M C Perianayagam1, N E Madias, B J G Pereira, B L Jaber. 1. Department of Medicine, Tufts University School of Medicine, and Department of Medicine, Caritas St. Elizabeth's Medical Center, Boston, MA 02135, USA.
Abstract
BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
BACKGROUND: 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. MATERIALS AND METHODS: The human promyelocytic leukaemia HL-60 cell line was differentiated into neutrophils using 1.25% DMSO. Differentiated cells were incubated with recombinant humanC5a 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. RESULTS: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). CONCLUSIONS: 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.
Authors: Anna Lenkiewicz; Kamila Bujko; Katarzyna Brzezniakiewicz-Janus; Bing Xu; Mariusz Z Ratajczak Journal: Front Immunol Date: 2019-06-07 Impact factor: 7.561
Authors: A Abdelbaset-Ismail; S Borkowska-Rzeszotek; E Kubis; K Bujko; K Brzeźniakiewicz-Janus; L Bolkun; J Kloczko; M Moniuszko; G W Basak; W Wiktor-Jedrzejczak; M Z Ratajczak Journal: Leukemia Date: 2016-07-25 Impact factor: 11.528