Migration of retinal pigment epithelium cells is regulated by protein kinase Cα in vitro.

Abstract

PURPOSE:

Retinal pigment epithelium (RPE) cell migration and proliferation are considered key elements in proliferative vitreoretinopathy (PVR). Downregulation of protein kinase Cα (PKCα) can inhibit RPE cell proliferation. Here, we sought to analyze whether PKCα affects the migration of RPE cells.

METHODS:

Human RPE (hRPE) cells were cultured, confirmed by immunofluorescence staining, and divided into four groups: control, thymeleatoxin, non-small interfering RNA (siRNA), and siRNA-PKCα. Thymeleatoxin was used to activate PKCα, and siRNA-PKCα was used to knock it down. Expression of PKCα was confirmed by quantitative RT-PCR (qRT-PCR). Cell migration ability was analyzed by wound healing assay and transwell chamber assay. Expression of zonula occludens (ZO)-1 and occludin was determined by immunofluorescence.

RESULTS:

Pure populations of hRPE cell cultures were observed using light and fluorescence microscopy. The mRNA levels of PKCα were not significantly increased by thymeleatoxin, but were reduced by siRNA-PKCα as determined by qRT-PCR assay. The wound healed faster in the thymeleatoxin group than in the control group at time points 12, 15, and 20 hours. The wound healed more slowly in the siRNA-PKCα group than in the non-siRNA group at the three time points. A similar tendency among the four groups was consistently observed in regard to cell numbers counted in the transwell chamber assay. The expression of ZO-1 was highest in the siRNA-PKCα group, similar in the control and non-siRNA groups, and lowest in the thymeleatoxin group. After migration, the fluorescence intensity of ZO-1 was reduced to similarly weak levels among the four groups.

CONCLUSIONS:

Retinal pigment epithelium cell migration is enhanced by a PKCα agonist and suppressed by a PKCα antagonist. The results suggest that a PKCα-mediated signal transduction pathway plays a crucial role in hRPE cell migration and may be a potential therapeutic target against hRPE cell migration and PVR disease.

• Authors: Suo Qiu, Zhaoxin Jiang, Zhen Huang, Xiaoqing Chen, Xiaobing Qian, Qianying Gao, Haihua Zheng
• Journal: Investigative ophthalmology & visual science (Invest Ophthalmol Vis Sci) Vol. 54 Issue 10 Pg. 7082-90 (Oct 29 2013) ISSN: 1552-5783 [Electronic] United States
• PMID: 24084091 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Phorbol Esters
• RNA, Small Interfering
• thymeleatoxin
• Protein Kinase C-alpha

Topics

• Cell Movement (physiology)
• Cells, Cultured
• Down-Regulation
• Gene Expression Regulation, Enzymologic
• Humans
• Phorbol Esters (pharmacology)
• Protein Kinase C-alpha (antagonists & inhibitors, metabolism, physiology)
• RNA, Small Interfering (physiology)
• Retinal Pigment Epithelium (cytology, physiology)
• Reverse Transcriptase Polymerase Chain Reaction
• Wound Healing (physiology)