Disruption of vitamin D receptor-retinoid X receptor heterodimer formation following ras transformation of human keratinocytes

A partial resistance to the growth inhibitory influence of 1,25-dihydroxyvitamin D-3 is apparent when immortalized keratinocytes are transformed by the ras oncogene. The vitamin D receptor (VDR) was isolated, analyzed, and found to be identical in normal, immortalized, and ras-transformed keratinocytes. Subsequently, nuclear extracts from immortalized and ras-transformed keratinocytes were analyzed in gel mobility shift assays utilizing labeled vitamin D response elements or thyroid hormone response elements. A specific protein DNA complex that was shown to contain VDR using an anti-VDR antibody was identified in both types of extracts; however, the addition of an anti-retinoid X receptor (RXR) antibody identified RXR in the complex of both normal and immortalized keratinocyte cell extracts, but not in ras-transformed keratinocytes. Furthermore, transfection of ras-transformed keratinocytes with wild-type human RXR alpha rescued VDR.RXR and thyroid hormone receptor.RXR complexes as demonstrated by a supershift in the presence of the anti-RXR antibody. Both cell lines were found to express RXR alpha message in equal amounts. Western blot analysis revealed that RXR alpha protein from ras-transformed keratinocytes was indistinguishable from that from immortalized keratinocytes and from control cells. These results suggest a causal relationship between resistance to the growth inhibitory influences of 1,25-dihydroxyvitamin D-3 and disruption of the VDR.RXR complex in malignant keratinocytes.