A clinical case documented a reversible change in airway epithelial differentiation that coincided with the initiation and discontinuation of trastuzumab, an anti-erbB2 antibody. This prompted the investigation into whether blocking the erbB2 receptor alters differentiation of the airway epithelium. To test this hypothesis, we treated an in vitro model of well-differentiated human airway epithelia with trastuzumab or heregulin-alpha, an erbB ligand. In addition, coculturing with human lung fibroblasts tested whether in vivo subepithelial fibroblasts function as an endogenous source of ligands able to activate erbB receptors expressed by the overlying epithelial cells. Epithelia were stained with hematoxylin and eosin and used for morphometric analysis. Trastuzumab treatment decreased the ciliated cell number by 49% and increased the metaplastic, flat cell number by 640%. Heregulin-alpha treatment increased epithelial height and decreased the number of metaplastic and nonciliated columnar cells, whereas it increased the goblet cell number. We found that normal human lung fibroblasts express transforming growth factor-alpha, heparin-binding epidermal-like growth factor, epiregulin, heregulin-alpha, and amphiregulin, all of which are erbB ligands. Cocultures of airway epithelia with primary fibroblasts increased epithelial height comparable to that achieved following heregulin-alpha treatment. These data show that erbB2 stimulation is required for maintaining epithelial differentiation. Furthermore, the mesenchyme underlying the airway epithelium secretes a variety of erbB ligands that may direct various pathways of epithelial differentiation.
Two enzymes, protein phosphatase 2A and atypical protein kinase C, are associated with the tight junction and regulate its function. For example, phosphorylation of the tight junction protein occludin is required for its incorporation into the junction. The association of a kinase and phosphatase with the tight junction suggests that a balance between their activities exists and is required for normal tight junction function. This hypothesis predicts that loss of epithelial integrity may disrupt this balance in such a way as to facilitate restoration of epithelial integrity. Our previous data have shown that apically localized growth factors segregate from their basolaterally localized erbB receptors. Loss of epithelial integrity allows ligand access to the basolateral membrane where it immediately binds to and activates erbB receptors. We found that activation of erbB1 leads to phosphorylation of protein phosphatase 2A, inhibiting its activity. Importantly, this phosphorylation event was dependent on factors in the overlying airway surface liquid; washing away this liquid prevented phosphorylation. erbB1-mediated inhibition of phosphatase activity would shift the balance in favor of the kinase such that tight junction proteins would regain their phosphorylation, allowing for their incorporation into the junction complex. This mechanism provides a rapid means of sensing the loss of epithelial integrity and subsequently restoring barrier function.Polarized epithelia line the cavities of the body, forming a physical barrier between the external environment and the body compartments. Essential to the function of an epithelium is its polarity. Cellular polarity is established and maintained by cell-cell adhesion junctions, including the tight junction and the adherens junction. We have previously demonstrated that maintenance of barrier function is required for silencing signaling cascades in differentiated primary cultures of human airway epithelia (1). Airway epithelia secrete growth factors into the airway surface liquid (ASL).2 Among these is heregulin-␣, a ligand for the family of erbB tyrosine kinase receptors that localize to the basolateral membrane in airway epithelia. This arrangement allows for receptor activation the instant epithelial integrity is compromised. In the presence of intact tight junctions, erbB receptors are silent (not phosphorylated). Disruption of cell-cell junctions allows ASL factors immediate access to the basolateral membrane, resulting in erbB receptor activation. Receptor activation triggers signaling cascades involved in injury repair. Following restoration of epithelial integrity, heregulin-␣ regains its restriction to the ASL, and erbB receptors are once again silenced. These data emphasize the importance of epithelial polarity and the cellcell adhesion junctions that maintain it. In the absence of tight junctions, polarity ceases to exist and erbB signaling cascades become activated. Such aberrant signaling can lead to many downstream effects, including cellular proliferation, ...
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