Involvement of P38MAPK in human corneal endothelial cell migration induced by TGF-β₂

Abstract

Because human corneal endothelial cells do not proliferate once the endothelial monolayer is formed, corneal wound healing is thought to be mediated by cell enlargement or migration rather than proliferation. However, the cellular mechanisms involved in corneal wound healing have not been fully determined. Because transforming growth factor-β₂ (TGF-β₂) isoform is present in high concentrations in normal human aqueous humor, it may play a role in human corneal endothelial cell wound healing. The purpose of this study was to determine the effect of TGF-β₂ on the proliferation and migration of cultured human corneal endothelial cells (HCECs). To achieve this, we first examined the effect of TGF-β₂ on the wound closure rate in an in vitro HCEC wound healing model. However, unexpectedly TGF-β₂ had no effect on the wound closure rate in this model. Therefore, a real-time cell electronic sensing (RT-CES) system and the BrdU incorporation assay were used to determine the effect of TGF-β₂ (0.1–10 ng/ml) on cultured HCEC proliferation during in vitro wound healing. The specificity of this effect was confirmed by adding the TGF-β receptor I kinase inhibitor. TGF-β₂ inhibited the proliferation of HCECs in a dose dependent way and was blocked by TGF-β receptor I kinase inhibitor. Next, the Boyden chamber assay was used to determine how TGF-β₂ (10 ng/ml) affect HCEC migration. Exposure to TGF-β₂ increased cell migration, and a synergistic effect was observed when FGF-2 was added. To determine whether the mitogen-activated protein kinase (MAPK) signaling pathway is involved in the migration of HCECs, western blot analysis and Bio-Plex™ suspension array were used to detect phosphorylation of Erk1/2, p38, and JNK in HCECs stimulated by TGF-β₂ and/or FGF-2. The effect of the p38 MAPK inhibitor, SB239063 (10 μM), on TGF-β₂ and/or FGF-2-induced cellular migration was determined by the Boyden chamber assay. Both TGF-β₂ and FGF-2-induced p38 phosphorylation, and a synergistic effect was observed with exposure to both growth factors. SB 239063 inhibited TGF-β₂ and FGF-2-induced migration of HCECs. These results indicate that TGF-β₂ reduces proliferation but stimulates migration of cultured HCECs. In addition, TGF-β₂ and FGF-2 may have synergistic effects on the migration of HCECs mediated by p38 MAPK phosphorylation.

Introduction

The corneal endothelium is a single layer of cells lying between the corneal stroma and the anterior chamber, which helps maintain corneal transparency by regulating corneal hydration. It is widely accepted that corneal endothelial cells do not proliferate in humans once the endothelial monolayer is formed (Murphy et al., 1984). Different types of corneal injuries including surgical stress during intraocular surgery, corneal trauma, and viral infections cause a decrease in corneal endothelial cell density. The damaged corneal endothelium is believed to be repaired by enlargement and/or migration of the remaining corneal endothelial cells rather than proliferation during human corneal endothelial wound healing (Joyce, 2003; Landshman et al., 1988; Ling et al., 1988; Treffers, 1982). However, the precise mechanisms involved in human corneal endothelial cell wound healing have not been fully determined.

Because the aqueous humor bathes the corneal endothelium, the cytokines present in the aqueous humor may contribute to the healing of endothelial injury. Among the growth factors in the aqueous humor, transforming growth factor-β₂ (TGF-β₂) is the main isoform that is present in relatively high concentrations in humans (Cousins et al., 1991; Jampel et al., 1990). In other tissues, TGF-β is known to be involved in regulating cell differentiation, cell proliferation, cell migration, and other cellular functions (Furuyama et al., 1999; Gailit et al., 1994; Saika, 2004). In addition, TGF-β₂ has been reported to be associated with the arrest of corneal endothelial cells at G1 by blocking the G1 to S transition (Harris and Joyce, 1999; Kim et al., 2001a,b). This is supported by the fact that rat and rabbit corneal endothelial cell proliferation is suppressed in vitro by exposure to TGF-β₂ (Chen et al., 1999; Harris and Joyce, 1999; Kim et al., 2001a). However, the effect of TGF-β₂ on the proliferation of human corneal endothelial cells has not been fully determined.

Three isoforms of TGF-β (β_1, β_2, and β₃) bind to the serine/threonine protein kinases (TGF-β type I and type II receptors). Both TGF-β type I and type II receptors are necessary for TGF-β signal transduction. When the TGF-βs bind to their receptors, multiple signaling cascades are activated such as the Smad proteins and mitogen-activated protein kinases (MAPKs), which include the extracellular signal-regulated kinases (Erk1/2), c-Jun N-terminal kinases (JNK), and p38 (Byfield and Roberts, 2004; Itoh and ten Dijke, 2007; Massague and Gomis, 2006). In the corneal epithelium, epidermal growth factor (EGF)-induced Erk1/2 and p38 phosphorylation have been demonstrated to induce corneal epithelial cell migration (Wang et al., 2006). Recent studies have demonstrated that TGF-β enhances migration of corneal epithelial cells through the p38 MAPK pathway, but not the Smad pathway (Saika et al., 2004; Terai et al., 2011).

Although corneal endothelial cells have been shown to express the mRNA and protein for all three receptor types (TGF-β type I, type II, and type III receptors) (Harris and Joyce, 1999; Joyce and Zieske, 1997), it is unclear whether the intracellular signaling mechanisms of TGF-β₂ induce migration of human corneal endothelial cells.

It is possible that human corneal endothelial cells in vivo are affected not only by TGF-β₂ but also by other cytokines in the aqueous humor and corneal endothelial cells. The cytokines present in normal human aqueous humor include basic-fibroblast growth factor (FGF-2), hepatocyte growth factor, insulin-like growth factor binding protein, and vascular endothelial growth factor. Epidermal growth factor and transforming growth factor-α are not present or below the detectable levels in normal human aqueous humor (van Setten et al., 1996). FGF-2 is present at particularly high concentrations in normal human aqueous humor and corneal endothelial cells (Hoppenreijs et al., 1994; Rieck et al., 1995; Wilson and Lloyd, 1991), and it has been reported that FGF-2 can stimulate the proliferation and migration of corneal endothelial cells (Hoppenreijs et al., 1994; Rieck et al., 1995, 2001).

The purpose of this study was to determine whether TGF-β₂, the main isoform of TGF-β in the human aqueous humor, is involved in the proliferation and migration of cultured human corneal endothelial cells (HCECs). Another aim of this study was to investigate whether the MAPKs, Erk1/2, JNK, and p38, are involved in the migration of HCECs induced by TGF-β₂.