Src Homology 2-containing Protein Tyrosine Research Articles

Inhibiting SHP2 reduces glycolysis, promotes microglial M1 polarization, and alleviates secondary inflammation following spinal cord injury in a mouse model.

JOURNAL/nrgr/04.03/01300535-202503000-00030/figure1/v/2024-06-17T092413Z/r/image-tiff Reducing the secondary inflammatory response, which is partly mediated by microglia, is a key focus in the treatment of spinal cord injury. Src homology 2-containing protein tyrosine phosphatase 2 (SHP2), encoded by PTPN11, is widely expressed in the human body and plays a role in inflammation through various mechanisms. Therefore, SHP2 is considered a potential target for the treatment of inflammation-related diseases. However, its role in secondary inflammation after spinal cord injury remains unclear. In this study, SHP2 was found to be abundantly expressed in microglia at the site of spinal cord injury. Inhibition of SHP2 expression using siRNA and SHP2 inhibitors attenuated the microglial inflammatory response in an in vitro lipopolysaccharide-induced model of inflammation. Notably, after treatment with SHP2 inhibitors, mice with spinal cord injury exhibited significantly improved hind limb locomotor function and reduced residual urine volume in the bladder. Subsequent in vitro experiments showed that, in microglia stimulated with lipopolysaccharide, inhibiting SHP2 expression promoted M2 polarization and inhibited M1 polarization. Finally, a co-culture experiment was conducted to assess the effect of microglia treated with SHP2 inhibitors on neuronal cells. The results demonstrated that inflammatory factors produced by microglia promoted neuronal apoptosis, while inhibiting SHP2 expression mitigated these effects. Collectively, our findings suggest that SHP2 enhances secondary inflammation and neuronal damage subsequent to spinal cord injury by modulating microglial phenotype. Therefore, inhibiting SHP2 alleviates the inflammatory response in mice with spinal cord injury and promotes functional recovery postinjury.

Abstract A094: Preclinical characterization of HMPL-415, a second-generation SHP2 inhibitor

Abstract Background: Oncogenic activation of the RAS/MAPK signaling pathway is one of the leading causes for driving a variety of cancers. Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) functions downstream of multiple RTKs, and integrates growth factor signals to promote RAS activation, making it an attractive drug target for cancers harboring oncogenic alterations in RAS/MAPK pathway. Herein, we present the preclinical characterization of HMPL-415, a highly potent, selective and non-competitive SHP2 inhibitor, discovered and being currently developed in phase I clinical trial (NCT05886374) by HUTCHMED. Methods: The inhibition of HMPL-415 on the full length and catalytic domain of SHP2 was detected by a biochemical DIFMUP pseudosubstracte-fluorgenic assay. For selectivity, HMPL-415 was assessed against a panel of 413 kinases and 21 phosphatases by Eurofins. The modulation of HMPL-415 on RAS/MAPK signaling cascades was evaluated by western blot. In vitro anti-proliferation activity was measured by CellTiter-Glo luminescent assay. Multiple human xenograft tumors with RAS/MAPK pathway activation were applied in immune-deficient mice to investigate the anti-tumor activity of HMPL-415. Results: In vitro, HMPL-415 displayed higher potency in enzyme (5 fold) and cellular target inhibition (> 20 fold) compared to first-generation SHP inhibitor TNO155. It strongly inhibited full length of human SHP2 with an IC50 of 0.4 nM, while had no inhibitory activity up to 10 µM against the catalytic domain of SHP2. In NCI-H358 (KrasG12C), NCI-H508 (class Ⅲ BRAFG596R), and NCI-H1838 (NF1N184fs) cells, HMPL-415 inhibited p-ERK with IC50s of 1~3 nM, and significantly suppressed downstream molecules p-RSK and p-S6. Through inhibition of RAS/MAPK signaling pathway, HMPL-415 robustly inhibited cell proliferation in a panel of human cancer cell lines with KRAS G12C/V, BRAF class Ⅲ, NF1 loss of function (LOF), RTK mutations or KRAS amplification (median GI50 =1 nM) compared to cell lines harboring KRAS G12D, G13 or Q61 mutations (median GI50 >1 µM), indicating a dependence on specific KRAS nucleotide-cycling for anti-tumor effect. Furthermore, the selectivity across panels of 413 kinases and 21 phosphatases revealed that HMPL-415 should have a low off-target risk. In vivo, oral administration of HMPL-415 showed prolonged and high tumor exposure and sustained inhibition on RAS/MAPK signaling after repeat dosing. In human xenograft models carrying KRAS, BRAF class Ⅲ, NF1LOF, and EGFR alterations, HMPL-415 dose-dependently induced tumor growth inhibition at continuous daily doses that were well tolerated. Tumor regression was observed in most tumor models tested at the dose of 3 mg/kg/day, which demonstrated much superior anti-tumor activity than that of TNO155 at 30 mg/kg/day. Conclusion: HMPL-415 is a highly potent, selective and non-competitive SHP2 inhibitor that demonstrates strong anti-tumor activity in vitro and in vivo, supporting further clinical evaluation. Citation Format: Jia Hu, Jun Ni, Zhihu Gao, Xiaoqing Liu, Hui Zhang, Pan Wang, Min Cheng, Guanglin Wang, Zeyu Zhong, Jian Wang, Yang Sai, Na Yang, Weiguo Qing, Yongxin Ren, Michael Shi, Weiguo Su. Preclinical characterization of HMPL-415, a second-generation SHP2 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A094.

Discovery of a Novel Series of Potent SHP2 Allosteric Inhibitors.

Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) is the first reported nonreceptor oncogenic tyrosine phosphatase connecting multiple signal transduction cascades and exerting immunoinhibitory function through the PD-1 checkpoint receptor. As part of a drug discovery program aimed at obtaining novel allosteric SHP2 inhibitors, a series of pyrazopyrazine derivatives bearing an original bicyclo[3.1.0]hexane basic moiety on the left-hand side region of the molecule were identified. We report herein the discovery process, the in vitro pharmacological profile, and the early developability features of compound 25, one of the most potent members of the series.

A comprehensive review of SHP2 and its role in cancer

Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) is a non-receptor protein tyrosine phosphatase ubiquitously expressed mainly in the cytoplasm of several tissues. SHP2 modulates diverse cell signaling events that control metabolism, cell growth, differentiation, cell migration, transcription and oncogenic transformation. It interacts with diverse molecules in the cell, and regulates key signaling events including RAS/ERK, PI3K/AKT, JAK/STAT and PD-1 pathways downstream of several receptor tyrosine kinases (RTKs) upon stimulation by growth factors and cytokines. SHP2 acts as both a phosphatase and a scaffold, and plays prominently oncogenic functions but can be tumor suppressor in a context-dependent manner. It typically acts as a positive regulator of RTKs signaling with some inhibitory functions reported as well. SHP2 expression and activity is regulated by such factors as allosteric autoinhibition, microRNAs, ubiquitination and SUMOylation. Dysregulation of SHP2 expression or activity causes many developmental diseases, and hematological and solid tumors. Moreover, upregulated SHP2 expression or activity also decreases sensitivity of cancer cells to anticancer drugs. SHP2 is now considered as a compelling anticancer drug target and several classes of SHP2 inhibitors with different mode of action are developed with some already in clinical trial phases. Moreover, novel SHP2 substrates and functions are rapidly growing both in cell and cancer. In view of this, we comprehensively and thoroughly reviewed literatures about SHP2 regulatory mechanisms, substrates and binding partners, biological functions, roles in human cancers, and different classes of small molecule inhibitors target this oncoprotein in cancer.

Hexachlorophene, a selective SHP2 inhibitor, suppresses proliferation and metastasis of KRAS-mutant NSCLC cells by inhibiting RAS/MEK/ERK and PI3K/AKT signaling pathways

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations account for 35% of the genetic alterations in non-small cell lung cancer (NSCLC). The Src-homology region 2-containing protein tyrosine phosphatase 2 (SHP2), encoded by PTPN11, is closely involved in RAS downstream pathways and development of many tumors by affecting cell proliferation, differentiation, and immunity. Targeting SHP2 with small molecules may be a promising avenue for the treatment of KRAS-mutant (mut) NSCLC. Herein, hexachlorophene (HCP) was identified as a SHP2 inhibitor with an IC50 value of 5.63 ± 0.75 μM through screening of the FDA-approved drug library. HCP specifically inhibited SHP2 rather than other phosphatases. Molecular docking showed that HCP displayed an orientation favorable for nucleophilic attack in the catalytic domain of SHP2. HCP suppressed viability of multiple KRAS-mut and KRAS-wild type cells and induced senescence and apoptosis in KRAS-mut cells. Moreover, HCP reversed epithelial-mesenchymal transition to suppress metastasis in KRAS-mut cells, and inhibited the RAS/MEK/ERK and PI3K/AKT signaling pathways by suppression of SHP2 phosphorylation and formation SHP2/Grb2/Gab1/SOS1 complex. In summary, HCP can act as a specific SHP2 inhibitor to inhibit KRAS-mut NSCLC cell proliferation and metastasis and induce senescence through suppression of the RAF/MEK/ERK and PI3K/AKT pathways. HCP warrants further investigation as a new compound skeleton for the development of selective SHP2 inhibitors for the treatment of NSCLC.

Targeting macrophagic SHP2 for ameliorating osteoarthritis via TLR signaling

Osteoarthritis (OA), in which M1 macrophage polarization in the synovium exacerbates disease progression, is a major cause of cartilage degeneration and functional disabilities. Therapeutic strategies of OA designed to interfere with the polarization of macrophages have rarely been reported. Here, we report that SHP099, as an allosteric inhibitor of src-homology 2-containing protein tyrosine phosphatase 2 (SHP2), attenuated osteoarthritis progression by inhibiting M1 macrophage polarization. We demonstrated that M1 macrophage polarization was accompanied by the overexpression of SHP2 in the synovial tissues of OA patients and OA model mice. Compared to wild-type (WT) mice, myeloid lineage conditional Shp2 knockout (cKO) mice showed decreased M1 macrophage polarization and attenuated severity of synovitis, an elevated expression of cartilage phenotype protein collagen II (COL2), and a decreased expression of cartilage degradation markers collagen X (COL10) and matrix metalloproteinase 3 (MMP3) in OA cartilage. Further mechanistic analysis showed thatSHP099 inhibited lipopolysaccharide (LPS)-induced Toll-like receptor (TLR) signaling mediated by nuclear factor kappa B (NF-κB) and PI3K–AKT signaling. Moreover, intra-articular injection of SHP099 also significantly attenuated OA progression, including joint synovitis and cartilage damage. These results indicated that allosteric inhibition of SHP2 might be a promising therapeutic strategy for the treatment of OA.

Grass carp (Ctenopharyngodon idellus) SHP2 suppresses IFN I expression via decreasing the phosphorylation of GSK3β in a non-contact manner

As a tyrosine phosphatase, Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) serves as an inhibitor in PI3K-Akt pathway. In mammals, SHP2 can phosphorylate GSK3β at Y216 site to control the expression of IFN. So far, the multiple functions of SHP2 have been reported in mammals. However, little is known about fish SHP2. In this study, we cloned and identified a grass carp (Ctenopharyngodon idellus) SHP2 gene (CiSHP2, MT373151). SHP2 is conserved among different vertebrates by amino acid sequences alignment and the phylogenetic tree analysis. CiSHP2 shared the closest homology with Danio rerio SHP2. Simultaneously, SHP2 was also tested in grass carp tissues and CIK (C. idellus kidney) cells. We found that it responded to poly I:C stimulation. CiSHP2 was located in the cytoplasm just as the same as those of mammals. Interestingly, it inhibited the phosphorylation level of GSK3β in a non-contact manner. Meanwhile CiGSK3β interacted with and directly phosphorylated CiTBK1. In addition, we found that CiSHP2 also reduced the phosphorylation level of CiTBK1 by CiGSK3β, and then it depressed the expression of IFN I via GSK3β-TBK1 axis. These results suggested that CiSHP2 was involved in CiGSK3β and CiTBK1 activity but not regulated their transcriptional level. At the same time, we also found that CiSHP2 also influenced the activity of CiIRF3. Therefore, fish SHP2 inhibited IFN I expression through blocking GSK3β-TBK1 signal axis.

Conditional deletion of SRC-homology 2-containing protein tyrosine phosphatase 2 attenuates the severity of experimental osteoarthritis

Purpose: To investigate the roles of the tyrosine phosphatase Src-homology 2-containing protein tyrosine phosphatase 2 (SHP2) in macrophage polarization during the process of osteoarthritis (OA) induced by destabilization of the medial meniscus (DMM) surgery in Mice.

Hepatic stellate cell autophagy inhibits extracellular vesicle release to attenuate liver fibrosis

Autophagy plays a crucial role in hepatic homeostasis and its deregulation has been associated with chronic liver disease. However, the effect of autophagy on the release of fibrogenic extracellular vesicles (EVs) by platelet-derived growth factor (PDGF)-stimulated hepatic stellate cells (HSCs) remains unknown. Herein, we aimed to elucidate the role of autophagy, specifically relating to fibrogenic EV release, in fibrosis. In vitroexperiments were conducted in primary human and murine HSCs as well as LX2 cells. Small EVs were purified by differential ultracentrifugation. Carbon tetrachloride (CCl4) or bile duct ligation (BDL) were used to induce fibrosis in our mouse model. Liver lysates from patients with cirrhosis or healthy controls were compared by RNA sequencing. Invitro, PDGF and its downstream molecule SHP2 (Src homology 2-containing protein tyrosine phosphatase 2) inhibited autophagy and increased HSC-derived EV release. We used this PDGF/SHP2 model to further investigate how autophagy affects fibrogenic EV release. RNA sequencing identified an mTOR (mammalian target of rapamycin) signaling molecule that was regulated by SHP2 and PDGF. Disruption of mTOR signaling abolished PDGF-dependent EV release. Activation of mTOR signaling induced the release of multivesicular body-derived exosomes (by inhibiting autophagy) and microvesicles (by activating ROCK1 signaling). These mTOR-dependent EVs promoted invitro HSC migration. To assess the importance of this mechanism invivo, SHP2 was selectively deleted in HSCs, which attenuated CCl4- or BDL-induced liver fibrosis. Furthermore, in the CCl4 model, mice receiving circulating EVs derived from mice with HSC-specific Shp2 deletion had less fibrosis than mice receiving EVs from control mice. Correspondingly, SHP2 was upregulated in patients with liver cirrhosis. These results demonstrate that autophagy in HSCs attenuates liver fibrosis by inhibiting the release of fibrogenic EVs. During liver fibrosis and cirrhosis, activated hepatic stellate cells (HSCs) are the key cell type responsible for fibrotic tissue deposition. Recently, we demonstrated that activated HSCs release nano-sized vesicles enriched with fibrogenic proteins. In the current study, we unveil the mechanism by which these fibrogenic vesicles are released, moving a step closer to the long-term goal of therapeutically targeting this process.

Upregulated SHP-2 expression in the epileptogenic zone of temporal lobe epilepsy and various effects of SHP099 treatment on a pilocarpine model.

Temporal lobe epilepsy (TLE) is defined as the sporadic occurrence of spontaneous recurrent seizures, and its pathogenesis is complex. SHP-2 (Src homology 2-containing protein tyrosine phosphatase 2) is a widely expressed cytosolic tyrosine phosphatase protein that participates in the regulation of inflammation, angiogenesis, gliosis, neurogenesis and apoptosis, suggesting a potential role of SHP-2 in TLE. Therefore, we investigated the expression patterns of SHP-2 in the epileptogenic brain tissue of intractable TLE patients and the various effects of treatment with the SHP-2-specific inhibitor SHP099 on a pilocarpine model. Western blotting and immunohistochemistry results confirmed that SHP-2 expression was upregulated in the temporal neocortex of patients with TLE. Double-labeling experiments revealed that SHP-2 was highly expressed in neurons, astrocytes, microglia and vascular endothelial cells in the epileptic foci of TLE patients. In the pilocarpine-induced C57BL/6 mouse model, SHP-2 upregulation in the hippocampus began one day after status epilepticus, reached a peak at 21days and then maintained a significantly high level until day 60. Similarly, we found a remarkable increase in SHP-2 expression at 1, 7, 21 and 60days post-SE in the temporal neocortex. In addition, we also showed that SHP099 increased reactive gliosis, the release of IL-1β, neuronal apoptosis and neuronal loss, while reduced neurogenesis and albumin leakage. Taken together, the increased expression of SHP-2 in the epileptic zone may be involved in the process of TLE.

Reciprocal regulation of TLR2-mediated IFN-\u03b2 production by SHP2 and Gsk3\u03b2

Toll-like receptor 2 (TLR2) mediates the innate immune response to bacterial lipopeptides and peptidoglycans by stimulating the production of inflammatory cytokines. However, the mechanisms by which TLR2 signaling regulates type I interferon (IFN)-β production are poorly understood. Here, we identified Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) as a negative regulator of TLR2-induced IFN-β production. Pharmacological inhibition or reduced expression of SHP2 potentiated TLR2 agonist-mediated IFN-β transcription and STAT1 activation, whereas overexpression of SHP2 impaired IFN-β transcription and STAT1 activation. SHP2 physically associated with the glycogen synthase kinase 3β (Gsk3β) in an agonist-dependent manner. Gsk3β positively regulates transcription of IFN-β following TLR2 stimulation by inhibiting the phosphorylation of SHP2. SHP2 inhibited the transcriptional activity of IRF-1 and IRF-8 at the IFN-β promoter. Remarkably, IRF-1 and IRF-8 are recruited to the IFN-β promoter in a SHP2 phosphatase activity-dependent manner. These findings provide insight into the mechanisms by which SHP2 and Gsk3β work together to modulate TLR2-mediated IFN-β production in macrophages.

The oncoprotein gankyrin promotes the development of colitis-associated cancer through activation of STAT3.

Although long-standing colonic inflammation due to refractory inflammatory bowel disease (IBD) promotes the development of colitis-associated cancer (CAC), the molecular mechanisms accounting for the development of CAC remains largely unknown. In this study, we investigated the role of gankyrin in the development of CAC since gankyrin is overexpressed in sporadic colorectal cancers. We analyzed gene expression of colon tissues obtained from 344 patients with IBD and CAC and found that expression of gankyrin was much higher in colonic mucosa of patients with refractory IBD than in those with IBD in remission. Expression of gankyrin was upregulated in inflammatory cells as well as tumor cells in colonic mucosa of patients with CAC. Over-expressing studies utilizing tagged ganlyrin-cDNA identified physical interaction between ganlyrin and Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1). Importantly, the interaction between ganlyrin and SHP-1 leads to inhibition of STAT3 activation and to enhancement of TNF-α and IL-17 in inflammatory cells. To further address the role of gankyrin in the development of CAC, we created mice with intestinal epithelial cell-specific gankyrin ablation (Vil-Cre;Gankyrinf/f) and deletion of gankyrin in myeloid and epithelial cells (Mx1-Cre;Gankyrinf/f). Gankyrin deficiency in myeloid cells, but not in epithelial cells, reduced the activity of mitogen activated protein kinase and the expression of stem cell markers, leading to attenuated tumorigenic potential. These findings provide important insights into the pathogenesis of CAC and suggest that gankyrin is a promising target for developing therapeutic and preventive strategies against CAC.

SHP-1 activation inhibits vascular smooth muscle cell proliferation and intimal hyperplasia in a rodent model of insulin resistance and diabetes

Aims/hypothesisAccelerated migration and proliferation of vascular smooth muscle cells (VSMCs) enhances arterial restenosis after angioplasty in insulin resistance and diabetes. Elevation of Src homology 2-containing protein tyrosine phosphatase 1 (SHP-1) induces apoptosis in the microvasculature. However, the role of SHP-1 in intimal hyperplasia and restenosis has not been clarified in insulin resistance and diabetes.MethodsWe used a femoral artery wire injury mouse model, rodent models with insulin resistance and diabetes, and patients with type 2 diabetes. Further, we modulated SHP-1 expression using a transgenic mouse that overexpresses SHP-1 in VSMCs (Shp-1-Tg). SHP-1 agonists were also employed to study the molecular mechanisms underlying the regulation of SHP-1 by oxidised lipids.ResultsMice fed a high-fat diet (HFD) exhibited increased femoral artery intimal hyperplasia and decreased arterial SHP-1 expression compared with mice fed a regular diet. Arterial SHP-1 expression was also decreased in Zucker fatty rats, Zucker diabetic fatty rats and in patients with type 2 diabetes. In primary cultured VSMCs, oxidised LDL suppressed SHP-1 expression by activating Mek-1 (also known as Map2k1) and increased DNA methylation of the Shp-1 promoter. VSMCs from Shp-1-Tg mice exhibited impaired platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation with a concomitant decrease in PDGF-stimulated VSMC proliferation and migration. Similarly, HFD-fed Shp-1-Tg mice and mice treated with the SHP-1 inducer, Icariside II, were protected from the development of intimal hyperplasia following wire injury.Conclusions/interpretationSuppression of SHP-1 by oxidised lipids may contribute to the excessive VSMC proliferation, inflammatory cytokine production and intimal hyperplasia observed in arteries from diabetes and insulin resistance. Augmenting SHP-1 levels is a potential therapeutic strategy to maintain stent patency in patients with insulin resistance and diabetes.

The Killer Cell Ig-like Receptor 2DL4 Expression in Human Mast Cells and Its Potential Role in Breast Cancer Invasion.

The killer-cell Ig-like receptor (KIR) 2DL4 (CD158d) acts as a receptor for human leukocyte antigen (HLA)-G and is expressed on almost all human natural killer (NK) cells. The expression and function of KIR2DL4 in other hematopoietic cells is poorly understood. Here, we focused on human mast cells, which exhibit cytotoxic activity similar to that of NK cells. KIR2DL4 was detected in all examined human cultured mast cells established from peripheral blood derived from healthy volunteers (PB-mast), the human mast cell line LAD2, and human nonneoplastic mast cells, including those on pathologic specimens. An agonistic antibody against KIR2DL4 decreased KIT-mediated and IgE-triggered responses, and enhanced the granzyme B production by PB-mast and LAD2 cells, by activating Src homology 2-containing protein tyrosine phosphatase (SHP-2). Next, we performed a coculture assay between LAD2 cells and the HLA-G(+) cancer cells, MCF-7 and JEG-3, and showed that KIR2DL4 on LAD2 cells enhanced MMP-9 production and the invasive activity of both cell lines via HLA-G. Immunohistochemical analysis revealed that the direct interaction between HLA-G(+) breast cancer cells and KIR2DL4(+) tissue mast cells (observed in 12 of 36 cases; 33.3%) was statistically correlated with the presence of lymph node metastasis or lymph-vascular invasion (observed in 11 of 12 cases; 91.7%; χ(2) = 7.439; P < 0.01; degrees of freedom, 1) in the clinical samples. These findings suggest that the KIR2DL4 on human mast cells facilitates HLA-G-expressing cancer invasion and the subsequent metastasis.

Protein-protein interaction between caveolin-1 and SHP-2 is dependent on the N-SH2 domain of SHP-2.

Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2) is known to protect neurons from neurodegeneration during ischemia/reperfusion injury. We recently reported that ROS-mediated oxidative stress promotes phosphorylation of endogenous SHP-2 in astrocytes and complex formation between caveolin-1 and SHP-2 in response to oxidative stress. To examine the region of SHP-2 participating in complex formation with caveolin-1, we generated three deletion mutant constructs and six point mutation constructs of SHP-2. Compared with wild-type SHP-2, binding of the N-SH2 domain deletion mutant of SHP-2 to p-caveolin-1 was reduced greatly, using flow cytometric competitive binding assays and surface plasmon resonance (SPR). Moreover, deletion of the N-SH2 domain of SHP-2 affected H2O2-mediated ERK phosphorylation and Src phosphorylation at Tyr 419 in primary astrocytes, suggesting that N-SH2 domain of SHP-2 is responsible for the binding of caveolin-1 and contributes to the regulation of Src phosphorylation and activation following ROS-induced oxidative stress in brain astrocytes. [BMB Reports 2015; 48(3): 184-189]

Blocking initial infiltration of pioneer CD8+T‐cells into the CNS via inhibition of SHP‐2 ameliorates experimental autoimmune encephalomyelitis in mice

In contrast to T-cell priming in the periphery, therapeutic strategies targeting the initiation step of T-cell trafficking into the CNS have not been extensively investigated. In this study, we examined the effect of NSC-87877, a potent Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2) inhibitor, on experimental autoimmune encephalomyelitis (EAE) and elucidated its unique mechanism of action. C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein35-55 and monitored for clinical severity of disease and histopathological features in the CNS. Levels of cytokines in serum were measured by elisa. Effects of NSC-87877 on expressions of chemokines and cytokines in the CNS were determined by quantitative PCR. NSC-87877-treated mice developed conventional TH 1 and TH 17 responses, but were highly resistant to the induction of EAE. NSC-87877 decreased the accumulation of lymphocytes in the CNS and increased the functional expression of chemokine receptor CXCR7 on CD8(+) T-cells. Adoptive transfer of T-cells from 2D2-transgenic mice restored EAE susceptibility in NSC-87877-treated mice, indicating that NSC-87877 only targets the initial migration of pioneer T-cells. Furthermore, T-cell-conditioned SHP-2-deficient mice treated with NSC-87877 were no longer resistant to EAE, suggesting that inhibition of SHP-2 contributes to the amelioration of EAE by NSC-87877. NSC-87877 almost completely abolished the development of EAE by blocking the initial infiltration of pioneer CD8(+) T-cells into the uninflamed CNS. These results reveal a critical role for SHP-2 in regulating EAE pathogenesis and indicate that NSC-87877 is a potential candidate for the treatment of relapsing-remitting multiple sclerosis.

B and T lymphocyte attenuator inhibits LPS-induced endotoxic shock by suppressing Toll-like receptor 4 signaling in innate immune cells

Although innate immune responses are necessary for the initiation of acquired immune responses and the subsequent successful elimination of pathogens, excessive responses occasionally result in lethal endotoxic shock accompanied by a cytokine storm. B and T lymphocyte attenuator (BTLA), a coinhibitory receptor with similarities to cytotoxic T-lymphocyte antigen (CTLA)-4 and programmed death (PD)-1, is expressed in not only B and T cells but also dendritic cells (DCs) and macrophages (Mϕs). Recently, several studies have reported that BTLA-deficient (BTLA(-/-)) mice show enhanced pathogen clearance compared with WT mice in early phase of infections. However, the roles of BTLA expressed on innate cells in overwhelming and uncontrolled immune responses remain unclear. Here, we found that BTLA(-/-) mice were highly susceptible to LPS-induced endotoxic shock. LPS-induced TNF-α and IL-12 production in DCs and Mϕs was significantly enhanced in BTLA(-/-) mice. BTLA(-/-) DCs also produced high levels of TNF-α on stimulation with Pam3CSK4 but not poly(I:C) or CpG, suggesting that BTLA functions as an inhibitory molecule on Toll-like receptor signaling at cell surface but not endosome. Moreover, BTLA(-/-) DCs showed enhanced MyD88- and toll/IL-1R domain-containing adaptor inducing IFN (TRIF)-dependent signaling on LPS stimulation, which is associated with impaired accumulation of Src homology 2-containing protein tyrosine phosphatase in lipid rafts. Finally, we found that an agonistic anti-BTLA antibody rescued mice from LPS-induced endotoxic shock, even if the antibody was given to mice that had developed a sign of endotoxic shock. These results suggest that BTLA directly inhibits LPS responses in DCs and Mϕs and that agonistic agents for BTLA might have therapeutic potential for LPS-induced endotoxic shock.

HPN, a Synthetic Analogue of Bromophenol from Red Alga Rhodomela confervoides: Synthesis and Anti-Diabetic Effects in C57BL/KsJ-db/db Mice

3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6-(isopropoxymethyl)benzyl)benzene-1,2-diol (HPN) is a synthetic analogue of 3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6-(ethoxymethyl)benzyl)benzene-1,2-diol (BPN), which is isolated from marine red alga Rhodomela confervoides with potent protein tyrosine phosphatase 1B (PTP1B) inhibition (IC50 = 0.84 μmol/L). The in vitro assay showed that HPN exhibited enhanced inhibitory activity against PTP1B with IC50 0.63 μmol/L and high selectivity against other PTPs (T cell protein tyrosine phosphatase (TCPTP), leucocyte antigen-related tyrosine phosphatase (LAR), Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) and SHP-2). The results of antihyperglycemic activity using db/db mouse model demonstrated that HPN significantly decreased plasma glucose (P < 0.01) after eight weeks treatment period. HPN lowered serum triglycerides and total cholesterol concentration in a dose-dependent manner. Besides, both of the high and medium dose groups of HPN remarkably decreased HbA1c levels (P < 0.05). HPN in the high dose group markedly lowered the insulin level compared to the model group (P < 0.05), whereas the effects were less potent than the positive drug rosiglitazone. Western blotting results showed that HPN decreased PTP1B levels in pancreatic tissue. Last but not least, the results of an intraperitoneal glucose tolerance test in Sprague–Dawley rats indicate that HPN have a similar antihyperglycemic activity as rosiglitazone. HPN therefore have potential for development as treatments for Type 2 diabetes.

Selective Sequestration of STAT1 in the Cytoplasm via Phosphorylated SHP-2 Ameliorates Murine Experimental Colitis

The side effects of current immunosuppressive drugs have impeded the development of therapies for immune diseases. Selective regulation of STAT signaling is an attractive strategy for treating immune disorders. In this study, we used a small-molecule compound to explore possible means of targeting STAT1 for the treatment of Th1-mediated inflammation. Selective regulation of STAT1 signaling in T cells from C57BL/6 mice was accomplished using fusaruside, a small-molecule compound that triggers the tyrosine phosphorylation of Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2). The interaction of tyrosine phosphorylated SHP-2 (pY-SHP-2) with cytosolic STAT1 prevented the recruitment of STAT1 to IFN-γR and specifically inhibited STAT1 signaling, resulting in a reduction in Th1 cytokine production and an improvement in 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis in mice. Blocking the pY-SHP-2-STAT1 interaction, with SHP-2 inhibitor NSC-87877 or using T cells from conditional SHP-2 knockout mice, reversed the effects of fusaruside, resulting in STAT1 activation and worsened colitis. The fusaruside-induced ability of pY-SHP-2 to selectively sequestrate STAT1 from recruitment to the receptor is independent of its function as a phosphatase, demonstrating a novel role for SHP-2 in regulating both STAT1 signaling and Th1-type immune responses. These findings could lead to increased options for the treatment of Crohn's disease and other Th1-mediated inflammatory diseases.

N-Acetyl–seryl–aspartyl–lysyl–proline inhibits ET-1-induced collagen production by preserving Src homology 2-containing protein tyrosine phosphatase-2 activity in cardiac fibroblasts

N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibits endothelin-1 (ET-1)-induced activation of p44/42 mitogen-activated protein kinase (p44/42 MAPK) and collagen production in cultured rat cardiac fibroblasts (RCFs). However, we do not know whether its inhibitory effect on p44/42 MAPK is due to the altered activity of protein tyrosine phosphatases (PTPs), which in turn downregulate the p44/42 MAPK signaling pathway. The activity of Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2) is downregulated by ET-1 in RCFs; thus, we hypothesized that Ac-SDKP inhibits ET-1-stimulated collagen production in part by preserving SHP-2 activity and thereby inhibiting p44/42 MAPK phosphorylation. When we stimulated RCFs with ET-1 in the presence or absence of Ac-SDKP, we found that (a) PTP activity was reduced by ET-1 and (b) this effect was counteracted by Ac-SDKP in a dose-dependent fashion. Next, we extracted SHP-2 from RCF lysates by immunoprecipitation and determined that (a) ET-1 inhibited SHP-2 by 40% and (b) this effect was prevented by Ac-SDKP. However, Ac-SDKP failed to inhibit ET-1-induced p44/42 MAPK phosphorylation in RCFs treated with SHP-2 short hairpin RNA (shRNA); in contrast, in cells transfected with control shRNA, Ac-SDKP's inhibitory effect on ET-1-induced p44/42 MAPK activation remained intact. Moreover, the inhibitory effect of Ac-SDKP on ET-1-stimulated collagen production was blunted in cells treated with the SHP-1/2 inhibitor NSC-87877. Thus, we concluded that the inhibitory effect of Ac-SDKP on ET-1-stimulated collagen production by RCFs is mediated in part by preserving SHP-2 activity and thereby preventing p44/42 MAPK activation. Ac-SDKP or its analogs could represent a new therapeutic tool to treat fibrotic diseases in the cardiovascular system.