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 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 RXRalpha 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 RXRalpha message in equal amounts. Western blot analysis revealed that RXRalpha 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 D3 and disruption of the VDR.RXR complex in malignant keratinocytes.
Solomon
C
C
Department of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada.
Sebag
M
M
White
J H
JH
Rhim
J
J
Kremer
R
R
D001483:Base Sequence; D002117:Calcitriol / Q000494:pharmacology; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D019281:Dimerization; D011905:Genes, ras; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D018167:Receptors, Calcitriol / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000378:metabolism*; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D002117:Calcitriol