Signal transduction mediated by adhesion of human trabecular meshwork cells to extracellular matrix

In this study we investigated the signaling event induced by adhesion of hu man trabecular meshwork (TM) cells to extracellular matrix (ECM) elements s uch as fibronectin, The role of tyrosine phosphorylation in adhesion was ev aluated. A number of intracellular entities involved in the adhesion-mediat ed pathways were identified, For the experiments, human TM cells were seede d onto fibronectin- or polylysine (negative control)-coated plates. Fifteen , 30, 90 and 240 min after the seeding, cell lysates were collected. Immuno blotting analysis revealed that tyrosine phosphorylation occurred within 15 min of adhesion of TM cells to fibronectin and the level increased with ti me. The phosphotyrosyl proteins had molecular masses 25-220 kDa. A much low er level of tyrosine phosphorylation was observed when cells were plated on polylysine. Immunoprecipitation experiments indicated that the phosphotyro sine-containing proteins included focal adhesion kinase, paxillin, phosphat idylinositol 3-kinase and mitogen activated protein kinase. Within 30 ruin of adherence to fibronectin, human TM cells immunostained for paxillin and phosphotyrosine and exhibited prominent focal contacts. When treated with t yrosine kinase inhibitors genistein and herbimycin A and a protein kinase C (PRC) pseudosubstrate peptide inhibitor, cell adhesion to fibronectin was compromised and focal contact formation was limited. These results demonstr ated that in human TM cells, tyrosine kinase was activated upon their adher ence to fibronectin. PKC also appeared to play a role in modulation of the cell-matrix adhesion process. The current study provides insight into the s ignaling pathways that are linked to the ECM-induced events in TM cells. El ucidation of the hierarchy of signal responses may help develop strategies manipulating the cell-matrix interactions in the TM system. (C) 2000 Academ ic Press.