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

Abstract

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 (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-κB activation. However, overexpression of a mutant lacking the C-terminal domain of A1 (A1ΔC) demonstrated a potent NF-κB inhibitory effect in these cells. Disparate effects of A1 and A1ΔC on NF-κB inhibition in human EC correlated with mitochondrial (A1) versus non-mitochondrial (A1ΔC) localization. In contrast, both full-length A1 and A1ΔC 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.

Constitutive expression of the anti-apoptotic Bcl-2 family member A1 in murine endothelial cells leads to transplant tolerance

“…However, expression of full length A1, using rAd.A1, in bovine ECs gave the same result as in human cells with respect to IKBα degradation [35]. These authors concluded that full length A1 does not inhibit NF-kB activation.…”

“…A1 has four Bcl-2 homology (BH) domains in addition to a C-terminal domain containing an amphipathic tail. Expression of fulllength A1 by recombinant adenovirus (rAd) has been shown to protect human ECs from staurosporine [35] and TNFinduced cell death [15]. This may be due to both the BH4 and C-terminal domains of this molecule, both of which have been reported to be involved in the anti-apoptotic function of A1 [36].…”

“…A1 has also been shown to inhibit NF-jB activation in ECs [10], suggesting that this molecule could have an antiinflammatory function in ECs. Interestingly however, Guedes et al [35] observed that when human HUVEC and HDMEC were transduced with recombinant adenovirus (rAd.) expressing full length human A1 (rAd.A1), TNFinduced up-regulation of ICAM-1 in ECs was not inhibited, reflecting a lack of inhibited of NF-jB activation, as measured by IKBa degradation.…”

“…has been shown to protect human ECs from staurosporine [35] and TNFinduced cell death [15]. This maybe due to both the BH4 and C-terminal domains of this molecule, both of which have been reported to be involved in the anti-apoptotic function of A1 [36].…”

“…A1 has also been shown to inhibit NF-kB activation in ECs [10] [18,35]. However, expression of full length A1, using rAd.A1, in bovine ECs gave the same result as in human cells with respect to IKBα degradation [35].…”