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We characterized the role of protein tyrosine phosphatase (PTP)-[alpha] in focal adhesion (FA) formation and remodeling using wild-type and PTP[alpha]deficient (PTP[[alpha].sup.-/-] ) cells. Compared with wild-type cells, spreading PTP[[alpha].sup.-/-] fibroblasts displayed fewer leading edges and formed elongated [alpha]-actinin-enriched FA at the cell periphery. These features suggest the presence of slowly remodeling cell adhesions and were phenocopied in human fibroblasts in which PTP[alpha] was knocked down using short interfering RNA (siRNA) or in NIH3T3 fibroblasts expressing catalytically inactive (C433S/C723S) PTP[alpha]. Fluorescence recovery after photobleaching showed slower green fluorescence protein-[alpha]-actinin recovery in the FA of PTP[[alpha].sup.-/-] than wild-type cells. These alterations correlated with reduced cell spreading, adhesion, and polarization and retarded contraction of extracellular matrices in PTP[[alpha].sup.-/-] fibroblasts. Activation of Rac1 and its recruitment to FA during spreading were diminished in cells expressing C433S/C723S PTP[alpha]. [Rac1.sup.-/-] cells also displayed abnormally elongated and peripherally distributed FA that failed to remodel. Conversely, expression of constitutively active Rac1 restored normal FA remodeling in PTPe[[alpha].sup.-/-] cells. We conclude that PTP[alpha] is required for remodeling of FA during cell spreading via a pathway involving Rac1. cell spreading; integrins; extracellular matrix; actin cytoskeleton

We characterized the role of protein tyrosine phosphatase (PTP)-alpha in focal adhesion (FA) formation and remodeling using wild-type and PTPalpha-deficient (PTPalpha(-/-)) cells. Compared with wild-type cells, spreading PTPalpha(-/-) fibroblasts displayed fewer leading edges and formed elongated alpha-actinin-enriched FA at the cell periphery. These features suggest the presence of slowly remodeling cell adhesions and were phenocopied in human fibroblasts in which PTPalpha was knocked down using short interfering RNA (siRNA) or in NIH-3T3 fibroblasts expressing catalytically inactive (C433S/C723S) PTPalpha. Fluorescence recovery after photobleaching showed slower green fluorescence protein-alpha-actinin recovery in the FA of PTPalpha(-/-) than wild-type cells. These alterations correlated with reduced cell spreading, adhesion, and polarization and retarded contraction of extracellular matrices in PTPalpha(-/-) fibroblasts. Activation of Rac1 and its recruitment to FA during spreading were diminished in cells expressing C433S/C723S PTPalpha. Rac1(-/-) cells also displayed abnormally elongated and peripherally distributed FA that failed to remodel. Conversely, expression of constitutively active Rac1 restored normal FA remodeling in PTPalpha(-/-) cells. We conclude that PTPalpha is required for remodeling of FA during cell spreading via a pathway involving Rac1.

During acute inflammation, neutrophil-mediated injury to epithelium may lead to disruption of epithelial function, including the induction of epithelial apoptosis. Herein, we report the effects of neutrophil transmigration and of purified leukocyte elastase on epithelial cell survival. Neutrophil transmigration induced apoptosis of epithelial cells [control monolayers: 5 +/- 1 cells/25 high-power fields (HPF) vs. neutrophil-treated monolayers: 29 +/- 10 cells/HPF, P < 0.05, n = 3 as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay] as did low concentrations (0.1 U/ml) of purified leukocyte elastase (control monolayers: 6.4 +/- 2.5% apoptotic vs. elastase: 26.2 +/- 2.9% apoptotic, P < 0.05, as determined by cytokeratin 18 cleavage). Treatment with elastase resulted in decreased mitochondrial membrane potential, release of cytochrome c to the cytosol, and cleavage of caspases-9 and -3 as determined by Western blot analysis, implicating altered mitochondrial membrane permeability as a primary mechanism for elastase-induced apoptosis. Additionally, incubation of epithelial cells with leukocyte elastase resulted in an early increase followed by a decrease in the phosphorylation of epithelial Akt, a serine/threonine kinase important in cell survival. Inhibition of epithelial Akt before elastase treatment potentiated epithelial cell apoptosis, suggesting that the initial activation of Akt represents a protective response by the epithelial cells to the proapoptotic effects of leukocyte elastase. Taken together, these observations suggest that epithelial cells exhibit a dual response to cellular stress imposed by leukocyte elastase with a proapoptotic response mediated via early alterations in mitochondrial membrane permeability countered by activation of the survival pathway involving Akt.