1-ALPHA,25-DIHYDROXYVITAMIN D-3 INDUCES SPHINGOMYELIN HYDROLYSIS IN HACAT CELLS VIA TUMOR-NECROSIS-FACTOR-ALPHA

Treatment of the human keratinocyte cell Line HaCaT with 1 alpha,25-di hydroxyvitamin D-3 (1,25-(OH)(2)D-3) resulted in the hydrolysis of sph ingomyelin with peak elevations of ceramide levels after 2-3 h (Geilen , C. C., Bektas, M., Wieder, Th., and Orfanos, C. E. (1996) FEBS Lett. 378; 88-92). In the present paper, the mechanism underlying the effec t of 1,25-(OH)(2)D-3 on sphingomyelin hydrolysis was investigated. Usi ng the cell culture supernatant of HaCaT cells treated with 1,25-(OH)( 2)D-3 for 2 h, it was possible to induce sphingomyelin hydrolysis as e arly as 30-60 min after addition to resting cells. Several lines of ex perimental evidence indicated that tumor necrosis factor alpha (TNF al pha) mediates sphingomyelin hydrolysis after 1,25-(OH)(2)D-3 treatment : (i) 1,25-(OH)(2)D-3 stimulated TNF alpha mRNA expression after 1 h, (ii) newly synthesized TNF alpha occurred 2 h after 1,25-(OH)(2)D-3 tr eatment, (iii) indirect activation of sphingomyelin hydrolysis by the supernatant of 1,25-(OH)(2)D-3-titrate HaCaT cells was abolished by pr eincubation of the supernatant with antibodies directed against TNF al pha, and (iv) preincubation of HaCaT cells with neutralizing antibodie s directed against the 55-kDa receptor of TNF alpha blocked the abilit y of 1,25-(OH)(2)D-3 to induce sphingomyelin hydrolysis in HaCaT cells . These data demonstrate that 1,25-(OH)(2)D-3 activated sphingomyelin hydrolysis by an autocrine mechanism via TNF alpha expression. Further more, this indirect mode of action may serve as an explanation for the delayed induction of sphingomyelin hydrolysis by vitamin D-3.