Triiodothyronine stimulates steroid and VEGF production in murine Leydig cells via cAMP-PKA pathway

Thyroid hormones affect testicular development as well as functions like spermatogenesis and steroidogenesis, thereby influencing male fertility. Our group earlier showed that the stimulatory role of the thyroid hormone, T₃ , on the production of vascular endothelial growth factor (VEGF) by murine Leydig cells is mediated by steroids and hypoxia-inducible factor-1 (HIF-1α). The current study further defines the signalling pathway(s) utilised by T₃ to stimulate the production of steroids, VEGF and HIF-1α in mouse Leydig tumour cell line (MLTC-1). Specific inhibitors for different signalling molecules were used to study the role of cyclic AMP (cAMP), and its downstream mediators. Expression of VEGF and HIF-1α mRNA were measured by quantitative RT-PCR; VEGF secretion by ELISA; steroid secretion by radioimmunoassay and HIF-1α protein levels by western blotting. Inhibitors of adenylate cyclase (AC), protein kinase A (PKA), sarcoma kinase (SrcK), phosphoinositide 3-kinase (PI3K) and MAP kinase kinase (MEK1/2) abolished the T₃ -induced increase in VEGF mRNA and protein levels. The same signalling molecules also mediated the increased production of steroids and HIF-1α protein in response to T₃ . Therefore, it was concluded that T₃ stimulates steroid secretion and HIF-1α protein in MLTC-1 cells through the AC-cAMP-PKA-PI3K-MEK pathway, which in turn stimulate VEGF production.