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

Abstract

A partial resistance to the growth inhibitory influence of 1,25- dihydroxyvitamin D3 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 RXRα 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 RXRα message in equal amounts. Western blot analysis revealed that RXRα 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(s) and disruption of the VDR·RXR complex in malignant keratinocytes.