Dataset Information

Regulation of osteoblast development by Bcl-2-associated athanogene-1 (BAG-1).

ABSTRACT: BCL-2-associated athanogene-1 (BAG-1) is expressed by osteoblast-lineage cells; early embryonic lethality in Bag-1 null mice, however, has limited the investigation of BAG-1 function in osteoblast development. In the present study, bone morphogenetic protein-2/BMP-2-directed osteogenic differentiation of bone marrow stromal cells (BMSCs) of Bag-1(+/-) (heterozygous) female mice was decreased significantly. Genes crucial for osteogenic differentiation, bone matrix formation and mineralisation were expressed at significantly lower levels in cultures of Bag-1(+/-) BMSCs supplemented with BMP-2, while genes with roles in inhibition of BMP-2-directed osteoblastogenesis were significantly upregulated. 17-?-estradiol (E2) enhanced responsiveness of BMSCs of wild-type and Bag-1(+/-) mice to BMP-2, and promoted robust BMP-2-stimulated osteogenic differentiation of BMSCs. BAG-1 can modulate cellular responses to E2 by regulating the establishment of functional estrogen receptors (ERs), crucially, via its interaction with heat shock proteins (HSC70/HSP70). Inhibition of BAG-1 binding to HSC70 by the small-molecule chemical inhibitor, Thioflavin-S, and a short peptide derived from the C-terminal BAG domain, which mediates binding with the ATPase domain of HSC70, resulted in significant downregulation of E2/ER-facilitated BMP-2-directed osteogenic differentiation of BMSCs. These studies demonstrate for the first time the significance of BAG-1-mediated protein-protein interactions, specifically, BAG-1-regulated activation of ER by HSC70, in modulation of E2-facilitated BMP-2-directed osteoblast development.

SUBMITTER: Greenhough J

PROVIDER: S-EPMC5025845 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Regulation of osteoblast development by Bcl-2-associated athanogene-1 (BAG-1).

Greenhough Joanna J Papadakis Emmanouil S ES Cutress Ramsey I RI Townsend Paul A PA Oreffo Richard O C ROC Tare Rahul S RS

Scientific reports 20160916

BCL-2-associated athanogene-1 (BAG-1) is expressed by osteoblast-lineage cells; early embryonic lethality in Bag-1 null mice, however, has limited the investigation of BAG-1 function in osteoblast development. In the present study, bone morphogenetic protein-2/BMP-2-directed osteogenic differentiation of bone marrow stromal cells (BMSCs) of Bag-1(+/-) (heterozygous) female mice was decreased significantly. Genes crucial for osteogenic differentiation, bone matrix formation and mineralisation wer ...[more]

PMID: 27633857

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Project description:Molecular chaperones, particularly the 70-kDa heat shock proteins (Hsp70s), are key orchestrators of the cellular stress response. To perform their critical functions, Hsp70s require the presence of specific co-chaperones, which include nucleotide exchange factors containing the BCL2-associated athanogene (BAG) domain. BAG-1 is one of these proteins that function in a wide range of cellular processes, including apoptosis, protein refolding, and degradation, as well as tumorigenesis. However, the origin of BAG-1 proteins and their evolution between and within species are mostly uncharacterized. This report investigated the macro- and micro-evolution of BAG-1 using orthologous sequences and single nucleotide polymorphisms (SNPs) to elucidate the evolution and understand how natural variation affects the cellular stress response. We first collected and analyzed several BAG-1 sequences across animals, plants, and fungi; mapped intron positions and phases; reconstructed phylogeny; and analyzed protein characteristics. These data indicated that BAG-1 originated before the animals, plants, and fungi split, yet most extant fungal species have lost BAG-1. Furthermore, although BAG-1's structure has remained relatively conserved, kingdom-specific conserved differences exist at sites of known function, suggesting functional specialization within each kingdom. We then analyzed SNPs from the 1000 genomes database to determine the evolutionary patterns within humans. These analyses revealed that the SNP density is unequally distributed within the BAG1 gene, and the ratio of non-synonymous/synonymous SNPs is significantly higher than 1 in the BAG domain region, which is an indication of positive selection. To further explore this notion, we performed several biochemical assays and found that only one out of five mutations tested altered the major co-chaperone properties of BAG-1. These data collectively suggest that although the co-chaperone functions of BAG-1 are highly conserved and can probably tolerate several radical mutations, BAG-1 might have acquired specialized and potentially unexplored functions during the evolutionary process.

Project description:BackgroundThe Bag (Bcl-2 associated athanogene) family of proteins consists of 6 members sharing a common, single-copied Bag domain through which they interact with the molecular chaperone Hsp70. Bag5 represents an exception in the Bag family since it consists of 5 Bag domains covering the whole protein. Bag proteins like Bag1 and Bag3 have been implicated in tumor growth and survival but it is not known whether Bag5 also exhibits this function.MethodsBag5 mRNA and protein expression levels were investigated in prostate cancer patient samples using real-time PCR and immunoblot analyses. In addition immunohistological studies were carried out to determine the expression of Bag5 in tissue arrays. Analysis of Bag5 gene expression was carried out using one-way ANOVA and Bonferroni's Multiple Comparison test. The mean values of the Bag5 stained cells in the tissue array was analyzed by Mann-Whitney test. Functional studies of the role of Bag5 in prostate cancer cell lines was performed using overexpression and RNA interference analyses.ResultsOur results show that Bag5 is overexpressed in malignant prostate tissue compared to benign samples. In addition we could show that Bag5 levels are increased following endoplasmic reticulum (ER)-stress induction, and Bag5 relocates from the cytoplasm to the ER during this process. We also demonstrate that Bag5 interacts with the ER-resident chaperone GRP78/BiP and enhances its ATPase activity. Bag5 overexpression in 22Rv.1 prostate cancer cells inhibited ER-stress induced apoptosis in the unfolded protein response by suppressing PERK-eIF2-ATF4 activity while enhancing the IRE1-Xbp1 axis of this pathway. Cells expressing high levels of Bag5 showed reduced sensitivity to apoptosis induced by different agents while Bag5 downregulation resulted in increased stress-induced cell death.ConclusionsWe have therefore shown that Bag5 is overexpressed in prostate cancer and plays a role in ER-stress induced apoptosis. Furthermore we have identified GRP78/BiP as a novel interaction partner of Bag5.

Dataset Information

Regulation of osteoblast development by Bcl-2-associated athanogene-1 (BAG-1).

Publications

Regulation of osteoblast development by Bcl-2-associated athanogene-1 (BAG-1).

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