Myocilin promotes substrate adhesion, spreading and formation of focal contacts in podocytes and mesangial cells

Abstract

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.

Differential Effects of Myocilin and Optineurin, Two Glaucoma Genes, on Neurite Outgrowth

“…This implies that myocilin may have more of an impact on the cells in the aqueous outflow pathway such as the TM than those in the optic nerve head and the retina such as RGCs, and vice versa in the case of optineurin. Myocilin has previously been shown to affect the actin cytoskeletal structure 45,55 and to lower the mitochondrial membrane potential 56 in human TM cells. The revelation that myocilin also impacts neurite outgrowth in neuronal cells is somewhat surprising.…”

The Glaucoma-associated Olfactomedin Domain of Myocilin Is a Novel Calcium Binding Protein

“…In studies of full-length myocilin, extracellular matrix interacting partners such as actin, laminin, fibronectin, and heparan sulfate, as well as cell-matrix adhesion properties, have been localized to the N-terminal coiled-coil, not the OLF domain (11)(12)(13)(14)(15). In addition, the explicit functions of non-myocilin OLF homologs, distributed throughout the body and commonly found in neural tissues, are also unknown (16).…”

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“…InterCluster, a tool developed to cluster protein-protein interactions, has helped in datamining the proteins involved in POAG and their primary interactions. Among 10500 proteins that were primary interactors of the 31 proteins involved in POAG we could datamine four proteins APOE, MYOC, ELANE and KNG1 in a cluster (Cluster 2) experimentally proved to be involved in heparin binding [21][22][23][24] . This would have been impossible without the aid of the tool.…”

“…From this analysis the 4 proteins APOE, MYOC, ELANE (UniProt ID: P08246) and KNG1 (UniProt ID: P01042) in this cluster are found to be involved in heparin binding. [21][22][23][24] Further, based on the localization of the proteins [25][26][27][28][29][30][31] in Cluster 2 ( Figure 3) it is most probable that STAT3 (UniProt ID: P40763) interacts with POAG proteins APOE and CYP1B1 in the cytoplasm. Also MYOC might interact with POAG proteins FN1 and CYP1B1 in the endoplasmic reticulum.…”