Reactive oxygen species (ROS) induce chemokines responsible for the recruitment of inflammatory cells to sites of injury or infection. Here we show that the plasma membrane Ca 2+ -permeable channel TRPM2 controls ROS-induced chemokine production in monocytes. In human U937 monocytes, hydrogen peroxide (H 2 O 2 ) evokes Ca 2+ influx through TRPM2 to activate Ca 2+ -dependent tyrosine kinase Pyk2 and amplify Erk signaling via Ras GTPase. This elicits nuclear translocation of nuclear factor-κB essential for the production of the chemokine interleukin-8 (CXCL8). In monocytes from Trpm2-deficient mice, H 2 O 2 -induced Ca 2+ influx and production of the macrophage inflammatory protein-2 (CXCL2), the mouse CXCL8 functional homolog, were impaired. In the dextran sulfate sodium-induced colitis inflammation model, CXCL2 expression, neutrophil infiltration and ulceration were attenuated by Trpm2 disruption. Thus, TRPM2 Ca 2+ influx controls the ROS-induced signaling cascade responsible for chemokine production, which aggravates inflammation. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases.

We have previously shown [Goto, Y., Calciano, L. J., & Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577] that beta-lactamase, cytochrome c, and apomyoglobin are maximally unfolded at pH 2 under conditions of low ionic strength, but a further decrease in pH, by increasing the concentration of HCl, refolds the proteins to the A state with properties similar to those of a molten globule state. To understand the mechanism of acid-induced refolding of protein structure, we studied the effects of various strong acids and their neutral salts on the acid-unfolded states of ferricytochrome c and apomyoglobin. The conformational transition of cytochrome c was monitored at 20 degrees C by using changes in the far-UV CD and in the Soret absorption at 394 nm, and that of apomyoglobin was monitored by changes in the far-UV CD. Various strong acids (i.e., sulfuric acid, perchloric acid, nitric acid, trichloroacetic acid, and trifluoroacetic acid) refolded the acid-unfolded cytochrome c and apomyoglobin to the A states as was the case with HCl. For both proteins neutral salts of these acids caused similar conformational transitions, confirming that the anions are responsible for bringing about the transition. The order of effectiveness of anions was shown to be ferricyanide greater than ferrocyanide greater than sulfate greater than thiocyanate greater than perchlorate greater than iodide greater than nitrate greater than trifluoroacetate greater than bromide greater than chloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient receptor potential (TRP) proteins form plasma-membrane cation channels that act as sensors for diverse cellular stimuli. Here, we report a novel activation mechanism mediated by cysteine S-nitrosylation in TRP channels. Recombinant TRPC1, TRPC4, TRPC5, TRPV1, TRPV3 and TRPV4 of the TRPC and TRPV families, which are commonly classified as receptor-activated channels and thermosensor channels, induce entry of Ca(2+) into cells in response to nitric oxide (NO). Labeling and functional assays using cysteine mutants, together with membrane sidedness in activating reactive disulfides, show that cytoplasmically accessible Cys553 and nearby Cys558 are nitrosylation sites mediating NO sensitivity in TRPC5. The responsive TRP proteins have conserved cysteines on the same N-terminal side of the pore region. Notably, nitrosylation of native TRPC5 upon G protein-coupled ATP receptor stimulation elicits entry of Ca(2+) into endothelial cells. These findings reveal the structural motif for the NO-sensitive activation gate in TRP channels and indicate that NO sensors are a new functional category of cellular receptors extending over different TRP families.

Protein tyrosine phosphatases (PTPases), such as SHP-1 and SHP-2, that contain Src homology 2 (SH2) domains play important roles in growth factor and cytokine signal transduction pathways. A protein of ϳ115 to 120 kDa that interacts with SHP-1 and SHP-2 was purified from v-src-transformed rat fibroblasts (SR-3Y1 cells), and the corresponding cDNA was cloned. The predicted amino acid sequence of the encoded protein, termed SHPS-1 (SHP substrate 1), suggests that it is a glycosylated receptor-like protein with three immunoglobulin-like domains in its extracellular region and four YXX(L/V/I) motifs, potential tyrosine phosphorylation and SH2-domain binding sites, in its cytoplasmic region. Various mitogens, including serum, insulin, and lysophosphatidic acid, or cell adhesion induced tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2 in cultured cells. Thus, SHPS-1 may be a direct substrate for both tyrosine kinases, such as the insulin receptor kinase or Src, and a specific docking protein for SH2-domain-containing PTPases. In addition, we suggest that SHPS-1 may be a potential substrate for SHP-2 and may function in both growth factor-and cell adhesion-induced cell signaling.

Oxygen (O(2)) is a prerequisite for cellular respiration in aerobic organisms but also elicits toxicity. To understand how animals cope with the ambivalent physiological nature of O(2), it is critical to elucidate the molecular mechanisms responsible for O(2) sensing. Here our systematic evaluation of transient receptor potential (TRP) cation channels using reactive disulfides with different redox potentials reveals the capability of TRPA1 to sense O(2). O(2) sensing is based upon disparate processes: whereas prolyl hydroxylases (PHDs) exert O(2)-dependent inhibition on TRPA1 activity in normoxia, direct O(2) action overrides the inhibition via the prominent sensitivity of TRPA1 to cysteine-mediated oxidation in hyperoxia. Unexpectedly, TRPA1 is activated through relief from the same PHD-mediated inhibition in hypoxia. In mice, disruption of the Trpa1 gene abolishes hyperoxia- and hypoxia-induced cationic currents in vagal and sensory neurons and thereby impedes enhancement of in vivo vagal discharges induced by hyperoxia and hypoxia. The results suggest a new O(2)-sensing mechanism mediated by TRPA1.

TRPA1 is a member of the transient receptor potential (TRP) cation channel family, and is predominantly expressed in nocicep-In TRPA1 responses to other cysteine-reactive inflammatory mediators, such as NO and H 2 O 2 , the extent of impairment by respective cysteine mutations differed from those in TRPA1 responses to 15d-PGJ 2 . Interestingly, the Cys421 mutation critically impaired the TRPA1 response to H + as well. Our findings suggest that TRPA1 channels are targeted by an array of inflammatory mediators to elicit inflammatory pain in the nervous system.

The following resources related to this article are available online at http://stke.sciencemag.org. Reactive oxygen species (ROS) are involved in many physiological and pathophysiological cellular processes. We used lymphocytes, which are exposed to highly oxidizing environments during inflammation, to study the influence of ROS on cellular function. Calcium ion (Ca 2+ ) influx through Ca 2+ release-activated Ca 2+ (CRAC) channels composed of proteins of the ORAI family is essential for the activation, proliferation, and differentiation of T lymphocytes, but whether and how ROS affect ORAI channel function have been unclear. Here, we combined Ca 2+ imaging, patch-clamp recordings and measurements of cell proliferation and cytokine secretion to determine the effects of hydrogen peroxide (H 2 O 2 ) on ORAI channel activity and human T helper lymphocyte (T H cell) function. ORAI1, but not ORAI3, channels were inhibited by oxidation by H 2 O 2 . The differential redox sensitivity of ORAI1 and ORAI3 channels depended mainly on an extracellularly located reactive cysteine, which is absent in ORAI3. T H cells became progressively less redox-sensitive after differentiation into effector cells, a shift that would allow them to proliferate, differentiate, and secrete cytokines in oxidizing environments. The decreased redox sensitivity of effector T H cells correlated with increased expression of Orai3 and increased abundance of several cytosolic antioxidants. Knockdown of ORAI3 with small-interfering RNA rendered effector T H cells more redox-sensitive. The differential expression of Orai isoforms between naïve and effector T H cells may tune cellular responses under oxidative stress. Article Toolshttp INTRODUCTION Intracellular Ca2+ is a second messenger involved in the regulation of a diverse range of functions (1). One of the major Ca 2+ entry pathways into nonexcitable cells, such as lymphocytes and epithelial cells, is through ubiquitously expressed Ca 2+ release-activated Ca 2+ (CRAC) channels that are localized in the plasma membrane. Ca 2+ influx through CRAC channels is activated when inositol 1,4,5-trisphosphate (IP 3 ) triggers Ca 2+ release from intracellular stores in the lumen of the endoplasmic reticulum (ER) (2-4).The concomitant decrease in ER luminal Ca 2+ triggers accumulation of the ER Ca 2+ sensor protein stromal interaction molecule (STIM1) into puncta close to the plasma membrane (5, 6). These clustered STIM1 proteins directly activate Ca 2+ influx through CRAC channels, which are encoded by the Orai gene family (7-11). ORAI proteins contain four transmembrane domains with both N-and C-terminal intracellular domains (12, 13) that contain putative N-terminal calmodulinbinding domains and C-terminal coiled-coil motifs. Orai family members are highly homologous, and Orai1 and 3 are widely expressed at the messenger RNA (mRNA) level, with Orai2 showing a somewhat more restricted expression pattern (14, 15). ORAI1 proteins contain the longest intracellular N termini with two proline-rich regions an...

The killer cell lectin-like receptor G1 (KLRG1) is the mouse homolog of the rat mast cell function-associated Ag and contains an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic domain. In this study we demonstrate that both pathogenic and nonpathogenic in vivo activation of NK cells induces the expression of KLRG1 on their cell surface. Upon infection with murine CMV, this induction peaks between days 5 and 7 with ∼90% of the NK cells expressing KLRG1. On day 1.5 post-murine CMV infection of C57BL/6 mice, the main producers of IFN-γ are the KLRG1-negative NK cells. This effect has been recapitulated in vitro as we show that engagement of KLRG1 on a transfected NK cell line inhibits both cytokine production and NK cell-mediated cytotoxicity. Taken together, these data illustrate the crucial role played by KLRG1 during the termination of mouse NK cell activation.