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TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility.

ABSTRACT: BACKGROUND:Mutations in the transient receptor potential channel 6 (TRPC6) gene are associated with an inherited form of FSGS. Despite widespread expression, patients with TRPC6 mutations do not present with any other pathologic phenotype, suggesting that this protein has a unique yet unidentified role within the target cell for FSGS, the kidney podocyte. METHODS:We generated a stable TRPC6 knockout podocyte cell line from TRPC6 knockout mice. These cells were engineered to express wild-type TRPC6, a dominant negative TRPC6 mutation, or either of two disease-causing mutations of TRPC6, G109S or K874*. We extensively characterized these cells using motility, detachment, and calpain activity assays; immunofluorescence; confocal or total internal reflection fluorescence microscopy; and western blotting. RESULTS:Compared with wild-type cells, TRPC6-/- podocytes are less motile and more adhesive, with an altered actin cytoskeleton. We found that TRPC6 binds to ERK1/2 and the actin regulatory proteins, caldesmon (a calmodulin- and actin-binding protein) and calpain 1 and 2 (calcium-dependent cysteine proteases that control the podocyte cytoskeleton, cell adhesion, and motility via cleavage of paxillin, focal adhesion kinase, and talin). Knockdown or expression of the truncated K874* mutation (but not expression of the gain-of-function G019S mutation or dominant negative mutant of TRPC6) results in the mislocalization of calpain 1 and 2 and significant downregulation of calpain activity; this leads to altered podocyte cytoskeleton, motility, and adhesion-characteristics of TRPC6 -/- podocytes. CONCLUSIONS:Our data demonstrate that independent of TRPC6 channel activity, the physical interaction between TRPC6 and calpain in the podocyte is important for cell motility and detachment and demonstrates a scaffolding role of the TRPC6 protein in disease.

SUBMITTER: Farmer LK

PROVIDER: S-EPMC6779362 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility.

Farmer Louise K LK Rollason Ruth R Whitcomb Daniel J DJ Ni Lan L Goodliff Alexander A Lay Abigail C AC Birnbaumer Lutz L Heesom Kate J KJ Xu Shang-Zhong SZ Saleem Moin A MA Welsh Gavin I GI

Journal of the American Society of Nephrology : JASN 20190815 10

<h4>Background</h4>Mutations in the transient receptor potential channel 6 (<i>TRPC6</i>) gene are associated with an inherited form of FSGS. Despite widespread expression, patients with <i>TRPC6</i> mutations do not present with any other pathologic phenotype, suggesting that this protein has a unique yet unidentified role within the target cell for FSGS, the kidney podocyte.<h4>Methods</h4>We generated a stable <i>TRPC6</i> knockout podocyte cell line from <i>TRPC6</i> knockout mice. These cel ...[more]

PMID: 31416818

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Project description:Transient receptor potential channel C6 (TRPC6) gain-of-function mutations and increased TRPC6 expression in podocytes induce glomerular injury and proteinuria. Sildenafil reduces TRPC6 expression and activity in nonrenal cell types, although the mechanism is unknown. Peroxisome proliferator-activated receptor γ (PPAR-γ) is a downstream target of sildenafil in the cyclic guanosine monophosphate (cGMP)-activated protein kinase G (PKG) axis. PPAR-γ agonists, like pioglitazone, appear antiproteinuric. We hypothesized that sildenafil inhibits TRPC6 expression in podocytes through PPAR-γ-dependent mechanisms, thereby counteracting podocyte injury and proteinuria. Treatment with sildenafil, the cGMP derivative 8-bromoguanosine 3',5'-cyclic monophosphate sodium salt (8-Br-cGMP), or pioglitazone dose-dependently downregulated podocyte injury-induced TRPC6 expression in vitro Knockdown or application of antagonists of PKG or PPAR-γ enhanced TRPC6 expression in podocytes and counteracted effects of sildenafil and 8-Br-cGMP. We observed similar effects on TRPC6 promoter activity and TRPC6-dependent calcium influx. Chromatin immunoprecipitation showed PPAR-γ binding to the TRPC6 promoter. Sildenafil or pioglitazone treatment prevented proteinuria and the increased TRPC6 expression in rats with adriamycin-induced nephropathy and mice with hyperglycemia-induced renal injury. Rats receiving PPAR-γ antagonists displayed proteinuria and increased podocyte TRPC6 expression, as did podocyte-specific PPAR-γ knockout mice, which were more sensitive to adriamycin and not protected by sildenafil. Thus, sildenafil ameliorates podocyte injury and prevents proteinuria through cGMP- and PKG-dependent binding of PPAR-γ to the TRPC6 promoter, which inhibits TRPC6 promoter activity, expression, and activity. Because sildenafil is approved for clinical use, our results suggest that additional clinical study of its antiproteinuric effect in glomerular disease is warranted.

Dataset Information

TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility.

Publications

TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility.

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