A major lipid raft protein raftlin modulates T cell receptor signaling and enhances th17-mediated autoimmune responses.

The membrane microdomains known as lipid rafts have been shown to act as platforms for the initiation of various receptor signals. Through proteomic analysis, we have identified a novel protein termed Raftlin (raft-linking protein) as a major protein in lipid rafts. To determine the physiological and immunological functions of Raftlin in mammals, we generated Raftlin-deficient mice, as well as Raftlin-transgenic (Tg) mice. Although Raftlin was originally identified in B cells, we observe no severe abnormalities in the B cells of these mice, presumably due to a high expression of Raftlin-homologue (Raftlin-2). T cells, in contrast, expressed a substantial amount of Raftlin but no Raftlin-2. In Raftlin-deficient mice, T cell-dependent Ab production was reduced, and experimental autoimmune encephalomyelitis, a Th17-dependent autoimmune disease model, was ameliorated. In Raftlin-Tg mice, in contrast, Ab production was enhanced and experimental autoimmune encephalomyelitis was more severe. Cytokine production, especially that of IL-17, was reduced in Raftlin-deficient T cells, while it was enhanced in Raftlin-Tg T cells. We found that these changes were associated with the strength of the TCR-mediated signals. Importantly, localization of Lck protein in the lipid rafts was enhanced by Raftlin overexpression and reduced by Raftlin deficiency. These data indicate that Raftlin modulates TCR signals and is necessary for the fine-tuning of T cell-mediated immune responses.

Spreds are essential for embryonic lymphangiogenesis by regulating vascular endothelial growth factor receptor 3 signaling.

Spred/Sprouty family proteins negatively regulate growth factor-induced ERK activation. Although the individual physiological roles of Spred-1 and Spred-2 have been investigated using gene-disrupted mice, the overlapping functions of Spred-1 and Spred-2 have not been clarified. Here, we demonstrate that the deletion of both Spred-1 and Spred-2 resulted in embryonic lethality at embryonic days 12.5 to 15.5 with marked subcutaneous hemorrhage, edema, and dilated lymphatic vessels filled with erythrocytes. This phenotype resembled that of Syk(-/-) and SLP-76(-/-) mice with defects in the separation of lymphatic vessels from blood vessels. The number of LYVE-1-positive lymphatic vessels and lymphatic endothelial cells increased markedly in Spred-1/2-deficient embryos compared with WT embryos, while the number of blood vessels was not different. Ex vivo colony assay revealed that Spred-1/2 suppressed lymphatic endothelial cell proliferation and/or differentiation. In cultured cells, the overexpression of Spred-1 or Spred-2 strongly suppressed vascular endothelial growth factor-C (VEGF-C)/VEGF receptor (VEGFR)-3-mediated ERK activation, while Spred-1/2-deficient cells were extremely sensitive to VEGFR-3 signaling. These data suggest that Spreds play an important role in lymphatic vessel development by negatively regulating VEGF-C/VEGFR-3 signaling.

Relative level of thymidylate synthase mRNA expression in primary tumors and normal tissues predicts survival of patients with oral tongue squamous cell carcinoma.

Thymidylate synthase (TS) is a major target of 5-fluorouracil (5-FU) and dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme in the degradation of 5-FU. There are no studies investigating the comparison of TS and DPD mRNA expressions in oral tongue SCC (OSCC) and nontumor tissues obtained from the same patients. In addition, increased interest has been focused on the biological roles of TS and DPD as the independent prognostic factors as well as responsive determinants for cancer patients with 5-FU based therapy. We determined the expression levels of TS and DPD in tumor (T) and nontumor squamous epithelial tissues (N) of OSCC using real-time reverse transcription-polymerase chain reaction and evaluated whether the T/N ratio would correlate with clinicopathological factors. The mRNA expressions of TS and DPD were significantly higher in tumor areas than in nontumor areas. No correlation was found between the T/N ratio of each mRNA expression and gender, clinical stage, T classification, N classification or differentiation. The T/N ratio of TS in patients that died of disease was significantly higher than in patients with free of disease, whereas there were no relationships between The T/N ratio of DPD and disease status. Clinical follow-up data showed shorter overall survival periods for cases with high T/N ratio of TS than for cases with low T/N ratio of TS with the statistically significant. Our study showed that TS but not DPD seems to have prognostic value in OSCC. These findings suggest that the assessment of TS activity may be useful both in the management and in the treatment of OSCC.

Silencing of SOCS1 in macrophages suppresses tumor development by enhancing antitumor inflammation.

Inflammation has been shown to contribute to both tumor development and antitumor immunity. However, conditions determining these opposing effects are not well understood. Suppressor of cytokine signaling 1 (SOCS1) has been shown to play an important role in regulating inflammation and tumor development. It has been reported that silencing of SOCS1 gene in dendritic cells potentiates antitumor immunity, while SOCS1-deficiency in whole organs except for T and B cells enhances inflammation-mediated colon tumor development. To determine which types of cells are important for the suppression of tumor development by SOCS1-deficiency, we employed the conditional knockout strategy. SOCS1 gene was deleted in macrophages and neutrophils by crossing SOCS1-flox/flox mice with LysM-cre mice. Resulting conditional knockout (cKO) mice showed enhanced sensitivity to endotoxin shock. SOCS1-cKO mice survived much longer than wild-type mice after B16 melanoma transplantation. Colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) plus dextran sulfate sodium (DSS) was also reduced in SOCS1-cKO mice. SOCS1-deficiency in monocytic cells enhanced tumor-killing activity of macrophages and tumor-specific cytotoxic T cell activity. These results suggest that inflammation induced by SOCS1-deficiency in monocytes potentiates antitumor immune responses rather than tumor-promoting inflammation.

Sprouty4 deficiency potentiates Ras-independent angiogenic signals and tumor growth.

Sprouty proteins have been shown to negatively regulate a variety of receptor tyrosine kinase (RTK) signaling pathways and are considered to be tumor suppressor proteins. The pathophysiological functions of Sproutys in vivo remain to be investigated. In this study, we examined the physiological function of Sprouty4 as an angiogenic regulator, using Sprouty4 knockout (KO) mice and cells. We found that transplanted tumor cells grow much faster in Sprouty4 KO mice than in wild type (WT) mice, which we associate with enhanced neovascularization in the tumors transplanted into Sprouty4 KO mice. Moreover, vascular endothelial growth factor (VEGF)-A-induced angiogenesis and vascular permeability in vivo were enhanced in Sprouty4 KO mice compared with WT mice. Ex vivo angiogenesis, which we induced by VEGF-A, basic fibroblast growth factor (bFGF), and sphingosine-1-phosphate (S1P), was also enhanced in the aortas of Sprouty4 KO mice. We demonstrated that Sprouty4 suppresses Ras-independent VEGF-A and S1P signaling, while it does not affect Ras-dependent VEGF-C signaling. These data indicate that Sprouty4 selectively suppresses Ras-independent angiogenic factor signals and is an important negative regulator of pathophysiological angiogenesis.

Correlations between thymidylate synthase expression and chemosensitivity to 5-fluorouracil, cell proliferation and clinical outcome in head and neck squamous cell carcinoma.

BACKGROUND: 5-Fluorouracil (5-FU) is a widely used drug in head and neck squamous cell carcinoma (HNSCC). Thymidylate synthase (TS), which is the target enzyme of 5-FU, has been demonstrated to be a key regulatory enzyme. In this study, we examined whether TS expression is correlated with chemosensitivity to 5-FU, cell proliferation and clinical outcome in HNSCC. METHODS: An antisense TS cDNA was constitutively expressed in the HNSCC cell line. The effects of TS expression on in vitro cell growth and 5-FU cytotoxicity were examined. We also evaluated the association between TS expression and cell proliferation in surgical specimens, and prognosis in HNSCC patients. RESULTS: Antisense TS transfection increases the cytotoxicity of 5-FU and inhibits cell proliferation in HNSCC cells in vitro. Immunohistochemical expression of TS may have prognostic value in patients with HNSCC. CONCLUSIONS: These results indicate that TS expression plays an important role in the sensitivity of HNSCC to 5-FU chemotherapy.

Clinical and mutational spectrum of neurofibromatosis type 1-like syndrome.

CONTEXT: Autosomal dominant inactivating sprouty-related EVH1 domain-containing protein 1 (SPRED1) mutations have recently been described in individuals presenting mainly with café au lait macules (CALMs), axillary freckling, and macrocephaly. The extent of the clinical spectrum of this new disorder needs further delineation. OBJECTIVE: To determine the frequency, mutational spectrum, and phenotype of neurofibromatosis type 1-like syndrome (NFLS) in a large cohort of patients. DESIGN, SETTING, AND PARTICIPANTS: In a cross-sectional study, 23 unrelated probands carrying a SPRED1 mutation identified through clinical testing participated with their families in a genotype-phenotype study (2007-2008). In a second cross-sectional study, 1318 unrelated anonymous samples collected in 2003-2007 from patients with a broad range of signs typically found in neurofibromatosis type 1 (NF1) but no detectable NF1 germline mutation underwent SPRED1 mutation analysis. MAIN OUTCOME MEASURES: Comparison of aggregated clinical features in patients with or without a SPRED1 or NF1 mutation. Functional assays were used to evaluate the pathogenicity of missense mutations. RESULTS: Among 42 SPRED1-positive individuals from the clinical cohort, 20 (48%; 95% confidence interval [CI], 32%-64%) fulfilled National Institutes of Health (NIH) NF1 diagnostic criteria based on the presence of more than 5 CALMs with or without freckling or an NF1-compatible family history. None of the 42 SPRED1-positive individuals (0%; 95% CI, 0%-7%) had discrete cutaneous or plexiform neurofibromas, typical NF1 osseous lesions, or symptomatic optic pathway gliomas. In the anonymous cohort of 1318 individuals, 34 different SPRED1 mutations in 43 probands were identified: 27 pathogenic mutations in 34 probands and 7 probable nonpathogenic missense mutations in 9 probands. Of 94 probands with familial CALMs with or without freckling and no other NF1 features, 69 (73%; 95% CI, 63%-80%) had an NF1 mutation and 18 (19%; 95% CI, 12%-29%) had a pathogenic SPRED1 mutation. In the anonymous cohort, 1.9% (95% CI, 1.2%-2.9%) of individuals with the clinical diagnosis of NF1 according to the NIH criteria had NFLS. CONCLUSIONS: A high SPRED1 mutation detection rate was found in NF1 mutation-negative families with an autosomal dominant phenotype of CALMs with or without freckling and no other NF1 features. Among individuals in this study, NFLS was not associated with the peripheral and central nervous system tumors seen in NF1.

[Blood concentration of laminin gamma2 chain in patients with head and neck cancer].

Laminin gamma2 chain (LN gamma2), expressed in human cancer cells and correlated with cancer malignancy, is cleaved by proteases and secreted into circulation. We measured the blood concentration of LN gamma2 in patients with head and neck cancer by an immuno-fluorescence assay using monoclonal antibodies against human LN gamma2. The normal blood concen- showed normal LN gamma2 concentration less than 50 ng/ml and 20 (33%) increased concentration exceeding 50 ng/ml. The relative ratio of the number of patients who showed increased LN gamma2 concentration correlated with the clinical stages of cancer. The blood concentration of LN gamma2 in 24 who initially showed normal concentrations did not change after radical treatments. Five who initially showed increased LN gamma2 concentration showed decreased concentration of less than 50 ng/ml after radical treatment. Four showed increased LN gamma2 concentration after treatment, and presented residual cancer, which killed them. Three of the 4 patients showed marked increase in LN gamma2 concentration exceeding 100 ng/ml and developed multiple distant metastases to the lung, liver, bone, and skin. The blood concentration of LN gamma2 in patients with head and neck cancer may thus indicate the amount of highly malignant cancer cells expressing LN gamma2. The blood concentration of LN gamma2 could therefore serve as a new tumor marker that might indicate the malignancy of and efficacy of treatments for head and neck cancer.

Suppression of Sproutys has a therapeutic effect for a mouse model of ischemia by enhancing angiogenesis.

Sprouty proteins (Sproutys) inhibit receptor tyrosine kinase signaling and control various aspects of branching morphogenesis. In this study, we examined the physiological function of Sproutys in angiogenesis, using gene targeting and short-hairpin RNA (shRNA) knockdown strategies. Sprouty2 and Sprouty4 double knockout (KO) (DKO) mice were embryonic-lethal around E12.5 due to cardiovascular defects. The number of peripheral blood vessels, but not that of lymphatic vessels, was increased in Sprouty4 KO mice compared with wild-type (WT) mice. Sprouty4 KO mice were more resistant to hind limb ischemia and soft tissue ischemia than WT mice were, because Sprouty4 deficiency causes accelerated neovascularization. Moreover, suppression of Sprouty2 and Sprouty4 expression in vivo by shRNA targeting accelerated angiogenesis and has a therapeutic effect in a mouse model of hind limb ischemia. These data suggest that Sproutys are physiologically important negative regulators of angiogenesis in vivo and novel therapeutic targets for treating peripheral ischemic diseases.

Sprouty2 and Sprouty4 are essential for embryonic morphogenesis and regulation of FGF signaling.

Sprouty genes encode cytoplasmic membrane-associated proteins that inhibit receptor tyrosine kinase signaling. Four orthologs of Drosophila Sprouty (dSpry) (Sprouty1-4) have been identified in mammals. Physiological function of Sprouty1 and Sprouty2 has been investigated using gene targeting approaches, however to date detailed examination of Sprouty4 knockout (KO) mice has not been reported. In this study, Sprouty4 KO mice were generated and characterized. Although a significant fraction of Sprouty4 KO mice died shortly after birth due to mandible defects, the remainder were viable and fertile. Growth retardation was observed for most Sprouty4-deficient mice, with nearly all Sprouty4 KO mice having polysyndactyly. ERK activation was sustained in Sprouty4 KO mouse embryonic fibroblasts (MEFs) in response to FGF, but not to EGF. Sprouty2 and Sprouty4 double KO (DKO) mice were embryonic lethal and showed severe defects in craniofacial, limb, and lung morphogenesis. These findings suggest both redundant and non-redundant functions for Sprouty2 and Sprouty4 on embryonic development and FGF signaling.