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

A mesenchymal-epithelial cell interaction exists in the testis between the Sertoli cells that form the seminiferous tubule and the mesenchym al-derived peritubular myoid cells that surround the tubule. Analysis of the mesenchymal-epithelial interactions between these cells reveale d the local production of a mesenchymal factor, PModS. PModS modulates the differentiated functions of Sertoli cells in vitro, including sti mulation of the iron-binding protein transferrin (Tf). Previous result s have indicated that PModS-induced Tf gene expression involves the ac tivation of immediate early genes. One of the immediate early genes wa s identified as c-fos. The importance of c-fos was demonstrated in the current study when a c-fos antisense oligonucleotide was found to inh ibit the ability of PModS to induce the expression of a Tf promoter-ch loramphenicol acetyltransferase (CAT) construct. The regulation of c-f os by PModS was investigated with various CAT constructs containing se gments of the c-fos promoter, such as the serum response element(SRE), sis-inducible element (SIE), cAMP response element (CRE), and phorbol ester/TPA response element (TRE), transfected into cultured Sertoli c ells. PModS has no effect on cAMP response element-CAT or TRE-CAT, sug gesting that PModS does not act through stimulation of cAMP and protei n kinase C pathways. PModS was found to activate the c-fos SEE-CAT con struct and the SIE-CAT construct. A construct containing both SIE and SRF, was stimulated to the same degree as either element alone. Gel mo bility shift assays using nuclear extracts from PModS-stimulated Serto li cells and a radiolabeled SRE oligonucleotide resulted in retarded m obility of a DNA-protein complex. A gel shift with a SRE oligonucleoti de containing an ETS domain resulted in a unique shift only detected i n 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 tha t PModS can activate the c-fos promoter through the SRE and SIE. PModS caused a labeled activating protein 1 (AP1) oligonucleotide to form a DNA-protein complex, indicating activation of the c-fos gene and bind ing of the c-fos/jun complex. To study the downstream regulation of Se rtoli cell differentiation, Tf gene expression was examined. CAT const ructs containing deletion mutants of a 3-kilobase (kb) mouse Tf promot er were used. When transfected into Sertoli cells the 581-base pair Tf minimal promoter had only a slight response to PModS, but was activat ed by FSH. The 2.6-kb Tf promoter construct responded to PModS. This r esponse was greater than that observed with the 1.6- or 3-kb Tf promot er constructs. These results suggest that an upstream enhancer located in the Tf promoter between -2.6 and -1.6 kb is responsive to PModS. G el retardation assays with two restriction fragments, designated SE1 a nd SE2, located at -2.4 and -1.9 kb, respectively, on the Tf promoter showed a retarded complex with PModS-stimulated Sertoli cell nuclear e xtracts. No other restriction enzyme fragment of the Tf promoter was f ound to cause a gel shift. These SE1 and SE2 domains are in a region w ith apparent enhancer activity and were found not to contain a c-fos/j un AP1-binding site. An immunoblot with c-fos antibodies of the SE1 an d SE2 gel shift indicated the absence of c-fos in the DNA-protein comp lex. Therefore, PModS-responsive cis-elements on the Tf promoter were identified (i.e. SE1 and SE2) that do not appear to involve a direct a ctivation by c-fos at AP1, 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-f os, which then influences an intermediate transcription factor(s) that regulates downstream Sertoli cell differentiated functions, such as T f expression. These studies have initiated an investigation of the tra nscriptional regulation of Sertoli cell differentiation.