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CIS3/SOCS3/SSI3 plays a negative regulatory role in STAT3 activation and intestinal inflammation.

Suzuki A, Hanada T, Mitsuyama K, Yoshida T, Kamizono S, Hoshino T, Kubo M, Yamashita A, Okabe M, Takeda K, Akira S, Matsumoto S, Toyonaga A, Sata M, Yoshimura A - J. Exp. Med. (2001)

Bottom Line: We have investigated the role of STATs and these JAK inhibitors in intestinal inflammation.Development of colitis as well as STAT3 activation was significantly reduced in IL-6-deficient mice treated with DSS, suggesting that STAT3 plays an important role in the perpetuation of colitis.These data suggest that hyperactivation of STAT3 results in severe colitis and that CIS3 plays a negative regulatory role in intestinal inflammation by downregulating STAT3 activity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Science, Kurume University, Kurume 839-0861, Japan.

ABSTRACT
Immune and inflammatory systems are controlled by multiple cytokines, including interleukins (ILs) and interferons. These cytokines exert their biological functions through Janus tyrosine kinases and signal transducer and activator of transcription (STAT) transcription factors. We recently identified two intrinsic Janus kinase (JAK) inhibitors, JAK binding protein (JAB; also referred to as suppressor of cytokine signaling [SOCS1]/STAT-induced STAT inhibitor [SSI1]) and cytokine-inducible SH2 protein (CIS)3 (or SOCS3/SSI3), which play an essential role in the negative regulation of cytokine signaling. We have investigated the role of STATs and these JAK inhibitors in intestinal inflammation. Among STAT family members, STAT3 was most strongly tyrosine phosphorylated in human ulcerative colitis and Crohn's disease patients as well as in dextran sulfate sodium (DSS)-induced colitis in mice. Development of colitis as well as STAT3 activation was significantly reduced in IL-6-deficient mice treated with DSS, suggesting that STAT3 plays an important role in the perpetuation of colitis. CIS3, but not JAB, was highly expressed in the colon of DSS-treated mice as well as several T cell-dependent colitis models. To define the physiological role of CIS3 induction in colitis, we developed a JAB mutant (F59D-JAB) that overcame the inhibitory effect of both JAB and CIS3 and created transgenic mice. DSS induced stronger STAT3 activation and more severe colitis in F59D-JAB transgenic mice than in their wild-type littermates. These data suggest that hyperactivation of STAT3 results in severe colitis and that CIS3 plays a negative regulatory role in intestinal inflammation by downregulating STAT3 activity.

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Expression and activation of STATs in human and mouse colon tissues. (A) Colon tissue extracts from the normal region (NR) of colon cancer patients (lane 1), those from UC or CD patients (lanes 2–4), or those from Balb/c mice treated without (day 0) or with 4% DSS for 7 d (lanes 5 and 6) were immunoblotted with antiphospho-STATs (PY-STAT) or anti-STAT antibodies. As a positive control (pos), IFN-γ–treated NIH-3T3 cells (for STAT1), LIF-treated CMT cells (STAT3), IL-3–treated Ba/F3 cells (STAT5), and IL-4–treated 32D cells (STAT6) are shown in lane 7. We analyzed more than seven patients and three DSS-treated mice, obtaining similar results. (B) STAT3 activation in UC and IC patients (lanes 1 and 2) as well as several mouse models (lanes 3–13). Colon samples were obtained from 10–15-wk-old WT C57BL6 mice (lanes 3 and 4), 16-wk-old IL-10−/− mice (lanes 5 and 6), 9-wk-old Mφ-STAT3−/− mice (lanes 7 and 8), 15-wk-old TCR−/− mouse (lane 9), TNBS-treated (lanes 11 and 12) or untreated (lane 10) mice, and CD4+ T cell–transplanted SCID mouse (lane 13). (C) Immunohistochemical detection of activated STAT3 in DSS-induced colitis. C57BL6 mice were treated with (b and d) or without (a and c) 2% DSS for 5 d, and colon tissues were fixed with 10% formalin. Slides with sectioned tissues of 7-μm thickness were immunostained with anti–phospho-STAT3–specific antibodies (c and d) or control IgG (a and b) and were lightly stained with hematoxylin.
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Figure 1: Expression and activation of STATs in human and mouse colon tissues. (A) Colon tissue extracts from the normal region (NR) of colon cancer patients (lane 1), those from UC or CD patients (lanes 2–4), or those from Balb/c mice treated without (day 0) or with 4% DSS for 7 d (lanes 5 and 6) were immunoblotted with antiphospho-STATs (PY-STAT) or anti-STAT antibodies. As a positive control (pos), IFN-γ–treated NIH-3T3 cells (for STAT1), LIF-treated CMT cells (STAT3), IL-3–treated Ba/F3 cells (STAT5), and IL-4–treated 32D cells (STAT6) are shown in lane 7. We analyzed more than seven patients and three DSS-treated mice, obtaining similar results. (B) STAT3 activation in UC and IC patients (lanes 1 and 2) as well as several mouse models (lanes 3–13). Colon samples were obtained from 10–15-wk-old WT C57BL6 mice (lanes 3 and 4), 16-wk-old IL-10−/− mice (lanes 5 and 6), 9-wk-old Mφ-STAT3−/− mice (lanes 7 and 8), 15-wk-old TCR−/− mouse (lane 9), TNBS-treated (lanes 11 and 12) or untreated (lane 10) mice, and CD4+ T cell–transplanted SCID mouse (lane 13). (C) Immunohistochemical detection of activated STAT3 in DSS-induced colitis. C57BL6 mice were treated with (b and d) or without (a and c) 2% DSS for 5 d, and colon tissues were fixed with 10% formalin. Slides with sectioned tissues of 7-μm thickness were immunostained with anti–phospho-STAT3–specific antibodies (c and d) or control IgG (a and b) and were lightly stained with hematoxylin.

Mentions: First, we examined which STAT is activated in colitis. Substantial amounts of STAT1, STAT3, STAT5, and STAT6 were detected in the colon of human UC and CD patients as well as DSS-treated mice. DSS-induced colitis is an experimental acute inflammatory bowel disease that is not dependent on T or B cells, as it occurs in SCID mice that lack lymphocytes 28. However, inflammatory cytokine production by macrophages and/or epithelial cells has been shown to contribute to the pathophysiology of colitis 5. Among STAT1, STAT3, STAT5, and STAT6, we could repeatedly detect the phosphorylation of STAT3 in the colon of DSS-treated mice as well as UC and CD patients (Fig. 1 A; PY-STAT), whereas the phosphorylation of STAT1, STAT5, or STAT6 was undetectable or very low by our assay. We also examined STAT3 activation in other types of intestinal inflammation. STAT3 phosphorylation was evident in a non-inflammatory bowel disease (IBD) infectious colitis patient (Fig. 1 B, lane 1). STAT3 activation was also detected in both Th1-dependent colitis in IL-10−/− mice (Fig. 1 B, lines 5 and 6) and macrophage- and neutrophil-specific STAT3 conditional knockout (Mφ-STAT3−/−) mice (reference 25; lines 7 and 8) as well as in IL-4–dependent colitis in TCR-α chain knockout (TCR−/−) mice (reference 32; lane 9). STAT3 activation was also observed in other T cell–dependent colitis induced by the haptenating reagent, TNBS (lanes 11 and 12) as well as by transplantation of CD45RBhighCD4+ T cells into SCID mice (line 13). Thus, STAT3 was activated not only in acute T cell–independent colitis but also in chronic T cell–dependent colitis. Clinical signs such as persistent diarrhea and the macroscopic examination of colon tissues indicated that the IL-10−/− mice suffered from much more severe colitis than the Mφ-STAT3−/− mice, probably due to the difference of age (IL-10−/− mice were 16 wk old, whereas Mφ-STAT3−/− mice were 9 wk old). Higher levels of STAT3 phosphorylation were observed in the IL-10−/− mice than in the Mφ-STAT3−/− mice (cf. lanes 5 and 6 and lanes 7 and 8). Furthermore, in the TNBS-induced colitis, one of the two mice (TNBS-1) showed much more severe symptoms along with stronger STAT3 phosphorylation than those of the other (TNBS-2; cf. lanes 11 and 12). The severity of colitis in the CD4+ T cell–transplanted SCID mouse was rather mild compared with other colitis that we examined in this study, and STAT3 phosphorylation in the colon was weak (lane 13). These data suggest that the levels of STAT3 phosphorylation were well correlated to the severity of colitis.


CIS3/SOCS3/SSI3 plays a negative regulatory role in STAT3 activation and intestinal inflammation.

Suzuki A, Hanada T, Mitsuyama K, Yoshida T, Kamizono S, Hoshino T, Kubo M, Yamashita A, Okabe M, Takeda K, Akira S, Matsumoto S, Toyonaga A, Sata M, Yoshimura A - J. Exp. Med. (2001)

Expression and activation of STATs in human and mouse colon tissues. (A) Colon tissue extracts from the normal region (NR) of colon cancer patients (lane 1), those from UC or CD patients (lanes 2–4), or those from Balb/c mice treated without (day 0) or with 4% DSS for 7 d (lanes 5 and 6) were immunoblotted with antiphospho-STATs (PY-STAT) or anti-STAT antibodies. As a positive control (pos), IFN-γ–treated NIH-3T3 cells (for STAT1), LIF-treated CMT cells (STAT3), IL-3–treated Ba/F3 cells (STAT5), and IL-4–treated 32D cells (STAT6) are shown in lane 7. We analyzed more than seven patients and three DSS-treated mice, obtaining similar results. (B) STAT3 activation in UC and IC patients (lanes 1 and 2) as well as several mouse models (lanes 3–13). Colon samples were obtained from 10–15-wk-old WT C57BL6 mice (lanes 3 and 4), 16-wk-old IL-10−/− mice (lanes 5 and 6), 9-wk-old Mφ-STAT3−/− mice (lanes 7 and 8), 15-wk-old TCR−/− mouse (lane 9), TNBS-treated (lanes 11 and 12) or untreated (lane 10) mice, and CD4+ T cell–transplanted SCID mouse (lane 13). (C) Immunohistochemical detection of activated STAT3 in DSS-induced colitis. C57BL6 mice were treated with (b and d) or without (a and c) 2% DSS for 5 d, and colon tissues were fixed with 10% formalin. Slides with sectioned tissues of 7-μm thickness were immunostained with anti–phospho-STAT3–specific antibodies (c and d) or control IgG (a and b) and were lightly stained with hematoxylin.
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Related In: Results  -  Collection

Show All Figures
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Figure 1: Expression and activation of STATs in human and mouse colon tissues. (A) Colon tissue extracts from the normal region (NR) of colon cancer patients (lane 1), those from UC or CD patients (lanes 2–4), or those from Balb/c mice treated without (day 0) or with 4% DSS for 7 d (lanes 5 and 6) were immunoblotted with antiphospho-STATs (PY-STAT) or anti-STAT antibodies. As a positive control (pos), IFN-γ–treated NIH-3T3 cells (for STAT1), LIF-treated CMT cells (STAT3), IL-3–treated Ba/F3 cells (STAT5), and IL-4–treated 32D cells (STAT6) are shown in lane 7. We analyzed more than seven patients and three DSS-treated mice, obtaining similar results. (B) STAT3 activation in UC and IC patients (lanes 1 and 2) as well as several mouse models (lanes 3–13). Colon samples were obtained from 10–15-wk-old WT C57BL6 mice (lanes 3 and 4), 16-wk-old IL-10−/− mice (lanes 5 and 6), 9-wk-old Mφ-STAT3−/− mice (lanes 7 and 8), 15-wk-old TCR−/− mouse (lane 9), TNBS-treated (lanes 11 and 12) or untreated (lane 10) mice, and CD4+ T cell–transplanted SCID mouse (lane 13). (C) Immunohistochemical detection of activated STAT3 in DSS-induced colitis. C57BL6 mice were treated with (b and d) or without (a and c) 2% DSS for 5 d, and colon tissues were fixed with 10% formalin. Slides with sectioned tissues of 7-μm thickness were immunostained with anti–phospho-STAT3–specific antibodies (c and d) or control IgG (a and b) and were lightly stained with hematoxylin.
Mentions: First, we examined which STAT is activated in colitis. Substantial amounts of STAT1, STAT3, STAT5, and STAT6 were detected in the colon of human UC and CD patients as well as DSS-treated mice. DSS-induced colitis is an experimental acute inflammatory bowel disease that is not dependent on T or B cells, as it occurs in SCID mice that lack lymphocytes 28. However, inflammatory cytokine production by macrophages and/or epithelial cells has been shown to contribute to the pathophysiology of colitis 5. Among STAT1, STAT3, STAT5, and STAT6, we could repeatedly detect the phosphorylation of STAT3 in the colon of DSS-treated mice as well as UC and CD patients (Fig. 1 A; PY-STAT), whereas the phosphorylation of STAT1, STAT5, or STAT6 was undetectable or very low by our assay. We also examined STAT3 activation in other types of intestinal inflammation. STAT3 phosphorylation was evident in a non-inflammatory bowel disease (IBD) infectious colitis patient (Fig. 1 B, lane 1). STAT3 activation was also detected in both Th1-dependent colitis in IL-10−/− mice (Fig. 1 B, lines 5 and 6) and macrophage- and neutrophil-specific STAT3 conditional knockout (Mφ-STAT3−/−) mice (reference 25; lines 7 and 8) as well as in IL-4–dependent colitis in TCR-α chain knockout (TCR−/−) mice (reference 32; lane 9). STAT3 activation was also observed in other T cell–dependent colitis induced by the haptenating reagent, TNBS (lanes 11 and 12) as well as by transplantation of CD45RBhighCD4+ T cells into SCID mice (line 13). Thus, STAT3 was activated not only in acute T cell–independent colitis but also in chronic T cell–dependent colitis. Clinical signs such as persistent diarrhea and the macroscopic examination of colon tissues indicated that the IL-10−/− mice suffered from much more severe colitis than the Mφ-STAT3−/− mice, probably due to the difference of age (IL-10−/− mice were 16 wk old, whereas Mφ-STAT3−/− mice were 9 wk old). Higher levels of STAT3 phosphorylation were observed in the IL-10−/− mice than in the Mφ-STAT3−/− mice (cf. lanes 5 and 6 and lanes 7 and 8). Furthermore, in the TNBS-induced colitis, one of the two mice (TNBS-1) showed much more severe symptoms along with stronger STAT3 phosphorylation than those of the other (TNBS-2; cf. lanes 11 and 12). The severity of colitis in the CD4+ T cell–transplanted SCID mouse was rather mild compared with other colitis that we examined in this study, and STAT3 phosphorylation in the colon was weak (lane 13). These data suggest that the levels of STAT3 phosphorylation were well correlated to the severity of colitis.

Bottom Line: We have investigated the role of STATs and these JAK inhibitors in intestinal inflammation.Development of colitis as well as STAT3 activation was significantly reduced in IL-6-deficient mice treated with DSS, suggesting that STAT3 plays an important role in the perpetuation of colitis.These data suggest that hyperactivation of STAT3 results in severe colitis and that CIS3 plays a negative regulatory role in intestinal inflammation by downregulating STAT3 activity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Science, Kurume University, Kurume 839-0861, Japan.

ABSTRACT
Immune and inflammatory systems are controlled by multiple cytokines, including interleukins (ILs) and interferons. These cytokines exert their biological functions through Janus tyrosine kinases and signal transducer and activator of transcription (STAT) transcription factors. We recently identified two intrinsic Janus kinase (JAK) inhibitors, JAK binding protein (JAB; also referred to as suppressor of cytokine signaling [SOCS1]/STAT-induced STAT inhibitor [SSI1]) and cytokine-inducible SH2 protein (CIS)3 (or SOCS3/SSI3), which play an essential role in the negative regulation of cytokine signaling. We have investigated the role of STATs and these JAK inhibitors in intestinal inflammation. Among STAT family members, STAT3 was most strongly tyrosine phosphorylated in human ulcerative colitis and Crohn's disease patients as well as in dextran sulfate sodium (DSS)-induced colitis in mice. Development of colitis as well as STAT3 activation was significantly reduced in IL-6-deficient mice treated with DSS, suggesting that STAT3 plays an important role in the perpetuation of colitis. CIS3, but not JAB, was highly expressed in the colon of DSS-treated mice as well as several T cell-dependent colitis models. To define the physiological role of CIS3 induction in colitis, we developed a JAB mutant (F59D-JAB) that overcame the inhibitory effect of both JAB and CIS3 and created transgenic mice. DSS induced stronger STAT3 activation and more severe colitis in F59D-JAB transgenic mice than in their wild-type littermates. These data suggest that hyperactivation of STAT3 results in severe colitis and that CIS3 plays a negative regulatory role in intestinal inflammation by downregulating STAT3 activity.

Show MeSH
Related in: MedlinePlus