Role of Protein-tyrosine Phosphatase SHP2 in Focal Adhesion Kinase Down-regulation during Neutrophil Cathepsin G-induced Cardiomyocytes Anoikis

Abstract

Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130Cas and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP 2 and PTEN phosphorylation; however, only SHP 2 forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP 2 mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP 2 expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP 2 activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.

c-Cbl Ubiquitin Ligase Regulates Focal Adhesion Protein Turnover and Myofibril Degeneration Induced by Neutrophil Protease Cathepsin G

“…Immunoprecipitation and Immunoblot Analysis-Extraction of proteins from cultured cells was performed as described previously (9). Briefly, lysates were cleared by centrifugation at 12,000 rpm and the supernatants (800 g of protein/ml) were subjected to immunoprecipitation with corresponding antibodies.…”

“…Neonatal Cardiomyocyte Isolation-Myocytes were isolated from the ventricles of neonatal Sprague-Dawley rats, wild-type (WT) or c-Cbl KO mice by collagenase digestion as previously described (9). After 30 min of preplating (to eliminate nonmyocyte cell contamination), myocytes were plated in collagen precoated dishes or in fibronectin (BD Biosciences)-precoated glass coverslips at a density of 160,000/cm 2 in 10% fetal bovine serum DMEM supplemented with 1 mmol/liter L-glutamine, antibiotic/antimycotic solution, and 100 mol/liter 5-bromo-2-deoxyuridine (BrdU).…”

“…Expression of Adenoviral Vectors-Production of recombinant adenovirus carrying wild-type human EGFR with the CAG (WT-EGFR) and dominant negative EGFR (EGFR-CD533, a mutant that lacks the kinase domain of EGFR) was described elsewhere (9). Wild-type c-Cbl (WT-Cbl) was provided by Dr. A. Sanjay, Temple University and ligase-deficient c-Cbl (C381A-Cbl), a mutant that lacks RING finger domain necessary for ubiquitin ligase activity, was provided by Dr. A. Y. Tsayganov, Temple University.…”

“…These effects of Cat.G on myocytes do not require PARs but involve MMP activation and epidermal growth factor receptor (EGFR) transactivation (8). Further studies have shown the role of focal adhesion (FA) signaling down-regulation as a mechanism whereby Cat.G modulates normal cell-cell or cell-ECM interactions necessary for myocyte survival (9). However, the mechanisms involved in FA signaling down-regulation are still poorly understood.…”

The role of autophagy in the heart

“…Activation of cathepsin causes apoptosis through cleavage/ activation of caspases, 63 Bid 64 and SHP2. 65 When the permeability of lysosomal membrane is increased, the lysosomal enzymes could spread into the cytoplasm and stimulate massive self-digestion. Chemical inhibitors of cathepsins have been shown to protect the heart from reperfusion injury in experimental animals.…”