Occludin is regulated by epidermal growth factor receptor activation in brain endothelial cells and brains of mice with acute liver failure

Tight junction (TJ) proteins regulate paracellular permeability in various organs, particularly in the brain neurovascular unit (1). In brain capillaries, the endothelial cell (EC) spreads itself over the entire capillary basal lamina with the two plasmalemmal surfaces aligning to form the TJ. The TJ proteins, including occludin, claudin-5, junctional associated molecules, and their intracellular accessory factors zona occludins (ZO), seal the paracellular space. Collectively, the EC and its TJ, basal lamina, and associated astrocyte end-feet form the blood-brain barrier (BBB) that tightly regulates what enters and exits the neurovascular unit of the brain (2). TJ proteins, particularly occludin and claudin-5, are important in the paracellular barrier function of the BBB, and their roles have been described in various pathological conditions (3-5). Occludin is a tetraspan membrane protein with two extracellular loops within the TJ and with the amino- and carboxy-terminal chains in the cytoplasm. The C-terminal domain binds to ZO-1 and -2 which serves as the link between occludin and the cytoskeleton (6). Claudin-5 has a similar distribution. Although the barrier function is typically compromised by structural breakdown of the BBB, recent evidence has suggested that subtle alterations in either occludin or claudin-5—without obvious structural changes—can result in selective permeability to small molecules (7-9). These findings support the concept that vasogenic edema might result from a subtle modification in TJ composition (10). In acute liver failure (ALF), brain edema is lethal and remains a major determinant of patient survival (11, 12). However, the exact alterations in BBB integrity that lead to brain edema in ALF are not known. In 2006, we reported that specific monoclonal antibodies against matrix metalloproteinase-9 (MMP-9) attenuate brain extravasation and edema in ALF mice (13). We recently showed that MMP-9 significantly alters the TJ proteins, particularly occludin, in brain ECs in vitro and in brains of mice that have experimentally induced ALF (5). However, the signal transductions associated with MMP-9 that mediate the alterations in occludin remain unknown. The role of epidermal growth factor receptor (EGFR) in cancer development and treatment is well known (14-16). EGFR belongs to the ErbB family of receptor tyrosine kinases. Upon ligand stimulation, they dimerize, and dimerization is then followed by receptor internalization and autophosphorylation of the intracytoplasmic EGFR tyrosine kinase domains, which serve as binding sites for recruiting signal transducers and activators of the intracellular signal transduction cascade. Recently EGFR was implicated in the regulation of cellular barrier function (17). EGFR has also been shown to participate in microvascular injury in diabetes (18), lung injury (19, 20), and intestinal permeability (21, 22). Ligation of EGFR activates the mitogen-activated protein kinase (MAPK) cascades (23, 24). MAPKs have been implicated in endothelial paracellular permeability associated with oxidative insults (25-27) and with alterations of TJ proteins (28); thus, MAPKs are important in maintaining cellular and TJ integrity (29). Previously, we observed that p38 MAPK is upregulated in isolated microvessels from the brains of ALF mice (30). However, the role of p38 MAPK and EGFR in BBB permeability in ALF has not been explored. In this study, we investigated the role of p38MAPK/NFκB signaling after EGFR transactivation by MMP-9 in altering the TJ element occludin in brain ECs in vitro and in brains of mice with ALF.