RhoA Activation Decreases Phagocytosis of Trabecular Meshwork Cells

ABSTRACT

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.

KEYWORDS:

• Phagocytosis
• trabecular meshwork cells
• RhoA
• Rho-associated kinase
• dexamethasone

Declaration of interest

T. Fujimoto, None; S. Sato-Ohira, none; H. Tanihara, Alcon Japan (F), Kowa (F, C), Merck Sharp & Dohme Corp. (C), Otsuka Pharmaceutical (F), Pfizer Japan (F), Santen Pharmaceutical (F), Senju Pharmaceutical (F), and T. Inoue, Alcon Japan (F), Kowa (F), Novartis Pharmaceutical (F), Otsuka Pharmaceutical (F), Pfizer Japan (F), Santen Pharmaceutical (F), Senju Pharmaceutical (F).

Supplemental material

Supplemental data for this article can be accessed on the publisher’s website.

Additional information

Funding

This work was supported by JSPS KAKENHI Grants Numbers 16K11289 (T.F.) and 17H04351 (H.T.). The sponsor or funding organization had no role in the design or conduct of this research.