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Previous studies on MCF-7 breast cancer cells have shown that the G-protein coupled receptor (GPCR) agonist carbachol increases intracellular calcium levels and the activation of extracellular signal-regulated kinase (ERK). Calcium and calmodulin regulate the calcium/calmodulin- dependent kinase (CaM kinase) family of proteins that have been proposed to regulate ERK and gene transcription. Our results suggest that both estrogen (E2) and carbachol treatment of MCF-7 breast cancer cells trigger phosphorylation of ERK I /2 and the transcription factor Elk-1. Carbachol and estrogen triggered nearly a four- to sixfold increase in MCF-7 cell proliferation by 96 h, respectively. Carbachol-stimulated ERK activation and cell growth was completely blocked by the Muscarinic M3- subtype GPCR inhibitor, 4-DAMP, and siRNA against the M3-subtype GPCR. Interestingly, blockade of CaM KK with the selective inhibitor ST0-609 prevented carbachol activation CaM KI, ERK, Elk-1 , and cell gro\vth. Consistent with these observations, knockdown of CaM KKa and CaM Kly with shRNA-containing plas1nids blocked ERK activation by carbachol. In addition, Elk-I phosphorylation and luciferase activity in response to carbachol treat1nent was also dependent upon CaM kinases and was inhibited by U0126, ST0-609, and siRNA knockdown of CaM kinases and ERK2. Finally, blockade of either CaM KK (with ST0-609) or ERK (with U0126) activities resulted in the inhibition of carbachol- and estrogen-mediated cyclin Dl expression and MCF-7 cell growth. Taken together, our results suggest that carbachol treatment of MCF-7 cells activates CaM KI, ERK, the transcription factor Elk-1 , cyclin D 1, and cell grovvth through CaM KK.


Originally published Molecular and Cell Biochemistry, 335(1); 155-171 (2010)