessed from references of relevant articles. Only primary data from original peer-reviewed articles was considered for this review. RESULTS: Different signaling pathways important for Leydig cells' functions are involved in the regulation of the activity of AP-1 members. These transcription factors participate in the regulation of genes related to different biological processes important for Leydig cells. CONCLUSIONS: We conclude that members of the AP-1 family of transcription factors play critical roles in the regulation of Leydig cell proliferation, steroidogenesis, and cell-to-cell communication.

Transcriptome modulation following administration of luteolin to bleomycin-etoposide-cisplatin chemotherapy on rat LC540 tumor Leydig cells.

Leydig cell tumours represent 1%-3% of all cases of testicular tumours in men. Such tumours respond poorly to radiation or chemotherapy, including bleomycin-etoposide-cisplatin (BEP) combinatorial therapy. In this study, we investigated an alternative approach involving luteolin to improve the efficacy of chemotherapy. LC540 tumour Leydig cells were treated with BEP (bleomycin 40 µg/ml, etoposide 4 µg/ml, cisplatin 8 µg/ml) and/or luteolin 10 µM for comparison with DMSO-treated cells. We performed a transcriptome analysis using RNA-Seq to characterise changes in biological processes and signalling pathways. Treatments of LC540 tumour Leydig cells with luteolin significantly decreased the expression of genes involved in cholesterol biosynthesis, while increasing the expression of genes related to glutathione conjugation (p < .05). Genes being significantly upregulated in response to BEP treatment were involved in the response to toxic substances and transcriptional regulation. Oppositely, genes being significantly downregulated by BEP treatment were enriched for intracellular signal transduction, cell migration, cell adhesion, reproductive system development and cholesterol biosynthesis. BEP chemotherapy proved to be effective in increasing gene expression related to apoptosis of tumour Leydig cells. However, addition of luteolin to BEP treatment had no other effects on biological processes or pathways related to cancer treatment.

Gigantol Improves Cholesterol Metabolism and Progesterone Biosynthesis in MA-10 Leydig Cells.

In aging males, androgen production by testicular Leydig cells decreases at a rate of approximately 1% per year. Phenolic compounds may enhance testosterone biosynthesis and delay the onset of male hypogonadism. Gigantol is a bibenzyl compound isolated from several types of orchids of the genus Dendrobium. This compound has various biological activities, including antioxidant activity. However, its capacity to regulate gene expression and steroid production in testicular Leydig cells has never been evaluated. We investigated the effect of gigantol on MA-10 Leydig cells' gene expression using an RNA-Seq approach. To further investigate the structure-function relationship of the hydroxy-methoxyphenyl moiety of gigantol, experiments were also performed with ferulic acid and isoferulic acid. According to transcriptomic analysis, all genes coding for cholesterol biosynthesis-related enzymes are increased in response to gigantol treatment, resulting in increased lipid droplets accumulation. Moreover, treatments with 10 µM gigantol increased StAR protein levels and progesterone production from MA-10 Leydig cells. However, neither ferulic acid nor isoferulic acid influenced StAR protein synthesis and progesterone production in MA-10 Leydig cells. Thus, our findings indicate that gigantol improves cholesterol and steroid biosynthesis within testicular Leydig cells.