Transcription of platelet-derived growth factor receptor in Leydig cells involves specificity protein 1 and 3

Abstract

Platelet-derived growth factor (PDGF) A is secreted by Sertoli cells and acts on Leydig precursor cells, which express the receptor PDGFRA, triggering their differentiation into steroidogenically active Leydig cells. There is, however, no information regarding the molecular mechanisms that govern Pdgfra expression in Leydig cells. In this study, we isolated and characterized a 2.2 kb fragment of the rat Pdgfra 5'-flanking sequence in the TM3 Leydig cell line, which endogenously expresses Pdgfra. A series of 5' progressive deletions of the Pdgfra promoter was generated and transfected in TM3 cells. Using this approach, two regions (-183/-154 and -154/-105), each conferring 46% of Pdgfra promoter activity, were identified. To better define the regulatory elements, trinucleotide mutations spanning the -154/-105 region were introduced by site-directed mutagenesis in the context of the -2.2 kb Pdgfra promoter. Mutations that altered the TCCGAGGGAAAC sequence at -138 bp significantly decreased Pdgfra promoter activity in TM3 cells. Several proteins from TM3 nuclear extracts were found to bind to this G(C/A) motif in electromobility shift assay. Two of the proteins were identified as the transcription factors SP1 and SP3. Using transient transfections of TM3 Leydig cells, SP1 and SP3 were found to activate the Pdgfra promoter by threefold. The SP1/SP3-dependent activation of the Pdgfra promoter was severely blunted when the G(C/A) motif was mutated. Our study provides new insights into the regulatory mechanisms of Pdgfra transcription in Leydig cells, which includes a role for the transcription factors SP1 and SP3.