Involvement Of MAPK Signal Transduction Pathways In The Regulatory Effects Of 1,25-dihydroxyvitamin D₃ On The Proliferation Of A Human Osteosarcoma Cell Line

The biologically active metabolite of vitamin D, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], exerts profound effects on cell proliferation, cell differentiation, and acts as modulator of immune and nervous system function, in addition to its classical effects on bone mineralization and calcium homeostasis.One mechanism by which 1,25(OH)₂D₃ transduces its signals in target cells is binding to its nuclear receptor, the vitamin D receptor (VDR). This receptor belongs to the superfamily of nuclear receptors, which includes steroid hormone receptors, thyroid hormone receptors, retinoic acid receptors (RAR), retinoid X receptors (RXR) and various orphan receptors with as yet undefined ligands. These receptors bind tospecific DNA sequences, the hormone response elements (HREs), and act as ligand-inducible transcriptional factors. VDR forms heterodimers with the promiscuous partner RXR and interacts with the specific vitamin D response element (VDRE), which consists of a hexameric direct repeat sequence separated by three base pairs (DR3 type element). Then participated with several transcription associated factors, vitamin D-VDR-RXR-VDRE modulate genes expression. This mechanism is referred to as hormone genomic actions. Another mechanism responsible for the effects of 1,25(OH)2D3 is to induce some quick effects through cross-membrane signals transducing system. These effects are always coupled with phosphorylation of some intracellular signals transducing factors, and this mechanism is referred to as hormone non-genomic actions.In order to investigate the effects of 1,25(OH)2D3 on proliferation and differentiation of a human osteosarcoma cell line(HOS-8603), and to further explore its possible molecular mechanism, we firstly assayed the regulatory effects of 1,25(OH)2D3 and its three novel analogues (EB1089, MC1288 and MC903) on proliferation of HOS-8603 cells. It was found that 1,25(OH)2D3 and its three novel analogues (EB1089, MC1288 and MC901) could inhibit the proliferation of HOS-8603 cells significantly.To explore whether the intracellular signal transduction systems are involved in regulatory effects of 1,25(OH)2D3 on the proliferation of HOS-8603 cells, we studied the inducing actions of 1,25(OH)2D3 on the activities of MAPK (ERK1/2, JNK1/2 and p38) and PKC by using the method of western blot analysis. The results showed that 1,25(OH)2D3 could quickly induce phosphorylation of JNK1/2, p38 and PKC in a dose-dependent manner. As for ERK1/2, the phosphorylation of ERK1/2 could not be induced by 1,25(OH)2D3, and in contrast, the phosphorylation of ERK1/2 induced byCBS was inhibited by 1,25(OH)2D3. (Phosphorylated ERK1/2, JNK1/2, p38 and PKC are their active forms.). Considering the compact relationship between MAPK signal transduction pathways and cell proliferation and differentiation, it was implied that the regulation of MAPK and PKC activity might be one of the mechanisms responsible for the inhibitory effect of 1,25(OH)2D3 on the proliferation of HOS-8603 cells. (PKC has a closer relationship with ERK1/2.).It is well known that many biological effects of 1,25(OH)2D3 are mediated by its specific nuclear receptor. Our previous works have demonstrated that VDR do express in HOS-8603 cells. Using the antisense oligonucleotides of VDR, we blocked the expression of VDR and found that the inhibitory effect of 1,25(OH)2D3 on the proliferation of HOS-8603 cells was weakened. To further clarify the pivotal roles of VDR and the relationship between VDR and MAPK signal transduction pathways in mediating the action of 1,25(OH)2D3 on proliferation of HOS-8603 cells, small interfering RNA for human VDR was cloned into expression vectors (pSilencer2.1-U6-VDRl/2-neo/hygro) and transfected into HOS-8603 cells. It was found that in HOS-8603 cells transiently transfected with all constructed vectors (including pSilencer2.1-U6-VDR1 -neo, pSilencer2.1 -U6-VDR2-neo, pSilencer2.1-U6-VDR1 -hygro and pSilencer2.1-U6-VDR2-hygro) and in cells stably transfected with pSilencer2.1-U6-VDRl-hygro and pSilencer2.1-U6-VDR2-hygro, the expression levels of VDR protein were all decreased significantly as compared with that in HOS-8603 cells transfected with control vector. These results demonstrated that the small interfering RNA we used could block the expression of VDR efficiently. As expected, the inhibitory effect of 1,25(OH)2D3 on the proliferation of HOS-8603 cells stably transfected with pSilencer2.1-U6-VDRl-hygro and pSilencer2.1-U6-VDR2-hygro were alleviated remarkably. Results stated above further confirmed that VDR...