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Heme oxygenase-1 induction attenuates imiquimod-induced psoriasiform inflammation by negative regulation of Stat3 signaling.

Zhang B, Xie S, Su Z, Song S, Xu H, Chen G, Cao W, Yin S, Gao Q, Wang H - Sci Rep (2016)

Bottom Line: Heme oxygenase-1 (HO-1), a stress-inducible protein with a potential anti-inflammatory effect, plays an important role in skin injury and wound healing.In this study, HO-1 activation significantly alleviated the disease-related pathogenesis abnormality.SHP-1-mediated suppression of Stat3 activation after HO-1 activation is a unique molecular mechanism for the regulation of Stat3 activation.

View Article: PubMed Central - PubMed

Affiliation: Center for Translational Medicine and Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing 210093, P.R. China.

ABSTRACT
Heme oxygenase-1 (HO-1), a stress-inducible protein with a potential anti-inflammatory effect, plays an important role in skin injury and wound healing. However, the function of HO-1 in cutaneous inflammatory diseases, such as psoriasis, remains unknown. The abnormal activation of Stat3, a known transcription factor that induces inflammation and regulates cell differentiation, is directly involved in the pathogenesis and development of psoriasis. Hence, targeting Stat3 is potentially beneficial in the treatment of psoriasis. In this study, HO-1 activation significantly alleviated the disease-related pathogenesis abnormality. To determine the mechanism by which HO-1 exerts immune protection on Th17-related cytokines, IL6/IL22-induced Stat3 activation was significantly suppressed, accompanied by decreased cell proliferation and reversed abnormal cell proliferation. Importantly, HO-1-induced Stat3 suppression was mediated through the activation of protein tyrosine phosphatase SHP-1. Overall, our study provides direct evidence indicating that HO-1 might be a useful therapeutic target for psoriasis. SHP-1-mediated suppression of Stat3 activation after HO-1 activation is a unique molecular mechanism for the regulation of Stat3 activation.

No MeSH data available.


Related in: MedlinePlus

Altered HO-1 expression levels by recombinant expression or RNAi also influence Stat3 activation in keratinocytes.(a) Altered HO-1 expression by RNAi increased Stat3 phosphorylation and downstream genes expression in HaCaT cells. (b) Altered HO-1 expression by RNAi increased Stat3 phosphorylation and downstream genes expression in A431 cells. In the RNAi experiments, HaCaT or A431 cells were cultured to 80% confluency in six-well plates. These cells were transfected with 100 nM specific HO-1 siRNA or control siRNA and treated with 25 μM Hemin for 24 h before exposure to 25 ng/mL IL-6 for 30 min. Stat3 phosphorylation levels and downstream proteins were determined by Western blot. (c) Enhanced HO-1 expression using the transfected recombinant plasmids decreased Stat3 activation in the presence or absence of IL-6 stimulus. (d) Enhanced HO-1 expression decreased the expression of Stat3 downstream proteins in the presence or absence of IL-6 stimulus. In gene overexpression experiments, HaCaT cells were transfected with GV230-HO-1 recombinant plasmid for 24 h before exposure to 25 ng/mL IL-6 for 30 min or 24 h, and the Stat3 phosphorylation levels and the expression of Stat3 downstream genes were determined by Western blot. Blots shown are derived from multiple gels. The gels were run under the same experimental conditions. The membrane was cut based on molecular weight and probed with antibody of interest. Band of interest is indicated with an arrow. All full-length blots are presented in Supplementary Fig. 2.
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f4: Altered HO-1 expression levels by recombinant expression or RNAi also influence Stat3 activation in keratinocytes.(a) Altered HO-1 expression by RNAi increased Stat3 phosphorylation and downstream genes expression in HaCaT cells. (b) Altered HO-1 expression by RNAi increased Stat3 phosphorylation and downstream genes expression in A431 cells. In the RNAi experiments, HaCaT or A431 cells were cultured to 80% confluency in six-well plates. These cells were transfected with 100 nM specific HO-1 siRNA or control siRNA and treated with 25 μM Hemin for 24 h before exposure to 25 ng/mL IL-6 for 30 min. Stat3 phosphorylation levels and downstream proteins were determined by Western blot. (c) Enhanced HO-1 expression using the transfected recombinant plasmids decreased Stat3 activation in the presence or absence of IL-6 stimulus. (d) Enhanced HO-1 expression decreased the expression of Stat3 downstream proteins in the presence or absence of IL-6 stimulus. In gene overexpression experiments, HaCaT cells were transfected with GV230-HO-1 recombinant plasmid for 24 h before exposure to 25 ng/mL IL-6 for 30 min or 24 h, and the Stat3 phosphorylation levels and the expression of Stat3 downstream genes were determined by Western blot. Blots shown are derived from multiple gels. The gels were run under the same experimental conditions. The membrane was cut based on molecular weight and probed with antibody of interest. Band of interest is indicated with an arrow. All full-length blots are presented in Supplementary Fig. 2.

Mentions: To further analyze the regulatory effect of HO-1 on Stat3, RNAi or gene overexpression was used to alter the HO-1 expression level. Various siRNA sequences were synthesized to fulfill RNAi, as mentioned in the Material and Methods section, and the gene knockdown efficiencies were compared. Figure 4a,b shows that two siRNAs were used in our experiments. When HO-1 was knocked down in HaCaT cells by using siRNAs, the IL-6-induced Stat3 phosphorylation significantly increased compared with the control group. The expression levels of Stat3 downstream genes, such as Survivin and Mcl-1, also considerably increased after 48 h of siRNA treatment (Fig. 4b). Analysis of the gene expression levels of keratin 16 and keratin 17 showed that the knockdown of HO-1 gene expression resulted in the increased expression of keratin 16 and keratin 17 in response to IL-6 treatment (Fig. 4a). The same results were procured by using a human epidermal carcinoma cell line, A431 (Fig. 4a). This result indicates that the knockdown of HO-1 gene expression enhanced Stat3 activation. The HO-1 recombinant plasmid was transferred to HaCaT cells in another experiment, and HO-1 overexpression was achieved (Fig. 4c,d). HO-1 overexpression markedly decreased Stat3 phosphorylation at the basal level, and similar results were observed when the cells were treated with IL-6. These results further confirmed that the increased HO-1 expression in keratinocytes inhibited Stat3 activation.


Heme oxygenase-1 induction attenuates imiquimod-induced psoriasiform inflammation by negative regulation of Stat3 signaling.

Zhang B, Xie S, Su Z, Song S, Xu H, Chen G, Cao W, Yin S, Gao Q, Wang H - Sci Rep (2016)

Altered HO-1 expression levels by recombinant expression or RNAi also influence Stat3 activation in keratinocytes.(a) Altered HO-1 expression by RNAi increased Stat3 phosphorylation and downstream genes expression in HaCaT cells. (b) Altered HO-1 expression by RNAi increased Stat3 phosphorylation and downstream genes expression in A431 cells. In the RNAi experiments, HaCaT or A431 cells were cultured to 80% confluency in six-well plates. These cells were transfected with 100 nM specific HO-1 siRNA or control siRNA and treated with 25 μM Hemin for 24 h before exposure to 25 ng/mL IL-6 for 30 min. Stat3 phosphorylation levels and downstream proteins were determined by Western blot. (c) Enhanced HO-1 expression using the transfected recombinant plasmids decreased Stat3 activation in the presence or absence of IL-6 stimulus. (d) Enhanced HO-1 expression decreased the expression of Stat3 downstream proteins in the presence or absence of IL-6 stimulus. In gene overexpression experiments, HaCaT cells were transfected with GV230-HO-1 recombinant plasmid for 24 h before exposure to 25 ng/mL IL-6 for 30 min or 24 h, and the Stat3 phosphorylation levels and the expression of Stat3 downstream genes were determined by Western blot. Blots shown are derived from multiple gels. The gels were run under the same experimental conditions. The membrane was cut based on molecular weight and probed with antibody of interest. Band of interest is indicated with an arrow. All full-length blots are presented in Supplementary Fig. 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4759695&req=5

f4: Altered HO-1 expression levels by recombinant expression or RNAi also influence Stat3 activation in keratinocytes.(a) Altered HO-1 expression by RNAi increased Stat3 phosphorylation and downstream genes expression in HaCaT cells. (b) Altered HO-1 expression by RNAi increased Stat3 phosphorylation and downstream genes expression in A431 cells. In the RNAi experiments, HaCaT or A431 cells were cultured to 80% confluency in six-well plates. These cells were transfected with 100 nM specific HO-1 siRNA or control siRNA and treated with 25 μM Hemin for 24 h before exposure to 25 ng/mL IL-6 for 30 min. Stat3 phosphorylation levels and downstream proteins were determined by Western blot. (c) Enhanced HO-1 expression using the transfected recombinant plasmids decreased Stat3 activation in the presence or absence of IL-6 stimulus. (d) Enhanced HO-1 expression decreased the expression of Stat3 downstream proteins in the presence or absence of IL-6 stimulus. In gene overexpression experiments, HaCaT cells were transfected with GV230-HO-1 recombinant plasmid for 24 h before exposure to 25 ng/mL IL-6 for 30 min or 24 h, and the Stat3 phosphorylation levels and the expression of Stat3 downstream genes were determined by Western blot. Blots shown are derived from multiple gels. The gels were run under the same experimental conditions. The membrane was cut based on molecular weight and probed with antibody of interest. Band of interest is indicated with an arrow. All full-length blots are presented in Supplementary Fig. 2.
Mentions: To further analyze the regulatory effect of HO-1 on Stat3, RNAi or gene overexpression was used to alter the HO-1 expression level. Various siRNA sequences were synthesized to fulfill RNAi, as mentioned in the Material and Methods section, and the gene knockdown efficiencies were compared. Figure 4a,b shows that two siRNAs were used in our experiments. When HO-1 was knocked down in HaCaT cells by using siRNAs, the IL-6-induced Stat3 phosphorylation significantly increased compared with the control group. The expression levels of Stat3 downstream genes, such as Survivin and Mcl-1, also considerably increased after 48 h of siRNA treatment (Fig. 4b). Analysis of the gene expression levels of keratin 16 and keratin 17 showed that the knockdown of HO-1 gene expression resulted in the increased expression of keratin 16 and keratin 17 in response to IL-6 treatment (Fig. 4a). The same results were procured by using a human epidermal carcinoma cell line, A431 (Fig. 4a). This result indicates that the knockdown of HO-1 gene expression enhanced Stat3 activation. The HO-1 recombinant plasmid was transferred to HaCaT cells in another experiment, and HO-1 overexpression was achieved (Fig. 4c,d). HO-1 overexpression markedly decreased Stat3 phosphorylation at the basal level, and similar results were observed when the cells were treated with IL-6. These results further confirmed that the increased HO-1 expression in keratinocytes inhibited Stat3 activation.

Bottom Line: Heme oxygenase-1 (HO-1), a stress-inducible protein with a potential anti-inflammatory effect, plays an important role in skin injury and wound healing.In this study, HO-1 activation significantly alleviated the disease-related pathogenesis abnormality.SHP-1-mediated suppression of Stat3 activation after HO-1 activation is a unique molecular mechanism for the regulation of Stat3 activation.

View Article: PubMed Central - PubMed

Affiliation: Center for Translational Medicine and Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing 210093, P.R. China.

ABSTRACT
Heme oxygenase-1 (HO-1), a stress-inducible protein with a potential anti-inflammatory effect, plays an important role in skin injury and wound healing. However, the function of HO-1 in cutaneous inflammatory diseases, such as psoriasis, remains unknown. The abnormal activation of Stat3, a known transcription factor that induces inflammation and regulates cell differentiation, is directly involved in the pathogenesis and development of psoriasis. Hence, targeting Stat3 is potentially beneficial in the treatment of psoriasis. In this study, HO-1 activation significantly alleviated the disease-related pathogenesis abnormality. To determine the mechanism by which HO-1 exerts immune protection on Th17-related cytokines, IL6/IL22-induced Stat3 activation was significantly suppressed, accompanied by decreased cell proliferation and reversed abnormal cell proliferation. Importantly, HO-1-induced Stat3 suppression was mediated through the activation of protein tyrosine phosphatase SHP-1. Overall, our study provides direct evidence indicating that HO-1 might be a useful therapeutic target for psoriasis. SHP-1-mediated suppression of Stat3 activation after HO-1 activation is a unique molecular mechanism for the regulation of Stat3 activation.

No MeSH data available.


Related in: MedlinePlus