Role of Specific Response Elements of the c-fos Promoter and Involvement of Intermediate Transcription Factor(s) in the Induction of Sertoli Cell Differentiation (Transferrin Promoter Activation) by the Testicular Paracrine Factor PModS*
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A mesenchymal-epithelial cell interaction exists in the testis between the Sertoli cells that form the seminiferous tubule and the mesenchymal-derived peritubular myoid cells that surround the tubule. Analysis of the mesenchymal-epithelial interactions between these cells revealed the local production of a mesenchymal factor, PModS. PModS modulates the differentiated functions of Sertoli cells in uitro, including stimulation of the iron-binding protein transferrin (Tf). Previous results have indicated that PModS-induced Tf gene expression involves the activation of immediate early genes. One of the immediate early genes was identified as c-fos. The importance of c-fos was demonstrated in the current study when a c-fos antisense oligonucleotide was found to inhibit the ability of PModS to induce the expression of a Tf promoter-chloramphenicol acetyltransferase (CAT) construct. The regulation of c-fos by PModS was investigated with various CAT constructs containing segments of the c-fos promoter, such as the serum response element (SRE), .&-inducible element (SIE), CAMP response element (CRE), and phorbol ester/TPA response element (TRE), transfected into cultured Sertoli cells. PModS has no effect on CAMP response element-CAT or TRE-CAT, suggesting that PModS does not act through stimulation of CAMP and protein kinase C pathways. PModS was found to activate the c-fos SRE-CAT construct and the SIE-CAT construct. A construct containing both SIE and SRE was stimulated to the same degree as either element alone. Gel mobility shift assays using nuclear extracts from PModSstimulated Sertoli cells and a radiolabeled SRE oligonucleotide resulted in retarded mobility of a DNA-protein complex. A gel shift with a SRE oligonucleotide containing an ETS domain resulted in a unique shift only detected in PModS-stimulated cells. PModS also promoted a gel shift with the SIE that is adjacent to the SRE on the c-fos promoter. The data imply that PModS can activate the c-fos promoter through the SRE and SIE. PModS caused a labeled activating protein 1 (APl) oligonucleotide to form a DNA-protein complex, indicating activation of the c-fos gene and binding of the c-fosljun complex. To study the downstream regulation of Sertoli cell differentiation, Tf gene expression was examined. CAT constructs containing deletion mutants of a 3-kilobase (kb) mouse Tf promoter were used. When transfected into Sertoli cells the 581-base pair Tf minimal promoter had only a slight response to PModS, but was activated by FSH. The 2.6-kb Tf promoter construct responded to PModS. This response was greater than that observed with the 1.6- or 3-kb Tf promoter constructs. These results suggest that an upstream enhancer located in the Tf promoter between -2.6 and -1.6 kb is responsive to PModS. Gel retardation assays with two restriction fragments, designated SE1 and SE2, located at -2.4 and -1.9 kb, respectively, on the Tf promoter showed a retarded complex with PModS-stimulated Sertoli cell nuclear extracts. No other restriction enzyme fragment of the Tf promoter was found to cause a gel shift. These SE1 and SE2 domains are in a region with apparent enhancer activity and were found not to contain a c-fosljun API-binding site. An immunoblot with c-fos antibodies of the SE1 and SE2 gel shift indicated the absence of c-fos in the DNA-protein complex. Therefore, PModS-responsive cis-elements on the Tf promoter were identified (i.e. SE1 and SE2) that do not appear to involve a direct activation by c-fos at APl, but involve an intermediate c-fos-regulated transcription factor(s). The findings suggest that PModS acts through activation of the SRE and SIE to induce the immediate early gene, c-fos, which then influences an intermediate transcription factor(s) that regulates downstream Sertoli cell differentiated functions, such as Tf expression. These studies have initiated an investigation of the transcriptional regulation of Sertoli cell differentiation.