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  • The interaction between intact AM and limbal

    2018-10-29

    The interaction between intact AM and limbal explants does not only degrade the matrix of the AM but also activate intracellular signaling cascades triggered by matrix components. By use of selective pharmacological inhibitors, we investigated the downstream effectors critical for intact AM-induced limbal epithelial outgrowth and secretion of MMP-9 in ex vivo expanded limbal epithelial cells. We found that ERK1/2, JNK1/2, and PI3-K/Akt signaling pathways were required for limbal explant outgrowth on intact AM (Fig. 3A and B). However, the MMP-9 activity and RNA transcription were only up-regulated by JNK1/2 and PI3-K/Akt in this model (Fig. 3C and D). The involvement of MAPKs and PI3-K/Akt in such explant culture model has also been demonstrated by others (He et al., 2006). In contrast to their results, our data show that JNK1/2 was required for limbal epithelial outgrowth on intact AM. A possible explanation might be that different JNK inhibitors were used between these two studies. SP600125 (anthrapyrazolone), a selective JNK 1, 2 and 3 inhibitor (Bennett et al., 2001), was used in this work, whereas JNK Inhibitor 1 (L-stereoisomer) was utilized by others (He et al., 2006). MAPKs phosphorylate specific serines and threonines of target proteins and regulate cellular activities including gene expression, cell proliferation, migration, metabolism, and programmed death (Johnson and Lapadat, 2002). Recent evidence suggests that MMP-9 is induced by TGF-β via activation of ERK1/2, p38 MAPK, and JNK1/2 pathways in corneal epithelial jnk pathway (Kim et al., 2005). However, it was intriguing to note that only the JNK1/2 pathway was involved in mediating MMP-9 expression in our study despite the essential roles of ERK1/2 and JNK1/2 in mediating limbal epithelial outgrowth (Fig. 3). The roles of individual MAPKs in MMPs expression vary with cell types and stimuli. For example, activation of p38 MAPK might play an important role in TGF-β-induced corneal epithelial migration in C57BL/6J mice (Saika et al., 2004). While JNK1/2 and ERK1/2, rather than p38 MAPK have been implicated in the regulation of MMPs expression induced by IL-1β in corneal fibroblasts (Lu et al., 2004). It is also interesting to note that MMP-9 expression was enhanced by U0126, a selective MEK1/2 inhibitor, in this model (Fig. 3C). This phenomenon let us jnk pathway to further delineate the role of ERK1/2 signaling and its downstream effectors in these responses. TIMP-1, an endogenous inhibitor of MMP-9, was shown to be attenuated by U0126 treatment (Fig. 3D), implying that a negative feedback regulation of gene expression and activity related to MMP-9 is mediated through ERK1/2 signaling in this study. By phosphorylation of its downstream target Akt, the PI3-K signaling pathway plays an important role in a wide array of cellular functions, including mitogenic signaling, cytoskeletal remodeling, metabolic control, and cell survival (Wymann and Pirola, 1998). In this report, inhibition of PI3-K or Akt activity by LY294002 or SH5 impaired expansion of limbal epithelial cells on intact AM as well as MMP-9 production (Fig. 3). It has been indicated that hepatocyte growth factor (HGF) protects corneal epithelial cells from apoptosis through the PI-3K/Akt-1 but not the ERK1/2-mediated signaling pathway (Kakazu et al., 2004). Recent study has also demonstrated that translational control mediated by PI3K/Akt/mTOR, but not Raf/MEK/ERK signaling, is critical in regulating proliferation and endothelial differentiation of lineage-restricted stem cell lines (Que et al., 2007). However, whether limbal epithelial progenitor cells preferentially adopt the PI3-K/Akt pathway to maintain their proliferation potential and stemness is largely unknown. Activation of PI3-K also plays an important role in insulin-like growth factor (IGF)-I-induced corneal epithelial cell migration (Lee et al., 2006). At present, the upstream components responsible for activation of PI3-K and MMP-9 production in the context of limbal epithelial cells and intact AM interactions are not clear. PI3-K activation is triggered by the binding of several growth factors such as HGF and IGF-1 to their cell surface receptors (Kakazu et al., 2004; Lee et al., 2006). In mammary epithelial cells, migration and adhesive interactions with the ECM might be mediated by the integrins, a large family of adhesion molecules composed of α and β heterodimers. Several lines of evidence suggest that α6β4 integrin alone or coupled with EGF receptor signaling is involved in PI3-K activation and epithelial cell migration (Mercurio et al., 2001). This finding is consistent with our recent work indicating that β4 integrin is preferentially expressed than other β integrins in intact AM-expanded limbal epithelial cells (unpublished data). Subsequent study should be directed to investigate the mechanisms by which integrins are activated and their crosstalk with other growth factors in this model.