Involvement of P38MAPK in human corneal endothelial cell migration induced by TGF-β2
Highlights
► The purpose of this study was to determine the effect of TGF-β2 on the proliferation and migration of human corneal endothelial cells. ► We examined the effect of TGF-β2 and FGF-2 on the cultured human corneal endothelial cells (HCECs) wound healing model. ► TGF-β2 reduces proliferation, but stimulates migration of HCECs through p38 MAPK. ► 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 (β1, β2, 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.
Section snippets
Materials and methods
All procedures including those involving human subjects were conducted in accordance with the principles of the Declaration of Helsinki, and this study protocol was approved by the Institutional Review Board of Ehime University.
RNA extraction and reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was isolated from the cultured HCECs and human corneal endothelium obtained from normal human corneas using TRIzol reagent according to the manufacturer's instructions (Invitrogen). Samples were further purified using the RNeasy kit (Qiagen, Valencia, CA). cDNA was prepared from 1 μg of total RNA by reverse transcription in a volume of 20 μl. cDNAs were synthesized with Superscript II reverse transcriptase according to the manufacturer's instructions (Invitrogen).
PCR amplification was
TGF-β receptor mRNA in cultured human corneal endothelial cells
The expression of TGF-β receptors I, II, and III in human corneal endothelium have been demonstrated in vivo (Joyce and Zieske, 1997), however their expression in cultured HCECs has not been determined. Therefore, we first confirmed that the mRNAs of TGF-β receptors I, II, and III were expressed in both subconfluent and confluent HCECs as well as in human corneal endothelium in situ before beginning our experiments. The results showed that the mRNA for all three TGF-β receptor types was
Effect of TGF-β2 on BrdU incorporation in cultured human corneal endothelial cell wound healing
The effect of TGF-β2 on the proliferation of HCECs during wound healing was investigated by culturing injured HCECs with serial concentrations of TGF-β2 followed by BrdU. No significant difference was detected between the number of BrdU-positive cells in control samples and those exposed to 0.1 ng/ml of TGF-β2. However, the number of BrdU-positive cells significantly decreased in a dose dependent way when cells were exposed to TGF-β2 at concentrations between 1.0 and 10.0 ng/ml (Fig. 4A).
When
Discussion
As best we know, our experiments are the first to examine the effect of TGF-β2 on cultured HCECs. In the initial experiments, we examined the effect of TGF-β2 in an in vitro HCEC wound healing model and found somewhat unexpectedly that TGF-β2 had no effect on the speed of wound healing of injured cultured HCECs.
Therefore, we further investigated the effect of TGF-β2 on injured HCEC proliferation using the BrdU incorporation assay, and cell migration with the Boyden chamber assay. The number of
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