Degradation of RXRs influences sensitivity of rat osteosarcoma cells to the antiproliferative effects of calcitriol.
Several cell lines, including ROS17/2.8 rat osteosarcoma (ROS) cells, contain functional VDRs and RXRs but are resistant to the antiproliferative effects of calcitriol and retinoids. We explored the role of receptor degradation in this hormone resistance. Results of transactivation assays indicated that ROS cells contain insufficient amounts of RXR to activate a DR-1 reporter, and Western blot analyses of cell extracts showed that the degradation of RXR is accelerated and produces an aberrant 45-kDa RXR. We stably expressed functional fluorescent chimeras of VDR and RXR [green fluorescent protein (GFP)-VDR; yellow fluorescent protein (YFP)-RXR] to evaluate degradation mechanisms and the impact of excess receptor expression on antiproliferative effects. Microscopy showed a diminished expression of YFP-RXR in ROS cells compared with the expression in CV-1 cells. Treatment with inhibitors of proteasomal degradation (lactacystin and MG132) selectively enhanced GFP-VDR and YFP-RXR expression and also increased the endogenous levels of VDR and RXR. Expression of GFP-VDR had no effect on the sensitivity of ROS cells to calcitriol. Increases of RXR levels by YFP-RXR expression, drug treatments, or the combination of the two, however, restored the growth-inhibitory effects of calcitriol and 9-cis-RA and restored p21 induction by calcitriol. These studies revealed that an accelerated and aberrant RXR degradation could cause resistance to the antiproliferative effects of calcitriol and retinoids in ROS cells.
Prüfer
Kirsten
K
Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Schröder
Claudia
C
Hegyi
Krisztina
K
Barsony
Julia
J
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D015153:Blotting, Western; D002117:Calcitriol / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004351:Drug Resistance; D015870:Gene Expression; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D012516:Osteosarcoma / Q000473:pathology*; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D018167:Receptors, Calcitriol / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; D012176:Retinoids / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; D012176:Retinoids; D014157:Transcription Factors; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D002117:Calcitriol