Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37

The closure of skin wounds is essential for resistance against microbial pathogens, and keratinocyte migration is an important step in skin wound healing. Cathelicidin hCAP18/LL-37 is an innate antimicrobial peptide that is expressed in the skin and acts to eliminate microbial pathogens. Because hCAP18/LL-37 is up-regulated at skin wound sites, we hypothesized that LL-37 induces keratinocyte migration. In this study, we found that 1 microg/ml LL-37 induced the maximum level of keratinocyte migration in the Boyden chamber assay. In addition, LL-37 phosphorylated the epidermal growth factor receptor (EGFR) after 10 min, which suggests that LL-37-induced keratinocyte migration occurs via EGFR transactivation. To test this assumption, we used inhibitors that block the sequential steps of EGFR transactivation, such as OSU8-1, CRM197, anti-EGFR no. 225 Ab, and AG1478. All of these inhibitors completely blocked LL-37-induced keratinocyte migration, which indicates that migration occurs via HB-EGF-mediated EGFR transactivation. Furthermore, CRM197, anti-EGFR no. 225, and AG1478 blocked the LL-37-induced phosphorylation of STAT3, and transfection with a dominant-negative mutant of STAT3 abolished LL-37-induced keratinocyte migration, indicating the involvement of the STAT3 pathway downstream of EGFR transactivation. Finally, we tested whether the suppressor of cytokine signaling (SOCS)/cytokine-inducible Src homology 2-containing protein (CIS) family of negative regulators of STAT3 regulates LL-37-induced keratinocyte migration. Transfection with SOCS1/Jak2 binding protein or SOCS3/CIS3 almost completely abolished LL-37-induced keratinocyte migration. In conclusion, LL-37 induces keratinocyte migration via heparin-binding-EGF-mediated transactivation of EGFR, and SOCS1/Jak 2 binding and SOCS3/CIS3 negatively regulate this migration. The results of this study suggest that LL-37 closes skin wounds by the induction of keratinocyte migration.     

HCV核心蛋白抑制干扰素α诱导的抗病毒基因表达及其机制研究

研究HCV核心蛋白对干扰素α诱导的抗病毒分子PKR和2′-5′OAS表达的影响及其机制。HCV核心蛋白表达质粒转染HepG2细胞,RT-PCR分析PKR和2′-5′OAS的mRNA水平变化,荧光素酶活性分析核心蛋白对ISRE介导的基因表达的影响;Western-blot分析SOCS3、STAT1及STAT1磷酸化水平的变化。在干扰素α刺激情况下,表达HCV核心蛋白的细胞中,PKR和2′-5′OAS的mRNA水平下降,ISRE介导的荧光素酶活性降低,STAT1磷酸化水平下降。此外,核心蛋白表达的细胞中SOCS3的mRNA和蛋白水平明显升高。结果表明,HCV核心蛋白可能通过激活SOCS3、抑制STAT1的磷酸化,从而下调干扰素α诱导的PKR和2′-5′OAS表达。     

Restoration of SOCS3 suppresses human lung adenocarcinoma cell growth by downregulating activation of Erk1/2, Akt apart from STAT3

SOCS3 is regarded as a major negative regulator of STAT3. Recent evidence indicates that SOCS3 regulates strength and duration of other signaling pathways including ras/ERK1/2/MAPK, PI3-K/Akt in non-malignant cells. The repression or silence of SOCS3 expression in a few tumor types has led to speculation that loss of SOCS3 gene is closely related to deregulation of multiple signal pathways during tumorigenesis. However, apart from STAT3, little is known in malignant cells about the mechanism by which SOCS3 modulates other intracellular signal cascades such as Erk1/2 and Akt, whose aberrant activation has been implicated in many human tumors. Expression of SOCS3 proved deficient in human lung adenocarcinoma A549 cells, and forced expression of SOCS3 resulted in growth inhibition. Growth suppression due to SOCS3 was associated with attenuated activation of Erk1/2, Akt as well as STAT3. The results suggested that SOCS3, as negative regulators of cytokine signaling, might maintain homeostasis by regulating multiple signaling pathways and reverse cell malignant behavior.     

SOCS3 regulates p21 expression and cell cycle arrest in response to DNA damage

Genotoxic agents such as ionizing radiation trigger cell cycle arrest at the G1/S and G2/M checkpoints, allowing cells to repair damaged DNA before entry into mitosis. DNA damage-induced G1 arrest involves p53-dependent expression of p21 (Cip1/Waf-1), which inhibits cyclin-dependent kinases and blocks S phase entry. While much of the core DNA damage response has been well-studied, other signaling proteins that intersect with and modulate this response remain uncharacterized. In this study, we identify Suppressor of Cytokine Signaling (SOCS)-3 as an important regulator of radiation-induced G1 arrest. SOCS3-deficient fibroblasts fail to undergo G1 arrest and accumulate in the G2/M phase of the cell cycle. SOCS3 knockout cells phosphorylate p53 and H2AX normally in response to radiation, but fail to upregulate p21 expression. In addition, STAT3 phosphorylation is elevated in SOCS3-deficient cells compared to WT cells. Normal G1 arrest can be restored in SOCS3 KO cells by retroviral transduction of WT SOCS3 or a dominant-negative mutant of STAT3. Our results suggest a novel function for SOCS3 in the control of genome stability by negatively regulating STAT3-dependent radioresistant DNA synthesis, and promoting p53-dependent p21 expression.     

miR-155靶向SOCS1对骨肉瘤Saos2细胞的增殖、侵袭和迁移能力的影响

目的:探讨microRNA-155(miR-155)对骨肉瘤Saos2细胞增殖、侵袭和迁移的影响以及其作用机制。方法:利用实时荧光定量(qRT-PCR)实验检测miR-155在正常成骨细胞与骨肉瘤Saos2细胞中的表达水平,以及miR-155-mimic、miR-155-inhibitor的转染效率。采用CCK-8实验检测细胞的增殖能力,Transwell实验和划痕实验分别检测Saos2细胞的侵袭和迁移能力,Western blot检测细胞内的STAT3磷酸化水平以及SOCS1表达水平,双荧光素酶报告基因实验进行靶基因验证。结果:miR-155在骨肉瘤Saos2细胞中表达明显高于正常成骨细胞(P0.001)。在分别转染miR-155-mimic和miR-155-inhibitor后,Saos2细胞内miR-155表达水平明显上调和下降(P0.001)。过表达miR-155可促进Saos2细胞增殖、侵袭和迁移,降低SOCS1的蛋白水平,上调STAT3的磷酸化水平,差异均具有统计学意义。相反,降低miR-155水平可抑制Saos2细胞的增殖、侵袭和迁移能力,差异均具有统计学意义。结论:骨肉瘤Saos2细胞中高表达的miR-155可以通过抑制SOCS1表达来激活STAT3信号通路进而促进细胞的增殖、侵袭和迁移,因此,靶向抑制miR-155表达可以作为潜在治疗骨肉瘤的途径。     

Ciliary Neurotrophic Factor Protects Mice Against Streptozotocin-induced Type 1 Diabetes through SOCS3: THE ROLE OF STAT1/STAT3 RATIO IN β-CELL DEATH*

Type 1 diabetes is characterized by a loss of islet β-cells. Ciliary neurotrophic factor (CNTF) protects pancreatic islets against cytokine-induced apoptosis. For this reason, we assessed whether CNTF protects mice against streptozotocin-induced diabetes (a model of type 1 diabetes) and the mechanism for this protection. WT and SOCS3 knockdown C57BL6 mice were treated for 5 days with citrate buffer or 0.1 mg/kg CNTF before receiving 80 mg/kg streptozotocin. Glycemia in non-fasted mice was measured weekly from days 0–28 after streptozotocin administration. Diabetes was defined as a blood glucose > 11.2 mmol/liter. Wild-type (WT) and SOCS3 knockdown MIN6 cells were cultured with CNTF, IL1β, or both. CNTF reduced diabetes incidence and islet apoptosis in WT but not in SOCS3kd mice. Likewise, CNTF inhibited apoptosis in WT but not in SOCS3kd MIN6 cells. CNTF increased STAT3 phosphorylation in WT and SOCS3kd mice and MIN6 cells but reduced STAT1 phosphorylation only in WT mice, in contrast to streptozotocin and IL1β. Moreover, CNTF reduced NFκB activation and required down-regulation of inducible NO synthase expression to exert its protective effects. In conclusion, CNTF protects mice against streptozotocin-induced diabetes by increasing pancreatic islet survival, and this protection depends on SOCS3. In addition, SOCS3 expression and β-cell fate are dependent on STAT1/STAT3 ratio.     

Taenia crassiceps infection and its excreted/secreted products inhibit STAT1 activation in response to IFN-γ

It is well understood that helminth infections modulate the immune responses of their hosts but the mechanisms involved in this modulation are not fully known. Macrophages and dendritic cells appear to be consistently affected during this type of infection and are common target cells for helminth-derived molecules. In this report, we show that macrophages obtained from chronically Taenia crassiceps-infected mice displayed an impaired response to recombinant murine IFN-γ, but not to recombinant murine IL-4, as measured based on the phosphorylation of STAT1 and STAT6, respectively. These macrophages expressed high levels of SOCS3. However, the inhibition of phosphatase activity by orthovanadate restored the IFN-γ response of these macrophages by increasing STAT1 phosphorylation without affecting SOCS3 expression. Therefore, we aimed to identify the phosphatases associated with IFN-γ signaling inhibition and found that macrophages from T. crassiceps-infected mice displayed enhanced SHP-1 expression. Interestingly, the exposure of naïve macrophages to T. crassiceps excreted/secreted products similarly interfered with IFN-γ-induced STAT1 phosphorylation. Moreover, macrophages exposed to T. crassiceps excreted/secreted products expressed high levels of SOCS3 as well as SHP-1. Strikingly, human peripheral blood mononuclear cells that were exposed to T. crassiceps excreted/secreted products in vitro also displayed impaired STAT1 phosphorylation in response to IFN-γ; again, phosphatase inhibition abrogated the T. crassiceps excreted/secreted product-altered IFN-γ signaling. These data demonstrate a new mechanism by which helminth infection and the products derived during this infection target intracellular pathways to block the response to inflammatory cytokines such as IFN-γ in both murine and human cells.     

Novel interferon-lambdas induce antiproliferative effects in neuroendocrine tumor cells

Interferon-alpha (IFN-alpha) is used for biotherapy of neuroendocrine carcinomas. The interferon-lambdas (IL-28A/B and IL-29) are a novel group of interferons. In this study, we investigated the effects of the IFN-lambdas IL-28A and IL-29 on human neuroendocrine BON1 tumor cells. Similar to IFN-alpha, incubation of BON1 cells with IL-28A (10 ng/ml) and IL-29 (10 ng/ml) induced phosphorylation of STAT1, STAT2, and STAT3, significantly decreased cell numbers in a proliferation assay, and induced apoptosis as demonstrated by poly(ADP-ribose) polymerase (PARP)-cleavage, caspase-3-cleavage, and DNA-fragmentation. Stable overexpression of suppressor of cytokine signaling proteins (SOCS1 and SOCS3) completely abolished the aforementioned effects indicating that SOCS proteins act as negative regulators of IFN-lambda signaling in BON1 cells. In conclusion, the novel IFN-lambdas IL-28A and IL-29 potently induce STAT signaling and antiproliferative effects in neuroendocrine BON1 tumor cells. Thus, IFN-lambdas may hint a promising new approach in the antiproliferative therapy of neuroendocrine tumors.     

Human primary keratinocytes show restricted ability to up-regulate suppressor of cytokine signaling (SOCS)3 protein compared with autologous macrophages

Suppressor of cytokine signaling (SOCS)3 belongs to a family of proteins that are known to exert important functions as inducible feedback inhibitors and are crucial for the balance of immune responses. There is evidence for a deregulated immune response in chronic inflammatory skin diseases. Thus, it was the aim of this study to investigate the regulation of SOCS proteins involved in intracellular signaling pathways occurring during inflammatory skin diseases and analyze their impact on the course of inflammatory responses. Because we and others have previously described that the cytokine IL-27 has an important impact on the chronic manifestation of inflammatory skin diseases, we focused here on the signaling induced by IL-27 in human primary keratinocytes compared with autologous blood-derived macrophages. Here, we demonstrate that SOCS3 is critically involved in regulating the cell-specific response to IL-27. SOCS3 was found to be significantly up-regulated by IL-27 in macrophages but not in keratinocytes. Other STAT3-activating cytokines investigated, including IL-6, IL-22, and oncostatin M, also failed to up-regulate SOCS3 in keratinocytes. Lack of SOCS3 up-regulation in skin epithelial cells was accompanied by prolonged STAT1 and STAT3 phosphorylation and enhanced CXCL10 production upon IL-27 stimulation compared with macrophages. Overexpression of SOCS3 in keratinocytes significantly diminished this enhanced CXCL10 production in response to IL-27. We conclude from our data that keratinocytes have a cell type-specific impaired capacity to up-regulate SOCS3 which may crucially determine the course of chronic inflammatory skin diseases.     

SOCS-1 inhibits expression of the antiviral proteins 2',5'-OAS and MxA induced by the novel interferon-lambdas IL-28A and IL-29

Recently, we have shown that SOCS-1/3 overexpression in hepatic cells abrogates signaling of type I interferons (IFN) which may contribute to the frequently observed IFN resistance of hepatitis C virus (HCV). IFN-lambdas (IL-28A/B and IL-29), a novel group of IFNs, also efficiently inhibit HCV replication in vitro with potentially less hematopoietic side effects than IFN-alpha because of limited receptor expression in hematopoietic cells. To further evaluate the potential of IFN-lambdas in chronic viral hepatitis, we examined the influence of SOCS protein expression on IFN-lambda signaling. First, we show that hepatic cell lines express the IFN-lambda receptor complex consisting of IFN-lambdaR1 (IL-28R1) and IL-10R2. Whereas in mock-transfected HepG2 cells, IL-28A and IL-29 induced STAT1 and STAT3 phosphorylation, overexpression of SOCS-1 completely abrogated IL-28A and IL-29-induced STAT1/3 phosphorylation. Similarly, IL-28A and IL-29 induced mRNA expression of the antiviral proteins 2',5'-OAS and MxA was abolished by overexpression of SOCS-1. In conclusion, we assume that despite antiviral properties of IFN-lambdas, their efficacy as antiviral agents may have similar limitations as IFN-alpha due to inhibition by SOCS proteins.     

pSTAT3和SOCS3在人乳腺癌组织中的表达及意义

目的 研究激活STAT3( pSTAT3)蛋白和SOCS3在人乳腺癌和乳腺良性病变组织中的蛋白表达及其临床意义.方法 应用免疫组织化学检测160例乳腺癌和36例乳腺良性病变组织pSTAT3和SOCS3蛋白的表达情况,分析它们与患者临床病理特征的关系.结果 人乳腺癌组织中pSTAT3和SOCS3蛋白表达阳性率分别为69.4％和40.0％,乳腺良性病变组织中pSTAT3和SOCS3蛋白表达阳性率分别为33.3％和22.2％,前者与后者相比具有统计学意义(P＜0.01和P＜0.05);乳腺癌pSTAT3蛋白表达与肿瘤的大小、淋巴结转移和临床分期均呈显著正相关(均P＜0.01),但与患者年龄、肿瘤的组织学分级、雌孕激素受体表达和c-erBb-2表达均无显著相关(均P＞0.05);SOCS3蛋白表达与肿瘤大小呈显著正相关(P＜0.05),但与患者年龄、淋巴结转移、临床分期、肿瘤的组织学分级、雌孕激素受体表达和c-erBb-2表达均无显著相关(均P＞0.05);乳腺癌pSTAT3和SOCS3表达呈显著正相关(r=0.237,P＜0.01).结论 乳腺癌pSTAT3和SOCS3表达状况与肿瘤生长、侵袭和转移等呈密切相关,提示STAT3和SOCS3可能在乳腺癌发生发展过程中发挥了重要作用.