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NOX4 Regulates CCR2 and CCL2 mRNA Stability in Alcoholic Liver Disease

View Article: PubMed Central - PubMed

ABSTRACT

Recruitment of inflammatory cells is a major feature of alcoholic liver injury however; the signals and cellular sources regulating this are not well defined. C-C chemokine receptor type 2 (CCR2) is expressed by active hepatic stellate cells (HSC) and is a key monocyte recruitment signal. Activated HSC are also important sources of hydrogen peroxide resulting from the activation of NADPH oxidase 4 (NOX4). As the role of this NOX in early alcoholic liver injury has not been addressed, we studied NOX4-mediated regulation of CCR2/CCL2 mRNA stability. NOX4 mRNA was significantly induced in patients with alcoholic liver injury, and was co-localized with αSMA-expressing activated HSC. We generated HSC-specific NOX4 KO mice and these were pair-fed on alcohol diet. Lipid peroxidation have not changed significantly however, the expression of CCR2, CCL2, Ly6C, TNFα, and IL-6 was significantly reduced in NOX4HSCKO compared to fl/fl mice. NOX4 promoter was induced in HSC by acetaldehyde treatment, and NOX4 has significantly increased mRNA half-life of CCR2 and CCL2 in conjunction with Ser221 phosphorylation and cytoplasmic shuttling of HuR. In conclusion, NOX4 is induced in early alcoholic liver injury and regulates CCR2/CCL2 mRNA stability thereby promoting recruitment of inflammatory cells and production of proinflammatory cytokines.

No MeSH data available.


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NOX4 is induced in patients with alcoholic liver disease.Liver biopsy samples from healthy individuals (N = 4, NL), and patients with alcoholic liver disease (ALD) (N = 4) were analyzed by RT qPCR for NOX4 expression (A). NOX4 was significantly upregulated in ALD (p < 0.05). Immunohistochemistry (IHC) showed increased expression of NOX4 in the biopsy samples of ALD patients both in hepatocytes and the periportal area (d, e, f, (B)). Confocal imaging following immunofluorescence staining for NOX4 (red) and αSMA (green) show enlarged area (box) for colocalization of NOX4 and αSMA in liver samples from patients with ALD (arrows, (C)). Bar = 100 μm.
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f1: NOX4 is induced in patients with alcoholic liver disease.Liver biopsy samples from healthy individuals (N = 4, NL), and patients with alcoholic liver disease (ALD) (N = 4) were analyzed by RT qPCR for NOX4 expression (A). NOX4 was significantly upregulated in ALD (p < 0.05). Immunohistochemistry (IHC) showed increased expression of NOX4 in the biopsy samples of ALD patients both in hepatocytes and the periportal area (d, e, f, (B)). Confocal imaging following immunofluorescence staining for NOX4 (red) and αSMA (green) show enlarged area (box) for colocalization of NOX4 and αSMA in liver samples from patients with ALD (arrows, (C)). Bar = 100 μm.

Mentions: To study NOX4 in patients with ALD, we performed real time qPCR (Fig. 1A), immunohistochemistry (Fig. 1B), and immunofluorescence studies to localize NOX4 and αSMA positive activated HSC with confocal microscopy (Fig. 1C). NOX4 was significantly induced in patients with ALD (p < 0.05). Immunohistochemistry demonstrated increased NOX4 signal in hepatocytes as well as in the periportal area in biopsy samples of ALD (Fig. 1B,d–f) while the periportal area in normal livers was devoid of NOX4 signal (Fig. 1B,c). To further delineate the histological localization of NOX4, confocal imaging was performed after immunofluorescence studies. These showed colocalization of NOX4 and αSMA in activated HSC (Fig. 1C).


NOX4 Regulates CCR2 and CCL2 mRNA Stability in Alcoholic Liver Disease
NOX4 is induced in patients with alcoholic liver disease.Liver biopsy samples from healthy individuals (N = 4, NL), and patients with alcoholic liver disease (ALD) (N = 4) were analyzed by RT qPCR for NOX4 expression (A). NOX4 was significantly upregulated in ALD (p < 0.05). Immunohistochemistry (IHC) showed increased expression of NOX4 in the biopsy samples of ALD patients both in hepatocytes and the periportal area (d, e, f, (B)). Confocal imaging following immunofluorescence staining for NOX4 (red) and αSMA (green) show enlarged area (box) for colocalization of NOX4 and αSMA in liver samples from patients with ALD (arrows, (C)). Bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5382722&req=5

f1: NOX4 is induced in patients with alcoholic liver disease.Liver biopsy samples from healthy individuals (N = 4, NL), and patients with alcoholic liver disease (ALD) (N = 4) were analyzed by RT qPCR for NOX4 expression (A). NOX4 was significantly upregulated in ALD (p < 0.05). Immunohistochemistry (IHC) showed increased expression of NOX4 in the biopsy samples of ALD patients both in hepatocytes and the periportal area (d, e, f, (B)). Confocal imaging following immunofluorescence staining for NOX4 (red) and αSMA (green) show enlarged area (box) for colocalization of NOX4 and αSMA in liver samples from patients with ALD (arrows, (C)). Bar = 100 μm.
Mentions: To study NOX4 in patients with ALD, we performed real time qPCR (Fig. 1A), immunohistochemistry (Fig. 1B), and immunofluorescence studies to localize NOX4 and αSMA positive activated HSC with confocal microscopy (Fig. 1C). NOX4 was significantly induced in patients with ALD (p < 0.05). Immunohistochemistry demonstrated increased NOX4 signal in hepatocytes as well as in the periportal area in biopsy samples of ALD (Fig. 1B,d–f) while the periportal area in normal livers was devoid of NOX4 signal (Fig. 1B,c). To further delineate the histological localization of NOX4, confocal imaging was performed after immunofluorescence studies. These showed colocalization of NOX4 and αSMA in activated HSC (Fig. 1C).

View Article: PubMed Central - PubMed

ABSTRACT

Recruitment of inflammatory cells is a major feature of alcoholic liver injury however; the signals and cellular sources regulating this are not well defined. C-C chemokine receptor type 2 (CCR2) is expressed by active hepatic stellate cells (HSC) and is a key monocyte recruitment signal. Activated HSC are also important sources of hydrogen peroxide resulting from the activation of NADPH oxidase 4 (NOX4). As the role of this NOX in early alcoholic liver injury has not been addressed, we studied NOX4-mediated regulation of CCR2/CCL2 mRNA stability. NOX4 mRNA was significantly induced in patients with alcoholic liver injury, and was co-localized with &alpha;SMA-expressing activated HSC. We generated HSC-specific NOX4 KO mice and these were pair-fed on alcohol diet. Lipid peroxidation have not changed significantly however, the expression of CCR2, CCL2, Ly6C, TNF&alpha;, and IL-6 was significantly reduced in NOX4HSCKO compared to fl/fl mice. NOX4 promoter was induced in HSC by acetaldehyde treatment, and NOX4 has significantly increased mRNA half-life of CCR2 and CCL2 in conjunction with Ser221 phosphorylation and cytoplasmic shuttling of HuR. In conclusion, NOX4 is induced in early alcoholic liver injury and regulates CCR2/CCL2 mRNA stability thereby promoting recruitment of inflammatory cells and production of proinflammatory cytokines.

No MeSH data available.


Related in: MedlinePlus