Protein kinase C independent activation of inducible nitric oxide synthase by tumor necrosis factor-α in TM4 Sertoli cells

To investigate the nitric oxide (NO) production and its signalling mechanism in TM4 Sertoli cells, the cells were treated with recombinant tumor necrosis factor-α (rTNF-α), recombinant interleukin-1 α (rIL-1α), or lipopolysaccharide (LPS), either alone or in combination with recombinant interferon-γ (rIFN-γ), and NO production was measured by using the Griess method. TM4 Sertoli cells produced a small amount of NO upon treatment with rIFN-γ. The effect of rIFN-γ was drastically increased by cotreatment with rTNF-α in a dose-dependent manner. However, combination of rIL-1α or LPS with rIFN-γ did not synergize to activate cells. RIFN-γ in combination with rTNF-α showed marked increase of the expression of iNOS protein. Protein kinase C inhibitors did not inhibit the production of NO induced by rIFN-γ plus rTNF-α. These results suggest that the role of TNF-α is to provide TM4 Sertoli cells with the active cofactor for NO production and TNF-α-induced signaling for induction of NO synthesis is not dependent on protein kinase C activation.