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Role of BCL2-associated athanogene 1 in differential sensitivity of human endothelial cells to glucocorticoids.

ABSTRACT: Chronic therapy with synthetic glucocorticoids has been associated with cardiovascular side effects, although differential interindividual susceptibility to glucocorticoids has been observed. The objective of this study was to identify the molecular mechanisms leading to differential glucocorticoid responses in endothelial cells.We tested the sensitivity of 42 human umbilical vein endothelial cells (HUVECs) to dexamethasone as determined by changes in gene expression, promoter transactivation, and procoagulant activity. We identified that 16 HUVECs were sensitive in every test, 14 HUVECs were sensitive in at least 1 test and 12 HUVECs were resistant in every test to dexamethasone. Nuclear translocation assays revealed that Dex-sensitive HUVECs have higher basal and Dex-stimulated levels of nuclear glucocorticoid receptor compared with Dex-resistant HUVECs. Cycloheximide assays revealed that Dex-resistant HUVECs have significantly shorter glucocorticoid receptor protein half-lives than Dex-sensitive HUVECs. Dex-resistant HUVECs have a stronger interaction of glucocorticoid receptor with the proteasomal recruiting protein, BCL2-associated athanogene 1 (BAG1), as shown by immunoprecipitation assays. Silencing BAG1 expression increased Dex-sensitivity in resistant HUVECs, whereas BAG1 overexpression decreased Dex-sensitivity in sensitive HUVECs. Finally, Dex-resistant HUVECs presented higher BAG1 expression than Dex-sensitive HUVECs.In vitro endothelial sensitivity to Dex varies within individuals and is inversely proportional to BAG1 protein expression and glucocorticoid receptor protein turnover.

SUBMITTER: Mata-Greenwood E

PROVIDER: S-EPMC5142448 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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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.

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Role of BCL2-associated athanogene 1 in differential sensitivity of human endothelial cells to glucocorticoids.

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