Occludin confers adhesiveness when expressed in fibroblasts.
Occludin is an integral membrane protein specifically associated with tight junctions. Previous studies suggest it is likely to function in forming the intercellular seal. In the present study, we expressed occludin under an inducible promotor in occludin-null fibroblasts to determine whether this protein confers intercellular adhesion. When human occludin is stably expressed in NRK and Rat-1 fibroblasts, which lack endogenous occludin and tight junctions but do have well developed ZO-1-containing adherens-like junctions, occludin colocalizes with ZO-1 to points of cell-cell contact. In contrast, L-cell fibroblasts which lack cadherin-based adherens junctions, target neither ZO-1 nor occludin to sites of cell contact. Occludin-induced adhesion was next quantified using a suspended cell assay. In NRK and Rat-1 cells, occludin expression induces adhesion in the absence of calcium, thus independent of cadherin-cadherin contacts. In contrast, L-cells are nonadhesive in this assay and show no increase in adhesion after induction of occludin expression. Binding of an antibody to the first of the putative extracellular loops of occludin confirmed that this sequence was exposed on the cell surface, and synthetic peptides containing the amino acid sequence of this loop inhibit adhesion induced by occludin expression. These results suggest that the extracellular surface of occludin is directly involved in cell-cell adhesion and the ability to confer adhesiveness correlates with the ability to colocalize with its cytoplasmic binding protein, ZO-1.
Van Itallie
C M
CM
Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510, USA.
Anderson
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D018938:Caco-2 Cells / Q000737:chemistry / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D004285:Dogs; D005347:Fibroblasts / Q000737:chemistry / Q000502:physiology; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D015151:Immunoblotting; D007686:Kidney Tubules, Distal / Q000166:cytology; D008565:Membrane Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D062793:Occludin; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D051381:Rats; D019108:Tight Junctions / Q000737:chemistry* / Q000502:physiology; D014162:Transfection
D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568987:Ocln protein, rat; D010455:Peptides