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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.


NOX4 stabilizes CCR2 and CCL2 mRNAs in stellate cells.24 hours after transducing LX-2 cells with adeno-NOX4 or control vector (adeno-CMV), cells were treated with actinomycin D (2 μg/ml) to inhibit RNA polymerase. CCR2 and CCL2 mRNA half-lives were calculated after 2, 4, 6, 8 and 10 hours of treatment as T1/2 = Ln(2)/λ. The result showed significantly prolonged CCR2 (A) and CCL2 (B) mRNA half-lives in cells transduced by adeno-NOX4 (compared to control vector) *p < 0.05.
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f4: NOX4 stabilizes CCR2 and CCL2 mRNAs in stellate cells.24 hours after transducing LX-2 cells with adeno-NOX4 or control vector (adeno-CMV), cells were treated with actinomycin D (2 μg/ml) to inhibit RNA polymerase. CCR2 and CCL2 mRNA half-lives were calculated after 2, 4, 6, 8 and 10 hours of treatment as T1/2 = Ln(2)/λ. The result showed significantly prolonged CCR2 (A) and CCL2 (B) mRNA half-lives in cells transduced by adeno-NOX4 (compared to control vector) *p < 0.05.

Mentions: LX-2 human stellate cell line has been shown to have all the key features of activated HSC and has been used in numerous studies in hepatic fibrogenesis1617, and it expresses NOX4 (Suppl. Fig. 4). As CCR2/CCL2 were downstream from NOX4, next we studied whether induction of NOX4 in HSC results in prolonged CCR2 and CCL2 mRNA stability. Steady-state levels of mRNA are regulated by multiple pathways including transcriptional, turnover and degradation events18. As mRNA stability was shown to be affected by oxidative regulation, LX-2 cells were transduced with adeno-NOX4 or control viral vector (ad-CMV) for 24 hours, followed by treatment with actinomycin D (2 μg/ml) to inhibit RNA polymerase and block transcription. CCR2 and CCL2 mRNA half-lives were calculated after 2, 4, 6, 8 and 10 hours. The results indicated that NOX4 induction in LX-2 cells resulted in a significantly prolonged CCR2 and CCL2 mRNA half-lives and therefore more stable mRNAs (Fig. 4).


NOX4 Regulates CCR2 and CCL2 mRNA Stability in Alcoholic Liver Disease
NOX4 stabilizes CCR2 and CCL2 mRNAs in stellate cells.24 hours after transducing LX-2 cells with adeno-NOX4 or control vector (adeno-CMV), cells were treated with actinomycin D (2 μg/ml) to inhibit RNA polymerase. CCR2 and CCL2 mRNA half-lives were calculated after 2, 4, 6, 8 and 10 hours of treatment as T1/2 = Ln(2)/λ. The result showed significantly prolonged CCR2 (A) and CCL2 (B) mRNA half-lives in cells transduced by adeno-NOX4 (compared to control vector) *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5382722&req=5

f4: NOX4 stabilizes CCR2 and CCL2 mRNAs in stellate cells.24 hours after transducing LX-2 cells with adeno-NOX4 or control vector (adeno-CMV), cells were treated with actinomycin D (2 μg/ml) to inhibit RNA polymerase. CCR2 and CCL2 mRNA half-lives were calculated after 2, 4, 6, 8 and 10 hours of treatment as T1/2 = Ln(2)/λ. The result showed significantly prolonged CCR2 (A) and CCL2 (B) mRNA half-lives in cells transduced by adeno-NOX4 (compared to control vector) *p < 0.05.
Mentions: LX-2 human stellate cell line has been shown to have all the key features of activated HSC and has been used in numerous studies in hepatic fibrogenesis1617, and it expresses NOX4 (Suppl. Fig. 4). As CCR2/CCL2 were downstream from NOX4, next we studied whether induction of NOX4 in HSC results in prolonged CCR2 and CCL2 mRNA stability. Steady-state levels of mRNA are regulated by multiple pathways including transcriptional, turnover and degradation events18. As mRNA stability was shown to be affected by oxidative regulation, LX-2 cells were transduced with adeno-NOX4 or control viral vector (ad-CMV) for 24 hours, followed by treatment with actinomycin D (2 μg/ml) to inhibit RNA polymerase and block transcription. CCR2 and CCL2 mRNA half-lives were calculated after 2, 4, 6, 8 and 10 hours. The results indicated that NOX4 induction in LX-2 cells resulted in a significantly prolonged CCR2 and CCL2 mRNA half-lives and therefore more stable mRNAs (Fig. 4).

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.