Triiodothyronine stimulates VEGF expression and secretion via steroids and HIF-1α in murine Leydig cells

Leydig cells are the principal steroidogenic cells of the testis. Leydig cells also secrete a number of growth factors including vascular endothelial growth factor (VEGF) which has been shown to regulate both testicular steroidogenesis and spermatogenesis. The thyroid hormone, T_3, is known to stimulate steroidogenesis in Leydig cells. T₃ has also been shown to stimulate VEGF production in a variety of cell lines. However, studies regarding the effect of T₃ on VEGF synthesis and secretion by the Leydig cells were lacking. Therefore, we investigated the effect of T₃ on VEGF synthesis and secretion in a mouse Leydig tumour cell line, MLTC-1. The effect of T₃ was compared with that of LH/cAMP and hypoxia, two known stimulators of Leydig cell functions. The cells were treated with T₃, 8-Br-cAMP (a cAMP analogue), or CoCl₂ (a hypoxia mimetic) and VEGF secreted in the cell supernatant was measured using ELISA. The mRNA levels of VEGF were measured by quantitative RT-PCR. In the MLTC-1 cells, T₃, 8-Br-cAMP, and CoCl₂ stimulated VEGF mRNA levels and the protein secretion. T₃ also increased steroid secretion as well as HIF-1α protein levels, two well-established upstream regulators of VEGF. Inhibitors of steroidogenesis as well as HIF-1α resulted in inhibition of T₃-stimulated VEGF secretion by the MLTC-1 cells. This suggested a mediatory role of steroids and HIF-1α protein in T₃-stimulated VEGF secretion by MLTC-1 cells. The mediation by steroids and HIF-1α were independent of each other.

8-Br-cAMP: 8-bromo - 3', 5' cyclic adenosine monophosphate; CoCl₂: cobalt chloride; HIF-1α: hypoxia inducible factor -1α; LH: luteinizing hormone; T₃: 3, 5, 3'-L-triiodothyronine; VEGF: vascular endothelial growth factor.