Hematopoietic Pyk2 regulates migration of differentiated HL-60 cells

<p>Abstract</p> <p>Background</p> <p>Pyk2 is a non-receptor cytoplasmic tyrosine kinase that belongs to the focal adhesion kinase family and has been implicated in neutrophil spreading and respiratory burst activity caused by TNF-α. However, the role of Pyk2 in neutrophil migration is incompletely defined. In this study, we tested the hypothesis that Pyk2 regulates the migration of neutrophil-like differentiated HL-60 cells subsequent to β2-integrin mediated cell adhesion.</p> <p>Methods</p> <p>HL-60 cells were induced to differentiate into neutrophil-like cells (dHL60) by incubation in medium containing 1.25% DMSO for up to 4 days. Pyk2 expression and tyrosine phosphorylation was measured by Western blot analysis. Adhesion of dHL60 cells to plated fibrinogen was measured by residual myeloperoxidase activity. dHL60 cell migration was evaluated using a 96-well chemoTx chamber.</p> <p>Results</p> <p>Western blot analysis demonstrated that hematopoietic Pyk2 was predominantly expressed after HL60 cell differentiation. Pyk2 was tyrosine phosphorylated upon adhesion of dHL60 cells to plated fibrinogen in the presence of fMLP. By contrast, tyrosine phosphorylation of Pyk2 was insignificant in dHL60 cells treated in suspension with fMLP. Antibodies against CD18 blocked both phosphorylation of Pyk2 and adhesion of dHL60 cells to fibrinogen, demonstrating that phosphorylation of Pyk2 was β₂-integrin dependent. TAT-Pyk2-CT, a dominant negative fusion protein in which the TAT protein transduction domain was fused to the c-terminal Pyk2, attenuated fMLP-stimulated spreading, migration and phosphorylation of endogenous Pyk2 without blocking adhesion of dHL-60 cells to fibrinogen. Similarly, silencing of Pyk2 expression by siRNA in dHL60 cells also attenuated dHL60 cell migration caused by fMLP. Phospho-Pyk2 was evenly distributed around cell membrane circumferentially in unstimulated dHL-60 cells adherent to plated fibrinogen. In dHL60 cells treated with fMLP to cause cell spreading and polarization, Pyk2 was concentrated at the leading edge of pseudopods or at the trailing edge of uropods during migration of neutrophilic dHL-60 cells.</p> <p>Conclusions</p> <p>We conclude that Pyk2 is activated by β2-integrin adhesion. The activated concentration of Pyk2 and colocalization with F-actin in pseudopodia suggests that Pyk2 may regulate cell spreading and migration in dHL60 cells.</p

Inhibition of Pyk2 blocks lung inflammation and injury in a mouse model of acute lung injury

<p>Abstract</p> <p>Background</p> <p>Proline-rich tyrosine kinase 2 (Pyk2) is essential in neutrophil degranulation and chemotaxis in vitro. However, its effect on the process of lung inflammation and edema formation during LPS induced acute lung injury (ALI) remains unknown. The goal of the present study was to determine the effect of inhibiting Pyk2 on LPS-induced acute lung inflammation and injury in vivo.</p> <p>Methods</p> <p>C57BL6 mice were given either 10 mg/kg LPS or saline intratracheally. Inhibition of Pyk2 was effected by intraperitoneal administration TAT-Pyk2-CT 1 h before challenge. Bronchoalveolar lavage analysis of cell counts, lung histology and protein concentration in BAL were analyzed at 18 h after LPS treatment. KC and MIP-2 concentrations in BAL were measured by a mouse cytokine multiplex kit. The static lung compliance was determined by pressure-volume curve using a computer-controlled small animal ventilator. The extravasated Evans blue concentration in lung homogenate was determined spectrophotometrically.</p> <p>Results</p> <p>Intratracheal instillation of LPS induced significant neutrophil infiltration into the lung interstitium and alveolar space, which was attenuated by pre-treatment with TAT-Pyk2-CT. TAT-Pyk2-CT pretreatment also attenuated 1) myeloperoxidase content in lung tissues, 2) vascular leakage as measured by Evans blue dye extravasation in the lungs and the increase in protein concentration in bronchoalveolar lavage, and 3) the decrease in lung compliance. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. By contrast, production of neutrophil chemokines MIP-2 and keratinocyte-derived chemokine in the bronchoalveolar lavage was not reduced by TAT-Pyk2-CT. Western blot analysis confirmed that tyrosine phosphorylation of Pyk2 in LPS-challenged lungs was reduced to control levels by TAT-Pyk2-CT pretreatment.</p> <p>Conclusions</p> <p>These results suggest that Pyk2 plays an important role in the development of acute lung injury in mice and that pharmacological inhibition of Pyk2 might provide a potential therapeutic strategy in the pretreatment for patients at imminent risk of developing acute lung injury.</p

Endoscopic management of Barrett’s dysplasia and early neoplasia: efficacy, safety and long-term outcomes in a UK tertiary centre

Background and Objectives: Endoscopic mucosal resection (EMR) and radiofrequency ablation (RFA) are effective treatments for dysplastic Barrett’s esophagus (BE). This study evaluates efficacy, durability and safety in a single high-volume UK tertiary centre with 15-years’ experience.Methods: Prospective data from Nottingham University Hospitals 2004-2019 for endotherapy of dysplastic BE or intramucosal adenocarcinoma. Procedural outcome measures: complete resection, complications and surgery rates. Efficacy outcomes: complete remission of dysplasia (CR-D) and intestinal metaplasia (CR-IM), recurrence, treatment failure rates, durability of RFA, median follow up and tumour associated mortality. Results: 319 lesions were resected. 671 RFAs were performed on 239 patients. Median age was 67(±9.5) years, male:female ratio was 5:1 and median BE length was C3(IQR:6) M6(IQR:5). The most common lesion was Paris IIa(64%) with a median size of 10mm(3-70). Final histology was adenocarcinoma in 50%. Complete resection rates were 96%. The multiband mucosectomy technique (91%) was most commonly used. The median number of RFA sessions was 3(IQR:2). The rates of CR-D and CR-IM were 90.4%% and 89.8% achieved after a median of 20.1(IQR:14) months. The most common complications: EMR was bleeding 2.2% and RFA was stricture (5.4%) requiring a median of 2 (range 1-7) dilatations. Median follow up post CR-IM/CR-D was 38 months(14-60). Metachronous lesions developed in 4.7% after CR-D and tumour related mortality was 0.8%. Dysplasia and intestinal metaplasia free survival at 5 years was 95% and 90% respectively. Conclusions: BE endotherapy is minimally invasive, effective, safe and deliverable in a day-case settin

Proline-Rich Tyrosine Kinase 2 Regulates Spreading and Migration of Eosinophils after β2-Integrin Adhesion

We examined the role of proline-rich tyrosine kinase (Pyk) 2 in the spreading and migration of human blood eosinophils after β2-integrin ligation. Western blot analysis showed that Pyk2 was activated by phosphorylation at Y402 after eosinophil adhesion to BSA-coated plates after activation with IL-5, platelet-activating factor (PAF), formyl-met-leu-phe (fMLP), or Mn2+. To determine the role of Pyk2 in regulating eosinophil migration, we used a transducable dominant-negative inhibitor of Pyk2, TAT-mediated protein transduction of dominant-negative C-terminal Pyk2 (TAT-Pyk2-CT), a fusion protein in which TAT peptide was fused to the C-terminal Pyk2. TAT-Pyk2-CT blocked tyrosine phosphorylation of Pyk2 caused by β2-integrin adhesion, but did not block adhesion of eosinophils to plated BSA. TAT-Pyk2-CT also blocked subsequent spreading and migration of eosinophils caused by IL-5, PAF, or fMLP. Spreading eosinophils stained with FITC-conjugated phalloidin showed elongation and formation of multiple fillopodia and lamellipodia, whereas nonspreading eosinophils were smaller and round. Treatment of eosinophils with TAT-Pyk2-CT had no effect on the initial cell polarization, but blocked the formation of fillopodia and lamellipodia in adherent cells. Migration of eosinophils through Transwell plates caused by IL-5, PAF, or fMLP was blocked significantly after inhibition of Pyk2. These data indicate that Pyk2, although not involved in β2-integrin adhesion, causes eosinophil spreading and regulates subsequent chemotactic migration after β2-integrin ligation to endothelial counter ligands. We conclude that Pyk2 is activated by β2-integrin adhesion and is a required signal for eosinophil spreading and subsequent chemotactic migration

TAT-Protein Blockade during Ischemia/Reperfusion Reveals Critical Role for p85 PI3K-PTEN Interaction in Cardiomyocyte Injury

Recent work shows that cooling protection after mouse cardiac arrest and cardiomyocyte ischemia is mediated by Akt activation. The PI3K p85 subunit can either augment or inhibit Akt activation depending on its binding to p110 or PTEN respectively. To further clarify the role of PI3K p85 in cardioprotection, we studied novel TAT-p85 fusion proteins that selectively inhibit PI3K p85 binding. We hypothesized that TAT fused p85 lacking the PTEN binding site (TAT-ΔPTEN p85) would enhance Akt phosphorylation to afford cardioprotection. Conversely, TAT fused p85 lacking the p110 binding site (TAT-Δp110p85) would decrease Akt phosphorylation and abrogate cardioprotection. Microscopy and Western blot analysis demonstrated that TAT fusion protein was transduced into cardiomyocytes within 5 min and remained more than 2 h. Inhibition of PI3K/Akt by TAT-Δp110 p85 significantly increased cell death from 44.6±2.7% to 92.5±3.4% after simulated ischemia and reperfusion. By contrast, PTEN inhibition using TAT-ΔPTEN p85 decreased cell death to 11.9±5.3%, a similar level of cardioprotection seen with past cooling studies. Additional studies with the small molecule PTEN inhibitor VO-OHpic confirmed that PTEN inhibition was highly protective against cell death induced by ischemia and reperfusion. We conclude that blockade of p85-PTEN interaction and PTEN inhibition may be promising strategies for rescuing the heart from ischemia and reperfusion injury.</p