Although sphingosine 1-phosphate (Sph-1-P) is reportedly involved in diverse cellular processes and the physiological roles of this bioactive sphingolipid have been strongly suggested, few studies have revealed the presence of Sph-1-P in human samples, including body fluids and cells, under physiological conditions. In this study, we identified Sph-1-P as a normal constituent of human plasma and serum. The Sph-1-P levels in plasma and serum were 191+/-79 and 484+/-82 pmol/ml (mean+/-SD, n=8), respectively. Furthermore, when Sph-1-P was measured in paired plasma and serum samples obtained from 6 healthy adults, the serum Sph-1-P/plasma Sph-1-P ratio was found to be 2.65+/-1.26 (mean+/-SD). It is most likely that the source of discharged Sph-1-P during blood clotting is platelets, because platelets abundantly store Sph-1-P compared with other blood cells, and release part of their stored Sph-1-P extracellularly upon stimulation. We also studied Sph-1-P-related metabolism in plasma. [3H]Sph was stable and not metabolized at all in plasma, but was rapidly incorporated into platelets and metabolized mainly to Sph-1-P in platelet-rich plasma. [3H]Sph-1-P was found to be unchanged in plasma, revealing that plasma does not contain the enzymes needed for Sph-1-P degradation. In summary, platelets can convert Sph into Sph-1-P, and are storage sites for the latter in the blood. In view of the diverse biological effects of Sph-1-P, the release of Sph-1-P from activated platelets may be involved in a variety of physiological and pathophysiological processes, including thrombosis, hemostasis, atherosclerosis and wound healing.
Several lines of evidence suggest nuclear factor of activated T-cells (NFAT) to control regulatory T cells: thymus-derived naturally occurring regulatory T cells (nTreg) depend on calcium signals, the Foxp3 gene harbors several NFAT binding sites, and the Foxp3 (Fork head box P3) protein interacts with NFAT. Therefore, we investigated the impact of NFAT on Foxp3 expression. Indeed, the generation of peripherally induced Treg (iTreg) by TGF-β was highly dependent on NFAT expression because the ability of CD4 + T cells to differentiate into iTreg diminished markedly with the number of NFAT family members missing. It can be concluded that the expression of Foxp3 in TGF-β-induced iTreg depends on the threshold value of NFAT rather than on an individual member present. This is specific for iTreg development, because frequency of nTreg remained unaltered in mice lacking NFAT1, NFAT2, or NFAT4 alone or in combination. Different from expectation, however, the function of both nTreg and iTreg was independent on robust NFAT levels, reflected by less nuclear NFAT in nTreg and iTreg. Accordingly, absence of one or two NFAT members did not alter suppressor activity in vitro or during colitis and transplantation in vivo. This scenario emphasizes an inhibition of high NFAT activity as treatment for autoimmune diseases and in transplantation, selectively targeting the proinflammatory conventional T cells, while keeping Treg functional.gene regulation | tolerance | autoimmunity
Introductionvon Willebrand factor (vWF) is a multimeric protein that mediates platelet adhesion to exposed subendothelium at sites of vascular injury. The adhesive property of vWF is tightly regulated so that plasma vWF does not normally interact with circulating platelets. However, after vWF is activated by binding to damaged vessel walls, it serves to bridge the constituents of subendothelium to glycoprotein Ib (GPIb) on the membrane of circulating platelets. 1,2 Although much is known about the molecular basis of the interaction between vWF and GPIb, little has been clarified about the intracellular signal transduction pathway in GPIb-mediated platelet activation.Events related to protein-tyrosine phosphorylation have emerged as important signals mediated by GPIb. [3][4][5][6][7] GPIb-mediated platelet activation in response to vWF plus botrocetin, shear stress, or vWF from patients with von Willebrand disease type IIb induces tyrosine phosphorylation of multiple proteins, [3][4][5] suggesting that the binding of vWF to GPIb causes the activation of tyrosine kinases. Studies have shown that the activation of Syk and Src and their association can be induced by GPIb stimulation. [6][7][8][9][10] To date, there is no report showing that tyrosine residues within the GPIb molecule can be phosphorylated, nor has GPIb itself intrinsic kinase activity. Thus, how clustering of GPIb induced by vWF mediates the activation of tyrosine kinases such as Src and Syk has become an important issue. Because Syk is activated by engagement of its tandem SH2 domains with phosphorylated tyrosine residues in proteins containing the immunoreceptor tyrosine-based activation motif (ITAM), 11 it is of interest whether ITAM-containing transmembrane molecules are also involved in GPIb signaling. Two ITAM-containing proteins have been identified in platelets, the low-affinity receptor for immunoglobulin (Ig) G, Fc␥RIIA, and the Fc receptor ␥-chain (FcR ␥-chain). Of particular note is the finding that Fc␥RIIA is physically associated with GPIb and that it mediates some signaling events. 12,13 However, the level of Fc␥RIIA tyrosine phosphorylation induced by GPIb clustering is much lower than that inducible by Fc␥RIIA clustering. Further, murine platelets lacking Fc␥RIIA on their membrane appear to have normal responses to GPIbrelated signals. Thus, to what extent Fc␥RIIA contributes to the GPIb signaling pathway remains to be determined.The other ITAM-containing protein, FcR ␥-chain, has been recognized for its role in signaling related to the high-affinity receptors for IgE (Fc⑀RI) 14 and IgG (Fc␥RI), 15 and the low-affinity IgG receptor (Fc␥RIII). 16 In platelets, FcR ␥-chain is colocalized with GPVI and is critical for GPVI-mediated platelet activation. 17,18 Clustering and activation of GPVI induces tyrosine phosphorylation of FcR ␥-chain ITAM, 19 which then facilitates the recruitment of Syk, resulting in its activation. 20 Moreover, FcR ␥-chain is required for tyrosine phosphorylation of phospholipase C ␥2 (PLC␥2). 20 Recently, Falati...
Background C-type lectin-like receptor 2 (CLEC-2) is a platelet activation receptor of sialoglycoprotein podoplanin, which is expressed on the surface of certain types of tumor cells. CLEC-2-podoplanin interactions facilitate hematogenous tumor metastasis. However, direct evidence of the role of CLEC-2 in hematogenous metastasis and cancer progression is lacking. Objective and methods We generated immunological CLEC-2-depleted mice by using anti-mouse CLEC-2 monoclonal antibody 2A2B10 and investigated whether CLEC-2 promoted hematogenous tumor metastasis and tumor growth and exacerbated the prognosis of mice bearing podoplanin-expressing B16F10 melanoma cells. Results Our results showed that hematogenous metastasis was significantly inhibited in CLEC-2-depleted mice. B16F10 cells co-cultured with wild-type platelets, but not with CLEC-2-deficient platelets, showed increased proliferation. However, B16F10 cell proliferation was not inhibited in CLEC-2-depleted mice. Histological analysis showed that thrombus formation in tumor vessels was significantly inhibited and functional vessel density was significantly increased in CLEC-2-depleted mice. These data suggest that CLEC-2 deficiency may inhibit thrombus formation in tumor vessels and increase the density of functional vessels, thus improving oxygen and nutrient supply to tumors, indirectly promoting tumor proliferation. Furthermore, the overall survival of CLEC-2-depleted mice was significantly prolonged, which may be due to the suppression of thrombus formation in the lungs and subsequent inhibition of systemic inflammation and cachexia. Conclusions These data provide a rationale for the targeted inhibition of CLEC-2 as a new strategy for preventing hematogenous tumor metastasis and for inhibiting cancer-related thromboembolism.
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