Integrin αVß1-activated PYK2 promotes the progression of non-small-cell lung cancer via the STAT3-VGF axis.

BACKGROUND: Non-small-cell lung cancer (NSCLC) accounts for 80-85% of all lung cancer and is the leading cause of cancer-related deaths globally. Although various treatment strategies have been introduced, the 5-year survival rate of patients with NSCLC is only 20-30%. Thus, it remains necessary to study the pathogenesis of NSCLC and develop new therapeutic drugs. Notably, PYK2 has been implicated in the progression of many tumors, including NSCLC, but its detailed mechanism remains unclear. In this study, we aimed to elucidate the mechanisms through which PYK2 promotes NSCLC progression. METHODS: The mRNA and protein levels of various molecules were measured using qRT-PCR, western blot (WB), and immunohistochemistry (IHC), respectively. We established stable PYK2 knockdown and overexpression cell lines, and CCK-8, EdU, and clonogenic assays; wound healing, transwell migration, and Matrigel invasion assays; and flow cytometry were employed to assess the phenotypes of tumor cells. Protein interactions were evaluated with co-immunoprecipitation (co-IP), immunofluorescence (IF)-based colocalization, and nucleocytoplasmic separation assays. RNA sequencing was performed to explore the transcriptional regulation mediated by PYK2. Secreted VGF levels were examined using ELISA. Dual-luciferase reporter system was used to detect transcriptional regulation site. PF4618433 (PYK2 inhibitor) and Stattic (STAT3 inhibitor) were used for rescue experiments. A public database was mined to analyze the effect of these molecules on NSCLC prognosis. To investigate the role of PYK2 in vivo, mouse xenograft models of lung carcinoma were established and examined. RESULTS: The protein level of PYK2 was higher in human NSCLC tumors than in the adjacent normal tissue, and higher PYK2 expression was associated with poorer prognosis. PYK2 knockdown inhibited the proliferation and motility of tumor cells and caused G1-S arrest and cyclinD1 downregulation in A549 and H460 cells. Meanwhile, PYK2 overexpression had the opposite effect in H1299 cells. The siRNA-induced inhibition of integrins alpha V and beta 1 led to the downregulation of p-PYK2(Tyr402). Activated PYK2 could bind to STAT3 and enhance its phosphorylation at Tyr705, regulating the nuclear accumulation of p-STAT3(Tyr705). This further promoted the expression of VGF, as confirmed by RNA sequencing in a PYK2-overexpressing H1299 cell line and validated by rescue experiments. Two sites in promoter region of VGF gene were confirmed as binding sites of STAT3 by Dual-luciferase assay. Data from the TGCA database showed that VGF was related to the poor prognosis of NSCLC. IHC revealed higher p-PYK2(Tyr402) and VGF expression in lung tumors than in adjacent normal tissues. Moreover, both proteins showed higher levels in advanced TNM stages than earlier ones. A positive linear correlation existed between the IHC score of p-PYK2(Tyr402) and VGF. Knockdown of VGF inhibited tumor progression and reversed the tumor promoting effect of PYK2 overexpression in NSCLC cells. Finally, the mouse model exhibited enhanced tumor growth when PYK2 was overexpressed, while the inhibitors PF4618433 and Stattic could attenuate this effect. CONCLUSIONS: The Integrin αVß1-PYK2-STAT3-VGF axis promotes NSCLC development, and the PYK2 inhibitor PF4618433 and STAT3 inhibitor Stattic can reverse the pro-tumorigenic effect of high PYK2 expression in mouse models. Our findings provide insights into NSCLC progression and could guide potential therapeutic strategies against NSCLC with high PYK2 expression levels.

Mitochondrial translation failure represses cholesterol gene expression via Pyk2-Gsk3ß-Srebp2 axis.

Neurodegenerative diseases and other age-related disorders are closely associated with mitochondrial dysfunction. We previously showed that mice with neuron-specific deficiency of mitochondrial translation exhibit leukoencephalopathy because of demyelination. Reduced cholesterol metabolism has been associated with demyelinating diseases of the brain such as Alzheimer's disease. However, the molecular mechanisms involved and relevance to the pathogenesis remained unknown. In this study, we show that inhibition of mitochondrial translation significantly reduced expression of the cholesterol synthase genes and degraded their sterol-regulated transcription factor, sterol regulatory element-binding protein 2 (Srebp2). Furthermore, the phosphorylation of Pyk2 and Gsk3ß was increased in the white matter of p32cKO mice. We observed that Pyk2 inhibitors reduced the phosphorylation of Gsk3ß and that GSK3ß inhibitors suppressed degradation of the transcription factor Srebp2. The Pyk2-Gsk3ß axis is involved in the ubiquitination of Srebp2 and reduced expression of cholesterol gene. These results suggest that inhibition of mitochondrial translation may be a causative mechanism of neurodegenerative diseases of aging. Improving the mitochondrial translation or effectiveness of Gsk3ß inhibitors is a potential therapeutic strategy for leukoencephalopathy.

Quantitative Prediction of Dissociation Rates of PYK2 Ligands Using Umbrella Sampling and Milestoning.

We used umbrella sampling and the milestoning simulation method to study the dissociation of multiple ligands from protein kinase PYK2. The activation barriers obtained from the potential of mean force of the umbrella sampling simulations correlated well with the experimental dissociation rates. Using the zero-temperature string method, we obtained optimized paths along the free-energy surfaces for milestoning simulations of three ligands with a similar structure. The milestoning simulations gave an absolute dissociation rate within 2 orders of magnitude of the experimental value for two ligands but at least 3 orders of magnitude too high for the third. Despite the similarity in their structures, the ligands took different pathways to exit from the binding site of PYK2, making contact with different sets of residues. In addition, the protein experienced different conformational changes for dissociation of the three ligands.

Homocysteine-induced sustained GluN2A NMDA receptor stimulation leads to mitochondrial ROS generation and neurotoxicity.

Homocysteine, a sulfur-containing amino acid derived from methionine metabolism, is a known agonist of N-methyl-D-aspartate receptor (NMDAR) and is involved in neurotoxicity. Our previous findings showed that neuronal exposure to elevated homocysteine levels leads to sustained low-level increase in intracellular Ca2+, which is dependent on GluN2A subunit-containing NMDAR (GluN2A-NMDAR) stimulation. These studies further showed a role of ERK MAPK in homocysteine-GluN2A-NMDAR-mediated neuronal death. However, the intracellular mechanisms associated with such sustained GluN2A-NMDAR stimulation and subsequent Ca2+ influx have remained unexplored. Using live-cell imaging with Fluo3-AM and biochemical approaches, we show that homocysteine-GluN2A NMDAR-induced initial Ca2+ influx triggers sequential phosphorylation and subsequent activation of the proline rich tyrosine kinase 2 (Pyk2) and Src family kinases, which in turn phosphorylates GluN2A-Tyr1325 residue of GluN2A-NMDARs to maintain channel activity. The continuity of this cycle of events leads to sustained Ca2+ influx through GluN2A-NMDAR. Our findings also show that lack of activation of the regulatory tyrosine phosphatase STEP, which can limit Pyk2 and Src family kinase activity further contributes to the maintenance of this cycle. Additional studies using live-cell imaging of neurons expressing a redox-sensitive GFP targeted to the mitochondrial matrix show that treatment with homocysteine leads to a progressive increase in mitochondrial reactive oxygen species generation, which is dependent on GluN2A-NMDAR-mediated sustained ERK MAPK activation. This later finding demonstrates a novel role of GluN2A-NMDAR in homocysteine-induced mitochondrial ROS generation and highlights the role of ERK MAPK as the intermediary signaling pathway between GluN2A-NMDAR stimulation and mitochondrial reactive oxygen species generation.

Hippocampal Pyk2 regulates specific social skills: Implications for schizophrenia.

Pyk2 has been shown previously to be involved in several psychological and cognitive alterations related to stress, Huntington's disease, and Alzheimer's disease. All these disorders are accompanied by different types of impairments in sociability, which has recently been linked to improper mitochondrial function. We hypothesize that Pyk2, which regulates mitochondria, could be associated with the regulation of mitochondrial dynamics and social skills. In the present manuscript, we report that a reduction of Pyk2 levels in mouse pyramidal neurons of the hippocampus decreased social dominance and aggressivity. Furthermore, social interactions induced robust Pyk2-dependent hippocampal changes in several oxidative phosphorylation complexes. We also observed that Pyk2 levels were increased in the CA1 pyramidal neurons of schizophrenic subjects, occurring alongside changes in different direct and indirect regulators of mitochondrial function including DISC1 and Grp75. Accordingly, overexpressing Pyk2 in hippocampal CA1 pyramidal cells mimicked some specific schizophrenia-like social behaviors in mice. In summary, our results indicate that Pyk2 might play a role in regulating specific social skills likely via mitochondrial dynamics and that there might be a link between Pyk2 levels in hippocampal neurons and social disturbances in schizophrenia.

Evaluation of Tyrosine Kinase Inhibitors Loaded Injectable Hydrogels for Improving Connexin43 Gap Junction Intercellular Communication.

Myocardial infarction (MI) is one of the leading causes of death in the developed world, and the loss of cardiomyocytes plays a critical role in the pathogenesis of heart failure. Implicated in this process is a decrease in gap junction intercellular communication due to remodeling of Connexin43 (Cx43). We previously identified that intraperitoneal injection of the Pyk2 inhibitor PF4618433 reduced infarct size, maintained Cx43 at the intercalated disc in left ventricle hypertrophic myocytes, and improved cardiac function in an MI animal model of heart failure. With the emergence of injectable hydrogels as a therapeutic toward the regeneration of cardiac tissue after MI, here, we provide proof of concept that the release of tyrosine kinase inhibitors from hydrogels could have beneficial effects on cardiomyocytes. We developed an injectable hydrogel consisting of thiolated hyaluronic acid and P123-maleimide micelles that can incorporate PF4618433 as well as the Src inhibitor Saracatinib and achieved sustained release (of note, Src activates Pyk2). Using neonatal rat ventricular myocytes in the presence of a phorbol ester, endothelin-1, or phenylephrine to stimulate cardiac hypertrophy, the release of PF4618433 from the hydrogel had the same ability to decrease Cx43 tyrosine phosphorylation and maintain Cx43 localization at the plasma membrane as when directly added to the growth media. Additional beneficial effects included decreases in apoptosis, the hypertrophic marker atrial natriuretic peptide (ANP), and serine kinases upregulated in hypertrophy. Finally, the presence of both PF4618433 and Saracatinib further decreased the level of ANP and apoptosis than each inhibitor alone, suggesting that a combinatorial approach may be most beneficial. These findings provide the groundwork to test if tyrosine kinase inhibitor release from hydrogels will have a beneficial effect in an animal model of MI-induced heart failure.

PYK2, a hub of signaling networks in breast cancer progression.

Breast cancer (BC) involves complex signaling networks characterized by extensive cross-communication and feedback loops between and within multiple signaling cascades. Many of these signaling pathways are driven by genetic alterations of oncogene and/or tumor-suppressor genes and are influenced by various environmental cues. We describe unique roles of the non-receptor tyrosine kinase (NRTK) PYK2 in signaling integration and feedback looping in BC. PYK2 functions as a signaling hub in various cascades, and its involvement in positive and negative feedback loops enhances signaling robustness, modulates signaling dynamics, and contributes to BC growth, epithelial-to-mesenchymal transition (EMT), stemness, migration, invasion, and metastasis. We also discuss the potential of PYK2 as a therapeutic target in various BC subtypes.

Blockade of DDR1/PYK2/ERK signaling suggesting SH2 superbinder as a novel autophagy inhibitor for pancreatic cancer.

Pancreatic cancer is highly lethal, of which 90% is pancreatic ductal adenocarcinoma (PDAC), with a 5-year survival rate of less than 12%, lacking effective treatment options and late diagnosis. Furthermore, the tumors show an intense resistance to cytotoxic chemotherapies. As autophagy is elevated in PDAC, targeting the autophagic pathway is regarded as a promising strategy for cancer treatment. Immunofluorescence and transmission electron microscopy were utilized to assess the autophagic flux. Label-free quantitative phosphoproteomics was used to figure out critically altered tyrosine phosphorylation of the proteins. Tumor-bearing mice were used to validate that SH2 TrM-(Arg)9 restrained the growth of tumor cells. SH2 TrM-(Arg)9 inhibited collagen-induced autophagy via blocking the DDR1/PYK2/ERK signaling cascades. SH2 TrM-(Arg)9 improved the sensitivity of PANC-1/GEM cells to gemcitabine (GEM). Inhibition of autophagy by SH2 TrM-(Arg)9 may synergized with chemotherapy and robusted tumor suppression in pancreatic cancer xenografts. SH2 TrM-(Arg)9 could enter into PDAC cells and blockade autophagy through inhibiting DDR1/PYK2/ERK signaling and may be a new treatment strategy for targeted therapy of PDAC.

Mettl3/Ythdf2 regulate macrophage inflammation and ROS generation by controlling Pyk2 mRNA stability.

As one of the most prevalent modifications on RNA, N6-methyladenosine (m6A) has been recently found implicated in various pathological processes. Emerging studies have demonstrated the role of m6A and its writer Mettl3 in fine-tuning the immune response, which now becomes a research hotspot owing to its potential therapeutic value. However, the results are inconsistent and even contradictory, suggesting that there might be multiple Mettl3 target genes involved in different pathways. To delve deeper into the function of Mettl3 in the cellular inflammatory response, we first conducted bioinformatics analysis using RNA-seq in Mettl3 ablation macrophages, and found that Mettl3 might attenuate LPS-induced proinflammatory pathways and reactive oxygen species (ROS) generation process. Mettl3 knockdown significantly increased the LPS-induced IL-6, TNF-α, NOXs (Nox1, Nox2, Ncf1, and Ncf2) expression, ROS generation, and the phosphorylation of MAPKs and AKT signaling. Combining the results of RNA-seq and m6A mapping, we found that Pyk2 might be the target gene of Mettl3 affecting the inflammatory response. Mettl3 and Ythdf2 depletion increased the expression and mRNA stability of Pyk2, and RIP-PCR showed that Ythdf2 directly targeting Pyk2 was Mettl3 dependent. Moreover, the upregulated expression of TNF-α, IL-6, NOXs, ROS generation, and the phosphorylation of MAPKs and AKT signaling were downregulated by Pyk2 inhibitor in Mettl3 knockdown cells. All of these results suggest that Mettl3 regulates the mRNA stability and expression of Pyk2 in a Ythdf2-dependent way, which consequently triggers the activation of MAPKs and AKT signaling and upregulation of NOXs, thus promoting the generation of proinflammatory cytokines and ROS.

PTK2B promotes TBK1 and STING oligomerization and enhances the STING-TBK1 signaling.

TANK-binding kinase 1 (TBK1) is a key kinase in regulating antiviral innate immune responses. While the oligomerization of TBK1 is critical for its full activation, the molecular mechanism of how TBK1 forms oligomers remains unclear. Here, we show that protein tyrosine kinase 2 beta (PTK2B) acts as a TBK1-interacting protein and regulates TBK1 oligomerization. Functional assays reveal that PTK2B depletion reduces antiviral signaling in mouse embryonic fibroblasts, macrophages and dendritic cells, and genetic experiments show that Ptk2b-deficient mice are more susceptible to viral infection than control mice. Mechanistically, we demonstrate that PTK2B directly phosphorylates residue Tyr591 of TBK1, which increases TBK1 oligomerization and activation. In addition, we find that PTK2B also interacts with the stimulator of interferon genes (STING) and can promote its oligomerization in a kinase-independent manner. Collectively, PTK2B enhances the oligomerization of TBK1 and STING via different mechanisms, subsequently regulating STING-TBK1 activation to ensure efficient antiviral innate immune responses.

ZC3H4 governs epithelial cell migration through ROCK/p-PYK2/p-MLC2 pathway in silica-induced pulmonary fibrosis.

BACKGROUND: Increased epithelial migration capacity is a key step accompanying epithelial-mesenchymal transition (EMT). Our lab has described that ZC3H4 mediated EMT in silicosis. Here, we aimed to explore the mechanisms of ZC3H4 by which to stimulate epithelial cell migration. METHODS: Silicon dioxide (SiO2)-induced pulmonary fibrosis (PF) animal models were administered by intratracheal instillation in C57BL/6 J mice. Pathological analysis and 2D migration assay were established to uncover the pulmonary fibrotic lesions and epithelial cell migration, respectively. Inhibitors targeting ROCK/p-PYK2/p-MLC2 and CRISPR/Cas9 plasmids targeting ZC3H4 were administrated to explore the signaling pathways. RESULTS: 1) SiO2 upregulated epithelial migration in pulmonary fibrotic lesions. 2) ZC3H4 modulated SiO2-induced epithelial migration. 3) ZC3H4 governed epithelial migration through ROCK/p-PYK2/p-MLC2 signaling pathway. CONCLUSIONS: ZC3H4 regulates epithelial migration through the ROCK/p-PYK2/p-MLC2 signaling pathway, providing the possibility that molecular drugs targeting ZC3H4-overexpression may exert effects on pulmonary fibrosis induced by silica.

Integrin signaling is critical for myeloid-mediated support of T-cell acute lymphoblastic leukemia.

We previously found that T-cell acute lymphoblastic leukemia (T-ALL) requires support from tumor-associated myeloid cells, which activate Insulin Like Growth Factor 1 Receptor (IGF1R) signaling in leukemic blasts. However, IGF1 is not sufficient to sustain T-ALL in vitro, implicating additional myeloid-mediated signals in leukemia progression. Here, we find that T-ALL cells require close contact with myeloid cells to survive. Transcriptional profiling and in vitro assays demonstrate that integrin-mediated cell adhesion activates downstream focal adhesion kinase (FAK)/ proline-rich tyrosine kinase 2 (PYK2), which are required for myeloid-mediated T-ALL support, partly through activation of IGF1R. Blocking integrin ligands or inhibiting FAK/PYK2 signaling diminishes leukemia burden in multiple organs and confers a survival advantage in a mouse model of T-ALL. Inhibiting integrin-mediated adhesion or FAK/PYK2 also reduces survival of primary patient T-ALL cells co-cultured with myeloid cells. Furthermore, elevated integrin pathway gene signatures correlate with higher FAK signaling and myeloid gene signatures and are associated with an inferior prognosis in pediatric T-ALL patients. Together, these findings demonstrate that integrin activation and downstream FAK/PYK2 signaling are important mechanisms underlying myeloid-mediated support of T-ALL progression.

Thrombin-Induced COX-2 Expression and PGE2 Synthesis in Human Tracheal Smooth Muscle Cells: Role of PKCδ/Pyk2-Dependent AP-1 Pathway Modulation.

In this study, we confirmed that thrombin significantly increases the production of COX-2 and PGE2 in human tracheal smooth muscle cells (HTSMCs), leading to inflammation in the airways and lungs. These molecules are well-known contributors to various inflammatory diseases. Here, we investigated in detail the involved signaling pathways using specific inhibitors and small interfering RNAs (siRNAs). Our results demonstrated that inhibitors targeting proteins such as protein kinase C (PKC)δ, proline-rich tyrosine kinase 2 (Pyk2), c-Src, epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), or activator protein-1 (AP-1) effectively reduced thrombin-induced COX-2 and PGE2 production. Additionally, transfection with siRNAs against PKCδ, Pyk2, c-Src, EGFR, protein kinase B (Akt), or c-Jun mitigated these responses. Furthermore, our observations revealed that thrombin stimulated the phosphorylation of key components of the signaling cascade, including PKCδ, Pyk2, c-Src, EGFR, Akt, and c-Jun. Thrombin activated COX-2 promoter activity through AP-1 activation, a process that was disrupted by a point-mutated AP-1 site within the COX-2 promoter. Finally, resveratrol (one of the most researched natural polyphenols) was found to effectively inhibit thrombin-induced COX-2 expression and PGE2 release in HTSMCs through blocking the activation of Pyk2, c-Src, EGFR, Akt, and c-Jun. In summary, our findings demonstrate that thrombin-induced COX-2 and PGE2 generation involves a PKCδ/Pyk2/c-Src/EGFR/PI3K/Akt-dependent AP-1 activation pathway. This study also suggests the potential use of resveratrol as an intervention for managing airway inflammation.

Lupenone attenuates thapsigargin-induced endoplasmic reticulum stress and apoptosis in pancreatic beta cells possibly through inhibition of protein tyrosine kinase 2 activity.

AIMS: Prolonged high levels of cytokines, glucose, or free fatty acids are associated with diabetes, elevation of cytosolic Ca2+ concentration ([Ca2+]C), and depletion of Ca2+ concentration in the endoplasmic reticulum (ER) of pancreatic beta cells. This Ca2+ imbalance induces ER stress and apoptosis. Lupenone, a lupan-type triterpenoid, is beneficial in diabetes; however, its mechanism of action is yet to be clarified. This study evaluated the protective mechanism of lupenone against thapsigargin-induced ER stress and apoptosis in pancreatic beta cells. MATERIALS AND METHODS: MIN6, INS-1, and native mouse islet cells were used. Western blot for protein expressions, measurement of [Ca2+]C, and in vivo glucose tolerance test were mainly performed. KEY FINDINGS: Thapsigargin increased the protein levels of cleaved caspase 3, cleaved PARP, and the phosphorylated form of JNK, ATF4, and CHOP. Thapsigargin increased the interaction between stromal interaction molecule1 (Stim1) and Orai1, enhancing store-operated calcium entry (SOCE). SOCE is further activated by protein tyrosine kinase 2 (Pyk2), which is Ca2+-dependent and phosphorylates the tyrosine residue at Y361 in Stim1. Lupenone inhibited thapsigargin-mediated Pyk2 activation, suppressed [Ca2+]C, ER stress, and apoptosis. Lupenone restored impaired glucose-stimulated insulin secretion effectuated by thapsigargin and glucose intolerance in a low-dose streptozotocin-induced diabetic mouse model. SIGNIFICANCE: These results suggested that lupenone attenuated thapsigargin-induced ER stress and apoptosis by inhibiting SOCE; this may be due to the hindrance of Pyk2-mediated Stim1 tyrosine phosphorylation. In beta cells that are inevitably exposed to frequent [Ca2+]C elevation, the attenuation of abnormally high SOCE would be beneficial for their survival.

MYD88L265P augments proximal B-cell receptor signaling in large B-cell lymphomas via an interaction with DOCK8.

Diffuse large B-cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease with at least 5 recognized molecular subtypes. Cluster 5 (C5)/MCD tumors frequently exhibit concurrent alterations in the toll-like receptor (TLR) and B-cell receptor (BCR) pathway members, MYD88L265P and CD79B, and have a less favorable prognosis. In healthy B cells, the synergy between TLR and BCR signaling pathways integrates innate and adaptive immune responses and augments downstream NF-κB activation. In addition, physiologic TLR9 pathway engagement via MYD88, protein tyrosine kinase 2 (PYK2), and dedicator of cytokinesis 8 (DOCK8) increases proximal BCR signaling in healthy murine B cells. Although C5/MCD DLBCLs are selectively sensitive to Bruton tyrosine kinase (BTK) inhibition in in vitro studies and certain clinical trials, the role of mutated MYD88 in proximal BCR signaling remains undefined. Using engineered DLBCL cell line models, we found that concurrent MYD88L265P and CD79B alterations significantly increased the magnitude and duration of proximal BCR signaling, at the level of spleen tyrosine kinase and BTK, and augmented PYK2-dependent DOCK8 phosphorylation. MYD88L265P DLBCLs have significantly increased colocalization of DOCK8 with both MYD88 and the proximal BCR-associated Src kinase, LYN, in comparison with MYD88WT DLBCLs, implicating DOCK8 in MYD88L265P/proximal BCR cross talk. Additionally, DOCK8 depletion selectively decreased proximal BCR signaling, cellular proliferation, and viability of DLBCLs with endogenous MYD88L265P/CD79BY196F alterations and increased the efficacy of BTK blockade in these lymphomas. Therefore, MYD88L265P/DOCK8-enhanced proximal BCR signaling is a likely mechanism for the increased sensitivity of C5/MCD DLBCLs to BTK blockade.

Tyrphostin A9 attenuates glioblastoma growth by suppressing PYK2/EGFR-ERK signaling pathway.

PURPOSE: Glioblastoma (GBM) is a fatal primary brain tumor with extremely poor clinical outcomes. The anticancer efficiency of tyrosine kinase inhibitors (TKIs) has been shown in GBM and other cancer, with limited therapeutic outcomes. In the current study, we aimed to investigate the clinical impact of active proline-rich tyrosine kinase-2 (PYK2) and epidermal growth factor receptor (EGFR) in GBM and evaluate its druggability by a synthetic TKI-Tyrphostin A9 (TYR A9). METHODS: The expression profile of PYK2 and EGFR in astrocytoma biopsies (n = 48) and GBM cell lines were evaluated through quantitative PCR, western blots, and immunohistochemistry. The clinical association of phospho-PYK2 and EGFR was analyzed with various clinicopathological features and the Kaplan-Meier survival curve. The phospho-PYK2 and EGFR druggability and subsequent anticancer efficacy of TYR A9 was evaluated in GBM cell lines and intracranial C6 glioma model. RESULTS: Our expression data revealed an increased phospho-PYK2, and EGFR expression aggravates astrocytoma malignancy and is associated with patients' poor survival. The mRNA and protein correlation analysis showed a positive association between phospho-PYK2 and EGFR in GBM tissues. The in-vitro studies demonstrated that TYR A9 reduced GBM cell growth, cell migration, and induced apoptosis by attenuating PYK2/EGFR-ERK signaling. The in-vivo data showed TYR A9 treatment dramatically reduced glioma growth with augmented animal survival by repressing PYK2/EGFR-ERK signaling. CONCLUSION: Altogether, this study report that increased phospho-PYK2 and EGFR expression in astrocytoma was associated with poor prognosis. The in-vitro and in-vivo evidence underlined translational implication of TYR A9 by suppressing PYK2/EGFR-ERK modulated signaling pathway. The schematic diagram displayed proof of concept of the current study indicating activated PYK2 either through the Ca2+/Calmodulin-dependent protein kinase II (CAMKII) signaling pathway or autophosphorylation at Tyr402 induces association to the SH2 domain of c-Src that leads to c-Src activation. Activated c-Src in turn activates PYK2 at other tyrosine residues that recruit Grb2/SOS complex and trigger ERK½ activation. Besides, PYK2 interaction with c-Src acts as an upstream of EGFR transactivator that can activate the ERK½ signaling pathway, which induces cell proliferation and cell survival by increasing anti-apoptotic proteins or inhibiting pro-apoptotic proteins. TYR A9 treatment attenuate GBM cell proliferation and migration; and induce GBM cell death by inhibiting PYK2 and EGFR-induced ERK activation.

Inhibition of Pyk2 Improves Cx43 Intercalated Disc Localization, Infarct Size, and Cardiac Function in Rats With Heart Failure.

BACKGROUND: Heart failure causes changes in Cx43 (Connexin43) regulation that are associated with arrhythmic heart disease. Pyk2 (proline-rich tyrosine kinase 2) is activated in cardiomyopathies and phosphorylates Cx43 to decrease intercellular communication. This study was designed to determine if Pyk2 inhibition improves cardiac function in a myocardial infarction (MI)-induced heart failure model in rats. METHODS: MI (ligation of left anterior descending artery) rats were treated with the Pyk2 inhibitor PF4618433. Hemodynamic and structural parameters were monitored in Sham (n=5), MI-vehicle (n=5), and MI-PF4618433 (n=8) groups. Heart tissues were collected after 6 weeks to assess Pyk2 and Cx43 protein level and localization. RESULTS: PF4618433 produced no observed adverse effects and inhibited ventricular Pyk2. PF4618433 reduced the MI infarct size from 34% to 17% (P=0.007). PF4618433 improved stroke volume (P=0.031) and cardiac output (P=0.009) in comparison to MI-vehicle with values similar to the Sham group. PF4618433 also led to an increase in the ejection fraction (P=0.002) and fractional shortening (P=0.006) when compared with the MI-vehicle (32% and 35% improvement, respectively) yet were lower in comparison with the Sham group. Pyk2 inhibition decreased Cx43 tyrosine phosphorylation (P=0.043) and maintained Cx43 at the intercalated disc in the distal ventricle 6 weeks post-MI. CONCLUSIONS: Unlike other attempts to decrease Cx43 remodeling after MI-induced heart failure, inhibition of Pyk2 activity maintained Cx43 at the intercalated disc. This may have aided in the reduced infarct size (acute time frame) and improved cardiac function (chronic time frame). Additionally, we provide evidence that Pyk2 is activated following MI in human left ventricle, implicating a novel potential target for therapy in patients with heart failure.

α1-Adrenergic receptor-PKC-Pyk2-Src signaling boosts L-type Ca2+ channel CaV1.2 activity and long-term potentiation in rodents.

The cellular mechanisms mediating norepinephrine (NE) functions in brain to result in behaviors are unknown. We identified the L-type Ca2+ channel (LTCC) CaV1.2 as a principal target for Gq-coupled α1-adrenergic receptors (ARs). α1AR signaling increased LTCC activity in hippocampal neurons. This regulation required protein kinase C (PKC)-mediated activation of the tyrosine kinases Pyk2 and, downstream, Src. Pyk2 and Src were associated with CaV1.2. In model neuroendocrine PC12 cells, stimulation of PKC induced tyrosine phosphorylation of CaV1.2, a modification abrogated by inhibition of Pyk2 and Src. Upregulation of LTCC activity by α1AR and formation of a signaling complex with PKC, Pyk2, and Src suggests that CaV1.2 is a central conduit for signaling by NE. Indeed, a form of hippocampal long-term potentiation (LTP) in young mice requires both the LTCC and α1AR stimulation. Inhibition of Pyk2 and Src blocked this LTP, indicating that enhancement of CaV1.2 activity via α1AR-Pyk2-Src signaling regulates synaptic strength.

PTK2B regulates immune responses of neutrophils and protects mucosal inflammation in ulcerative colitis.

Neutrophils participate in the pathogenesis of ulcerative colitis (UC) through regulating the intestinal homeostasis. Several inflammatory diseases are reported to be regulated by proline-rich tyrosine kinase 2B (PTK2B). However, the role of PTK2B in regulating the function of neutrophils and the pathogenesis of UC remains unknown. In this study, the mRNA and protein levels of PTK2B in the colonic tissues from UC patients were measured by using quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. TAE226, a PTK2B inhibitor, was used to inhibit the activity of PTK2B in neutrophils, and then, the pro-inflammatory factors were analyzed by using qRT-PCR and ELISA. To determine the role of PTK2B in intestinal inflammation, a dextran sulfate sodium (DSS)-induced colitis model was established in PTK2B gene knockout (PTK2B KO) and wild-type (WT) mice. We found that compared with healthy donor controls, the expression level of PTK2B was significantly elevated in inflamed mucosa from UC patients. In addition, expression of PTK2B was positively correlated with the severity of disease. Pharmacological inhibition of PTK2B could markedly reduce the generation of reactive oxygen species (ROS), myeloperoxidase (MPO), and antimicrobial peptides (S100a8 and S100a9) in neutrophils. The vitro study showed that tumor necrosis factor (TNF)-α is involved in promoting the expression of PTK2B in neutrophils. As expected, UC patients treated with infliximab, an anti-TNF-α agent, showed significantly reduced level of PTK2B in neutrophils, as well as in the intestinal mucosa. Of note, compared with DSS-treated WT mice, DSS-treated PTK2B KO mice showed more severe colitis symptoms. Mechanistically, PTK2B could enhance neutrophil migration by regulating CXCR2 and GRK2 expression via the p38 MAPK pathway. Additionally, mice treated with TAE226 exhibited the same effects. In conclusion, PTK2B is involved in the pathogenesis of UC by promoting the migration of neutrophils and inhibiting mucosal inflammation, highlighting PTK2B as a new potential therapeutic target to treat UC.