CHARACTERIZATION OF THE PLATELET-DERIVED GROWTH FACTOR RECEPTOR ALPHA (PDGF-Rα) PROMOTER IN MOUSE LEYDIG CELLS

Leydig cells present in the testicular interstitium are the main site of testosterone production in males. During fetal and neonatal life, a specific population of Leydig cells (fetal Leydig cells, FLC) is responsible for androgen production and masculinization of the male foetus. During postnatal life, the FLC population regresses and is substituted by a distinct population (adult Leydig cells, ALC) derived from undifferentiated stem cells. ALC provide the testosterone essential for completion of male sex differentiation and male reproductive function. Platelet-derived growth factors (PDGF) and their tyrosine kinase receptors (PDGF-R) are known to be implicated in various functions in numerous tissues including the testis. In the testis, expression of PDGF and PDGF-R is developmentally regulated and is cell-specific. The PDGF-A ligand is produced by Sertoli cells while Leydig cells express PDGF-Rα. The PDGF-A/PDGF-Rα system is essential for normal testis development and for differentiation of both populations of Leydig cells since in the absence of PDGF-A or PDGFRα, Leydig cells fail to differentiate. No information, however, is available regarding the molecular mechanisms regulating PDGF-Rα expression in Leydig cells. The aim of this study was to characterize the mouse PDGFRα promoter. We have isolated 4 kb of the PDGF-Rα promoter from mouse genomic DNA, a fragment previously shown to be sufficient for proper expression in Leydig cells in vivo. Since we found that PDGF-Rα is expressed in the TM3 Leydig cell line both at the mRNA and protein levels, we used this cell line to map the regulatory elements important for PDGF-Rα promoter activity. A series of 5′ progressive deletion constructs (−2540 bp, −859 bp, −478 bp, −203 bp, −134 bp, −113 bp, −70 bp, −52 bp) were generated and transfected in TM3 cells. Using this approach, we found that a 60 bp region located between −134 bp to −70 bp is responsible for conferring about 80 % of PDGF-Rα promoter activity in TM3 Leydig cells. Sequence analysis of this region revealed the presence of potential binding sites for several transcription factors previously reported to be present in the testis. These include Pax5, FoxJ2, Smad3, Mok2, Ik2, and members of the RUSH family. To better define the regulatory elements within the 60 bp region, we have used a linker-scanning approach to mutate by sitedirected mutagenesis the 60 bp region 3 bp at a time in the context of the - 4 kb promoter. A total of 20 site-directed mutants were thus generated and transfected in TM3 Leydig cells. We found that 2 mutants had a dramatic effect on PDGF-Rα promoter activity, decreasing it by 75–80 %. The first mutant is located at −103 bp and eliminates a putative binding site for the transcription factors Pax5 and Mok2 while the second mutant, at −87 bp, abolishes a site for members of the Fox family (FoxJ2). The implication of these transcription factors, however, remains to be established. In conclusion, we have mapped the regulatory regions essential for transcription of the PDGF-Rα gene which encodes a critical regulator of Leydig cell differentiation and function. C. Dubé is recipient of CIHR/Wyeth Pharmaceuticals postdoctoral fellowship. Supported by CHIR. (poster)

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