TRANSCRIPTIONAL REGULATION OF SERTOLI-CELL DIFFERENTIATION BY FOLLICLE-STIMULATING-HORMONE AT THE LEVEL OF THE C-FOS AND TRANSFERRIN PROMOTERS

One of the primary endocrine hormones that influence the onset of Sert oli cell differentiation at puberty and help maintain differentiation in the adult testis is FSH. FSH can modulate the majority of Sertoli c ell differentiated functions, including stimulation of the iron-bindin g protein transferrin. Previous studies have shown that FSH alters the levels of cAMP and the immediate early gene c-fos. The current study was designed to investigate the transcriptional regulation of Sertoli cell differentiation by examining the actions of FSH on the promoter o f the immediate early gene c-fos and the promoter of the downstream di fferentiated function gene transferrin. The regulation of c-fos by FSH was investigated with various chloramphenicol acetyltransferase (CAT) constructs containing segments of the c-fos promoter, such as the ser um response element (SRE), cAMP response element (CRE), and AP1/phorbo l ester/TPA response element (TRE), that were transfected into culture d Sertoli cells. Observations indicate that FSH can stimulate all thre e response elements, as well as a whole c-fos promoter construct. Inte restingly, FSH was found to have a more dramatic effect on the SRE-CAT than a cAMP analog, suggesting a difference in the actions of the two agents. Gel mobility shift assays were performed to confirm the repor ter gene results. Nuclear extracts of FSH-stimulated Sertoli cells cau sed a labeled AP1 oligonucleotide to form a DNA/protein complex (i.e., gel shift), indicating activation of the c-fos gene and binding of th e c-fos/jun complex. Nuclear extracts from both FSH- and cAMP-stimulat ed Sertoli cells promoted similar gel shifts with SRE and CRE oligonuc leotides. This observation supports the reporter gene data in indicati ng that FSH can influence both the SRE and CRE. A gel mobility shift a ssay was also performed with an oligonucleotide containing the 5'-flan king ETS domain of the SRE (ETS-SRE) that allows the formation of a te rnary complex. FSH-stimulated Sertoli cell nuclear extracts were found to promote a unique ETS-SRE gel shift not present in cAMP-stimulated cells. The observations imply that FSH actions on the SRE are in pari distinct from the actions of cAMP. Transferrin gene expression was exa mined to study the downstream regulation of Sertoli cell differentiati on. CAT constructs containing deletion mutants of a 3-kb mouse transfe rrin promoter were used. When transfected into Sertoli cells, the 581- bp transferrin minimal promoter, previously shown to contain a CRE, ha d a significant response to cAMP and FSH. The 1.6-, 2.6-, and 3-kb tra nsferrin promoter constructs also responded to FSH and cAMP to the sam e extent as, or to a lesser extent than, the 581-bp minimal promoter. Interestingly, the actions of FSH on the 581-bp minimal transferrin pr omoter were more dramatic than those of cAMP. The importance of FSH-in duced c-fos in the regulation of transferrin expression was demonstrat ed in the current study when a c-fos antisense oligonucleotide was fou nd to partially inhibit (50%) the ability of FSH to induce the express ion of a transferrin promoter (CAT) construct. Therefore, FSH appears to act through multiple transcriptional activation pathways. The first involves cAMP and the CRE at both early-event genes (e.g., c-fos) and downstream genes (e.g., transferrin). It is likely that other pathway s involve alternate signal transduction events (e.g. , calcium mobiliz ation) and promoter response elements (e.g., SRE). These multiple path ways may act in a compensatory manner to assure the ability of FSH to influence Sertoli cell differentiation and/or in a synergistic manner to amplify FSH actions.