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Journal of Cell Science, Vol 108, Issue 11 3531-3540, Copyright © 1995 by Company of Biologists
JOURNAL ARTICLES |
EU Saelman, PJ Keely and SA Santoro
Department of Pathology, Washington University School of Medicine, St Louis, MO 63110-1093, USA.
Cellular interactions with collagen in a model of kidney tubulogenesis were investigated using Madin-Darby canine kidney (MDCK) cells in an in vitro morphogenetic system. MDCK cells adhered to collagen types I and IV in a Mg(2+)-dependent manner, typical of the alpha 2 beta 1 integrin. Collagen-Sepharose affinity chromatography and immunoblotting demonstrated the presence and collagen binding activity of the alpha 2 beta 1 integrin on MDCK cells. To assess the function of alpha 2 beta 1 integrin, MDCK cells were transfected with a plasmid pRSV alpha 2' which allowed the expression of alpha 2-integrin subunit antisense RNA. Three G418-resistant clones showing reduced adhesion to collagen, stable genomic integration of the antisense construct, decreased alpha 2-integrin subunit mRNA and decreased alpha 2-integrin subunit protein expression were selected for analysis in morphogenetic experiments. MDCK cells and plasmid-only control transfectants, cultured in three-dimensional collagen type I gels, showed normal cyst formation, whereas the antisense RNA transfectants showed increased apoptosis and formed small rudimentary cysts. Stimulation with hepatocyte growth factor/scatter factor-containing 3T3 fibroblast-conditioned medium or recombinant hepatocyte growth factor/scatter factor resulted in extensive branching of the preformed control cysts whereas the surviving small cysts formed by antisense expressing cells increased in size but failed to elongate and branch upon stimulation. We conclude that alpha 2 beta 1 integrin collagen interactions play a crucial role in the hepatocyte growth factor/scatter factor-induced tubulogenesis and branching morphogenesis of MDCK cells in collagen gels as well as an important role in cell survival.
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