J Bacteriol Virol.  2009 Sep;39(3):205-216. 10.4167/jbv.2009.39.3.205.

Expression of Endothelin-1 by Stimulation with CXCL8 in Mouse Peritoneal Macrophages

• Bae JJ
• Kim JH
• Kim H
• Kim HS

• KMID: 1474163
• DOI: http://doi.org/10.4167/jbv.2009.39.3.205

Abstract

Endothelin-1 (ET-1) has been characterized as a potent vasoconstrictor secreted by the endothelium, and play a major role in the regulation of vascular tone. It has been also known to participate in inflammatory reactions. The production of ET-1 by macrophages during infection and inflammation is related to tissue perfusion and leukocyte extravasation. The aim of this study is to investigate the role of IL-8/CXCL8, as a major inflammatory chemokine, for ET-1 expression in macrophges. Expression of ET-1 mRNA in mouse peritoneal macrophages (PeM phi) was weaker than that in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). However, expression of IL-8/CXCL8-induced ET-1 mRNA in PeM phi was much more stronger than that in SHR and WKY VSMCs. Maximum expression of ET-1 mRNA was observed at 50 ng/ml dose of IL-8/CXCL8 and occurred at 2 h after addition of IL-8/CXCL8. Expression of ET-1 by IL-8/CXCL8 was dependent on NF-kappaB activation and ERK1/2 phosphorylation. Baicalein, a 12-lipoxygenase (LO) inhibitor, inhibited the expression of IL-8/CXCL8-induced ET-1 mRNA. This inhibitory action of baicalein was mediated via ERK1/2 inactivation. Induction of 12-LO mRNA by IL-8/CXCL8 and expression of ET-1 mRNA by 12-LO metabolite, 12(S)-HETE were also detected. The expression of IL-8/CXCL8-induced ET-1 mRNA was not detected in PeM phi transfected with 12-LO siRNA. These results suggest that IL-8/CXCL8 can act as one of main inducers of ET-1 in vascular inflammatory reactions, and ET-1 expression by IL-8/CXCL8 is related to 12-LO pathway in PeM phi.

Keyword

Endothelin-1; IL-8/CXCL8; Mouse peritoneal macrophages; 12-lipoxygenase

MeSH Terms

Animals
Arachidonate 12-Lipoxygenase
Endothelin-1
Endothelium
Flavanones
Inflammation
Leukocytes
Macrophages
Macrophages, Peritoneal
Mice
Muscle, Smooth, Vascular
NF-kappa B
Perfusion
Phosphorylation
Rats
Rats, Inbred SHR
RNA, Messenger
RNA, Small Interfering
Arachidonate 12-Lipoxygenase
Endothelin-1
Flavanones
NF-kappa B
RNA, Messenger
RNA, Small Interfering

Figure

• 

  Figure 1. Expression of ET-1 mRNA in mouse peritoneal macro-phages. A: Real-time PCR for constitutive expression of ET-1 mRNA in thioglycollate-elicited mouse peritoneal macrophages (TG-PeMφ) and vascular smooth muscle cells from SHR (SHR-V) and WKY (WKY-V). B: TG-PeMφ, SHR-V and WKY-V were untreated (NT) or treated with IL-8/CXCL8 (50 ng/ml) for 2 h. The total RNAs were isolated and real time PCR was performed. Bars represent means ± SD from three independent experiments ∗p < 0.05 vs. untreated PeMφ.

• 

  Figure 2. Dose response of IL-8/CXCL8 on the expression of ET-1 mRNA and time course of IL-8/CXCL8-induced ET-1 mRNA expression in mouse peritoneal macrophages. TG-PeMφ were untreated (NT) or treated with 0, 10, 50, 100 or 200 ng/ml of IL-8/CXCL8 for 2 h (A), and TG-PeMφ were treated with IL-8/CXCL8 (50 ng/ml) for 0, 1, 2, 4, or 8 h (B). Total RNAs were isolated and real-time PCR was performed. Bars represent means ± SD from three separate experiments.

• 

  Figure 3. Production of IL-8/CXCL8-induced ET-1 is dependent on NF-κB activation in mouse peritoneal macrophages. A: TG-PeMφ were untreated or treated with IL-8/CXCL8 (50 ng/ml) in the absence or presence of Bay11-7082 (10 μM) for 2 h. Total RNAs were prepared, and real-time PCR was performed. B: Specific binding activity of NF-κB from nuclear extracts was assessed by electrophoretic mobility shift assay (EMSA). For positive or negative control, aliquots of nuclear extract were incubated with a 100-fold excess of mutant probe (m) or with 2 μg of anti NF-κB Ig (Ab) before EMSA. C: Cell lysates were separated on 10% SDS-polyacrylamide gels and then immunoblotted with ET-1 antibody. Data shown are representatives of three independent experiments. Bars represent means ± SD from three independent experiments. ∗p < 0.05 vs. treated with IL-8/CXCL8.

• 

  Figure 4. Expression of IL-8/CXCL8-induced ET-1 mRNA is mediated by 12-LO pathway in mouse peritoneal macrophages. A: TG-PeMφ were untreated (NT) or treated with IL-8/CXCL8 (50 ng/ml) for 2 h. After total mRNAs were isolated, real-time PCR for 12-LO mRNA expression was performed. Bars represent means ± SD from three independent experiments. ∗p < 0.05 vs. untreated cells. B: TG-PeMφ were untreated or treated with IL-8/CXCL8 (50 ng/ml) in the presence or absence of baicalein (10 μM) for 2 h. After total mRNAs were isolated, real-time PCR was performed. Bars represent means ± SD from three independent experiments. ∗p < 0.05 vs. cells treated with IL-8/CXCL8. C: TG-PeMφ were untreated (NT) or treated with IL-8/CXCL8 (50 ng/ml) or 12(S)-HETE (500 nM) for 2h. After total mRNAs were isolated, real-time PCR was performed. D: TG-PeMφ were plated on 24-well plates, grown to 90% confluence and then transfected with 12-LO siRNA oligomers (50 nmol/ℓ). TG-PeMφ were then untreated or treated with IL-8/CXCL8 (50 ng/ml) for 2 h. After total RNAs were isolated, RT-PCR and real-time PCR were performed. non TF; non-transfected cells. TF; 12-LO siRNA-transfected cells. Bars represent means ± SD from three independent experiments.

• 

  Figure 5. Expression of IL-8/CXCL8-induced ET-1 mRNA is dependent on ERK1/2 activation, and inhibitory action of baicalein on the expression of IL-8/CXCL8-induced ET-1 mRNA is mediated via ERK1/2 inactivation in mouse peritoneal macrophages. A: TG-PeMφ were untreated or treated with IL-8/CXCL8 (50 ng/ml) and/or baicalein (10 μM) for 2 h. Cell lysates were separated on 10% SDS-polyacrylamide gels and then immunoblotted with phospho-ERK1/2 antibody. B: TG-PeMφ were untreated or pretreated with PD98059 (MEK1/2 inhibitor, 10 μM) for 30 min. Cells were left untreated or treated with IL-8/CXCL8 (50 ng/ml) and/or baicalein (10 μM) for 2 h. Cell lysates were separated on 10% SDS-polyacrylamide gels and then immunoblotted with ET-1 antibody. Data shown are representatives of three independent experiments.

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