Redox signal-mediated sensitization of transient receptor potential melastatin 2 (TRPM2) to temperature affects macrophage functions Makiko Kashioa, Takaaki Sokabea, Kenji Shintakua,b, Takayuki Uematsuc, Naomi Fukutaa, Noritada Kobayashic, Yasuo Morid, and Makoto Tominagaa,b,1 aDivision of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), National Institutes of Natural Sciences, Okazaki 444-8787, Japan; bDepartment of Physiological Sciences, Graduate University for Advanced Studies, Okazaki 444-8585, Japan; cBiomedical Laboratory, Division of Biomedical Research, Kitasato Institute Medical Center Hospital, Kitasato University, Saitama 108-8641, Japan; and dDepartment of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8530, Japan Edited* by David Julius, University of California, San Francisco, CA, and approved March 15, 2012 (received for review August 30, 2011) The ability to sense temperature is essential for organism survival H2O2, a reactive oxygen species (ROS) produced by NADPH and efficient metabolism. Body temperatures profoundly affect oxidase (Nox), is crucial for microorganism removal, given that many physiological functions, including immunity. Transient re- defects in H2O2 production lead to persistent infections (21). As ceptor potential melastatin 2 (TRPM2) is a thermosensitive, Ca2+- the first line of defense against infections, the Toll-like receptors permeable cation channel expressed in a wide range of immuno- (TLRs) of phagocytes, including macrophages, recognize com- cytes. TRPM2 is activated by adenosine diphosphate ribose and hy- mon microbial components, such as pathogen-associated mo- drogen peroxide (H2O2), although the activation mechanism by lecular patterns. Then infective organisms are phagocytosed and cleared by systems in which Nox activity is engaged. Along with H2O2 is not well understood. Here we report a unique activation H O ’s important role in microbicidal function inside the phag- mechanism in which H2O2 lowers the temperature threshold for 2 2 TRPM2 activation, termed “sensitization,” through Met oxidation osomes, membrane-diffusible H2O2 also could play roles in cell and adenosine diphosphate ribose production. This sensitization is signaling outside the phagosomes by acting on various proteins PHYSIOLOGY completely abolished by a single mutation at Met-214, indicating (22). ROS such as H2O2 are now considered to be signaling molecules, in parallel with reactive nitrogen species. These cel- that the temperature threshold of TRPM2 activation is regulated by “ ” redox signals that enable channel activity at physiological body lular redox signals play important roles in a wide range of physiological functions, including ion channel activity (23). We temperatures. Loss of TRPM2 attenuates zymosan-evoked macro- hypothesized that redox signals generated by microbicidal ac- phage functions, including cytokine release and fever-enhanced tivity in macrophages could regulate the function of TRPM2, phagocytic activity. These findings suggest that redox signals sen- which is expressed in macrophages (8). To test the hypothesis, we sitize TRPM2 downstream of NADPH oxidase activity and make investigated the regulation mechanisms of TRPM2. TRPM2 active at physiological body temperature, leading to in- Here we describe a unique mechanism for TRPM2 activation in creased cytosolic Ca2+ concentrations. Our results suggest that which its temperature threshold is regulated dynamically by H2O2, TRPM2 sensitization plays important roles in macrophage functions. termed “sensitization.” Sensitization of TRPM2 is caused by a re- duction in its temperature threshold through oxidation of a sin- calcium | immune cells gle methionine at Met-214, and is partially attenuated by a poly (ADP ribose) polymerase (PARP) inhibitor. The loss of TRPM2 he capacity to sense temperature is essential for organism attenuates macrophage functions such as cytokine release at 37 °C Tsurvival and efficient metabolism, and body temperature has and enhancement of phagocytic activity at febrile temperatures. profound effects on many physiological functions, including im- We suggest that TRPM2 is sensitized by redox signals downstream munity. Paradoxically, lowering body temperature with cyclo- of Nox activity, and contributes to macrophage functions. oxygenase inhibitors worsens survival rates for bacterial infection (1), whereas fever elevates immune reactivity (2). Together, these Results fi H2O2 Sensitizes TRPM2 to Heat. We first examined the effects of effects suggest that elevated body temperature has bene cial 2+ H2O2 on heat-evoked TRPM2 activities using a Ca -imaging effects for the immune system, although the molecular mecha- ∼ nisms underlying these effects remain largely unknown. method. Heat stimulation of up to 41 °C was applied before and after H2O2 treatment of mouse TRPM2-expressing HEK293 cells. Transient receptor potential melastatin 2 (TRPM2) is a ther- 2+ mosensitive, Ca2+-permeable cation channel expressed by a wide Heat-evoked [Ca ]i increases were dramatically enhanced by H O treatment in a dose-dependent manner, whereas heat range of immunocytes, including macrophages, whose function is 2 2 stimulation without H O treatment caused only slight activation gradually being clarified (3–9). We previously reported that heat 2 2 (Fig. 1 A and B). In addition to the concentration dependence, the stimulation activates TRPM2 in the presence of low concen- duration of H O treatment also affected the responses; increasing trations of agonists, such as adenosine diphosphate ribose 2 2 H2O2 (30 μM) treatment from 1 min to 5 min proportionally en- (ADPR) and related molecules (10). These agonists are believed hanced heat (∼41 °C)-evoked responses (Fig. 1 C–E). We observed to act on a unique C-terminal pyrophosphatase domain in TRPM2 (Nudix-like domain) (11–13). Temperature-dependent activation fi of TRPM2 plays signi cant roles in cellular functions, including Author contributions: M.K., T.S., and M.T. designed research; M.K., K.S., and N.F. per- insulin release from pancreatic β cells (10, 14). TRPM2 channels formed research; Y.M. contributed new reagents/analytic tools; M.K., K.S., T.U., and N.K. analyzed data; and M.K., T.S., and M.T. wrote the paper. can be activated by hydrogen peroxide (H2O2) and are reported to be involved in cell death caused by oxidative stress via mechanisms The authors declare no conflict of interest. that remain to be clarified (15, 16). ADPR released from in- *This Direct Submission article had a prearranged editor. tracellular organelles, such as the nucleus and mitochondria, may Freely available online through the PNAS open access option. play a primary role in TRPM2 activation by H2O2 (17–19), al- 1To whom correspondence should be sent. E-mail: [email protected]. though one report suggests involvement of an ADPR-independent This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. activation mechanism (20). 1073/pnas.1114193109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1114193109 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 2+ temperatures induced potent [Ca ]i increases even without H2O2 treatment (Fig. 2A, upper trace). When temperature thresholds 2+ were determined from temperatures causing [Ca ]i increases in excess of those observed for DsRed-negative cells, the average threshold was 47.2 ± 0.2 °C (n = 5) (Fig. 2B). Treatment with H2O2 for 1 min significantly lowered this threshold [100 μM: 41.7 ± 0.1 °C (n = 5); 3 mM: 36.3 ± 0.4 °C (n = 8); P < 0.001 vs. H2O2 untreated] in a dose-dependent manner (Fig. 2 A and B). Similar to the time dependence of heat-evoked responses (Fig. 1 C–E), temperature threshold reductions also depended on the duration of H2O2 treatment (Fig. 2B). To more precisely determine the temperature thresholds, we used the heat-evoked currents observed in whole- cell patch-clamp recordings to generate Arrhenius plots, which displayed an explicit flex point during heating (Fig. 2C). The reductions in temperature thresholds were recapitulated in whole- cell patch-clamp recordings in which cells were exposed to H2O2 in the pipette solution [100 μM: 40.2 ± 1.3 °C (n = 11); 3 mM: 36.3 ± 0.6 °C (n = 10); P < 0.01] (Fig. 2 C and D). Of note, the sensitization of heat-evoked currents was more easily reproduced by lower concentrations when H2O2 was applied in the pipette solution rather than extracellularly (Fig. 2C and Fig. S1). In the whole-cell recordings, higher concentrations of H2O2 are needed when H2O2 is applied extracellularly, because H2O2 entering the cell can be diluted by the pipette solution. This suggests an intracellular site for H2O2 action. H2O2-mediated reduction in the temperature threshold for TRPM2 activation could explain the increased TRPM2 activity under physiological temperatures, as shown in Fig. S2A. Therefore, the effect of H2O2 on TRPM2 can be viewed as a “sensitization” to physiological body temperature. Molecular Mechanism of TRPM2 Sensitization to Heat. Most previous studies have suggested that TRPM2 activation by H2O2 is caused by ADPR release from intracellular organelles (17–19). To test this possibility, we evaluated the effects of H2O2 in inside-out Fig. 1. Heat-evoked responses of TRPM2 were elevated by H2O2 in a concen- single-channel recordings in which intracellular components are tration- and time-dependent manner. (A)H2O2 (100 μM) enhanced heat-evoked absent.