title: Gain-of-function mutations in transient receptor potential C6 (TRPC6) activate extracellular signal-regulated kinases 1/2 (ERK1/2).
abstract: Gain-of-function mutations in the canonical transient receptor potential 6 (TRPC6) gene are a cause of autosomal dominant focal segmental glomerulosclerosis (FSGS). The mechanisms whereby abnormal TRPC6 activity results in proteinuria remain unknown. The ERK1/2 MAPKs are activated in glomeruli and podocytes in several proteinuric disease models. We therefore examined whether FSGS-associated mutations in TRPC6 result in activation of these kinases. In 293T cells and cultured podocytes, overexpression of gain-of-function TRPC6 mutants resulted in increased ERK1/2 phosphorylation, an effect dependent upon channel function. Pharmacologic inhibitor studies implicated several signaling mediators, including calmodulin and calcineurin, supporting the importance of TRPC6-mediated calcium influx in this process. Through medium transfer experiments, we uncovered two distinct mechanisms for ERK activation by mutant TRPC6, a cell-autonomous, EGF receptor-independent mechanism and a non-cell-autonomous mechanism involving metalloprotease-mediated release of a presumed EGF receptor ligand. The inhibitors KN-92 and H89 were able to block both pathways in mutant TRPC6 expressing cells as well as the prolonged elevation of intracellular calcium levels upon carbachol stimulation seen in these cells. However, these effects appear to be independent of their effects on calcium/calmodulin-dependent protein kinase II and PKA, respectively. Phosphorylation of Thr-70, Ser-282, and Tyr-31/285 were not necessary for ERK activation by mutant TRPC6, although a phosphomimetic TRPC6 S282E mutant was capable of ERK activation. Taken together, these results identify two pathways downstream of mutant TRPC6 leading to ERK activation that may play a role in the development of FSGS.
authors: Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.
authors: David
authors: D
authors: Chiluiza
authors: Sneha
authors: S
authors: Krishna
authors: Valérie A
authors: VA
authors: Schumacher
authors: Johannes
authors: J
authors: Schlöndorff
mesh_terms: D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D019703:Calcineurin / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D005923:Glomerulosclerosis, Focal Segmental / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D007546:Isoquinolines / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D050199:Podocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology; D050052:TRPC Cation Channels / Q000235:genetics / Q000502:physiology*; D000074287:TRPC6 Cation Channel
chemical_list: D001596:Benzylamines; D002147:Calmodulin; D004791:Enzyme Inhibitors; D007546:Isoquinolines; C425780:KN 92; D013449:Sulfonamides; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019703:Calcineurin; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide
keywords: Calcium; Epidermal Growth Factor Receptor (EGFR); Focal Segmental Glomerulosclerosis; Kidney; Protein Kinase A (PKA); TRP Channels
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