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The C-terminal domain of A1/Bfl-1 regulates its anti-inflammatory function in human endothelial cells.

ABSTRACT: A1/Bfl-1 is a NF-?B dependent, anti-apoptotic Bcl-2 family member that contains four Bcl-2 homology domains (BH) and an amphipathic C-terminal domain, and is expressed in endothelial cells (EC). Based on NF-?B reporter assays in bovine aortic EC, we have previously demonstrated that A1, like Bcl-2 and Bcl-xL, inhibits NF-?B activation. These results, however, do not fully translate when evaluating the cell's own NF-?B machinery in human EC overexpressing A1 by means of recombinant adenovirus (rAd.) mediated gene transfer. Indeed, overexpression of full-length A1 in human umbilical vein EC (HUVEC), and human dermal microvascular EC (HDMEC) failed to inhibit NF-?B activation. However, overexpression of a mutant lacking the C-terminal domain of A1 (A1?C) demonstrated a potent NF-?B inhibitory effect in these cells. Disparate effects of A1 and A1?C on NF-?B inhibition in human EC correlated with mitochondrial (A1) versus non-mitochondrial (A1?C) localization. In contrast, both full-length A1 and A1?C protected EC from staurosporine (STS)-induced cell death, indicating that mitochondrial localization was not necessary for A1's cytoprotective function in human EC. In conclusion, our data uncover a regulatory role for the C-terminal domain of A1 in human EC: anchoring A1 to the mitochondrion, which conserves but is not necessary for its cytoprotective function, or by its absence freeing A1 from the mitochondrion and uncovering an additional anti-inflammatory effect.

SUBMITTER: Guedes RP

PROVIDER: S-EPMC3690303 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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The C-terminal domain of A1/Bfl-1 regulates its anti-inflammatory function in human endothelial cells.

Guedes Renata P RP Rocha Eduardo E Mahiou Jerome J Moll Herwig P HP Arvelo Maria B MB Taube Janis M JM Peterson Clayton R CR Kaczmarek Elzbieta E Longo Christopher R CR da Silva Cleide G CG Ferran Christiane C

Biochimica et biophysica acta 20130313 6

A1/Bfl-1 is a NF-κB dependent, anti-apoptotic Bcl-2 family member that contains four Bcl-2 homology domains (BH) and an amphipathic C-terminal domain, and is expressed in endothelial cells (EC). Based on NF-κB reporter assays in bovine aortic EC, we have previously demonstrated that A1, like Bcl-2 and Bcl-xL, inhibits NF-κB activation. These results, however, do not fully translate when evaluating the cell's own NF-κB machinery in human EC overexpressing A1 by means of recombinant adenovirus (rA ...[more]

PMID: 23499873

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Project description:Neutrophil granulocytes are innate effector cells of the first line of defense against pyogenic bacteria. Neutrophil lifespan is short, is prolonged by pro-inflammatory stimuli, controls functionality of the cells and can determine tissue damage. Experimental analysis of primary neutrophils is difficult because of their short lifespan and lack of possibilities of genetic manipulation. The Hoxb8 system of neutrophil differentiation from immortalized progenitor cells offers the advantage of unlimited production of neutrophils in vitro as well as easy genetic modification. We here use this system to analyze the role of the poorly characterized anti-apoptotic B-cell lymphoma protein 2 (Bcl-2) family member A1/Bfl-1 (Bcl-2-related protein A1) for survival and homeostasis of neutrophils and of neutrophil progenitors. Low constitutive mRNA and protein expression of A1 was detected, while A1 was transiently upregulated early during differentiation. Pro-inflammatory stimuli caused strong, mainly transcriptional, A1 upregulation, in contrast to posttranscriptional regulation of Mcl-1 (induced myeloid leukemia cell differentiation protein). Inhibitor studies showed that phosphoinositide-3 kinase (PI3K)/Akt and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) is required for A1 expression and survival of progenitors and mature neutrophils. ShRNA-mediated constitutive A1 knockdown (KD) impaired maintenance of progenitors. ShRNA experiments further showed that A1 was required early during neutrophil differentiation as well as in mature neutrophils upon pro-inflammatory stimulation. Our data further indicate differential regulation of the two anti-apoptotic proteins A1 and Mcl-1. Relevant findings were confirmed in primary human neutrophils. Our data indicate that A1, in addition to the well-established Mcl-1, substantially contributes to neutrophil survival and homeostasis. A1 may thus be a promising target for anti-inflammatory therapy.

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The C-terminal domain of A1/Bfl-1 regulates its anti-inflammatory function in human endothelial cells.

Publications

The C-terminal domain of A1/Bfl-1 regulates its anti-inflammatory function in human endothelial cells.

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