The ability to sense temperature is essential for organism survival and efficient metabolism. Body temperatures profoundly affect many physiological functions, including immunity. Transient receptor potential melastatin 2 (TRPM2) is a thermosensitive, Ca 2+ -permeable cation channel expressed in a wide range of immunocytes. TRPM2 is activated by adenosine diphosphate ribose and hydrogen peroxide (H 2 O 2 ), although the activation mechanism by H 2 O 2 is not well understood. Here we report a unique activation mechanism in which H 2 O 2 lowers the temperature threshold for TRPM2 activation, termed “sensitization,” through Met oxidation and adenosine diphosphate ribose production. This sensitization is completely abolished by a single mutation at Met-214, indicating that the temperature threshold of TRPM2 activation is regulated by redox signals that enable channel activity at physiological body temperatures. Loss of TRPM2 attenuates zymosan-evoked macrophage functions, including cytokine release and fever-enhanced phagocytic activity. These findings suggest that redox signals sensitize TRPM2 downstream of NADPH oxidase activity and make TRPM2 active at physiological body temperature, leading to increased cytosolic Ca 2+ concentrations. Our results suggest that TRPM2 sensitization plays important roles in macrophage functions.

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Membrane-type 1 matrix metalloproteinase (MT1-MMP), a powerful modulator of the pericellular environment, promotes migration, invasion, and proliferation of cells. To perform its potent proteolytic activity in a controlled manner, MT1-MMP has to be regulated precisely. However, our knowledge about substrates and regulatory proteins is still very limited. In this study we identify a catalog of proteins that directly or indirectly interact with MT1-MMP. We expressed a FLAG-tagged MT1-MMP stably in human malignant melanoma A375 cells. We prepared cell lysate using Brij98 and MT1-MMP was affinity purified together with associating proteins using an anti-FLAG antibody. A distinct set of membrane proteins was found to copurify with MT1-MMP when biotin-labeled proteins were monitored. The proteins were analyzed with an integrated system composed of nano-flow liquid chromatography and tandem mass spectrometry. We identified 158 proteins including several previously reported to bind MT1-MMP, although most had not previously been identified. (1-5) MT1-MMP deficiency in mice causes early postnatal death associated with severe phenotypes such as dwarfism, osteopenia, arthritis, connective tissue disorder, and disturbed angiogenesis.(6,7) The physiological roles of MT1-MMP are mediated by its proteolytic activity in the pericellular space. Possible substrates include extracellular matrix (ECM) proteins (type I collagen, fibronectin, vitronectin, laminin-1, -5, etc.), cell adhesion molecules (CD44, syndecan-1, αv integrin), cytokines (SDF-1, TGF-β, etc.), and latent forms of proMMPs (proMMP-2, proMMP-13).(1,2,8,9) However, our knowledge of the physiological substrates of MT1-MMP is still limited and identification of these substrates should enable a better understanding of the biological functions of MT1-MMP.Proper physiological function of MT1-MMP requires that its cell surface protease activity be active at the appropriate site and time. Uncontrolled protease activity is rather hazardous to cell physiology even in the presence of natural inhibitors such as TIMPs and reversion-inducing cysteine-rich protein with kazal motif (RECK).(5,10) During the migration and invasion of cells, MT1-MMP localizes to the leading edges and invadopodia of migrating and invading cells. (11,12) Although the exact mechanisms that determine the localization of MT1-MMP are not well understood, the binding of MT1-MMP to cellular proteins linked to the actin cytoskeleton, such as CD44 or integrin, is thought to be a factor determining its localization. (11) Thus, identification of the membrane proteins that interact with MT1-MMP on the cell surface is important to understanding the mechanism of tumor cell invasion promoted by MT1-MMP.CD44 is also a substrate of MT1-MMP and cleaves at different sites from those for ectodomain shedding by the A disintegrin and metalloproteinase (ADAM) family of proteases.(13) Since the binding of proteins to MT1-MMP brings them into close proximity to the catalytic site of the enzyme, such proteins are obvious candida...

Erythropoietin has been thought to be secreted to plasma soon after the production because of the difficulty of Western blot analysis and immunohistochemistry. We established the new methods of Western blot analysis and immunohistochemistry. Using the new methods, we investigated the effects of aldosterone and fludrocortisone, an analogue of aldosterone on erythropoietin mRNA and protein production by the kidneys. Aldosterone stimulated Epo and HIF2α mRNA expressions in tubule suspensions and microdissected medullary thick ascending limbs and outer medullary collecting ducts. Western blot analysis showed a recombinant erythropoietin at 34-45 kDa and kidney erythropoietin at 36-40 and 42 kDa, both of which shifted to 22 kDa by deglycosylation. Erythropoietin protein expression was observed in the nephrons but not in the interstitial cells in control condition. Fludrocortisone stimulated erythropoietin mRNA and protein expressions in the distal nephrons, particularly in the intercalated cells of the collecting ducts. These data show that erythropoietin is produced by the nephrons by the regulation of renin-angiotensin-aldosterone system and not by the renal interstitial cells in control condition.