TRPM2 is a calcium-permeable channel and a member of the transient receptor potential (TRP) superfamily, which share six putative transmembrane domains. This channel is part of a physiological pathway through which oxidative stress and tumor necrosis factorα (TNFα)induce susceptibility to cell death. TRPM2 is activated by micromolar levels of H2O2 and other agents which produce reactive oxygen species, resulting in an increase in the intracellular free oxygen concentration. Alternative splice variants of TRPM2 have been identified including a short isoform (TRPM2S), which consists of the TRPM2 N terminus and the first two predicted transmembrane domains. Inclusion of a stop codon (TAG) at the splice junction between exons 16 and 17 results in deletion of the four C-terminal transmembrane domains, the putative calcium pore, and the entire C-terminus. Heterologous expression of full length TRPM2 (TRPM2-L) in 293T cells resulted in susceptibility to H2O2-induced cell death, which correlated with elevation of the intracellular calcium concentration. In contrast, TRPM2-S expression inhibited calcium influx through TRPM2L and susceptibility to cell death induced by H2O2 in cells expressing TRPM2-L [1]. Experiments with confocal microscopy demonstrated that TRPM2-L and TRPM2-S colocalize on the plasma membrane. Immunoprecipitation experiments demonstrated that TRPM2-L and TRPM2-S directly associate, suggesting that this may be a mechanism through which TRPM2S modulates TRPM2-L activation [1]. Here, the physiological role of TRPM2-L and TRPM2-S in the proliferation and susceptibility to cell death of hematopoietic cells was examined. This approach was taken because TRPM2-S was cloned from human bone marrow, and TRPM2-L and TRPM2-S are widely expressed in primary hematopoietic cells and cell lines. Exposure of the human monocytic cell line U937 to H2O2 or TNFα results in significantly reduced cell viability. TRPM2-L or TRPM2-S were overexpressed by retroviral infection of U937 cells stabley expressing ecotrophic retroviral receptor. The retrovirus used also expresses GFP, and high efficiency of cell infection was documented by GFP fluorescence. TRPM2 expression was confirmed by RT-PCR and Western blotting. (1) The effect of increased expression of TRPM2L or TRPM2-S on cell proliferation was examined using a colorimetric method to identify metabolically active cells (CellTiter 96 Aqueous One Cell Proliferation Assay, Promega, Madison, WI). For untreated U937 cells, increased expression of TRPM2-L had no significant influence on cell proliferation compared to control cells. In contrast, increased expression of TRPM2-S in U937 cells resulted in significantly greater numbers of proliferating cells compared to cells infected with empty retrovirus or expressing TRPM2-L. (2) The effect of increased TRPM2-L or TRPM2-S expression on cell viability following treatment with H2O2 or TNFαwas examined by trypan blue exclusion. Treatment of U937 cells expressing increased levels of TRPM2-L with H2O2 or TNFα resulted in a significant decrease in cell viability, compared to cells infected with retrovirus alone. In contrast, increased TRPM2-S expression resulted in significantly enhanced cell viability after treatment with H2O2 or TNFα,compared to control cells infected with empty retrovirus or TRPM2-L. (3) The effect of increased TRPM2-L or TRPM2-S expression on induction of apoptosis by H2O2 or TNFα was examined by Alexa Fluor 488/annexin V staining. In U937 cells overexpressing TRPM2-L, treatment with H2O2 or TNFα resulted in an increase in cells undergoing apoptosis, compared to cells infected with retrovirus alone. Expression of TRPM2-S reduced the number of apoptotic cells observed following treatment with H2O2 or TNFα.These data suggest that TRPM2-L is an important modulator of hematopoietic cell death and that TRPM2-S can antagonize this pathway. To further explore the physiological role of TRPM2 in hematopoietic cell survival, endogenous TRPM2 was down regulated by infection of U937 cells with retrovirus (pSuppressorRetro, Imgenex, San Diego, CA) expressing RNAi oligonucleotides targeted to TRPM2. Infection with retrovirus and decreased expression of TRPM2 mRNA were confirmed by RTPCR. Reduced expression of TRPM2 in U937 cells was associated with significantly enhanced cell viability after treatment with H2O2 or TNFα,compared to control cell infected with empty retrovirus or retrovirus with RNAi targeted to luciferase. These data demonstrate that TRPM2 isoforms are important modulators of hematopoietic cell survival following oxidative stress and exposure to TNFα.They also suggest that the stoichiometry of TRPM2-L and TRPM2-S expression is an important determinant of hematopoietic cell fate.