Expression ofMISin the Testis Is Downregulated by Tumor Necrosis Factor Alpha through the Negative Regulation of SF-1 Transactivation by NF-κB

Abstract

The expression of Mullerian inhibiting substance (MIS),Mullerian inhibiting substance (MIS), also known as antiMullerian hormone (AMH), is essential in normal sex differentiation and reproductive function (for a review, see reference 48). Its expression is tightly regulated in tissue-specific and development-specific manners. MIS expression in the testis is restricted to Sertoli cells and is high in fetal-to-prepubertal mice, but it becomes low in pubertal-to-adult mice. Previous studies have suggested that MIS expression is regulated independently by meiotic germ cells (1, 38), androgens (39), and gonadotropins (24), although the factors that govern the complex expression of the MIS gene have not been fully understood. The MIS promoter contains a number of evolutionarily conserved elements, including those for steroidogenic factor 1 (SF-1), Sox9, and GATA-4, and these conserved elements and corresponding transcription factors have been shown to be required for the activation of MIS transcription (11, 43, 53).SF-1, an orphan nuclear receptor, plays an important role in development and differentiation of the endocrine and reproductive systems (for reviews, see references 37 and 41). Molecular studies have revealed that many genes encoding steroidogenic enzymes and regulators of endocrine function are governed by SF-1 for their expression and contain SF-1 response elements within their proximal promoters. In testicular Sertoli cells, SF-1 is required for the expression of MIS (12, 43). Diverse coregulators have been reported to modulate the effect of SF-1 on gene transcription. WT-1 and GATA-4 activate SF-1-mediated MIS expression (34, 51), whereas DAX-1 represses MIS expression (20). Other proteins, such as steroid receptor coactivator 1 (SRC-1) (7), c-Jun (26), and DP103 (35), have also been shown to modulate the transactivation activity of SF-1, although the significance of these interactions remains elusive in terms of reproductive development and function.NF-B is a pivotal transcription factor governing the expression of early response genes involved in numerous cellular responses to a wide range of signals. The major form of NF-B is a heterodimer of the p65 and p50 subunits. In the inactivated state, NF-B is sequestered in the cytoplasm through its association with the inhibitor protein IB. Activation of the NF-B signaling cascade results in phosphorylation and subsequent degradation of IB, allowing the translocation of NF-B to the nucleus, where it induces transcription by binding to specific response elements (4). Previous studies have shown that NF-B cross-talks with other proteins. The NF-B transactivation function is modulated by proteins such as C/EBP, SRC-1, glucocorticoid receptor (GR), and RXR, resulting in the regulation of promoters with B enhancer motifs (31)(32)(33)45). Furthermore, NF-B itself is able to repress the activity of steroid receptors, modulating a number of gene responses to hormonal stimuli (17,31,36).There have been reports of an inverse relationship between

Molecular Mechanism of Suppression of Testicular Steroidogenesis by Proinflammatory Cytokine Tumor Necrosis Factor Alpha

“…7A). On the other hand, the SF-1 activity repressed by p65 was recovered by TSA treatment, as previously reported (22). We confirmed the TSA insensitivity of the NF-B repression of Nur77 transactivation with a native steroidogenic-enzyme P450c17 gene promoter.…”

“…Whole-cell extracts were prepared after a 6-h treatment with TNF-␣, and immunoprecipitations were carried out using anti-Nur77 and anti-SF-1 antibodies. Western blot analysis of the immunoprecipitated material revealed that, in response to TNF-␣, the Nur77-p65 complex did not recruit HDACs, whereas the SF-1-p65 complex recruited HDAC4 but not HDAC1, as previously reported (22).…”

“…The mammalian expression vectors of p65, p50, and IB and the glutathione S-transferase (GST)-p65, GST-p50, and Gal4-p65 constructs were described previously (39). The mammalian expression vector of SF-1 and the reporter SFRE-Luc were previously described (22). Mammalian expression constructs of Nur77, Nurr-1, NOR-1, and Nur77⌬AF2 and the reporter plasmids NurRE-Luc and NBRE-tk-Luc were previously described (48,49).…”

“…TNF-␣ activates the transcription factor NF-B, its major downstream effector. Furthermore, it was previously demonstrated that the p65 subunit of NF-B interacts with SF-1 protein and inhibits SF-1 transactivation to regulate gene expression (22). Thus, we first investigated the involvement of NF-B in the TNF-␣-mediated suppression of steroidogenic enzyme gene promoters using pyrrolidine dithiocarbamate (PDTC), an antioxidant that has been widely used as an NF-B inhibitor (4).…”

Effects of Trichostatin A, a Histone Deacetylase Inhibitor, on Mouse Gonadal Development In Vitro

“…Hsp86 immunohistochemisty showed that the number of male germ cells were significantly decreased in the TSA treated gonadal explants. Testicular germ cells affect MIS expression in Sertoli cells by TNF-α [25]. However, in this study, MIS and SF-1 expressions revealed no change.…”