Pinto 2013

Inhibition of Ca2+/Calmodulin Activates RAS and Enhances cAMP-dependent ERK Activation in Human ADPKD Cells

Cibele S. Pinto, Gail A. Reif  and Darren P. Wallace.

Background: cAMP plays a central role in the pathogenesis of ADPKD by stimulating cyst epithelial cell proliferation through activation of B-Raf and MEK/ERK signaling. By contrast, in normal renal cells (NHK), cAMP inhibits Raf-1, ERK and cell proliferation. The molecular mechanism for this phenotypic difference in the mitogenic response to cAMP remains unclear. Activated RAS (GTP bound) recruits Raf to the plasma membrane and mediates Raf activation. Ca2+ /calmodulin (CaM) can stimulate or inhibit RAS, depending on cell type. Previously, we showed that CaM inhibition with W-7, alone or in the presence of AVP, significantly increased ERK phosphorylation (P-ERK) in ADPKD cells, while having no effect in NHK cells. Here, we determined if Ca2+/CaM differentially regulates RAS activity to account for differences in cAMP-dependent ERK activation between ADPKD and NHK cells

Method:  ADPKD and NHK cells were treated with W-7 ± AVP and RAS activity was determined by RAS-GTP affinity pull-down assay. NHK cells were treated with a Ca2+ chelator BAPTA to restrict intracellular Ca2+, mimicking ADPKD cells.

Results: cAMP stimulated RAS to similar levels in ADPKD and NHK cells; however, it only increased P-ERK and proliferation of ADPKD cells. In ADPKD cells, CaM inhibition with W-7 alone increased RAS activity and P-ERK. cAMP caused a further increase in P-ERK, while having only a modest effect on RAS activation, suggesting cAMP had an effect downstream of RAS. In NHK cells, W-7 increased RAS activity, but did not stimulate ERK. In the presence of W-7, cAMP had no additional effect on RAS and failed to stimulate ERK. Restriction of intracellular Ca2+ with BAPTA increased RAS activity 3-fold and stimulated ERK in NHK cells. cAMP had no further effect of RAS but stimulated ERK and cell proliferation, suggesting that the Ca2+-induced phenotypic switch in the cAMP mitogenic response was below the level of RAS.

Conclusion: These data support the hypothesis that the differential effect of cAMP on the ERK signaling pathway between NHK and ADPKD cells is due to regulation of components downstream of RAS, notably the Raf kinases.