Role of sphingosine in the tumor necrosis factor alpha stimulatory effect on lactate dehydrogenase A expression and activity in porcine sertoli cells

In this study, the intracellular signaling mechanisms through which TNF alp ha increases LDH(AB) activity/expression in primary cultures of porcine tes ticular Sertoli cells were investigated. Studies were focused on sphingomye lin hydrolysis pathway. Treatment of [C-14]serine-labeled cells with TNF al pha (15 ng/ml, 0.8 nM) resulted in a transient decrease (similar to 20%) in cellular [C-14]sphingomyelin and in an increase (similar to 27%) in [C-14] sphingosine that remained elevated for at least 75 min. In the same experim ents, no significant changes were detected in ceramide levels. Exogenous sp hingosine stimulated LDH(A4) activity and LDHA expression in a dose-depende nt manner (ED50 = 8 muM of sphingosine). Such an increase in LDHA messenger RNA levels and LDH(A4) activity was detected at 24 h and was maximal after 48 h of treatment. Kinetically, the increase in LDH(A4) activity was simil ar whether Sertoli cells were treated with sphingosine (12 muM) or with TNF alpha (20 ng/ml). Although sphingosine mimicked the action of TNF alpha on Sertoli cells LDH(A4) activity and expression, the maximal stimulatory eff ect represented about 30% of TNF alpha maximal activity. Sphingomyelinase, C2 ceramide, sphingosine l-phosphate, N,N-dimethylsphingosine, and phosphor ylcholine had no significant effect on LDHA expression/LDH(A4) activity. Ex ogenous C2 ceramide increased LDH(A4) activity only in cytokine-treated cel ls, suggesting its involvement as sphingosine precursor in TNF alpha -stimu lated LDH(A4) activity via the sphingomyelin hydrolysis pathway. The LDH(A4 ) activity stimulated by TNF alpha was decreased by 36.2% by an inhibitor o f sphingosine formation, NH4Cl (4 mM), supporting a role of sphingosine in the TNF alpha effect. Moreover, bisindolylmaleimide (100 nM), a protein kin ase C (PKC) inhibitor decreased significantly by 28.7% the TNF alpha effect on LDH(A4) activity but had no effect on the stimulating action of sphingo sine, suggesting that if PKC is involved in TNF alpha action, the sphingosi ne effect on LDH(A4) is unrelated to the PKC activity or inhibition. Togeth er, the present data suggest that in primary Sertoli cell cultures, TNF alp ha stimulating action on LDHA expression is partly exerted via sphingomyeli n hydrolysis pathway, sphingosine being the active metabolite.