Induced Signal Transduction Research Articles

Understanding thermosensitive transient receptor potential channels as versatile polymodal cellular sensors.

Transient receptor potential (TRP) ion channels are eukaryotic polymodal sensors that function as molecular cellular signal integrators. TRP family members sense and are modulated by a wide array of inputs, including temperature, pressure, pH, voltage, chemicals, lipids, and other proteins. These inputs induce signal transduction events mediated by nonselective cation passage through TRP channels. In this review, we focus on the thermosensitive TRP channels and highlight the emerging view that these channels play a variety of significant roles in physiology and pathophysiology in addition to sensory biology. We attempt to use this viewpoint as a framework to understand the complexity and controversy of TRP channel modulation and ultimately suggest that the complex functional behavior arises inherently because this class of protein is exquisitely sensitive to many diverse and distinct signal inputs. To illustrate this idea, we primarily focus on TRP channel thermosensing. We also offer a structural, biochemical, biophysical, and computational perspective that may help to bring more coherence and consensus in understanding the function of this important class of proteins.

Expression of Bioactive Callithrix jacchus Follicle-Stimulating Hormone in Pichia pastoris

Callithrix jacchus (common marmoset) is a New World primate monkey, used as an animal model in biomedical research. Marmoset-specific follicle-stimulating hormone (FSH) preparation is required to improve superovulation protocols and to develop homologous FSH monitoring assays in these monkeys. In this study, we document the large-scale expression of recombinant marmoset FSH in methylotropic yeast, Pichia pastoris. The recombinant preparation was found to be immunologically active in Western blotting and radioimmunoassay. The preparation displayed receptor binding ability in radioreceptor assay. Based on the receptor binding ability, the yield of fermentation was estimated to be 7.2 mg/L. FSH-induced cAMP assay and estradiol assay revealed that the recombinant hormone is able to induce signal transduction. Both immunological and in vitro biological activity of marmoset FSH was found to be comparable to purified human pituitary FSH, which served as reference hormone for these assays. Thus, the study suggests that a Pichia expression system can be used for large-scale expression of bioactive recombinant marmoset FSH.

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction.

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.

Overdominance effect of the bovine ghrelin receptor (GHSR1a)-DelR242 locus on growth in Japanese Shorthorn weaner bulls: heterozygote advantage in bull selection and molecular mechanisms.

Ghrelin and the ghrelin receptor (GHSR1a) are involved in growth hormone secretion, food intake, and several other important functions. Ghrelin acts on GHSR1a and induces signal transduction via the Gαq subunit. In our previous study, we identified the DelR242 (3R) allele, a truncated 3-arginine residue (3R) [major type: 4 arginine residues (4R)] of the third intracellular loop of GHSR1a, with a high frequency in Japanese Shorthorn bulls (0.43) but with a low frequency in other cattle breeds (0.00–0.09). To further investigate the reasons for the higher frequency of the 3R allele, we performed several experiments. In this study, we found a significant sex difference in the frequency of the 3R allele. Statistical analysis revealed a significant overdominance effect of the DelR242 locus on growth in Japanese Shorthorn weaner bulls. However, additive/dominance/overdominance effects of the 3R allele on carcass traits in adult steers and dams were not significant. The mode of the overdominance effect was estimated to be solely controlled by the single DelR242 locus without any other linked loci using linkage disequilibrium analysis in GHSR1a. These results indicated that 4R/3R heterozygotes had a selective advantage in weaner bulls because of their higher average daily gain than homozygotes. We discussed possible molecular mechanisms involved in the overdominance effect of the DelR242 locus on these traits in weaner bulls using a structural model of the complex consisting of a GHSR1a dimer and Gαq.

Overexpression of Nodal induces a metastatic phenotype in pancreatic cancer cells via the Smad2/3 pathway.

Metastasis is the major cause for the high mortality rate of pancreatic cancer. Human embryonic stem cell (hESC) associated genes frequently correlate with malignant disease progression. Recent studies have demonstrated that the embryonic protein Nodal, which plays a critical role during embryonic development, is re-expressed in several types of tumors and promotes cancers progression. However, little is known about the role of Nodal in pancreatic cancer. Here, we show that Nodal expression is upregulated in human pancreatic cancer tissues. Moreover, Nodal expression levels correlate well with the grade of pancreatic cancer differentiation. In addition, we present clear evidence that Nodal induces signal transduction through the Smad2/3-dependent pathway in vitro. Furthermore, we show that Nodal promotes pancreatic cancer cell migration and invasion, induces epithelial-mesenchymal transition (EMT) and enhances the expression of matrix metalloproteinase-2 (MMP2) and CXC chemokine receptor 4 (CXCR4). Using an in vivo liver metastasis model of pancreatic cancer, we observed that blocking Nodal signaling activity with the small-molecule inhibitor SB431542 decreases the number and size of liver metastases. Taken together, our results suggest that Nodal overexpression induces a metastatic phenotype in pancreatic cancer cells, and that targeting Nodal signaling may be a promising therapeutic strategy for pancreatic cancer.

The p38 MAPK inhibitor SB203580 differentially modulates LPS-induced interleukin 6 expression in macrophages.

The p38 mitogen-activated protein kinase (MAPK) plays a key role in lipopolysaccharide (LPS)-induced signal transduction pathways that lead to inflammatory cytokine synthesis in macrophages; however, whether the inhibition of p38 MAPK regulates LPS-induced inflammatory cytokine expression in different types of macrophages remains the subject of debate. Herein, we assessed whether the inhibition of p38 MAPK by SB203580 regulates LPS-induced expression of the inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) in RAW264.7 and resident peritoneal macrophages. Lipopolysaccharide stimulation of RAW264.7 macrophages or mouse resident peritoneal macrophages significantly increased TNF-α and IL-6 production. The addition of SB203580 to cultures dramatically blocked LPS-induced TNF-α production in RAW264.7 and mouse resident peritoneal macrophages, and dramatically blocked LPS-induced IL-6 production in RAW264.7 macrophages, but not in mouse resident peritoneal macrophages. Additionally, high concentrations of SB203580 resulted in increased IL-6 production. However, LPS-stimulation significantly up-regulated the mRNA transcript levels of TNF-α and IL-6 in RAW264.7 and mouse resident peritoneal macrophages, whereas pretreatment with SB203580 dramatically down-regulated LPS-induced mRNA transcript levels of TNF-α and IL-6 in these cells. Our data show that SB203580 differentially modulates LPS-induced production of the inflammatory cytokine IL-6 in two different sources of macrophages, and that this course of regulation occurs at the IL-6 mRNA post-transcriptional stage.

Bone Morphogenetic Proteins: Promising Molecules for Bone Healing, Bioengineering, and Regenerative Medicine.

Bone morphogenetic proteins (BMPs), glycoproteins secreted by some cells, are members of the TGF-β superfamily that have been implicated in a wide variety of roles. Currently, about 20 different BMPs have been identified and grouped into subfamilies, according to similarities with respect to their amino acid sequences. It has been shown that BMPs are secreted growth factors involved in mesenchymal stem cell differentiation, also being reported to control the differentiation of cancer stem cells. BMPs initiate signaling from the cell surface by binding to two different receptors (R: Type I and II). The heterodimeric formation of type I R and II R may occur before or after BMP binding, inducing signal transduction pathways through SMADs. BMPs may also signal through SMAD-independent pathways via mitogen-activated protein kinases (ERK, p38MAPKs, JNK). BMPs may act in an autocrine or paracrine manner, being regulated by specific antagonists, namely: noggin and chordin. Genetic engineering allows the production of large amounts of BMPs for clinical use, and clinical trials have shown the benefits of FDA-approved recombinant human BMPs 2 and 7. Several materials from synthetic to natural sources have been tested as BMP carriers, ranging from hydroxyapatite, and organic polymers to collagen. Bioactive membranes doped with BMPs are promising options, acting to accelerate and enhance osteointegration. The development of smart materials, mainly based on biopolymers and bone-like calcium phosphates, appears to provide an attractive alternative for delivering BMPs in an adequately controlled fashion. BMPs have revealed a promising future for the fields of Bioengineering and Regenerative Medicine. In this chapter, we review and discuss the data on BMP structure, mechanisms of action, and possible clinical applications.

Phosphoinositide 3-kinase/Akt Pathway Mediates Fip1-like1-platelet-derived Growth Factor Receptor α-induced Cell Infiltration and Activation: Possible Molecular Mechanism for the Malignant Phenotype of Chronic Eosinophilic Leukemia

The fip1-like1/platelet-derived growth factor receptor-α fusion gene (F/P) is responsible for 14-60% cases of hypereosinophilia syndrome (HES), also known as F/P-positive chronic eosinophilic leukemia (F/P(+) CEL). The major pathogenesis of F/P(+) CEL is known to involve migration and activation of mast cells and eosinophils, leading to severe multi-organ dysfunction, but the mechanism was still unclear. Phosphoinositide 3-kinase (PI3K) and serine-threonine protein kinase Akt have been reported to be targets of F/P in the F/P-promoted cell proliferation. They are extensively involved in the migration and adhesion of hematopoietic stem/progenitor cells, and also control cell invasion in some leukemias. The PI3K/Akt pathway is involved in eosinophil/neutrophil activation and infiltration; its possible role in regulating F/P induced cytotoxicity and upregulation of A4-integrin in eosinophils, and inducing eosinophil activation through controlling F/P-induced Nuclear factor-kB activity. Akt was recently shown to be stimulated by F/P, synergistically with stem cell factor, resulting in the induction of MCs migration and excessive activation. PI3K/Akt pathway is also a principal mediator of interleukin-5 (IL-5)-induced signal transduction promoting eosinophil trafficking and degranulation, whereas IL-5 is a necessary cytokine for F/P-mediated CEL development. We, therefore, propose the hypothesis that the PI3K/Akt pathway might be vital downstream of F/P to induce target cell activation and tissue infiltration, resulting in the malignant phenotype seen in F/P(+) CEL.

UPAR Directly Interacts with LDLR-like Proteins to Induce Angiogenic Endothelial Cell Behavior

In this study we characterized a conserved motif of domain 3 of the urokinase-type plasminogen activator receptor (uPAR) to directly interact with low-density lipoprotein receptor (LDLR)-related protein (LRP) family proteins, thereby affecting endothelial cell motility and angiogenesis in vitro and in vivo.There is increasing evidence that uPAR plays a central role in growth factor induced endothelial cell activation. Beside its proteolytic role, urokinase-type plasminogen activator (uPA) / uPAR-complex formation induces intracellular signal transduction, which leads to endothelial cell migration and invasion. Since uPAR is a GPI-anchored protein, an interaction with transmembrane proteins - such as members of the LDL-receptor family - is required, inducing signal transduction but also regulating distribution of uPAR via its internalization and recycling to the leading edge. Recently, a direct interaction between uPAR and LRP-family members has been suggested to be sufficient to mediate internalization of uPAR-complex. A crystal structure analysis revealed a small sequence of domain 3 (D3) of uPAR, to be highly exposed upon uPA binding to its receptor.Applying affinity chromatography analysis as well as mutation expression studies, we identified the sequence as an LRP-binding motif, which affects endothelial cell spreading, migration and invasion upon VEGF in vivo as well as in vitro.In detail, matrigel-filled angioreactors with embedded retroviral constructs, carrying wild-type or modified uPAR genes, were implanted subcutaneously into uPAR deficient C57BL/6 mice. After explantation, blood vessel in-growth analysis revealed that only angioreactors with reconstituted wild-type uPAR but not reactors with modified uPAR, being deficient in LDLR interaction, showed angiogenesis.To test a therapeutic impact, peptides mimicking the binding motif and competitive for LDLR binding were used. We found that in a dose dependent manner the peptides did not only block uPAR/LDLR-like protein interaction, but were also capable of blocking VEGF-induced endothelial cell migration in vitro.In summary, our data show that a conserved motif of uPAR domain 3 is capable to interact with LDLR-like proteins, which is required for efficient growth-factor induced endothelial cell behavior. Preliminary functional data suggest that this extracellular motif might be a potential therapeutic target in angiogenesis dependent diseases such as cancer. DisclosuresNo relevant conflicts of interest to declare.

T cell immune abnormalities in immune thrombocytopenia.

Immune thrombocytopenia is an autoimmune disease with abnormal T cell immunity. Cytotoxic T cells, abnormal T regulatory cells, helper T cell imbalance, megakaryocyte maturation abnormalities and abnormal T cell anergy are involved in the pathogenesis of this condition. The loss of T cell-mediated immune tolerance to platelet auto-antigens plays a crucial role in immune thrombocytopenia. The induction of T cell tolerance is an important mechanism by which the pathogenesis and treatment of immune thrombocytopenia can be studied. Studies regarding the roles of the new inducible costimulator signal transduction pathway, the ubiquitin proteasome pathway, and the nuclear factor kappa B signal transduction pathway in the induction of T cell tolerance can help improve our understanding of immune theory and may provide a new theoretical basis for studying the pathogenesis and treatment of immune thrombocytopenia.

Recombinant p35 from bacteria can form Interleukin (IL-)12, but Not IL-35.

The Interleukin (IL)-12 family contains several heterodimeric composite cytokines which share subunits among each other. IL-12 consists of the subunits p40 (shared with IL-23) and p35. p35 is shared with the composite cytokine IL-35 which comprises of the p35/EBI3 heterodimer (EBI3 shared with IL-27). IL-35 signals via homo- or heterodimers of IL-12Rβ2, gp130 and WSX-1, which are shared with IL-12 and IL-27 receptor complexes, respectively. p35 was efficiently secreted in complex with p40 as IL-12 but not with EBI3 as IL-35 in several transfected cell lines tested which complicates the analysis of IL-35 signal transduction. p35 and p40 but not p35 and EBI3 form an inter-chain disulfide bridge. Mutation of the responsible cysteine residue (p40C197A) reduced IL-12 formation and activity only slightly. Importantly, the p40C197A mutation prevented the formation of antagonistic p40 homodimers which enabled the in vitro reconstitution of biologically active IL-12 with p35 produced in bacteria (p35bac). Reconstitution of IL-35 with p35bac and EBI3 did, however, fail to induce signal transduction in Ba/F3 cells expressing IL-12Rβ2 and gp130. In summary, we describe the in vitro reconstitution of IL-12, but fail to produce recombinant IL-35 by this novel approach.

131: Pomalidomide disrupts the association between Cereblon and Rabex-5 resulting in the suppression of TLR induced cytokine production

The Teratogenic properties of Thalidomide, in addition to its efficacy in the treatment of Multiple Myeloma, are dependent on Cereblon. Importantly however, the role of Cereblon in mediating Thalidomide’s anti-inflammatory properties remains unclear. Using shRNA knockdown we show that Cereblon functions critically in TLR induced signal transduction. Furthermore, we show that under physiological conditions a proportion of total cellular Cereblon exists in complex with Rabex-5, a mediator of endosomal transport. This interaction was found to be dependent on the Rabex-5N terminal ubiquitin binding domain. Significantly, the Thalidomide derivative Pomalidomide was shown to reduce the association of Cereblon with Rabex-5. We further show that Pomalidomide inhibits TRIF/IRF3 dependent gene induction and that both Cereblon and Rabex-5 function critically in this pathway. These findings clearly indicate that the anti-inflammatory properties of Thalidomide derivatives result from interference with the function of Cereblon and Rabex-5 in TLR induced signal transduction. We anticipate that further investigation of the relationship between Cereblon and Rabex-5 will inform our understanding of Thalidomide and its derivatives in other contexts. Moreover, the identification of Cereblon as a critical regulator of the TLR signalling apparatus will reveal novel aspects of signal transduction and contribute to our understanding of innate immunity.

A model integration approach linking signalling and gene-regulatory logic with kinetic metabolic models

Systems biology has to increasingly cope with large- and multi-scale biological systems. Many successful in silico representations and simulations of various cellular modules proved mathematical modelling to be an important tool in gaining a solid understanding of biological phenomena. However, models spanning different functional layers (e.g. metabolism, signalling and gene regulation) are still scarce. Consequently, model integration methods capable of fusing different types of biological networks and various model formalisms become a key methodology to increase the scope of cellular processes covered by mathematical models. Here we propose a new integration approach to couple logical models of signalling or/and gene-regulatory networks with kinetic models of metabolic processes. The procedure ends up with an integrated dynamic model of both layers relying on differential equations. The feasibility of the approach is shown in an illustrative case study integrating a kinetic model of central metabolic pathways in hepatocytes with a Boolean logical network depicting the hormonally induced signal transduction and gene regulation events involved. In silico simulations demonstrate the integrated model to qualitatively describe the physiological switch-like behaviour of hepatocytes in response to nutritionally regulated changes in extracellular glucagon and insulin levels. A simulated failure mode scenario addressing insulin resistance furthermore illustrates the pharmacological potential of a model covering interactions between signalling, gene regulation and metabolism.

Role of advanced glycation end product (AGE)-induced receptor (RAGE) expression in diabetic vascular complications

AimsVascular complications are the major causes of morbidity and mortality in diabetic subjects. Interaction of advanced glycation end products (AGEs) with their receptor (RAGE) induces signal transduction that culminates in vascular complications. Therefore, in the present study we investigated the dependence of RAGE expression on circulating AGEs and evaluated the outcome of AGE–RAGE interaction by the oxidative stress and nature of vascular complications in type 2 diabetes mellitus (T2DM) patients. MethodsRAGE expression was determined by quantitative real-time PCR and western blotting, serum AGEs were estimated by ELISA and spectrofluorometry and oxidative stress markers namely protein carbonyl (PCO), advanced oxidation protein products (AOPP) and lipid peroxidation (MDA) were assayed spectrophotometerically in 75 T2DM patients (DM without vascular complication n=25; DM with microvascular complications n=25; DM with macrovascular complications n=25) and 25 healthy controls. ResultsSerum AGE level was significantly higher in diabetic patients having vascular complications as compared to T2DM without complications (p<0.01). RAGE m-RNA expression level in PBMCs assayed by quantitative real time PCR was four times higher in diabetic subjects without vascular complications while DM patients having microvascular and macrovascular complications showed 12 fold and 8 fold higher RAGE m-RNA expression respectively compared to healthy controls. Circulating AGE level showed significant positive correlation with RAGE m-RNA expression and oxidative stress markers. ConclusionAGE-mediated exacerbation of RAGE expression may contribute to oxidative stress generation that plays a key role in pathogenesis of vascular complications in diabetes.

Visualization of the pH-dependent dynamic distribution of G2A in living cells.

G2A (from G2 accumulation) receptor is a member of the proton-sensing G-protein coupled receptor (GPCR) family and induces signal transduction events that regulate the cell cycle, proliferation, oncogenesis, and immunity. The mechanism by which G2A-mediated signal transduction is regulated by the extracellular pH remains unresolved. Here, we first visualize the pH-dependent G2A distribution change in living cells by a sortase A-mediated pulse labeling technology: the short-peptide tag-fused human G2A on human embryo kidney HEK293T cell surfaces was labeled with a small fluorescent dye in the presence of lysophosphatidylcholine, and the labeled G2A was chased at acidic and neutral pHs in real time by microscope time course observations. G2A internalization from cell surfaces into intracellular compartments was observed to be inhibited under acidic pH conditions, and this inhibition was relieved at neutral pH. Additionally, the internalized G2A was redistributed onto cell surfaces by jumping from a neutral to an acidic pH. From quantitative image analysis data, we conclude the amount of G2A on the cell surface was controlled by suppressing the G2A internalization rate by one-tenth in response to the extracellular acidic pH, and this acidic pH-induced G2A accumulation on cell surfaces may be explained by proton-induced dissociation of G2A from endocytic machinery.

Preclinical Therapeutic Potential of a Nitrosylating Agent in the Treatment of Ovarian Cancer

This study examines the role of s-nitrosylation in the growth of ovarian cancer using cell culture based and in vivo approaches. Using the nitrosylating agent, S-nitrosoglutathione (GSNO), a physiological nitric oxide molecule, we show that GSNO treatment inhibited proliferation of chemoresponsive and chemoresistant ovarian cancer cell lines (A2780, C200, SKVO3, ID8, OVCAR3, OVCAR4, OVCAR5, OVCAR7, OVCAR8, OVCAR10, PE01 and PE04) in a dose dependent manner. GSNO treatment abrogated growth factor (HB-EGF) induced signal transduction including phosphorylation of Akt, p42/44 and STAT3, which are known to play critical roles in ovarian cancer growth and progression. To examine the therapeutic potential of GSNO in vivo, nude mice bearing intra-peritoneal xenografts of human A2780 ovarian carcinoma cell line (2×106) were orally administered GSNO at the dose of 1 mg/kg body weight. Daily oral administration of GSNO significantly attenuated tumor mass (p<0.001) in the peritoneal cavity compared to vehicle (phosphate buffered saline) treated group at 4 weeks. GSNO also potentiated cisplatin mediated tumor toxicity in an A2780 ovarian carcinoma nude mouse model. GSNO’s nitrosylating ability was reflected in the induced nitrosylation of various known proteins including NFκB p65, Akt and EGFR. As a novel finding, we observed that GSNO also induced nitrosylation with inverse relationship at tyrosine 705 phosphorylation of STAT3, an established player in chemoresistance and cell proliferation in ovarian cancer and in cancer in general. Overall, our study underlines the significance of S-nitrosylation of key cancer promoting proteins in modulating ovarian cancer and proposes the therapeutic potential of nitrosylating agents (like GSNO) for the treatment of ovarian cancer alone or in combination with chemotherapeutic drugs.

Expression of Nemo-like kinase after spinal cord injury in rats

Wnt can induce signal transduction via the canonical pathway, which was involved in many processes in the nervous system. Nemo-like kinase (NLK) acts as a negative regulator of β-catenin/T-cell factor/lymphoid enhancer factor (LEF) and functions downstream of transforming growth factor β-activated kinase-1 in the Wnt signaling pathway. In this study, we performed a spinal cord injury (SCI) test in adult Sprague-Dawley rats and investigated the dynamic changes and role of NLK expression in the spinal cord. Western blot analysis revealed that NLK expression was low in normal spinal cord. It then increased markedly, peaked at 3days, and declined to basal levels from 5days after injury. Immunohistochemistry confirmed that NLK immunoactivity was expressed at low levels in gray and white matter under normal conditions and increased prominently in gray matter after the SCI test. Double immunofluorescent staining for NLK, caspase-3, β-catenin, and NeuN (neuronal nuclei) revealed that NLK and β-catenin were markedly increased and colocalized in apoptotic neurons. Coimmunoprecipitation data demonstrated that overexpression of NLK protein reduced β-catenin binding to LEF-1. Our results suggested that NLK was associated with neuronal apoptosis through attenuating the Wnt/β-catenin signaling pathway after SCIs.

RAGE gene polymorphism and expression: risk factor for vascular complications in type 2 diabetes mellitus

Background Advanced glycation end products (AGEs) are formed due to hyperglycemia in T2DM. Interaction of AGEs with its receptor RAGE induces signal transduction that culminates in vascular complications, the major cause of morbidity and mortality in diabetic subjects. Some functional polymorphism of this gene show differential activity of this receptor and therefore may be associated with the development of diabetic complications. In the present study we investigated the association of expression of RAGE gene and its polymorphism namely -374T/A and -429T/C in the promoter region and Gly82Ser polymorphism in the exon 3 region with vascular complications in T2DM patients.

TIM-3, a Leukemia Stem Cell Marker, Plays a Role In Leukemic Transformation Through Autocrine Stimulatory Signaling By Its Ligand, Galectin-9

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. We have reported that the T-cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML but not on normal hematopoietic stem cells (HSCs) (Kikushige et al, Cell Stem Cell, 2010). We extended the analysis of TIM-3 expression into various types of human hematological malignancies, and found that human TIM-3 is expressed in the vast majority of CD34+CD38- LSCs of human myeloid malignancies including chronic myeloid leukemia, chronic myelomonocytic leukemia and myelodysplastic syndromes (MDS). Although CD34+CD38- normal bone marrow stem cells do not express TIM-3, TIM-3 is expressed in the CD34+CD38- population in MDS, and is further up-regulated with progression into leukemia. The average percentages of TIM-3+ cells in the CD34+CD38- population was 7.8% in RCMD (n=10), 19.2% in RAEB-1 (n=10), 84.0% in RAEB-2 (n=10) and 92.2% in overt AML (n=10). The close association of TIM-3 expression with transformation into AML led us to hypothesize that TIM-3 itself has a function in AML stem cell development. TIM-3 is a type 1 cell-surface glycoprotein and has a structure that includes an N-terminal immunoglobulin variable domain followed by a mucin domain, a transmembrane domain and a cytoplasmic tail. Tyrosine residues are clustered in the cytoplasmic tail, suggesting that TIM-3 can induce signal transduction in TIM-3+ AML cells. To understand the function of TIM-3, we investigated the interaction between TIM-3 and its ligand galectin-9 in AML LSCs. We found that AML patients showed significantly higher serum galectin-9 concentration than healthy individuals (healthy controls: 18.3+4.3 pg/ml, AML patients: 139.1+33.4 pg/ml, P<0.05). Unexpectedly, we found that leukemic cells expressed a high level of galectin-9 protein, as compared to other hematopoietic cells including T cells, B cells and monocytes. Using KASUMI-3 (TIM-3+ AML cell line) and primary AML samples, we confirmed that AML cells could secrete galectin-9 after TLR stimulation in vitro. Furthermore, microarray analysis demonstrated that TIM-3 stimulation by the physiological concentration of galectin-9 induced significant gene expression changes toward pro-survival axis including up-regulation of MCL-1, the important survival factor for HSCs and LSCs. These results collectively suggest that AML cells can produce and secrete galectin-9, and galectin-9 can bind and stimulate TIM-3-expressing AML cells including LSCs in an autocrine manner to support their survival or leukemia progression. Disclosures:Miyamoto:Kyushu University Hospital: Employment.

Hypodermal delivery of cosmetic actives for improved facial skin morphology and functionality

Skin compartments traditionally targeted by cosmetic actives - epidermis and dermis - are anchored and nourished by the underlying hypodermis, which therefore should be a key target for skin-rejuvenating formulations. However, given the difficulty to reach even the superficial layers of the skin, and to its 'unglamorous' fatty composition, the regenerative potential of hypodermis remains largely untapped. Therefore, this study was to investigate the capacity of a cosmetic material to trigger a regenerative response in dermis and epidermis through a selective action on hypodermis. Furthermore, it aimed to establish the effect of such cosmetic material in transbuccal hypodermal delivery form, on the hypodermal precursor cells - the preadipocytes. A combination of grape seed extract and soy phospholipids was formulated and standardized for elastase activity and free radical inhibition. This formulation was then used to contact the hypodermal layer of human skin biopsies and - under a transbuccal delivery vehicle form - the 3T3-L1 preadipocytes, and its effects were quantified using PCR arrays and histochemistry. Application of the standardized grape/soy material to the hypodermal layer of skin triggered modulation of gene expression in the upper layers of the skin and resulted in the clear morphological improvement at the dermal and epidermal levels. Furthermore, when this material was formulated in a mucoadhesive, intraoral film and applied on 3T3-L1 preadipocytes, the resulting modulation of gene expression in these cells was consistent with differentiation and detoxification effects. These results suggest that transbuccal formulations of nutraceutical grade cosmetics have potential to induce signal transduction pathways in facial hypodermis, resulting in anti-aging effects throughout all skin compartments, including dermal and epidermal layers.