Src Homology-2 Domain-containing Protein Tyrosine Research Articles

The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

Mutations in the tyrosine phosphatase Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting autoinhibitory interactions between its phosphatase domain and N-terminal SH2 (phosphotyrosine recognition) domain. By contrast, some disease-associated mutations located in the ligand-binding pockets of the N- or C-terminal SH2 domains do not increase basal activity and likely exert their pathogenicity through alternative mechanisms. We lack a molecular understanding of how these SH2 mutations impact SHP2 structure, activity, and signaling. Here, we characterize five SHP2 SH2 domain ligand-binding pocket mutants through a combination of high-throughput biochemical screens, biophysical and biochemical measurements, and molecular dynamics simulations. We show that while some of these mutations alter binding affinity to phosphorylation sites, the T42A mutation in the N-SH2 domain is unique in that it also substantially alters ligand-binding specificity, despite being 8 to 10 Å from the specificity-determining region of the SH2 domain. This mutation exerts its effect on sequence specificity by remodeling the phosphotyrosine-binding pocket, altering the mode of engagement of both the phosphotyrosine and surrounding residues on the ligand. The functional consequence of this altered specificity is that the T42A mutant has biased sensitivity toward a subset of activating ligands and enhances downstream signaling. Our study highlights an example of a nuanced mechanism of action for a disease-associated mutation, characterized by a change in protein-protein interaction specificity that alters enzyme activation.

Allosteric modulation of SHP2: Quest from known to unknown.

Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) is a key regulatory factor in the cell cycle and its activating mutations play an important role in the development of various cancers, making it an important target for antitumor drugs. Due to the highly conserved amino acid sequence and positively charged nature of the active site of SHP2, it is difficult to discover inhibitors with high affinity for the catalytic site of SHP2 and sufficient cell permeability, making it considered an "undruggable" target. However, the discovery of allosteric regulation mechanisms provides new opportunities for transforming undruggable targets into druggable ones. Given the limitations of orthosteric inhibitors, SHP2 allosteric inhibitors have become a more selective and safer research direction. In this review, we elucidate the oncogenic mechanism of SHP2 and summarize the discovery methods of SHP2 allosteric inhibitors, providing new strategies for the design and improvement of SHP2 allosteric inhibitors.

SHP2 is involved in the occurrence, development and prognosis of cancer.

Src homology-2 domain-containing protein tyrosine phosphatase (SHP2), encoded by protein tyrosine phosphatase non-receptor type 11 (PTPN11), is widely expressed in several human tissue types, and plays an important role in a variety of diseases. The present study assessed the impact of SHP2 on the occurrence, development and prognosis of solid tumors. The transcriptome sequencing data of 33 cancer types were downloaded from The Cancer Genome Atlas database. Clinical information of the corresponding patients, tumor mutational burden and information pertinent to microsatellite instability were also downloaded. The log-rank test and univariate Cox's regression test were used to evaluate patient survival. The 'ESTIMATE' method was used to assess the tumor microenvironment, and the 'CIBERSORT' algorithm was used to evaluate tumor immune cell infiltration. Spearman's correlation analysis was used to evaluate the correlation between SHP2 expression and the targets identified. ELISA was used to assess the SHP2 expression levels in peripheral blood samples of patients with breast, ovarian, endometrial and cervical cancer. The data indicated that the expression levels of SHP2 were increased in a variety of tumor tissues, and were associated with tumor progression and prognosis. In peripheral blood, the positive rates of SHP2 expression in breast cancer (71.43%) and ovarian cancer (58.82%) were significantly higher than those in the corresponding control groups. However, the positive rates of SHP2 expression in patients with endometrial cancer (31.03%) and cervical cancer (41.30%) were significantly lower than those in the corresponding control groups. Increased SHP2 expression improved overall survival (OS) and disease free survival (DFS) time in patients with kidney renal clear cell carcinoma. However, increased SHP2 expression reduced OS and DFS in patients with urothelial carcinoma, and cervical and endocervical cancer types. Moreover, the elevated expression of SHP2 could also reduce the OS of patients with breast invasive carcinoma, mesothelioma and liver hepatocellular carcinoma. PTPN11 expression was associated with the tumor microenvironment of various tumor types. The tumor mutational burden of various tumor types was associated with microsatellite instability. PTPN11 inhibited T-cell activation and promoted M2 macrophage activation in several tumors. Therefore, SHP2 may be used in the evaluation of tumor progression and prognosis, and it may be an optimal potential biological target for cancer therapy.

Inhibition of SHP2 by the Small Molecule Drug SHP099 Prevents Lipopolysaccharide-Induced Acute Lung Injury in Mice.

Excessive pulmonary inflammation in acute lung injury (ALI) causes high patient mortality. Anti-inflammatory therapy, combined with infection resistance, can help to prevent ALI and save lives. The expression of Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP2) was found to be significantly higher in macrophages and lung tissues with ALI, and SHP2-associated MAPK pathways were activated by lipopolysaccharide (LPS). The knockdown of the SHP2 gene suppressed the LPS-induced release of inflammatory factors and the phosphorylation of regulators in the NF-κB pathways in macrophages. Our findings showed crosstalk between the LPS-induced inflammatory pathway and the SHP2-associated MAPK pathways. SHP2 inhibition could be a valuable therapeutic approach for inhibiting excessive inflammation in ALI. We discovered that giving SHP099, a specific allosteric inhibitor of SHP2, to mice with ALI and sepsis relieves ALI and significantly increases animal survival. Our study highlights the important role of SHP2 in ALI development and demonstrates the potential application of SHP099 for treating ALI.

Discovery of allosteric SHP2 inhibitors through ensemble-based consensus molecular docking, endpoint and absolute binding free energy calculations

SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase-2) is a cytoplasmic protein -tyrosine phosphatase encoded by the gene PTPN11. It plays a crucial role in regulating cell growth and differentiation. Specifically, SHP2 is an oncoprotein associated with developmental pathologies and several different cancer types, including gastric, leukemia and breast cancer and is of great therapeutic interest. Given these roles, current research efforts have focused on developing SHP2 inhibitors. Allosteric SHP2 inhibitors have been shown to be more selective and pharmacologically appealing compared to competitive catalytic inhibitors targeting SHP2. Nevertheless, there remains a need for novel allosteric inhibitor scaffolds targeting SHP2 to develop compounds with improved selectivity, cell permeability, and bioavailability. Towards this goal, this study applied various computational tools to screen over 6 million compounds against the allosteric site within SHP2. The top-ranked hits from our in-silico screening were validated using protein thermal shift and biolayer interferometry assays, revealing three potent compounds. Kinetic binding assays were employed to measure the binding affinities of the top-ranked compounds and demonstrated that they all bind to SHP2 with a nanomolar affinity. Hence the compounds and the computational workflow described herein provide an effective approach for identifying and designing a generation of improved allosteric inhibitors of SHP2.

Role of SHP2/PTPN11 in the occurrence and prognosis of cancer: A systematic review and meta-analysis.

Src homology-2 domain-containing protein tyrosine phosphatase (SHP2)/PTP non-receptor type 11 (PTPN11) have been reported to be expressed in a variety of solid tumors, though its role in tumors remains controversial. The aim of the present study was to explore the role of SHP2/PTPN11 in the occurrence and prognosis of cancer. Literature on the relationship between SHP2/PTPN11 expression and tumor was searched in PubMed, China National Knowledge Infrastructure and Cochrane Library electronic database, following which the Stata 12.0 software was used for meta-analysis. A total of 23 articles were included in the present statistical analysis. Higher expression levels of SHP2/PTPN11 can significantly increase the risk of non-small-cell lung cancer [NSCLC; odds ratio (OR)=3.41, 95% confidence interval (CI)=1.07-10.80; P=0.037] while reducing the overall survival (OS) time of patients with NSCLC [hazards ratio (HR)=2.83, 95% CI=1.97-4.07; P<0.001]. In addition, increased expression of SHP2/PTPN11 can significantly increase the risk of gastric (OR=5.35, 95% CI=1.81-15.77; P=0.002) and cervical cancer (OR=12.04, 95% CI=3.45-42.01; P<0.001). However, no significant difference could be found in the expression level of SHP2/PTPN11 in liver cancer (OR=1.47, 95% CI=0.37-5.84; P=0.582) compared with that in the adjacent normal tissues. Taken together, SHP2/PTPN11 was found to be expressed highly in a number of tumors, which was in turn associated with tumorigenesis and patient prognosis. In particular, increased expression of SHP2/PTPN11 can increase the risk of NSCLC, gastric cancer and cervical cancer, whereas higher expression levels of SHP2/PTPN11 can reduce OS of NSCLC.

Sciatic nerve block downregulates the BDNF pathway to alleviate the neonatal incision-induced exaggeration of incisional pain via decreasing microglial activation

Sciatic nerve block is under investigation as a possible therapeutic strategy for neonatal injury-induced exaggeration of pain responses to reinjury. Spinal microglial priming, brain-derived neurotrophic factor (BDNF) and Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) participate in exaggerated incisional pain induced by neonatal incision. However, effects of sciatic nerve block on exacerbated incisional pain and underlying mechanisms remain unclear. Here, we demonstrated that sciatic nerve block alleviates pain hypersensitivity and microglial activation in rats subjected to neonatal incision and adult incision (nIN-IN). Chemogenetic activation or inhibition of spinal microglia attenuates or mimics effects of sciatic nerve block on pain hypersensitivity, respectively. Moreover, α-amino-3-hydroxy- 5-methy- 4-isoxazole propionate (AMPA) receptor subunit GluA1 contributes to the exaggeration of incisional pain. The inhibition of BDNF or SHP2 blocks upregulations of downstream molecules in nIN-IN rats. Knockdown of SHP2 attenuates the increase of GluA1 induced by injection of BDNF in adult rats with only neonatal incision. The inhibition of microglia or ablation of microglial BDNF attenuates upregulations of SHP2 and GluA1. Additionally, sciatic nerve block downregulates the expression of these three molecules. Upregulation of BDNF, SHP2 or AMPA receptor attenuates sciatic nerve block-induced reductions of downstream molecules and pain hypersensitivity. Microglial activation abrogates reductions of these three molecules induced by sciatic nerve block. These results suggest that decreased activation of spinal microglia contributes to beneficial effects of sciatic nerve block on the neonatal incision-induced exaggeration of incisional pain via downregulating BDNF/SHP2/GluA1-containing AMPA receptor signaling. Thus, sciatic nerve block may be a promising therapy.

Abstract 1298: An allosteric SHP2 inhibitor RMC-4550 induces immunogenicity in pancreatic adenocarcinoma (PDA) and enhances the cytotoxicity of CD8+ effector T cells

Abstract Background: Immunotherapy is ineffective in MSS PDA. Src homology-2 domain-containing protein tyrosine phosphatase (SHP2) mediates multiple immunosuppressive mechanisms in tumor cells and tumor microenvironment (TME). SHP2 plays an important role in oncogenic RAS signaling, the hallmark of PDA, that reduces tumor MHC-I expression and promotes immunotherapy resistance by stabilizing PD-L1. It modulates multiple T cell inhibitory pathways including PD-1 signaling via dephosphorylation of ITIM and ITSM. We hypothesize that SHP2i will increase anti-PDA immunity and help overcome resistance to PD-1 blockade when combined with gemcitabine, an active cytotoxic agent against PDA with pleiotropic immunomodulatory effects on its TME. Methods: Mouse KPC-luc cells (derived from LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre GEMM) and human RAS mutant PDA cells were treated in vitro with various concentrations of RMC-4550, a potent and selective allosteric SHP2 inhibitor, to test its cytotoxicity using cell viability assay and measure tumor MHC-I and PD-L1 expression upon IFNy stimulation. Human PBMC were used for T cells in vitro experiments to examine the effect of RMC-4550 on T-cell proliferation inhibition by PD-1 ligand (PD-L1) stimulation. In vivo antitumor activity of RMC-4550 combined with αPD-1 or with αPD-1 and gemcitabine was investigated in a syngeneic KPC-luc subcutaneous model. After treatment, tumors and spleens were harvested, digested to single cell suspensions, and immune infiltrates quantified using flow cytometry. Results: In vitro, there was no direct cytotoxicity of RMC-4550 on mouse KPC or human PDAC cells and no additive effect to gemcitabine. SHP2i increases IFNy mediated upregulation of MHC-I, and downregulates PD-L1 in both KPC mouse and MiaPaca-2 human PDA cells. In vitro, RMC-4550 reverses the PD-L1-mediated inhibition of T cell proliferation. In vivo, SHP2i with αPD-1 has a combinatorial effect on tumor growth inhibition. Gemcitabine seems to augment this effect. Preliminary data revealed no significant changes in number of immune cells in treatment groups when compared to the control group while an increase in proportion of Granzyme B+ CD8+ T cells and Ki-67+ CD8+ T cells in spleen and a decrease in proportion of PD1+ CD8+ T cells in TME was observed in RMC-4550 plus αPD-1 plus gemcitabine group compared to the control group. Conclusion: SHP2i RMC-4550 and αPD-1 combination induces significant anti-tumor benefit in an in vivo PDA tumor model, an effect that is enhanced by adding gemcitabine. SHP2i may promote antitumor immunity in part by increasing MHC-I expression and reducing PD-L1 expression in RAS mutant PDA cells. PD-L1 mediated suppression of T cell proliferation was overcome by RMC-4550. In vivo, RMC-4550 may potentiate anti-PD-1 therapy by enhancing cytotoxicity of CD8+ effector T cells. Citation Format: Agnieszka Looney, Uma Giri, Yezi Zhu, Alex Somma, Nisha Holay, Milad Soleimani, Heta Gandhi, Anna Capasso, Jeanne Kowalski, William Matsui, Carla Van Berg, S Gail Eckhardt, Todd Triplett, Kyaw Lwin Aung. An allosteric SHP2 inhibitor RMC-4550 induces immunogenicity in pancreatic adenocarcinoma (PDA) and enhances the cytotoxicity of CD8+ effector T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1298.

Tyrosine phosphatase SHP2 exacerbates psoriasis-like skin inflammation in mice via ERK5-dependent NETosis.

Psoriasis is a chronic inflammatory skin disease, often accompanied by increased infiltration of immune cells, especially neutrophils. However, the detailed mechanism of the neutrophil function in psoriasis progression remains unclear. Here, we found that both Src homology‐2 domain‐containing protein tyrosine phosphatase‐2 (SHP2) and neutrophils were highly correlated to developing psoriasis by single‐cell ribonucleic acid (RNA) sequencing and experiment verification. The deficiency of SHP2 in neutrophils significantly alleviated psoriasis‐like phenotype in an imiquimod‐induced murine model. Interestingly, high levels of neutrophil extracellular traps (NETs) were produced in the inflamed lesions of psoriatic patients. In addition, imiquimod‐induced psoriasis‐like symptoms were remarkably ameliorated in peptidyl arginine deiminase 4 (PAD4) knockout mice, which cannot form NETs. Mechanistically, RNA‐seq analysis revealed that SHP2 promoted the formation of NETs in neutrophils via the ERK5 pathway. Functionally, this mechanism resulted in the infiltration of pro‐inflammatory cytokines such as TNF‐α, IL‐1β, IL‐6, IL‐17A, and CXCL‐15, which enhances the inflammatory response in skin lesions and reinforces the cross‐talk between neutrophils and keratinocytes, ultimately aggravating psoriasis. Our findings uncover a role for SHP2 in NET release and subsequent cell death known as NETosis in the progression of psoriasis and suggest that SHP2 may be a promising therapeutic target for psoriasis.

Knockdown of RNF183 suppressed proliferation of lung adenocarcinoma cells via inactivating the STAT3 signaling pathway

ABSTRACT Proteins of the RNF183 (RING finger 183) family proteins have been reported to be of great importance in tumor the initiation and progression. However, the biological role and regulatory mechanism of RNF183 in non small cell lung cancer (NSCLC) development and progression are poorly defined. Hence, lung adenocarcinoma (LUAD) cell proliferation, cell apoptosis and cell cycle were measured using Cell Counting Kit-8 and flow cytometry analysis, respectively. The correlation between RNF183 and SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase) was measured using coimmunoprecipitation and ubiquitination analysis in vitro. Tumor growth of NSCLC cells in vivo was measured using the nude mouse xenograft model. In this study, we verify that elevated RNF183 expression in tumor tissues of LUAD, origin from the TCGA, GEPIA, TIMER, and UALCAN database. RNF183 regulates apoptosis and cell cycle in vitro and tumor growth in vivo by activating the STAT3 pathway through ubiquitination of SHP2, a negative feedback regulator of the STAT3 pathway. Taken together, our results demonstrate that RNF183 regulates proliferation, apoptosis, and cell cycle in LUAD cells via modulation of SHP2/STAT3 signaling, suggesting the potential for targeting the RNF183-SHP2/STAT3 pathway for use in LUAD treatment.

MiR-514a-3p: a novel SHP-2 regulatory miRNA that modulates human cytotrophoblast proliferation.

Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP-2), encoded by the PTPN11 gene, forms a central component of multiple signalling pathways and is required for insulin-like growth factor (IGF)-induced placental growth. Altered expression of SHP-2 is associated with aberrant placental and fetal growth indicating that drugs modulating SHP-2 expression may improve adverse pregnancy outcome associated with altered placental growth. We have previously demonstrated that placental PTPN11/SHP-2 expression is controlled by miRNAs. SHP-2 regulatory miRNAs may have therapeutic potential; however, the individual miRNA(s) that regulate SHP-2 expression in the placenta remain to be established. We performed in silico analysis of 3’UTR target prediction databases to identify libraries of Hela cells transfected with individual miRNA mimetics, enriched in potential SHP-2 regulatory miRNAs. Analysis of PTPN11 levels by quantitative (q) PCR revealed that miR-758-3p increased, while miR-514a-3p reduced PTPN11 expression. The expression of miR-514a-3p and miR-758-3p within the human placenta was confirmed by qPCR; miR-514a-3p (but not miR-758-3p) levels inversely correlated with PTPN11 expression. To assess the interaction between these miRNAs and PTPN11/SHP-2, specific mimetics were transfected into first-trimester human placental explants and then cultured for up to 4 days. Overexpression of miR-514a-3p, but not miR-758-3p, significantly reduced PTPN11 and SHP-2 expression. microRNA-ribonucleoprotein complex (miRNP)-associated mRNA assays confirmed that this interaction was direct. miR-514a-3p overexpression attenuated IGF-I-induced trophoblast proliferation (BrdU incorporation). miR-758-3p did not alter trophoblast proliferation. These data demonstrate that by modulating SHP-2 expression, miR-514a-3p is a novel regulator of IGF signalling and proliferation in the human placenta and may have therapeutic potential in pregnancies complicated by altered placental growth.

Synthesis and biological evaluation of 2,5-diaryl-1,3,4-oxadiazole derivatives as novel Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) inhibitors

The Src homology-2 domain containing-protein tyrosine phosphatase-2 (SHP2) is a convergent node for oncogenic cell-signaling cascades including the PD-L1/PD-1 pathway. As an oncoprotein as well as a potential immunomodulator, SHP2 has now emerged as an attractive target for novel anti-cancer agents. Although significant progress has been made in identifying chemotypes of SHP2 inhibitors, these specific compounds might not be clinically useful to inhibit frequently encountered mutated SHP2 variants. Consequently, it is highly desirable to develop chemically different SHP2 inhibitors sensitive to SHP2 mutants. This work developed a new type of SHP2 inhibitors with 2,5-diaryl-1,3,4-oxadiazole scaffold. The representative compound 6l exhibited SHP2 inhibitory activity with IC50 of 2.73 ± 0.20 µM, showed about 1.56-fold, 5.26-fold, and 7.36-fold selectivity for SHP2 over SHP1, PTP1B and TCPTP respectively. Further investigations confirmed that 6l behaved as mixed-type inhibitor sensitive to leukemia cell TF-1 and inhibited SHP2 mediated cell signaling and proliferation. Molecular dynamics simulation provided more detailed information on the binding modes of compounds and SHP2 protein. These preliminary results could provide a possible opportunity for the development of novel SHP2 inhibitors sensitive to SHP2 mutants with optimal potency and improved pharmacological properties.

SHP2-Mediated Inhibition of DNA Repair Contributes to cGAS-STING Activation and Chemotherapeutic Sensitivity in Colon Cancer.

As a cytoplasmic sensor of double-stranded DNA (dsDNA), the cyclic GMP-AMP synthase-stimulator of IFN genes (STING) pathway plays an important role in antitumor immunity. In this study, we investigated the effect of Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) on tumor cell-intrinsic STING pathway activity and DNA repair in colon cancer. SHP2 interacted with and dephosphorylated PARP1 after DNA damage. PARP1 inhibition by SHP2 resulted in reduced DNA repair and accumulation of dsDNA in cells, thus promoting hyperactivation of the STING pathway. The SHP2 agonist lovastatin was able to enhance SHP2 activity and promote STING pathway activation. Moreover, lovastatin significantly enhanced the efficacy of chemotherapy in colon cancer models, in part via STING pathway-mediated antitumor immunity. These findings suggest that SHP2 exacerbates STING pathway activation by restricting PARP1-mediated DNA repair in tumor cells, providing a basis for the combined use of lovastatin and chemotherapy in the treatment of colon cancer. SIGNIFICANCE: Dephosphorylation of PARP1 by SHP2 simultaneously suppresses DNA repair and enhances STING pathway-mediated antitumor immunity, highlighting SHP2 activation as a potential therapeutic approach in colon cancer.

Exploring the Allosteric Mechanism of Src Homology-2 Domain-Containing Protein Tyrosine Phosphatase 2 (SHP2) by Molecular Dynamics Simulations.

The Src homology-2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP2, encoded by PTPN11) is a critical allosteric phosphatase for many signaling pathways. Programmed cell death 1 (PD-1) could be phosphorylated at its immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) and can bind to SHP2 to initiate T cell inactivation. Although the interaction of SHP2-PD-1 plays an important role in the immune process, the complex structure and the allosteric regulation mechanism remain unknown. In this study, molecular dynamics (MD) simulations were performed to study the binding details of SHP2 and PD-1, and explore the allosteric regulation mechanism of SHP2. The results show that ITIM has a preference to bind to the N-SH2 domain and ITSM has almost the same binding affinity to the N-SH2 and C-SH2 domain. Only when ITIM binds to the N-SH2 domain and ITSM binds to the C-SH2 domain can the full activation of SHP2 be obtained. The binding of ITIM and ITSM could change the motion mode of SHP2 and switch it to the activated state.

Recent Advances of SHP2 Inhibitors in Cancer Therapy: Current Development and Clinical Application

SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase-2) is a non-receptor protein tyrosine phosphatase that removes tyrosine phosphorylation. Functionally, SHP2 serves as an important hub to connect several intracellular oncogenic signaling pathways, such as Jak/STAT, PI3K/AKT, RAS/Raf/MAPK, and PD-1/PD-L1 pathways. Mutations and/or overexpression of SHP2 has been associated with genetic developmental diseases and cancers. Because of the role of SHP2 plays in many diseases, the development of inhibitors targeting the catalytic site in SHP2 has been pursued for more than a decade, but none has advanced to clinical development. Recent discovery of allosteric inhibitors has inspired a novel approach to selectively target SHP2 via the noncatalytic site. To date, four SHP2 allosteric inhibitors have entered clinical trials for the treatment of solid tumors. This review will provide a summary of the physiological and biological functions of SHP2 and discuss the development of nonallosteric/allosteric SHP2 inhibitors in recent years.

P0-Related Protein Accelerates Human Mesenchymal Stromal Cell Migration by Modulating VLA-5 Interactions with Fibronectin.

P0-related protein (PZR), a Noonan and LEOPARD syndrome target, is a member of the transmembrane Immunoglobulin superfamily. Its cytoplasmic tail contains two immune-receptor tyrosine-based inhibitory motifs (ITIMs), implicated in adhesion-dependent signaling and regulating cell adhesion and motility. PZR promotes cell migration on the extracellular matrix (ECM) molecule, fibronectin, by interacting with SHP-2 (Src homology-2 domain-containing protein tyrosine phosphatase-2), a molecule essential for skeletal development and often mutated in Noonan and LEOPARD syndrome patients sharing overlapping musculoskeletal abnormalities and cardiac defects. To further explore the role of PZR, we assessed the expression of PZR and its ITIM-less isoform, PZRb, in human bone marrow mesenchymal stromal cells (hBM MSC), and its ability to facilitate adhesion to and spreading and migration on various ECM molecules. Furthermore, using siRNA knockdown, confocal microscopy, and immunoprecipitation assays, we assessed PZR and PZRb interactions with β1 integrins. PZR was the predominant isoform in hBM MSC. Migrating hBM MSCs interacted most effectively with fibronectin and required the association of PZR, but not PZRb, with the integrin, VLA-5(α5β1), leading to modulation of focal adhesion kinase phosphorylation and vinculin levels. This raises the possibility that dysregulation of PZR function may modify hBM MSC migratory behavior, potentially contributing to skeletal abnormalities.

Spinal SHP2 Contributes to Exaggerated Incisional Pain in Adult Rats Subjected to Neonatal and Adult Incisions via PI3K

Neonatal injury-induced exaggeration of pain hypersensitivity after adult trauma is a significant clinical challenge. However, the underlying mechanisms remain poorly understood. Growing evidence shows that spinal Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) contributes to chronic pain in adult rodents. Here we demonstrated that the phosphorylation and expression of SHP2 in synaptosomal fraction of the spinal dorsal horn are elevated in adult rats subjected to neonatal and adult incisions (nIN-IN), and the upregulation of SHP2 is highly correlated with pain hypersensitivity. Intrathecal blockade of SHP2 phosphorylation using a SHP2 protein tyrosine phosphatase inhibitor NSC-87877, or knockdown of SHP2 by intrathecal delivery of small interfering RNA (siRNA), ameliorates mechanical allodynia and heat hyperalgesia in nIN-IN rats. Moreover, the expression of phosphatidylinositol 3-kinase (PI3K) in the spinal dorsal horn is significantly increased in nIN-IN rats. Intrathecal application of PI3K inhibitor, LY294002 or wortmannin, alleviates pain hypersensitivity in nIN-IN rats. Additionally, intrathecal administration of NSC-87877 or SHP2 siRNA attenuates the upregulation of PI3K. Finally, no alternation of SHP2 phosphorylation in the dorsal root ganglion and dorsal root of nIN-IN rats as well as PI3K expression in the dorsal root of nIN-IN rats intrathecally treated with NSC-87877 or SHP2 siRNA is observed. These results suggest that the phosphorylation and expression of SHP2 in the spinal dorsal horn play vital roles in neonatal incision-induced exaggeration of adult incisional pain via PI3K. Thus, SHP2 and PI3K may serve as potential therapeutic targets for exaggerated incisional pain induced by neonatal and adult injuries.

SIRPα1-SHP2 Interaction Regulates Complete Freund Adjuvant-Induced Inflammatory Pain via Src-Dependent GluN2B Phosphorylation in Rats.

The elusiveness of pain mechanisms is a major impediment in developing effective clinical treatments. We examined whether the signal regulatory protein α1 (SIRPα1)-activated spinal Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP2)/Src cascade and the downstream GluN2B phosphorylation play a role in inflammatory pain. At hour 3 and days 1, 3, 5, and 10 after the intraplantar injection of complete Freund adjuvant (CFA), we assessed paw withdrawal latency using the Hargreaves test and analyzed dorsal horn samples (L4-L5) by Western blotting and immunoprecipitation. Intraplantar CFA injection provoked the behavioral hyperalgesia in the ipsilateral hind-paw along with SIRPα1, phosphorylated SHP2 (pSHP2), phosphorylated Src (pSrc), and phosphorylated GluN2B expressions and total SHP2 (tSHP2)-SIRPα1/pSHP2/pSrc and total Src (tSrc)-SIRPα1/pSHP2/pSrc coprecipitation in the ipsilateral dorsal horn. Although both of them failed to show an effect on CFA-enhanced SIRPα1 expression, spinal administration with SIRPα1-neutralizing antibody (10, 50, and 100 μg, 10 μL) and 8-Hydroxy-7-[(6-sulfo-2-naphthyl)azo]-5-quinolinesulfonic acid (NSC 8787; an SHP2 antagonist, 1, 10, and 100 μM, 10 μL) dose-dependently attenuated the behavioral hyperalgesia, SHP2 and Src phosphorylation, and tSHP2-SIRPα1/pSHP2/pSrc coprecipitation at day 1 after CFA injection. Intrathecal application of 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2; a Src-family kinase inhibitor, 10, 30, and 50 nM, 10 μL) exhibited a similar effect as these agents, except that it failed to ameliorate CFA-enhanced SHP2 phosphorylation and tSHP2-SIRPα1/pSHP2 coprecipitation. CFA-induced spinal SIRPα1 expression, which triggers SHP2, and Src phosphorylation, which subsequently induced pSrc-GluN2B interaction to mediate the GluN2B activation, contribute to spinal plasticity underlying the maintenance of inflammatory pain. These findings provide a possible strategy for pain relief by targeting to spinal SIRPα1-SHP2 coupling.

BDNF contributes to the development of neuropathic pain by induction of spinal long-term potentiation via SHP2 associated GluN2B-containing NMDA receptors activation in rats with spinal nerve ligation

The pathogenic mechanisms underlying neuropathic pain still remain largely unknown. In this study, we investigated whether spinal BDNF contributes to dorsal horn LTP induction and neuropathic pain development by activation of GluN2B-NMDA receptors via Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) phosphorylation in rats following spinal nerve ligation (SNL). We first demonstrated that spinal BDNF participates in the development of long-lasting hyperexcitability of dorsal horn WDR neurons (i.e. central sensitization) as well as pain allodynia in both intact and SNL rats. Second, we revealed that BDNF induces spinal LTP at C-fiber synapses via functional up-regulation of GluN2B-NMDA receptors in the spinal dorsal horn, and this BDNF-mediated LTP-like state is responsible for the occlusion of spinal LTP elicited by subsequent high-frequency electrical stimulation (HFS) of the sciatic nerve in SNL rats. Finally, we validated that BDNF-evoked SHP2 phosphorylation is required for subsequent GluN2B-NMDA receptors up-regulation and spinal LTP induction, and also for pain allodynia development. Blockade of SHP2 phosphorylation in the spinal dorsal horn using a potent SHP2 protein tyrosine phosphatase inhibitor NSC-87877, or knockdown of spinal SHP2 by intrathecal delivery of SHP2 siRNA, not only prevents BDNF-mediated GluN2B-NMDA receptors activation as well as spinal LTP induction and pain allodynia elicitation in intact rats, but also reduces the SNL-evoked GluN2B-NMDA receptors up-regulation and spinal LTP occlusion, and ultimately alleviates pain allodynia in neuropathic rats. Taken together, these results suggest that the BDNF/SHP2/GluN2B-NMDA signaling cascade plays a vital role in the development of central sensitization and neuropathic pain after peripheral nerve injury.

From an orphan disease to a generalized molecular mechanism: PTPN11 loss-of-function mutations in the pathogenesis of metachondromatosis.

Recently, loss-of-function mutations in PTPN11 were linked to the cartilage tumor syndrome metachondromatosis (MC), a rare inherited disorder featuring osteochondromas, endochondromas and skeletal deformation. However, the underlying molecular and cellular mechanism for MC remained incompletely understood. By studying the role of the Src homology-2 domain-containing protein tyrosine phosphatase Shp2 (encoded by mouse Ptpn11) in cathepsin K-expressing cells, we identified a novel cell population in the perichondrial groove of Ranvier. In the absence of Shp2, these cells exhibit elevated Indian hedgehog (Ihh) signaling, proliferate excessively and cause ectopic cartilage formation and tumors. Our findings establish a critical role for a protein-tyrosine phosphatase (PTP) family member, in addition to the well-known roles of receptor tyrosine kinases (RTKs), in cartilage development and homeostasis. However, whether Shp2 deficiency in other epiphyseal chondroid cells and whether signaling pathways in addition to the IHH/Parathyroid Hormone–related Peptide (PTHrP) axis attribute to the formation of enchondromas and osteochondromas remains elusive. Understanding how chondrogenic events are regulated by SHP2 could aid in the development of novel therapeutic approaches to prevent and treat cartilage diseases, such as MC and osteoarthritis (OA).