Nonreceptor Tyrosine Kinase Pyk2 Research Articles

TMIC-51. THE RELEASE OF INFLAMMATORY CYTOKINES ARE REGULATED BY PYK2 SIGNALING IN GBM CELLS

Abstract Glioblastoma (GBM) is a highly aggressive brain cancer that is typically fatal within a year of diagnosis. Previous research has shown a positive relationship between the activation of a protein called proline-rich non-receptor tyrosine kinase (Pyk2) in GBM cells in addition to the expression of cytokines by tumor-infiltrating myeloid (TIM cells). Leading to a sustained increase in the expression levels of monocyte chemoattractant protein 1 (CCL2), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL10, IL12, and vascular endothelial growth factor (VEGF), as well as an increase in the CD86+/CD206+ TIM population. We hypothesize that Pyk2 signaling in GBM cells is involved in the regulation of the release of inflammatory cytokines, which in turn suppresses the immune response in the tumor microenvironment. We used CRISPR/Cas9 knockout Pyk2 (Pyk2KO) and wild type (Pyk2WT) GL261 mouse glioma cells in this study. Analysis of the cytokines present in the cell culture medium conditioned by Pyk2KO and Pyk2WT cells identified a decrease in the release of CCL2 and CCL5 in the Pyk2KO cells compared to the Pyk2WT cells. RT-PCR analysis of myeloid cell population, isolated from Pyk2WT and Pyk2KO tumors in the GL261-C57Bl/6 GBM model, identified a significant down-regulation of key pro-tumorigenic factors such as CCL2, CCL12, CCL5, tumor necrosis factor (TNF), VEGF and epidermal growth factor (EGF) in the Pyk2KO tumors compared to the Pyk2WT tumors. Additionally, flow cytometry analysis revealed a reduction in myeloid-derived suppressor cells (MDSCs), an increase in lymphoid dendritic cells (DC), and up-regulation in TNF/IFNg expressing and antigen-specific CD8+ T cells in Pyk2KO tumors. Furthermore, a significant decrease in pro-tumorigenic Ly6C+/CD206+ and an up-regulation of inflammatory Ly6C+/CD86+ myeloid cells were found in the Pyk2KO tumors. These findings suggest that Pyk2KO tumors display an immunocompetent microenvironment with enhanced phagocytic and cytotoxic function compared to Pyk2WT tumors. NIH Grant 1SC1GM122691 and PRSTRT2022 supported this study

A novel Pyk2-derived peptide inhibits invadopodia-mediated breast cancer metastasis.

Dissemination of cancer cells from the primary tumor into distant body tissues and organs is the leading cause of death in cancer patients. While most clinical strategies aim to reduce or impede the growth of the primary tumor, no treatment to eradicate metastatic cancer exists at present. Metastasis is mediated by feet-like cytoskeletal structures called invadopodia which allow cells to penetrate through the basement membrane and intravasate into blood vessels during their spread to distant tissues and organs. The non-receptor tyrosine kinase Pyk2 is highly expressed in breast cancer, where it mediates invadopodia formation and function via interaction with the actin-nucleation-promoting factor cortactin. Here, we designed a cell-permeable peptide inhibitor that contains the second proline-rich region (PRR2) sequence of Pyk2, which binds to the SH3 domain of cortactin and inhibits the interaction between Pyk2 and cortactin in invadopodia. The Pyk2-PRR2 peptide blocks spontaneous lung metastasis in immune-competent mice by inhibiting cortactin tyrosine phosphorylation and actin polymerization-mediated maturation and activation of invadopodia, leading to reduced MMP-dependent tumor cell invasiveness. The native structure of the Pyk2-PRR2:cortactin-SH3 complex was determined using nuclear magnetic resonance (NMR), revealing an extended class II interaction surface spanning the canonical binding groove and a second hydrophobic surface which significantly contributes to ligand affinity. Using structure-guided design, we created a mutant peptide lacking critical residues involved in binding that failed to inhibit invadopodia maturation and function and consequent metastatic dissemination in mice. Our findings shed light on the specific molecular interactions between Pyk2 and cortactin and may lead to the development of novel strategies for preventing dissemination of primary breast tumors predicted at the time of diagnosis to be highly metastatic, and of secondary tumors that have already spread to other parts of the body.

The Non-receptor Tyrosine Kinase Pyk2 in Brain Function and Neurological and Psychiatric Diseases.

Pyk2 is a non-receptor tyrosine kinase highly enriched in forebrain neurons. Pyk2 is closely related to focal adhesion kinase (FAK), which plays an important role in sensing cell contacts with extracellular matrix and other extracellular signals controlling adhesion and survival. Pyk2 shares some of FAK’s characteristics including recruitment of Src-family kinases after autophosphorylation, scaffolding by interacting with multiple partners, and activation of downstream signaling pathways. Pyk2, however, has the unique property to respond to increases in intracellular free Ca2+, which triggers its autophosphorylation following stimulation of various receptors including glutamate NMDA receptors. Pyk2 is dephosphorylated by the striatal-enriched phosphatase (STEP) that is highly expressed in the same neuronal populations. Pyk2 localization in neurons is dynamic, and altered following stimulation, with post-synaptic and nuclear enrichment. As a signaling protein Pyk2 is involved in multiple pathways resulting in sometimes opposing functions depending on experimental models. Thus Pyk2 has a dual role on neurites and dendritic spines. With Src family kinases Pyk2 participates in postsynaptic regulations including of NMDA receptors and is necessary for specific types of synaptic plasticity and spatial memory tasks. The diverse functions of Pyk2 are also illustrated by its role in pathology. Pyk2 is activated following epileptic seizures or ischemia-reperfusion and may contribute to the consequences of these insults whereas Pyk2 deficit may contribute to the hippocampal phenotype of Huntington’s disease. Pyk2 gene, PTK2B, is associated with the risk for late-onset Alzheimer’s disease. Studies of underlying mechanisms indicate a complex contribution with involvement in amyloid toxicity and tauopathy, combined with possible functional deficits in neurons and contribution in microglia. A role of Pyk2 has also been proposed in stress-induced depression and cocaine addiction. Pyk2 is also important for the mobility of astrocytes and glioblastoma cells. The implication of Pyk2 in various pathological conditions supports its potential interest for therapeutic interventions. This is possible through molecules inhibiting its activity or increasing it through inhibition of STEP or other means, depending on a precise evaluation of the balance between positive and negative consequences of Pyk2 actions.

Pyk2 suppresses contextual fear memory in an autophosphorylation-independent manner.

Clustered protocadherins (Pcdhs) are a large family of cadherin-like cell adhesion proteins that are central for neurite self-avoidance and neuronal connectivity in the brain. Their downstream nonreceptor tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2, also known as Ptk2b, Cakb, Raftk, Fak2, and Cadtk) is predominantly expressed in the hippocampus. We constructed Pyk2-null mouse lines and found that these mutant mice showed enhancement in contextual fear memory, without significant change in auditory-cued and spatial-referenced learning and memory. In addition, by preparing Y402F mutant mice, we observed that Pyk2 suppressed contextual fear memory in an autophosphorylation-independent manner. Moreover, using high-throughput RNA sequencing, we found that immediate early genes, such as Npas4, cFos, Zif268/Egr1, Arc, and Nr4a1, were enhanced in Pyk2-null mice. We further showed that Pyk2 disruption affected pyramidal neuronal complexity and spine dynamics. Thus, we demonstrated that Pyk2 is a novel fear memory suppressor molecule and Pyk2-null mice provide a model for understanding fear-related disorders. These findings have interesting implications regarding dysregulation of the Pcdh‒Pyk2 axis in neuropsychiatric disorders.

The Caveolin-1 Scaffolding Domain Peptide Reverses Aging-Associated Deleterious Changes in Multiple Organs.

Aging is a progressive, multifactorial, degenerative process in which deleterious changes occur in the biochemistry and function of organs. We showed that angiotensin II (AngII)-induced pathologies in the heart and kidney of young (3-month-old) mice are suppressed by the caveolin-1 scaffolding domain (CSD) peptide. Because AngII mediates many aging-associated changes, we explored whether CSD could reverse pre-existing pathologies and improve organ function in aged mice. Using 18-month-old mice (similar to 60-year-old humans), we found that >5-fold increases in leakage of serum proteins and >2-fold increases in fibrosis are associated with aging in the heart, kidney, and brain. Because tyrosine phosphorylation of cell junction proteins leads to the loss of microvascular barrier function, we analyzed the activation of the receptor tyrosine kinase PDGFR and the nonreceptor tyrosine kinases c-Src and Pyk2. We observed 5-fold activation of PDGFR and 2- to 3-fold activation of c-Src and Pyk2 in aged mice. Treatment with CSD for 4 weeks reversed these pathologic changes (microvascular leakage, fibrosis, kinase activation) in all organs almost down to the levels in healthy, young mice. In studies of heart function, CSD reduced the aging-associated increase in cardiomyocyte cross-sectional area and enhanced ventricular compliance in that echocardiographic studies demonstrated improved ejection fraction and fractional shortening and reduced isovolumic relation time. These results suggest that versions of CSD may be developed as treatments for aging-associated diseases in human patients based on the concept that CSD inhibits tyrosine kinases, leading to the inhibition of microvascular leakage and associated fibrosis, thereby improving organ function. SIGNIFICANCE STATEMENT: The caveolin-1 scaffolding domain (CSD) peptide reverses aging-associated fibrosis, microvascular leakage, and organ dysfunction in the heart, kidneys, and brain via a mechanism that involves the suppression of the activity of multiple tyrosine kinases, suggesting that CSD can be developed as a treatment for a wide range of diseases found primarily in the aged.

Acute stress induces visceral hypersensitivity via glucocorticoid receptor-mediated membrane insertion of TRPM8: Involvement of a non-receptor tyrosine kinase Pyk2.

Psychological stress is an important factor for the development and recurrence of irritable bowel syndrome (IBS). The mechanisms underlying stress-induced visceral hypersensitivity (VH), a key pathophysiological component in IBS, are still incompletely understood. We aimed to test whether transient receptor potential melastatin 8 (TRPM8) participates in acute stress-induced VH. Rats were subjected to 1-hour water avoidance stress (WAS). Visceral sensitivity was measured with visceromotor response to colorectal distension. Western blot and immunofluorescence were applied to evaluate the expression of GR and TRPM8 and activation of PKA, Akt, and PKC pathways. WAS-caused VH depended on glucocorticoid receptors (GRs) and TRPM8 channels. In a dorsal root ganglion (DRG)-derived cell line, corticosterone rapidly (within 30minutes) induced membrane expression of TRPM8. This effect was inhibited by GR antagonism and was mimicked by membrane-impermeable corticosterone. PKA, PI3K/Akt, and PKC pathways, which lied downstream of GR and acted in parallel to promote membrane expression of TRPM8, contributed to WAS-induced VH. The non-receptor tyrosine kinase Pyk2, which may serve as a convergence point for PKA, PI3K/Akt, and PKC pathways, facilitated membrane insertion of TRPM8 via tyrosine-phosphorylating TRPM8 in L6-S2 DRGs and participated in WAS-induced VH. Collectively, acute stress-induced VH could involve membrane-bound GR-dependent enhancement of TRPM8 function in nociceptive DRG neurons. Mechanistically, Pyk2 could act as a key mediator that coordinates multiple protein kinase signaling and triggers phosphorylation and membrane insertion of TRPM8.

The non-receptor tyrosine kinase Pyk2 modulates acute locomotor effects of cocaine in D1 receptor-expressing neurons of the nucleus accumbens

The striatum is critical for cocaine-induced locomotor responses. Although the role of D1 receptor-expressing neurons is established, underlying molecular pathways are not fully understood. We studied the role of Pyk2, a non-receptor, calcium-dependent protein-tyrosine kinase. The locomotor coordination and basal activity of Pyk2 knock-out mice were not altered and major striatal protein markers were normal. Cocaine injection increased Pyk2 tyrosine phosphorylation in mouse striatum. Pyk2-deficient mice displayed decreased locomotor response to acute cocaine injection. In contrast, locomotor sensitization and conditioned place preference were normal. Cocaine-activated ERK phosphorylation, a signaling pathway essential for these late responses, was unaltered. Conditional deletion of Pyk2 in the nucleus accumbens or in D1 neurons reproduced decreased locomotor response to cocaine, whereas deletion of Pyk2 in the dorsal striatum or in A2A receptor-expressing neurons did not. In mice lacking Pyk2 in D1-neurons locomotor response to D1 agonist SKF-81297, but not to an anticholinergic drug, was blunted. Our results identify Pyk2 as a regulator of acute locomotor responses to psychostimulants. They highlight the role of tyrosine phosphorylation pathways in striatal neurons and suggest that changes in Pyk2 expression or activation may alter specific responses to drugs of abuse, or possibly other behavioral responses linked to dopamine action.

Hydrogen peroxide inducible clone-5 sustains NADPH oxidase-dependent reactive oxygen species-c-jun N-terminal kinase signaling in hepatocellular carcinoma

Target therapy aiming at critical molecules within the metastatic signal pathways is essential for prevention of hepatocellular carcinoma (HCC) progression. Hic-5 (hydrogen peroxide inducible clone-5) which belongs to the paxillin superfamily, can be stimulated by a lot of metastatic factors, such as transforming growth factor (TGF-β), hepatocyte growth factor (HGF), and reactive oxygen species (ROS). Previous studies implicated Hic-5 cross-talks with the ROS-c-jun N-terminal kinase (JNK) signal cascade in a positive feedback manner. In this report, we addressed this issue in a comprehensive manner. By RNA interference and ectopic Hic-5 expression, we demonstrated Hic-5 was essential for activation of NADPH oxidase and ROS generation leading to activation of downstream JNK and c-jun transcription factor. This was initiated by interaction of Hic-5 with the regulator and adaptor of NADPH oxidase, Rac1 and Traf4, respectively, which may further phosphorylate the nonreceptor tyrosine kinase Pyk2 at Tyr881. On the other hand, promoter activity assay coupled with deletion mapping and site directed mutagenesis strategies demonstrated the distal c-jun and AP4 putative binding regions (943–1126 bp upstream of translational start site) were required for transcriptional activation of Hic-5. Thus Hic-5 was both downstream and upstream of NADPH oxidase-ROS-JNK-c-jun cascade. This signal circuit was essential for regulating the expression of epithelial mesenchymal transition (EMT) factors, such as Snail, Zeb1, E-cadherin, and matrix metalloproteinase 9, involved in HCC cell migration and metastasis. Due to the limited expression of Hic-5 in normal tissue, it can be a promising therapeutic target for preventing HCC metastasis.

PYK2 negatively regulates the Hippo pathway in TNBC by stabilizing TAZ protein

The tumor suppressor Hippo pathway negatively regulates the transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) to inhibit cell growth and control organ size, whereas activation of YAP and TAZ is implicated in tumorigenesis and cancer metastasis. Here, we report that the nonreceptor tyrosine kinase PYK2 positively regulates TAZ and YAP transcriptional activity in triple-negative breast cancer (TNBC). We found that inhibition of PYK2 expression or its kinase activity substantially affects the steady-state level of TAZ and markedly facilitates its proteasomal degradation. This effect was specific to PYK2 inhibition and was not obtained by inhibition of FAK. Destabilization of TAZ was associated with profound effect of PYK2 inhibition on cell growth at low-density concomitant with reduced expression of TAZ-target genes and induction of cell apoptosis. We further show that PYK2 enhances the tyrosine phosphorylation of both TAZ and LATS1/2 and concomitantly TAZ stability, and that PYK2 protein level correlates with the level of TAZ protein in primary breast tumors. Together these observations suggest that PYK2 is an important regulator of the Hippo pathway, and its tyrosine kinase activity has a striking effect on TAZ stabilization and activation in TNBC.

Systems modelling of the EGFR-PYK2-c-Met interaction network predicts and prioritizes synergistic drug combinations for triple-negative breast cancer.

Prediction of drug combinations that effectively target cancer cells is a critical challenge for cancer therapy, in particular for triple-negative breast cancer (TNBC), a highly aggressive breast cancer subtype with no effective targeted treatment. As signalling pathway networks critically control cancer cell behaviour, analysis of signalling network activity and crosstalk can help predict potent drug combinations and rational stratification of patients, thus bringing therapeutic and prognostic values. We have previously showed that the non-receptor tyrosine kinase PYK2 is a downstream effector of EGFR and c-Met and demonstrated their crosstalk signalling in basal-like TNBC. Here we applied a systems modelling approach and developed a mechanistic model of the integrated EGFR-PYK2-c-Met signalling network to identify and prioritize potent drug combinations for TNBC. Model predictions validated by experimental data revealed that among six potential combinations of drug pairs targeting the central nodes of the network, including EGFR, c-Met, PYK2 and STAT3, co-targeting of EGFR and PYK2 and to a lesser extent of EGFR and c-Met yielded strongest synergistic effect. Importantly, the synergy in co-targeting EGFR and PYK2 was linked to switch-like cell proliferation-associated responses. Moreover, simulations of patient-specific models using public gene expression data of TNBC patients led to predictive stratification of patients into subgroups displaying distinct susceptibility to specific drug combinations. These results suggest that mechanistic systems modelling is a powerful approach for the rational design, prediction and prioritization of potent combination therapies for individual patients, thus providing a concrete step towards personalized treatment for TNBC and other tumour types.

PO-139 Identify rational drug combinations for triple negative breast cancer by integrative network modelling

Introduction Triple negative breast cancer (TNBC) is a subtype of breast cancer that accounts for up to 20% of all newly diagnosed cases. Due to its recalcitrance to available targeted agents, there is an urgent need to optimise therapy in order to improve patient prognosis. We previously demonstrated that the non-receptor tyrosine kinase PYK2 promotes tumour growth in a subtype of TNBC and in a xenograft mouse model and that PYK2 is a downstream effector of EGFR, c-Met and their crosstalk signalling. Here we use a system biology approach to identify and prioritise potent drug combinations for TNBC with the help of a mathematical model we developed from the integrated EGFR-PYK2-c-Met signalling network. Material and methods A quantitative, kinetic model of the EGFR-PYK2-c-Met signalling interaction network was developed and experimentally validated using two TNBC cell lines, MDA-MB-468 and BT-20. Signalling induction and dose response experiments for potential combinations of drug pairs targeting the central nodes of the network, including EGFR, c-Met, PYK2 and STAT3 were performed and evaluated by western blot and cell viability assays to calculate drug synergism. The mathematical model was implemented in MATLAB. For model calibration and parameter estimation MATLAB’s Global Optimisation toolbox was utilised. High-performance super-computers at Monash University were employed for parameter estimation. The Chou-Talalay’s Combination Index, (CI), Bliss Independence (BI) and Coefficient of Drug Interaction (CDI) were applied to numerically assess drug combination effects. Moreover, patient-specific models were generated through the incorporation of gene and protein expression data from individual TNBC patients using a discovery patient cohort, containing 108 patients with both patient-specific transcriptomic and proteomic data. For validation, an independent cohort was obtained from the European-Genome Phenome Archive. Results and discussions Model predictions backed up by experimental in vitro data revealed that co-targeting of EGFR and PYK2 has the most synergistic effect. Furthermore, simulations of patient-specific models led to stratification of patients into subgroups showing discrete susceptibility to specific drug combinations. Conclusion These results suggest that mechanistic systems modelling is a powerful tool for the rational design, prediction and prioritisation of potent combinatorial therapies for individual patients, hence providing a concrete step towards personalised medicine for TNBC and other tumour types.

Pyk2 and FAK differentially regulate invadopodia formation and function in breast cancer cells.

The nonreceptor tyrosine kinase Pyk2 is highly expressed in invasive breast cancer, but the mechanism by which it potentiates tumor cell invasiveness is unclear at present. Using high-throughput protein array screening and bioinformatic analysis, we identified cortactin as a novel substrate and interactor of proline-rich tyrosine kinase 2 (Pyk2). Pyk2 colocalizes with cortactin to invadopodia of invasive breast cancer cells, where it mediates epidermal growth factor-induced cortactin tyrosine phosphorylation both directly and indirectly via Src-mediated Abl-related gene (Arg) activation, leading to actin polymerization in invadopodia, extracellular matrix degradation, and tumor cell invasion. Both Pyk2 and the closely related focal adhesion kinase (FAK) regulate tumor cell invasion, albeit via distinct mechanisms. Although Pyk2 regulates tumor cell invasion by controlling invadopodium-mediated functions, FAK controls invasiveness of tumor cells by regulating focal adhesion-mediated motility. Collectively, our findings identify Pyk2 as a unique mediator of invadopodium formation and function and also provide a novel insight into the mechanisms by which Pyk2 mediates tumor cell invasion.

The nonreceptor protein tyrosine kinase Pyk2 promotes the turnover of monocytes at steady state.

Monocytes are short-lived myeloid cells that perform functions essential for tissue homeostasis and disease resolution. However, the cellular mechanisms controlling the maintenance and turnover of monocyte populations are largely undefined. Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase that regulates numerous immune cell functions, but its role in monocytes is currently unknown. In this study, we sought to characterize the expression and function of Pyk2 in lineage-committed monocyte populations. Here, we report that Pyk2 protein expression is increased in the Ly6C- monocyte population. Using a Pyk2 knockout mouse model (Pyk2-/-), we show that Pyk2 regulates the relative proportion of monocyte subsets normally represented in the bone marrow (BM) at steady state. In support of this conclusion, a similar phenotype was observed in the peripheral blood and spleen. Data from reciprocal BM chimera experiments indicate that the alterations in monocyte populations exhibited by Pyk2-/- mice are due to factors intrinsic to the monocytes. Lineage-tracing of monocyte populations suggests that Pyk2 promotes apoptosis in BM monocytes, thereby acting as an important homeostatic regulator of turnover in these short-lived, innate immune cells.

The non-receptor protein tyrosine kinase Pyk2 promotes the turnover of mouse monocyte populations at steady-state

Abstract Peripheral monocytes, consisting of distinct Ly6C+ and Ly6Cneg subsets in mice, are innate immune effector cells that exhibit a relatively short half-life of 1–3 days. While the differentiation and function of these monocyte subpopulations have been explored extensively, the cellular mechanisms regulating the maintenance and turnover of these populations are not completely understood. The focus of our current study is to investigate the role of proline-rich tyrosine kinase 2 (Pyk2) in the establishment and/or maintenance of monocyte populations at steady-state. Using a Pyk2 knock-out mouse model (Pyk2−/−), we show for the first time that Pyk2−/− mice exhibit reduced bone marrow (BM) cellularity, resulting in a reduction of total BM monocytes in these mice. Accordingly, Pyk2−/− mice display reduced numbers of BM monocyte progenitors and Ly6C+ monocytes; however, there is no reduction in the representation of Ly6Cneg monocytes in the absence of Pyk2, suggesting that Pyk2 regulates the relative proportion of monocyte subsets normally represented in the BM at steady state. In support of this conclusion, a similar phenotype was observed in the peripheral blood and spleen. Data from reciprocal and mixed BM chimera experiments indicate that the alterations in monocyte populations exhibited by Pyk2−/− mice are due to factors intrinsic to the monocytes. Pyk2 does not appear to regulate the proliferation, differentiation, or egress of monocyte subsets. Instead, experiments using annexin V and cleaved caspase as markers for apoptosis indicate that Pyk2 may promote apoptosis in BM monocytes, thereby acting as an important regulator of turnover in these short-lived effector cells.

An in silico high-throughput screen identifies potential selective inhibitors for the non-receptor tyrosine kinase Pyk2.

The non-receptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2) is a critical mediator of signaling from cell surface growth factor and adhesion receptors to cell migration, proliferation, and survival. Emerging evidence indicates that signaling by Pyk2 regulates hematopoietic cell response, bone density, neuronal degeneration, angiogenesis, and cancer. These physiological and pathological roles of Pyk2 warrant it as a valuable therapeutic target for invasive cancers, osteoporosis, Alzheimer’s disease, and inflammatory cellular response. Despite its potential as a therapeutic target, no potent and selective inhibitor of Pyk2 is available at present. As a first step toward discovering specific potential inhibitors of Pyk2, we used an in silico high-throughput screening approach. A virtual library of six million lead-like compounds was docked against four different high-resolution Pyk2 kinase domain crystal structures and further selected for predicted potency and ligand efficiency. Ligand selectivity for Pyk2 over focal adhesion kinase (FAK) was evaluated by comparative docking of ligands and measurement of binding free energy so as to obtain 40 potential candidates. Finally, the structural flexibility of a subset of the docking complexes was evaluated by molecular dynamics simulation, followed by intermolecular interaction analysis. These compounds may be considered as promising leads for further development of highly selective Pyk2 inhibitors.

Reversal of maladaptive fibrosis and compromised ventricular function in the pressure overloaded heart by a caveolin-1 surrogate peptide

Chronic ventricular pressure overload (PO) results in congestive heart failure (CHF) in which myocardial fibrosis develops in concert with ventricular dysfunction. Caveolin-1 is important in fibrosis in various tissues due to its decreased expression in fibroblasts and monocytes. The profibrotic effects of low caveolin-1 can be blocked with the caveolin-1 scaffolding domain peptide (CSD, a caveolin-1 surrogate) using both mouse models and human cells. We have studied the beneficial effects of CSD on mice in which PO was induced by trans-aortic constriction (TAC). Beneficial effects observed in TAC mice receiving CSD injections daily included: improved ventricular function (increased ejection fraction, stroke volume, and cardiac output; reduced wall thickness); decreased collagen I, collagen chaperone HSP47, fibronectin, and CTGF levels; decreased activation of non-receptor tyrosine kinases Pyk2 and Src; and decreased activation of eNOS. To determine the source of cells that contribute to fibrosis in CHF, flow cytometric studies were performed that suggested that myofibroblasts in the heart are in large part bone marrow-derived. Two CD45+ cell populations were observed. One (Zone 1) contained CD45+/HSP47−/macrophage marker+ cells (macrophages). The second (Zone 2) contained CD45moderate/HSP47+/macrophage marker− cells often defined as fibrocytes. TAC increased the number of cells in Zones 1 and 2 and the level of HSP47 in Zone 2. These studies are a first step in elucidating the mechanism of action of CSD in heart fibrosis and promoting the development of CSD as a novel treatment to reduce fibrosis and improve ventricular function in CHF patients.

Ethanol preconditioning of rat cerebellar cultures targets NMDA receptors to the synapse and enhances peroxiredoxin 2 expression

Epidemiological studies indicate that light-moderate alcohol (ethanol) consumers tend to have reduced risks of cognitive impairment and progression to dementia during aging. Exploring possible mechanisms, we previously found that moderate ethanol preconditioning (MEP, 20–30mM) of rat brain cultures for several days instigated neuroprotection against β-amyloid peptides. Our biochemical evidence implicated the NMDA receptor (NMDAR) as a potential neuroprotective “sensor”, specifically via synaptic NMDAR signaling. It remains unclear how ethanol modulates the receptor and its downstream targets to engender neuroprotection. Here we confirm with deconvolution microscopy that MEP of rat mixed cerebellar cultures robustly increases synaptic NMDAR localization. Phospho-activation of the non-receptor tyrosine kinases Src and Pyk2, known to be linked to synaptic NMDAR, is also demonstrated. Additionally, the preconditioning enhances levels of an antioxidant protein, peroxiredoxin 2 (Prx2), reported to be downstream of synaptic NMDAR signaling, and NMDAR antagonism with memantine (earlier found to abrogate MEP neuroprotection) blocks the Prx2 elevations. To further link Prx2 with antioxidant-based neuroprotection, we circumvented the ethanol preconditioning-NMDAR pathway by pharmacologically increasing Prx2 with the naturally-occurring cruciferous compound, 3H-1,2-dithiole-3-thione (D3T). Thus, D3T pretreatment elevated Prx2 expression to a similar extent as MEP, while concomitantly preventing β-amyloid neurotoxicity; D3T also protected the cultures from hydrogen peroxide toxicity. The findings support a mechanism that couples synaptic NMDAR signaling, Prx2 expression and augmented antioxidant defenses in ethanol preconditioning-induced neuroprotection. That this mechanism can be emulated by a cruciferous vegetable constituent suggests that such naturally-occurring “neutraceuticals” may be useful in therapy for oxidative stress-related dementias.

A Dual Role for the Nonreceptor Tyrosine Kinase Pyk2 during the Intracellular Trafficking of Human Papillomavirus 16.

The infectious process of human papillomaviruses (HPVs) has been studied considerably, and many cellular components required for viral entry and trafficking continue to be revealed. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during HPV16 pseudovirion infection of human keratinocytes. We found that Pyk2 is necessary for infection and appears to be involved in the intracellular trafficking of the virus. Small interfering RNA-mediated reduction of Pyk2 resulted in a significant decrease in infection but did not prevent viral entry at the plasma membrane. Pyk2 depletion resulted in altered endolysosomal trafficking of HPV16 and accelerated unfolding of the viral capsid. Furthermore, we observed retention of the HPV16 pseudogenome in the trans-Golgi network (TGN) in Pyk2-depleted cells, suggesting that the kinase could be required for the viral DNA to exit the TGN. While Pyk2 has previously been shown to function during the entry of enveloped viruses at the plasma membrane, the kinase has not yet been implicated in the intracellular trafficking of a nonenveloped virus such as HPV. Additionally, these data enrich the current literature on Pyk2's function in human keratinocytes. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during human papillomavirus (HPV) infection of human skin cells. Infections with high-risk types of HPV such as HPV16 are the leading cause of cervical cancer and a major cause of genital and oropharyngeal cancer. As a nonenveloped virus, HPV enters cells by interacting with cellular receptors and established cellular trafficking routes to ensure that the viral DNA reaches the nucleus for productive infection. This study identified Pyk2 as a cellular component required for the intracellular trafficking of HPV16 during infection. Understanding the infectious pathways of HPVs is critical for developing additional preventive therapies. Furthermore, this study advances our knowledge of intracellular trafficking processes in keratinocytes.

PYK2 integrates growth factor and cytokine receptors signaling and potentiates breast cancer invasion via a positive feedback loop.

The involvement of ErbB family members in breast cancer progression and metastasis has been demonstrated by many studies. However, the downstream effectors that mediate their migratory and invasive responses have not been fully explored. In this study, we show that the non-receptor tyrosine kinase PYK2 is a key effector of EGFR and HER2 signaling in human breast carcinoma. We found that PYK2 is activated by both EGF and heregulin (HRG) in breast cancer cells, and positively regulates EGF/HRG-induced cell spreading, migration and invasion. PYK2 depletion markedly affects ERK1/2 and STAT3 phosphorylation in response to EGF/HRG as well as to IL8 treatment. Importantly, PYK2 depletion also reduced EGF/HRG-induced MMP9 and IL8 transcription, while IL8 inhibition abrogated EGF-induced MMP9 transcription and attenuated cell invasion. IL8, which is transcriptionally regulated by STAT3 and induces PYK2 activation, prolonged EGF-induced PYK2, STAT3 and ERK1/2 phosphorylation suggesting that IL8 acts through an autocrine loop to reinforce EGF-induced signals. Collectively our studies suggest that PYK2 is a common downstream effector of ErbB and IL8 receptors, and that PYK2 integrates their signaling pathways through a positive feedback loop to potentiate breast cancer invasion. Hence, PYK2 could be a potential therapeutic target for a subset of breast cancer patients.

Sphingomyelin Synthase 2, but Not Sphingomyelin Synthase 1, Is Involved in HIV-1 Envelope-mediated Membrane Fusion

Membrane fusion between the viral envelope and plasma membranes of target cells has previously been correlated with HIV-1 infection. Lipids in the plasma membrane, including sphingomyelin, may be crucially involved in HIV-1 infection; however, the role of lipid-metabolic enzymes in membrane fusion remains unclear. In this study, we examined the roles of sphingomyelin synthase (SMS) in HIV-1 Env-mediated membrane fusion using a cell-cell fusion assay with HIV-1 mimetics and their target cells. We employed reconstituted cells as target cells that stably express Sms1 or Sms2 in Sms-deficient cells. Fusion susceptibility was ∼5-fold higher in Sms2-expressing cells (not in Sms1-expressing cells) than in Sms-deficient cells. The enhancement of fusion susceptibility observed in Sms2-expressing cells was reversed and reduced by Sms2 knockdown. We also found that catalytically nonactive Sms2 promoted membrane fusion susceptibility. Moreover, SMS2 co-localized and was constitutively associated with the HIV receptor·co-receptor complex in the plasma membrane. In addition, HIV-1 Env treatment resulted in a transient increase in nonreceptor tyrosine kinase (Pyk2) phosphorylation in Sms2-expressing and catalytically nonactive Sms2-expressing cells. We observed that F-actin polymerization in the region of membrane fusion was more prominent in Sms2-expressing cells than Sms-deficient cells. Taken together, our research provides insight into a novel function of SMS2 which is the regulation of HIV-1 Env-mediated membrane fusion via actin rearrangement.