Sanna Ryhänen

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

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 t1/2 > 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 (t1/2 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 RXRβ‐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. J. Cell. Biochem. 76:548–558, 2000.

Inhibition of MG-63 cell cycle progression by synthetic vitamin D3 analogs mediated by p27, Cdk2, cyclin E, and the retinoblastoma protein

Progression through eukaryotic cell division cycle is regulated by synergistic activities of both positive and negative regulatory factors. The active form of vitamin D(3) (1alpha,25(OH)(2)D(3), 1,25D) and a number of its synthetic analogs have been shown to arrest cells in the G(1) phase of the cell cycle. In the present study, 1alpha,25(OH)(2)D(3) and the analogs KH1060, EB1089, and CB1093 were used to study the mechanism of the cell cycle arrest and to compare the effectiveness of these compounds in human MG-63 osteosarcoma cells. The 20-epi analogs KH1060 and CB1093, as well as the 20-normal analog EB1089, were found to be more potent than 1alpha,25(OH)(2)D(3) in inhibiting cell proliferation and arresting the MG-63 cells in the G(1) phase. These analogs were more active than 1alpha,25(OH)(2)D(3) in increasing the cyclin dependent kinase inhibitor p27 protein levels (approximately 2.3-2.5-fold compared to 1alpha,25(OH)(2)D(3)) by both increasing its formation and decreasing its degradation rate. The increased p27 formation was accompanied by stabilization of binding of nuclear proteins to the Sp1+NF-Y responsive promoter region of the p27 gene. The increase in p27 protein levels and the simultaneous decrease in cyclin E protein levels was accompanied by decreased Cdk2 kinase activity, retinoblastoma (Rb) protein hypophosphorylation and, finally, cell cycle arrest in the G(1) phase. In summary, the analogs KH1060, EB1089, and CB1093 keep Rb protein in its growth-suppressing, hypophosphorylated form and prevent cell cycle progression through the restriction point. Therefore, these synthetic vitamin D(3) analogs may be potential candidates for treating diseases, where cell cycle regulation is needed.

Inhibition of proliferation and induction of differentiation of osteoblastic cells by a novel 1,25-dihydroxyvitamin D3 analog with an extensively modified side chain (CB1093)

1,25‐Dihydroxyvitamin D3 (1,25D) is involved in the regulation of proliferation and differentiation of a variety of cell types including cancer cells. In recent years, numerous new vitamin D3 analogs have been developed in order to obtain favorable therapeutic properties. The effects of a new 20‐epi analog, CB1093 (20‐epi‐22‐ethoxy‐23‐yne‐24a,26a,27a‐trihomo‐1α,25(OH)2D3), on the proliferation and differentiation of human MG‐63 osteosarcoma cell line were compared here with those of the parent compound 1,25D. Proliferation of the MG‐63 cells was inhibited similarly by 22%, 50% and 59% after treatment with 0.1 μM 1,25D or CB1093 for 48 h, 96 h, and 144 h, respectively. In transfection experiments, the compounds were equipotent in stimulating reporter gene activity under the control of human osteocalcin gene promoter. In cell culture experiments, however, CB1093 was more potent than 1,25D at low concentrations and more effective for a longer period of time in activating the osteocalcin gene expression at mRNA and protein levels. Also, a 6‐h pretreatment and subsequent culture for up to 120 h without 1,25D or CB1093 yielded higher osteocalcin mRNA and protein levels with analog‐treated cells than with 1,25D‐treated cells. The electrophoretic mobility shift assay (EMSA) revealed stronger VDR‐VDRE binding with analog‐treated MG‐63 cells than with 1,25D‐treated cells. The differences in the DNA binding of 1,25D‐bound vs. analog‐bound VDR, however, largely disappeared when the binding reactions were performed with recombinant hVDR and hRXRβ proteins. These results demonstrate that the new analog CB1093 was equally or even more effective than 1,25D in regulating all human osteosarcoma cell functions ranging from growth inhibition to marker gene expression and that the differences in effectivity most probably resulted from interactions of the hVDR:hRXRβ‐complex with additional nuclear proteins. J. Cell. Biochem. 70:414–424, 1998.

Vitamin D3 analogs (MC 1288, KH 1060, EB 1089, GS 1558, and CB 1093): studies on their mechanism of action

Selected 20-epi and 20-normal vitamin D3 analogs were studied. First, point mutations were introduced into human vitamin D receptor (VDR) to identify residues important for ligand binding. In helices three, four and five, His229, Asp232, Ser237 and Arg274 seem to have an important role in the binding of calcitriol. Surprisingly, the 20-epi analog MC 1288 did not bind to Ser237. Second, the effects of analogs on VDR degradation were studied. The transcriptionally active 20-epi analogs protected VDR against degradation more efficiently than the 20-normal analogs and calcitriol. With proteasome inhibitor MG-132 formation of Sug-1-RXRβ-VDR-VDRE complex was detected. The 20-epi analogs effectively prevented its formation. Thus, the 20-epi analogs induce a VDR conformation, which prevents binding of factors mediating VDR degradation. Third, the analogs were found to be powerful regulators of cell cycle progression in MG-63 cells. They arrested cell cycle in the G0/G1 phase at lower concentrations and earlier time points than calcitriol. This was accompanied by hypophosphorylation of Rb followed by strong inhibition of Cdk2 activity. This correlated with increased levels of p27. Cdk2 and cyclin E levels were downregulated but those of p21 and cyclin D1 were not affected. Thus, a similar sequence of events with calcitriol and the analogs in inhibiting MG-63 cell growth was detected but the analogs had much longer lasting and stronger effects than calcitriol. A unifying scheme for the varying effects of vitamin D3 analogs is presented.

State of methylation of the human osteocalcin gene in bone‐derived and other types of cells

DNA methylation is a general mechanism of controlling tissue‐specific gene expression. Osteocalcin is a bone matrix protein whose expression is limited almost entirely to osteoblasts. We were interested in determining whether the state of methylation of the osteocalcin gene plays a role in its expression by studying human bone‐derived (MG‐63, U2‐Os, SaOs‐2) and other types (normal lymphocytes, A‐498, Hep G2) of cells. Reverse transcription–polymerase chain reaction (RT‐PCR) analysis revealed that osteocalcin mRNA production is stimulated by 1,25(OH)2D3 in MG‐63 and induced in SaOs‐2 but not in U2‐Os osteoblast‐like osteosarcoma cells. Genomic analysis of the human osteocalcin gene showed that the local surroundings of this single‐copy gene are identical in all cell lines studied. Using an isoschizomeric pair of restriction enzymes and Southern analysis, we found that the osteocalcin gene is identically methylated in all three osteosarcoma cell lines. The same sites are also methylated in human normal lymphocytes and A‐498 kidney cells, whereas the degree of methylation is higher in Hep G2 human hepatocellular carcinoma cells. Furthermore, the osteocalcin gene was identically protected against enzymatic digestion at the chromatin level in normal lymphocytes and in all cell lines studied. Induction of hypomethylation of DNA by 5‐azacytidine treatment did not cause an induction of osteocalcin synthesis in these cell lines. On the contrary, it attenuated the induction by 1,25(OH)2D3 in MG‐63 cells. In gel mobility shift assays, human vitamin D receptor and the AP‐1 transcription factor bound to an unmethylated response element oligonucleotide of the osteocalcin gene with greater affinity than to an in vitro methylated response element. These results indicate that the in vivo methylation state of the osteocalcin gene at sites determined in this study does not correlate with the inducibility of this gene. Nevertheless, the in vitro results clearly indicated that hypomethylation of critical regions of the osteocalcin gene promoter is a potential mechanism influencing effective binding of specific nuclear factors and, consequently, gene expression. J. Cell. Biochem. 66:404–412, 1997.

9‐cis retinoic acid accelerates calcitriol‐induced osteocalcin production and promotes degradation of both vitamin D receptor and retinoid X receptor in human osteoblastic cells

Abstract vitamin D receptor (VDR) and retinoid X receptor (RXR) heterodimerize to mediate the genomic actions of 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3, calcitriol), the biologically active form of vitamin D3. In this study, we show that 9‐cis retinoic acid (9‐cisRA), the ligand for RXR, accelerates calcitriol‐induced expression of osteocalcin gene, the marker for mature osteoblasts. Calcitriol and its synthetic analog KH1060 (1 nM) induced osteocalcin secretion after a 96‐h incubation period as detected by radioimmunoassay. When these compounds were used together with 9‐cisRA, osteocalcin protein secretion was, however, detected already after 72 and 48 h, respectively. Detection of osteocalcin mRNA with quantitative PCR revealed elevated mRNA levels already after a 4‐h treatment of the cells with calcitriol, KH1060, or 9‐cisRA compared with untreated cells. In combination treatments, 9‐cisRA rapidly stimulated osteocalcin mRNA synthesis induced by the different vitamin D3 compounds. In MG‐63 cells treated with calcitriol or KH1060, the stimulation was maximal after the first 4 h and diminished thereafter. In fact, after the 48‐h incubation 9‐cisRA reduced osteocalcin mRNA levels in KH1060‐treated cells, the amount of mRNA being only 44% of the levels obtained with KH1060 alone. The reduction was accompanied by an increased degradation rate of both VDR and RXRβ in the presence of 9‐cisRA. Furthermore, 9‐cisRA increased the formation of RXRβ–VDR–VDRE complex on the osteocalcin gene VDRE. These results suggest that 9‐cisRA accelerates calcitriol‐induced osteocalcin production in human osteoblastic cells through increased formation of transcriptionally active chromatin complexes and, subsequently, promotes degradation of the heterodimeric complex of VDR and RXR.