However, the inhibition of Erk resulted in a stronger suppression of proliferation compared with Akt inhibition. decreased total cellular phosphotyrosine levels, specifically inhibited phosphorylation of Erk1/2 and Akt\1, and dampened the proliferation response of epithelial cells adherent to LN\2. Inhibition of SN 2 Erk or Akt activation inhibited cell proliferation in a dose\dependent manner. However, the inhibition of Erk resulted in a stronger suppression of proliferation compared with Akt inhibition. From these results, it is usually concluded that human conjunctival epithelial cells adhere to immobilized LN\2 using 31 integrin. 31 integrin/LN\2 signalling, transduced primarily through an Erk pathway, enhances epithelial cell proliferation. These results demonstrate that LN\2 can impact on epithelial cell biology in addition to nerve and muscle, and provide information regarding the role of this isoform in ocular surface epithelial cells. INTRODUCTION Laminins are a family of extracellular matrix glycoproteins typically localized to basement membranes underlying epithelia, nerve and muscle. Laminin isoforms are crucial to cell\extracellular matrix integrity and, in general, play roles in epithelial proliferation, adhesion, migration, differentiation and survival (Ekblom 1998; Colognato & Yurchenco 2000). Laminin\2 (LN\2, merosin) is composed of a heterotrimer of 211 chains. LN\2 and LN\4 (221) are the most abundant isoforms in muscle and peripheral nerve basement membranes and are vital for normal functioning of these tissues (Sunada 1994; Xu 1994; Frost 1996; Colognato 1997; Kuang 1998). For example LN\2 has been implicated in aspects of bronchial myogenesis by stimulation of peribronchial mesenchymal cell spreading and elongation (Relan 1999). The majority of studies regarding LN\2 have focused on localization and function in developing and adult muscle and peripheral nerve tissues. However, this isoform is present in other tissues as well (Vuolteenaho 1994), including the basement membrane associated with epidermis (Schuler & Sorokin 1995; Miner 1997; Qin 1997) and intestine (Orian\Rousseau 1996). LN\2 is also a component of the basement membrane associated with conjunctival epithelium of the ocular surface (Ljubimov 1995; SN 2 Touri 1996; Qin 1997; Ljubimov 1998). With the exception of LN\2 localization in normal and damaged eye, essentially nothing is currently known regarding its function in ocular surface epithelium. In a study assessing laminin chain function Hoffman (1998) exhibited that a peptide fragment of the 2 2 chain stimulated multicellular morphological organization of human submandibular gland cells in suspension culture. The authors surmised that different laminin chains might be involved in different morphological events during development. This notion is usually supported by our work showing that during embryonic development of the ocular surface of the mouse eye LN\1 (111) expression was ubiquitous. In contrast, expression of the laminin 2 chain was restricted to the conjunctival basement membrane (Qin 1997). Expression of the laminin 2 chain was first detected during the same developmental period in which differentiated (keratin K4\positive) conjunctival epithelial cells were initially observed. We hypothesized from our developmental work that laminin isoforms play vital roles in the regulation of ocular surface epithelial cell adhesion, survival and differentiation. Rabbit Polyclonal to RPAB1 To test the hypothesis, we exhibited that this dynamics of tyrosine phosphorylation of signalling intermediates, including focal adhesion kinase (FAK), extracellular signal\related kinase (Erk\1) and Erk\2, were different in human conjunctival epithelial cells adherent to laminin\10 (LN\10, 511) compared with laminin\1 (LN\1, 111) (Lin 2000). Additionally, we exhibited that laminin signalling via Erk is usually mediated by integrins, cell surface heterodimers that function as receptors for extracellular matrix proteins (Hynes 1987; Yamada & Miyamoto 1995). Integrins can also activate the phosphatidylinositol 3\kinase (PI 3\kinase) signalling pathway leading to the activation of protein kinase?B, also known as Akt (Rameh & Cantley SN 2 1999). Thus, in addition to Erk, Akt\mediated signalling can also play a role in transducing signals in SN 2 response to integrin\induced epithelial cell adhesion to extracellular matrix (Khwaja 1997). We have extended our analyses of laminin isoform function in epithelial cells by determining the role of LN\2 in conjunctival epithelium. We hypothesize that LN\2, although usually considered a component of muscle and nerve basement membranes, can also impact on epithelial cell biology. In this report, we provide evidence that LN\2 plays roles in regulating conjunctival epithelial cell adhesion to extracellular matrix via 31 integrin. We also provide evidence that LN\2 ligation to epithelial cells via 3 integrin activates both Erk1/2 and Akt\1 signalling intermediates. Studies using signalling intermediate blockers suggest that LN\2/31 integrin modulates the proliferation of conjunctival epithelial cells primarily via Erk1/2. MATERIALS AND METHODS Antibodies Monoclonal antibodies (mAb) directed against the integrin 3 chain (Clone P1B5) were purchased from Gibco\BRL Life Technologies (Gaithersburg, MD, USA). P1B5 mAb has been documented to inhibit 3 integrin function in cultured epithelial cells (Carter 1990). mAb to integrin 1.
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