Migration of RPE cells into the vitreous body plays an important role in the onset and development of PVR. Protein kinase C has been demonstrated to regulate both the migration and proliferation of RPE cells.
21 Ten isoforms are present in cultured human RPE cells, and PKCα has been confirmed to regulate RPE cell cycle progression and proliferation. Here, we further demonstrated that PKCα regulates the migration of RPE cells in vitro.
Protein kinase C is a family of isoenzymes that play a central role in cellular processes such as proliferation, differentiation, mitosis, and inflammation.
13 The differences in the function of specific PKC isoforms are mainly due to their subcellular localization, activation, or inhibition by different stimuli and transcriptional regulation.
33 Murphy et al.
21 reported that migration of hRPE cells, stimulated by phorbol 12-myristate 13-acetate (PMA) and inhibited by calphostin C, is regulated by PKC in vitro. Our previous study characterized the expression pattern of all 12 PKC isoforms and showed that 10 of these are present in cultured human RPE cells.
28 Additionally, we found that PKCα was both necessary and sufficient to promote cell cycle progression after being stimulated with PMA; that is, downregulation of PKCα can inhibit RPE cell proliferation.
29 Moreover, siRNA-PKCα could be released sustainably from a novel foldable capsular vitreous body (FCVB) to inhibit PKCα expression in RPE cells in vitro.
34 Therefore, the role of PKCα in migration will be an interesting and essential research point after its proliferative role is ascertained.
The mechanism through which PKCα stimulates migration relies on its structure. Protein kinase Cα is a conventional PKC that has a C2 domain containing a putative Ca
2+ binding site. The binding of Ca
2+ to the five aspartates orients the bulky aromatics to interact with the membrane.
35 Thymeleatoxin, a chemically related phorbol compound, selectively activates PKCα, β, and γ and increases the calcium sensitivity in intact cells. Moreover, 100 nM thymeleatoxin effectively activates PKCα.
36 Ryves et al.
37 demonstrated that thymeleatoxin enhances the kinase activity of PKCα through kinase activation assays. Nishizuka
38 and Miyawaki and Ashraf
39 showed that translocation is a key index of PKC activation as it enables the interaction of PKC with membrane phospholipids. Newton
33 and Zidovetzki and Lester
40 showed that PKCα was activated through interactions with the cell membrane, with allosteric effects of phosphatidylserine, DAG, and Ca
2+ on the enzyme. Western blot analysis confirmed that PKCα was specifically translocated by thymeleatoxin from the soluble cytosolic fraction to the membranous fraction,
41–43 while our previous and current studies both showed that the PKCα mRNA level was not significantly upregulated by thymeleatoxin treatment.
29 Based on this evidence, we speculate that thymeleatoxin did not increase the mRNA levels of PKCα but instead translocated it from the cytosolic fraction to the membranous fraction and enhanced its kinase activity.
However, the roles of PKCα in proliferation and migration might overlap and interact with each other. Murphy et al.
21 reported that PMA and calphostin C affected the migration of RPE cells equally in growth-inhibited and growth-dependent migration. Similarly, mitomycin C treatment in this study enabled us to inhibit proliferation and thereby evaluate migration primarily based on cell motility. Protein kinase Cα is knocked down by siRNA, and the non-siRNA group was set as the negative control to redress the influences of siRNA. Small interfering RNAs, achieving target-specific gene silencing via double-stranded RNA-mediated RNA interference, have attracted much attention as a new therapeutic technique.
44,45 Previous research has demonstrated that 100 nM siRNA-PKCα has the most effective inhibitive influence among doses in the 10 to 200 nM range, with no significant toxicity (data not shown). However, the wound healed faster in the non-siRNA group than in the control group. Similar patterns were observed in the transwell chamber assay. The reasons are difficult to identify, and more in-depth research will be conducted in the future.
Tight junction proteins play key roles in migration and can be divided into two groups.
46,47 Occludin belongs to the first group (integral membrane proteins),
48,49 and ZO-1 belongs to the second group (plaque proteins).
50–52 The classical junctional scaffold protein ZO-1 is widely recognized for its vital role in the assembly of cell–cell adhesion complexes.
53 Zonula occludens-1 associates with the subcellular C-terminal tail of occludin, and interaction between these proteins is crucial for tight junctions.
54 In our study, the expression of ZO-1 was highest in the siRNA-PKCα group, moderately intense in the control and non-siRNA groups, and lowest in the thymeleatoxin group, indicating that a PKCα agonist might decrease the expression of ZO-1. After migration, fluorescence intensity of ZO-1 was reduced to similarly weak levels among the four groups, indicating that migration might decrease the expression of ZO-1.
Some effective therapy involving PKCα has recently been studied. A new siRNA-based therapeutic strategy targeting the PKCα gene has been designed to overcome the chemoresistance of ovarian cancer.
55 Berberine, an isoquinoline alkaloid inhibiting PKCα in breast cancer cells, may be used as a candidate drug for the inhibition of metastasis of human breast cancer.
56 We have demonstrated that siRNA-PKCα can be released sustainably from a novel FCVB to inhibit PKCα expression in RPE cells in vitro.
34 As PKCα is the only isoform associated with the proliferation of RPE cells, combining its role in migration and inhibition of PKCα might be a rational approach for therapy against PVR disease.
In conclusion, RPE cell migration is enhanced by a PKCα agonist and is suppressed by a PKCα antagonist. These results suggest that a PKCα-mediated signal transduction pathway plays a crucial role in RPE cell migration and might be useful as a potential therapeutic target against RPE cell migration and PVR disease.