RhoA Activation Decreases Phagocytosis of Trabecular Meshwork Cells.

PURPOSE: RhoA signaling is important for the regulation of intraocular pressure through the trabecular meshwork (TM). However, the relationship between RhoA signaling and phagocytosis in TM cells is unclear. The purpose of this study was to investigate the effects of RhoA signaling on the phagocytosis of TM cells. MATERIALS AND METHODS: TM cells were isolated from enucleated porcine eyes and treated with lysophosphatidic acid (LPA) or calpeptin to activate RhoA to determine phagocytic activity. To assess phagocytic activity, TM cells were incubated with pHrodo® Red S. aureus bioparticles, and the fluorescence intensity was measured using a cell sorter. The phagocytic activity of RhoA knockdown TM cells was also assessed using small interfering RNA (siRNA). To resolve the effects of dexamethasone on phagocytosis, TM cells were treated with dexamethasone for 72 h. The immunocytochemistry of vinculin and F-actin were evaluated in LPA- and dexamethasone-treated TM cells. RESULTS: RhoA activities after treatment with 10 µM LPA and 100 µM calpeptin were 1.38 ± 0.026-fold and 1.47 ± 0.070-fold higher, respectively, compared with the control. The phagocytic activity was reduced by LPA (0.67 ± 0.099) and calpeptin (0.57 ± 0.016), compared with the control. C3 transferase (Rho inhibitor) and Y-27632 (Rho-associated kinase inhibitor) prevented the effects of LPA on phagocytosis, and C3 partially inhibited the effects of calpeptin on phagocytosis. Knockdown of RhoA prevented the effect of LPA on phagocytosis. By immunostaining, LPA-induced stress fiber and focal adhesion formation was prevented by C3 and Y-27632 treatment. Moreover, RhoA knockdown prevented the effects of LPA on F-actin and focal adhesion. Dexamethasone treatment decreased phagocytic activity and increased stress fiber and focal adhesion. Y-27632 prevented the effects of dexamethasone on phagocytosis, and on stress fiber and focal adhesion fomation. CONCLUSIONS: These results suggest that the RhoA signal pathway regulates the phagocytic activity of TM cells.Abbreviations: TM: trabecular meshwork; LPA: lysophosphatidic acid; C3: C3 transferase; ROCK: Rho-associated kinase; siRNA: small interfering RNA.

Interleukin-6-mediated trans-signaling inhibits transforming growth factor-ß signaling in trabecular meshwork cells.

Glaucoma is one of the major causes of blindness, and transforming growth factor-ß2 (TGF-ß2) has been found to be elevated in the aqueous humor of eyes with primary open-angle glaucoma (POAG). TGF-ß2 in aqueous humor causes the glaucoma-related fibrosis of human trabecular meshwork (HTM), suggesting an important role of TGF-ß in POAG pathogenesis. Here, we sought to elucidate the effects of IL-6 trans-signaling on TGF-ß signaling in HTM cells. Using a multiplex immunoassay, POAG patients decreased IL-6 levels and increased soluble IL-6 receptor (sIL-6R) levels compared with the controls. In in vitro experiments, we observed that the IL-6 level was increased in the conditioned medium of HTM cells after TGF-ß2 stimulation. To elucidate the relationship between TGF-ß2 and IL-6 in HTM cells, we conducted Western blotting and immunohistochemical analyses, and we noted that the combination of IL-6 and sIL-6R (IL6/sIL-6R) suppressed TGF-ß-induced up-regulation of α-smooth muscle actin in HTM cells, whereas IL-6 alone did not. This suggests that trans-signaling, not classic signaling, of IL-6 suppresses TGF-ß-induced fibrosis of HTM. IL6/sIL-6R also suppressed TGF-ß-mediated activation of myosin light chain 2 (MLC2), Smad2, and p38. Of note, these inhibitory effects of IL6/sIL-6R on TGF-ß were partly reduced by siRNA-mediated knockdown of STAT3. Moreover, IL-6/sIL-6R partly inhibited TGF-ß-induced activation of the Smad-sensitive promoter detected with luciferase reporter gene assays and up-regulation of TGFRI and TGFRII, evaluated by quantitative real-time RT-PCR. Strikingly, overexpression of TGFRI and TGFRII diminished these inhibitory effects of IL-6/sIL-6R. We conclude that of IL-6-mediated trans-signaling potently represses TGF-ß signaling in HTM cells.