Negative Regulation of Hepatic Inflammation by the Soluble Resistance-Related Calcium-Binding Protein via Signal Transducer and Activator of Transcription 3

Figures

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  Figure 1. Generation of sorcin−/− mice. (A) Homologous recombination strategy. The neomycin cassette served as the positive selection marker during the ES targeting step. Exons 3–6 were deleted by replacing exon 3–6 (~4.5 kb) with a loxp-pgkneo-loxp cassette. Two ES cell clones were identified by Southern Blot, which had one copy of the sorcin gene replaced by the neomycin resistance gene cassette. The mutant ES cells were injected into 4-day-old C57BL/6J mouse blastocysts. The resultant chimeric male offsprings were crossed with 129S6/SvEvTac females for germline transmission. (B) Examination of sorcin expression in splenocytes from sorcin+/+ (WT), sorcin+/− (HT), or sorcin−/− (KO) mice by Western Blot. Splenocytes were isolated from the freshly harvested spleens of sorcin+/+, sorcin+/−, and sorcin−/− mice and lysed with lysis buffer, followed by examination with Western Blot using anti-sorcin antibodies. Endogenous β-actin expressions were used as internal controls. (C) The density of bands in (B) was quantitated by densitometry. The relative levels of sorcin were calculated as follows: the band density of sorcin/the band density of actin in the same sample. Results are representative of…

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  Figure 2. Enhanced ConA-induced hepatitis in sorcin−/− mice. (A) Enhanced inflammation in the liver of sorcin−/− mice post ConA treatment. Sorcin−/− mice (n = 9) and the littermate WT controls (sorcin+/+) (n = 9) were injected with a single dose of ConA (20 mg/kg of body weight) to induce hepatitis as described in Section “Materials and Methods.” Mice were sacrificed 24 h post-ConA injection. The livers were harvested and pictures of the organs were taken. (B,C) Enhanced spleen mass in sorcin−/− mice. Mice were treated with ConA as in (A), and the spleens were harvested and weighed (B), and the spleen weight vs. body weight of the same mouse was calculated (C). (D) H&E straining of liver section from sorcin+/+ and sorcin−/− mice, respectively, 24 h after ConA injection. Mice were treated with ConA as in (A). The arrowhead indicated massive hepatic cell death and inflammatory infiltrates in sorcin−/− liver sections. Original magnification, ×100 or ×400. The scale bars represent 20 or 100 µm, respectively. (E) Quantitative analysis of inflammatory lesions in the liver. Mice were treated with ConA as in (A), and the degree of inflammation in the liver was graded as described in Section…

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  Figure 3. Enhanced T cell response in sorcin−/− mice. (A) Splenocytes from sorcin+/+ (n = 4) and sorcin−/− mice (n = 4) were stimulated with plate-bound anti-CD3 with or without soluble anti-CD28 McAbs or with Con A. Ninety-six hours post stimulation, the proliferation of splenic cells was measured by MTT assay. (B–F) Splenocytes from sorcin+/+ and sorcin−/− mice were stimulated with plate-bound anti-CD3 with or without soluble anti-CD28 McAbs or with Con A. Forty-eight hours post stimulation, the culture supernatants were collected and cytokine concentrations were determined by CBA. (A–F) Results are representative of three independent experiments with similar results. Error bars are presented as mean ± SD. The differences between the two groups are statistically significant as determined by two-way ANOVA (* stands for p < 0.05, ** for p < 0.01, and *** for p < 0.001).

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  Figure 4. The interaction of sorcin with signal transducer and activator of transcription 3 (STAT3). (A–D) Yeast two-hybrid screening of sorcin-associated proteins. (A) pGBKT7-p53 + pGADT7-T antigen as positive control. (B) pGBKT7-sorcin + pGADT7-lectin. (C) pGBKT7-sorcin + pGADT7-STAT3. (D) pGBKT7-Lamin C + pGADT7-T antigen as negative control. (E) The interaction of sorcin with exogenous STAT3. HEK293T cells (8 × 105/well) were transfected with 2.5 µg of pEGFP-sorcin and/or pRK5-flag-STAT3 or empty vector as controls, and co-immunoprecipitation assay was performed with anti-flag monoclonal antibody. Sorcin and STAT3 in the immune complex were examined with Western Blot using anti-GFP and anti-flag monoclonal antibodies, respectively. (F) The interaction of STAT3 with endogenous sorcin in HEK293T cells. HEK293T cells were transfected with 5 µg of pRK5-flag-STAT3 or empty vector, and co-immunoprecipitation assay was performed with anti-flag monoclonal antibody. Sorcin in the immune complex was examined with Western Blot using anti-sorcin monoclonal antibody. (G) Chemical crosslinking of endogenous STAT3 and endogenous sorcin. HepG2 cells were cultured, washed with PBS and treated with…

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  Figure 5. Determination of the binding domains of sorcin for signal transducer and activator of transcription 3 (STAT3). (A) Schematics represent the genes encoding the full length and truncated sorcin molecules. (B) STAT3 interacts with different truncated sorcin proteins. HEK293T cells were transfected with the full length or truncated sorcin plasmids or empty vectors and together with the full-length flag-STAT3. Twenty-four hours after transfection, cell lysates were immunoprecipitated by anti-GFP monoclonal antibodies. The pellets were subjected to Western Blot analysis using anti-flag monoclonal antibody. Results are representative of three independent experiments with similar results. (C) A schematic represents the sorcin-binding domain (aa 1–100 of sorcin) for STAT3.

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  Figure 6. Determination of the binding domain of signal transducer and activator of transcription 3 (STAT3) for sorcin. (A) Schematics represent the genes encoding the full length and truncated STAT3 molecules. (B) Sorcin interacts with different truncated STAT3 proteins. HEK293T cells were transfected with the full length or truncated STAT3 plasmids or empty vectors and together with pEGFP-sorcin. Twenty-four hours after tansfection, cell lysates were immunoprecipitated by anti-flag monoclonal antibodies. The pellets were subjected to Western Blot analysis using anti-GFP monoclonal antibody. Results are representative of three independent experiments with similar results. (C) A schematic represents the binding domain of STAT3 (aa 581–770 of STAT3) for sorcin.

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  Figure 7. Roles of sorcin and signal transducer and activator of transcription 3 (STAT3) in TNF-α- or SeV-induced activation of NF-κB promoters. (A,B) Overexpression of sorcin or STAT3 inhibits TNF-α- or SeV-induced activation of NF-κB promoters. HEK293T cells were transfected with pRK5-sorcin or pRK5-STAT3 or empty vector together with indicated reporter plasmids. Eighteen hours after transfection, cells were treated with TNF-α or SeV. After treatment, the activities of NF-κB promoter were determined by luciferase assays. (C,D) Effects of STAT3 RNAi on the expression of endogenous STAT3. HEK293T cells were transfected with siRNA (#1–3) or controls as described in Section “Materials and Methods.” Twenty-four hours after the second transfection, cell lysates were prepared and examined by Western Blot with anti-STAT3 antibody (C). Endogenous β-actin expressions were used as internal controls. The density of bands in (C) was quantitated by densitometry (D). The relative levels of STAT3 in STAT3 RNAi cells were calculated as follows: the ratio (the band density of STAT3/that of actin in the same sample) of STAT3 RNAi/the ratio (the band density of STAT3/that of actin) of RNAi control. (E,F)…

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  Figure 8. Overexpression or knockdown of sorcin influences phosphorylation of signal transducer and activator of transcription 3 (STAT3) on Tyr705 in hepatoma cells. (A) Overexpression of sorcin enhances phosphorylation of STAT3 on Tyr705 but not Ser727 in hepatoma cells. Hep2G cells were transfected with pEGFP-N1 or pEGFP-sorcin, and the cells were stimulated with IL-6 (20 ng/mL) for the indicated time when cells were lysed and subjected to Western Blot analysis using anti-pSTAT3 (Tyr705), anti-pSTAT3 (Ser727), anti-STAT3, anti-GFP, and anti-actin antibodies. (B) Densitometric quantification of the results in (A) was shown. The relative intensity of pSTAT3 (Tyr705) was normalized to the endogenous actin of the same sample. (C) Knockdown of sorcin reduced phosphorylation of STAT3 on Tyr705 but not Ser727 in hepatoma cells. Hep2G cells were transfected with sorcin RNAi constructs or RNAi controls. Twenty-four hours after the second transfection, cells were stimulated with IL-6 (20 ng/mL) for the indicated time, and cells were lysed and subjected to Western Blot analysis using with anti-pSTAT3(Tyr705), anti-pSTAT3(Ser727), anti-STAT3, anti-sorcin, or anti-actin antibodies. (D) Densitometric…

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  Figure 9. Colocalization of sorcin with signal transducer and activator of transcription 3 (STAT3). (A–C) Colocalization of sorcin with STAT3 in normal HepG2 cells. HepG2 cells were fixed and probed with mouse anti-sorcin antibody and rabbit anti-STAT3 antibody, followed by the FITC-conjugated goat anti-mouse antibody (green) and TRITC-conjugated goat anti-rabbit antibody (red). Nuclei were counterstained with DAPI (blue). The cell samples were observed with a laser confocal scanning microscope. All scale bars represent 10 µm. (D–K) Overexpression of sorcin enhanced IL-6-induced phosphorylation of STAT3. HepG2 cells were transfected with the pEGFP-N1 (D–G) or pEGFP-sorcin (H–K), and the cells were stimulated with IL-6 (20 ng/mL) for 30 min. Then the cells were fixed with 1% paraformaldehyde. After washes, the fixed cells were permeabilized with 0.1% triton X-100, and probed with anti-sorcin and anti-pSTAT3 (Tyr705) antibodies, and followed by the FITC- and TRITC-conjugated secondary antibodies. Nuclei were counterstained with DAPI (Blue). (L–S) Knockdown of sorcin suppressed IL-6-induced phosphorylation of STAT3. HepG2 cells were transfected with the control RNAi or sorcin RNAi construct,…