Mouse methylation profiles for leukocyte cell types, and estimation of leukocyte fractions in inflamed gastrointestinal DNA samples.

Precise analysis of tissue DNA and RNA samples is often hampered by contaminating non-target cells whose amounts are highly variable. DNA methylation profiles are specific to cell types, and can be utilized for assessment of the fraction of such contaminating non-target cells. Here, we aimed 1) to identify methylation profiles specific to multiple types of mouse leukocytes, and 2) to estimate the fraction of leukocytes infiltrating inflamed tissues using DNA samples. First, genome-wide DNA methylation analysis was conducted for three myeloid-lineage cells and four lymphoid-lineage cells isolated by fluorescence-activated cell sorting after magnetic-activated cell sorting from leukocytes in the spleen. Clustering analysis using CpG sites within enhancers separated the three myeloid-lineage cells and four lymphoid-lineage cells while that using promoter CpG islands (TSS200CGIs) did not. Among the 266,108 CpG sites analyzed, one CpG site was specifically hypermethylated (ß value ≥ 0.7) in B cells, and four, seven, 183, and 34 CpG sites were specifically hypomethylated (ß value < 0.2) in CD4+ T cells, CD8+ T cells, B cells, and NK cells, respectively. Importantly, cell type-specific hypomethylated CpG sites were located at genes involved in cell type-specific biological functions. Then, marker CpG sites to estimate the leukocyte fraction in a tissue with leukocyte infiltration were selected, and an estimation algorithm was established. The fractions of infiltrating leukocytes were estimated to be 1.6-12.4% in the stomach (n = 10) with Helicobacter pylori-induced inflammation and 1.5-4.3% in the colon with dextran sulfate sodium-induced colitis (n = 4), and the fractions were highly correlated with those estimated histologically using Cd45-stained tissue sections [R = 0.811 (p = 0.004)]. These results showed that mouse methylation profiles at CpG sites within enhancers reflected leukocyte cell lineages, and the use of marker CpG sites successfully estimated the leukocyte fraction in inflamed gastric and colon tissues.

Lactic acid promotes PD-1 expression in regulatory T cells in highly glycolytic tumor microenvironments.

The balance of programmed death-1 (PD-1)-expressing CD8+ T cells and regulatory T (Treg) cells in the tumor microenvironment (TME) determines the clinical efficacy of PD-1 blockade therapy through the competition of their reactivation. However, factors that determine this balance remain unknown. Here, we show that Treg cells gain higher PD-1 expression than effector T cells in highly glycolytic tumors, including MYC-amplified tumors and liver tumors. Under low-glucose environments via glucose consumption by tumor cells, Treg cells actively absorbed lactic acid (LA) through monocarboxylate transporter 1 (MCT1), promoting NFAT1 translocation into the nucleus, thereby enhancing the expression of PD-1, whereas PD-1 expression by effector T cells was dampened. PD-1 blockade invigorated the PD-1-expressing Treg cells, resulting in treatment failure. We propose that LA in the highly glycolytic TME is an active checkpoint for the function of Treg cells in the TME via upregulation of PD-1 expression.

HLA Class I Analysis Provides Insight Into the Genetic and Epigenetic Background of Immune Evasion in Colorectal Cancer With High Microsatellite Instability.

BACKGROUND & AIMS: A detailed understanding of antitumor immunity is essential for optimal cancer immune therapy. Although defective mutations in the B2M and HLA-ABC genes, which encode molecules essential for antigen presentation, have been reported in several studies, the effects of these defects on tumor immunity have not been quantitatively evaluated. METHODS: Mutations in HLA-ABC genes were analyzed in 114 microsatellite instability-high colorectal cancers using a long-read sequencer. The data were further analyzed in combination with whole-exome sequencing, transcriptome sequencing, DNA methylation array, and immunohistochemistry data. RESULTS: We detected 101 truncating mutations in 57 tumors (50%) and loss of 61 alleles in 21 tumors (18%). Based on the integrated analysis that enabled the immunologic subclassification of microsatellite instability-high colorectal cancers, we identified a subtype of tumors in which lymphocyte infiltration was reduced, partly due to reduced expression of HLA-ABC genes in the absence of apparent genetic alterations. Survival time of patients with such tumors was shorter than in patients with other tumor types. Paradoxically, tumor mutation burden was highest in the subtype, suggesting that the immunogenic effect of accumulating mutations was counterbalanced by mutations that weakened immunoreactivity. Various genetic and epigenetic alterations, including frameshift mutations in RFX5 and promoter methylation of PSMB8 and HLA-A, converged on reduced expression of HLA-ABC genes. CONCLUSIONS: Our detailed immunogenomic analysis provides information that will facilitate the improvement and development of cancer immunotherapy.

The PD-1 expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1 blockade therapies.

Immune checkpoint blockade has provided a paradigm shift in cancer therapy, but the success of this approach is very variable; therefore, biomarkers predictive of clinical efficacy are urgently required. Here, we show that the frequency of PD-1+CD8+ T cells relative to that of PD-1+ regulatory T (Treg) cells in the tumor microenvironment can predict the clinical efficacy of programmed cell death protein 1 (PD-1) blockade therapies and is superior to other predictors, including PD ligand 1 (PD-L1) expression or tumor mutational burden. PD-1 expression by CD8+ T cells and Treg cells negatively impacts effector and immunosuppressive functions, respectively. PD-1 blockade induces both recovery of dysfunctional PD-1+CD8+ T cells and enhanced PD-1+ Treg cell-mediated immunosuppression. A profound reactivation of effector PD-1+CD8+ T cells rather than PD-1+ Treg cells by PD-1 blockade is necessary for tumor regression. These findings provide a promising predictive biomarker for PD-1 blockade therapies.

The Spleen Is an Ideal Site for Inducing Transplanted Islet Graft Expansion in Mice.

Alternative islet transplantation sites have the potential to reduce the marginal number of islets required to ameliorate hyperglycemia in recipients with diabetes. Previously, we reported that T cell leukemia homeobox 1 (Tlx1)+ stem cells in the spleen effectively regenerated into insulin-producing cells in the pancreas of non-obese diabetic mice with end-stage disease. Thus, we investigated the spleen as a potential alternative islet transplantation site. Streptozotocin-induced diabetic C57BL/6 mice received syngeneic islets into the portal vein (PV), beneath the kidney capsule (KC), or into the spleen (SP). The marginal number of islets by PV, KC, or SP was 200, 100, and 50, respectively. Some plasma inflammatory cytokine levels in the SP group were significantly lower than those of the PV group after receiving a marginal number of islets, indicating reduced inflammation in the SP group. Insulin contents were increased 280 days after islet transplantation compared with those immediately following transplantation (p<0.05). Additionally, Tlx1-related genes, including Rrm2b and Pla2g2d, were up-regulated, which indicates that islet grafts expanded in the spleen. The spleen is an ideal candidate for an alternative islet transplantation site because of the resulting reduced inflammation and expansion of the islet graft.

Pretreatment of donor islets with papain improves allograft survival without systemic immunosuppression in mice.

Although current immunosuppression protocols improve the efficacy of clinical allogenic islet transplantation, T cell-mediated allorejection remains unresolved, and major histocompatibility complexes (MHCs) play a crucial role in this process. Papain, a cysteine protease, has the unique ability to cleave the extracellular domain of the MHC class I structure. We hypothesized that pretreatment of donor islets with papain would diminish the expression of MHC class I on islets, reducing allograft immunogenicity and contributing to prolongation of islet allograft survival. BALB/c islets pretreated with papain were transplanted into C57BL/6J mice as an acute allorejection model. Treatment with 1 mg/mL papain significantly prolonged islet allograft survival. In vitro, to determine the inhibitory effect on T cell-mediated alloreactions, we performed lymphocyte proliferation assays and mixed lymphocyte reactions. Host T cell activation against allogenic islet cells was remarkably suppressed by pretreatment of donor islet cells with 10 mg/mL papain. Flow cytometric analysis was also performed to investigate the effect of papain treatment on the expression of MHC class I on islets. One or 10 mg/mL papain treatment reduced MHC class I expression on the islet cell surface. Pretreatment of donor islets with papain suppresses MHC class I-mediated allograft rejection in mice and contributes to prolongation of islet allograft survival without administration of systemic immunosuppressants. These results suggest that pretreatment of human donor islets with papain may reduce the immunogenicity of the donor islets and minimize the dosage of systemic immunosuppressants required in a clinical setting.

Islet-derived damage-associated molecular pattern molecule contributes to immune responses following microencapsulated neonatal porcine islet xenotransplantation in mice.

BACKGROUND: Clinical allogeneic islet transplantation has become an attractive procedure for type 1 diabetes mellitus treatment. However, there is a severe shortage of human donors. Microencapsulated neonatal porcine islet (NPI) xenotransplantation may be an alternative transplantation procedure. Currently, the efficacy of microencapsulated NPI xenotransplantation into the peritoneal cavity is limited because of early non-function resulting from inflammation, which is a serious hindrance to promoting this procedure as a standard therapy. Previously, we have demonstrated that high-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecule, was released from transplanted islets and triggered inflammatory reactions leading to early loss of intrahepatic syngeneic islet grafts in mice. In this study, we hypothesized that the inflammatory reaction in the peritoneal cavity following the transplantation of microencapsulated NPIs is more severe than that of empty capsules. Additionally, we predicted that HMGB1 released from transplanted microencapsulated NPIs triggers further inflammatory reactions in mice. Finally, we hypothesized that microencapsulated NPI xenotransplantation efficacy would be improved by treatment-targeting inflammatory reactions in a mouse model. METHODS: A total of 10 000 empty capsules (alginate-poly-L-ornithine-alginate) or 10 000 IEQ microencapsulated NPIs were transplanted into the peritoneal cavity of streptozotocin-induced diabetic C57BL/6 mice. RESULTS: The numbers of mononuclear cells in the peritoneal cavity following empty capsule or microencapsulated NPI transplantation were 4.8 × 10(6)  ± 0.9 × 10(6) and 13.6 × 10(6)  ± 3.0 × 10(6) , respectively (P < 0.05). Fluorescence-activated cell sorting (FACS) analysis revealed that tumor necrosis factor (TNF)-α-, interleukin (IL)-6-, interferon (IFN)-γ-, and/or IL-12-positive macrophages, neutrophils, and dendritic cells had infiltrated the peritoneal cavity after empty capsules or microencapsulated NPIs administration. IL-6 concentrations in the peritoneal lavage fluids on 7 days after empty capsule or microencapsulated NPI transplantation were 18.5 ± 10.0 and 157.4 ± 46.3 pg/ml, respectively (P < 0.001), while TNF-α concentrations were 4.6 ± 1.4 and 19.8 ± 8.4 pg/ml, respectively (P < 0.01). In addition, HMGB1 concentrations were 37.6 ± 6.6 and 117.4 ± 8.1 ng/ml, respectively (P < 0.0001). In vitro experiments revealed that the total amount of released HMGB1 into the culture medium of empty capsule (200 capsules/dish) and microencapsulated NPI (200 IEQ/dish) after hypoxic culture (1% O2 , 5% CO2 , and 94% N2 ) was 0 and 8.6 ± 2.2 ng, respectively (P < 0.001). FACS analysis revealed that TNF-α- and IL-6-positive macrophages were also observed in the peritoneal cavity following intraperitoneal injection of HMGB1 itself. Anti-TNF-α antibody treatment was associated with slightly prolonged graft survival and improved glucose tolerance 30 days after transplantation, but none of the recipients were remained normoglycemic. CONCLUSIONS: In conclusion, early inflammatory reactions might be therapeutic targets for the prolongation of microencapsulated NPIs graft survival. Thus, treatment-targeting inflammation might improve the efficiency of clinical microencapsulated NPI xenotransplantation.

HMGB1-Mediated Early Loss of Transplanted Islets Is Prevented by Anti-IL-6R Antibody in Mice.

OBJECTIVES: The limited success in achieving insulin independence of patients with type 1 diabetes mellitus after islet transplantation from a single donor, mainly due to early loss of transplanted islets, hampers clinical application of islet transplantation. Previously, we have shown in mice that the early loss of transplanted islets in the liver, the site of islet transplantation, is caused by innate immune rejection triggered by high-mobility group box 1 (HMGB1) protein released from transplanted islets. We herein determined whether the HMGB1-mediated early loss of transplanted mouse islets is prevented by anti-interleukin-6 receptor (IL-6R) antibody. METHODS: The effect of anti-IL-6R antibody on amelioration of hyperglycemia in streptozocin-induced diabetic mice receiving 200 islets into the liver from a single donor was evaluated in association with HMGB1-stimulated interferon-γ production of hepatic mononuclear cells. RESULTS: Hyperglycemia of diabetic mice receiving 200 syngeneic islets was ameliorated with down-regulation of interferon-γ production of hepatic natural killer T cells and neutrophils when anti-IL-6R was administered at the time of transplantation. This beneficial effect was also seen in allografts when alloimmune rejection was prevented by anti-CD4 antibody. CONCLUSIONS: These findings demonstrate that anti-IL-6R antibody prevented the early loss of intrahepatic islet grafts with inhibiting HMGB1-induced immune activation after islet transplantation.

GM-CSF treated F4/80+ BMCs improve murine hind limb ischemia similar to M-CSF differentiated macrophages.

Novel cell therapy is required to treat critical limb ischemia (CLI) as many current approaches require repeated aspiration of bone marrow cells (BMCs). The use of cultured BMCs can reduce the total number of injections required and were shown to induce therapeutic angiogenesis in a murine model of hind limb ischemia. Blood flow recovery was significantly improved in mice treated with granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent BMCs that secreted inflammatory cytokines. Angiogenesis, lymphangiogenesis, and blood flow recovery ratio were significantly higher in the GM-CSF-cultured F4/80+ macrophage (GM-Mø)-treated group compared with controls. Furthermore, Foxp3+ cell numbers and tissue IL-10 concentrations were significantly increased compared with controls. There was no significant difference in blood flow recovery between GM-Mø and M-CSF-cultured F4/80+ macrophages (M-Mø). Thus, GM-Mø were associated with improved blood flow in hind limb ischemia similar to M-Mø. The selective methods of culturing and treating GM-Mø cells similar to M-Mø cells could be used clinically to help resolve the large number of cells required for BMC treatment of CLI. This study demonstrates a novel cell therapy for CLI that can be used in conjunction with conventional therapy including percutaneous intervention and surgical bypass.

Edaravone, a free radical scavenger, accelerates wound healing in diabetic mice.

UNLABELLED: Refractory wound healing is a major complication of diabetes, which restricts wound healing by interfering with the inflammatory response, decreasing granulation, causing peripheral neuropathy, and inhibiting angiogenesis. Oxidative stress has been proposed as an important pathogenic factor in diabetic wound complications. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a strong free radical scavenger that suppresses the effect of oxidative stress. MATERIAL AND METHODS: Streptozotocin-induced diabetes was established in adult C57BL/6 mice, and full-thickness skin was then removed from the dorsomedial back using an 8-mm biopsy punch. Edaravone or vehicle alone was applied to the wound on day 0 and day 4 after wound creation. The wound was monitored with a digital camera and analyzed on days 0, 4, and 7 after wound creation. RESULTS: This study investigated whether accelerated wound closure occurred in the edaravone group (n = 24) compared with the vehicle-alone group (n = 15). On day 7, wound closure between the 2 groups was statistically different (P = 0.0019). Angiogenesis and lymphangiogenesis were markedly promoted. The possibility of an inhibitory effect of edaravone characterized by suppression of oxidative stress was explored. Edaravone-induced upregulation of endothelial nitric oxide synthase (eNOS) mRNA expression, and eNOS protein was immunohistochemically detected. CONCLUSION: Edaravone upregulates eNOS expression and accelerates wound healing.

Vascular endothelial growth factor-C derived from CD11b+ cells induces therapeutic improvements in a murine model of hind limb ischemia.

OBJECTIVE: The use of bone marrow cells (BMCs) in therapeutic angiogenesis has been studied extensively. However, the critical paracrine effects of this treatment are still unclear. Therefore, we studied autotransfusable cells that produce vascular endothelial growth factor (VEGF), especially VEGF-C. METHODS: Male C57BL/6 mice with hind limb ischemia were administered intramuscular injections of phosphate-buffered saline as controls, or unsorted BMCs, sorted CD11b(+), or CD11b(-) cells from BMCs, and recombinant VEGF-C. To evaluate the treatments, perfusion was measured by laser Doppler scanning performed on days 0, 1, 3, 7, 14, 21, and 28. A functional assay was performed in parallel, with mice traversing an enclosed walkway. Capillary density was determined by directly counting vessels stained positive with von Willebrand factor at individual time points. Lymphangiogenesis was assessed by LYVE-1 positive cells. RESULTS: Postischemic recovery of hind limb perfusion significantly improved in BMC, CD11b(+), and VEGF-C treatment groups compared with the control groups, as assessed by laser Doppler scanning. On early operative days 1 and 3, the blood flow recovery ratio was higher in the CD11b(+)-treated group compared with BMC and VEGF-C treatment groups. In the functional assay, the VEGF-C group dramatically recovered compared with the control group. The capillary/myofiber ratio in the thigh muscle and number of LYVE-1 positive cells was higher in the CD11b(+) and VEGF-C groups than in controls. Furthermore, expression of VEGF-A, VEGF-C, and VEGF receptor messenger ribonucleic acid and protein was observed in CD11b(+) cells. CONCLUSIONS: The VEGF-C derived from CD11b(+) cells play a critical role in angiogenesis and lymphangiogenesis in a murine model of hind limb ischemia. Consequently, treatment with self-CD11b(+) cells accelerated recovery from ischemia and may be a promising therapeutic strategy for peripheral arterial disease patients.

Prevention of high-mobility group box 1-mediated early loss of transplanted mouse islets in the liver by antithrombin III.

BACKGROUND: The low efficiency of pancreatic islet transplantation mainly because of the early loss of transplanted islets hampers its clinical application. Previously, we have shown in mice that the early loss of transplanted islets in the liver is caused by innate immune rejection in concert with dendritic cells, natural killer T cells, and neutrophils to produce interferon (IFN)-γ, which is triggered by high-mobility group box 1 (HMGB1) released from transplanted islets. We herein determined whether the HMGB1-mediated early loss of transplanted mouse islets is prevented by antithrombin (ATIII). METHODS: The effect of ATIII on in vitro and in vivo HMGB1-stimulated IFN-γ production of hepatic mononuclear cells was examined. Then, the effect of ATIII on amelioration of hyperglycemia in streptozotocin-induced diabetic mice receiving 200 syngeneic islets from a single donor was determined. RESULTS: In vitro and in vivo IFN-γ production of mononuclear cells in the liver of mice in response to HMGB1 was suppressed by ATIII. Hyperglycemia of streptozotocin-induced diabetic mice receiving 200 syngeneic islets into the liver from a single donor was ameliorated with down-regulation of IFN-γ production of natural killer T cells and neutrophils in the liver when ATIII but not vehicle was administered once at the time of islet transplantation. The favorable effect of ATIII was similarly achieved in mice receiving islet allografts when rejection was prevented with anti-CD4 antibody treatment. CONCLUSIONS: These findings demonstrate that ATIII prevents HMGB1-mediated early loss of transplanted islets caused by innate immune rejection, suggesting a potential application of ATIII to improve efficiency of clinical islet transplantation.

An RNA-binding protein alphaCP-1 is involved in the STAT3-mediated suppression of NF-kappaB transcriptional activity.

Signal transducer and activator of transcription 3 (STAT3) has been shown to mediate the anti-inflammatory effect of IL-10. Activated STAT3 suppresses LPS-induced IL-6, tumor necrosis factor-alpha and IL-12 gene expression in macrophages and dendritic cells. However, the mechanism of Toll-like receptor (TLR) signal suppression by STAT3 has not been clarified. In this study, we investigated the effect of constitutively activated STAT3 (STAT3C) on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The forced expression of STAT3C in HEK293/TLR4 cells, but neither wild-type STAT3 nor dominant-negative form of STAT3, suppressed LPS-TLR4-mediated NF-kappaB reporter activation. The over-expression of STAT3C did not affect the signal transduction of TLR4, such as the phosphorylation of inhibitory nuclear factor-kappaBalpha and mitogen-activated protein kinases and the DNA-binding activity of NF-kappaB. Thus, STAT3C could suppress the transcriptional and/or translational activity of NF-kappaB. To define the molecular mechanism, we searched STAT3C-binding proteins by using a proteomic approach and found that a novel RNA-binding protein, alphaCP-1, interacted with STAT3C. alphaCP-1 is a K-homology domain-containing RNA-binding protein with specificity for C-rich pyrimidine tracts. Such proteins play pivotal roles in a broad-spectrum of transcriptional and translational events. The over-expression of alphaCP-1 augmented the suppressive effect of STAT3C on NF-kappaB activation in HEK293/TLR4 cells. Furthermore, the forced expression of alphaCP-1 enhanced the antagonistic effect of IL-10 on IL-6 production in RAW264.7 cells, while small interfering RNA against alphaCP-1 reduced it. These data suggest that alphaCP-1 is involved in the STAT3-mediated suppression of NF-kappaB activity.

Negative regulation of cytokine signaling and immune responses by SOCS proteins.

Immune and inflammatory systems are controlled by multiple cytokines, including interleukins and interferons. Many of these cytokines exert their biological functions through JAKs (Janus tyrosine kinases) and STAT (signal transduction and activators of transcription) transcription factors. CIS (cytokine-inducible SH2 (Src homology 2) protein) and SOCS (suppressor of cytokine signaling) are a family of intracellular proteins, several of which have emerged as key physiological regulators of cytokine-mediated homeostasis, including innate and adaptive immunity. In this review we focus on the molecular mechanism of the action of CIS/SOCS family proteins and their roles in immune regulation and inflammatory diseases including rheumatoid arthritis.

Induction of hyper Th1 cell-type immune responses by dendritic cells lacking the suppressor of cytokine signaling-1 gene.

Suppressor of cytokine signaling (SOCS1/JAB) has been shown to play an important role in regulating dendritic cell (DC) function and suppressing inflammatory diseases and systemic autoimmunity. However, role of SOCS1 in DCs for the initiation of Th cell response has not been clarified. Here we demonstrate that SOCS1-deficient DCs induce stronger Th1-type responses both in vitro and in vivo. SOCS1-deficient DCs induced higher IFN-gamma production from naive T cells than wild-type (WT) DCs in vitro. Lymph node T cells also produced a higher amount of IFN-gamma when SOCS1-deficient bone marrow-derived DCs (BMDCs) were transferred in vivo. Moreover, SOCS1(-/-) BMDCs raised more effective anti-tumor immunity than WT BMDCs. Microarray analysis revealed that IFN-inducible genes were highly expressed in SOCS1-deficient DCs without IFN stimulation, suggesting hyper STAT1 activation in SOCS1(-/-) DCs. These phenotypes of SOCS1-deficient DCs were similar to those of CD8alpha(+) DCs, and in the WT spleen, SOCS1 is expressed at higher levels in the Th2-inducing CD4(+) DC subset, relative to the Th1-inducing CD8alpha(+) DC subset. We propose that reduction of the SOCS1 gene expression in DCs leads to CD8alpha(+) DC-like phenotype which promotes Th1-type hyperresponses.

Socs3 deficiency in the brain elevates leptin sensitivity and confers resistance to diet-induced obesity.

Leptin is an adipocyte-derived hormone that plays a key role in energy homeostasis, yet resistance to leptin is a feature of most cases of obesity in humans and rodents. In vitro analysis suggested that the suppressor of cytokine signaling-3 (Socs3) is a negative-feedback regulator of leptin signaling involved in leptin resistance. To determine the functional significance of Socs3 in vivo, we generated neural cell-specific SOCS3 conditional knockout mice using the Cre-loxP system. Compared to their wild-type littermates, Socs3-deficient mice showed enhanced leptin-induced hypothalamic Stat3 tyrosine phosphorylation as well as pro-opiomelanocortin (POMC) induction, and this resulted in a greater body weight loss and suppression of food intake. Moreover, the Socs3-deficient mice were resistant to high fat diet-induced weight gain and hyperleptinemia, and insulin-sensitivity was retained. These data indicate that Socs3 is a key regulator of diet-induced leptin as well as insulin resistance. Our study demonstrates the negative regulatory role of Socs3 in leptin signaling in vivo, and thus suppression of Socs3 in the brain is a potential therapy for leptin-resistance in obesity.

The Sprouty-related protein, Spred, inhibits cell motility, metastasis, and Rho-mediated actin reorganization.

Sprouty and the Sprouty-related protein, Spred (Sprouty-related Ena/vasodilator-stimulated phosphoprotein homology-1 (EVH1) domain-containing protein), inhibit Ras-dependent extracellular signal-regulated kinase (ERK) signaling induced by a variety of growth factors. Since Sprouty proteins have been shown to inhibit not only ERK activation but also cell migration, we postulated that Spreds also inhibit cellular migration. Using stably highly metastatic LM8 cells infected with the Spred1-Sendai virus vector, we demonstrated that Spred1 inhibits the metastasis of LM8 cells in nude mice. Spred1 overexpression also inhibited migration of cells in vitro in response to chemokines, CCL19 and CCL21. We also found that Spred1 overexpression dissolved actin-stress fibers. Both EVH1 domain and C-terminal Sprouty-related domain were required for actin reassembly. Spred1 and Spred2 suppressed constitutively activated RhoA (V14RhoA)-induced stress fiber formation and serum response factor activation. Spred1 bound to activated RhoA, but not cdc42 and Rac. Spred1 also inhibited chemokine-induced RhoA activation and active RhoA-induced Rho-kinase activation. These data suggest that Spreds are key regulators of RhoA-mediated cell motility and signal transduction. Furthermore, our study suggests that the induction of Spreds could be a novel strategy for preventing cancer cell metastasis.

Engagement of CD44 promotes Rac activation and CD44 cleavage during tumor cell migration.

CD44 is a major cell surface adhesion molecule for hyaluronan, a component of the extracellular matrix, and is implicated in tumor metastasis and invasion. We reported previously that hyaluronan oligosaccharides induce CD44 cleavage from tumor cells. Here we show that engagement of CD44 promotes CD44 cleavage and tumor cell migration, both of which were suppressed by a metalloproteinase inhibitor KB-R7785 and tissue inhibitor of metalloproteinases-1 (TIMP-1) but not by TIMP-2. We also present evidence that blockade of metalloproteinase-disintegrin ADAM10 (a disintegrin and metalloproteinase 10) by RNA interference suppresses CD44 cleavage induced by its ligation. Engagement of CD44 concurrently induced activation of the small GTPase Rac1 and led to drastic changes in cell morphology and actin cytoskeleton with redistribution of CD44 to newly generated membrane ruffling areas. A fluorescence resonance energy transfer approach to visualize GTP-bound Rac1 in living cells revealed the localization of the active Rac1 in the leading edge of the membrane ruffling areas upon ligation of CD44. Taken together, our results indicate that the cleavage of CD44 catalyzed by ADAM10 is augmented by the intracellular signaling elicited by engagement of CD44, through Rac-mediated cytoskeletal rearrangement, and suggest that CD44 cleavage contributes to the migration and invasion of tumor cells.

Hyaluronan oligosaccharides induce CD44 cleavage and promote cell migration in CD44-expressing tumor cells.

CD44 is an adhesion molecule that serves as a cell surface receptor for several extracellular matrix components, including hyaluronan (HA). The proteolytic cleavage of CD44 from the cell surface plays a critical role in the migration of tumor cells. Although this cleavage can be induced by certain stimuli such as phorbol ester and anti-CD44 antibodies in vitro, the physiological inducer of CD44 cleavage in vivo is unknown. Here, we demonstrate that HA oligosaccharides of a specific size range induce CD44 cleavage from tumor cells. Fragmented HA containing 6-mers to 14-mers enhanced CD44 cleavage dose-dependently by interacting with CD44, whereas a large polymer HA failed to enhance CD44 cleavage, although it bound to CD44. Examination using uniformly sized HA oligosaccharides revealed that HAs smaller than 36 kDa significantly enhanced CD44 cleavage. In particular, the 6.9-kDa HA (36-mers) not only enhanced CD44 cleavage but also promoted tumor cell motility, which was completely inhibited by an anti-CD44 monoclonal antibody. These results raise the possibility that small HA oligosaccharides, which are known to occur in various tumor tissues, promote tumor invasion by enhancing the tumor cell motility that may be driven by CD44 cleavage.