THE EFFECT OF TUMOR-NECROSIS-FACTOR-ALPHA AND CAMP ON INDUCTION OF AP-1 ACTIVITY IN MA-10 TUMOR LEYDIG-CELLS

The immunostimulant tumor necrosis factor-alpha (TNF alpha), produced by monocytes/macrophages in response to inflammatory disorders, regula tes gene expression in part through induction of mitogen-activated pro tein kinases (MAPKs), including the stress-activated protein kinase (S APK) (c-jun N-terminal kinase [JNK]) and the extracellular signal-regu lated kinases (ERKs). In testicular Leydig cells, the induction of ste roidogenesis by cAMP is inhibited by TNF alpha. To examine the potenti al mechanisms governing the mutual inhibition between cAMP and TNF alp ha in Leydig cells, the intracellular signaling pathways that contribu te to AP-l-dependent gene expression were examined in the mouse MA-10 Leydig cell line. TNF alpha induced SAPK activity sixfold at 15 min, a nd the PKC inhibitor calphostin C reduced the induction of SAPK by 30% . cAMP induced SAPK activity twofold but reduced TNF alpha-induced SAP K activity, ERK activity was inhibited by both cAMP and TNF alpha, TNF alpha increased c-Jun protein, but only weakly induced FOS proteins ( c-Fos, FosB, Fra-1, and Fra-2) whereas cAMP increased the abundance of several FOS proteins (c-Fos, FosB, Fra-1, and Fra-2), with little eff ect on c-Jun levels. AP-1 binding activity, assessed using electrophor etic mobility shift assays, was increased twofold by TNF alpha and fiv efold by cAMP. Cyclic AMP alone induced AP-1-responsive reporter (p3TP LUX) activity threefold after 2 h with peak effect of 4-fold at 4 hr. AP-1 reporter was not induced by TNF alpha alone but in the presence o f cAMP, TNF alpha induced AP-1 reporter activity 12-fold. In conclusio n, TNF alpha and cAMP induce distinct components that separately contr ibute to the modulation of AP-1 activity in MA-10 cells.