Gain-of-function mutations in transient receptor potential C6 (TRPC6) activate extracellular signal-regulated kinases...

Area of Science:

• Nephrology
• Molecular Biology
• Cell Signaling

Background:

• Gain-of-function mutations in the TRPC6 gene are linked to autosomal dominant focal segmental glomerulosclerosis (FSGS).
• The precise mechanisms by which aberrant TRPC6 activity leads to proteinuria are not fully understood.
• ERK1/2 MAP kinases are known to be activated in glomerular and podocyte injury models.

Purpose of the Study:

• To investigate whether FSGS-associated TRPC6 mutations activate ERK1/2 MAP kinases.
• To elucidate the signaling pathways downstream of mutant TRPC6 that lead to ERK activation.

Main Methods:

• Overexpression of gain-of-function TRPC6 mutants in 293T cells and cultured podocytes.
• Pharmacologic inhibition studies using specific signaling mediators.
• Medium transfer experiments to differentiate cell-autonomous and non-cell-autonomous mechanisms.
• Analysis of specific phosphorylation sites on TRPC6.

Main Results:

• Overexpression of mutant TRPC6 led to increased ERK1/2 phosphorylation, dependent on channel function.
• TRPC6-mediated calcium influx, involving calmodulin and calcineurin, is crucial for ERK activation.
• Two pathways were identified: a cell-autonomous, EGF receptor-independent pathway and a non-cell-autonomous pathway involving metalloprotease-mediated ligand release.
• Inhibitors KN-92 and H89 blocked both pathways and prolonged calcium elevation, but effects were independent of CaMKII and PKA.
• Specific phosphorylation sites on TRPC6 were not essential for ERK activation, though a phosphomimetic mutant showed activity.

Conclusions:

• Mutant TRPC6 activates ERK1/2 MAP kinases via two distinct downstream pathways.
• These pathways, involving TRPC6-mediated calcium influx, may contribute to the pathogenesis of FSGS.
• Understanding these mechanisms could offer new therapeutic targets for FSGS.