PubMed · 9867580
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E F Elst, A P Oranje, L W van Suijlekom-Smit. 1998-07-04. [Linear morphea].. https://pubmed.ncbi.nlm.nih.gov/9867580/
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We and others have previously shown that selected vitamin D analogs potentiate the vitamin D receptor (VDR) mediated transcription much more efficiently than the natural hormone itself. Here we show that the transcriptionally active 20-epi analogs, namely KH 1060 and MC 1288, protect VDR against degradation 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), but retained the VDR levels over longer periods of time than calcitriol. The transcriptionally weak analog GS 1558, on the other hand, enhanced VDR degradation 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 treated with the vehicle or GS 1558 (2.5-fold and 2.7-fold, respectively), more 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 the VDR levels in cells treated with KH 1060 or MC 1288. The electrophoretic mobility shift assay (EMSA) with nuclear extracts from MG-132-treated cells revealed formation of a high-molecular-weight RXRbeta-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 effectively prevented the formation of this complex, since, in this case, only 16% of total VDR was found in this complex. These results suggest that KH 1060 and MC 1288 induce a VDR conformation, which prevents binding of proteins mediating receptor degradation. As a result, the regulation of VDR degradation differs from that found with the calcitriol-VDR complex resulting in superactive transcriptional action of the analogs.
1alpha,25-dihydroxyvitamin D(3) (VD) is a pleiotropic nuclear hormone that also has effects on cell cycle regulation. VD and its synthetic analogues are known inhibitors of cellular growth and inducers of apoptosis, however, the primary mediator genes of these effects largely remain unknown. In order to identify novel targets for VD, that may be involved in the regulation of the cell cycle, a differential display PCR (ddPCR) approach was applied to the MCF-7 human breast cancer cell line, which provided the gene for cyclin C as an interesting candidate. Quantitative assessment of cyclin C expression showed that the gene was significantly upregulated by VD and its analogues, EB1089 and CB1093 both on the level of mRNA expression and more so on the level of protein expression in MCF-7 cells. Upregulation of cyclin C protein expression could also be confirmed in MeWo human melanoma and in U937 human promyelocytic leukemia cells. This observation adds a new gene candidate to the list of primary VD responding genes. Cyclin C is not a typical cyclin, as it apparently modulates the activity of the RNA polymerase II complex, which provides fresh insight into the mechanisms of cell cycle and general transcriptional regulation by VD and its analogues.
The influence of interferential current (IFC) on the release of four cytokines was investigated. IFC is an amplitude-modulated 4 kHz current used in therapeutic applications. Human promyelocytes (HL-60) were differentiated to monocytes/macrophages by treatment with calcitriol. Release of tumor necrosis factor alpha (TNFalpha) and interleukines 1beta, 6, and 8 (IL-1beta, IL-6, and IL-8) into the supernatant was measured after exposure to IFC at different modulation frequencies. TNFalpha release was stimulated about twofold by 4 kHz sine waves alone. The influences of exposure time (5-30 min) and current density (2.5-2500 microA/c m(2)) were tested. A maximum field effect was found at an exposure time of 15 min and a current density of 250 microA/cm(2). With these exposure conditions (15 min and 250 microA/cm(2) ), cells were treated at different modulation frequencies and reacted for TNFalpha, IL-1beta, and IL-8 release in a complex manner. Within the frequencies studied (0-125 Hz), we found stimulation as well as depression of the release. In a second run the cells were activated by pretreatment with 10 microg/ml lipopolysaccharide (LPS) and exposed in the same way as the nonactivated cells. Again the modulation frequency influenced, in a complex way, the induction of TNFalpha, IL-1beta, and IL-8, resulting in a pattern of stimulation and depression of release different from that found in nonactivated cells. For IL-6 production no significant changes were detected in activated or non-activated cells.