Cell adhesion to the ECM appears to

Cell adhesion to the ECM appears to trigger various intracellular signaling pathways. In addition to proteolytic degradation of amniotic basement membrane components by proteases such as MMP-9, intracellular signaling cascades stimulated by growth factors or cytokines released from cell-cell matrix interactions might participate in migration and proliferation of intact AM-expanded limbal outgrowth. It has been reported that epidermal growth factor (EGF) and nerve growth factor (NGF) are required for limbal explant culture on AM (Yokoo et al., 2008). These factors may, when coupling with their respective receptors, activate intracellular signaling pathways, primarily through the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3-K)/Akt cascades (He et al., 2006). There are three major superfamilies of MAPKs in several cell types, including extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, and c-Jun N-terminal kinases (JNKs, also termed stress-activated protein kinases) (Johnson and Lapadat, 2002). Activation of MAPKs exerts distinct cellular responses such as cell proliferation and migration by phosphorylation of specific target proteins (Johnson and Lapadat, 2002). PI3-K is an important enzyme that promotes cell survival and growth (Datta et al., 1999) by phosphorylating the downstream core component Akt/protein kinase B (PKB), first identified as the cellular homologue of the transforming oncogene (Staal, 1987). Accumulating evidences demonstrate the involvement of both MAPK and PI3-K/Akt pathways in regulation of MMP-9 production (Lin et al., 2009; Ellerbroek et al., 2001). However, the association between the activation of these intracellular signaling pathways and intact AM-expanded limbal epithelial tsh receptor is not completely understood. Therefore, the purpose of the current study is to investigate the signaling cascades involved in the outgrowth of limbal explants and the regulatory mechanisms of MMP-9 expression in this model. Our results demonstrate that both MAPKs and PI3-K/Akt are required for limbal epithelial outgrowth on intact AM, only the PI3-K/Akt/JNK is essential for MMP-9 expression mediated through activation of transcriptional factor NF-κB in this model.
Results
Discussion MMPs are proteolytic enzymes that degrade almost all components of the ECM and basement membrane proteins which are associated with physiological and pathological processes under various situations. In fact, several lines of evidence suggest that MMP-9 is implicated in migration and axonal outgrowth of neural SCs (Lin et al., 2009; Tonti et al., 2009), as well as in transmigration of hematopoietic progenitor cells (Rao et al., 2004). Like the SCs in other tissues, limbal epithelial progenitor cells are in continuous contact and interact with their surrounding environment, termed the stem cell niche, which is a network composed of several ECM components, cytokines and growth factors (Grueterich et al., 2003). Previous reports have demonstrated that intact AM-expanded limbal epithelial cells behave as a limbal epithelial phenotype (Grueterich et al., 2002) and maintain in a less apoptotic state (Sun et al., 2006). Therefore, the intact AM may serve as an ideal “niche” to support ex vivo expansion of human limbal epithelial progenitor cells (Grueterich et al., 2003), this is the reason why we used an intact AM as the culture carrier in this study. In the present study, we demonstrate that in situ MMP-9 zymographic activity and protein expression were co-localized and accumulated at the leading edge of expanded human limbal epithelial cells on intact AM (Fig. 2). The focally active MMP-9 location at the front of migration could be explained, at least in part, by the fact that much more ECM components were left on intact AM, necessitating more proteases to degrade them. This notion was supported by the observation that in situ MMP-9 activity was more prominently distributed in intact AM-expanded limbal epithelial cells than on denuded AM (Fig. 1). On the other hand, accumulating evidence demonstrates that protein fragments cleaved by MMPs could gain activities that are inactive in the intact molecule. For instance, MMP-9 was involved in the release and activation of ECM-sequestered transforming growth factor–β (TGF-β) (Yu and Stamenkovic, 2000). Another example is laminin-5, which is cleaved by MMP-2 to expose a cryptic site that stimulates mammary epithelial cell migration (Giannelli et al., 1997). These results were in accordance with our recent work which indicates that MMP-9, together with urokinase plasminogen activator (uPA), could process human laminin-5 γ2-chain into several protein fragments implicated in limbal epithelial outgrowth on intact AM (Cheng et al., 2009). However, the structural characteristics and functional significance of these protein fragments derived from laminin-5 remain to be clarified.