Sustained activation of Erk1/2 MAPK and cell growth suppression by the insert-negative, but not the insert-positive isoform of the human calcitonin receptor

Recently we reported that calcitonin (CT) induces growth arrest at the G2 stage of the cell cycle in HEK-293 cell lines expressing the most abundant, insert-negative, isoform of the human CT receptor (insert (-)ve hCTR). The present study investigates the involvement of the MAPK signalling pathway in the anti-proliferative actions of CT and compares the activity of an isoform of the hCTR that contains a 16 amino acid insert in the first putative intracellular loop (insert +ve hCTR). Comparison of HEK-293 cells stably transfected with the insert (-)ve or the insert +ve hCTR, showed that accumulation of cAMP and intracellular free calcium in response to CT were specific for the insert (-)ve receptor isoform. However, a novel acidification of the extracellular medium was mediated by both isoforms. Treatment with CT of cells expressing the insert (-)ve hCTR, caused a decrease in cell growth associated with an induction of p21(WAF1/CIP1) Analysis by fluorescence-activated cell scanning showed that growth inhibition was associated with an accumulation of cells in G2. CT treatment of cells expressing the insert (-)ve, but not insert +ve hCTR, induced the phosphorylation of Erk1/2 MAPK, which persisted for at least 72 h. Treatment of cells expressing the insert (-)ve hCTR with the MAPK kinase (MEK) inhibitor, PD-98059, inhibited the phosphorylation of Erk1/2 and abrogated the growth inhibitory effects of salmon CT, the accumulation of cells in G2, and the associated induction of p21(WAF1/CIP1). These data suggest that activation of Erk1/2 are downstream effecters of the insert (-)ve hCTR in modulating cell cycle progression.