Study On The Role Of Poly(ADP-ribose)polymerase-1 In The Transcriptional Regulation Of Vitamin D Receptor In Rat Thoracic Aortic Smooth Muscle Cells

Objective: To investigate whether poly(ADP-ribose)polymerase-1 interacts with vitamin D receptor and the interaction mechanisms between them in the cultured rat thoracic aortic smooth muscle cells (VSMCs).Methods: Explants culture of vascular tissue from 2 month-old Sprague-Dawley rats was used to obtain primary thoracic aortic VSMCs. Western blotting (WB) was used to detect the protein level of PARP-1 and VDR in the nuclear extra of cells. Co-immunoprecipitation (co-IP), far-WB and the poly(ADP-ribosyl)ation of nuclear extracts or recombinant VDR protein were used to study the interaction mechanisms of PARP-1 and VDR [whether PARP-1 interacts with VDR as a co-transcription factor or/and as a VDR modifier by poly(ADP-ribosyl)ation]. VSMCs were treated with VDR agonist 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3 ] and different concentration PARP-1 activity inhibitor 3AB or PJ-34, then polymerase chain reaction (PCR) was used to detect the transcription level of VDR target genes in the treated cells.Results: VSMCs were successfully cultured with modified known methods and could be used until number of passage of 15. In the VSMCs treated with 75nM 1α,25(OH)2D3 for 24h, VDR not only failed to bind to non-auto-modified PARP-1 or auto-modified [auto-poly(ADP-ribosyl)ation] PARP-1, but also couldn't be poly(ADP-ribosyl)ated by PARP-1 intracelluar. VDR from the unclear extract of the same treated cells failed to bind to recombined non-auto-modified PARP-1 or auto-modified PARP-1 and couldn't be poly(ADP-ribosyl)ated by recombined PARP-1. Recombined VDR protein failed to bind to recombined non-auto-modified PARP-1 or auto-modified PARP-1 and couldn't be poly(ADP-ribosyl)ated by recombined PARP-1. Real-time RT-PCR detection showed that in the cells treated with PARP-1 activity inhibitor 3AB or PJ-34, or inhibitor following 1α,25(OH)2D3, the transcription levels of VDR target genes Cyp24a1, Vegfa and Hgf were not inhibitor dose dependent, but the mRNA levels of Vegfa and Hgf did elevate more than 1α,25(OH)2D3 treated group alone (P<0.05).Conclusion: In the cultured VSMCs treated with 75nM 1α,25(OH)2D3, the transcriptional regulation of VDR target genes by VDR is independent of PARP-1 direct interaction or poly(ADP-ribosyl)ation effect by PARP-1. The mRNA level changes of VDR target genes Cyp24a1, Vegfa, Hgf may be regulated through other nuclear pathways. The VSMCc phenotype changed toward proliferation under the PARP-1 activity inhibiton condition indicated that, major proliferation genes transcriptional level in the VSMCs regulated by VDR may not be repressed by PARP-1 activity inhibition, or even be elevated, and this effect is not induced by VDR-PARP-1 interaction or VDR poly(ADP-ribosyl)ation by PARP-1.