Enhanced expression of extracellular calcium sensing receptor in monocyte-differentiated versus undifferentiated HL-60 cells: Potential role in regulation of a nonselective cation channel

Human promyelocytic leukemia cells (HL-60) have been used widely as a model for studying the differentiation of hematopoietic progenitor cells in vitr o. After treatment with phorbol-12-myristate-13-acetate (PMA) or 1,25-dihyd roxyvitamin D-3 [1,25(OH)(2)D-3], HL-60 cells differentiate into cells with the phenotype of monocytes/ macrophages. We previously showed that periphe ral blood monocytes and the murine J774 monocytic cell line express the CaR , and myeloid progenitors in the bone marrow and myeloid cells in periphera l blood other than monocytes express lower levels of the CaR. Therefore, we investigated whether undifferentiated HL-60 cells express a functional G p rotein-coupled, extracellular calcium (Ca2+,)-sensing receptor (CaR) and if the expression of the CaR increases as these cells differentiate along the monocytic lineage. The use of reverse transcription-polymerase chain react ion (RT-PCR) with CaR-specific primers, followed by sequencing of the ampli fied products, identified an authentic CaR transcript in undifferentiated H L-60 cells. Both immunocytochemistry and Western blot analysis using a CaR- specific antiserum detected low levels of CaR protein expression in undiffe rentiated HL-60 cells. The levels of CaR protein increased considerably fol lowing treatment of the cells with PMA (50 nM) or 1,25(OH)(2)D-3 (100 nM) f or 5 days. Northern analysis using a CaR-specific riboprobe identified CaR transcripts in undifferentiated HL-60 cells, but CaR mRNA levels did not ch ange appreciably after treatment with either agent, suggesting that upregul ation of CaR protein occurs at a translational level. PMA-treated HL-60 cel ls expressed a nonselective cation channel (NCC), and the calcimimetic CaR activator, NPS R-467, but not its less active stereoisomer, NPS S-467, as w ell as the polycationic CaR agonist, neomycin, activated this NCC, demonstr ating that the CaR expressed in these cells is functionally active. Therefo re, HL-60 cells exhibit an increase in CaR protein expression, occurring at a translational level during their differentiation into cells with a monoc yte/macrophage phenotype in response to treatment with PMA or 1,25(OH)(2)D- 3, which is functionally linked to activation of a nonselective cation chan nel.