Ubiquitin/proteasome pathway regulates levels of retinoic acid receptor gamma and retinoid X receptor alpha in human keratinocytes

Repeated exposure of human skin to solar UV radiation leads to premature ag ing (photoaging) and skin cancer. UV-induced skin damage can be ameliorated by all-trans retinoic acid treatment. The actions of retinoic acid in skin keratinocytes are mediated primarily by nuclear retinoic acid receptor gam ma (RAR gamma) and retinoid X receptor alpha (RXR alpha). We found that exp osure of cultured primary human keratinocytes to UV irradiation (30 mJ/cm(2 )) substantially reduced (50-90%) RAR gamma and RXR alpha mRNA and protein within 8 h. The rates of disappearance of RAR gamma and RXR alpha proteins after UV exposure or treatment with the protein synthesis inhibitor cyclohe ximide were similar. UV irradiation did not increase the rate of breakdown of RAR gamma or RXR alpha but rather reduced their rate of synthesis. The a ddition of proteasome inhibitors MG132 and LLvL, but not the lysosomal inhi bitor E64, prevented loss of RAR gamma and RXR alpha proteins after exposur e of keratinocytes to either UV radiation or cycloheximide, Soluble extract s from nonirradiated or UV-irradiated keratinocytes possessed similar level s of proteasome activity that degraded RAR gamma and RXR alpha proteins in vitro. Furthermore, RAR gamma and RXR alpha were polyubiquitinated in intac t cells. RXR alpha was found to contain two proline, glutamate/aspartate, s erine, and threonine (PEST) motifs, which confer rapid turnover of many sho rt-lived regulatory proteins that are degraded by the ubiquitin/proteasome pathway. However, the PEST motifs in RXR alpha did not function to regulate its stability, because deletion of the PEST motifs individually or togethe r did not alter ubiquitination or proteasome-mediated degradation of RXR al pha. These results demonstrate that loss of RAR gamma and RXR alpha protein s after UV irradiation results from degradation via the ubiquitin/proteasom e pathway. Taken together, the data here indicate that ubiquitin/proteasome -mediated breakdown is an important mechanism regulating the levels of nucl ear retinoid receptors.