Rho-Rho kinase pathway in the actomyosin contraction and cell-matrix adhesion in immortalized human trabecular meshwork cells

[摘要] Purpose: The outflow facility foraqueous humor across the trabecular meshwork (TM) is enhanced by agentsthat oppose the actomyosin contraction of its resident cells.Phosphorylation of MYPT1 (myosin light chain [MLC] phosphatase complexof Type 1) at Thr853 and Thr696 inhibits dephosphorylation of MLC,leading to an increase in actomyosin contraction. In this study, weexamined the effects of Rho kinase (ROCK) inhibitors on the relativedephosphorylation of the two sites of MYPT1 using human TM cells(GTM3). Methods: Dephosphorylation of MYPT1 atThr853 and Thr696 was determined by western blot analysis followingexposure to selective inhibitors of ROCK, namely Y-27632 and Y-39983.Consequent dephosphorylation of MLC and decreases in actomyosincontraction were assessed by western blot analysis and collagen gelcontraction assay, respectively. Changes in the cell-matrix adhesionwere measured in real time by electric cell-substrate impedance sensingand also assessed by staining for paxillin, vinculin, and focaladhesion kinase (FAK). Results: Both ROCK inhibitors produced aconcentration-dependent dephosphorylation at Thr853 and Thr696 of MYPT1in adherent GTM3 cells. IC50 values for Y-39983 were15 nM and 177 nM for dephosphorylation at Thr853 and Thr696,respectively. Corresponding values for Y-27632 were 658 nM and2270 nM. Analysis of the same samples showed a decrease in MLCphosphorylation with IC50 values of 14 nM and1065 nM for Y-39983 and Y-27632, respectively. Consistent withthese changes, both inhibitors opposed contraction of collagen gelsinduced by TM cells. Exposure of cells to the inhibitors led to adecrease in the electrical cell-substrate resistance, with the effectof Y-39983 being more pronounced than Y-27632. Treatment with theseROCK inhibitors also showed a loss of stress fibers and a concomitantdecrease in tyrosine phosphorylation of paxillin and FAK. Conclusions: Y-39983 and Y-27632 opposeROCK-dependent phosphorylation of MYPT1 predominantly at Thr853 with acorresponding decrease in MLC phosphorylation. A relatively low effectof both ROCK inhibitors at Thr696 suggests a role for other Ser/Thrkinases at this site. Y-39983 was several-fold more potent whencompared with Y-27632 at inhibiting the phosphorylation of MYPT1 ateither Thr853 or Thr696 commensurate with its greater potency atinhibiting the activity of human ROCK-I and ROCK-II enzymes.

[学科分类] 生物化学/生物物理