Figure 3

Signaling cascades that mediate the effects of TNF-α, TPA, and Cer on the PPARβ gene in keratinocytes. (A) (Overall scheme) The solid arrows represent the major TNFα-activated, FAN-mediated, pathway regulating PPARβ expression. The cascade invoked by TPA is depicted by the dashed arrows. (B) TNF-α induces PPARβ gene expression through FAN and Cer. Keratinocytes were cotransfected with expression vectors for wild-type FAN (wtFAN, second panel) or a dominant-negative form of FAN (dnFAN, third and fourth panels) and pCMV-β-galactosidase as internal control. Transfected keratinocytes were treated with TNF-α and RPA were performed at the indicated times. (fourth panel) Suppression of PPARβ expression by dnFAN was rescued by addition of exogenous ceramide at 12 h post-transfection. (first panel) Control experiment with TNF-α alone; (fifth panel) effect of ceramide in absence of TNF-α treatment. (C) Regulation of PPARβ via the stress-associated kinase pathway. Keratinocytes were transfected with a CAT reporter driven by the pPPARβ(−445) promoter region together with expression vectors encoding dominant negative (dn) or constitutively active (ca) kinases. The empty vector pCDNA 3.1 was used as a control (vector). Transfected keratinocytes were treated with an inducer (TNF-α, ceramide, or TPA) and CAT activity was measured 24 h post-treatment. SB203580 (10 μM) is a specific inhibitor of p38 and was added 1 h prior to treatment with the inducers. Values are means of six independent experiments.