Interaction of the anti-apoptotic protein BAG-1 with the vitamin D receptor

Abstract

BAG-1 (BCL-2 associated anti-death gene) is a multi-isoform, BCL-2 binding protein having anti-apoptotic capabilities. In addition to binding BCL-2, BAG-1 has been found to bind and regulate the function of various steroid hormone receptors. It is postulated that BAG-l's ability to inhibit apoptosis and proliferation induced by hormone receptors could lead to tumor growth if BAG-1 were to become overexpressed. In fact, BAG-1 has been found to be overexpressed in cervical and breast tumors indicating that BAG-1 may be a proto-oncogene. To further investigate BAG-1's role in steroid hormone regulation, I decided to investigate whether or not BAG-1 could bind and regulate another member of the steroid hormone receptor super family - the vitamin D 3 receptor. -- Far Western blot analysis and glutathione S-transferase -BAGp50 pull-down assays revealed that the full length 50 KDa isoform of BAG-1 could interact with the vitamin D receptor (VDR). The shorter isoforms however, could not. Gel shift assays using cell extracts from BAGp50 stably transfected U87 glioblastoma cells (U87BAG-1) showed that BAG-1 could inhibit the VDR from binding to its consenus response element, as well as a vitamin D response element (VDRE) from the P21wafl promoter. Furthermore, overexpression of BAGp50 not only resulted in an increased rate of proliferation but rendered the cells resistant to vitamin D-induced growth inhibition. A CAT construct containing a osteocalcin VDRE was employed to demonstrate that BAGp50 could also inhibit vitamin D-mediated gene expression. This was further illustrated by the fact that the presence of BAGp50 in U87 cells blocked the induction of VDR protein levels in response to vitamin D3. -- Because BAGp50 could block vitamin D3-mediated transcription of the osteocalcin VDRE it was decided to investigate whether B AGp50 could block vitamin D3-mediated upregulation of p21wafl transcription. Using p21wafl luciferase constructs, it was found that BAGp50 could inhibit vitamin D3 activation of p21wafl transcription. Interestingly, the p46 isoform of BAG-1 decreased the level of P21wafl transcription through an unknown mechanism. -- These results demonstrate for the first time that BAGp50 can bind and regulate the function of the VDR. It was also found that BAG-1 may act as a regulator of proliferation. BAGp50 and p46 could also regulate the transcription of p21wafl through two separate mechanisms, suggesting that different isoforms of BAG-1 may work together to achieve the common goal of promoting cell proliferation.