Shu-jen Chen

The second member of transient receptor potential-melastatin channel family protects hearts from ischemia-reperfusion injury.

The second member of the transient receptor potential-melastatin channel family (TRPM2) is expressed in the heart and vasculature. TRPM2 channels were expressed in the sarcolemma and transverse tubules of adult left ventricular (LV) myocytes. Cardiac TRPM2 channels were functional since activation with H2O2 resulted in Ca(2+) influx that was dependent on extracellular Ca(2+), was significantly higher in wild-type (WT) myocytes compared with TRPM2 knockout (KO) myocytes, and inhibited by clotrimazole in WT myocytes. At rest, there were no differences in LV mass, heart rate, fractional shortening, and +dP/dt between WT and KO hearts. At 2-3 days after ischemia-reperfusion (I/R), despite similar areas at risk and infarct sizes, KO hearts had lower fractional shortening and +dP/dt compared with WT hearts. Compared with WT I/R myocytes, expression of the Na(+)/Ca(2+) exchanger (NCX1) and NCX1 current were increased, expression of the α1-subunit of Na(+)-K(+)-ATPase and Na(+) pump current were decreased, and action potential duration was prolonged in KO I/R myocytes. Post-I/R, intracellular Ca(2+) concentration transients and contraction amplitudes were equally depressed in WT and KO myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation, levels of ROS were significantly higher in KO compared with WT LV myocytes. Compared with WT I/R hearts, oxygen radical scavenging enzymes (SODs) and their upstream regulators (forkhead box transcription factors and hypoxia-inducible factor) were lower, whereas NADPH oxidase was higher, in KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by decreasing generation and enhancing scavenging of ROS, thereby reducing I/R-induced oxidative stress.

A Splice Variant of the Human Ion Channel TRPM2 Modulates Neuroblastoma Tumor Growth through Hypoxia-inducible Factor (HIF)-1/2α

Background: TRPM2 channels play an essential role in cell death following oxidative stress. Results: Dominant negative TRPM2-S decreases growth of neuroblastoma xenografts and increases doxorubicin sensitivity through modulation of HIF-1/2α expression, mitophagy, and mitochondrial function. Conclusion: TRPM2 is important for neuroblastoma growth and viability through modulation of HIF-1/2α. Significance: Modulation of TRPM2 may be a novel approach in cancer therapeutics. The calcium-permeable ion channel TRPM2 is highly expressed in a number of cancers. In neuroblastoma, full-length TRPM2 (TRPM2-L) protected cells from moderate oxidative stress through increased levels of forkhead box transcription factor 3a (FOXO3a) and superoxide dismutase 2. Cells expressing the dominant negative short isoform (TRPM2-S) had reduced FOXO3a and superoxide dismutase 2 levels, reduced calcium influx in response to oxidative stress, and enhanced reactive oxygen species, leading to decreased cell viability. Here, in xenografts generated with SH-SY5Y neuroblastoma cells stably expressing TRPM2 isoforms, growth of tumors expressing TRPM2-S was significantly reduced compared with tumors expressing TRPM2-L. Expression of hypoxia-inducible factor (HIF)-1/2α was significantly reduced in TRPM2-S-expressing tumor cells as was expression of target proteins regulated by HIF-1/2α including those involved in glycolysis (lactate dehydrogenase A and enolase 2), oxidant stress (FOXO3a), angiogenesis (VEGF), mitophagy and mitochondrial function (BNIP3 and NDUFA4L2), and mitochondrial electron transport chain activity (cytochrome oxidase 4.1/4.2 in complex IV). The reduction in HIF-1/2α was mediated through both significantly reduced HIF-1/2α mRNA levels and increased levels of von Hippel-Lindau E3 ligase in TRPM2-S-expressing cells. Inhibition of TRPM2-L by pretreatment with clotrimazole or expression of TRPM2-S significantly increased sensitivity of cells to doxorubicin. Reduced survival of TRPM2-S-expressing cells after doxorubicin treatment was rescued by gain of HIF-1 or -2α function. These data suggest that TRPM2 activity is important for tumor growth and for cell viability and survival following doxorubicin treatment and that interference with TRPM2-L function may be a novel approach to reduce tumor growth through modulation of HIF-1/2α, mitochondrial function, and mitophagy.

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.

The human ion channel TRPM2 modulates neuroblastoma cell survival and mitochondrial function through Pyk2, CREB, and MCU activation

Transient receptor potential melastatin channel subfamily member 2 (TRPM2) has an essential function in cell survival and is highly expressed in many cancers. Inhibition of TRPM2 in neuroblastoma by depletion with CRISPR technology or expression of dominant negative TRPM2-S has been shown to significantly reduce cell viability. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in TRPM2 modulation of neuroblastoma viability was explored. In TRPM2-depleted cells, phosphorylation and expression of Pyk2 and cAMP-responsive element-binding protein (CREB), a downstream target, were significantly reduced after application of the chemotherapeutic agent doxorubicin. Overexpression of wild-type Pyk2 rescued cell viability. Reduction of Pyk2 expression with shRNA decreased cell viability and CREB phosphorylation and expression, demonstrating Pyk2 modulates CREB activation. TRPM2 depletion impaired phosphorylation of Src, an activator of Pyk2, and this may be a mechanism to reduce Pyk2 phosphorylation. TRPM2 inhibition was previously demonstrated to decrease mitochondrial function. Here, CREB, Pyk2, and phosphorylated Src were reduced in mitochondria of TRPM2-depleted cells, consistent with their role in modulating expression and activation of mitochondrial proteins. Phosphorylated Src and phosphorylated and total CREB were reduced in TRPM2-depleted nuclei. Expression and function of mitochondrial calcium uniporter (MCU), a target of phosphorylated Pyk2 and CREB, were significantly reduced. Wild-type TRPM2 but not Ca2+-impermeable mutant E960D reconstituted phosphorylation and expression of Pyk2 and CREB in TRPM2-depleted cells exposed to doxorubicin. Results demonstrate that TRPM2 expression protects the viability of neuroblastoma through Src, Pyk2, CREB, and MCU activation, which play key roles in maintaining mitochondrial function and cellular bioenergetics.

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