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

Witcher, Michael (1999) Interaction of the anti-apoptotic protein BAG-1 with the vitamin D receptor. Masters thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (13MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

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.

Actions (login required)

View Item