FIG. 4.

Inhibition of RAR function by stress signals is mediated through JNK. (A) MEKK-1 activation inhibits ligand-induced RARE activation. COS-1 cells were transiently cotransfected with expression plasmids containing β-Gal, MEKK1Δ, or empty vector (−), and reporter constructs (DR5TK-luc or TK-luc control). After 36 h, cells were treated or not treated with 1 μM RA and subjected to luciferase assays. After correction for differences in transfection efficiency with β-Gal values, luciferase values were expressed as means ± standard deviations from three identical wells. (B) UV radiation activates JNK and p38 but not AKT or ERK. HeLa cells were serum starved overnight and treated (+) or not treated (−) with UV, insulin-like growth factor 1 (100 ng/ml), or PMA (500 nM). Lysates were subjected to Western blotting with antibodies to the indicated proteins. (C and D) MKK4 activation decreases RARα levels by activating JNK. HeLa cells (10⁶) were treated with medium alone or incubated with Ad expressing constitutively active mutant MKK4 (Ad-MKK4) or control empty vector (Ad-EV) (200 particles/cell). After 24 h, cells were lysed, and lysates were subjected to Western analysis of MKK4 and actin (C) or RARα and β (D) and in vitro kinase assays (C). For the kinase assays, JNK and p38 were immunoprecipitated from the lysates and subjected to immune complex kinase assays with GST-c-Jun as a JNK substrate and GST-ATF2 as a p38 substrate. (E and F) UV-induced RARα loss is blocked by inhibition of JNK but not PI3K or ERK. HeLa cells were treated (+) or not treated (−) with LY294002 (50 μM), U0126 (10 μM), or SP600125 (30 μM); subjected to UV treatment; and lysed. The lysates were subjected to Western blot analysis (RARα, RARβ, P-c-Jun, c-Jun, and poly-ADP ribose polymerase as a loading control) and MAPKAP-K2 in vitro kinase assays. For the kinase assays, lysates were immunoprecipitated to isolate MAPKAP-K2, a p38 substrate, which was subjected to in vitro kinase assays with HSP27 as a substrate.