Myocilin promotes substrate adhesion, spreading and formation of focal contacts in podocytes and mesangial cells.
Myocilin, a secreted glycoprotein of the olfactomedin family, is constitutively expressed in podocytes of the rat kidney and induced in mesangial cells during mesangioproliferative glomerulonephritis. As myocilin has been found to be associated with fibrillar components of the extracellular matrix, and adhesive properties have been shown for other members of the olfactomedin family, we hypothesized that myocilin might play a role in cell-matrix interactions in the glomerulus. To elucidate functional properties of myocilin, recombinant myocilin was expressed in 293 EBNA cells and purified by Ni-chelate and heparin chromatography. Culture plates were coated with myocilin, and primary rat mesangial cells and cells from an immortal murine podocyte cell line were seeded onto the plates in serum free conditions. Both cell types showed concentration-dependant attachment to myocilin, an effect that was statistically significant and could be blocked with specific antibodies. When compared to equal amounts of fibronectin or collagen 1, myocilin was less effective in promoting substrate adhesion. Synergistic effects in substrate adhesion were observed when myocilin was added to low concentrations of fibronectin. Twenty-five percent of cells that had attached to myocilin substrates showed spreading and expressed focal contacts which were labeled by vinculin/phalloidin staining. Comparable findings were observed when human or murine trabecular meshwork cells were seeded on myocilin substrates. Adhesive properties of myocilin required multimer formation, and were not observed when culture plates were coated with a C-terminal fragment of myocilin, containing the olfactomedin domain. We conclude that myocilin promotes substrate adhesion of podocytes and mesangial cells, and might contribute to cell-matrix adhesion of both cell types in vivo.
Institute of Anatomy, University of Erlangen-Nürnberg, Erlangen, Germany.
Andreas
A
Goldwich
Michael
M
Scholz
Ernst R
ER
Tamm
D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000494:pharmacology*; D005136:Eye Proteins / Q000494:pharmacology*; D005353:Fibronectins / Q000494:pharmacology; D022001:Focal Adhesions / Q000648:ultrastructure; D006023:Glycoproteins / Q000494:pharmacology*; D006801:Humans; D050527:Mesangial Cells / Q000166:cytology / Q000187:drug effects*; D051379:Mice; D050199:Podocytes / Q000166:cytology / Q000187:drug effects*; D051381:Rats; D014162:Transfection; D000099179:Myocilin
D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D005353:Fibronectins; D006023:Glycoproteins; D000099179:Myocilin