Fibroblast growth factor-1 (FGF-1) is a well characterized growth factor among the 22 members of the FGF superfamily in humans. It binds to all the four known FGF receptors and regulates a plethora of functions including cell growth, proliferation, migration, differentiation, and survival in different cell types. FGF-1 is involved in the regulation of diverse physiological processes such as development, angiogenesis, wound healing, adipogenesis, and neurogenesis. Deregulation of FGF-1 signaling is not only implicated in tumorigenesis but also is associated with tumor invasion and metastasis. Given the biomedical significance of FGFs and the fact that individual FGFs have different roles in diverse physiological processes, the analysis of signaling pathways induced by the binding of specific FGFs to their cognate receptors demands more focused efforts. Currently, there are no resources in the public domain that facilitate the analysis of signaling pathways induced by individual FGFs in the FGF/FGFR signaling system. Towards this, we have developed a resource of signaling reactions triggered by FGF-1/FGFR system in various cell types/tissues. The pathway data and the reaction map are made available for download in different community standard data exchange formats through NetPath and NetSlim signaling pathway resources. Fibroblast growth factor (FGF) superfamily consists of structurally related polypeptides most of which function through its high affinity fibroblast growth factor receptors (FGFRs). In addition to FGFRs, they also bind to heparan sulfate proteoglycans (HPSGs) and their analog, heparin. These interactions influence the stability of FGFs in the extracellular matrix and also regulate their binding and activation of FGFRs –9. In hu2+ ions [Most FGFs have an N-terminal signal peptide and are thus secreted. FGFs 1, 2, 9, 16, and 20 do not have signal peptides. FGFs 9, 16, and 20 may be released through classical secretory pathway; however, FGF-1 and FGF-2 are released from damaged cells or through endoplasmic reticulum-golgi independent exocytotic pathway . FGF-1 a2+ ions –37. FGF-2+ ions –40. Admi2+ ions  and isch2+ ions –44. Incr2+ ions  and pros2+ ions . Taken t2+ ions , 48. A phttp://pid.nci.nih.gov/), Database of Cell Signaling (http://stke.sciencemag.org/cm/), KEGG Pathway Database (http://www.genome.jp/kegg/pathway.html), and INOH Pathway Database (http://inoh.org/) have cataloged basic components of FGF signaling. We have expanded the scope of this by providing a comprehensive representation of FGF1 signaling pathway and its diverse roles in regulating various cellular processes.Curated pathway maps are invaluable resources for scientific community. Such comprehensive pathway datasets are being increasingly used in bioinformatics efforts directed towards analysis of high-throughput datasets from various disease contexts. Repositories including Pathway Interaction Database of the National Cancer Institute  physical associations between proteins, (b) posttranslational modifications (PTMs), (c) change in subcellular localization of proteins, (d) activation or inhibition of specific proteins, and (e) regulation of gene expression. Relevant information from research articles were manually documented using the curation tool, PathBuilder. To streamline and organize data collection from literature, we followed the previously described criteria for the inclusion/exclusion of pathway specific reactions , 50. TheCanonical FGF/FGFR signaling reactions have been documented in a few public repositories and review articles. Vast amount of literature in the last few years have revealed several novel pathway intermediates of FGF/FGFR signaling system. In order to generate a comprehensive view of FGF/FGFR signaling pathway, we carried out extensive literature search on PubMed for articles pertaining to FGF-1 signaling. Of a total of 3275 articles that were screened, 237 of them had molecular reactions reported downstream of FGF-1 in various cell types/tissues. Manual curation from these research articles revealed 109 molecules involved in FGF-1 induced physical associations, modulation by PTMs, activity, and subcellular or cell surface translocation events. Of the 42 physical associations that were cataloged, 29 were “binary” and 13 were “complex” interactions inclusive of the ligand/receptor interactors. We could record a total of 87 catalysis events, 15 activation/inhibition, and 21 translocation events. The 87 catalysis events include 19 events, where the enzymes directly catalyzing the reactions were studied and reported, and 68 events for which the enzymes which post-translationally modified the proteins are not studied under FGF-1 stimulation. Apart from these molecular reactions, we have also cataloged 117 genes whose expression is reported to be either upregulated or downregulated by FGF-1 treatment. However, only a total of 25 genes were reported to be differentially regulated at mRNA level by FGF-1 stimulation in different human cell types. A list of genes reported to be regulated by FGF-1 in different mammalian systems at the mRNA and/or the protein level is provided in Signaling modules comprise a well-characterized group of molecules and their interactions downstream of activation of a receptor. We documented the following signaling modules to be activated upon stimulation with FGF-1.The Ras/Raf/Mek/Erk pathway has been implicated in cellular processes including cell growth, proliferation, and migration. Stimulation of different cell types with FGF-1 resulted in the formation of multiple complexes involving FRS2, GAB1, SOS1, PTPN11, SHC1, SH2B1, and GRB2 –60. ThesThe complexes mentioned above also lead to the activation of Pi3k/Akt pathway, another signaling module that regulates various processes including cell growth, survival, cell proliferation, and cell migration . A numbe (Jnk) pathway is implicated in the regulation of cell cycle, cell survival and apoptosis. FGF-1 stimulates the phosphorylation of p38 Mapk (MAPK14) as well as Jnk1/2 (MAPK8/9). The Jnk1/2 was also found to be crucial to neurogenesis and vascular remodeling [The c-jun N-terminal kinasemodeling , 74. Themodeling –77.FGF-1 also stimulates STATs (STAT1 and STAT3) and Nf-kB signaling modules. FGFR signaling is reported to be regulated through several downstream molecules including JAK2, SRC, SH2B1, MAPK1/3, MAPK8/9, and STAT3. This signaling axis is known to regulate various cellular processes including neurite outgrowth, cell proliferation, and increased cancer cell invasion –80. In aFGF-1 was found to be involved in a number of biological processes. It is associated with the development of heart , lens 8, lung, ahttp://www.netpath.org/index.html). Additionally, we have drawn a map of FGF-1/FGFR signaling using the data accumulated in NetPath. This network map represents the molecules and their reactions organized by topology and excludes the molecules identified through phosphoproteomics approaches for which topology could not be assigned (http://www.netpath.org/netslim/FGF-1_pathway.html. Each node in the map is linked to their molecule page in NetPath, thereby to other pathways in NetPath, and to HPRD [User-friendly visualization of pathways is an important aspect to provide a concise view. A number of tools are available for visualization and analysis of pathway data including Cytoscape , ChisioBassigned . The map to HPRD  and RefSAvailability of specific ligand-receptor mediated signaling data in community approved formats is crucial to the understanding of proteins and their reactions in diverse biological processes. Analysis of high-throughput data obtained from microarray- and mass spectrometry-based platforms essentially relies on enrichment of biological function or signaling pathways available in databases to obtain insights into their physiological functions. Although some resources have cataloged FGF signaling in general, this is the first attempt to provide a comprehensive view of FGF-1 signaling. This will be extended to other FGF ligands and/or specific FGFRs in the future to facilitate the analysis of differences between different FGFs and/or FGFRs. The pathway information has been made available through NetPath and NetSlim resources in multiple community standard data formats. The FGF-1 signaling pathway data will be periodically updated in NetPath. We have cataloged multiple signaling modules that are activated upon activation of FGFR and their implications in diverse physiological and pathophysiological processes. We believe that the data presented here will boost further research in this area and will help identify novel therapeutically important molecules that could be targeted in pathological conditions involving aberrant FGF-1 signaling.


HAM/TSP patients show increased HTLV-1 proviral load in peripheral blood mononuclear cells (PBMCs), however, little is known about the character of HTLV-1-infected cells. It has been considered that the immune system of HTLV-1-infected individuals are impaired, however, the details are unknown. Here, we investigated HTLV-1-infected cells from HAM/TSP patients for their surface markers and immune function. PBMCs were obtained from the patients and cultured for several hours. HTLV-1-infected cells were identified by detection of intracellular HTLV-1 Tax protein. The Tax-positive, HTLV-1-infected cells showed a phenotype of CD2+CD4+CD5+CD26- CD45RO+CD45RA- CCR4+CCR7- and a reduced expression of T cell receptor (TCR) and CD3 antigen. Next, PBMCs were stimulated with CD3 antibody and TCR signaling was detected by phospho-specific antibodies for Lck and ZAP70, which are early signaling molecules of TCR. The degree of phosphorylation in whole CD4+ cells from HAM/TSP patients were lower than that from normal controls. In addition, Tax-positive CD4+ cells showed reduced phosphorylation of these molecules compared to Tax-negative CD4+ cells in HAM/TSP patients. Finally, cytomegalovirus (CMV)-specific, HTLV-1-infected CD4+ cells showed a decreased production of interferon-gamma by stimulation of CMV antigens compared to CMV-specific, non-infected cells in the patients. These results indicate that HTLV-1-infected cells reduced expression of TCR/CD3 complex in HAM/TSP patients, resulting in a reduction in TCR signaling and impaired immune function.


Saccharomyces cerevisiae BT0510 was exposed to 50 MPa for 30 min at room temperature, followed by incubation at room pressure with aeration for 15 min. Samples of total RNA were collected every 5 min for transcriptional analysis by DNA microarray technique. Bioinformatics analysis demonstrated the upregulation (≥ 2 fold) by HHP treatment of genes related to the sulfur amino acids synthesis, methionine and cysteine. The HHP treatment induced the genes MET3, MET10, MET14 and MET16, which are correlated with the conversion of intracellular sulfate in sulfide. MET2, related to the conversion of homoserine to O-acetylhomoserine, was also induced by HHP, as well as the gene that codes for Met17p, responsible for the incorporation of sulfide in O-acetylhomoserine to form homocysteine, that will be directed to methionine or cysteine synthesis. These amino acids are directly correlated with sulfur assimilation in yeast cells. Methionine is the S-adenosylmethionine precursor, which participates in the biosynthesis of lipids and polyamines, and is also involved in methylation reactions, being a methyl group donor [MET6) was upregulated by HHP, while the gene related to the biosynthesis of cysteine (CYS4) was unaffected. Five minutes after pressure release MET6 was repressed. The genes related to the conversion of methionine to S-adenosylmethionine, SAM1 and SAM2, were downregulated. Methionine residues are important against reactive oxygen species (ROS) [MXR1 and MXR2) were induced by HHP treatment, suggesting that methionine plays an important role in the reduction of ROS resulting from stress caused by HHP [MET28 was strongly induced during the entire HHP and post treatment. Other factors, such as the transcription factor encoded by MET4 were not affected by HHP, and also MET30 that negatively regulates Met4p. Met28p appears to play an important role in the biosynthesis of sulfur amino acids in response to HHP. It seems that this protein participates in the Met4 complexes-DNA stabilization. Methionine biosynthesis upregulation is not related to other stresses, such as heat and osmotic stresses, and appears to be specific to HHP, which reinforces the use of this treatment to study the stress response in microorganisms.High hydrostatic pressure (HHP) is successfully applied in several industrial segments, as in vaccine production and food conservation. The response of microorganisms to HHP treatment resemble the responses of other stresses with industrial relevance, like osmotic, temperature and ethanol, which make the HHP a valuable tool in biotechnology research, as in the ethanol production . Amino alation ≥  fold by lation ≥  fold by


Aspergillus fumigatus is the most important airborne fungal pathogen, causing severe infections with invasive growth in immunocompromised patients. The fungal cell wall (CW) prevents the cell from lysing and protects the fungus against environmental stress conditions. Because it is absent in humans and because of its essentiality, the fungal CW is a promising target for antifungal drugs. Nowadays, compounds acting on the CW, i.e., echinocandin derivatives, are used to treat A. fumigatus infections. However, studies demonstrating the clinical effectiveness of echinocandins in comparison with antifungals currently recommended for first-line treatment of invasive aspergillosis are still lacking. Therefore, it is important to elucidate CW biosynthesis pathways and their signal transduction cascades, which potentially compensate the inhibition caused by CW- perturbing compounds. Like in other fungi, the central core of the cell wall integrity (CWI) signaling pathway in A. fumigatus is composed of three mitogen activated protein kinases. Deletion of these genes resulted in severely enhanced sensitivity of the mutants against CW-disturbing compounds and in drastic alterations of the fungal morphology. Additionally, several cross-talk interactions between the CWI pathways and other signaling pathways are emerging, raising the question about their role in the CW compensatory mechanisms. In this review we focused on recent advances in understanding the CWI signaling pathway in A. fumigatus and its role during drug stress response and virulence. The fungal cell wall (CW) is the exoskeleton of fungal cells. Besides its starkness and firmness, it has a very flexible structure, which, therefore, can alter rapidly and efficiently in response to external and internal stimuli. In particular, filamentous fungi adapt their internal pressure and turgor in concert with CW-biosynthesis enzymes, in order to direct hyphal growth, following gradients of nutrients, and chemo attractants , or to avoid adverse habitats .Aspergillus fumigatus is completed by the addition of sugar-chains composed of α-1,3-glucan, galactofuran, and mannan, which make the structure rather variable compared to other fungi  plays the major role . Fks1 ise lethal . Howeverhenotype .A. fumigatus genome harbors many putative genes responsible for CW modifications. Different β-glucanases and branching enzymes were identified  anchored proteins . However, it is increasingly acknowledged that defense mechanisms against these drugs have common elements. Consistently, a genome-wide screen conducted in Schizosaccharomyces pombe indicated that different ergosterol biosynthesis deficient mutants were also sensitive to β-1,3-glucan synthase inhibitor echinocandins and β-glucanase . The fungicidal activity of AmB is potentially based on the formation of channels in the cell membrane, which lead to increased membrane permeability and leakage of small molecules, inhibition of aerobic and anaerobic respiration, and accumulation of reactive oxygen species (ROS) . It was suggested that eight AmB molecules are assembled to a ring-like structure, showing both a hydrophobic interaction to ergosterol with its conjugated double-bond system and forming a hydrophilic pore with its inward directed hydroxyl groups , first isolated from fections . AmB posl groups . Thus, tCandida , which are characterized by an intrinsically low ergosterol content . In A. fumigatus, two genes have been assigned to this enzymatic function: cyp51A and cyp51B (erg11) . However, single deletions of genes involved in either demethylation or desaturation of ergosterol intermediates such as cyp51A, cyp51B, and erg3A, erg3B and erg3C, respectively, showed differences in relative sterol composition, but further tests indicated that neither of these genes is individually essential for A. fumigatus survival and virulence  . The dedirulence .A. fumigatus against azole antifungal drugs is based on a mutation of the cyp51A gene locus with a conversion of leucine at position 98 into histidine (L98H) in combination with the appearance of a 34-bp tandem repeat in cyp51A promoter region (TR34). In addition to TR34/L98H, also other hot spots for point mutations in the cyp51A gene have been reported, e.g., the exchange of glycine at pos. 54, with different impact on azole resistance in clinical isolates  pathway, and elements acting in the calcineurin pathway , Mkk2 (MAPK kinase), and MpkA (MAPK), which sequentially phosphorylate each other .Among the different signaling pathways, the MAPK CWI signaling plays a major role in CW maintenance. The CWI signal pathway in The lack of phosphorylation of MpkA yields mutants with a typically impaired CW, showing compact colonies and reduced filamentation . AdditiompkA mutant of A. fumigatus showed that the deletion of this important kinase globally changed the expression of genes putatively involved in primary metabolism. Many genes involved in sugar and amino acid metabolism were differentially regulated, whereas many well-known CW biosynthesis genes were not affected  melanin, which is responsible for the characteristic gray-greenish color of the conidia , and downstream (TFs) elements are more diverse . In partstress response components)  , which al stress . BLAST agprC and gprD genes in A. fumigatus produced mutant strains impaired in CW-related stress response . The number of these receptors also varies between different species . Deletioresponse .S. cerevisiae, receptors activating the MAPK signaling cascade are connected to the MAPK module via different signaling elements. BLAST analysis revealed that those elements are also conserved in the A. fumigatus genome , there are no studies describing its function in tabolism . Howeverntil now .S. cerevisiae were identified by BLAST analysis also in the A. fumigatus genome  and the CWI signaling pathway has been elucidated in detail. Under CW stress, the pathways can display co-regulatory roles depending on the stress-inducing agent. It was shown that Hog1 (the yeast SakA ortholog) was activated in the absence of Slt2 (the yeast MpkA ortholog) under conditions causing CW stress  in A. fumigatus resulted in the loss of the paradoxical growth phenotype under caspofungin stress, which was attributed to the transcriptional regulation of chitin synthase-encoding genes by this signal transduction pathway  can in part restore the wild-type phenotype of a ΔcnaA deletion mutant . Surprisingly, many mutant strains with defects in CW biosynthesis enzymes were still virulent in the applied infection models. As an example, both the α-1,3-glucan synthase mutant strains, Δags1, and Δags2, were still pathogenic, although their α-1,3-glucan content was reduced by 50% compared to the wild-type strain (ags3 mutant was reported to be even hyper-virulent (gel2) and a α-1,2-mannosyltransferase (mnt1) resulted in a decrease of virulence (It is common praxis in infection biology to test the virulence of mutant strains in mouse infection models, in order to define whether genes have a potential function as virulence determinant. Among the different e strain . Moreovevirulent . To our irulence .calA, ΔrasA, and B, and ΔpkaC mutants (ptcB, a putative protein phosphatase 2C, positively affected the phosphorylation status of both MpkA and SakA, resulting in decreased virulence (As reported above, structuring of the CW depends on different signaling pathways. The observation that these pathways are connected to each other is increasingly acknowledged. Deletion of a single gene often affected more than one specific signaling pathway. In fact, many of the reported mutants with impaired CW, which displayed decreased virulence, were obtained by deleting genes putatively involved in signaling, such as Δ mutants . Recentlirulence . Similarirulence . Howeverirulence .A. fumigatus target either enzymatic steps involved in cell membrane/CW biosynthesis or ergosterol as part of the cytoplasmic membrane. These drugs show some limitations either because they cannot really clear A. fumigatus infection, or because resistant strains were isolated and are therefore of major clinical concern. The increase of life-threatening mycoses accompanied with the lack of effective drugs, has fostered the search for new, broad-spectrum fungicidal agents. Drug efficacy could also be increased by the reformulation of existing antifungals as well as the search for synergistically acting compounds that can be used for treatment and prophylaxis. As an example, the effectiveness of caspofungin in prophylaxis is still a matter of debate, and there are studies indicating that caspofungin did not decrease the mortality rate of patients with diagnosed invasive aspergillosis (in vitro (A major question concerns the selection of suitable and effective targets for future antifungal drugs. The three clinically important classes of antifungal drugs used against gillosis . In futugillosis . In linein vitro . Additioin vitro .The fungal CW still remains a very powerful target for antifungal drugs. However, recent studies suggest that the signaling pathways responsible for CW formation have not been completely elucidated. It remains to be shown which signaling pathways act as compensatory pathways that decrease the effectiveness of drug treatments. The identification of such pathways could lead to the discovery of new targets and new modes of action that can be exploited to potentiate the efficiency of known drugs, and to improve prophylaxis against invasive mycoses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The involvement of Wnt in carcinogenesis and progression of pancreatic cancer is currently intensely discussed. We evaluated activation of the Wnt signaling pathway by using a Wnt reporter mouse strain expressing β-galactosidase under the control of the Axin2 promotor during pancreatitis induced formation of precancerous lesions. We also evaluated activation of Wnt signaling during interaction of pancreatic cancer with the tumor stroma.Activation of Wnt signaling was observed during acinar-to-ductal metaplasia after chronic as well as acute pancreatitis. Activation of Wnt signaling was also noticed during growth of pancreatic cancer in an orthotopic syngeneic pancreas cancer model. Activation of Wnt signaling was, however, not observed in carcinoma associated fibroblasts, but was detected in few cell clusters inside the tumor. Genetic ablation of Axin2 significantly reduced body weight without having a major impact on blood glucose concentration. However, ablation of Axin2 had no influence on the observed β-galactosidase positive cell clusters or on tumor weight.These data demonstrate that the Wnt signaling pathway is activated during acinar-to-ductal metaplasia after injury to the pancreas. However these data do not support a major role of Wnt signaling or of Axin2 in carcinoma associated fibroblasts and tumor growth.The online version of this article (doi:10.1186/s13578-016-0116-4) contains supplementary material, which is available to authorized users. This can result in prolonged Wnt signaling in vivo [The expression of Axin2 is induced by canonical Wnt signaling during cancerogenesis but also during many other Wnt-regulated physiological and pathophysiological processes . The intctivated . Axin2 fctivated . In Axin in vivo .It is well established that Wnt signaling is involved in the cancerogenesis of multiple gastrointestinal carcinomas . In coloHowever, it is still controversial how important this signaling pathway is during carcinogenesis of pancreatic cancer . About 2Only few publications exist that suggest that Wnt signaling might also contribute to tumor stroma interaction , 9. ThisThus, the purpose of this study was to evaluate the activation of the Wnt signaling pathway in precancerous lesions such as tubular complexes after acinar-to-ductal metaplasia (ADM) and during the tumor stroma interaction of fully established PDA.+/lacZ mice without pancreatitis no β-galactosidase stained tubular complexes were observed, whereas a strong β-galactosidase staining was observed in tubular complexes during chronic pancreatitis  or hyperglycemic mice (STZ+Cer) and the pancreas was compared to normoglycemic (Sham) or hyperglycemic mice (STZ) without pancreatitis  Activation of Wnt signaling has no effect on cancerogenesis, but is a concomitant phenomenon when cells are stuck in dedifferentiation, (b) Wnt signaling inhibits cancerogenesis, (c) Wnt signaling promotes cancerogenesis. Possibly, the function of Wnt signaling is completely different during cancerogenesis leading to PDA or other subtypes of pancreatic cancer.In respect to cancerogenesis of PDA most data support the first or second hypothesis and disagree with the third hypothesis. For example, activation of Wnt signaling does not cooperate with oncogenic Ras to induce PDA formation in mice . MoreoveIn respect to cancerogenesis of SPNs or ACCs most data disagree with the first and second hypothesis and support the third hypothesis. For example, activation of Wnt signaling in mice results in the formation of tumors resembling human SPNs , whereasACTB-eGFP1Osb/J as well as Axin2+/lacZ mice  or in blood glucose concentration  between mice lacking one or none allele of Axin2. A blood cell count did not reveal major differences in the number of leukocytes , lymphocytes , or monocytes plus neutrophil granulocytes  dependent on the mouse genotype. We did also not observe an obvious influence of Axin2 function on the tumor weight when comparing tumors grown in Axin2+/lacZ to tumors grown in AxinlacZ/lacZ  between mice lacking one or none allele of Axin2. Thus, no major function of Wnt signaling on the growth of pancreatic cancer could be observed in this syngeneic orthotopic animal model.In order to evaluate if Axin2 influences the growth of pancreatic cancer we compared the tumor weight in Axin2ion Fig. a, b. We


The expanded NK cells in the peripheral blood are considered to be reactive, to maintain the confinement of the HTLV-1-positive CD4+ cells in the skin, and to prevent the progression of the disease.Natural killer (NK) cells were proposed to play an important role in the pathogenesis of human T-cell lymphotropic virus type 1- (HTLV-1-) associated neurologic disease. Our patient was a 77-year-old Japanese man, who had been treated for infective dermatitis associated with HTLV-1 for nearly 10 years. When referred to us, he had facial eczema/edema as well as extensive dermatitis at the neck/upper chest and nuchal area/upper back regions. Dermal lesions had CD3+CD4+ cells, but no NK cells. Flow cytometry of his peripheral blood showed a phenotype of CD2+ (97%), CD3+ (17%), CD4+ (12%), CD7+ (94%), CD8+ (6%), CD11c+ (70%), CD16+ (82%), CD19+ (0%), CD20+ (0%), CD56+ (67%), HLA-DR+ (68%), and NKp46+ (36%). Absolute numbers of CD56+NK cells in the peripheral blood were in a range of 986/ Among human T-cell lymphotropic virus type 1 (HTLV-1) infected individuals, adult T-cell leukemia/lymphoma (ATLL), and a chronic neurological disease, the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are two major symptomatic disorders. In addition, dermatological disorders are noted in 5–10% of the HTLV-1 infected patients . The infμL, Hb 12.3 g/dL, platelet counts 91,000/μL, AST 74 U/L, ALT 45 U/L, LDH 882 U/L, BUN 16.5 mg/dL, creatinine 0.94 mg/dL, CRP 2.39 mg/dL, sIL-2R 1,280 U/mL, beta-2-microglobulin 6.5 mg/L, and ANA x40 positive, but complements were within normal. The patient had 22 copy/10e4 PBMCs of proviral HTLV-1 load , with positive anti-HTLV-1 antibody, consisting of western blot pattern of GP46 (+), p53 (+), p24 (+), and p19 (+). He showed a marked increase of granular lymphocytes with atypical nuclei in this peripheral blood . Thus, during the pretreatment period, absolute CD56+ NK cell counts in the peripheral blood were in a range of 986/μL–1,270/μL . Bone marrow smear showed hypocellular and hypoplastic marrow, with decreased numbers of megakaryocytes; however no abnormal features were noted. Flow cytometry of bone marrow revealed NK cell dominance as seen in the peripheral blood. Analysis of his peripheral blood as well as bone marrow showed a normal karyotype. On the other hand, infiltrating mononuclear cells in his dermis obtained by the skin biopsy of facial eczema were shown to be CD3+CD4+CD56-EBNA-. Only a few cells in this dermal area were stained with TIA-1 and Granzyme B  for nearly 10 years. When he was referred to us, he had fatigue, loss of appetite, and exacerbated facial eczema/edema as well as dermatitis at the neck/upper chest and nuchal area and upper back regions . He did al blood  which weanzyme B . These fβ1 rearrangement in these cells confirmed that they are actually NK cells.The clinical and pathological features of HTLV-1-associated cutaneous disease are diverse . Our pat in vitro culture assay of 7 days, CD56+ NK cells spontaneously proliferated in response to HTLV-1. This spontaneous NK cell proliferation positively correlated with HTLV-1 proviral load, but not with the presence of HAM/TSP [ in vitro phenomenon that HTLV-1 primarily drives expansion of CD56+ NK cells.In the past, Norris et al. demonstrated that in HAM/TSP . Coelho- HAM/TSP . By cont HAM/TSP . Conside HAM/TSP . In our  HAM/TSP , it is a


The results showed that normal keratinocytes in the upper epidermis preferentially expressed TSLP receptors and conversely lacked ΔNp63, which has an inhibitory effect on the expression of TSLP receptors. Interestingly, the epidermis of AD lesions was found to abundantly contain keratinocytes with low or undetectable levels of ΔNp63 (ΔNp63lo/-). Moreover, in the absence of ΔNp63, keratinocytes readily presented TSLP and other cytokines by stimuli through Toll-like receptor 3 (TLR3). Together with the evidence that extrinsic TSLP itself augments TSLP production by keratinocytes without ΔNp63, the results indicate that ΔNp63lo/- keratinocytes generate TSLP through a putative autocrine and/or paracrine pathway upon TLR3 stimulation within AD lesions, since moieties of damaged cells and pathogens stimulate TLR3.In the skin lesions of atopic dermatitis (AD), keratinocytes release large quantities of thymic stromal lymphopoietin (TSLP), causing unfavorable inflammation along with skin damage. Nevertheless, how TSLP influences keratinocytes themselves is still unknown. In this study, we showed that ΔNp63, a p53-homologue, predominantly expressed in keratinocytes regulated the receptor complex of TSLP, which determines susceptibility to self-derived TSLP. Expression of TSLP receptors in skin tissues and keratinocytes was assessed by immunohistochemistry and quantitative RT-PCR, and  The prevalence of allergic disorders has been significantly increasing in many countries in the past few decades. The fact that over 10% of children and 6% of adults suffer from atopic dermatitis (AD) has become a worldwide health issue + T cells into T helper type 2 (Th2) cells secreting IL-4, IL-5 and IL-13, in a manner similar to that in allergic asthma. TSLP is also able to stimulate eosinophils, macrophages, invariant natural killer T cells and innate lymphoid cells, eventually resulting in chronic inflammation. Recent genome-wide association studies on AD patients have revealed somatic mutations of genes regulating keratinocytes such as those for filaggrin, a natural skin moisturizer, and claudin-1, a representative barrier protein Extensive investigations have revealed a salient abnormality of AD skin lesions in which an interleukin (IL)-7-like cytokine, thymic stromal lymphopoietin (TSLP), produced by keratinocytes provokes a hyperreactive immune state Transcription regulator p63 , a p53 homologue, plays a primary role in the construction of fully functional epidermal layers lo/- keratinocytes) within AD lesions and an interesting role of ΔNp63 in the production of TSLP. Studies of normal epidermal tissues and keratinocyte-derived cells have demonstrated that loss of ΔNp63 increased the level of TSLP receptors, which are composed of a heterodimer of TSLP-binding chain (TSLPR) and interleukin-7 receptor α (IL-7Rα) chain. Interestingly, stimulation of TLR3 by poly I:C downregulated ΔNp63 and upregulated TSLP receptors in keratinocytes, which concomitantly produced TSLP. This process related to TLR3 was found to be dependent on nuclear factor κB (NF-κB). Given that damaged cell-derived self-RNA can stimulate TLR3 signaling, these findings imply that ΔNp63lo/- keratinocytes in AD lesions may cause chronic inflammation through a vicious cycle of their own TSLP-TSLPR axis loop induced by RNA moieties derived from surrounding damaged keratinocytes Here we report the abundance of unique keratinocytes that expressed ΔNp63 at low or undetectable levels (collectively named ΔNp63Skin tissues were obtained from patients with AD or systemic lupus erythematosus (SLE) and patients presenting without pathological findings who underwent skin biopsies at Sapporo Medical University Hospital. The diagnosis of AD was established by both dermatologists and pathologists. All tissues were obtained with written informed consent according to the guidelines of the Declaration of Helsinki and with approval of the Institutional Review Board (IRB) of Sapporo Medical University Hospital under permit number 25–136 titled investigation of immunologic abnormality in inflammatory skin diseases.2.Human primary keratinocytes were purchased from DS Pharma Biomedical  and cultured in a serum-free medium for human keratinocytes . Human HaCaT epidermal cells  were maintained in DMEM  supplemented with 100 U/ml penicillin G, 100 µg/ml streptomycin and 5% fetal bovine serum. For stimulation of cells, culture media were supplemented with the following reagents: 10 µg/ml zymozan , 5 to 25 µg/ml polyinosine-polycytidylic acid , 100 ng/ml lipopolysaccharide , 5 µM Type B CpG oligonucleotide , 10 ng/ml TNFα , or 1 to 10 ng/ml TSLP (R&D Systems). All cells were cultured at 37°C in a humidified atmosphere in 5% COCells were treated with poly I:C at 5 µg/ml (for primary keratinocytes) or 10 µg/ml (for HaCaT keratinocytes) and TSLP  simultaneously. The culture media was changed into the media with poly I:C and TSLP at 16 hr after p63 siRNA transfection. For detection of TSLP protein by ELISA, we removed the media and washed cells with PBS twice at 24 hr after poly I:C and TSLP stimulation. And then the cells were cultured in the media containing only poly I:C at the same concentration for 48 hr. For detection of GM-CSF or IL-6 protein by ELISA, culture supernatant were collected at 48 hr after poly I:C and TSLP treatment.The primary antibodies used were a mouse monoclonal antibody (mAb) to detect all isoforms of p63 , a mouse anti-TSLPR mAb , a mouse anti-β-actin mAb , a rabbit polyclonal antibody (pAb) to detect ΔNp63 , a rabbit anti-IL-7Rα pAb , a rabbit anti-TSLP pAb , a sheep anti-TSLP pAb (R&D Systems), a rabbit anti-cytokeratin pAb , a rabbit anti-NF-κB p65 mAb  and a rabbit anti-phospho NF-κB p65 (Ser536) mAb . Alexa 488 (green)- and Alexa 594 (red)-conjugated anti-mouse and anti-rabbit IgGs were purchased from Invitrogen . Peroxidase-conjugated goat anti-mouse and anti-rabbit IgGs were obtained from KPL .Total RNA was extracted and reverse-transcribed into cDNA as described previously A cocktail of three siRNAs for human p63 (siTrio full set) was purchased from B-Bridge International  and transfected at 100 nmol/L. The sense sequences of the siRNAs for human p63 were 5′-CAGAAGAUGGUGCGACAAATT-3′, 5′-GUGAAUUCAACGAGGGACATT-3′, and 5′-GCAAAAAAGAGUUGGGUGUTT-3′. Negative control siRNA was obtained from Invitrogen. Transfections were carried out using Lipofectamine RNAi max (Invitrogen) in Opti-MEM  according to the manufacturer's instructions. Briefly, 7.5 µl of Lipofectamine RNAi max and siRNA for p63 were suspended into 500 µl Opti-MEM each. After mixing them together, Opti-MEM containing Lipofectamine RNAi max and the siRNA was incubated at room temperature for 15 min and then added into 2 ml culture medium. The culture medium was changed at 16 hr after transfection.hi, ΔNp63lo and ΔNp63−. Three pathologists independently examined a specimen and determined the ratio of ΔNp63lo/- cells per total cells in at least 5 high-power fields. Final scores of a specimen were defined by averages.Formalin-fixed and paraffin-embedded and frozen tissue sections were used for immunostaining analysis as described previously Procedures of cell lysis and immunoblotting were as described previously 5 cells/well in a 12-well plate under the condition. Concentrations of TSLP, GM-CSF and IL-6 in culture supernatants were analyzed in triplicate with specific ELISA kits (R&D Systems) as described in the manufacturer's protocol. Detection limit of TSLP, GM-CSF and IL-6 are 9.87 pg/ml, 3 pg/ml and 10 pg/ml, respectively.Cells were cultured at a density of 1×105 cells/well in a 12-well plate before stimulation. The cells were treated with 2 µg/ml anti-TSLP pAb (R&D Systems) and/or 5 µg/ml poly I:C simultaneously 24 hr after plating them and incubated for 24 hr. They were harvested for total RNA isolation. Sheep IgG was used as a negative control.Cells were cultured at a density of 5×106 cells/well in a 6-well plate. After 24 hr, medium was changed and the cells were treated with 5 µM TRIF inhibitory peptide or 5 µM control peptide (Invivogen) for 16 hr. The cells were irradiated with 60 mJ/cm2 of UVB. The cells were harvested and total RNA was isolated at 24 hr after irradiation.HaCaT keratinocytes were plated at a density of 1×10t test or one-way ANOVA followed by Tukey's multiple-comparison test. Graph bars in the figures present means ± SD.Statistical significance was examined using the unpaired two-tailed Student's in vitro studies using HaCaT and primary keratinocytes with p63-specific siRNA revealed that downregulation of ΔNp63 could increase the expression of TSLPR and IL-7Rα, even though keratinocytes presented certain levels of TSLPR and IL-7Rα  family members for the emergence of efficient immunity Next, we examined the effects of exogenous TSLP on keratinocytes stimulated with TLR3. Results demonstrated that exogenous TSLP helped the keratinocytes under the condition of TLR3 stimulation to produce intrinsic TSLP , as wellhi, ΔNp63lo or ΔNp63−, respectively) in the epidermal areas. In contrast, the epidermal areas of normal skin tissues as well as skin lesions of patients with SLE were composed of keratinocytes steadily expressing ΔNp63 at high levels (lo and ΔNp63− (ΔNp63lo/-) keratinocytes to total keratinocytes of the AD skin lesions were much higher than those in normal epidermal tissues  Immunoblot analysis showing the expressions of ΔNp63 and TAp63 in epidermal keratinocytes. Epidermal keratinocytes predominantly express ΔNp63, but not TAp63. Other isoforms of p63 in the keratinocytes were undetectable by immunoblot analysis. β-actin was used as a loading control. (b) Phase-contrast microscopy demonstrating the morphological features of intercellular adhesive properties of ΔNp63-deficient HaCaT keratinocytes dramatically changed a cohesive pattern into a scattered pattern. Bar  = 100 µm.(TIFF)Click here for additional data file.Figure S2Expression of TSLPR and IL-7Rα is regulated by ΔNp63 in primary keratinocytes. Quantitative RT-PCR demonstrating the levels of ΔNp63, TSLPR and IL-7Rα mRNAs in control siRNA (siControl) and p63-specific siRNA (sip63)-transfected primary keratinocytes. Downregulation of ΔNp63 by specific siRNA increased the levels of TSLPR and IL-7Rα mRNAs. Student's t test. *P<0.05 and ***P<0.005 versus control. Data are representative of three independent experiments.(TIFF)Click here for additional data file.Figure S3Effects of ultraviolet B (UVB) irradiation on HaCaT keratinocytes. (a) Quatitative RT-PCR showing the levels of ΔNp63, TSLPR and IL-7Rα mRNAs in UVB-exposed HaCaT keratinocytes. UVB exposure simply reduced the level of ΔNp63 and concomitantly upregulated the levels of TSLPR and IL-7Rα mRNAs. (b) Quantitative RT-PCR confirming whether inhibition of Toll/interleukin-1 receptor domain-containing adaptor protein inducing interferon β (TRIF) rescues downregulation of ΔNp63 mRNA by UVB exposure in HaCaT keratinocytes. TRIF inhibitor rescued ΔNp63 downregulation by UVB exposure. One-way ANOVA followed by Tukey's multiple-comparison test.*P<0.05, **P<0.01 and ***P<0.005 versus control. Data are representative of at least three independent experiments.(TIFF)Click here for additional data file.Figure S4Expression of inflammatory cytokines in ΔNp63-deficient keratinocytes is enhanced by TSLP stimulation. (a) Quantitative RT-PCR demonstrating the levels of granulocyte colony-stimulating factor (GM-CSF) and IL-6 mRNAs at 24 hr after treatment with exogenous TSLP (1 or 10 ng/ml) under the condition of TLR3 stimulation (5 µg/ml poly I:C) in primary keratinocytes. Treatment with exogenous TSLP increased the expressions of these cytokines under the condition of TLR3 stimulation. (b) Quantitative RT-PCR showing the levels of GM-CSF and IL-6 mRNAs at 24 hr after stimulation with 10 µg/ml poly I:C and 10 ng/ml TSLP in siControl and sip63 HaCaT keratinocytes. Stimulation with TLR3 and TSLP enhanced the expressions of these cytokines in ΔNp63-deficient HaCaT keratinocytes. (c) ELISA demonstrating GM-CSF and IL-6 release at 48 hr after treatment with 5 µg/ml poly I:C and 10 ng/ml TSLP in culture supernatant from siControl and sip63 primary keratinocytes. Down regulation of ΔNp63 and/or exogenous TSLP increase GM-CSF and IL-6 release under the condition of TLR3 stimulation. (d) Quantitative RT-PCR confirming whether neutralization of TSLP inhibits the generation of GM-CSF and IL-6 under the condition of TLR3 stimulation (5 µg/ml poly I:C) in primary keratinocytes. Neutralization of TSLP inhibits the expressions of these cytokines under the condition of TLR3 stimulation. One-way ANOVA followed by Tukey's multiple-comparison test. *P<0.05, ***P<0.005 and N.S., not significant. Data are representative of at least three independent experiments.(TIFF)Click here for additional data file.Figure S5Representative figures of ΔNp63-immunostaining of AD skin lesions. Paraffin-embedded tissue sections of 10 cases of AD lesions (AD1 to AD10) were examined for the distribution of ΔNp63. AD10 is depicted in (TIFF)Click here for additional data file.Figure S6Immunofluorescence labeling of ΔNp63 and TSLP in AD and normal skin. TSLP was overexpressed in the skin from a patient with AD compared to the level in normal skin. On the other hand, keratinocytes in the skin from a patient with AD expressed a lower level of ΔNp63 than that in normal keratinocytes. Bar  = 20 µm.(TIFF)Click here for additional data file.


Previous studies have suggested a positive link between serum uric acid (UA) and bone mineral density (BMD). In this study, we re-examined the association between UA and BMD and further explored whether this was mediated by skeletal muscle mass in a general Chinese population.2, kg/m2) for the total limbs, arms, and legs was then calculated.This community-based cross-sectional study was conducted among 3079 (963 men and 2116 women) Chinese adults aged 40–75 years. Face-to-face interviews and laboratory analyses were performed to determine serum UA and various covariates. Dual-energy X-ray absorptiometry was used to assess the BMD and appendicular skeletal muscle mass. The skeletal muscle mass index , 4.1%(lumbar spine), 2.4%, and 2.0%  greater BMDs. The mean SMIs in the highest (vs. lowest) quartile increased by 2.7% , 2.5% (arm), 2.7% (leg) respectively. In addition, path analysis suggested that the favorable association between UA and BMD might be mediated by increasing SMI.The elevated serum UA was associated with a higher BMD and a greater muscle mass in a middle-aged and elderly Chinese population and the UA-BMD association was partly mediated by muscle mass. Osteoporosis and sarcopenia, the results of musculoskeletal aging, are a tremendous and growing public health problem for the aging society , 2. MuscIn vitro, reactive oxygen species (ROS) are observed to suppress osteoblast generation and differentiation, to enhance osteoclast development and activity, and to promote myofibrillar proteolysis  according to serum UA value. The continuous variables were expressed as the mean and standard deviation (SD). A comparison of the means across the quartiles was performed using a one-way ANOVA, or the Mann-Whitney nonparametric test in the case of non-normal distribution. The categorical variable was presented as a number or a percentage, and the Pearson chi-square test was used to examine the difference in frequency.etc.), chronic hepatitis, diabetes, and cardiovascular diseases. Multiple comparisons between quartile groups were conducted using the Bonferroni test. Path analysis, via structural equation modeling, including the variables serum UA, mediator (SMI), and BMDs at all sites, was performed to examine and quantify the proposed theoretical model, that is, the direct or the indirect effect via SMI of UA on BMD variance. To attenuate the short-term fluctuation of a single assessment of UA, we additionally examined in retrospect baseline serum UA among participants successfully followed up and utilized an average value of baseline and follow-up UA as exposure parameter, and further re-examined the relationship between UA and BMDs, SMI.Analysis of covariance (ANCOVA) was used to examine the quartile differences in BMD and SMI. The multiplicative interactions between UA and gender were estimated by adding the interaction terms. As no significant interactions were found between the UA levels and gender (P for interactions: 0.153–0.860), all of the data analyses were then performed for men and women combined. In the ANCOVA models, we adjusted for age, gender, height, weight, blood pressure, education level, years since menopause, physical exercise, smoking, alcohol drinking, use of calcium and multivitamin supplements, medications affecting bone metabolism , except for the path analysis, which was performed using AMOS 17.0. The two-sided value was considered statistically significant at p < 0.05.There was a significantly dose-dependent and positive association between the serum UA and the BMDs at all sites after adjusting for age and gender in Model 1 . The mean BMDs were 3.0% (whole body), 6.8% (lumbar spine), 4.8% , and 4.9%  greater in Q4 compared to in Q1. After further adjustment for other potential confounding factors in Model 2, the significant association remained. The mean BMDs in Q4 were 2.3% (whole body), 4.1% (lumbar spine), 2.4% , and 2.0%  higher than those in Q1 .When anaA similarly positive and linear association between serum UA and muscle mass was found, after adjusting for a variety of potential covariates . The mean values for total SMI and arm and leg SMIs were 5.5%, 4.5%, and 5.9% greater in quartile 4 (vs. 1) for UA in Model 2, respectively. Further controlling for whole-body fat mass in Model 3 , those sAt whole body and lumber, serum UA showed both directly and indirectly positive associations with the BMDs in the path analysis  among 1,705 older Australian men (70 years) , in 7,50vs. longitudinal) studies, in those using indices of BMD [vs. fractures [In overall, our findings were consistent with the results from the majority of the previous studies, which supported the hypothesis that UA is beneficial to bone health in human. The between-study heterogeneity among previous results might be explained by the variations in population characteristics , study design, study size, and covariates adjusted for. The favorable association appeared to be more frequently observed in Asians –28, 30 ts of BMD  (vs. fraractures ), and inractures .et al. reported that the lean body mass and the mid-arm muscle circumference increased with an increase in UA tertiles in 216 maintenance hemodialysis patients [Kappa = 0.48) [A limited number of population studies have tested the relationship between UA and skeletal muscle, generating inconsistent results. Similar to our results, Beberashvili patients . In addipatients –43. Howepatients . A possi = 0.48) . More stIn vitro experiments showed that UA treatment significantly decreases the ROS content of osteoclast precursors and osteoclastogenesis [A possible mechanism explaining the potentially beneficial effect of serum UA on bone and muscle might be related to its antioxidant capacity. UA, as a reactive water-soluble antioxidant, reacts with various oxidants and is thought to be responsible for over half amount of the antioxidant properties of plasma . Accumulogenesis  and induogenesis . Thus, set al., lean mass had a significantly favorable association with BMDs at the total body, spine, and femur in 1,475 Chinese men and 1,534 women [In this study, our findings showed that the beneficial UA-BMD association might be partly mediated by muscle mass. It is well established that greater muscle mass is associated with better bone health in population-based studies. A cross-sectional study showed a positive association between lean mass and BMD in 6,249 Italian women . In the This study has several strengths. To the best of our knowledge, this is the first study to report a favorable association between UA and muscle mass and to show that the UA-BMD association is partly mediated by muscle mass. Secondly, among participants successfully followed up, we additionally retrospectively examined their baseline serum UA value, then calculated an average value of baseline and follow-up UA as exposure parameter and re-examined the relationship between UA, BMDs and SMI, attenuating the short-term fluctuation of a single assessment of UA. Thirdly, a relatively large study size of this present study allowed us to achieve more precise results.R2 = 0.96) [The limitations of this study also deserve attention. First, the largest limitation is absence of muscle performance measurements, which hindered a further exploration of the relationship between UA, muscle performance and bone. Second, the cross-sectional study design did not allow us to infer a cause–effect relationship, although an average value of baseline and follow-up UA in supplemental data might represent a long-term exposure level and then to some extent reduce the possibility of reverse causality. Third, we measured the skeletal muscle mass by the DXA technique rather than the golden methods by MRI or CT. However, previous studies have showed an excellent validity between results determined by DXA as compared to those by MRI and CT ( = 0.96) , 55. LasUA has long been known for its harmful effects such as gouts and uric lithiasis, as well as its potential association with hypertension, metabolism syndrome, renal disease and cardiovascular diseases. A large number of studies have been focusing on the clinical significance of lowering circulating UA to prevent/improve the chronic diseases via diet or relevent medications –58. HoweIn summary, our study revealed that the serum UA level was positively and significantly associated with both the BMD and the skeletal muscle mass, and suggested that the favorable UA-BMD association might be partly mediated by skeletal muscle mass in Chinese middle-aged and elderly adults. Our findings support the hypothesis that UA exerts a beneficial effect on bone and muscle metabolism. Due to limited experimental evidence available, further interventional studies (particularly in humans) are needed to clarify if the favorable associations are causal or not.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


An important immunopathological hallmark of allergic disease is tissue eosinophilic and basophilic inflammation, a phenomenon which originates from hemopoietic progenitors (HP). The fate of HP is determined by local inflammatory cytokines that permit “in situ hemopoiesis,” which leads to the accumulation of eosinophils and basophils (Eo/B). Given that recent evidence supports a critical immunomodulatory role for thymic stromal lymphopoietin (TSLP) in allergic inflammation, as well as TSLP effects on CD34+ progenitor cytokine and chemokine secretion, we investigated the role of TSLP in mediating eosinophilo- and basophilopoiesis, the mechanisms involved, and the association of these processes with atopic sensitisation. In the studies presented herein, we demonstrate a direct role for TSLP in Eo/B differentiation from human peripheral blood CD34+ cells. In the presence of IL-3, TSLP significantly promoted the formation of Eo/B colony forming units (CFU) (including both eosinophils and basophils) from human HP (HHP), which was dependent on TSLP–TSLPR interactions. IL-3/TSLP-stimulated HHP actively secreted an array of cytokines/chemokines, key among which was TNFα, which, together with IL-3, enhanced surface expression of TSLPR. Moreover, pre-stimulation of HHP with IL-3/TNFα further promoted TSLP-dependent Eo/B CFU formation. HHP isolated from atopic individuals were functionally and phenotypically more responsive to TSLP than those from nonatopic individuals. This is the first study to demonstrate enhanced TSLP-mediated hemopoiesis ex vivo in relation to clinical atopic status. The capacity of HHP to participate in TSLP-driven allergic inflammation points to the potential importance of “in situ hemopoiesis” in allergic inflammation initiated at the epithelial surface. The airway epithelium is an important initiator of the allergic response; it secretes cytokines/chemokines, which regulate innate immune cells . One succommitted CD34+ hemopoietic progenitor cell, the eosinophil–basophil (Eo/B) progenitor, found in bone marrow, cord blood, and peripheral blood (PB) [Human eosinophils and basophils differentiate from a common ood (PB) . We haveood (PB) –20. Thisood (PB) , demonstood (PB) , reporteood (PB) , Kim et ood (PB) , and Dorood (PB)  collectiood (PB) ,24; furtood (PB)  demonstrRecent evidence supports a critical immunomodulatory role for TSLP in allergic inflammation, as well as TSLP effects on CD34+ progenitor cytokine and chemokine secretion , but then = 10) or without (n = 10) atopy were recruited  and all subjects provided written informed consent. Atopy-unattributable subjects were initially recruited for the study , followiecruited . Atopy wOne hundred mL of blood were collected through direct venipuncture into heparinized vacutainer tubes . Peripheral blood mononuclear cells were isolated by density centrifugation and CD34+ progenitors were enriched using EasySep™ Human Progenitor Cell Enrichment Kit with Platelet Depletion  as per manufacturer's instructions.2). Eo/B CFU were enumerated using inverted light microscopy .Enriched CD34+ progenitors (8000 cells/well) were cultured in duplicates in 0.9% methylcellulose  with Iscove's 2+  supplemented with FBS, penicillin–streptomycin, and 2-ME) and IL-3 (1 ng/mL), IL-5 (1 ng/mL), or GM-CSF  in the presence or absence of TSLP  in 12-well plates . In some experiments, cells were treated with anti-TSLP , anti-TSLPR , anti-TNFα (R&D), or isotype control (each at 10 µg/mL). Treatment with the indicated stimulatory/inhibitory conditions had no effects on cell viability as determined by trypan blue exclusion. Cultures were incubated for 14 days  were stimulated overnight as indicated. Following which, CD34+ progenitors were stained as previously described with modification [5 cells/tube) and incubated in the dark . Next, optimal amounts of isotype controls or test antibodies were added and incubated in the dark . Cells were then washed, fixed in cytofix (BD), and stored in the dark at 4°C until ready for acquisition.Isolated CD34+ progenitors (10fication . BrieflyStained cells were acquired with a LSR II flow cytometer (BD Biosciences) using the FACSDiva software (BD Biosciences). Offline analysis was performed using FlowJo software . CD34+ cells were enumerated using a previously established multi-parameter sequential gating strategy . CD34+ p6 cells/mL) were stimulated overnight as indicated and cell-free supernatant was harvested and IL-1β, IL-4, IL-6, IL-9, IL-10, IL-13, GM-CSF, IFNγ, TNFα, CXCL8, eotaxin, CCL2 (MCP-1), CCL5 (RANTES), and CCL17 (TARC) were assessed using Bio-Plex assays  according to manufacturer's recommendation. The detection limits for these cytokines were 3.2 (IL-1β), 2.2 (IL-4), 2.3 (IL-6), 2.1 (IL-9), 2.2 (IL-10), 3.7 (IL-13), 2.2 (GM-CSF), 92.6 (IFNγ), 5.8 (TNFα), 1.0 (CXCL8), 40.9 (eotaxin), 2.1 (CCL2), 2.2 (CCL5), and 1.7 pg/mL (CCL17).CD34+ cells . Eosinophils were identified using Diff-Quik  and basophils identified using toluidine blue stain (Sigma).The total number of cells in each individual colony was enumerated using inverted light microscopy before being picked from methylcellulose and placed into PBS, boiled , centrifuged, and cell-free supernatant harvested and measured for histamine content using Histamine Enzyme Immunoassay Kit  according to manufacturer's recommendation. The detection limit of this assay is 55 pg/mL.P < 0.05. All analyses were performed with Prism version 5  using non-parametric tests. Differences within groups were assessed by Friedman test with Dunnett post hoc test. Between-group comparisons (nonatopic vs. atopic) were made using the Mann–Whitney U-test.All data are expressed as the mean ± SEM. Significance was assumed at P < 0.001; P < 0.05) and anti-TSLPR . TSLP did not have any effects on either IL-5- or GM-CSF-responsive Eo/B CFU (Supplementary The addition of TSLP significantly increased the formation of IL-3-responsive Eo/B CFU from 7.56 ± 0.9 to 15.8 ± 1.6 (P < 0.001) and TSLP-stimulated  and TSLP-stimulated HHP (P < 0.05). A similar trend was seen for IL-7Rα expression, although not significant . In the presence of neutralizing anti-TSLP or neutralizing anti-TNFα, the histamine content was significantly lower compared to IL-3/TSLP stimulated colonies . PB HHP failed to secrete detectable levels of IL-4, IL-9, IL-10, GM-CSF, IFNγ, and eotaxin; while CCL5 was highly secreted by PB HHP cells under all conditions.TSLP alone induced significant levels of IL-1β, IL-6, TNFα, CXCL8, and CCL17 from PB HHP, compared to unstimulated controls  and IL-3/TSLP  and IL-3/TSLP , IL-13 (P < 0.05), TNFα (P < 0.01), CXCL8 (P < 0.05), and CCl17 (P < 0.05), compared to PB HHP from nonatopic individuals, post IL-3/TSLP-stimulation  and chemokines  . Of the Although we have not herein reported on which signaling pathways are involved in TSLP-induced Eo/B differentiation, we do have findings implicating the preferential dependence of p38MAPK signaling pathways in IL-3/TSLP-mediated Eo/B differentiation  differentiation within tissues. The enhanced TSLP-mediated hemopoiesis ex vivo in relation to clinical atopic status may help explain increased eosinophils and basophils at sites of inflammation in atopic individuals.


Supplementing miR-140-5p activity induced stromal cell ST2 and preadipocyte 3T3-L1 to differentiate into mature adipocytes. Conversely, inhibition of the endogenous miR-140-5p repressed ST2 and 3T3-L1 to fully differentiate. By contrast, knockdown of the endogenous miR-140-5p enhanced osteoblast differentiation. Transforming growth factor-β receptor I (Tgfbr1) was shown to be a direct target of miR-140-5p. Supplementing miR-140-5p in ST2 reduced the level of TGFBR1 protein, while suppression of endogenous miR-140-5p increased TGFBR1. Overexpression of Tgfbr1 inhibited, whereas knockdown of Tgfbr1 promoted adipogenic differentiation of ST2 cells. Further investigation of mechanisms that control miR-140-5p expression revealed that C/EBPα induced transcriptional activity of the miR-140-5p promoter. Removal of the putative response element of C/EBP from the promoter abolished the enhancement of the promoter activity by C/EBPα, suggesting that C/EBPα transcriptionally controls miR-140-5p expression. Taken together, our study provides evidences that miR-140-5p regulates adipocyte differentiation through a C/EBP/miR-140-5p/TGFBR1 regulatory feedback loop.Recent emerging studies of miRNAs in adipocyte commitment provide new insights to understand the molecular basis of adipogenesis. The current study indicated that miR-140-5p was altered in primary cultured marrow stromal cells and established progenitor lines after adipogenic and/or osteogenic treatment. miR-140-5p was increased in adipose tissue in db/db obese mice  In vivo and in vitro studies have shown that PPARγ is critical both for adipocyte differentiation and for major functions of mature adipocytes, including lipid metabolism, adipokine secretion, and insulin sensitivity689101113It is believed that adipocyte and osteoblast originate from a common mesenchymal stem cell (MSC), thus a reciprocal and inverse relationship exists between adipogenesis and osteogenesis234Recent emerging studies have shown that miRNAs are able to indirectly regulate adipogenic and/or osteoblast differentiation of MSCs by targeting various genes that may be involved in balancing self-renewal and stem cell differentiation. miRNA family 27 (miR-27a and miR-27b) has been found to be able to negatively regulate adipogenic differentiation of 3T3-L1 preadipocytes151920Mir140 gene, was previously believed to be abundantly and relatively specifically expressed in chondrocytes. The in vivo function of miR-140 was demonstrated by Miyaki’s group, who created miR-140 knockout and transgenic miceAdamts5, which was shown to be a direct target of miR-140. Conversely, the transgenic mouse overexpressing miR-140 in cartilage displayed no skeletal phenotype during development but was resistant to antigen-induced arthritis. Recently, miR-140 was identified as a direct downstream component of the BMP4 signaling pathway during the commitment of C3H10T1/2 cells to adipocyte lineagemicroRNA-140 (miR-140), generated from the in vivo, we examined the expression of miR-140-5p in the genetically obese db/db mice. Compared with the genetically matched lean mice, the expression level of miR-140-5p was increased in both epididymal and inguinal adipose tissue of the db/db mice  . In 3T3-Transfection of miR-140-5p inhibitor substantially blocked the differentiation of ST2 cells into adipocytes in presence of adipogenic treatment. Oil-red O quantification revealed a 35% decrease after miR-140-5p inhibitor transfection compared to control transfection . ConsistFurthermore, miR-140-5p inhibitor also blocked adipocyte formation from 3T3-L1 cells as compared to control transfection . ConsistTo investigate if miR-140-5p plays a role in regulating osteoblast differentiation, we altered the level of miR-140-5p in stromal ST2 by transfecting the miR-140-5p inhibitor into the cells. Compared to the controls, miR-140-5p inhibitor enhanced alkaline phosphatase (ALP) staining 14 days after osteogenic treatment , suggestet al. demonstrated that the site 1674–1680 of Ostm1 3′ UTR was the direct targeting site of miR-140, but they did not examine the site 596–603It is well known that miRNAs function through blocking the translation of their target mRNAs. Several regulators of adipocyte and osteoblast differentiation including wingless-type MMTV integration site family member 11 (Wnt11), Tgfbr1 and Ostm1 were predicted to be the potential targets of miR-140-5p by using online programs. The reporter constructs of their 3′-UTRs were made and their potential pairings with miR-140-5p are shown in These results suggested that Tgfbr1 is the direct target of miR-140-5p. Furthermore, supplementing miR-140-5p activity in ST2 cells using the synthetic mimics reduced the level of TGFBR1 protein . In contin vitro and in vivoIt is well known that TGF-β signaling inhibits adipogenesis miR140/Luc had transcriptional activity in 3T3-L1 cells, showing 11-fold increase vs. the promoterless pGL3-basic. The prediction of transcription factor binding sites was performed with TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html) and TFBIND (http://tfbind.hgc.jp/), which revealed a putative C/EBP binding motif within the proximal promoter at −281 nt (CCCTTTCACCAGCC)  , indicatpromoter . We furtpromoter . Consistpromoter .in vitro in stromal cells during adipogenesis, or in vivo in adipose tissue of db/db obese mice. Moreover, it was decreased in primary stromal cells during osteogensis, suggesting that miR-140-5p might play a role in adipogenesis and/or osteogenesis.In the present study, we found that the expression of miR-140-5p was increased either To delineate the precise role of miR-140-5p in cell fate decision, we tested the effects of miR-140-5p on adipocyte differentiation. Our data showed that supplementing miR-140-5p in undifferentiated adipogenic progenitors using synthetic miR-140-5p mimics potentiated the formation of adipocytes in presence of adipogenic treatment. Conversely, inactivating miR-140-5p in adipogenic progenitors using miR-140-5p inhibitor blocked differentiation of adipocytes. Concomitantly, knockdown of miR-140-5p using the inhibitor in stromal ST2 potentiated osteoblast differentiation. These findings provided evidences that miR-140-5p reciprocally regulates adipocyte and osteoblast differentiation from progenitor cells.et al. demonstrated that the poorly conserved site 1674–1680 of Ostm1 3′ UTR was the direct targeting site of miR-140miRNAs finely modulate osteoblast/adipocyte differentiation through their direct targeting of diverse signaling molecules and pathways that include bone morphogenetic protein 2 (BMP2)/Smad, Wnt/β-catenin and Runx2 signaling pathways262728et al. demonstrated that Wnt11 overexpression in preosteoblast increases β-catenin accumulation and promotes bone morphogenetic protein (BMP)-induced expression of alkaline phosphatase and mineralizationWnt11 signals through both canonical and noncanonical pathwaysOf note, Wnt11 mRNA remains at a quite low expression level in the adipogenic cell lines we used before and after adipogenic treatment (data not shown). Thus it might not be critical during adipogenesis. By contrast, Tgfbr1 has much higher expression level in the adipogenic cell lines (data not shown). Furthermore, supplementing miR-142-5p in ST2 cells reduced, while silencing miR-142-5p induced the protein levels of TGFBR1. These data identified Tgfbr1 as a direct target of miR-140-5p.TGF-β can provide competence for early stages of chondroblastic and osteoblastic differentiation, but it inhibits adipogenesis and late-stage osteogenesis24To gain further insight into the control of miR-140-5p expression and function, we cloned the promoter of miR-140-5p and examined cis-acting elements within the proximal promoter using TFSEARCH and TFBIND programs and found a potential binding site for C/EBPs within the proximal promoter. In our study, the WT promoter construct that harbored the C/EBP binding sequence showed transcriptional activity in 3T3-L1 cells, which was further stimulated following C/EBPα overexpression. Consistently, dominant negative form of C/EBPα downregulated the expression level of miR-140-5p. Moreover, point mutation of the C/EBP binding sequence at −281 nt was made and the mutant construct showed less transcriptional activity. Of more interest, the mutant failed to respond to C/EBPα transcription factor. We thus draw the conclusion that C/EBPs bind to the proximal promoter of miR-140-5p to positively regulate the expression of miR-140-5p.The data we have shown in this paper tends to develop a model for miR-140-5p expression and function. Adipogenic signals lead to the activation of C/EBPs transcription factors in progenitor cells. In addition to working together with PPARγ to induce the coding genes essential for adipocyte differentiation, C/EBPs may also transactivate miR-140-5p promoter to increase the miR-140-5p level. In turn, miR-140-5p may further promote adipocyte differentiation and maintain the levels of C/EBPs mRNA and protein levels via repressing TGFBR1. Thus, we propose that a unique autoregulatory feedback loop exists among C/EBPs, miR-140-5p and TGFBR1. The current study also suggests that miR-140-5p is an attractive potential target for new therapies aimed at controlling metabolic disorders like osteoporosis and obesity.10-week-old db/db mice and their genetically matched lean littermates were purchased from Model Animal Research Center of Nanjing University . RNA was extracted from epididymal and inguinal adipose tissue. qRT-PCR was done to examine the expression level of miR-140-5p. All the experiments involving animals were carried out in accordance with the Chinese guidelines for animal welfare and experimental protocol, and were approved by the Animal Care and Use Committee of the Metabolic Diseases Hospital, Tianjin Medical University.C57 mice and cultured in α-MEM containing 10% FBS in 25 cm2 flasks as previously described19α-MEM containing 10% FBS, 50 μg/mL ascorbic acid and 5 mmol/L β-glycerophosphate) for 3 days followed by miRNA isolation. For adipogenic differentiation, confluent cells were cultured in adipogenic medium  for 3 days followed by miRNA isolation.Bone marrow stromal cells were isolated from femurs and tibias of 4-week-old 3T3-L1 and ST2 cells were maintained in DMEM supplemented with 10% FBS. When the cells reached 100% confluence, AIM was added to induce adipocyte formation. When ST2 cells reached 80% confluence, OIM was added to induce osteoblast differentiation.We previously examined the miRNA expression profiles in primary cultured marrow stromal cells after adipogenic and osteogenic treatmentFor mRNA expression analysis, total cellular RNA was isolated and reverse transcribed to cDNA. Real-time PCR amplifications were performed using SYBR green real-time PCR kit . The primers are listed in 50 nM mimics or 75 nM inhibitor of miR-140-5p  was transfected into cell cultures using lipofectamine RNAi-max  to activate or inactivate miR-140-5p activity, respectively. Negative controls (Scramble) were used for both reactions. 12 h following transfection, the transfection medium was replaced with complete medium and was then replaced with AIM when the cells reached 100% confluence to induce adipocyte differentiation. 48 h following adipogenic treatment, the cells were subjected to RNA extraction and qRT-PCR analysis. 72 h following adipogenic treatment, the cells were subjected to protein isolation and Western blotting analysis. For oil-red O staining, the cells were treated with AIM for 72 h, then with insulin alone for additional 48 h.For the Tgfbr1 loss of function study, we transfected ST2 cells for 12 h with either 30 nmol/L Tgfbr1 siRNAs or negative control siRNA  using lipofectamine RNAi-max. For the Tgfbr1 gain of function studies, the ST2 cells were transfected with Tgfbr1 expression plasmid or the empty vector for 12 h using lipofectamine 3000 . AIM was added to the cultures to allow the cells to differentiate when reaching 100% confluence.http://www.targetscan.org/), PicTar (http://pictar.mdc-berlin.de/) and miRDB (mirdb.org/miRDB) were used to predict the potential target genes of miR-140-5p. We PCR-amplified the 3′-UTR sequences of the potential target genes carrying the putative miR-140-5p binding sites using mouse DNA as a template. The primers are listed in The online programs TargetScan (miR140/Luc) was made as described by YangKpnI and HindIII sites, respectively. Point mutation to the C/EBP binding sequence  was made. To study the involvement of C/EBPs in the regulation, the wild-type or mutant miR-140-5p promoter construct was co-transfected with C/EBPα expression plasmid , or the empty vector into 3T3-L1 cells using lipofectamine 3000 reagent. pRL-SV40 was also included in the co-transfection mixture. 36 h after transfection, the cell extracts were harvested and the luciferase activity was measured.For the miR-140-5p promoter study, the wild-type mouse miR-140-5p promoter construct , and then fixed in 4% paraformaldehyde for 10 min. The samples were then washed twice with deionized water, and staining was carried out in 60% saturated oil-red O solution for 5 min. For oil-red O quantification, isopropanol was added to each well. Light absorbance was measured at 520 nm.The ST2 cells were cultured for 14 days followed by alkaline phosphatase (ALP) staining as described in previous studiesCells were lysed and proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane. The membranes were incubated overnight with rabbit monoclonal anti-C/EBPα, , rabbit polyclonal anti-TGFBR1 , rabbit polyclonal anti-PPARγ, anti-aP2, or anti-β-actin antibodies . This was then followed by incubation with the corresponding horseradish peroxide-labeled IgG (1:3000) for 1 h. Finally, chemiluminescence reagent  was used to visualize the results.Data are expressed as mean ± SD. For the relative mRNA and luciferase activity quantification, the means of the control groups are set to 1. Statistical analysis was performed using the independent t test. A p value of < 0.05 was considered to be statistically significant.How to cite this article: Zhang, X. et al. miR-140-5p regulates adipocyte differentiation by targeting transforming growth factor-β signaling. Sci. Rep.5, 18118; doi: 10.1038/srep18118 (2015).


NTR, a member of the TNF receptor superfamily member, and through receptor tyrosine kinases , often with opposite functional outcomes. The two classes of receptors are activated preferentially by proneurotrophins and mature processed neurotrophins, respectively. However, both receptor classes also possess neurotrophin-independent signaling functions. Signaling functions of p75NTR and Trk receptors are each influenced by the other class of receptors. This review focuses on the mechanisms responsible for the functional interplay between the two neurotrophin receptor signaling systems.The nerve growth factor family of growth factors, collectively known as neurotrophins, are evolutionarily ancient regulators with an enormous range of biological functions. Reflecting this long history and functional diversity, mechanisms for cellular responses to neurotrophins are exceptionally complex. Neurotrophins signal through p75 Nerve growth factor (NGF) and its orthologs are collectively known as neurotrophins. Mammals have four neurotrophins – NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin 4 . Neurotrophins, functioning as homodimers, have a wide variety of functions in both neural and non-neural tissues, and they control adult physiology as well as embryonic development.NTR), which is a member of the death domain-containing receptor subgroup  of the TNF receptor superfamily. While p75NTR is activated by all four neurotrophins, the Trk receptors are more selective, as shown in the table.The 4 neurotrophins signal through three paralogous receptor tyrosine kinases  and the 75 kDa neurotrophin receptor  and p75NTR signaling promoting long term depression (LTD)6. Functional interactions between the two receptor systems produce multiple levels of complexity, while several different mechanisms control the balance between the yin and the yang of neurotrophin signaling.It has been said that there is a yin and yang relationship between p75NTR binds both mature and proneurotrophins, and is more effectively activated by proneurotrophins, while only mature neurotrophins activate Trk receptors9. The enhanced action of proneurotrophins binding to p75NTR is dependent on association of p75NTR with sortilin or SorCS2, Vps10p-domain proteins which bind a conserved motif in proneurotrophin prodomains11.Like many biologically active polypeptides, neurotrophins are synthesized as precursors (pro-neurotrophins), which are cleaved to release an N-terminal prodomain peptide and a C-terminal mature neurotrophin. This cleavage event may occur either within the secretory pathway or following secretion, so that receptors may be exposed to both proneurotrophins and mature neurotrophins. Importantly, p75NTR, but not TrkB. For these neurons, proNGF, which activates p75NTR but not TrkA, promotes cell death. Mature NGF, which activates both p75NTR and TrkA, promotes cell survival. ProBDNF or mature BDNF promotes cell death, because these ligands bind p75NTR but not TrkA14.The complexity of function that can be generated by these relationships is well illustrated by sympathetic neurons, which express TrkA and p7515. However, alternatively spliced forms of TrkB and TrkC (misleadingly known as truncated TrkB and truncated TrkC) lack a tyrosine kinase domain, but possess alternative cytoplasmic domain sequences that signal by less extensively characterized mechanisms17.The canonical mode of signaling by Trk receptors is similar to signaling by other receptor tyrosine kinases. Neurotrophin binding promotes formation of Trk dimers, and induces trans-phosphorylation of Trk cytoplasmic domain tyrosine residues, initiating recruitment of signaling adapter proteins that foster signaling by ras/ERK1/2, PI3 kinase/Akt STAT and phospholipase Cγ pathways21. The exquisite complexity associated with this mode of signaling is nicely illustrated by sympathetic neurons, where NT3 and NGF differently control axonal TrkA signaling functions because of differences in the pH-dependence for NT3 and NGF binding. Sympathetic axons encounter NT3 on the route to their target. NT3 activates TrkA and achieves local control of axonal growth cone dynamics, but does not engage signaling to the cell soma because NT3 dissociates from TrkA at the acidic pH within endosomes, causing TrkA receptors to recycle to the local plasma membrane without production of axonally transported signaling endosomes. In contrast, NGF/TrkA complexes remain intact as endosomes acidify, allowing TrkA to engage the motor systems that mediate retrograde axonal transport of TrkA-bearing endosomes22.One feature of canonical signaling by Trk receptors that differs from many other receptor tyrosine kinases is the use of so-called signaling endosomes to achieve retrograde axonal signaling. For most receptor tyrosine kinases, ligand-mediated activation of the receptor leads to receptor endocytosis, followed either by lysosomal degradation of the receptor or recycling back to the cell surface. However, in many physiological scenarios, the survival and/or differentiated state of neurons is regulated by neurotrophins secreted by the target tissues those neurons innervate. In this context, endocytosis of the neurotrophin/Trk complex generates signaling endosomes, which undergo retrograde axonal transport, delivering the activated neurotrophin/receptor complex to the somatic compartment in order to permit control of nuclear transactivation of genesNTR signaling shares several features with other death receptors. A juxta-membrane region of the cytoplasmic domain binds TRAF6, which engages signaling pathways leading to activation of NF-κB and JNK24. The death domain interacts with RhoGDI, which controls RhoA activation, and RIP2 kinase, which contributes to NF-κB and JNK activation25. Neurotrophin binding to p75NTR inhibits RhoA activation26, while enhancing JNK activation28. However, TRAF6, RhoGDI and RIP2 are only a few of the bewildering array of p75NTR-binding signaling adapter proteins that have been reported to mediate p75NTR signaling. Other notable examples include NRIF, which promotes JNK activation29, MAGE proteins including NRAGE, which promote Rac1 and JNK activation30, and Bex1, which negatively affects NF-κB signaling32. Further, p75NTR has been reported to influence glucose uptake in adipocytes via Glut4 by directly binding the trafficking regulators Rab5 and Rab3133, to controls energy expenditure in obese mice on a high-fat diet by inhibiting cAMP signaling in adipocytes via direct association of p75NTR with protein kinase A34, and to promote fibrinolysis in nerve injury and lung fibrosis by binding and enhancing the cAMP degradative activity of phosphodiesterase PDE4A4/535.p75NTR function is unique, so far, among known receptors. A cysteinyl residue in the membrane spanning domain of p75NTR forms a disulfide bond, within the lipid bilayer, creating a covalently linked-dimeric form of p75NTR, and this covalent linkage is required for neurotrophin-dependent JNK activation, but not inhibition of RhoA activity36. Mutation of the single cysteinyl residue required to form this disulfide bond eliminates p75NTR-dependent death signaling in neuronsin vitro andin vivo37. A physiologically occurring disulfide bond within a lipid bilayer has never been described previously in any membrane protein, and the mechanism by which a disulfide forms in such an unusual environment is unclear. However, our unpublished evidence (Leslayann Schecterson and Mark Bothwell) demonstrates that this linkage forms within 2 minutes when cells are exposed to minute concentrations of hydrogen peroxide, indicating that oxidative stress may control p75NTR signaling, as we have reported previously38.One feature of p75NTR signaling25. In the absence of bound neurotrophin, the death domains of p75NTR dimers form a homodimeric complex. The RhoGDI binding site is not occluded by this interaction, so non-liganded p75NTR engages RhoGDI-dependent RhoA activation. Binding of a neurotrophin dimer to the extracellular domain of a disulfide-linked p75NTR dimer, causes a scissoring action (or more accurately a snail-tong action) around the disulfide pivot-point, separating originally juxtaposed death domains, and allowing access of RIP2 to a binding site that was previously partially occluded by the death domain/death domain interaction. The RIP2 binding site partially overlaps with the RhoGDI binding site, so RIP2 binding displaces RhoGDI, initiating JNK activation and terminating RhoA activation.A detailed model, illustrated inNTR oligomer observed on non-reducing SDS gels is a trimer, rather than a dimer40. This conclusion relies primarily on the ratio of the apparent molecular weights of p75NTR monomer and oligomer on non-reducing SDS gels. A caveat for such analysis is that the theoretical basis for the proportionality of electrophoretic mobility and protein mass on SDS gels assumes that SDS-induced denaturation fully unfolds the protein and causes the protein to form linear structures with length proportional to mass41. This assumption does not apply to p75NTR, which contains multiple intra-chain disulfide linkages if disulfide bonds are not reduced before electrophoresis. p75NTR function as a trimer is inconsistent with X-ray crystallographic and/or NMR generated three-dimensional structures indicating that the extracellular domain of p75NTR42, the death domain region of the intracellular domain of p75NTR25, and the membrane spanning domain of p75NTR43 each forms dimers, not trimers. Application of emerging technologies such as cryo-EM will be required to provide definitive evidence about the stoichiometry of intact p75NTR.Challenging the elegant simplicity of this model, an element of controversy has been introduced by the suggestion that the p75NTR signaling, and its manner of influence by Trk receptors, provides substantial additional complexity, as summarized inNTR/Trk heterodimeric complexes could form, enhancing the affinity of NGF binding to TrkA44, and causing TrkA and TrkB to be less effectively activated by NT315. Although the physiological importance of these p75NTR effects on Trk signaling remain uncertain, recently Trk-dependent effects on p75NTR signaling have emerged that seem likely to have physiological relevance. p75NTR has an alternative signaling pathway that resembles the mode of signaling of Notch. ADAM10 or ADAM17-dependent cleavage of the p75NTR extracellular domain near the membrane, followed by γ-secretase mediated release of the intracellular domain into the cytoplasm, fosters signaling47. Interestingly, a similar mode of signaling by another TNF receptor superfamily member, TNFR1, has been described recently48. Differential cleavage of p75NTR in different types of neurons produced different signaling outcomes49. Signaling effects attributed to the mobilized p75NTR intracellular domain include nuclear accumulation of NRIF47, association with the ubiquitin ligase siah2 controlling degradation of the transcription factor Hif1α50, and association with nuclear pore complexes promoting nuclear uptake of the SMAD2 transcription factor51. Neurotrophin binding to p75NTR does not directly influence the rate of ADAM protease-mediated cleavage of p75NTR52 although signaling pathways initiated by neurotrophin binding may enhance p75NTR cleavage many hours after neurotrophin exposure47. Interestingly, activation of Trk receptors promotes the p75NTR cleavage pathway53.Although the reader may think that the preceding account is already quite complicated enough, another mode of p75NTR cleavage signaling pathwaysin vivo. However, the recently reported function of TrkA and TrkC as dependence receptors may reflect a consequence of this mode of interaction. Dependence receptors are receptors that signal constitutively until ligand binding terminates signaling. Although each of the three Trk paralogs was originally found to promote neuronal survival, in some neuronal populations, neurotrophin-independent effects of TrkA and TrkC were reported to promote neuronal cell death, and the cleavage mediated signaling pathway of p75NTR has been implicated as a mediator of this effect55.It remains to be determined definitively whether Trk activity represents a major mode of regulation of the p75NTR receptors. For Trk receptors, the most common mechanism for signaling in response to non-neurotrophin ligands involves receptor transactivation, most commonly of TrkB. A variety of G protein-coupled receptors, including PACAP and A2a adenosine receptors, activate TrkB via Gsα-dependent activation of Src family kinases 57. In the context of embryonic cerebral cortex, where developing neurons express abundant TrkB receptors, EGF-dependent activation of EGF receptors engages Src-dependent TrkB activation58. Src family kinase-mediated Trk transactivation also is induced by ligand-dependent activation of Low-density lipoprotein receptor-related protein 1 (LRP1)59, and by zinc ion, which is co-released during glutamatergic neurotransmission61. One interesting feature of transactivation of TrkB is that activation commonly occurs in the ERGIC or Golgi compartments, rather than at the cell surface. Signaling from intracellular sites may not be functionally equivalent to signaling from the plasma membrane. For example, PACAP-dependent transactivation of TrkB in cultured hippocampal neurons, by coupling to pathways that otherwise control Golgi dynamics during cell division, induces fragmentation of the Golgi apparatus and alters Golgi-dependent processing of other membrane proteins62.The foregoing paragraphs have focused on neurotrophin-dependent signaling by neurotrophin receptors, but other ligands importantly engage signaling by both Trk and p75NTR have been reported. p75NTR is one of several receptors that bind Aβ peptide and putatively engage in pathogenic signaling in Alzheimer’s disease63. Other scenarios in which non-neurotrophin ligands control p75NTR signaling involve association of p75NTR with co-receptors that bind the activating ligand. Axon--repellant signaling by CNS myelin proteins such as Nogo, MAG, or OMgp, mediated by association of NgR1 with p75NTR,65 or the p75NTR homolog, Troy66 while ephrin-A/p75NTR complexes have been implicated as mediators of EPH-dependent reverse signaling67.A variety of modes of neurotrophin-independent activation of p75in vivo is sadly lacking. It is a daunting task to understand how this extraordinarily rich palette of neurotrophin receptor signaling modalities is controlled physiologically.Although great progress has been made in elucidating the signaling pathways employed by neurotrophin receptors, a systems level understanding of how these signaling pathways are selectively engagedNTR, 75 kDa neurotrophin receptor; RhoGDI, Rho GDP dissociation inhibitor; TNF, tumor necrosis factor; Trk, tropomyosin related kinase.BDNF, brain-derived neurotrophic factor; EGF, epidermal growth factor; ERGIC, endoplasmic reticulum Golgi intermediate compartment; JNK, Jun kinase; MAG, myelin associated glycoprotein; NGF, nerve growth factor; NMR, nuclear magnetic resonance; NT3, neurotrophin 3; NT4, neurotrophin 4; OMgp, oligomyelin glycoprotein; p75


Human T-cell leukemia virus type 1 (HTLV-1) causes both neoplastic and inflammatory diseases: adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Since these disabling and/or life threatening diseases are not yet curable, it is important to prevent new infections. In this study, we have established a simple humanized mouse model of HTLV-1 infection for evaluating therapeutic and immunomodulatory interventions. Using this model, we tested the effect of HTLV-1 specific neutralizing antibodies. HTLV-1-negative normal human peripheral blood mononuclear cells (PBMCs) were transplanted directly into the spleens of severely immunodeficient mice (NOD/SCID/γCnull: NOG) together with the mitomycin-treated HTLV-1 producing T cells (ILT-M1). Before (one hour) and after (24 hours) transplantation of human PBMCs, monoclonal antibodies against HTLV-1 as well as human IgG isolated from both HTLV-1 infected and non-infected individuals were inoculated intraperitonealy. On day 14, human PBMCs were isolated from mouse spleen, and tested for HTLV-1 infection by real time PCR and flow cytometry. Similar to the naturally HTLV-1 infected PBMCs, both CD4+ and CD8+ T cells isolated from untreated or isotype antibody treated mice were found to be HTLV-1 infected, and the CD8+ T cells harbored HTLV-1 to a lesser extent. Also, HTLV-1 Tax expression was negative in isolated human PBMCs but became positive after 16 hours of culture. Although non-neutralizing monoclonal antibodies to gp46, monoclonal antibody to gag p19, and normal human IgG did not block the infection, neutralizing monoclonal antibody to gp46 and human anti-HTLV-1 IgG completely blocked the infection. Our findings provide a new strategy for preventing initial HTLV-1 infection and blocking further spread in vivo. The potential mechanisms involved in the antibody effect will also be discussed.