PARENT SESSION
STEROIDOGENESIS AND HORMONE ACTION IN THE TESTIS
Kent
7:30 AM-10:00 AM

(104) REGULATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR GENE EXPRESSION IN LEYDIG CELLS.

Mukhopadhyay, Amal¹, Anand, Ravinder¹, Schwarzenbach, Heidi¹, Chakrabarti, Gopa¹, Paust, Hans-Joachim¹, ¹ Institute for Hormone and Fertility Research, Hamburg, Germany

ABSTRACT- VEGF is known to be a potent regulator of vascular permeability acting on resting endothelial cells, besides having angiogenic effects during embryonic development, wound healing, tumor growth, etc. Expression of VEGF and its receptors flt-1 and flk-1(KDR) have been previously demonstrated in human Leydig and Sertoli cells. The present study has been conceived to understand the underlying mechanisms of hormonal regulation of VEGF expression by Leydig cells. Experiments have been carried out with Leydig cells purified from mouse testes and with murine tumor Leydig, MA-10, cells. VEGF production by both primary Leydig and MA-10 cells, as measured by a commercially available ELISA, was stimulated by hCG and 8Br-cAMP in a time- and concentration-dependent manner. In MA-10 cells stimulated with 1 mM 8-Br-cAMP for various duration, Northernblot analyses revealed an increased level of VEGF mRNA expression at 4 hours after the addition of 8-Br-cAMP and a maximum level of expression was achieved at 20 hours, and declined thereafter. Interestingly, an additional band at 2.2 kb could only be detected after 20 hour of stimulation with 8Br-cAMP. This RNA transcript might correspond in size to a mRNA encoding the low-abundance VEGF splice variant of 144 amino acids. H-89, a protein kinase A (PKA) inhibitor, inhibited both hCG and 8Br-cAMP-stimulated VEGF production. Phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, had no effect on VEGF production by Leydig cells, although hCG-stimulated testosterone production was inhibited by PMA. Furthermore, MEK 1/2 inhibitors, PD98059 and U0126 could inhibit both hCG and 8Br-cAMP-mediated VEGF secretion. The results obtained so far suggest that hCG/LH is able to stimulate VEGF expression in Leydig cells via a protein kinase A dependent mechanism. Protein kinase C appears not play a role. Interestingly, the MAP kinase pathway also appear to play an essential role suggesting an integrated signaling mechanism involving both PKA and MAP kinase pathways in the regulation of VEGF expression by gonadotrophin in Leydig cells.

KEY WORDS: VEGF, Leydig Cells, Gonadotrophin, MAP kinase