E-box and cyclic adenosine monophosphate response elements are both required for follicle-stimulating hormone-induced transferrin promoter activation in Sertoli cells

Sertoli cells are the epithelial cells responsible for the onset of pubertal development and maintenance of spermatogenesis in the adult. Transferrin is one of the major secretory products expressed by differentiated Sertoli cells. Investigation of the transcriptional control of transferrin gene expression provides insight into the regulation of Sertoli cell differentiation. Analysis of the mouse transferrin (mTf) promoter reveals the presence of a number of conserved response elements that have previously been shown to regulate cell specific expression of the human transferrin (hTf) promoter. One of these elements is the human PRII region, which is a cAMP response element (CRE)-like element that is more than 80% conserved in the mTf promoter. The activation of the hTf promoter by FSH and cAMP in rat Sertoli cells has been shown to be mediated in part through the CRE-like PRII region and binding of the CRE binding protein (CREB). The present study investigates the role of PRII in the activation of mTf promoter by FSH and cAMP in rat Sertoli cells. Mutations in the PRII of the mTf promoter reduced FSH activation by only 50% and cAMP activation by more than 90%. In contrast, the mutant PRII mTf promoter construct was fully activated by a partially purified testicular paracrine activity PModS(S300). Gel shift experiments demonstrated that proteins that can bind a consensus CRE oligonucleotide also bind the PRII region of the mTf promoter. An immunoblot confirmed that CREB binds the PRII and promotes the gel shift observed. The hypothesis developed was that another cis-acting element in addition to the CRE-like PRII is also involved in FSH actions. A conserved response element in both the mTf and hTf promoters is the basic helix-loop-helix (bHLH) responsive E-box sequence. Both FSH and PModS (S300) activity were found to promote a mTf E-box gel shift that contained the E2A gene product the bHLH protein E47. Interestingly, mutations in the E-box of the mTf promoter completely abolished the PModS(S300) activation and partially (52%) inhibited the activation by FSH. In contrast, the mutant E-box mTf promoter construct was fully activated by cAMP. Finally a double mutation of both the PRII and the E-box completely abolished FSH activation of the mTf promoter. These results suggest that optimal activation of the mouse transferrin promoter by FSH requires both CREB binding to the CRE-like PRII region and bHLH binding to the E-box. Information is provided that indicates a number of Sertoli cell promoters contain a close association of E-box and CRE-like elements. Observations are discussed in regards to the potential interactions of the CRE and E-box response elements in mediating FSH actions in Sertoli cells.