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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.


HuR shuttling to the cytoplasm is induced by NOX4 activation in HSC in response to acetaldehyde, and HuR controls CCR2 and CCL2 mRNA stability.Primary HSC from WT and NOX4KO mice were isolated and treated with 100 μM AC for 24 hours. Immunofluorescence studies using HuR antibody (green) were performed with results showing that in response to AC treatment HuR is translocated to the cytoplasm in WT HSC but not in NOX4KO cells. (A Blue: DAPI, nucleus. Scale: 10 μm). LX-2 cells were transduced by adeno-NOX4, or control vector. Western blot analysis showed phosphorylation of HuR at S221 in response to NOX4 activation in the cytoplasmic extracts of the cells transduced by Ad-NOX4 (B). mRNA levels of HuR did not change following NOX4 transduction (C). To address the role of HuR in regulating CCR2/CCL2 mRNA stabilities, LX-2 cells were transfected by control siRNA or siHuR, and 24 hours later cells were treated with actinomycin D (2 μg/ml) to inhibit RNA polymerase. CCR2 and CCL2 mRNA half-lives were calculated after 2, 4, and 8 hours of treatment as T1/2 = Ln(2)/λ. Both CCR2/CCL2 were regulate by HuR (D, p < 0.05).
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f5: HuR shuttling to the cytoplasm is induced by NOX4 activation in HSC in response to acetaldehyde, and HuR controls CCR2 and CCL2 mRNA stability.Primary HSC from WT and NOX4KO mice were isolated and treated with 100 μM AC for 24 hours. Immunofluorescence studies using HuR antibody (green) were performed with results showing that in response to AC treatment HuR is translocated to the cytoplasm in WT HSC but not in NOX4KO cells. (A Blue: DAPI, nucleus. Scale: 10 μm). LX-2 cells were transduced by adeno-NOX4, or control vector. Western blot analysis showed phosphorylation of HuR at S221 in response to NOX4 activation in the cytoplasmic extracts of the cells transduced by Ad-NOX4 (B). mRNA levels of HuR did not change following NOX4 transduction (C). To address the role of HuR in regulating CCR2/CCL2 mRNA stabilities, LX-2 cells were transfected by control siRNA or siHuR, and 24 hours later cells were treated with actinomycin D (2 μg/ml) to inhibit RNA polymerase. CCR2 and CCL2 mRNA half-lives were calculated after 2, 4, and 8 hours of treatment as T1/2 = Ln(2)/λ. Both CCR2/CCL2 were regulate by HuR (D, p < 0.05).

Mentions: HuR mRNA binding protein was shown to be phosphorylated at S221 by protein kinase C in response to oxidative stress, resulting in its translocation to the cytoplasm, and stabilization of target mRNAs1920. HuR is expressed in activated HSC, and HuR silencing attenuated oxidative stress and hepatic fibrosis in the BDL model21. To show the effects of acetaldehyde on HuR shuttling, we treated the primary HSC from fl/fl (wt) and NOX4KO mice with acetaldehyde. We found that HuR translocated from the nucleus to the cytoplasm in wt but not in NOX4KO HSC (Fig. 5A). In addition, when compared to control vector-transduced HSC, adeno-NOX4 induced the phosphorylation of HuR (S221) as detected in the cytoplasmic fraction (Fig. 5B, full length gel is included in the Suppl. files). HuR mRNA expression as assessed by real-time qPCR however, was not affected in Ad-NOX4-transfected cells (Fig. 5C). To study if HuR is indeed a key to regulate CCR2/CCL2 mRNA stability in HSC, LX-2 cells were transfected with scrambled siRNA or siHuR, and mRNA stabilities were assessed as described above. In cells transfected by siHuR, CCR2/CCL2 mRNA stabilities have decreased significantly (Fig. 5D).


NOX4 Regulates CCR2 and CCL2 mRNA Stability in Alcoholic Liver Disease
HuR shuttling to the cytoplasm is induced by NOX4 activation in HSC in response to acetaldehyde, and HuR controls CCR2 and CCL2 mRNA stability.Primary HSC from WT and NOX4KO mice were isolated and treated with 100 μM AC for 24 hours. Immunofluorescence studies using HuR antibody (green) were performed with results showing that in response to AC treatment HuR is translocated to the cytoplasm in WT HSC but not in NOX4KO cells. (A Blue: DAPI, nucleus. Scale: 10 μm). LX-2 cells were transduced by adeno-NOX4, or control vector. Western blot analysis showed phosphorylation of HuR at S221 in response to NOX4 activation in the cytoplasmic extracts of the cells transduced by Ad-NOX4 (B). mRNA levels of HuR did not change following NOX4 transduction (C). To address the role of HuR in regulating CCR2/CCL2 mRNA stabilities, LX-2 cells were transfected by control siRNA or siHuR, and 24 hours later cells were treated with actinomycin D (2 μg/ml) to inhibit RNA polymerase. CCR2 and CCL2 mRNA half-lives were calculated after 2, 4, and 8 hours of treatment as T1/2 = Ln(2)/λ. Both CCR2/CCL2 were regulate by HuR (D, p < 0.05).
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f5: HuR shuttling to the cytoplasm is induced by NOX4 activation in HSC in response to acetaldehyde, and HuR controls CCR2 and CCL2 mRNA stability.Primary HSC from WT and NOX4KO mice were isolated and treated with 100 μM AC for 24 hours. Immunofluorescence studies using HuR antibody (green) were performed with results showing that in response to AC treatment HuR is translocated to the cytoplasm in WT HSC but not in NOX4KO cells. (A Blue: DAPI, nucleus. Scale: 10 μm). LX-2 cells were transduced by adeno-NOX4, or control vector. Western blot analysis showed phosphorylation of HuR at S221 in response to NOX4 activation in the cytoplasmic extracts of the cells transduced by Ad-NOX4 (B). mRNA levels of HuR did not change following NOX4 transduction (C). To address the role of HuR in regulating CCR2/CCL2 mRNA stabilities, LX-2 cells were transfected by control siRNA or siHuR, and 24 hours later cells were treated with actinomycin D (2 μg/ml) to inhibit RNA polymerase. CCR2 and CCL2 mRNA half-lives were calculated after 2, 4, and 8 hours of treatment as T1/2 = Ln(2)/λ. Both CCR2/CCL2 were regulate by HuR (D, p < 0.05).
Mentions: HuR mRNA binding protein was shown to be phosphorylated at S221 by protein kinase C in response to oxidative stress, resulting in its translocation to the cytoplasm, and stabilization of target mRNAs1920. HuR is expressed in activated HSC, and HuR silencing attenuated oxidative stress and hepatic fibrosis in the BDL model21. To show the effects of acetaldehyde on HuR shuttling, we treated the primary HSC from fl/fl (wt) and NOX4KO mice with acetaldehyde. We found that HuR translocated from the nucleus to the cytoplasm in wt but not in NOX4KO HSC (Fig. 5A). In addition, when compared to control vector-transduced HSC, adeno-NOX4 induced the phosphorylation of HuR (S221) as detected in the cytoplasmic fraction (Fig. 5B, full length gel is included in the Suppl. files). HuR mRNA expression as assessed by real-time qPCR however, was not affected in Ad-NOX4-transfected cells (Fig. 5C). To study if HuR is indeed a key to regulate CCR2/CCL2 mRNA stability in HSC, LX-2 cells were transfected with scrambled siRNA or siHuR, and mRNA stabilities were assessed as described above. In cells transfected by siHuR, CCR2/CCL2 mRNA stabilities have decreased significantly (Fig. 5D).

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.