title: Juxtacrine activation of EGFR regulates claudin expression and increases transepithelial resistance.
abstract: Heparin-binding (HB)-EGF, a ligand for EGF receptors, is synthesized as a membrane-anchored precursor that is potentially capable of juxtacrine activation of EGF receptors. However, the physiological importance of such juxtacrine signaling remains poorly described, due to frequent inability to distinguish effects mediated by membrane-anchored HB-EGF vs. mature "secreted HB-EGF." In our studies, using stable expression of a noncleavable, membrane-anchored rat HB-EGF isoform (MDCK(rat5aa) cells) in Madin-Darby canine kidney (MDCK) II cells, we observed a significant increase in transepithelial resistance (TER). Similar significant increases in TER were observed on stable expression of an analogous, noncleavable, membrane-anchored human HB-EGF construct (MDCK(human5aa) cells). The presence of noncleavable, membrane-anchored HB-EGF led to alterations in the expression of selected claudin family members, including a marked decrease in claudin-2 in MDCK(rat5aa) cells compared with the control MDCK cells. Reexpression of claudin-2 in MDCK(rat5aa) cells largely prevented the increases in TER. Ion substitution studies indicated decreased paracellular ionic permeability of Na(+) in MDCK(rat5aa) cells, further indicating that the altered claudin-2 expression mediated the increased TER seen in these cells. In a Ca(2+)-switch model, increased phosphorylation of EGF receptor and Akt was observed in MDCK(rat5aa) cells compared with the control MDCK cells, and inhibition of these pathways inhibited TER changes specifically in MDCK(rat5aa) cells. Therefore, we hypothesize that juxtacrine activation of EGFR by membrane-anchored HB-EGF may play an important role in the regulation of tight junction proteins and TER.
authors.affiliation: Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232-4794, USA. amar.singh@vanderbilt.edu
authors.forename: Amar B
authors.initials: AB
authors.lastname: Singh
authors.forename: Keisuke
authors.initials: K
authors.lastname: Sugimoto
authors.forename: Punita
authors.initials: P
authors.lastname: Dhawan
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Harris
mesh_terms: D000818:Animals; D000906:Antibodies; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D019898:Autocrine Communication; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability; D004285:Dogs; D017097:Electric Impedance; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D019012:Integrin beta1 / Q000276:immunology / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007444:Inulin / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000378:metabolism; D060245:Tetraspanin 29; D019108:Tight Junctions / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014316:Tritium
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D002118:Calcium; D066246:ErbB Receptors; D066257:Heparin-binding EGF-like Growth Factor; D019012:Integrin beta1; D036341:Intercellular Signaling Peptides and Proteins; D007444:Inulin; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D012964:Sodium; D060245:Tetraspanin 29; D014316:Tritium; C554869:CD9 protein, human; C587883:HBEGF protein, human; C587885:Hbegf protein, rat