SOX9 and SF1 as alternative pathways in cyclic AMP-mediated anti-Müllerian hormone gene transactivation

LASALA CELINA; BEDECARRÁS PATRICIA; SCHTEINGART HELENA FDEDORA; PELLIZZARI ELIANA HERMINIA; DI CLEMENTE NATALIE; REY RODOLFO

Naantali, Finlandia

Workshop; 15th European Testis Workshop; 2008

We previously showed that in Sertoli cells FSH increases anti-Müllerian (AMH) gene expression exclusively through the pathway involving Gsa protein, adenylyl cyclase, cyclic AMP (cAMP) and protein kinase A (Lukas-Croisier et al., 2003). Since no classical cAMP response element (CRE) is present in the 3.6 kb sequences upstream of the AMH gene transcription start site, we searched for alternative pathways. We found that transcription factors NFkB and AP2, binding to specific response elements existing in the distal (> 1.9 kb) AMH promoter, are involved in cAMP-mediated response to FSH. However, site-directed mutagenesis of NFkB and AP2 sites did not completely abolish response to cAMP. Our aim was to find other transcription factors involved in cAMP-mediated FSH regulation of AMH gene expression. Our hypothesis was that AP1, GATA4, SF1 and/or SOX9, all known to be activated by cAMP and having response elements on the AMH promoter, could be alternative mediators. Materials and Methods Firefly luciferase reporter vectors using pGL2B (Promega) and 3063 bp of the human AMH promoter containing mutated sites for GATA (-74, -168 and -408), AP1 (-203), SOX9 (-141) or SF1 (-92 and -218) were transfected into an immortalized prepubertal Sertoli cell line (SMAT1). Incubations were performed in the presence or absence of 1 mM dbcAMP. pRL-TK (renilla luciferase) vector was used for transfection efficiency control. Luciferase activity was measured as an estimation of the AMH promoter activity and expressed in relative luciferase units (RLU). Results Mutations in GATA and AP1 sites did not affect AMH promoter activity in response to dbcAMP (left panel). Conversely, mutations in SOX9 and SF1 sites abolished AMH promoter response to dbcAMP (right panel). Note the different scales used in both panels for illustrative purpose. The dotted line shows the activity of the wild-type AMH promoter in basal conditions Conclusions SF1 and SOX9 binding sites are essential for AMH promoter transactivation in response to cAMP, and could be the main mediators of FSH action on AMH expression.