Control of cooperative integrin signaling in human trabecular meshwork cells

In the eye, human trabecular meshwork (HTM) cells, use cytoskeletal organization and cell-ECM adhesive forces to regulate intraocular pressure. Chemical agents that disrupt the cytoskeleton or the signaling pathways that maintain the actomyosin network decrease intraocular pressure. Understanding the signaling pathways that mediate these processes is critical for understanding how intraocular pressure is maintained. The work presented here begins to characterize how proliferating and quiescent HTM cells control cooperative integrin signaling to mediate cell spreading.;Proliferating cells use alpha5beta1 integrin to adhere and spread on the III 7-10 repeats of fibronectin with a bipolar morphology with few focal adhesions and stress fibers. Addition of soluble Hep II domain increases cell spreading and the numbers of focal adhesions and stress fibers and this is mediated by alpha4beta1 integrin rather than heparan sulfate proteoglycans. This is the first demonstration that the Hep II domain can induce stress fiber and focal adhesion formation through alpha4beta1 integrin and shows that alpha5beta1 and alpha4beta1 integrins can act cooperatively to mediate cell spreading.;In contrast, quiescent cells use alpha5beta1 integrin to adhere and spread on the III 7-10 repeats with cortical actin structures and focal adhesions. Cell spreading is mediated by integrin linked kinase (ILK). When ILK is inhibited, cell spreading and focal adhesion formation is significantly reduced as is focal adhesion kinase phosphorylation. This could be rescued by inducing alpha4beta1 integrin signaling with soluble Hep II domain. Without ILK inhibition, the Hep II domain has no effect on quiescent cell spreading, suggesting that alpha4beta1 integrin signaling may be regulated by ILK trans-dominantly.;Proliferating and quiescent cells spread with different morphologies on matrix proteins. Proliferating cells exhibit stress fibers while quiescent cells have cortical actin structures. When plated on fibronectin, proliferating cells require alpha5beta1/alpha4beta1 integrin co-signaling while quiescent cells only require alpha5beta1 integrin. alpha4beta1 integrin activity appears to be downregulated in quiescent cells as they do not spread on the IIICS domain of fibronectin (alpha4beta1 integrin ligand). These studies indicate that integrin mediated TM cell spreading is cell cycle dependent.