Wenlong Lin

Phosphatase Holoenzyme PP1/GADD34 Negatively Regulates TLR Response by Inhibiting TAK1 Serine 412 Phosphorylation

The molecular mechanisms that fine tune TLRs responses need to be fully elucidated. Protein phosphatase-1 (PP1) has been shown to be important in cell death and differentiation. However, the roles of PP1 in TLR-triggered immune response remain unclear. In this study, we demonstrate that PP1 inhibits the activation of the MAPK and NF-κB pathway and the production of TNF-α, IL-6 in macrophages triggered by TLR3, TLR4, and TLR9 in a phosphatase-dependent manner. Conversely, PP1 knockdown increases TLRs-triggered signaling and proinflammatory cytokine production. Tautomycetin, a specific inhibitor of PP1, aggravates LPS-induced endotoxin shock in mice. We further demonstrate that PP1 negatively regulates TLR-triggered signaling by targeting TGF-β–activated kinase 1 (TAK1) serine 412 (Ser412) phosphorylation, which is required for activation of TAK1-mediated IL-1R and TLR signaling. Mutation of TAK1 Serine 412 to alanine (S412A) significantly inhibits TLR/IL-1R–triggered NF-κB and MAPK activation and induction of proinflammatory cytokines in macrophage and murine embryonic fibroblast cells. DNA damage-inducible protein 34 (GADD34) specifies PP1 to dephosphorylate TAK1 at Ser412. GADD34 depletion abolished the interaction between TAK1 and PP1, and it relieved PP1 overexpression-induced inhibition of TLRs signaling and proinflammatory cytokine production. In addition, knockdown of GADD34 significantly promotes TLR-induced TAK1 Ser412 phosphorylation, downstream NF-κB and MAPK activation, and proinflammatory cytokine production. Therefore, PP1, as a physiologic inhibitor, together with its regulatory subunit GADD34, tightly controls TLR-induced TAK1 Ser412 phosphorylation, preventing excessive activation of TLRs and protecting the host from overwhelmed inflammatory immune responses.

Protein phosphatase PP1 negatively regulates the Toll-like receptor- and RIG-I-like receptor-triggered production of type I interferon by inhibiting IRF3 phosphorylation at serines 396 and 385 in macrophage.

The production of type I interferon must be tightly regulated, and the aberrant production of this protein is harmful or even fatal to the host. The transcription factor IRF3 phosphorylation is a central regulator of type I interferon meditated antiviral response. Protein phosphatase-1 (PP1) has been reported to be important in many cell functions, including development, differentiation, and tumorigenesis. However, the roles of PP1 in Toll-like receptor (TLR)- or retinoic acid-inducible gene I like receptor (RLR)-triggered IRF-3 activation remain unclear. Here, we show that the activity of PP1 is downregulated in macrophages upon stimulation with TLR or RLR ligands, including lipopolysaccharide, and poly(I:C), or vesicular stomatitis virus (VSV), respectively. The overexpression of PP1 selectively inhibits TLR- and VSV-induced interferon regulatory factor 3 (IRF3) activation but has no substantial effect on TANK-binding kinase 1 (TBK1),ΚB kinase ε (IKKε) activation. Conversely, RNA interference of PP1 significantly promotes IRF3 activation. Consistently, The overexpression of PP1 inhibits TLR- and VSV-triggered IFN-β production while PP1 knockdown significantly increases the production of IFN-β in macrophages. We further demonstrate that PP1 directly interacts with IRF3 and dephosphorylates IRF3 at Ser385 and Ser396, resulting in the suppression of TLR- and RLR-triggered IFN-β production. Thus, PP1 functions as a negative feedback regulator of TLR- and RLR-triggered antiviral immune responses by acting as an IRF3 phosphatase.

Raf kinase inhibitor protein mediates intestinal epithelial cell apoptosis and promotes IBDs in humans and mice.

Objective Raf kinase inhibitor protein (RKIP) appears to control cancer cell metastasis and its expression in colonic tissue is related to colonic cancer development. We sought to identify the roles of RKIP in maintaining homeostasis of GI tract. Design The expression of RKIP was determined by immunohistochemistry and western blot analysis. RKIP knockout and wild-type mice were administered dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce experimental colitis, and the mice were assessed based on colitis symptoms and biochemical approaches. The mechanism was analysed using immunoprecipitation and pull-down experiments. Results The RKIP expression is positively correlated with the severity of IBD. RKIP deficiency protects mice from DSS-induced or TNBS-induced colitis and accelerated recovery from colitis. RKIP deficiency inhibits DSS-induced infiltration of acute-phase immune cells and reduces production of proinflammatory cytokines and chemokines in colon. RKIP deficiency inhibits DSS-induced or TNBS-induced colonic epithelial barrier damage and intestinal epithelial cell (IEC) apoptosis. RKIP deficiency also inhibits tumour necrosis factor-alpha-induced IEC apoptosis and colitis. Mechanistically, RKIP enhances the induction of P53-upregulated modulator of apoptosis by interacting with TGF-β-activated kinase 1 (TAK1) and promoting TAK1-mediated NF-κB activation. This is supported by the observation that TAK1 activation is positively correlated with the expression of RKIP in human clinical samples and the development of IBD. Conclusions RKIP contributes to colitis development by promoting inflammation and mediating IEC apoptosis and might represent a therapeutic target of IBD.

NDR1 protein kinase promotes IL‐17‐ and TNF‐α‐mediated inflammation by competitively binding TRAF3

Interleukin 17 (IL‐17) is an important inducer of tissue inflammation and is involved in numerous autoimmune diseases. However, how its signal transduction is regulated is not well understood. Here, we report that nuclear Dbf2‐related kinase 1 (NDR1) functions as a positive regulator of IL‐17 signal transduction and IL‐17‐induced inflammation. NDR1 deficiency or knockdown inhibits the IL‐17‐induced phosphorylation of p38, ERK1/2, and p65 and the expression of chemokines and cytokines, whereas the overexpression of NDR1 promotes IL‐17‐induced signaling independent of its kinase activity. Mechanistically, NDR1 interacts with TRAF3 and prevents its binding to IL‐17R, which promotes the formation of an IL‐17R‐Act1‐TRAF6 complex and downstream signaling. Consistent with this, IL‐17‐induced inflammation is significantly reduced in NDR1‐deficient mice, and NDR1 deficiency significantly protects mice from MOG‐induced experimental autoimmune encephalomyelitis (EAE) and 2,4,6‐trinitrobenzenesulfonic acid (TNBS)‐induced colitis likely by its inhibition of IL‐17‐mediated signaling pathway. NDR1 expression is increased in the colons of ulcerative colitis (UC) patients. Taken together, these findings suggest that NDR1 is involved in the development of autoimmune diseases.

RKIP and TBK1 form a positive feedback loop to promote type I interferon production in innate immunity

TANK‐binding kinase 1 (TBK1) activation is a central event in type I interferon production in anti‐virus innate immunity. However, the regulatory mechanism underlying TBK1 activation remains unclear. Here we report that Raf kinase inhibitory protein (RKIP) is essential for TBK1 activation and type I interferon production triggered by viral infection. Upon viral infection, RKIP is phosphorylated at serine 109 (S109) by TBK1. Phosphorylation of RKIP enhances its interaction with TBK1 and in turn promotes TBK1 autophosphorylation. Mutation of RKIP S109 to alanine abrogates the interaction between RKIP and TBK1, and the anti‐viral function of RKIP. RKIP deficiency inhibits intracellular double‐stranded RNA‐ or DNA‐induced type I interferon production. Consistently, RKIP deficiency renders the mice more susceptible to vesicular stomatitis virus (VSV) and herpes simplex virus (HSV) infections. This study reveals a previously unrecognized positive feedback loop between RKIP and TBK1 that is essential for type I interferon production in anti‐viral innate immunity.

Raf Kinase Inhibitor Protein Preferentially Promotes TLR3-Triggered Signaling and Inflammation

Raf kinase inhibitor protein (RKIP) protects against host immunological responses in nematodes and Drosophila. Whether RKIP functions in innate immune responses in mammals remains unknown. In this article, we report that RKIP preferentially regulates the TLR3-mediated immune response in macrophages. RKIP deficiency or silencing significantly decreases polyinosinic:polycytidylic acid [Poly(I:C)]-induced IFN-β, IL-6, and TNF-α production without affecting the counterpart induced by LPS or CpG. Compared with their wild-type counterparts, RKIP-deficient mice produce less IFN-β, IL-6, and TNF-α in serum and display decreased lethality upon peritoneal Poly(I:C) plus d-galactosamine injection. Mechanistically, RKIP interacts with TBK1 and promotes the Poly(I:C)-induced TANK-binding kinase 1/IRF3 activation. Simultaneously, RKIP enhances the Poly(I:C)-induced interaction between TGF-β–activated kinase 1 and MAPK kinase 3 (MKK3), thus promoting MKK3/6 and p38 activation. We further demonstrated that Poly(I:C) treatment, but not LPS treatment, induces RKIP phosphorylation at S109. This action is required for RKIP to promote TANK-binding kinase 1 activation, as well as the interaction between TGF-β–activated kinase 1 and MKK3, which lead to activation of the downstream IRF3 and p38, respectively. Therefore, RKIP acts as a positive-feedback regulator of the TLR3-induced inflammatory response and may be a potential therapeutic target for inflammatory disease.

RKIP mediates autoimmune inflammation by positively regulating IL‐17R signaling

Th17 cells contribute to the development of autoimmune diseases by secreting interleukin‐17 (IL‐17), which activates its receptor (IL‐17R) that is expressed on epithelial cells, macrophages, microglia, and resident neuroectodermal cells. However, the mechanisms through which IL‐17R‐mediated signaling contributes to the development of autoimmune disease have not been completely elucidated. Here, we demonstrate that Raf‐1 kinase inhibitor protein (RKIP) deficiency in mice ameliorates the symptoms of experimental autoimmune encephalomyelitis (EAE). Adoptive T‐cell‐transfer experiments demonstrate that RKIP plays a predominant role in Th17‐mediated, but not in Th1‐mediated immune responses. RKIP deficiency has no effect on Th17‐cell differentiation ex vivo, nor does it affect Th17‐cell differentiation in EAE mice. However, RKIP significantly promotes IL‐17R‐induced proinflammatory cytokine and chemokine production. Mechanistically, RKIP directly interacts with IL‐17RA and Act1 to promote the formation of an IL‐17R‐Act1 complex, resulting in enhanced MAPK‐ and P65‐mediated NF‐κB activation and downstream cytokine production. Together, these findings indicate that RKIP functions as an essential modulator of the IL‐17R‐Act1 axis in IL‐17R signaling, which promotes IL‐17‐induced inflammation and autoimmune neuroinflammation.

Raf kinase inhibitor protein negatively regulates FcεRI-mediated mast cell activation and allergic response

Significance Mast cell activation contributes to multiple allergic disorders, such as asthma, rhinitis, and atopic dermatitis. Here, we demonstrate that the Raf kinase inhibitor protein (RKIP) functions as an inhibitor of mast cell activation. RKIP negatively regulates the pathogeneses of the mast cell-mediated anaphylactic response and allergic asthma in vivo. Furthermore, the expression of RKIP was significantly down-regulated in peripheral blood from asthma patients. Collectively, our findings not only suggest that RKIP plays an important role in controlling mast cell-mediated allergic responses but also provide insight into therapeutic targets for mast cell-related allergic diseases. The signaling cascades triggered by the cross-linkage of immunoglobulin E (IgE) with its high-affinity receptor (FcεRI) on mast cells contribute to multiple allergic disorders, such as asthma, rhinitis, and atopic dermatitis. Restraint of intracellular signals for mast cell activation is essential to restore homeostasis. In this study, we found that Raf kinase inhibitor protein (RKIP) negatively regulated mast cell activation. RKIP-deficient mast cells showed greater IgE−FcεRI-mediated activation than wild-type mast cells. Consistently, RKIP deficiency in mast cells rendered mice more sensitive to IgE−FcεRI-mediated allergic responses and ovalbumin-induced airway inflammation. Mechanistically, RKIP interacts with the p85 subunit of PI3K, prevents it from binding to GRB2-associated binding protein 2 (Gab2), and eventually inhibits the activation of the PI3K/Akt/NF-κB complex and its downstream signaling. Furthermore, the expression of RKIP was significantly down-regulated in the peripheral blood of asthma patients and in the IgE−FcεRI-stimulated mast cells. Collectively, our findings not only suggest that RKIP plays an important role in controlling mast cell-mediated allergic responses but also provide insight into therapeutic targets for mast cell-related allergic diseases.

Protein Kinase Serine/Threonine Kinase 24 Positively Regulates Interleukin 17-Induced Inflammation by Promoting IKK Complex Activation

Interleukin 17 (IL-17) is a key inflammatory cytokine that plays a critical role in tissue inflammation and autoimmune diseases. However, its signaling remains poorly understood. In this study, we identified serine/threonine kinase 24 (Stk24) as a positive modulator of IL-17-mediated signaling and inflammation. Stk24 deficiency or knockdown markedly inhibited IL-17-induced phosphorylation of NF-κB and impaired IL-17-induced chemokines and cytokines expression. Stk24 overexpression greatly enhanced IL-17-induced NF-κB activation and expression of chemokines and cytokines in a kinase activity-independent manner. The IL-17-induced inflammatory response was significantly reduced in Stk24-deficient mice. In addition, the severity of experimental autoimmune encephalomyelitis was markedly reduced in mice with a deficiency of Stk24 in non-hematopoietic cells. We further demonstrated that Stk24 directly interacts with TAK1 and IKKβ and promotes the formation of TAK1/IKK complexes, leading to enhanced IKKβ/NF-κB activation and downstream cytokines and chemokines induction. Collectively, our findings suggest that Stk24 plays an important role in controlling IL-17-triggered inflammation and autoimmune diseases and provides new insight into the therapeutic targets of IL-17-mediated inflammatory disease.