Qin Tong

Regulation of the transient receptor potential channel TRPM2 by the Ca2+ sensor calmodulin.

TRPM2, a member of the transient receptor potential (TRP) superfamily, is a Ca2+-permeable channel activated by oxidative stress or tumor necrosis factorα involved in susceptibility to cell death. TRPM2 activation is dependent on the level of intracellular Ca2+. We explored whether calmodulin (CaM) is the Ca2+ sensor for TRPM2. HEK 293T cells were transfected with TRPM2 and wild type CaM or mutant CaM (CaMMUT) with substitutions of all four EF hands. Treatment of cells expressing TRPM2 with H2O2 or tumor necrosis factor α resulted in a significant increase in intracellular calcium ([Ca2+]i). This was not affected by coexpression of CaM, suggesting that endogenous CaM levels are sufficient for maximal response. Cotransfection of CaMMUT with TRPM2 dramatically inhibited the increase in [Ca2+]i, demonstrating the requirement for CaM in TRPM2 activation. Immunoprecipitation confirmed direct interaction of CaM and CaMMUT with TRPM2, and the Ca2+ dependence of this association. CaM bound strongly to the TRPM2 N terminus (amino acids 1–730), but weakly to the C terminus (amino acids 1060–1503). CaM binding to an IQ-like motif (amino acids 406–416) in the TRPM2 N terminus was demonstrated utilizing gel shift, immunoprecipitation, biotinylated CaM overlay, and pull-down assays. A substitution mutant of the IQ-like motif of TRPM2 (TRPM2-IQMUT1) reduced but did not eliminate CaM binding to TRPM2, suggesting the presence of at least one other CaM binding site. The functional importance of the TRPM2 IQ-like motif was demonstrated by treatment of TRPM2-IQMUT1-expressing cells with H2O2. The increase in [Ca2+]i observed with wild type TRPM2 was absent and cell viability was preserved. These data demonstrate the requirement for CaM in TRPM2 activation. They suggest that Ca2+ entering through TRPM2 enhances interaction of CaM with TRPM2 at the IQ-like motif in the N terminus, providing crucial positive feedback for channel activation.

TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells

Erythropoietin (Epo) stimulates a significant increase in the intracellular calcium concentration ([Ca2+]i) through activation of the murine transient receptor potential channel TRPC2, but TRPC2 is a pseudogene in humans. TRPC3 expression increases on normal human erythroid progenitors during differentiation. Here, we determined that erythropoietin regulates calcium influx through TRPC3. Epo stimulation of HEK 293T cells transfected with Epo receptor and TRPC3 resulted in a dose-dependent increase in [Ca2+]i, which required extracellular calcium influx. Treatment with the phospholipase C (PLC) inhibitor U-73122 or down-regulation of PLCγ1 by RNA interference inhibited the Epo-stimulated increase in [Ca2+]i in TRPC3-transfected HEK 293T cells and in primary human erythroid precursors, demonstrating a requirement for PLC. TRPC3 associated with PLCγ, and substitution of predicted PLCγ Src homology 2 binding sites (Y226F, Y555F, Y648F, and Y674F) on TRPC3 reduced the interaction of TRPC3 with PLCγ and inhibited the rise in [Ca2+]i. Substitution of Tyr226 alone with phenylalanine significantly reduced the Epo-stimulated increase in [Ca2+]i but not the association of PLCγ with TRPC3. PLC activation results in production of inositol 1,4,5-trisphosphate (IP3). To determine whether IP3 is involved in Epo activation of TRPC3, TRPC3 mutants were prepared with substitution or deletion of COOH-terminal IP3 receptor (IP3R) binding domains. In cells expressing TRPC3 with mutant IP3R binding sites and Epo receptor, interaction of IP3R with TRPC3 was abolished, and Epo-modulated increase in [Ca2+]i was reduced. Our data demonstrate that Epo modulates TRPC3 activation through a PLCγ-mediated process that requires interaction of PLCγ and IP3R with TRPC3. They also show that TRPC3 Tyr226 is critical in Epo-dependent activation of TRPC3. These data demonstrate a redundancy of TRPC channel activation mechanisms by widely different agonists.

Role of TRPM2 in cell proliferation and susceptibility to oxidative stress

The transient receptor potential (TRP) channel TRPM2 is an ion channel that modulates cell survival. We report here that full-length (TRPM2-L) and short (TRPM2-S) isoform expression was significantly increased in human neuroblastoma compared with adrenal gland. To differentiate the roles of TRPM2-L and TRPM2-S in cell proliferation and survival, we established neuroblastoma SH-SY5Y cell lines stably expressing either TRPM2 isoform or empty vector. Cells expressing TRPM2-S showed significantly enhanced proliferation, downregulation of phosphatase and tensin homolog (PTEN), and increased protein kinase B (Akt) phosphorylation and cell surface glucose transporter 1 (Glut1) compared with cells expressing TRPM2-L or empty vector. ERK phosphorylation was increased, and forkhead box O 3a (FOXO3a) levels were decreased. Inhibitor studies demonstrated that enhanced proliferation was dependent on phosphatidylinositol 3-kinase/Akt, ERK, and NADPH oxidase activation. On the other hand, TRPM2-S-expressing cells were significantly more susceptible to cell death induced by low H2O2 concentrations (50-100 μM), whereas TRPM2-L-expressing cells were protected. This was associated with a significant increase in FOXO3a, MnSOD (SOD2), and membrane Glut1 in TRPM2-L-expressing cells compared with TRPM2-S expressing cells. We conclude that TRPM2 channels occupy a key role in cell proliferation and survival following oxidative stress in neuroblastoma. Our results suggest that overexpression of TRPM2-S results in increased proliferation through phosphatidylinositol 3-kinase/Akt and ERK pathways, while overexpression of TRPM2-L confers protection against oxidative stress-induced cell death through FOXO3a and SOD. TRPM2 channels may represent a novel future therapeutic target in diseases involving oxidative stress.

TRPC3 activation by erythropoietin is modulated by TRPC6.

Regulation of intracellular calcium ([Ca2+]i) by erythropoietin (Epo) is an essential part of signaling pathways controlling proliferation and differentiation of erythroid progenitors, but regulatory mechanisms are largely unknown. TRPC3 and the homologous TRPC6 are two members of the transient receptor potential channel (TRPC) superfamily that are expressed on normal human erythroid precursors. Here we show that TRPC3 expression increases but TRPC6 decreases during erythroid differentiation. This is associated with a significantly greater increase in [Ca2+]i in response to Epo stimulation, suggesting that the ratio of TRPC3/TRPC6 is physiologically important. In HEK 293T cells heterologously expressing TRPC and erythropoietin receptor (Epo-R), Epo stimulated an increase in [Ca2+]i through TRPC3 but not TRPC6. Replacement of the C terminus of TRPC3 with the TRPC6 C terminus (TRPC3-C6C) resulted in loss of activation by Epo. In contrast, substitution of the C terminus of TRPC6 with that of TRPC3 (TRPC6-C3C) resulted in an increase in [Ca2+]i in response to Epo. Substitution of the N termini had no effect. Domains in the TRPC3 C terminus between amino acids 671 and 746 are critical for the response to Epo. Epo-R and phospholipase Cγ associated with TRPC3, and these interactions were significantly reduced with TRPC6 and TRPC3-C6C chimeras. TRPC3 and TRPC6 form heterotetramers. Coexpression of TRPC6 or C3/C6 chimeras with TRPC3 and Epo-R inhibited the Epo-stimulated increase in [Ca2+]i. In a heterologous expression system, Epo stimulation increased cell surface expression of TRPC3, which was inhibited by TRPC6. However, in primary erythroblasts, an increase in TRPC3 cell surface expression was not observed in erythroblasts in which Epo stimulated an increase in [Ca2+]i, demonstrating that increased membrane insertion of TRPC3 is not required. These data demonstrate that TRPC6 regulates TRPC3 activation by Epo. Endogenously, regulation of TRPC3 by TRPC6 may primarily be through modulation of signaling mechanisms, including reduced interaction of TRPC6 with phospholipase Cγ and Epo-R.

The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin

Modulation of intracellular calcium ([Ca2+]i) by erythropoietin (Epo) is an important signaling pathway controlling erythroid proliferation and differentiation. Transient receptor potential (TRP) channels TRPC3 and homologous TRPC6 are expressed on normal human erythroid precursors, but Epo stimulates an increase in [Ca2+]i through TRPC3 but not TRPC6. Here, the role of specific domains in the different responsiveness of TRPC3 and TRPC6 to erythropoietin was explored. TRPC3 and TRPC6 TRP domains differ in seven amino acids. Substitution of five amino acids (DDKPS) in the TRPC3 TRP domain with those of TRPC6 (EERVN) abolished the Epo-stimulated increase in [Ca2+]i. Substitution of EERVN in TRPC6 TRP domain with DDKPS in TRPC3 did not confer Epo responsiveness. However, substitution of TRPC6 TRP with DDKPS from TRPC3 TRP, as well as swapping the TRPC6 distal C terminus (C2) with that of TRPC3, resulted in a chimeric TRPC6 channel with Epo responsiveness similar to TRPC3. Substitution of TRPC6 with TRPC3 TRP and the putative TRPC3 C-terminal AMP-activated protein kinase (AMPK) binding site straddling TRPC3 C1/C2 also resulted in TRPC6 activation. In contrast, substitution of the TRPC3 C-terminal leucine zipper motif or TRPC3 phosphorylation sites Ser-681, Ser-708, or Ser-764 with TRPC6 sequence did not affect TRPC3 Epo responsiveness. TRPC3, but not TRPC6, and TRPC6 chimeras expressing TRPC3 C2 showed significantly increased plasma membrane insertion following Epo stimulation and substantial cytoskeletal association. The TRPC3 TRP domain, distal C terminus (C2), and AMPK binding site are critical elements that confer Epo responsiveness. In particular, the TRPC3 C2 and AMPK site are essential for association of TRPC3 with the cytoskeleton and increased channel translocation to the cell surface in response to Epo stimulation.

Trpc2 depletion protects red blood cells from oxidative stress-induced hemolysis.

Transient receptor potential (TRP) channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca(2+)](i)) through TRPC2 and TRPC3. Because modulation of [Ca(2+)](i) is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild-type littermate controls. Although the erythropoietin-induced increase in [Ca(2+)](i) was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenylhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with a significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. Although erythropoietin-induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis, which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion.

Abstract 1089: Two nonselective calcium channels, TRPC3 and TRPC6, play roles in erythropoietin signaling

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The signal transduction pathways of erythropoietin (Epo), a glycoprotein regulating production of red blood cells, have been a model for understanding growth factor stimulated cell proliferation and differentiation. Erythropoietin receptors (Epo-R) are also expressed on non-hematopoietic cells, and recently Epo was reported to have antiapoptotic effects on these cells including a potential role in specific cancers. Two calcium channels were identified which are involved in Epo signaling, TRPC3 and TRPC6. These are homologous, nonselective calcium channels and are members of the canonical transient receptor potential (TRPC) subfamily expressed on human erythroblasts. Both channels have also been implicated in stimulating proliferation of cancer cells; TRPC3 in ovarian cancer, TRPC6 in gliomas, and both channels in breast cancer. Here TRPC3 and TRPC6 function was studied in transfected human embryonic kidney (HEK)-293T cells, and in the human leukemia UT-7/Epo cell line. Measurement of [Ca2+]i was performed on single cells with Digital Video Imaging. TRPC3 is activated by erythropoietin, whereas TRPC6 is not. In fact, TRPC6 can inhibit Epo-induced activation of TRPC3. Domains regulating channel activation were studied here. The TRP domain of TRPC3 is required for its activation, and substitution of five amino acids in the TRPC3 TRP domain with those of TRPC6 significantly reduced the Epo-stimulated increase in [Ca2+]i in TRPC3-expressing cells. Substitution of the same amino acids in the TRPC6 TRP with those of TRPC3 did not enhance the response of TRPC6-expressing cells to Epo stimulation. However, substitution of the distal TRPC6 C-terminus (C2) with that of TRPC3 in addition to substitution of TRPC6 TRP with TRPC3 TRP resulted in a TRPC6 channel with Epo responsiveness similar to TRPC3. In contrast, substitution of the TRPC3 C-terminal leucine zipper motif or TRPC3 phosphorylation sites S681, S708, or S764 with corresponding TRPC6 sequence did not affect TRPC3 Epo-responsiveness. TRPC3, but not TRPC6, and chimeras expressing the distal TRPC3 C terminus showed significantly increased membrane insertion following Epo stimulation. This was associated with substantial cytosketetal localization of TRPC3 and chimeras expressing TRPC3C2 but not TRPC6. These data demonstrate the important role of the TRP domain in specificity of channel responsiveness to agonist stimulation, and the requirement for other sites for optimal response to agonists, such as those found in the distal C-terminus of TRPC3. A few mutations in critical channel sites could have an important effect on proliferation and survival of cells expressing Epo-R. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1089. doi:10.1158/1538-7445.AM2011-1089

Abstract 2079: The role of TRPM2 splice variants in enhanced cell proliferation and sensitivity to oxidative-stress mediated apoptosis in neuroblastoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Neuroblastoma is the most frequent and deadly solid tumors of childhood. The cure rate for advanced disease is only 30%. Understanding the mechanisms of cell proliferation and apoptosis in neuroblastoma is desperately needed in order to develop new anti-cancer drugs. The transient receptor potential channel TRPM2 is an ion channel which regulates critical cellular processes and survival in response to oxidative stress in many cell types. Isoforms include TRPM2-L (full length or wild type) and TRPM2-S (short, containing only the first two transmembrane domains and no pore) and they are expressed in primary neuroblastoma. In this study, we investigated the expression and function of TRPM2-L and -S isoforms in cell proliferation and sensitivity to apoptosis induced by oxidative-stress in neuroblastoma. Three neuroblastoma cell lines which stably express TRPM2-L, TRPM2-S or vector were established by G418 selection. Cell proliferation and viability were measured by XTT assay. Mechanisms of enhanced proliferation and apoptosis were studied by western blotting, subcellular fractionation and treatment with inhibitors. [Ca2+]i was evaluated by digital video image analysis of single cells. Our data demonstrated that TRPM2-S-expressing cells have significantly enhanced proliferation compared to TRPM2-L- or vector-expressing cells. TRPM2-S expressing cells showed increased Akt phosphorylation which correlated with increased proliferation. LY294002, a PI3K inhibitor, abolished the enhanced cell proliferation in TRPM2-S-expressing cells, demonstrating that enhanced proliferation involved PI3K/Akt activation. Consisted with activation of Akt, glucose transporter 1 (GLUT1) expression was elevated on the membrane of TRPM2-S expressing cells and correlated with increased cell growth. The viability and apoptosis of all three cell lines was reduced in response to H2O2 or doxorubicin in a time- and dose- dependent manner. However, TRPM2-S-expressing cells were the most sensitive and demonstrated the greatest cleavage and activation of apoptotic molecules. Akt is a major mediator in neuroblastoma proliferation and apoptosis. After treatment with physiological concentrations H2O2 (100 µM) or doxorubicin (0.1 μM), pAkt decreased in TRPM2-S-expressing cells, but increased in TRPM2-L-expressing cells. These results demonstrate that differential expression of TRPM2 isoforms influences a cells proliferative potential and responsiveness to oxidative stress and chemotherapy. The increased growth of TRPM2-S expressing cells and sensitivity to chemotherapy involves the PI3K/Akt signaling pathway. TRPM2 isoforms may be future therapeutic targets for selective cell killing in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2079. doi:10.1158/1538-7445.AM2011-2079