Abstract
Purpose :
Cellular contractility and adhesion, mechanotransduction and extracellular matrix (ECM) organization of the trabecular meshwork are all presumed to interdependently influence aqueous humor outflow and intraocular pressure. This study uses mechanistic approaches to explore whether ECM protein phosphorylation plays a role in modulating these events
Methods :
Experimental studies have been designed using human TM cell primary cultures to quantitatively assess the effects of vertebrate lonesome kinase (VLk), a secretory tyrosine kinase on ECM protein phosphorylation, the influence of ECM protein phosphorylation on TM cell focal adhesion kinase (FAK) activity, focal adhesions and actin stress fibers, and the effects of pharmacological inhibition of FAK in TM cells using siRNA, pharmacological agents, immunoblot and immunofluorescence analyses.
Results :
siRNA-mediated suppression of VLK expression in human TM cells led to a dramatic decrease in ECM protein tyrosine phosphorylation, and was associated with a significant decrease in actin stress fibers and focal adhesions. Importantly, the effects of ECM protein phosphorylation on TM cell focal adhesions and actin stress fibers were found to be regulated partly through activation of FAK activity. Moreover, pharmacological inhibition of FAK in the TM cells triggered cellular changes that were identical to those caused by suppression of VLK-mediated ECM phosphorylation
Conclusions :
Taken together, these observations reveal novel and close mechanistic and functional interactions between VLK-regulated ECM protein tyrosine phosphorylation and FAK activation, and a collective influence of these two events on the regulation of TM cell adhesive activity, mechanotransduction and contractile response, all of which are expected to impact aqueous humor outflow and intraocular pressure. These results also support the conclusion that VLK may prove to be a potentially useful therapeutic target for lowering of intraocular pressure in glaucoma patients.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.