TRPM7-mediated spontaneous Ca²⁺ entry regulates the proliferation and differentiation of human leukemia cell line...

Continuous Ca²⁺ influx is essential to maintain intracellular Ca²⁺ homeostasis and its dysregulation leads to a variety of cellular dysfunctions. In this study, we explored the functional roles of spontaneous Ca²⁺ influx for the proliferation and differentiation of a human erythromyeloid leukemia cell line K562. mRNA/protein expressions were assessed by the real-time RT-PCR, western blotting, and immunocytochemical staining. Intracellular Ca²⁺ concentration ([Ca²⁺ ]_i ) and ionic currents were measured by fluorescent imaging and patch clamping techniques, respectively. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and hemoglobin synthesis, respectively. Elimination of extracellular Ca²⁺ decreased basal [Ca²⁺ ]_i in proliferating K562 cells. Cation channel blockers such as SK&F96365, 2-APB, Gd³⁺ , and FTY720 dose dependently decreased basal [Ca²⁺ ]_i . A spontaneously active inward current (I_spont ) contributive to basal [Ca²⁺ ]_i was identified by the nystatin-perforated whole-cell recording. I_spont permeated Ca²⁺ comparably to Na⁺ , and was greatly eliminated by siRNA targeting TRPM7, a melastatin member of the transient receptor potential (TRP) superfamily. Consistent with these findings, TRPM7 immune reactivity was detected by western blotting, and immunofluorescence representing TRPM7 was found localized to the K562 cell membrane. Strikingly, all these procedures, that is, Ca²⁺ removal, TRPM7 blockers and siRNA-mediated TRPM7 knockdown significantly retarded the growth and suppressed hemin-induced γ-globin and hemoglobin syntheses in K562 cells, respectively, both of which appeared associated with the inhibition of ERK activation. These results collectively suggest that spontaneous Ca²⁺ influx through constitutively active TRPM7 channels may critically regulate both proliferative and erythroid differentiation potentials of K562 cells.