Mechanism of action of superactive vitamin D analogs through regulated receptor degradation

We and others have previously shown that selected vitamin D analogs potenti ate the vitamin D receptor (VDR) mediated transcription much more efficient ly than the natural hormone itself. Here we show that the transcriptionally active 20-epi analogs, namely KH 1060 and MC 1288, protect VDR against deg radation more efficiently than calcitriol at 10(-10) M concentration (VDR t (1/2) > 48 h, 17 h, and 10 h, respectively). The conformationally epi-like analog EB 1089 did not significantly alter the half-life of VDR (10.3 h), b ur retained the VDR levels over longer periods of time than calcitriol. The transcriptionally weak analog CS 1558, on the other hand, enhanced VDR deg radation even more than what was observed with the unliganded receptor (t(1 /2) 4.5 h and 5 h, respectively). Inhibition of proteasome activity by the inhibitor MG-132 resulted in a marked increase in the VDR levels in cells t reated with the vehicle or CS 1558 (2.5-fold and 2.7-fold, respectively), m ore than twice the levels observed in the presence of calcitriol or EB 1089 (1.2-fold and 1.1-fold, respectively). MG-132 treatment did not increase t he VDR levels in cei Is treated with KH 1060 or MC 1288. The electrophoreti c mobility shift assay (EMSA) with nuclear extracts from MG-132-treated cel ls revealed formation of a high-molecular-weight RXR beta-VDR-VDRE complex, which also contained Sug1. In the presence of calcitriol. 34% of total VDR in its DNA binding state was present in this complex. The 20-epi analogs e ffectively prevented the formation of this complex, since, in this case, on ly 16% of total VDR was found in this complex. These results suggest that K H 1060 and MC 1288 induce a VDR conformation, which prevents binding of pro teins mediating receptor degradation. As a result, the regulation of VDR de gradation differs from that found with the calcitriol-VDR complex resulting in superactive transcriptional action of the analogs. (C) 2000 Wiley-Liss, Inc.