TNFalpha reduces the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) via the production of ceramide and activation of atypical PKC

Although tumor necrosis factor alpha (TNFalpha) decreases the expression of peroxisome proliferator-activated receptor gamma (PPARgamma), its mechanism is not understood. We evaluated the effect of ceramide, the second messenger of TNFalpha, on the expression of PPARgamma in primary cultured adipocytes. PPARgamma mRNA and aP2 mRNA levels were measured with real-time PCR. The PPARgamma protein level was measured with immunoblot. C6- and C2-ceramide, but not dihydroC6-ceramide, reduced the expression of PPARgamma in a time and concentration dependent manner. The application of 1 microM C6-ceramide for 36 h reduced PPARgamma mRNA level, aP2 mRNA level, and PPARgamma protein level to 56.3%, 80.4% and 62.1%, respectively. Since ceramide is known to activate atypical PKC, we also studied the role of atypical PKC on the PPARgamma reducing effect. Overexpression of wild type PKCzeta magnified and accelerated the effect of TNFalpha and C6-ceramide on PPARgamma mRNA levels, whereas overexpression of dominant negative PKCzeta abolished the effect. We also found that the overexpression of constitutive active PKCzeta reduced PPARgamma mRNA level, aP2 mRNA level, and PPARgamma protein level to 61.4%, 70.3% and 81.6%, respectively. Furthermore, TNFalpha activated nuclear factor-kappaB (NF-kappaB), known as a downstream effector of PKCzeta to 256.6%, which was enhanced with overexpression of wild-type PKCzeta. On the other hand, treatment with phorbol 12-myristate 13-acetate, another activator of NF-kappaB, also reduced the expression of PPARgamma to 57.8%. These results indicate that the reducing effect of TNFalpha is mediated through ceramide, atypical PKC and NF-kappaB pathway.