N-Glycosylation of ß4 Integrin Controls the Adhesion and Motility of Keratinocytes

α6ß4 integrin is an essential component of hemidesmosomes and modulates cell migration in wound healing and cancer invasion. To elucidate the role of N-glycosylation on ß4 integrin, we investigated keratinocyte adhesion and migration through the re-expression of wild-type or N-glycosylation-defective ß4 integrin (ΔNß4) in ß4 integrin null keratinocytes. N-glycosylation of ß4 integrin was not essential for the heterodimer formation of ß4 integrin with α6 integrin and its expression on a cell surface, but N-glycosylation was required for integrin-mediated cell adhesion and migration. Concomitantly with the reduction of ß4 integrin in the membrane microdomain, the intracellular signals of Akt and ERK activation were decreased in cells expressing ΔNß4 integrin. Forced cross-linking of ß4 integrin rescued the decreased ERK activation in ΔNß4 integrin-expressing cells to a similar extent in wild-type ß4 integrin-expressing cells. Surprisingly, compared with cells expressing wild-type ß4 integrin, an alternation in N-glycan structures expressed on epidermal growth factor receptor (EGFR), and the induction of a stronger association between EGFR and ß4 integrin were observed in ΔNß4 integrin-expressing cells. These results clearly demonstrated that N-glycosylation on ß4 integrin plays an essential role in keratinocyte cellular function by allowing the appropriate complex formation on cell surfaces

Hypertonic saline increases lung epithelial lining fluid glutathione and thiocyanate: two protective CFTR-dependent thiols against oxidative injury

<p>Abstract</p> <p>Background</p> <p>Cystic fibrosis is a debilitating lung disease due to mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) and is associated with chronic infections resulting in elevated myeloperoxidase activity and generation of hypochlorous acid (HOCl). CFTR mutations lead to decreased levels of glutathione (GSH) and thiocyanate (SCN) in the epithelial lining fluid (ELF). Hypertonic saline is used to improve lung function however the mechanism is uncertain.</p> <p>Methods</p> <p>In the present study, the effect of GSH and SCN on HOCl-mediated cell injury and their changes in the ELF after hypertonic saline nebulization in wild type (WT) and CFTR KO mice was examined. CFTR sufficient and deficient lung cells were assessed for GSH, SCN and corresponding sensitivity towards HOCl-mediated injury, in vitro.</p> <p>Results</p> <p>CFTR (-) cells had lower extracellular levels of both GSH and SCN and were more sensitive to HOCl-mediated injury. In vivo, hypertonic saline increased ELF GSH in the WT and to a lesser extent in the CFTR KO mice but only SCN in the WT ELF. Finally, potential protective effects of GSH and SCN at concentrations found in the ELF against HOCl toxicity were examined in vitro.</p> <p>Conclusions</p> <p>While the concentrations of GSH and SCN associated with the WT ELF protect against HOCl toxicity, those found in the CFTR KO mice were less sufficient to inhibit cell injury. These data suggests that CFTR has important roles in exporting GSH and SCN which are protective against oxidants and that hypertonic saline treatment may have beneficial effects by increasing their levels in the lung.</p

Classical, novel and atypical isoforms of PKC stimulate ANF- and TRE/AP-1-regulated-promoter activity in ventricular cardiomyocytes

Cultured neonatal rat ventricular myocytes were co-transfected with expression plasmids encoding protein kinase C (PKC) isoforms from each of the PKC subfamilies (classical PKC-α, novel PKC-ε or atypical PKC-ξ) together with an atrial natriuretic factor (ANF) reporter plasmid. Each PKC had been rendered constitutively active by a single Ala→Glu mutation or a small deletion in the inhibitory pseudosubstrate site. cPKC-α, nPKC-ε or aPKC-ξ expression plasmids each stimulated ANF-promoter activity and expression of a reporter gene under the control of a 12-O-tetradecanoylphorbol 13-acetate-response element (TRE). Upregulation of the ANF promoter is characteristic of the hypertrophic response in the heart ventricle and a TRE is present in the ANF promoter. Thus all subfamilies of PKC may have the potential to contribute to hypertrophic response in cardiomyocytes