CREB transcription factor modulates Bcl2 transcription in response to C5a in HL‐60‐derived neutrophils

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Abstract
Complement fragment C5a and neutrophils have been implicated in the pathogenesis of renal disease and C5a has also been shown to delay apoptosis of human neutrophils via a transcription-independent pathway. However, transcription-dependent pathways have not been well described. The present study examined whether activation of HL-60-derived neutrophils by C5a modulates the transcription of two members of the Bcl2 family, Bax (pro-apoptotic) and Bcl2 (anti-apoptotic) molecules, and whether the cAMP-response element-binding protein (CREB) transcription factor mediates these effects through the phosphatidylinositol 3-kinase (PI3K)/Akt and extra-cellular signal-regulated kinase (ERK) signalling pathways. The human promyelocytic leukaemia HL-60 cell line was differentiated into neutrophils using 1.25% DMSO. Differentiated cells were incubated with recombinant human C5a for 30-120 min with, or without, pretreatment with wortmannin or PD98059. The cells were lysed and quantified for gene-specific Bax and Bcl2 mRNA. In separate experiments, cells were incubated with C5a for 5-30 min with, or without, pretreatment with wortmannin, PD98059, or alkaline phosphatase. Cells were then lysed and immunoblotted using antihuman phospho-CREB (Ser133) antibody. Apoptosis was assessed by measuring active caspase-3 in differentiated HL-60 cells. C5a inhibited caspase-3 activation in HL-60-derived neutrophils (P=0.003). C5a significantly increased the expression of Bcl2 mRNA (P=0.028), which was time-dependent, peaking at 30 min, and was abrogated in the presence of either wortmannin or PD98059 (both P=0.028). The C5a had no impact on Bax mRNA expression. The Bax : Bcl2 mRNA ratio markedly decreased at 30 min (P=0.028). Time-dependent effect of C5a on CREB phosphorylation was demonstrable and rapid, peaking at 5 min, and was abrogated by either wortmannin or PD98059 (both P=0.028). Phosphorylation of CREB, but not of Akt and ERK, was inhibited by alkaline phosphatase (P=0.028). The effect of C5a on Bcl2 mRNA expression was abrogated by alkaline phosphatase (P=0.028). The Bax : Bcl2 mRNA ratio markedly increased in the presence of alkaline phosphatase (P=0.046). This study demonstrates that C5a induces Bcl2 mRNA transcription in HL-60-derived neutrophils, which is mediated in part by CREB through the convergence of the PI3K/Akt and ERK-signalling pathways.