ANTISENSE EXPRESSION OF A DESMOCOLLIN GENE IN MDCK CELLS ALTERS DESMOSOME PLAQUE ASSEMBLY BUT DOES NOT AFFECT DESMOGLEIN EXPRESSION

The desmocollins are one of two types of putative adhesive proteins pr esent in the desmosome type of cell junctions, the other type being th e desmogleins; both are members of the cadherin superfamily. Each type of desmosomal cadherin occurs as a number of isoforms which have diff ering tissue distribution; within stratifying epithelia some isoforms occur only suprabasally. We have sought to analyse desmocollin functio n by reducing the amount of protein using antisense gene expression in the widely studied Madin-Darby canine kidney (MDCK) cell line, Althou gh this is a simple epithelial cell line, we show by Northern blot ana lysis that it expresses multiple isoforms of the desmosomal cadherins, Desmocollins DSC2 and DSC3 and desmogleins DSG2 and DSG3 (the pemphig us vulgaris antigen PVA) were detected, but DSC1 and DSG1, which are p resent exclusively in the suprabasal layers of the epidermis, were abs ent. The major desmocollin isoform was the type 2 (DSC2). A DSC2 clone isolated from a MDCK cDNA library had the same cell adhesion recognit ion sequence (Phe-Ala-Thr) as human, bovine and mouse type 2 isoforms, This sequence appears diagnostic for the three desmocollin isoforms, This cDNA clone was used to isolate a genomic DSC2 clone; antisense ex pression of this clone in MDCK cells resulted in a drastic reduction o f desmocollin protein as judged by Western blots; Dsc3 was not upregul ated to compensate for the loss of Dsc2, This antisense expression sig nificantly altered desmosome assembly There was a loss of punctate sta ining evident when using a desmosome plaque protein (desmoplakin) anti body, Electron microscopy revealed that there was a reduction in the n umber of desmosomes and a notable increase in the asymmetry of plaques between adjacent cells, Immunolabelling showed that similar levels of desmogleins and E-cadherin were present, Immunoelectron microscopy al so showed that many vesicular structures were label led, at intervals along the lateral membranes between cells. The distinctive loose organ ization of the remaining desmosomes may originate in modifications to the targeting and incorporation of proteins into fully assembled plaqu es. Other junctions were unaffected and the cells maintained their int egrity as a confluent monolayer.