Here, potassium usnate (KU) was prepared by the salinization of UA to enhance its water solubility. KU showed increased bioavailability compared with UA in the tumor, liver, and plasma of a CT26 syngeneic mouse tumor xenograft model after oral administration, as determined by LC-MS/MS analysis. KU exhibited potent anticancer effects on colorectal cancer cells and inhibited liver metastasis in an orthotopic murine colorectal cancer model. KU treatment downregulated the epithelial-mesenchymal markers Twist, Snail, and Slug and the metastasis-related genes CAPN1, CDC42, CFL1, IGF1, WASF1, and WASL in cells and tumor tissues. The present results suggest the potential application of the water-soluble form of UA, KU, in anticancer therapy.Usnic acid (UA), a lichen secondary substance, has considerable anticancer activity  Excellent solubility and a satisfactory dissolution rate are essential conditions for the clinical application of candidate drugs.Drug oral bioavailability is positively related to reduced molecular flexibility and low polar surface area or total hydrogen bond count3. UA was first shown to have inhibitory activity against lung cancer cells5, and its effect on inhibiting proliferation was subsequently demonstrated in a wide variety of cancer cell lines6. Despite the promising anticancer activity of UA, it has not been developed for clinical application because of poor water solubility7 and high hepatotoxicity9. Therefore, the development of a bioavailable form of UA is an important issue in clinical research. Several strategies were proposed to improve the anticancer activity of UA in vivo by developing modified forms with high water solubility and potent antitumor activity. Studies focused on finding a suitable solvent for the solubilization of UA that showed no direct effects on any of the commonly used cell lines. 2-Hydroxypropyl-beta-cyclodextrin was identified as a solubilizing agent for UA that does not affect its antiproliferative activity against the human leukemia cell line K-56210. Nanoencapsulation was investigated as a method to improve the antitumor activity and reduce the hepatotoxicity of UA, and the encapsulation of UA into PLGA-nanocapsules produced a 26% increase in ascitic tumor formation (Sarcoma-180) in inoculated Swiss mice and a reduction in drug hepatotoxicity11. However, the underlying mechanism remains unclear. Also, Polyacrylamide complex formation with UA by establishment of strong acidic-base interactions made UA water soluble and showed enhanced antimicrobial activity12, and Nanocrystal suspensions of UA prepared by wet milling method showed higher bioavailability in rats13. Another study demonstrated that the cytotoxic activity of UA against L1210 cells can be improved by conjugation to a polyamine chain14. UA was derivatized with various amine moieties to improve drug cellular uptake by targeting the polyamine transport system (PTS). Although the results showed an increase in drug cytotoxicity against human cancer cell lines, drug targeting to the PTS was unsuccessful. In the present study, potassium usnate (KU) was generated using a previously described method, and its anticancer activity was examined in vitro and in vivo experimental model of colorectal cancer as colorectal cancer is one of the most common cancer types and, despite of advances in developing chemotherapy regimen, patients with the cancer suffer from local recurrence with chemoresistance and/or metastasis with 40–80% to liver15. The oral bioavailability of KU was enhanced significantly after oral administration in a CT26 syngeneic mouse xenograft model, as determined by LC-MS/MS detection. KU showed potent anticancer activity against colorectal cancer cell lines, and KU administration suppressed tumor growth in a mouse liver metastasis model. The findings of the present study suggest a promising solution for the low water solubility-related limitation to the use of UA in clinical anticancer therapy.Usnic acid (UA) is one of the most widely studied bioactive lichen secondary metabolites17. Also, it was reported that UA has cytotoxic activity against HCT116, human colorectal cancer cells18. Here, to further examine the anticancer effect of UA on colorectal cancer, human colorectal cancer cell lines including HCT116, DLD1, SW480, HT29, SW620, Caco2, and COLO320 and the CT26 mouse colon carcinoma cell line were exposed to UA at concentrations of 12.5–100 μM, and cell viability was assessed with the MTT assay. As shown in Fig. In a previous study, we demonstrated the cytotoxic, anti-motility, and anticancer activity of UA against several cancer cellsin vivo efficacy of UA was then assessed in orthotopic liver metastasis mouse models. Firefly luciferase-expressing CT26 cells were inoculated via splenic injection to form multiple tumor foci in the livers of syngeneic BALB/c mice. On day 3 after tumor establishment, 5 or 10 mg/kg UA in DMSO in a total volume of 200 μL of PBS was administered via intraperitoneal injection (six or ten times within 2 weeks), and each mouse was analyzed by optical imaging on days 2, 9, and 16. Due to the solubility limit, the maximum dosage of UA is 10 mg/kg. Immediately after final imaging analysis, the liver and disseminated peritoneal tumors were excised and counted. Isolated mouse liver tissues from the control and treatment groups showed different numbers of tumor nodules of different sizes  at 25 °C, pH 7.4 by μSol Method with DMSO stock solution; the structures and the physical properties of KU and UA are shown in Figs in vivo, and KU represents a potential form for in vivo administration as its oral bioavailability was higher than that of UA.As low solubility may account for the poor effect of UA 50 values were calculated for UA and KU in the tested cells. The results showed a significantly lower IC50 value for KU than for UA except in SW480 and CT26 cells  in a total volume of 200 μL were administered via intraperitoneal injection (six times within 2 weeks). Immediately after the final imaging analysis, the liver and disseminated peritoneal tumors were excised and counted. The number of tumor nodules was significantly lower in the KU-treated group than in the control group as determined from isolated liver tissue images  and alanine transaminase (ALT) were measured in the sham and liver metastasis mouse groups. These enzymes are released from liver cells when the liver is injured or damaged, and increased AST and ALT levels in the plasma indicate hepatic toxicity or disease. AST and ALT levels were higher in orthotopic liver metastasis mice than in the sham control group  markers in A549 lung cancer cells2 profilerTM PCR assay was performed to screen for cell motility-related target genes of KU in Caco2 cells. The results showed that CAPN1, CDC42, CFL1, IGF1, WASF1, and WASL were significantly downregulated by KU treatment  was partially dissolved in 40 mL of water at 40 °C and then mixed with 10% KOH until the compound was completely solubilized. The solution was frozen at −80 °C and lyophilized, and the dried powder was dissolved in autoclaved distilled water (DW) before use. The structure of KU was confirmed by nuclear magnetic resonance.KU was prepared following a previously published method22. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin solution under a humidified 5% CO2 atmosphere at 37 °C in an incubator.The human colorectal cancer cell lines HCT116, DLD1, SW480, HT29, SW620, Caco2, and COLO320, and the mouse colorectal cancer cell line CT26, were used in this study23. Briefly, cells were seeded and cultured in 96-well plates (2.5 × 103 cells/well), and treated with UA or KU for 48 h. After DMSO dissolution, the absorbance at 540 nm was determined using a microplate reader with Gen 5 (2.03.1) software .Colorectal cancer cell growth and survival were detected with the colorimetric quantification of 3--2,5-diphenyltetrazolium bromide using the MTT assay16. A 120 μL Cells (2 × 106) suspended in medium (DMEM containing 0.2% BSA dissolved in PBS) treated with or without UA or KU were seeded in the upper transwell chamber  coated with gelatin. Then, fibronectin (10 μg/mL) was added into the lower chamber medium (400 μL) as a chemotactic agent.Then invaded cells adhering to the underneath of upper chamber were fixed and stained with the Diff Quik kit (Sysmex) after 24 h of incubation. The quantitation of the cells were performed under an upright microscope (5 fields/chamber). Each invasion assay was repeated in three independent experiments.Invasion assays were performed in Boyden chambers in vivo experiments were performed. The Chonnam National University Medical School Research Institutional Animal Care and Use Committee approved the experimental protocol.Five-week-old male BALB/c mice with pathogen-free condition were obtained from Orient Company . According to the Guiding Principles in the Care and Use of Animals , the handling of animals and all 24. Briefly, a left subcostal incision was performed under isoflurane anesthesia, and CT26 cells expressing firefly luciferase (CT26-Fluc) (1 × 105 cells) in 50 μL of PBS were inoculated by intrasplenic injection. For optical live imaging in this model, a splenectomy was performed immediately after inoculation of tumor cell, and the firefly luciferase activity was analyzed at several time points with the Xenogen system. After imaging analysis, the liver and disseminated peritoneal tumors were excised and weighed. Weight of tumor was determined by subtracting the average weight of liver of normal mice (n = 4) from the weight of total tumor-bearing liver with addition of the weight of total extrahepatic tumors. UA or KU  was given via intraperitoneal injection 3 days after inoculation of tumor cell. To evaluate inhibition of tumor growth, mice were analyzed every 7 days with the use of optical imaging. On day 21 after inoculation of tumor, mice were sacrificed and a laparotomy was performed. Metastatic tumor nodules in the liver with a diameter of >1.0 mm were counted using a microscope, and a metastasis score was assigned based on nodule size as follows: 0 (no gross metastasis), 1 (tumor size >1 mm), and 10 (tumor size >5 mm). The metastasis score was multiplied by the number and the score of nodules.A syngeneic mouse model of colorectal cancer metastasis to the liver was established by infusion of tumor cells into the portal system via intrasplenic injectionin vitro imaging, CT26-Fluc cells subjected to lentiviral vector transduction were confirmed with the Xenogen system at 2 min after addition of D-luciferin. For in vivo imaging, anesthetization of mice were indueded with ketamine (100 mg/kg) and xylazine (10 mg/kg), and 2 min after intraperitoneal (ip) administration with D-luciferin (2 mg/mouse), analysis of bioluminescent signals were performed with the Xenogen system with a 1 min acquisition time.Examination of bioluminescence in cultured cells or live mice was performed by optical imaging using a cooled CCD system (Xenogen IVIS). Photographic images of the tissues with gray-scale background were overlaid with color images of bioluminescent signals using Living Image and IGOR-PRO image analysis software (Wave Metrics). For Stock solutions (10 mM) of UA or KU were prepared by dissolution in DMSO. Stock standard solutions were stored at −20 °C. A working standard solution was prepared by dilution of the stock standard solution with a mixture of acetonitrile-water . The working standard solution was serially diluted to prepare a concentration series of 0.975, 3.9, 15.6, 62.5, 250, 1000, and 4000 nM in 50% acetonitrile.25. After 7 days of inoculation and gross tumor formation, UA or KU at 30 mg/kg was administered orally, and tumor, liver, and plasma samples were collected after 16 h (n = 9 each). Mouse tumor and liver samples were homogenized in four volumes of ice-cold deionized water. Plasma and homogenized tissue samples were prepared for analysis in a 96-well cluster tube plate by protein precipitation. A volume of 30 µL of each sample was transferred to eight-well tube strips placed in an 8 × 12 rack . Four volumes of ice-cold extraction solution (acetonitrile) containing 4-methylumbelliferone  were added and vortexed for 10 min. After sonication for 30 min, samples were placed on ice for 1 h. The 96-well cluster tube plate was centrifuged at 1000 × g for 10 min, and the supernatant was analyzed by LC-MS/MS. Standard samples were prepared in the same manner using a blank matrix.Skin tumor xenografts were established using CT26 cells18 column  and maintained at 30 °C. The column was pre-equilibrated in 100% v/v solvent A (deionized water containing 0.1% v/v formic acid)/0% v/v solvent B (acetonitrile containing 0.1% v/v formic acid) at a flow rate of 0.3 mL/min. The optimized LC elution conditions were 0.0–1.0 min, 0% B; 1.1–2.0 min, 50% B; 2.1–6.0 min, 98% B; 6.0–6.01 min, 0% B; and 6.01–8.0 min, 0% B. The overall chromatographic run time was 8 min. The autosampler compartment was maintained at 10 °C throughout the analysis. The retention times of UA, KU, and IS (4-MUF) were 4.52, 4.52, and 3.76 min, respectively. The ESI source was operated at −4500 V and 500 °C in a negative mode. Quadrupoles Q1 and Q3 were set on unity resolution. The samples were analyzed via multiple reaction monitoring. The monitoring ions were set as m/z 343 → 839 for UA and m/z 175 → 133 for 4-MUF. The acquisition and analysis of data were performed with Analyst™ software .Samples were analyzed with the Prominence UPLC system  equipped with an API 3200 QTRAP™ LC-MS/MS system . Sample volumes of 10 μL were injected into an Aquity HSS UPLC CResected livers were fixed in 10% neutral-buffered formalin for 3 days. Then, the organs were dissected, embedded in paraffin, and stained with hematoxylin for histopathological evaluation. Tissue sections were immunostained with specific antibodies against phospho-Histone H3 , Slug , Snail , and Twist  using a Bond-max system . Programmed heat-induced epitope retrieval was performed for 15 min using citrate-based pH 6.0 BOND epitope retrieval solution 1 (Slug) or EDTA-based pH 9.0 epitope retrieval solution 2 (Snail and Twist).26. Antibodies against E-cadherin , N-cadherin , Snail/Slug , Twist , PARP , Caspase-3 , α-Tubulin , and ZEB2  were detected with horseradish peroxidase-conjugated secondary antibody  with the use of Immobilon Western Chemiluminescent HRP Substrate Kit  and luminescence imaging (Image Quant LAS 4000 mini). Multi-Gauge 3.0 was used to measure bands, and relative density was calculated based on the density of the α-tubulin bands in each sample. Expression of values were as arbitrary densitometric units corresponding to signal intensity.Cells treated with UA or KU for 24 h were washed twice with ice-cold PBS and lysed in lysisbuffer27. The manufacturer’s instructions were strictly followed. Gene expression levels were analyzed using the web-based software ‘RT2 Profiler PCR Array Data Analysis version 3.5′. Six cell motility-related genes, including CAPN1, CDC42, CFL1, IGF1, WASF1, and WASL, were selected as candidates.The RT² Profiler™ PCR Array  was used to examine the expression patterns of 84 genes involved in human cell motility as previouslydescribed23. Briefly, total RNA (1 mg) isolated from UA- or KU-treated Caco2 cells using RNAiso Plus  was used to synthesize cDNA using a M-MLV reverse transcriptase kit  and SYBR green . The primers used for real-time PCR were as follows: E-cadherin (forward) 5′-cagaaagttttccaccaaag-3′ and (reverse) 5′-aaatgtgagcaattctgctt-3′; N-cadherin (forward) 5′-ctcctatgagtggaacaggaacg-3′ and (reverse) 5′-ttggatcaatgtcataatcaagtgctgta-3′; Snail (forward) 5′-tcccgggcaatttaacaatg-3′ and (reverse) 5′-tgggagacacatcggtcga-3′; Twist (forward) 5′-cgggagtccgcagtctta-3′ and (reverse) 5′-tgaatcttgctcagcttgtc-3′; Slug (forward) 5′-cgaactggacacacatacagtg-3′ and (reverse) 5′-ctgaggatctctggttgtggt-3′; ZEB1(forward) 5′-atgacacaggaaaggaagg-3′ and (reverse) 5′-agcagtgtcttgttgtag-3′; ZEB2 (forward) 5′-caagaggcgcaaacaagcc-3′ and (reverse) 5′-ggttggcaataccgtcatcc-3′; CAPN1 (forward) 5′-cctgcttgagaaggcctatg-3′ and (reverse) 5′-ggtccacgttgttccactct-3′; CDC42 (forward) 5′-aggctctctagtttaataaaaatcatgg-3′ and (reverse) 5′-gtttgtttaatacatctgaaaagaatgc-3′; CFL1 (forward) 5′-caaggatgccatcaagaa-3′ and (reverse) 5′-atccttagcctcctcgta-3′; IGF1 (forward) 5′-gatacacatcatgtcgtcttcaca-3′ and (reverse) 5′-cagtacatctccagtctcctcaga-3′; WASF1 (forward) 5′-tcctgatgttttaaaagaagaaacact-3′ and (reverse) 5′-aaaagtttttaactcctataggcaagc-3′; WASL (forward) 5′-agtggaggtctctgattggcc-3′ and (reverse) 5′-tctcctttcagggtctccca-3′; and GAPDH (forward) 5′-atcaccatcttccaggagcga-3′ and (reverse) 5′-agttgtcatggatgaccttggc-3′. qRT-PCR reactions and analyses were performed using CFX .Quantitative RT-PCR (qRT-PCR) was performed as previously describedt-test was utilized to determine statistical significance between two groups, and analysis of variance was utilized between three or more groups, respectively. P-values of < 0.05 are considered statistically significant.Data are presented as the mean ± standard error of the mean obtained from three independent experiments unless otherwise indicated. The Student’s Dataset 1


Models of G protein-coupled receptor (GPCR) signaling have dramatically altered over the past two decades. Indeed, GPCRs such as the follicle-stimulating hormone receptor (FSHR) have contributed to these new emerging models. We now understand that receptor signaling is highly organized at a spatial level, whereby signaling not only occurs from the plasma membrane but distinct intracellular compartments. Recent studies in the role of membrane trafficking and spatial organization of GPCR signaling in regulating gonadotropin hormone receptor activity has identified novel intracellular compartments, which are tightly linked with receptor signaling and reciprocally regulated by the cellular trafficking machinery. Understanding the impact of these cell biological mechanisms to physiology and pathophysiology is emerging for certain GPCRs. However, for FSHR, the potential impact in both health and disease and the therapeutic possibilities of these newly identified systems is currently unknown, but offers the potential to reassess prior strategies, or unveil novel opportunities, in targeting this receptor. The follicle-stimulating hormone receptor (FSHR) belongs to the superfamily of G protein-coupled receptors (GPCRs). With more than 800 members in humans they represent the largest family of signaling receptors and a major, successful, drug target . The canThis review will discuss our current understanding of the molecular mechanisms and signaling roles of membrane trafficking of FSHR, and how gonadotropin hormone receptors have shed light on novel cell biological pathways potentially applicable to many GPCRs. We will primarily focus on post-endocytic intracellular trafficking, and then discuss how this novel cell biology could shed light on specific facets of FSH/FSHR function and its implications to endocrine function.21/18 and the fully glycosylated FSH24     . This labinding) . While pFollowing internalization, GPCRs are trafficked to endosomes where they are sorted to either a plasma membrane recycling pathway, or to the lysosomal pathway for degradation. Such pathways program the temporal profile of G protein signaling, by regulating resensitization/hormone recovery (recycling) or permanent signal termination (degradation), Additionally, the sorting fate of a GPCR pharmacologically manipulated and altered in disease . HoweverThe textbook model of cargo sorting depicts the Rab5 early endosome (EE) as the common post-endocytic compartment from which receptors are first sorted to opposing fates. GPCRs sorted to a degradative pathway following internalization are trafficked from EEs to Rab7 positive late endosomes. Receptors are involuted into vesicles within the lumen of these endosomes, to form multivesicular bodies (MVBs). MVBs will then fuse with lysosomes resulting in protein degradation. GPCRs will engage with this pathway with distinct kinetics and for those receptors targeted to the recycling pathway, chronic ligand stimulation will reroute receptors to this degradative pathway as part of the mechanism of downregulation. Classically, lysosomal targeting of different receptors is via ubiquitination at lysine residues and engagement with endosomal sorting complex required for transport (ESCRT)-dependent degradation, however, GPCRs exhibit ubiquitin-independent and ESCRT-independence in their mechanisms of degradation . GPWhile the above describes that the recycling pathway of FSHR, and other GPCRs, involves a one-step mechanism with its recycling sequence and interacting partner, which may be unique to a given GPCR, we now know that both common and receptor-specific post-endocytic mechanisms exist. Furthermore, sequence-directed recycling  occur via a complex, multi-step system, with the GPCR's own signaling playing a key role in driving receptors to these distinct cellular fates, in addition to trafficking regulating the signal from that receptor. This interconnected property of post-endocytic trafficking and GPCR signaling has been highlighted recently through studies of the gonadotropin hormone receptors and will be discussed next.The internalization of GPCRs into the endocytic network is no longer viewed as a mechanism to only control plasma membrane signaling but to also provide additional platforms to continue or reactivate signaling from intracellular compartments, including G protein signaling. Furthermore, this spatial control of signaling has been shown to be important for cells to decode common second messenger signaling molecules, such as cAMP signaling, activated by many GPCRs, into specific downstream cellular functions –61. IntrMany GPCRs, including the human FSHR and LHR undergo the regulated recycling pathway described in Section Post-endocytic intracellular trafficking pathways of FSHR. Our studies first identifying the VEE with the gonadotropin hormone receptors were initially driven as way to understand why GPCRs have distinct recycling sequences if there is a common primary function, i.e., sorting to the recycling pathway. Thus, comparisons were first made between the β2AR and LHR that initially seem quite similar in their signaling and trafficking profiles . Both ar3 production and calcium release  to possibly distinct functional consequences. It also raises the intriguing possibility that GPCR activity, can be reprogrammed by the cell to alter the compartment the receptor is targeted to via altering expression of key proteins in this pathway, e.g., GIPC, APPL1 Figure . Such a These recent advances in FSHR function from its distinct roles in extragonadal tissues and novel cell biological functions demonstrates that FSHR is a good example of a GPCR that achieves signal diversification via multiple strategies and a prototype receptor for understanding novel facets of GPCR function. Whether such pathways are then perturbed in disease and if we can harness these properties of GPCRs therapeutically needs to be addressed.While much has been uncovered about novel intracellular trafficking systems, there are numerous outstanding questions at the molecular level. A major one to be tackled in the future is the identifying the molecular composition (both protein and lipid content of the membrane) of the VEEs, as so far APPL1 is the only known protein to reside there and is only present in ~50% of the VEE population . While nThe ability to pharmacologically exploit these new GPCR signaling models and target the intracellular signaling receptor specifically has been recently demonstrated for three distinct GPCRs. Employing cholestenol-conjugated antagonists, which accumulate in the lumen of endosomal compartments, can block endosomal signaling from the neurokinin 1 receptor and calcitonin-gene related peptide receptor and has been shown to be an effective nociceptive target in animal models , 97. Whiin vivo. This would provide the opportunity to be able to target these intracellular signaling modalities with high precision, in order to create the next generation of therapeutics for reproductive medicine.In summary, our understanding of the complexity of GPCR signaling pathways via the tight control of their intracellular location has been advanced through studies on the gonadotropin hormone receptors. Such mechanisms have highlighted the interconnected nature of these intracellular systems, and thus a primary future goal is to further understand the significance of these molecular systems to health and disease if they are to be of therapeutic value. The critical nature of intracellular sorting of FSHR to signaling has been demonstrated, so it is not a question of is it important, but rather how the intricacies of modulating receptor from one intracellular compartment to another impact specific functions All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Psychiatric illnesses may be qualified to the cellular impairments of the function for survival or death in neurons, which may consequently appear as abnormalities in the neuroplasticity. The molecular mechanism has not been well understood, however, it seems that PI3K, AKT, GSK3, and their downstream molecules have crucial roles in the pathogenesis. Through transducing cell surviving signal, the PI3K/AKT/GSK3 pathway may organize an intracellular central network for the action of the synaptic neuroplasticity. In addition, the pathways may also regulate cell proliferation, cell migration, and apoptosis. Several lines of evidence have supported a role for this signaling network underlying the development and treatment for psychiatric illnesses. Indeed, the discovery of molecular biochemical phenotypes would represent a breakthrough in the research for effective treatment. In this review, we summarize advances on the involvement of the PI3K/AKT/GSK3 pathways in cell signaling of neuronal cells. This study may provide novel insights on the mechanism of mental disorder involved in psychiatric illnesses and would open future opportunity for contributions suggesting new targets for diagnostic and/or therapeutic procedures.  Psychiatric illnesses are conditions for which the precise underlying reason remains unknown; however, roles of dysregulation of the signaling related to neurotransmitters, intracellular signal transduction, and neural development have been emphasized in the pathogenesis of these illnesses . For exaNeuron survival mechanisms ordinarily depend on activation of phosphatidylinositol 3′-kinase (PI3K), which exists as a dimer comprising of a 110 kDa catalytic subunit (p110) and an 85 kDa (p85) regulatory subunit . The p11The tumor suppressor PTEN, phosphatase and tensin homolog on chromosome 10, is a dual-specificity phosphatase with protein phosphatase activity and lipid phosphatase activity. Cells that lack PTEN have constitutively high levels of PIP3 and could activate downstream PI3K/AKT . ConversThe GSK3 family is composed of two isoenzymes termed GSK3α and GSK3β, which have been initially recognized for the roles in insulin receptor signaling. GSK3 is constitutively active serine/threonine kinase in cells. The activity of GSK3 is positively regulated by phosphorylation on tyrosine residues (Thy 279 for GSK3α and Thy 216 for GSK3β)  and negaIn general, high mortality of diseases is mostly due to a lack of effective treatments and efficient markers for early diagnosis. The PI3K/AKT/GSK3 signaling cascade may be a center for psychiatric illnesses. If alteration of the signaling activities in brain neurons should be also detected in peripheral blood lymphocytes of illnesses patients, it could work for efficient diagnosis of the illnesses. In human lymphocytes, levels of PI3K subunit p110 have been impaired in patients with schizophrenia . On the GSK3 knock-in mice have revealed high susceptibility to depressive behaviors . In addiNoncoding 20–25-nucleotide-long RNAs termed microRNAs (miRNAs) have biological functions such as cellular proliferation and apoptosis, which could modulate gene expression by miRNA-induced silencing. The potential application of miRNAs has been considered as an early detection biomarker for illnesses. Genome studies have revealed genetic variants adjoining a miR-137 region may contribute to schizophrenia risk , suggestMood stabilizers, antidepressants and antipsychotics all may upturn the PI3K/AKT/GSK3 signaling , resultiCurcuma longa used as culinary turmeric, curcumin, can improve synaptic plasticity and enhance memory abilities [As the efficacy of pharmacological treatments has been imperfect and had unexpected side effects, psychiatric illnesses represent a significant public health problem. A number of preventive factors have been suggested by epidemiological research including lifestyle factors such as diet and physical exercise. Among them, dietary choices could play certain roles in the neuroprotection. In addition, some involvements of food ingredients might be promising in the prevention of brain dysfunction by modifying the PI3K/AKT/GSK3 signaling. For example, an active ingredient derived from the root of bilities . The neubilities . In addibilities . Severalbilities . Kaempfebilities . On the bilities . Serine bilities . Fatty abilities . Supplembilities . In addibilities . Fatty abilities . Anti-debilities .Neuroprotection by inhibiting PTEN tumor suppressor with food ingredients has been described by activating the PI3K/AKT/GSK3 signaling in neuronal cells. For example, a traditional medicinal herb ingredient, Icariin, inhibits the PTEN expression following the AKT activation . In addiLifestyle interventions such as dietary education could be promising and cost-effective for people with psychiatric illnesses. However, despite those experimental observations, the precise mechanisms for food ingredients remain elusive for clinical uses. A number of studies have examined the association between diet and behavioral states, but the findings have been inadequate. In addition, the relation between nutrient intake and neuroprotection activity is intricate. Whereas many questions remain to be answered about the roles of PI3K/AKT/GSK3 signaling in psychiatric disorders, it is possible that the PI3K/AKT/GSK3 signaling of neuronal populations in a certain brain area could be associated with distinct behavioral outcomes. It seems that both activation and inhibition of those molecules, if they drive one-sidedly, may not contribute to the improvement of the neuronal disorders . It seemIn conclusion, progress in understanding molecular key targets in order to develop new therapeutics for psychiatric illnesses would undoubtedly benefit this research field. The involvement of PI3K/AKT/GSK3 signaling in schizophrenia and mood disorders is highly relevant. Some diets may contribute to the neuro-protection in psychiatric illnesses via the modulation of the intracellular neuronal signaling. Therefore, improving treatments for human neuropsychiatric disorders by dietary approach is particularly challenging. Further investigation on the neuronal signaling could lead to a better understanding of the molecular basis implicated in neuropsychiatric illnesses.


Understanding the contribution of peripartum infection to the burden of disease will be important to gauge the risk-benefit of interventions in this area. Few studies have examined the impact of HTLV-1 infection on fertility or pregnancy outcomes nor the susceptibility of the mother to infection during pregnancy and lactation. Whilst breast-feeding is strongly associated with transmission and avoidance of breast-feeding a proven intervention little is known about the mechanism of transmission from the breast milk to the infant and there have been no clinical trials of antiretroviral therapy (ARV) to prevent this route of transmission.Mother-to-child transmission (MTCT) of Human T-cell lymphotropic virus type 1 (HTLV-1) causes lifelong infection. At least 5–10 million individuals worldwide are currently living with HTLV-1. Studies of regional variation are required to better understand the contribution of MTCT to the global burden of infection. Although most infected individuals remain asymptomatic ∼10% develop high morbidity, high mortality disease. Infection early in life is associated with a higher risk of disease development. Adult T-cell leukemia (ATL), which is caused by HTLV-1 and has a median survival of 8 months is linked to MTCT, indeed evidence of ATL following infection as an adult is sparse. Infective dermatitis also only occurs following neonatal infection. Whilst HTLV-1-associated myelopathy (HAM) follows sexual and iatrogenic infection approximately 30% of patients presenting with HAM/TSP acquired the infection through their mothers. HAM/TSP is a disabling neurodegenerative disease that greatly impact patient’s quality of life. To date there is no cure for HTLV-1 infection other than bone marrow transplantation for ATL nor any measure to prevent HTLV-1 associated diseases in an infected individual. In this context, prevention of MTCT is expected to contribute disproportionately to reducing both the incidence of HTLV-1 and the burden of HTLV-1 associated diseases. In order to successfully avoid HTLV-1 MTCT, it is important to understand all the variables involved in this route of infection. Questions remain regarding frequency and risk factors for  Early after the first identification of HTLV-1 the possibility of mother-to-child transmission (MTCT) was supported by several pieces of evidence including: (1) high incidence of HTLV-1 infection in children born to HTLV infected mothers; (2) high prevalence of infection in mothers of HTLV-1 seropositive children; (3) presence of approximately 10ed women ; (5) lowed women . MoreoveWhat We Need to Know About HTLV-1 Infection in Pregnant Women•What is the prevalence of HTLV-1 infection in pregnant women in different countries/regions?•What is the susceptibility to HTLV-1 infection during pregnancy and lactation?•What is the impact of pregnancy on HTLV-1 infection – is viral control affected?•vice-versa?What is the impact of pregnancy in HTLV-1 associated diseases and •Does HTLV-1 infection impact fertility, miscarriage rates or 3rd trimester pregnancy outcomes including birth weight and gestational age at delivery?•What is the impact of HTLV-1 infection and/or its diagnosis on psychological health of pregnant women and parturient?HTLV-1 prevalence is considered more representative of the general population than prevalence studies among blood donors due to the impact of pre-screening on donor self-selection. However, many cross-sectional studies also show a significant increase in prevalence with age, particularly in women and most notably after age 40 . This isThe published prevalence of HTLV-1 infection among pregnant women is represented in the map  and presThe various risk factors for HTLV-1 infection that have been reported in pregnant women or in women of reproductive age are listed in Few studies have examined the impact of pregnancy on HTLV-1 infection. HTLV-1 proviral load (PVL), which is until now, the main predictor of disease outcome, seems to be stable during pregnancy, but increase after delivery in a recent Japanese study of 36 women . Howevervice-versa. ATL in pregnancy has been reported. The majority were acute ATL and most patients died soon after delivery (by pre labor cesarean section [PLCS]), despite treatment  when comparing to non-infected children born from seropositive mothers (n = 42) and may raise the question if underweight babies or preterm births are risk factors for increased HTLV-1 susceptibility (A number of studies have explored the impact of HTLV-1 infection on pregnancy. In a study from Mato Grosso do Sul (Brazil)  6.7% 11. In the tibility . In a catibility . Moreovetibility . Clearlytibility  As pro-itibility , it is pThere are limited data on the impact of HTLV-1 on fertility and on assisted reproduction. While HTLV-1 infection can be associated with erectile dysfunction in up to 55.2% men  and withAnother point that needs more attention is about the impact of HTLV-1 infection in the psychological health of pregnant women and parturient . PregnanWhat we need to know about HTLV-1 mother-to-child transmission•Does intra-uterine transmission occur? If so, why it is so rare?•Which mechanisms are involved in the protection of intra-uterine transmission?•What factors are important in HTLV-1 MTCT?•What is the impact, if any, of co-infections in the risk of MTCT?•Which cells are involved with HTLV-1 transmission via oral route?•What is the mechanism of HTLV-1 transmission across the digestive tract?•Is the cellular component in breast milk from HTLV-1 infected women similar to healthy uninfected women over time?•What is the role of different breast milk constituents in the efficiency of infection?•There is a need to identify maternal neutralizing antibodies and their role in protection.The overall MTCT rates observed in different areas are presented in HTLV-1 proviral DNA was detected in 2/9 placentas from HTLV-1 positive mothers by nested PCR. HTLV-1 antigen (p19) was also identified in placental villous cells cultured from these women . Others,in vitro study, malignantly transformed trophoblast cells (choriocarcinoma) were infected by HTLV-1, however, the infection was essentially non-productive, as viral protein expression could not be detected  cells from HTLV-1 carriers. Notably none of them identified the presence of anti-HTLV IgM in cord blood, which is often considered a marker of intra-uterine infection . The preantibody .Strongyloides stercoralis, respectively, even with similar duration of breastfeeding . Moreover, infected breast luminal epithelial cells can activate primary T cells, which is important to enhance HTLV-1 infection . In the other hand, they did not express a complete DC phenotype (lack of expression of CD14 and CD11b). The transmissibility of HTLV-1 to T cells by infected BrMMφ was more efficient than from MT-2 cells. Acid medium did not alter the capacity of HTLV-1 transmission by these cells, indicating that they can transmit HTLV-1 to other cells even after exposure to acid  is associated with low transmission rates, breastfeeding for longer periods increase the risk of HTLV-1 transmission . This cain vitro in a mechanism that seems to involve NK cells  of children (including those who later become infected through late breastfeeding). Among those who were infected, seroconversion occurred 6–24 months after birth with only IgG and not IgM detectable . TherefoNK cells . Anti-HTNK cells , 1987.in vitro, both TGF-β and PGE2 were able to enhance HTLV-1 transmission to CB lymphocytes  promoter and, interestingly, this activation is more prominent in CB mononuclear cells than in PBMCs. In the other hand, the same protein suppressed viral entry, although to a lesser degree than was observed for HIV . Moriuchphocytes . HTLV-1 phocytes , while Tphocytes .Maternal anti-HTLV-1 antibody titer has also been associated with MTCT risk. However, the association between antibody titer and risk of HTLV-1 transmission may be related to the strong correlation observed with antibody titer and PVL . This coWhen PBMC and BMMC of HTLV-1 infected mothers are cultured they produce HTLV-1 antigen positive cells. Interestingly, 27.9% can be considered high HTLV-1 antigen producing mothers, while 72.1% are considered low antigen producing mothers. The transmission rate of HTLV-1 was 37.5% among high and 3.2% for low antigen producing mothers, independent of duration of breastfeeding. While the production observed was parallel in PBMC and BMMC from the same patient, it did not correlate with maternal anti-HTLV-1 antibody titers, indicating that the cytotoxic T cell response could be more important than humoral response to control HTLV-1 load and, consequently, vertical transmission .Human Leukocyte Antigen (HLA) concordance between mothers and child is another recognized risk factor for HTLV-1 transmission. In Jamaica, HTLV-1 transmission risk increased (1.75-fold) with each increase in concordance in HLA class I types . Maternain utero transplacental or the post-partum gastro-intestinal trans-epithelial route will be important for the design of interventions, particularly post-exposure antiretroviral prophylaxis.Understanding the persistence of HTLV-1 infected maternal lymphocytes in the neonates, whether through the What we need to know about neonatal infection•What are the consequences of neonatal infection in early life as well as in adult life?•in utero?Are these consequences different if infection occurred •What is the relative contribution of HTLV-1 MTCT to the global burden of HTLV-1 associated disease?•How and when should HTLV-1 infection be diagnosed in children?Orally infected young rats present higher PVL in spleen cells when comparing to intraperitoneal infection. In this animal model, oral infection was associated with impaired HTLV-1 specific cellular immune response  and no production of HTLV-1 specific antibodies . TherefoData on the impact of HTLV-1 on child development are scarce. HTLV-1 neonatal infection does not seem to impact the early children development, as no difference in reaching the development milestones was detected in a total of 68 HTLV-1 infected compared with 80 uninfected Peruvian children born to HTLV-1 infected mothers . As the p = 0.06). The anemia was microcytic hypochromic, indicative of iron deficiency, which can also result from parasitic diseases and malnutrition. Both of which can be linked with poor socioeconomic status (c status . The plac status . Interesc status .p < 0.001), which strongly correlated with time since seroconversion  and the percentage of CD25CD4 T cells   . Childre = 0.02) .Infective dermatitis associated with HTLV-1 (IDH) is also observed in children infected early in infancy, and is associated with a high PVL and increased risk for the development of other diseases such as ATL and HAM/TSP . VarandaChildren infected with HTLV-1 report more frequently leg weakness, lumbar pain, paraesthesia/dysesthesia, urinary incontinence and constipation than those uninfected children. Clonus and lower extremity hyperreflexia were associated with HTLV-1 infection, Therefore, it is important to perform neurological evaluation of HTLV-1 infected children, once it can detect otherwise-unrecognized neurological disease . HTLV-1 There are many reports of early onset of HTLV-1 associated diseases, such as HAM/TSP and ATL . A revieJuvenile HAM/TSP occurs predominantly in females and is frequently associated with IDH (history or co-incidence in up to 55.6%) . In JapaA descriptive study of 5 patients with juvenile HAM/TSP showed normal development among all patients, who reached the development milestone at normal age . HoweverATL usually occurs in adults  and the in utero infection alters HTLV-1 infection outcomes in the short or long term.There are no data on whether Until now, there is no cure to the lifelong HTLV-1 infection nor any preventive measure to avoid the onset of HTLV-1 associated diseases in patients living with HTLV-1. Thus, infection prevention is extremely important. While almost all ATL and IDH cases are associated with maternal transmission , MTCT caThe diagnosis of HTLV-1 neonatal infection can be challenging. Passively transmitted antibodies can be detected in new-borns from HTLV-1 positive mothers. These antibodies decrease gradually and at 12 months the great majority of children do not have anti-HTLV-1 maternal antibodies anymore. In the other hand, seroconversion of infected children can occur from months to more than 2 years after birth. No seroconversion after 3 years of age was reported . MoleculWhat we need to know about prophylactic measures•Does pregnancy impair the detection of HTLV-1 infection?•Should we recommend avoidance of breastfeeding to all HTLV-1 seropositive women?•Cost-effectiveness analysis of HTLV-1 antenatal screening in different scenarios•Should short-term breast feeding, and freeze and thawed milk be recommended as a prophylactic measure to MTCT?•Safety and efficacy of antiretroviral therapy (ARV) as a prophylactic method for MTCT•What is the role of neutralizing antibodies in the prevention of MTCT?•What is the impact, if any, of pre-labor elective cesarean section in the prevention of MTCT?•Approaches to a successful antenatal screening program: education of general population, doctors, pregnant womenIn order to consider any measure to avoid MTCT, it is essential to acknowledge HTLV infection in pregnant women. However, HTLV-1 antenatal screening is only mandatory in Japan and part of clinical practice in other areas, such as French Guiana and some Brazilian States, as Bahia.In general population serological tests, such as ELISA, are widely recommended as a screening test, followed by confirmatory assay . More recently, chemiluminescent microparticle immunoassay (CMIA), electrochemiluminescent assays (ECLIA), and line immunoassays (LIA) became available for HTLV-1 diagnosis. Such tests have proven robust for the detection of other blood borne infections in the ante-natal population without loss of sensitivity. However, the National Screening Committee in the United Kingdom have cited lack of evidence that current HTLV assays detect this infection in pregnant women as one reason not to recommend the addition of HTLV-1 testing to the existing national antenatal screening programme .Therefore, the questions which arise are how we should test pregnant women and when? The use of pooled samples to reduce the costs of antenatal screening can be considered , but thiSeveral studies have used different techniques in order to diagnosis HTLV-1 infection during pregnancy . Tsuji eIn a scenario of limited resources and many demands in public health, cost-effectiveness analysis is required to guide policy makers. In Nagasaki (Japan), in the late 80’s, with 4% prevalence, the cost for detecting a HTLV-1 infected person, for preventing an infection and preventing a case of ATL was 50,000, 250,000, and 5,000,000 (JYE), respectively. At the time of the national screening program implementation in Japan (2010), considering the prevalence of 0.1% the estimated cost was 40-fold higher, according to Even if antenatal screening program is implemented in a country, success will depend on many factors including education of obstetrics, pediatrics, and general community . The majThe avoidance of breastfeeding has conclusively been shown to be an efficient measure to prevent post-partum HTLV-1 MTCT  and is tIn this context, it is also important to consider if the presence of risk factors should be taken into account to establish who should avoid breastfeeding. For example, the PVL in mother‘s blood or in breast milk, could be considered to guide this recommendation? The availability of adequate substitutes of breastmilk should also be considered . FinallyTherefore, it is necessary to discuss whether the benefits of breastfeeding will outweigh or not the risk of HTLV-1 infection, and if this is applicable to all scenarios. This remains one of the main questions regarding HTLV-1 MTCT.Early weaning is associated with low risk of HTLV-1 transmission  and therIn fact, The process of freezing and subsequently thawing the breast milk results in morphological changes and loss of function of cells present in milk . Babies in vitro studies demonstrated that drugs, such as Zidovudine, Adefovir, and the prodrugs Adefovir dipivoxil and Tenofovir disoproxil can inhibit HTLV-1 infection of lymphocytes in vitro  . As HTLVin vitro and protected the infants against intra-peritoneum HTLV-1 infection. Moreover, similar results were observed when using humanized LAT-27 antibodies in humanized immunodeficient mice (NOG mice). Both in vitro neutralization of HTLV-1 and in vivo resistance for HTLV-1 infection were acquired  reduced HTLV-1 transmission to rabbit offspring (42.9% vs. 8.3%) . HTLV-1 acquired . Unfortuacquired . This reA study conducted in 1995 evaluated the presence of proviral DNA in maternal blood at delivery, cord blood and oral aspirates from babies delivered by vaginal route and cesarean section. They found that all maternal bloods were positive while all cord bloods were negative. Interestingly, 4/10 new-borns from vaginal route had HTLV-1 proviral DNA in oral aspirates while only one out of seven born by cesarean section had, indicating HTLV exposure during passage through the birth canal .In a cohort of 41 HTLV-1 infected pregnant women in Brazil, 81% delivered by elective cesarean section and all babies were bottle-feed. None of those children were HTLV-1 positive when tested by PCR from 3 to 39 months. The authors hypothesized that the delivery method, together with the avoidance of breastfeeding, contributed to the prevention of vertical transmission .In Okinawa (Japan) a decrease in the transmission rate among non-breastfeed children was observed between 1986–1991 (12.8%) and 1995–1999 (3.2%). Although the difference was not statically significant, the authors highlighted that since 1991, doctors have been advised to protect new-borns against HTLV-1 nosocomial infection at delivery. Therefore, obstetricians and gynecologists have taken precautions to avoid new-borns contamination at the time of delivery, ensuring that they do not swallow blood during birth. They hypothesize that this action contributed to the observed decrease . More stSoon after the discovery of HTLV-1, MTCT has been implicated as one of the most important modes for HTLV transmission and for maintaining HTLV-1 within a population. However, little has been done in the world to avoid HTLV transmission from mother to child. Despite the success observed, Japan remains the only country to include HTLV-1 antenatal screening in their national programme. Therefore, although many research questions remain , the mosCR and GT contributed to conception and design of the study. CR wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.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.


To investigate the role of HTLV-1 subgroups in viral pathogenesis, we studied the functional difference in the subgroup-specific viral transcriptional regulators Tax and HBZ using microarray analysis, reporter gene assays, and evaluation of viral-host protein–protein interaction.Among human T cell leukemia virus type 1 (HTLV-1)-infected individuals, there is an association between HTLV-1 (1) Transcriptional changes in Jurkat Tet-On human T-cells that express each subgroup of Tax or HBZ protein under the control of an inducible promoter revealed different target gene profiles; (2) the number of differentially regulated genes induced by HBZ was 2–3 times higher than that induced by Tax; (3) Tax and HBZ induced the expression of different classes of non-coding RNAs (ncRNAs); (4) the chemokine CXCL10, which has been proposed as a prognostic biomarker for HAM/TSP, was more efficiently induced by subgroup-A Tax (Tax-A) than subgroup-B Tax (Tax-B), in vitro as well as in unmanipulated (ex vivo) PBMCs obtained from HAM/TSP patients; (5) reporter gene assays indicated that although transient Tax expression in an HTLV-1-negative human T-cell line activated the CXCL10 gene promoter through the NF-κB pathway, there was no difference in the ability of each subgroup of Tax to activate the CXCL10 promoter; however, (6) chromatin immunoprecipitation assays showed that the ternary complex containing Tax-A is more efficiently recruited onto the promoter region of CXCL10, which contains two NF-κB binding sites, than that containing Tax-B.Our results indicate that different HTLV-1 subgroups are characterized by different patterns of host gene expression. Differential expression of pathogenesis-related genes by subgroup-specific Tax or HBZ may be associated with the onset of HAM/TSP.The online version of this article (10.1186/s12977-018-0454-x) contains supplementary material, which is available to authorized users. All samples were examined in triplicate. Antibody titers to HTLV-1 were determined using a particle agglutination method , according to the manufacturer’s instructions.To examine the HTLV-1 proviral load (PVL), quantitative PCR was performed using 100 ng of genomic DNA  was performed as previously reported  and 1 mM EDTA) successively. Then, bound proteins were eluted from the agarose beads in elution buffer (1% SDS and 100 mM NaHCO3) by incubation at room temperature for 15 min. Crosslinking was reversed by incubation at 65 °C overnight. All samples were treated with 45 μg/ml of proteinase K  at 50 °C for 2 h, then 40 µg of glycogen  was added, followed by extraction with phenol:chloroform:isoamyl alcohol and precipitation with ethanol. DNA fragments were subjected to PCR or quantitative real-time PCR using EmeraldAmp Master Mix  or SYBR Premix EX Taq II  with specific primer sets: 5′-GAA AGT GAA ACC TAA TTC ACT ATT ACC AA-3′ and 5′-ACT TAG CAA AAC CTG CTG GCT GTT-3′ for genomic DNA fragment containing both NF-κB and AP-1 binding sites. Three independent experiments were performed, and the data represent the mean ± SD.Jurkat cells , the data were statistically analyzed using one-way analysis of variance (ANOVA). Inter-group comparisons were done by Scheffe’s post hoc multiple comparisons test. The Mann–Whitney U test was used to compare data between two groups  Table , or HBZ,CXCL10 mRNA, since CXCL10 was preferentially induced by Tax-A rather than by Tax-B (Table CXCL10 in HAM/TSP patients with subgroup-A were significantly higher than in HAM/TSP patients with subgroup-B (p = 0.0334 by Scheffe’s post hoc multiple comparisons test.) , 20 HCs, and 20 NCs  to the culture medium . Tax protein was almost undetectable in JPX-9 before the induction but became detectable 6 h after the addition of CdCl2 to the culture medium , indicating that increased CXCL10 expression is independent of tax expression. Thus, our data indicated that increased expression of CXCL10 mRNA is associated with “having HTLV-1 subgroup-A or subgroup-B”, not “tax expression in infected cells” both in vitro and ex vivo .We measured the levels of CXCL10 mRNA and protein expression in HTLV-1-infected and -uninfected human T-cell lines. As shown in Fig. low Fig. b, high llow Fig. a and prolow Fig. c were obion Fig. d. ImportWe further examined whether there were any differences in the ability of subgroup-specific Tax to activate the human CXCL10 promoter . Jurkat While HTLV-1 is the causative agent of ATL and HAM/TSP, the closely related virus HTLV-2 is not clearly associated with a known clinical disease . It has To investigate whether HTLV-1 subgroups affect the binding of Tax to the endogenous CXCL10 promoter, we performed ChIP assays Fig. . ChromatIn this study, to investigate the role of HTLV-1 subgroups in viral pathogenesis, we first performed a microarray-based study to define the gene expression profiles of each subgroup-specific Tax or HBZ. Our results showed that some Tax-responsive genes identified by microarray, such as EBI3 , VCAM-1 Our microarray analysis showed that out of a total of 50,599 genes including Entrez genes and lincRNA genes screened, approximately 2619 and 8074 were differentially expressed upon Tax or HBZ induction, respectively. The list of the top 50 genes up-regulated by each subgroup of Tax  or HBZ  showed that the genes that were differentially expressed in the presence of Tax included those related to apoptosis (BCL2-related protein A1: BCL2A1), the cell cycle and DNA repair , cytokines , chemokines , and adhesion molecules (vascular cell adhesion molecule 1: VCAM1). In contrast, the genes that were differentially expressed in the presence of HBZ include those related to RNA-splicing , the Notch signaling pathway (NOTCH-regulated ankyrin repeat protein: NRARP) and chemokine (CCL21). These results suggest that Tax mainly induces expression of genes related to the activation and transformation of CD4+ T cells, whereas HBZ modulates a variety of cellular signaling pathways which are related to the immune response, differentiation, and growth of T-cells.Our microarray results also showed that various kinds of ncRNAs frequently appeared among both Tax- and HBZ-regulated genes. It is now well established that the majority of transcribed genomic sequences are associated with ncRNAs rather than protein-coding RNAs, and such ncRNAs, including regulatory miRNAs and lncRNAs that are functionally involved in a variety of host immune responses and in the pathogenesis of human diseases . It is, A previous report suggested that serum CXCL10 is significantly higher in HAM/TSP patients than in HCs, and the CSF level of CXCL10 was strongly correlated with disease severity . Thus, CIn clear contrast to HTLV-1, HTLV-2 has not been associated with ATL or other types of malignancies . As HTLVTo further investigate how the subgroup-specific Tax regulates the CXCL10 gene expression in T-cells, we performed the ChIP assay using Jurkat T-cells. The results showed that DNA fragments bound by ternary protein complexes including Tax-A are more abundant than those including Tax-B in the CXCL10 promoter, suggesting that Tax-A is more frequently recruited at this genomic region than Tax-B.In conclusion, we demonstrate that different HTLV-1 subgroups are characterized by different patterns of host gene expression. Differential expression of pathogenesis-related genes regulated directly or indirectly by subgroup-specific Tax or HBZ may be associated with the onset of HAM/TSP. To better understand the pathogenesis of HAM/TSP, further studies are needed to elucidate the role of subgroup-specific viral transcription factors and its target genes in more detail.Additional file 1: Table S1. Osame Motor Disability Score (OMDS).Additional file 2: Table S2. Primer sequences for plasmid construction.Additional file 3: Fig. S1. Subgroup-specific Tax molecules do not differ in the transcriptional activation of the reporter construct containing a luciferase gene under control of the NF-κB binding sequence of the IL-2Ra gene. The reporter construct containing a luciferase gene fused to five repeats of the NF-κB site of the IL-2Ra gene was independently transfected into Jurkat human T-cells with or without the Tax expression plasmid. Luciferase assays were performed 24 h after transfection. There was no difference between Tax-A and Tax-B with respect to transcriptional activity. Three independent experiments were performed. Data shown as mean ± SD, n  =  3.


Human osteoclast formation from mononuclear phagocyte precursors involves interactions between members of the tumor necrosis factor (TNF) ligand superfamily and their receptors. Recent evidence indicated that TNF-α-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation via a TRAF6-dependent signaling pathway; but paradoxically, it inhibits RANK ligand (RANKL)-induced osteoclast differentiation. Although a number of signaling pathways were linked to the RANK and osteoclastogenesis, it is not known how TRAIL regulates RANK signaling. In this study, we demonstrate that TRAIL regulates RANK-induced osteoclastogenesis in terms of the assembly of lipid raft-associated signaling complexes. RANKL stimulation induced recruitment of TRAF6, c-Src, and DAP-12 into lipid rafts. However, the RANKL-induced assembly of lipid raft-associated signaling complexes and TRAF6 recruitment was abolished in the presence of TRAIL. TRAIL-induced dissociation of RANKL-induced lipid raft signaling complexes was reversed by treatment with TRAIL receptor (TRAIL-R) siRNA or an anti-TRAIL-R blocking antibody, indicating that TRAIL mediates suppression of RANKL-induced lipid raft signaling via interactions with TRAIL-R. Finally, we demonstrated that TRAIL suppressed inflammation-induced bone resorption and osteoclastogenesis in a collagen-induced arthritis (CIA) rat animal model. Our results provide a novel apoptosis-independent role of TRAIL in regulating RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment. RANK provokes biochemical signaling via the recruitment of intracellular TNF receptor-associated factors (TRAFs) after ligand binding and receptor oligomerization. Accumulating evidence from various laboratories indicates that TRAFs, most importantly TRAF6, are key to understanding how RANK ligand (RANKL) links cytoplasmic signaling to the nuclear transcriptional program9. It is likely that the RANK/RANKL/osteoprotegerin (OPG) system is the central and primary regulator of bone remodeling; however, it is clear that this is not the only mechanism involved. Many of the cytokines and growth factors implicated in inflammatory processes in inflammatory diseases were also demonstrated to impact osteoclast differentiation and function either directly, by acting on cells of the osteoclast lineage, or indirectly, by acting on other cell types that modulate expressions of the key osteoclastogenic factor, RANKL, and/or its inhibitor, OPG13. In addition to RANKL, recent studies demonstrated there are several TNF family molecules which promote osteoclast differentiation, including TNF14, FasL15, decoy receptor 3 (DcR3)16, and TRAIL18, indicating that activated T cells and inflammatory responses can remodel bone homeostasis via these effector molecules. TRAIL, a member of the TNF ligand superfamily, induces apoptosis in diverse tumor cell lines19, and its expression is upregulated in activated T cells. Previous studies demonstrated that in addition to triggering apoptosis, TRAIL induces osteoclast differentiation in mononuclear phagocyte precursors18, and is also able to suppress osteoclastic differentiation induced by RANKL plus M-CSF20, suggesting that TRAIL may play a role in regulating osteoclast differentiation, and this may implicate it in osteoimmunology in immune response-associated bone absorption. However, the mechanism and signaling pathways of how TRAIL regulates RANKL-induced osteoclast differentiation are still not clear.Osteoclasts are multinucleated cells, derived from precursors of monocyte/macrophage lineages, and are specialized for bone absorption and remodeling. It is already known that normal differentiation of osteoclasts requires TNF family receptors, such as RANK22. Lipid rafts are dynamic assemblies of proteins and lipids that harbor many receptors and regulatory molecules, and so act as a platform for signal transduction. In T cell antigen receptor signaling, raft domains function as signaling platforms where selective signaling molecules are recruited24, which initiate downstream signaling cascades by phosphorylating tyrosine residues on receptor complexes. It was demonstrated that RANK-mediated signaling and osteoclast function are dependent on the expression and integrity of lipid rafts25. It is still not clear whether lipid raft-associated signaling is critical for RANK signaling, or whether TRAIL regulates RANK signaling at lipid raft-associated signaling. To understand the TRAIL-mediated regulation of RANK signal transduction osteoclastogenesis, we studied the roles of lipid raft-associated signaling in RANKL-induced osteoclast differentiation and bone resorption. We demonstrated that RANKL stimulation induced recruitment of TRAF6, c-Src, and DAP-12 into lipid rafts. However, the RANKL-induced assembly of lipid raft-associated signaling complexes was abolished in the presence of TRAIL. Our results indicated that lipid raft-associated signaling is essential for RANKL-induced osteoclast differentiation, and TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment. Our study results suggest that TRAIL modifies RANK signaling by interacting with lipid raft-associated signaling. This provides new insights into the molecular mechanism that may implicate osteoimmunology in the immune response associated with bone absorption.Rafts are specialized membrane microdomains enriched in cholesterol, glycosphingolipids, and glycosylphosphatidylinositol (GPI)-anchored proteinsTrail-r−/−) mice were obtained from Henning Walczak . All animal work was conducted according to guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care. All animal experiments were approved by the Animal Ethics Committee of the National Taiwan University Medical Center.Sprague–Dawley (SD) rats  and C57BL/6 mice  were housed under specific pathogen-free conditions and provided with standard food and water. TRAIL receptor (TRAIL-R) knockout  in an ice-cold water bath. Male SD rats were first subcutaneously immunized (day 0) at the base of the tail with 0.2 ml of this emulsion. On day 7, rats were given a booster injection of 0.2 ml of the emulsion. Clinical signs of arthritis in each hind paw were assessed under blinded conditions every day. The hind paw thickness was measured with Vernier calipers. The severity of arthritis in each hind paw was monitored and scored according to the following criteria: 0 = no evidence of erythema and swelling; 1 = erythema and mild swelling confined to the tarsals or ankle joint; 2 = erythema and mild swelling extending from the ankle to the tarsals; 3 = erythema and moderate swelling extending from the ankle to metatarsal joints; and 4 = erythema and severe swelling encompassing the ankle, foot, and digits. Each hind paw was graded and an average score of ≥ 3 was defined as severe arthritis.Bovine collagen type II (bCII) was solubilized at 2 mg/ml in 0.05 M acetic acid, and then emulsified in an equal volume of complete Freund’s adjuvant (CFA) containing 4 mg/ml of heat-killed Recombinant murine TRAIL was purchased from PeproTech  and diluted with phosphate-buffered saline (PBS) to produce a dose of 1.5 μg/kg body weight (BW)/rat. The soluble TNF-α receptor, etanercept (Enbrel), was purchased from Pfizer  and diluted with PBS to produce a dose of 6 mg/kg BW/rat. Mouse immunoglobulin G (IgG) was purchased from R&D Systems  and diluted with PBS to produce a dose of 200 ng/rat. Rats were injected subcutaneously at the hind paws with PBS, IgG, TRAIL, or etanercept starting on day 4 after the booster immunization (day 11 after the bCII immunization).5/cells and cultured for 7 days in the presence of 100 ng/ml RANKL and 50 ng/ml M-CSF, or 100 ng/ml TRAIL. The medium was replaced every three days.We used bone marrow-derived macrophages (BMMs) and the RAW 264.7 murine monocytic/macrophagic cell line as model systems of osteoclastogenesis. Both cell types differentiate into osteoclast-like cells in the presence of RANKL plus M-CSF. Bone marrow cells from C57BL/6 mice were cultured in minimum essential medium (MEM) supplemented with 50 ng/ml M-CSF, 10% FCS, 2 nM L-glutamine, and 100 U/ml penicillin/streptomycin for 3 days. Adherent cells were referred to as BMMs. For induction of osteoclast differentiation, RAW 264.7 cells, mouse macrophage cell line, and BMMs were seeded in a 96-well plate at a density of 2 × 10siRNA oligonucleotides against TRAIL messenger (m)RNA were purchased from Dharmacon . siRNA oligos were resuspended and diluted to a concentration of 80 nmol/ml. RAW 264.7 cells or BMMs from mice were transfected with 4 nmol siRNA oligo using a Bio-Rad Gene Pulser II system at 280 V and 975 μF.+ multinuclear cells (MNCs) (with more than three nuclei) were regarded as osteoclasts and counted under an inverted-phase contrast microscope. Morphological features of osteoclasts were photographed with a photomicroscope. For detecting the osteoclasts in the tissue sections, osteoclast numbers were measured by quantifying cells positively stained for TRAP . Briefly, specimens were fixed for 30 s and then stained with naphthol AS-BI phosphate and a tartrate solution for 1 h at 37 °C, followed by counterstaining with a hematoxylin solution. TRAP-positive multinuclear cells with three or more nuclei were regarded as osteoclasts and counted under an inverted-phase contrast microscope. The total number of TRAP-positive cells and the number of nuclei per TRAP-positive cells in each well were counted.Cells were fixed and stained with TRAP  for 1 h at 37 °C, followed by counterstaining with a hematoxylin solution. TRAP6) in 30 ml of hypotonic lysis buffer  by gentle pipetting, then incubation on ice for 5 min. The homogenate was centrifuged at 6000 rpm for 5 min at 4 °C. The supernatant representing the cytosolic fraction was collected and stored at –80 °C, and the pellet containing cell nuclei was lysed in 30 ml RIPA lysis buffer. The nuclear extract (supernatant) was retained after centrifugation at 13,000 × g for 5 min at 4 °C for subsequent western blotting with anti-NFATc1  and anti-histone deacetylase (HDAC) antibodies .To detect activation of NFATc1, RAW 264.7 cells were treated with the TRAIL (500 ng/ml), M-CSF (20 ng/ml), and RANKL (50 ng/ml) in the presence or absence of TRAIL-R siRNA or anti-TRAIL-R. After stimulation, cell lysates of the nuclear fraction were prepared. Nuclear and cytosolic proteins were separated upon resuspension of pelleted cells (107) were treated with 375 μl of cold lysis TNE buffer  and incubated for 30 min on ice. TNE buffer containing 0.5% Brij-58 was used for TRAF6 and c-Src isolation. Lysates were then mixed with 375 μl of 85% sucrose in TNE buffer. The solution was overlaid with 2.25 ml of 30% sucrose in TNE buffer, followed by 1.5 ml of 5% sucrose in TNE buffer. The sucrose gradients were centrifuged at 2 × 105× g in a SW55Ti rotor  overnight at 4 °C. Twelve fractions of proteins were added to sodium dodecylsulfate (SDS) sample buffer and resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting.Lipid rafts are defined by their solubility in cold, non-ionic detergent. Stimulated RAW cells . For nuclear translocation of transcription factors, stimulated cells were lysed with hypotonic buffer and fractionated into cytoplasmic or nuclear fractions, followed by immunoblotting with specific antibodies for NFATc1 and HDAC1 .5) were cultured on coverslips. Cells were cultured with M-CSF for 1 day and treated with RANKL for another 4 h. After washing, cells were incubated with PE-conjugated cholera toxin (Ctx) B subunit for 30 min at 4 °C. Cells were fixed with 4% formaldehyde for 30 min and washed with 1% bovine serum albumin (BSA)/PBS. Cells were then permeabilized with 0.5% saponin in 1% BSA dissolved in PBS for 30 min. Cells were incubated with an anti-RANK, anti-TRAF6 , anti-c-Src, or anti-DAP-12 antibody  for 30 min, followed by washing and incubation with an FITC-conjugated secondary antibody for 30 min. Confocal microscopic scanning was performed with a Zeiss LSM-510 META laser scanning confocal microscope .RAW 264.7 cells  kits according to the manufacturer’s instructions . Briefly, conjugated DPD-alkaline phosphatase was added to rat sera from each group as the antibody, and the enzyme activity was detected with a p-nitrophenyl phosphate substrate, the reaction products of which could be detected at absorbance at 405 nm. Elevated levels of serum DPD indicate increased bone resorption in individuals.p-value < 0.05 was considered statistically significant. All analyses were conducted using SPSS software, vers. 16.0 .Non-parametric Mann–Whitney U test was used for comparison of different parameters between two groups. A Trail-r−/−) mice  plus M-CSF 20 ng/ml) or TRAIL (500 ng/ml) alone could induce BMMs into osteoclasts; but paradoxically when treated with both RANKL plus M-CSF and TRAIL together, the ability to induce osteoclast differentiation by BMMs was markedly inhibited Fig. . TRAIL sice Fig. . To furtice Fig.  and S2, 0 ng/ml oice Fig.  and S4. 27. Lipid rafts are dynamic assemblies of proteins and lipids that harbor many receptors and regulatory molecules and so act as a platform for signal transduction24 in immune signaling. It was demonstrated that RANK-mediated signaling and osteoclast function are dependent on the expression and integrity of lipid rafts25  or treated with a TRAIL-R-blocking antibody (2 μg) during induction of osteoclast differentiation. As results demonstrate in Fig. To understand whether TRAIL is able to prevent RANKL-induced lipid raft assembly or dissociate the assembly after its formation, we stimulated cells with RANKL plus M-CSF for four days to establish the signaling and then treated them with TRAIL after RANKL stimulation, and studied the composition of lipid raft components after TRAIL treatment at each time point. As per results shown in Fig. To further confirm the spatial reorganization of lipid raft-associated components on the cell surface of osteoclast precursors after RANKL stimulation, co-localization of these signaling molecules with the raft marker, GM-1, was further analyzed by confocal microscopic imaging. Localization of RANK, TRAF6, c-Src, and DAP-12 in lipid rafts was analyzed in RAW246.7 cells after RANKL stimulation in the presence and absence of TRAIL. Results in Fig. We further investigated the role of the TRAIL in arthritis-induced bone erosion. Etanercept, a soluble TNF receptor used clinically for treating rheumatoid arthritis, was used as a positive control. A micro-CT analysis revealed that rats with CIA exhibited profound osteolytic lesions with a markedly decreased bone volume density in inflamed paws and femurs on day 29 post-collagen injection Fig. . SignifiTo further address the in vivo effects of TRAIL on osteoclast differentiation in inflammatory arthritis, we next investigated impacts of TRAIL on osteoclast activation during CIA. As illustrated in Fig. 29. TRAIL administration induced anti-inflammation in several autoimmune animal models32. All of those studies and our results emphasize the regulatory role of the TRAIL in modulating autoimmune immune responses, and raise potential therapeutic implications of the TRAIL in autoimmune diseases. In the present study, we demonstrated that the TRAIL effectively restored arthritis-induced joint erosion and inhibited bone resorption by suppressing osteoclastogenesis in CIA. These results indicate that the TRAIL is not only potent in inhibiting joint inflammation, but also efficacious in protecting against joint erosion and bone loss in inflammatory arthritis. It evidences a novel role for the TRAIL in regulating osteoclast activation and bone erosion in vivo. Therefore in addition to regulation of immune responses, the TRAIL also regulates osteoclast activation during immune responses, suggesting that the TRAIL may play an important role in osteoimmunology.In this study, we demonstrate that the TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment. This suggests a novel apoptosis-independent role of the TRAIL in regulating RANK signaling and suppressing osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment. Although the TRAIL induces apoptosis in many tumor cell lines, almost all primary cells are resistant to TRAIL-induced cell death, and the actual biological role of the TRAIL remains to be elucidated. In recent years, accumulating evidence has demonstrated that the TRAIL can modulate immune responses in autoimmune inflammatory diseases via an apoptosis-independent pathway18; and the TRAIL is also able to suppress osteoclastic differentiation induced by the RANKL plus M-CSF20, suggesting that the TRAIL may play a role in regulating osteoclast differentiation. In this study, we further demonstrated that RANKL stimulation induced recruitment of TRAF6, c-Src, and DAP-12 into lipid rafts. However, the RANKL-induced assembly of TRAF6, c-Src, and DAP-12 into lipid rafts was abolished in the presence of the TRAIL. The results also demonstrated that the TRAIL is able to disassemble RANKL-induced signaling complexes in lipid rafts even after they had been formed. This study indicates that the TRAIL disrupts RANKL-induced lipid raft signaling complexes either by preventing their formation or dissociating them from lipid rafts after their formation. The involvement of rafts has been implicated in many important cellular processes, which include cell surface receptor signaling, and the generation and maintenance of cellular polarity. For T cell and B lymphocyte antigen receptors, raft domains function as signaling platforms where selective signaling molecules are recruited24. Osteoclast formation from mononuclear phagocyte precursors involves interactions between TNF ligand superfamily members and their receptors4. However, the signaling pathways and whether lipid raft-associated signaling is essential for RANKL-induced osteoclast differentiation are still not clear. Previous studies demonstrated that raft expression increases during osteoclastogenesis and that TRAF6 is recruited to rafts by RANKL stimulation in osteoclasts25. All these findings demonstrate a crucial role for membrane lipid rafts in the function, and potentially differentiation, of osteoclasts. Our study is the first to demonstrate that lipid raft assembly and TRAF6 recruitment are critical for TRAIL-induced suppression of RANK signaling in osteoclast differentiation and bone resorption activity. TRAF6 is the critical RANK-associated signaling molecule regulating osteoclast differentiation and activity. TRAF6-knockout mice displayed a defect in NF-κB signaling and consequently developed osteopetrosis, indicating the essential role of TRAF6 in osteoclast function33. This study suggested that lipid raft assembly and TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and the TRAIL modifies RANK signaling by interacting with lipid raft-associated signaling.It is still not clear how the TRAIL regulates RANK signaling in osteoclast differentiation. In this study, we demonstrated that the TRAIL regulates RANK-induced osteoclastogenesis of the assembly of lipid raft-associated signaling complexes. Previous studies demonstrated that the TRAIL itself induced osteoclast differentiation via activation of a TRAF6-dependent signaling pathway29. These results suggest that the TRAIL-R could transduce signals to modulate the assembly of lipid raft-associated signaling. It provides a novel mechanism of TRAIL-R signaling in regulating osteoclast differentiation, a novel signaling pathway distinct from traditional death receptor signaling.In this study, we demonstrated that TRAIL/TRAIL-R interactions transduce signaling, a novel pathway distinct from apoptosis signaling via a caspase cascade, leading to interruption of RANKL-induced lipid raft assembly and TRAF6 recruitment. It is still not clear how the TRAIL-R transduces signals that affect lipid raft-associated signaling. Previous studies by our group demonstrated that the TRAIL directly inhibits T cell activation, and TRAIL/TRAIL-R interactions transduce signals that inhibit phosphorylation of proximal T cell receptor (TCR) signaling-associated molecules, including ZAP70, Lck, LAT, and PLCγ1, with repressed recruitment of these signaling molecules into lipid raftsFigure S1Figure S2Figure S3Figure S4Figure S5


Few major advances in fighting parasitic diseases have been made in China since the development of new methods for prevention, control, and elimination. However, the proportion of immunocompromised individuals has increased due to the growth of chronic diseases, population aging, and more frequent cases of patients with AIDS and cancer. All these problems can promote development of parasitic infections, which is commonly associated with manipulation of host signaling pathways and the innate immune system. Mitogen-activated protein kinase (MAPK) signaling pathways are evolutionarily conserved in metazoan organisms, which play critical roles in the cell cycle, gene expression, growth, differentiation, apoptosis, and parasite–host interactions. Recent discoveries of the MAPK components involved in activation, regulation, and signal transduction appeared to be promising for the diagnosis, prevention, and treatment of parasitic diseases in the future. This review summarizes the involvement and critical role of the MAPK family in parasitic disease development and maintenance in the host. Moreover, it highlights recent studies concerning the mechanisms and novel drug development for inhibition and regulation of MAPK pathways in order to prevent parasitic disease. In addition, we discuss some antigenic proteins as prospective inhibitory molecules or vaccines for the regulation and control of MAPK signaling involved in parasite physiological activity. Significant advances have been made in the control and elimination of human parasitic diseases in China since the growth of central and local government attention in developing a range of control programs, as well as improvements in hygienic conditions. Therefore, recent improvements resulted in significant decrease of clinical incidents associated with the hookworms, roundworms, and other soil-transmitted infections. However, the increased prevalence of foodborne parasitic diseases in some provinces of China remains a challenging issue. For instance, the abundance of hydatidosis is still a serious problem in Western China . FurtherMany pathogens target a variety of cell signaling pathways to inhibit or modulate the host’s immune response . BesidesToxoplasma gondii has an ability to deliver a protein, GRA24, into cells, which establishes an interaction with a host protein, p38αMAPK(MAPK14). Consequently this interaction results in activation and nuclear translocation of the host kinase, which triggers an inflammatory response ..LeishmanE. multilocularis and contributes to larval growth. Targeting the signaling pathways involved in regulating germinative cells may provide a novel therapeutic strategy against echinococcosis and other human cestodiasis (Echinococcus activity by specifically targeting the p38 MAPK family [Zhe Cheng et al.  found thtodiasis . In factK family .MAPK signaling pathway is a critical mechanism, which is involved in cell growth, proliferation, and differentiation. Numerous studies focusing on gene cloning, sequence analysis, and functional identification of molecules related to MAPK signaling pathway provided a better understanding about the mechanisms involved in the interaction between parasite and host, which cause various pathological processes. These findings provide novel avenues for development of new methods for prevention, diagnosis, treatment, and clinical application against parasitic diseases. Recently, significant progress has been made in the study of MAPK signaling in parasitic diseases. However, further studies are required for better understanding of the signal molecules and mechanism involved in it. Therefore, it has a great potential in control of parasitic diseases, clinical applications including diagnosis, prevention, and treatment.


Vascular calcification is a major health risk and is highly correlated with atherosclerosis, diabetes, and chronic kidney disease. The development of vascular calcification is an active and complex process linked with a multitude of signaling pathways, which regulate promoters and inhibitors of osteogenesis, the balance of which become deregulated in disease conditions. SIRT1, a protein deacetylase, known to be protective in inhibiting oxidative stress and inflammation within the vessel wall, has been shown as a possible key player in modulating the cell-fate determining canonical Wnt signaling pathways. Suppression of SIRT1 has been reported in patients suffering with cardiovascular pathologies, suggesting that the sustained acetylation of osteogenic factors could contribute to their activation and in turn, lead to the progression of calcification. There is clear evidence of the synergy between β-Catenin and elevated Runx2, and with Wnt signaling being β-Catenin dependent, further understanding is needed as to how these molecular pathways converge and interact, in order to provide novel insight into the mechanism by which smooth muscle cells switch to an osteogenic differentiation programme. Therefore, this review will describe the current concepts of pathological soft tissue mineralization, with a focus on the contribution of SIRT1 as a regulator of Wnt signaling and its targets, discussing SIRT1 as a potential target for manipulation and therapy. In vitro models use glucose, calcium and inorganic phosphate as inducers of calcification within vSMCs, with deposition of calcium on the extracellular matrix, and an upregulation of osteogenic markers including alkaline phosphatase (ALP), Runt-relative transcription factor (Runx2), and osteocalcin  has been identified as a highly conserved nicotinamide adenine dinucleotide-dependent deacetylase, interacting with a range of protein targets involved in Wnt signaling, glucose homeostasis, insulin regulation, and calcium signaling , making ess gene . Subsequ in 1991 . Being h in 1991 . Recent  in 1991 , 25, actThe development of smooth muscle calcification occurs in the presence of hyperphosphatemia, often coupled with hyperglycaemia, in patients with diabetes and CKD. The histone deacetylase SIRT1, known to ameliorate calcification , is showin vitro   regulateRunx2; a key osteoblastic transcription factor , is esseSclerostin, a selective inhibitor of Low-density lipoprotein Receptor-related Protein (LRPs), a member of the Frz membrane complex downstream of Wnt, is increased during CVC differentiation . IncreasRunx2 expression is controlled by Wnt signaling via the direct binding of TCF/LCF co-transcription factors. Direct β-Catenin binding to TCF/LEF at the Runx2 promotor site is enhanced by p300 acetylation , propagaActivation of Sirtuins, in particular SIRT1, has been shown to decrease Wnt signaling and reduce the risk of cardiovascular disease in both animal and human models –105, andSirtuin activators have generally been described for SIRT1, and resveratrol, a natural compound found as a constituent of grapes and red wine , is the With an increasingly aging population throughout the Western world, vascular calcification has become a major health concern, correlating with cardiovascular disease development and mortality. Although, the molecular mechanism underpinning vascular calcification remains closely linked to bone formation, the association between loss of SIRT1, activation of Wnt signaling and the upregulation of major osteogenic factors add to the growing armament of deranged osteogenic signaling pathways occurring under pathological conditions in the vessel wall. Additionally, SIRT1, a longevity factor and deacetylase, may act at an epigenetic level to control these converging pathways and cardiovascular risk factors. Furthermore, SIRT7 and SIRT1 have been shown to co-ordinately enhance Sp7/Osx activity and support orthotropic bone formation, and thus further studies could add to the understanding of the role of osteotropic signals during mineralization in skeletal and vascular environments. Indeed, it may be, that other members of the sirtuin family, may contribute to cardiovascular Wnt signaling  and eithAlthough the hyperglycaemia and hyperphosphatemia present in many CVD patients is known to suppress SIRT1 expression, there is a growing need for a comprehensive single-cell differentiation pathway for vSMC phenotypic switching, to identify the temporal activity of relevant signaling pathways, and the importance of their early inhibition and reactivation at later time-points. Only then, will we be able to extend our current knowledge of osteogenic vSMC differentiation, which potentially could have implications for future research and clinical application in this field.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.