In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo.The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-μ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4 The Human T cell leukemia virus type-I (HTLV-I) is the first human retrovirus that is etiologically associated with adult T cell leukemia (ATL) and HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) ,2,3. HTL+ cytotoxic T lymphocyte (CTL) responses and readily detectable levels of antibodies specific for HTLV-I antigens in not only ATL and HAM/TSP patients but also asymptomatic HTLV-I carriers clearly indicate that repeated production of HTLV-I must occur in vivo. In accordance, once fresh peripheral blood mononuclear cells (PBMCs) of HTLV-I infected individuals are cultured in vitro for a short time, they begin to produce HTLV-I antigens. Little is known, however, about the mechanism(s) for HTLV-I activation in vivo. So far, several lines of in vitro studies have shown that HTLV-I is activated upon host cell activation by a variety of stimuli. These include lactoferrin [HTLV-I is dormant in vivo at least in peripheral blood or lymph nodes, so that freshly isolated lymphoid cells from HTLV-I infected individuals do not express detectable levels of mRNA or proteins of HTLV-I . Howevertoferrin , prostagtoferrin , T cell toferrin ,12,13, atoferrin ,15.It is well established that heat shock proteins (HSPs) play roles not only in housekeeping functions by serving as molecular chaperones for cell survival but also in supporting gene expression of various DNA and RNA viruses . Accordi+ T cells. We report herein that HS significantly enhanced the expression of Tax followed by enhanced expression of gp46 along with Tax-inducible host proteins. These results reported herein suggest a role for environmental heat stress on HTLV-I reactivation in vivo.Using our library of anti-HTLV-I monoclonal antibodies (mAbs), and ATL patients’ and HTLV-I carriers’ blood samples, we determined the effect of HS in more detail by studying the expression of HTLV-I antigens in various HTLV-I-infected T cell lines and primary human CD42O3) and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich, Inc. . Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) .The medium used throughout the studies consisted of RPMI 1640 medium , supplemented with 10% fetal calf serum, 100 U/mL penicillin and 100 μg/mL streptomycin . The IL-2-dependent T cell lines utilized herein were maintained in vitro in RPMI medium containing 20 U/mL of recombinant human IL-2. Our mouse and rat monoclonal antibodies (mAbs) utilized in this study included mouse IgG3 anti-HTLV-I Tax (clone Lt-4) , rat IgG+ ILT-H2 and the HAM/TSP patient-derived CD8+ ILT-M1 [+ T cell lines utilized were established in our laboratory from ATL patients and included ATL-026i, ATL-056i, ATL-083i, and the cell line YT/cM1 that was established from CD4+ T cells isolated from a normal donor. The HTLV-I-unrelated T cell line that was used for the syncytium formation assay was the Jurkat cell line  [The IL-2-dependent T cell lines utilized herein included an ATL patient-derived CD4+ ILT-M1 . Other CMD, USA) .For HS treatment, cells in growth media in a volume of 1 mL in 15 mL plastic conical tubes  were heated in a water bath at various temperatures for various times as indicated in the text. After heating, these cells were dispensed into 24-well culture plates  and cultured at 37 °C for 12~72 h. The protocol for the use of human PBMCs and animals were approved by the Human Institutional Review Board and the Institutional Animal Care and Use Committee on clinical and animal research of the University of the Ryukyus, prior to initiation of the present study.The polychromatic phenotypic analysis of cells was performed as described previously ,26. BrieThe syncytium formation assay was performed as reported previously . BrieflyTax antigen concentration in whole cell lysates were determined using our in-house formulated and standardized enzyme-linked immunosorbent assay (ELISA) kit using a pair of anti-Tax mAbs that included the capture TAXY-7 mAb  and the 7 cells/mouse) from HTLV-I-negative donors were transplanted into the peritoneal cavity of NOG mice together with MMC-treated HTLV-I producing ATL-040 cell line (1 × 107 cells/mouse). Two weeks later, cells in the peritoneal lavage were collected, either untreated or heat-treated, and cultured for 1~2 days. The expression of Tax antigen in CD4+ human T cells was analyzed using flow cytometry (FCM) on day 1 and 2. In addition, whole heparinized peripheral blood of HTLV-I carriers was diluted 1:4 in RPMI medium, aliquoted, and an aliquot heat treated and another sham treated, and both cultured. The cells in the cultures were stained with anti-CD4, anti-Tax and anti-gp46 mAbs, and analyzed using FCM after hypotonic lysis of the red blood cells.The protocol for infection of humanized mice with HTLV-I has been described previously . Brieflyt test using Prism software . Data from more than three-armed experiments were analyzed by one-way analysis of variance (ANOVA) with post hoc Holm test and Tukey test.Data were tested for statistical significance by either paired or unpaired Student’s + T cell lines generated from acute ATL patients, ATL-026i and ATL-056i. Aliquots of these cell lines were heated at various temperatures, 37, 39, 41, 43 and 45 °C for 30 min and cultured for 24 h. The intra-cellular expression of Tax and HSP70 antigens was analyzed by FCM. At first, in order to determine whether HS affects the expression of Tax antigen in HTLV-I-infected T cells, we examined two IL-2-dependent CD4+ cells also slightly increased under HS at both bulk and single cell levels as shown in Next, we determined the optimum exposure time for enhanced Tax expression. As shown in + T cell lines and an in vitro- HTLV-I-immortalized CD4+ T cell line prepared from a normal donor (YT/cM1). These data thus confirm the fact that exposure to HS treatment increases the total amount of Tax antigen per culture during the 24 h culture period.The intra-cellular localization of Tax has been shown to be altered in response to various forms of cellular stress, such as HS and ultra violet (UV) light, resulting in an increase in cytoplasmic Tax about 1~2 h after treatment and a decrease in Tax speckled structures , which m+ and CD8+ T cell line (ILT-M1). In + Tax− cells also increased after HS. We speculate that the gp46 on Tax-negative cells may represent biofilm of HTLV-I particles produced from Tax-positive cells, or alternatively, gp46+ Tax− cells may be at a resting phase after a productive infection phase. It is noteworthy that syncytium-forming capacity which is an indicator of HTLV-I infectivity, was enhanced significantly by the exposure of YT/cM1 cells to HS . As shown in We examined the kinetics by which exposure to HS leads to optimal expression of HSP70, Tax and gp46 using two HTLV-I+ and HSP70+ cells in the absence of HS. On the other hand, PFT treatment inhibited the increase in the frequencies of Tax+ cells by HS , that returned to control levels by 48 h. + T cells from three HTLV-I carriers (p < 0.05). Representative dot blots are shown in Finally, we examined the effect of HS exposure on HTLV-I-infected primary human CD4+ T cells. We conclude from these observations that HS is one of the environmental factors that can potentially activate integrated HTLV-I provirus in vitro in latently infected T cells, although in vivo studies are yet to be performed.In the present study, we demonstrated that exposure to HS up-regulates the expression of HTLV-I Tax antigen during in vitro culture for 12~48 h along with induction of functional HTLV-I gp46 and the Tax-inducible host antigens CD83 and OX40 in not only human HTLV-I-infected T cell lines but also in primary human CD4In the literature, Andrew et al. reported that heat stress enhances the expression of HTLV-I envelope and gag antigens in long-term cultured HTLV-I-transformed T cells lines, such as HUT-102 and MT-2 cells ,32,33, aThe precise mechanism by which Tax antigen synthesis is up-regulated by exposure to HS is yet to be defined. On the basis of our preliminary data (data not shown) indicating that HS up-regulated total levels of Tax mRNA, it is possible that HS directly controls transcription. Thus, further studies are in progress to determine this possibility by measuring nuclear RNA levels for Tax RNA and genomic RNA in the cytoplasm. Since Tax operates by the activation of transcriptional factors such as the cAMP-response element-binding protein (CREB) for activation of not only viral but also host cellular genes, it can be speculated that CREB is also activated by exposure to HS in HTLV-I-infected cells. The finding that ER stress is induced by exposure to HS  and in tAs it is the case that the cellular onco-protein c-Myc protein is up-regulated following HS , rapid i2O3. It has been utilized for ATL therapy as it induces the apoptosis of HTLV-I-infected cells [2O3 on HTLV-I synthesis has been a subject of controversy. Thus, while Andrew et al. reported an enhancing effect [2O3 mediated enhancement of Tax and gp46 expression in various conditions and various types of cell lines (2O3-mediated oxidative stress may utilize distinct pathways on Tax expression by HTLV-I infected cells.Another representative cellular stress that is critical for Tax expression is oxidative stress, such as Ased cells . The speg effect ,32,33, Ng effect . These dll lines . Thus, iThe involvement of HSPs in viral infection has been reported for several viruses ,45,46 in2 within 15 min [+ T cell lines via CD3 molecule further enhanced the effect of HS on Tax expression (Infrared sunlight is known to generate heat and for their ability to increase skin temperature depending on exposure dose and time. It has been reported that intra-dermal-temperature reaches up to 44 °C when irradiated with IR at 970 nm at 80 mW/cmn 15 min . Thus, in 15 min . Althougpression , it is aIn this way, it is likely that, in subtropical and tropical areas, repeated skin exposure to strong sunlight for a prolonged time can cause HTLV-I reactivation in vivo.In conclusion, results of the present study indicate that a mild exposure to heat shock may be one of the natural environmental factors that stimulate Tax antigen expression leading to not only reactivation of HTLV-I in latently infected T cells but also induction of expression of Tax-inducible host antigens, which may favor expansion of HTLV-I infection in vivo. It can be speculated that exposure to strong sunlight in subtropical and tropical areas is involved in the geographic bias of increased HTLV-I prevalence within these regions, and that, for HTLV-I carriers, precaution against exposure to strong sunlight for prolonged periods of time might be beneficial to prevent reactivation of HTLV-I.


Adult T cell leukemia and lymphoma (ATL) is a highly aggressive form of hematological malignancies, which is caused by chronic infection of human T cell leukemia virus type 1 (HTLV-1). The viral genome encodes an oncogenic protein Tax that plays a key role in trans activating viral gene transcription and in deregulating cellular oncogenic signaling to promote survival and proliferation of virally infected T cells. Hence, Tax is a desirable therapeutic target, particularly at early stage of HTLV-1-mediated oncogenesis. We here show that niclosamide, an anti-helminthic molecule, induced apoptosis of HTLV-1-transformed T cells. Niclosamide promoted formation of polyubiquitinated Tax protein aggregate, facilitating its subsequent degradation in proteasome. Consistent with niclosamide-mediated Tax degradation, the transcription of HTLV-1 viral genes is suppressed. Furthermore, niclosamide inhibited the activities of MAPK/ERK and Stat3, and down regulated pro-survival Bcl-2 family members such as Mcl-1. Since Tax, Stat3 and Mcl-1 are growth-promoting molecules in HTLV-1-transformed T cells, our data demonstrated a novel mechanism of niclosamide in inducing polyubiquitination-dependent degradation of Tax and certain cellular pro-survival molecules, thereby promoting apoptosis of HTLV-1-associated leukemia.


HTLV-1 infected T cells and blood dentritic cells (DCs) can be found in HTLV-1 carriers. It is currently believed that T cell infection requires cell-cell contact, while DCs could also be infected with cell-free virus. However, a comparative study using different modes of viral preparation for infecting human primary autologous T and DC cells has not been performed. Supernatant from HTLV-1 chronically infected T cells, cell conjugates between HTLV-1 infected cells and target cells, or purified viral biofilm were used side-by-side to infect autologous human primary lymphocytes and monocytes-derived dendritic cells. To monitor the early steps of viral infection, we also used pseudotyped viruses carrying either the HTLV-1 or the VSV envelopes. Infection was then followed by flow cytometry, ELISA, immunofluorescence, real-time PCR and alu-PCR. Our first data show that, depending on the source of viruses, lymphocytes have distinct susceptibility to HTLV-1 infection. The impact of the viral source in the spread of HTLV-1 infection will be discussed.


Elevated uric acid (UA) is associated with the presence of the metabolic syndrome (MetS). In a prospective cohort study, we assessed whether baseline and longitudinal change in UA were risk factors for development of MetS and its individual components.We included 3087 women and 2996 men who had UA measured in the population based Tromsø Study 1994–95. The participants were stratified according to body mass index (BMI). Endpoints were MetS and each component of the syndrome after 7 years, according to the revised National Cholesterol Education Program’s Adult Treatment Panel III (NCEP-ATP III) definition.2, odds ratio [OR] per 59 μmol/L UA increase 1.44, 95 % confidence interval [CI] = 1.17–1.77, P = 0.001), but not in normal-weight subjects . Overweight also modified the association between baseline UA and the development of elevated fasting glucose (P for interaction = 0.01). UA was a predictor of MetS in all subjects . Furthermore, longitudinal UA change was independently associated with the development of MetS in all subjects .Multiple logistic regression analyses showed that higher baseline UA was associated with higher odds of developing elevated blood pressure in overweight subjects (BMI ≥ 25 kg/mIncreased levels of baseline UA independently predicted development of elevated blood pressure and higher fasting glycemia in the overweight, but not the normal-weight subjects. Baseline UA and longitudinal increase in UA over 7 years was associated with the development of MetS in all subjects. Whether increased UA should be treated differently in normal-weight and overweight persons needs further study. High levels of serum uric acid (UA) are prevalent in the general population. In the National Health and Nutrition Examination Survey (NHANES) 2007–2008 UA levels higher than 339 μmol/L were found in 21.6 % of the women, and among men 21.2 % had UA levels higher than 416 μmol/L [The metabolic syndrome (MetS) is a constellation of interrelated risk factors that increases the risk of cardiovascular disease and type 2 diabetes . MetS isUA has been reported to be a risk factor for cardiovascular disease or cardiovascular death in many studies –14, but n = 405), prevalent diabetes at baseline , and the under-weight subjects . Thus, the final study cohort consisted of 6083 subjects at baseline. The University of Tromsø conducted The Tromsø Study in cooperation with The National Health Screening Service. The Regional Committee for Medical Research Ethics approved the study, and all participants gave their written consent.The Tromsø Study is a series of population-based, prospective surveys of inhabitants of the municipality of Tromsø, Norway . In 19942. BMI was dichotomized into normal-weight (BMI < 25 kg/m2) and overweight (BMI ≥ 25 kg/m2). Blood pressure was recorded in triplet after 5-min seating; the mean of the second and third measurement was used. Physical activity was classified as active  or inactive . Smoking habits were classified as non-smokers or current smokers. Alcohol intake was classified as teetotalers, 1–7 units/week and >7 units a week. Non-fasting blood samples were drawn and time since last meal was recorded. Serum UA was measured by photometry with COBAS® instruments  using an enzymatic colorimetric test, the uricase/PAP method. Change in UA (ΔUA) was calculated as serum UA in 2001–2002 minus serum UA in 1994–1995. Creatinine was originally analyzed by a modified Jaffe reaction, but because of a possible drift in the results between baseline and follow-up, 111 plasma samples from the 1994–1995 survey and 142 samples from the 2001–2002 survey were thawed and reanalyzed with an enzymatic method (Modular P/Roche Diagnostics) in 2006, as previously described [All participants provided information on diabetes, alcohol and smoking habits, current use of medication and physical activity through a self-administered questionnaire. Experienced nurses made anthropometric measurements. We calculated BMI as weight (kg)/height (m)escribed . Values escribed . Detaileescribed .Our definition of MetS was based on the revised National Cholesterol Education Program’s Adult Treatment Panel III (NCEP-ATP III) criteria as published by the American Heart Association . Because2 and BMI ≥ 25 kg/m2) at baseline, and interaction between UA and the BMI group was tested for. We also checked for interactions between UA and gender and UA and a BMI-cutoff of obesity (BMI < 30 kg/m2 and BMI ≥ 30 kg/m2) for MetS and each of the MetS criteria. The logistic regression analyses were repeated for the group of subjects who did not have MetS at baseline. Finally, we assessed whether ΔUA was associated with MetS and its components by adding ΔUA as an independent variable to each model. These analyses were also run in the group without MetS at baseline. Two-sided P values < 0.05 were considered statistically significant. We did all the analyses using SPSS software version 22.0 .Data are given as mean ± standard deviation (SD). Independent sample t-tests and chi square tests were applied to compare baseline variables between participants with normal-weight and overweight. In each of these groups, we assessed Pearson’s correlation coefficient between UA and the baseline variables. We conducted multiple binary logistic regression analyses with each single criterion of MetS and MetS (any three criteria or more) in 2001–2002 as dependent variables and uric acid as an independent variable in separate models. Covariates were sex, age, systolic blood pressure, total cholesterol, current smoking, physical activity, Hba1c, eGFR, alcohol consumption, use of diuretics and waist circumference at baseline. In each of these analyses, we only included the subjects who did not fulfill the MetS criterion of interest at baseline. We ran the analyses both with the entire cohort and stratified into normal-weight and overweight (BMI < 25 kg/m2) and overweight (BMI ≥ 25 kg/m2) are shown in Table 2) and 13.5 % were obese (BMI ≥ 30 kg/m2).Baseline characteristics of the cohort divided into normal-weight (BMI < 25 kg/mP = 0.001). The interaction between the BMI-cutoff and UA for the prediction of new cases of elevated blood pressure was significant (P = 0.04). There were no statistically significant interactions between neither UA and gender nor UA and a BMI-cutoff of obesity (BMI < 30 kg/m2 and BMI ≥ 30 kg/m2) for any of the outcomes.Figure P < 0.001), whereas baseline UA was not significantly associated with new onset fasting glucose elevation for subjects with normal-weight (P for interaction = 0.01). There was no interaction between UA and the BMI cut-off for elevated triglycerides and low HDL cholesterol according to MetS criteria (P for interaction = 0.39 for both), and UA did not significantly predict the development of central obesity according to the MetS criteria in either group.BMI also modified the association between UA at baseline and new cases of elevated fasting glucose after 7 years Fig. . In the P < 0.001).Results of unstratified multivariable logistic regression analyses of the subjects without MetS at baseline are displayed in Table Change in UA over 7 years as a predictor of MetS and its single components in 2001–2002 was assessed in multiple logistic regression models. The results are shown in Table In this large prospective study of subjects without diabetes from the general population, elevated UA at baseline was independently associated with increased risk of elevated blood pressure in the overweight individuals 7 years later. We found no association between UA and future elevated blood pressure in the normal-weight subjects. Moreover, UA at baseline predicted new-onset impaired fasting glucose in the overweight persons, but not in the normal-weight group. Baseline UA was a predictor of MetS in all subjects. Finally, a longitudinal increase in UA of 59 μmol/L over 7 years raised the odds of developing MetS by 28 %.The association between UA and MetS is in accordance with previous prospective studies , 30, 31.n = 69) from the United Arab Emirates examined the univariable relationship between a set of biomarkers, among them UA, and components of MetS in healthy, young females, stratified into normal-weight (BMI ≤ 25 kg/m2), overweight , and obese (BMI ≥ 30 kg/m2) [2 and BMI ≥ 30 kg/m2) for neither MetS nor any of its components. This may be due to a small group of obese in our cohort.To the best of our knowledge, there are no other studies of this scale where the population is stratified into normal-weight/overweight before examining the association between UA and MetS and its components. A small study (0 kg/m2) . This st2), compared to the hypertensive normal-weight (BMI < 25 kg/m2), in the presence of hyperuricemia [The association between hypertension and UA was first noted in the 1870s and has been demonstrated in numerous publications. In a recent meta-analysis, UA increase was reported to be associated with a statistically significant elevation in incident hypertension . It has uricemia , and a ruricemia . UA mighuricemia . These cEpidemiologically, UA is associated with insulin resistance , and theThe present study has important strengths: the large size, solid attendance rate, long follow-up time, use of UA as a continuous variable, and the ability to correct for confounders such as eGFR, use of diuretics and all the traditional cardiovascular risk factors. However, a major shortcoming of our study is the lack of fasting blood samples. In particular, glucose and triglycerides, and thereby the definition of MetS, are affected by this. The incorporation of time since last meal and adjustment of the cut-offs in the definition of elevated fasting glucose and elevated triglycerides compensated in part, but not fully, for this limitation. In addition, only a single measurement of serum UA was done in each survey. Another shortcoming of this study may be the fact that our baseline data were collected 21–22 years ago, and 14–15 years have passed since follow-up. Both lifestyles and pharmacological treatment have changed in that time. However, if the effects of overweight on UA’s association with MetS can be reproduced in studies on newer data, our findings may be even more relevant as overweight and obesity is an even greater challenge in the world of today. That our study population comprised largely of healthy, middle-aged to elderly Caucasians can be viewed as both a weakness and a strength; the results may not be generalizable to dissimilar populations, but the homogeneity of our cohort may have prevented dilution of our findings due to important diversities in baseline properties.In a large cohort from the general population, baseline UA was independently associated with future cases of elevated blood pressure and elevated fasting glucose in overweight subjects, but not normal-weight individuals. Both elevated baseline UA and longitudinal increase in UA over 7 years from baseline was associated with the development of MetS in all subjects. These findings warrant further studies to examine the exact causal relationship between UA and MetS, in overweight as well as in normal-weight individuals, and to assess whether treatment strategies need to be targeted differentially according to BMI.The Regional Committee for Medical Research Ethics approved the study (committee’s reference number 2009/2536-3), and all participants gave their written consent to participate. The Tromsø Study complies with the Declaration of Helsinki.Not applicable.http://tromsoundersokelsen.uit.no/tromso.The dataset supporting the conclusions of this article is legally restricted. It will therefore not be shared publicly. However, meta-data as well as general information about the study is available at


In this review, we consider data from recent in vivo studies analyzing the role of TCR signal strength in controlling the outcome of CD4+ T cell differentiation and discuss the role of TCR in controlling the critical nature of CD4+ T cell interactions with dendritic cells during activation. We further propose a model whereby TCR signal strength controls the temporal aspects of T–DC interactions and the implications for this in mediating the downstream signaling events, which influence the transcriptional and epigenetic regulation of effector differentiation.The ability of CD4+ T cells to differentiate into effector subsets underpins their ability to shape the immune response and mediate host protection. During T cell receptor-induced activation of CD4+ T cells, both the quality and quantity of specific activatory peptide/MHC ligands have been shown to control the polarization of naive CD4+ T cells in addition to co-stimulatory and cytokine-based signals. Recently, advances in two-photon microscopy and tetramer-based cell tracking methods have allowed investigators to greatly extend the study of the role of TCR signaling in effector differentiation under  CD4+ T cells constitute a key population in the adaptive arm of the immune response, due to their ability to differentiate into various distinct effector populations such as Th1, Th2, Th17, T follicular helper (Tfh) cells, and induced regulatory (iTreg) T cells, which help to orchestrate host defense against various classes of pathogens , 4. HoweThis review aims to discuss the evidence that supports the strength of signal-based mechanisms for inducing differentiation in parallel with qualitative mechanisms and argues that TCR signaling dynamics include a temporal component that regulates the interactions between APC and CD4+ T cells, which explains much of the observed qualitative and quantitative phenomena previously described.in vivo conditions  induce a specific pathway of differentiation following antigen-dependent activation of CD4+ T cells. Following the activation of APC by pathogen-associated molecular patterns (PAMPs), APCs begin to upregulate the levels of both MHCII and co-activation molecules allowing for the activation of CD4+ T cells in an antigen-specific manner. PAMPs additionally induce the production of specific cytokines by activated APCs, which have been shown to induce the differentiation of specialized T-helper populations . Cytokinnditions , 14. Addnditions . Thus, i pathway . Th17 cenditions .in vitro or in vivo, the quantitative/TCR signal strength-based component is generally not considered giving the appearance that cytokines dominate. However, in practice, this conclusion requires a more explicit analysis to assess the relationship and possible hierarchical and temporal dominance of qualitative vs. quantitative aspects of control.These studies form a large body of compelling evidence indicating that the cytokine milieu represents a highly deterministic cellular cue during the polarization process. However, it should be noted that cell fate decisions occur due to a number of factors, and as the majority of qualitative/cytokine-based studies have been conducted at single antigen concentrations in vitro conditions, a strong TCR signal leads to a predominance of Th1 differentiation, whereas weak TCR signaling leads to Th2 differentiation  peptide   and the However, responsiveness to IL-2 signaling has been shown to be regulated by TCR signal strength; activation by both weak and strong stimuli leads to an increased expression of IL-2R, but strong stimuli also cause an attenuated ability to signal via IL-2R through inhibition of phosphorylation of the transcription factor STAT5 , inducedin vitro and in vivo conditions, TCR signal strength controlled downstream IL-12 receptor expression, with IL-12Rβ2 directly correlating with signal strength. Linking TCR signaling to the ability of CD4+ T cells to respond to cytokine signaling and establishing TCR-induced signaling play a dominant role in the signaling hierarchy pMHC:TCR interactions, which would be agonistic under steady-state conditions. It has been proposed that once activated ERK , 80 and/2 +  of activation where APC encounters consist of brief serial contacts, before transitioning to phase II where long-lasting contacts with APCs are maintained (in vitro activation T cells may proceed directly to phase II with the formation of an IS. It is possible that in vivo, the priming phase is used as a mechanism to calculate the level of pMHC being presented, leading to a decision whether to form an IS, while under in vitro conditions, the pMHC concentrations used to study IS formation are sufficient to bypass phase I. Alternatively, as the physiological signals that normally drive naive T cell migration are not present in vitro, this may lead to a reduction in cellular momentum and thus a decreased requirement for a brake-inducing signal. To account for the observed T cell behavior, such a stopping mechanism must be both quantitative for pMHC encountered and iterative with each APC interaction. One possibility for such an integrative function is the IP3-mediated release of intracellular Ca2+, whereby receptor signal spreading leads to an exponential increase in the quantity of TCR signaling complexes available to recruit and activate PLCγ causing a concomitant increase in IP3 production and signal strength-dependent release of Ca2+ from the ER. Huse et al. (2+ release, which suggests that a threshold level of IP3 is required for Ca2+ flux. Additionally, it has been demonstrated that in vivo activation of naive T cells with a low potency APL can induce upregulation of CD69 expression without inducing a sustained Ca2+ release, indicating the ability of T cells to be activated in the absence of sufficient TCR signaling to trigger Ca2+ release (While T cells are able to calculate the level of signal imparted to them by a specific APC, a mechanism for this has not yet been fully ascertained. However, it is known that naive T cells are able to form a stable long-lasting IS within minutes when forced to interact with lipid bilayers or APCs in culture, indicating that ormation . In vivointained , 93. Howe et al.  observed release .2+ are reached, store-operated calcium entry is then triggered via activation of STIM1 and STIM2, which sense Ca2+ release via low-affinity Ca2+-binding EF hand domains located in the ER lumen. Translocation of STIM molecules to the plasma membrane causes opening of CRAC channels, a sustained increase in intracellular Ca2+ levels, and initiation of a cellular brake response allowing for IS formation (2+ levels must reach the threshold for CRAC channel activation within a predetermined period of time in order to induce a T cell braking response; otherwise, migration will continue. As in the absence of CRAC opening, the T cell fails to arrest, and the kinaptic interaction will be broken, with ER Ca2+ slowly returning to baseline, such that, ER Ca2+ levels may form an intracellular clock based on Ca2+ reuptake vs. [pMHC] stimulation.Once threshold levels of intra-ER Caormation , as oppo2+ signaling following encounter with APC, although a mechanism for this decreased motility has not been completely described (2+ levels following TCR-induced CRAC opening marks the transition from a migratory phase to a primary signaling phase, which may better account for the T cell ability to quantitate [pMHC]. It has been reported (2+] are associated with a decrease in T cell motility, with Ca2+ flux signaling initiating the reorganization of f-actin function, from providing the mechanical force necessary for migration (2+ level followed by a period of heightened intracellular [Ca2+] as Ca2+ ions are removed by specific pumps. At this time, the IP3R is unresponsive to further signaling as bound Ca2+ on the cytosolic side of the ER inhibits IP3 binding (2+ to sufficient levels, PKCθ activity induces a redirection of f-actin polarization away from the T cell–APC interface, destabilizing the connection and allowing the resumption of migration.Several studies , 90, 95 escribed , 96, 97.reported  that folreported  suggest igration  to one wigration . F-actin binding . During in vivo priming (2+-associated gene targets is more efficiently activated by oscillating calcium levels rather than a sustained increase in Ca2+ levels (Studies have shown that LFA-1 is required for optimal  priming  and that priming . Thus, a+ levels , 104.It is now well established that the stimulation of naive CD4+ T cells with a strong agonist or high dose of peptide leads to the induction of Th1 differentiation, while low dose or weak APL stimulation leads to the induction of Th2 differentiation , 24, 105In addition to low-dose/affinity peptide stimulation inducing Th2 differentiation, alterations to the TCR-associated signaling machinery can lead to the induction of Th2 differentiation. Inhibition of ZAP70 , a proxiTh2-associated signaling has also been observed to be more dependent on activation of co-stimulatory pathways with defects in CD4 , CD28 1, Lck 2121, and POther transcription factors known to affect differentiation include the AP1 binding elements fos:jun and jun:jun, which induce Th1 and Th2 differentiation, respectively . While jHere, a temporally based mechanism of differentiation is suggested that accounts for many of the observed aspects of strength of signal-induced differentiation and the downstream signaling pathways associated with Th1 and Th2 differentiation. In this model, long-term interactions induce a program of Th1 differentiation and short-term interactions induce a program of Th2 differentiation. Integrating TCR signal strength through downstream signaling machinery such as calcium release allows for the influence of additional signaling systems such as co-stimulatory molecule activation and potentially cytokine signals to be combined to dictate the length of interaction between DC and CD4+ T cells and thus contributes to downstream effector differentiation . By varyFrom a biological point of view, this temporal model helps explain several phenomena. As interaction time is relative to total signal strength, a model where marginal signaling such as engagement of only a few pMHC or use of very weak APL or activation in the absence of co-stimulation leads to the induction of an anergic program, and weak signaling induces Th2 differentiation and strong signaling causes Th1 induction. This provides a potential explanation for how tolerance to environmental antigens is induced, how the immune system detects a parasitic infection, and how environmental allergens induce Th2-associated allergic disease, whereas self-antigen recognition leads to Th1-associated autoimmune disease. Tolerance to common environmental antigens is generally induced as a normal response to the presentation of low levels of antigen in the absence of co-stimulation and thus induces a weak TCR response and an anergic program due to abrogated signaling and interactions . NematodIncorporating a temporal component into our models of how CD4+ T cells interact with DC during Treg, Th17, and Tfh cell differentiation also allows for an explanation of some of the additional phenomena that have been reported to occur during differentiation, which are not fully explained by a purely qualitative model of cellular decision-making. A distinct parallel can be drawn between the differentiation of Th1–Th2 cells and Th17–Treg cells, with both Th1 and Th17 cells requiring a strong TCR signal and have been observed to interact for long periods of time with DC, whereas both Th2 and Treg cells require weak TCR signals and only interact for brief periods of time. Further, Th2 and Treg cells are able to differentiate either via endogenous signals or from cytokines that are secreted in a diffuse fashion, whereas Th1 and Th17 cells require additional cues that are delivered at the cellular synapse and require a strong and/or ongoing signal in order to regulate their ability to respond to the cytokines that direct their differentiation. Tfh cell differentiation has also been observed to have a temporal component with ongoing interactions being required for differentiation, and as such, it is appealing to hypothesize that their expression of Bcl-6 may allow for this ongoing interaction in a non-deleterious fashion, while they acquire additional interaction-based signals from DC in the form of factors such as ICOSL  and CD40In summary, a temporal signaling model would explain how many of the observed alterations in TCR signaling modulate differentiation by linking TCR signal strength, co-stimulation, and cytokine inputs through a mechanism that allows for the integration of multiple signaling inputs into an overall measure of signal strength and outputs these as divergent programs of behavior during priming, which ultimately leads to alterations in cell fate and effector function.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tolerance of plants to abiotic stressors such as drought and salinity is triggered by complex multicomponent signaling pathways to restore cellular homeostasis and promote survival. Major plant transcription factor families such as bZIP, NAC, AP2/ERF, and MYB orchestrate regulatory networks underlying abiotic stress tolerance. Sucrose non-fermenting 1-related protein kinase 2 and mitogen-activated protein kinase pathways contribute to initiation of stress adaptive downstream responses and promote plant growth and development. As a convergent point of multiple abiotic cues, cellular effects of environmental stresses are not only imbalances of ionic and osmotic homeostasis but also impaired photosynthesis, cellular energy depletion, and redox imbalances. Recent evidence of regulatory systems that link sensing and signaling of environmental conditions and the intracellular redox status have shed light on interfaces of stress and energy signaling. ROS (reactive oxygen species) cause severe cellular damage by peroxidation and de-esterification of membrane-lipids, however, current models also define a pivotal signaling function of ROS in triggering tolerance against stress. Recent research advances suggest and support a regulatory role of ROS in the cross talks of stress triggered hormonal signaling such as the abscisic acid pathway and endogenously induced redox and metabolite signals. Here, we discuss and review the versatile molecular convergence in the abiotic stress responsive signaling networks in the context of ROS and lipid-derived signals and the specific role of stomatal signaling. Survival of plants under adverse environmental conditions relies on integration of stress adaptive metabolic and structural changes into endogenous developmental programs. Abiotic environmental factors such as drought and salinity are significant plant stressors with major impact on plant development and productivity thus causing serious agricultural yield losses . The comPlant hormones function as central integrators that link and re-program the complex developmental and stress adaptive signaling cascades. The phytohormone abscisic acid (ABA) functions as a key regulator in the activation of plant cellular adaptation to drought and salinity and has a pivotal function as a growth inhibitor . AdditioArabidopsis has been shown  bind ABA and inhibit type 2C protein phosphatases (PP2Cs) such as ABI1 and ABI2 . Inactiven shown . Bindingeceptors . Extendireported . Thus, lreported . These freported .Arabidopsis GA signaling is mediated by binding of GA to GID1a/b/c that are GA receptor orthologs of the rice GA receptor gene OsGID1 , REPRESSOR of ga1-3 (RGA), SCARECROW (SCR)] transcription factors function as major regulators in plant GA-controlled development. Cellular accumulation of the GRAS protein subgroup of DELLA proteins  represses GA signaling and restrains growth and development  has a key regulatory function in the plant cellular network of stress and developmental signaling . Accordielopment . Interacelopment . GA signbidopsis . Subsequbidopsis .rga, gai, rgl1, and rgl2  and of key regulators of JA signaling OsJAZ (jasmonic acid ZIM-domain proteins) indicate significant integration of JA metabolism and signaling in plant abiotic stress responses .Recently, interesting evidence has been also provided for a convergence and crosstalk of GA and ABA signaling with the developmental regulator jasmonate in plant responses to drought. Jasmonates are membrane-lipid derived metabolites that originate from linolenic acid and have signaling functions in plant growth and biotic stress responses e.g., . Droughtesponses . Importaesponses . These rcis element  transcription factor SlNAC1 was responsive to multiple abiotic and biotic stresses (VNI2 (VND-INTERACTING1) is a repressor of xylem vessel formation and has additional functions in leaf aging thus integrating plant senescence to ABA signaling  links leaf senescence to hyperosmotic salinity response and is involved in H2O2 signaling  such as glutathione S-transferase GSTU11, peroxidases, and lipoxygenase LOX1  targets genes with function in biosynthesis of phytohormones and of the cell-wall . Interestingly, vice versa ABA transcriptionally downstream targets lipid metabolism and lipid transfer proteins suggesting tight interaction of ABA-dependent signaling and lipid metabolic pathways to maintain structure and function of cellular membranes  suggests lipid-based adaptive changes of membranes and the plant cuticle to regulate water loss and transpiration under drought  have been suggested to stabilize and maintain lamellar bilayer structure and thus the function of chloroplasts under drought stress (Craterostigma plantagineum during desiccation are likely to contribute to membrane stabilization and to the maintenance of photosynthetic energy supply (Arabidopsis cold-responsive SFR2 (SENSITIVE TO FREEZING 2) mediates removal of monogalactolipids from the chloroplast envelope membrane and stabilizes membranes during freezing indicating that structural re-shaping of chloroplast membranes is an essential and general mechanism of plant cellular dehydration responses  and digalactosyldiacylglycerol (DGDG) contents in the chloroplast envelope and in thylakoid membranes in cowpea t stress . In suppy supply . The Araesponses .Arabidopsis has provided considerable insight in the drought stress related lipid signaling by identifying links of phosphoinositides to the DREB2 pathway , PA (phosphatidic acid), and DGPP (diacylglycerolpyrophosphate) increased upon salt stress . Based o pathway .A role of lipid-derived messengers in ABA signaling was also evident by ACBP1 (acyl-CoA-binding protein 1) regulated expression of PHOSPHOLIPASE Dα1 PLDα1; . PHOSPHOFigure 1). Accepted models emphasize hub functions of yeast sucrose non-fermenting 1 (SNF1) serine-threonine protein kinase, homologous mammalian AMP-activated protein kinase (AMPK) and plant SnRKs [Snf (sucrose non-fermenting)-1-related protein kinases] in the cellular carbon and energy metabolism  regulates stomatal closure by modulating the cellular production of H2O2 via NADPH oxidases  mediated signal transduction cascades have provided another pivotal understanding of the integration of physiological and cellular responses to environmental adversity. MAPK cascades functionally link MAP3Ks (MAP2K kinase) serine/threonine kinases, MAP2K (MAPK kinase) dual-specificity kinases and MAPK serine/threonine kinases . As an and SIMKK . Recent +/H+ antiporter SOS1 by NaCl and by PA supporting relationships of lipids to MAPK signaling in plant salt stress responses . Significantly, environmental adversity such as drought and salinity impairs cellular ionic and osmotic homeostasis but additionally compromises photosynthesis, cellular energy depletion, and redox imbalances  linked ABA and H2O2 signaling in stomatal closure  is a central regulator of stomatal aperture and links guard cell movement to the ABA signaling network  mediates potassium fluxes and has a dual function in regulating both cellular auxin transport and stomatal closure (Recent research uncovered co-regulation of ABA-induced stomatal closure, guard cell Hit CHLH; . CHLH/ABit CHLH; . In Arabgulation . These folerance . Interesubunit A . Again, ubunit A . Intrigu closure .In conclusion, recent research advances have elucidated a molecular cellular signaling network for the understanding how plants control and regulate adaptation to the abiotic stresses drought and salinity. Essentially, molecular signaling components in plant adaptation to environmental adversity have been connected to hub transcription factors, MAPK pathways, ROS and lipid-derived pathways. Importantly, it is expected that further and perspective advances in the network modeling of cellular abiotic stress signaling will provide new and efficient strategies for improving environmental tolerance in crops.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 Wnt signaling pathway plays a key role in many biological aspects, such as cellular proliferation, tissue regeneration, embryonic development, and other systemic effects. Under a physiological condition, it is tightly controlled at different layers and arrays, and a dysregulated activation of this signaling has been implicated into the pathogenesis of various human disorders, including autoimmune diseases. Despite the fact that therapeutic interventions are available for ameliorating disease manifestations, there is no curative therapy currently available for autoimmune disorders. Increasing lines of evidence have suggested a crucial role of Wnt signaling during the pathogenesis of many autoimmune diseases; in addition, some of microRNAs (miRNAs), a class of small, noncoding RNA molecules capable of transcriptionally regulating gene expression, have also recently been demonstrated to possess both physiological and pathological roles in autoimmune diseases by regulating the Wnt signaling pathway. This review summarizes currently our understanding of the pathogenic roles of Wnt signaling in several major autoimmune disorders and miRNAs, those targeting Wnt signaling in autoimmune diseases, with a focus on the implication of the Wnt signaling as potential biomarkers and therapeutic targets in immune diseases, as well as miRNA-mediated regulation of Wnt signaling activation in the development of autoimmune diseases. The evolutionary process of organisms leads to development of immune system enabling the body to identify “self-” and “nonself”-components to maintain an immune homeostasis, and a malfunction of immune system may cause abnormally low activity or over activity of the immune system. The low activity is a cause of immune deficiency diseases that decrease an ability to eliminate invaded pathogens, and the overwhelming activity leads the body to attack and damage its own tissues and cause autoimmune diseases (ADs).An autoimmune disease (AD) can affect one or many different types of body tissues, which can be characterized by self-immune disorders involving chronic inflammation, causing multiple major organ damages and failures, as well as the accumulation of autoantibodies in genetically susceptible individuals. They can generally be further grouped into two categories: systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and organ- or tissue-specific autoimmune diseases including rheumatoid arthritis (RA) and inflammatory bowel disease (IBD) . There aThe Wnt signaling has been demonstrated to play crucial roles in several biological aspects, including cellular proliferation, embryonic development, tissue homeostasis, development of immune system, and other systemic effects . In addiβ-catenin, planer cell polarity (PCP), c-Jun N-terminal protein kinases (JNK), protein kinase C/calcium (PCK/Ca2+), receptor-like tyrosine kinase (RYK), and receptor tyrosine kinase-like orphan receptor (Ror) pathways . In. Inβ-catli (APC) . In thiset genes .β-catenin-TCF/LEF is classified into the “noncanonical signaling pathway”  and PKC and sequentially leads to the release of intracellular Ca2+. The evoked Ca2+ concentration activates the phosphatase calcineurin, which leads to dephosphorylation of transcription factor, nuclear factor of activated T cells (NFAT) to regulate the transcription of genes controlling cell fate and cell migration . Bel. Bel1]. β-catenin has been shown to stagnate anagen initiation and disable stem cells to differentiation to hair keratinocytes rather than epidermal cells instead [β-catenin activity may be attributed to an upregulation of canonical Wnt signaling inhibitors and activation of noncanonical Wnt pathway, leading to an UDP-mediated β-catenin degradation [Alopecia areata (AA) is an autoimmune disease manifested hair loss, which is caused by a cell-mediated immune attack of the cycling hair follicle . Impaire instead , 136. Thradation . These fradation  and an iradation .β-cells in islets and abolish endogenous insulin production [β showed a potential for the regeneration of β-cell function and mass in patients with diabetes [β small molecule inhibitor or siRNA showed an increased proliferative capacity and resistance to high concentrations of glucose and the saturated fatty acid palmitate [β-catenin signaling by overexpression of Wnt3a could promote porcine pancreatic stem cells (PSCs) proliferation and enhance the tolerance of cells to ROS-induced mitochondria injury and cell apoptosis [Type 1 diabetes mellitus (T1DM) is believed to be an autoimmune disease where the immune system attacks pancreatic oduction . An enhadiabetes . In thisalmitate . In linepoptosis . These sα, TNF-α, IFN-γ, and TGF-α in cultured keratinocytes. Of interest, a reduced expression of Axin2 and lack of nuclear β-catenin suggested a suppression of canonical Wnt signaling in lesional skins, implying that the canonical Wnt signaling was shifted toward noncanonical pathways by activation Wnt5a-mediated signaling and impaired homeostatic inhibition of Wnt/β-catenin signaling by WIF-1 and DKK in psoriasis [Psoriasis is a disease characterized by chronic inflammation and altered differentiation and hyperproliferation of keratinocytes . Wnt5a hsoriasis .MicroRNAs (miRNAs) are a class of noncoding, small RNA molecules found in both prokaryotes and eukaryotes. The biogenesis of a miRNA is begun from the transcription of the miRNA gene by RNA polymerase II, to a primary miRNA (pri-miRNA) containing hundreds of nucleotides length of RNA with a stem loop structure. The pri-miRNA is then cleaved by Drosha enzyme from the nonloop end to form the microRNA precursor (pre-miRNA), a double-stranded hairpin structure of RNA with 60–70 bp in length . The preβ-catenin, TGF-β, NF-κB, hepatocyte nuclear factor 4-alpha (HNF4A), and STAT3. In addition, the kallikrein-related peptidase 4 (KLK4) was further identified as a target of miR-422a, an upregulated miRNA in LN tissues [Recently, an increasing number of studies demonstrate that miRNA expression is related to various diseases including cancer, inflammatory, and autoimmune diseases . Immunol tissues .The production of autoantibodies by uncontrolled hyperactivated B cells is a hallmark of SLE, and the early B cell factor 1 (EBF1) has been shown to play a key role in the development, activation, and proliferation of B cells through the AKT signaling pathway , which hα showed an upregulated expression of miR-155 and this miRNA is constitutively more highly expressed in RA FLS than in synovial fibroblasts of osteoarthritis (OA) patients. Functionally, the elevated expression of miR-155 suppressed the expression of MMP-3 and MMP-1 induced by cytokines and Toll-like receptor ligands, suggesting that miR-155 might have an important role in modulation of destructive behavior of RA FLS [ In vivo administration of miR-146a mimics by intravenous injection led to the suppression of cartilage and bone destruction [RA is another autoimmune and progressive systemic disease with high incidence. Recent reports show that altered expression and function of miRNAs might play an important role in the regulation of inflammatory innate immune responses in RA, which implied therapeutic potentials of miRNAs for RA. For instance, more abundance of miR-155 transcripts was found in PMBCs of RA patients compared with normal subjects. In addition, FLS exposed to TNF-f RA FLS . The miRtruction .β-catenin [β-catenin in FLS. Conversely, increasing miR-663 expression induced the FLS proliferation and the expression MMP3 and fibronectin during disease development [Recently, a series of investigations conducted by Miao et al. demonstrated that several miRNAs, including miR-152, miR-375, and miR-663, were involved in the pathogenesis of RA by targeting Wnt signaling pathways , 77, 78.-catenin . In thiselopment .β-catenin signaling were putative targets of these miRNAs. Furthermore, the NOTCH signaling was identified as a target of miR-449a in CD [Apart from the RA and SLE, differential expression of miRNAs participating in Wnt signaling pathways was also reported in other ADs, such as CD and primary biliary cirrhosis (PBC). Capuano et al. quantified 365 human miRNAs expression in human small intestinal tissues, and 20% of the examined miRNAs exhibited differential expression between CD and control children. Bioinformatics analysis predicted the NOTCH1 and 9a in CD . Qin et 9a in CD . Bioinfo9a in CD .The Wnt signaling pathway plays a fundamental role in the embryonic development, cell proliferation, differentiation, stem cell maintenance, and tissue homeostasis, including the immune system development and immune regulation. A large body of study shows that a dysregulated Wnt signaling may lead to malfunctions of immune system and develop autoimmune diseases. Equally noteworthy, accumulating evidences have suggested that miRNAs are important regulators in pathogenesis of diseases, including the autoimmune diseases, through a mechanism by targeting Wnt signaling molecules. Indeed, increasing lines of evidence have indicated involvements of miRNAs in pathogenesis of autoimmune diseases by dysregulating Wnt signaling pathways, despite the fact that the precise underlying mechanisms remain unclear.Although the regulatory roles of both the Wnt signaling and miRNAs in the pathogenesis of autoimmune diseases are now recognized, mechanisms by which miRNAs target Wnt signaling pathways in the autoimmune diseases remain largely unknown. Particularly, the miRNA expression profiling analysis of many autoimmune diseases has been performed, and signaling networks involving the targets of these miRNAs have also been predicted using bioinformatics tools. However, these targets need to be validated experimentally and clinically. The knowledge on the sequential molecular mechanisms of miRNAs targeting Wnt signaling cascade in autoimmune diseases may thus provide new diagnostic/prognostic markers and therapeutic targets for developing novel strategies and/or agents for treatments of immune diseases, through targeting Wnt signaling pathways.


Kiss1r (mRNA) was recently documented in the mouse uterus on D4 of pregnancy (the day of embryo implantation) suggesting that the uterine-based kisspeptin (KP)/kisspeptin receptor (KISS1R) signaling system regulates embryo implantation. Despite this important suggestion, it was never demonstrated that the uterus actually exhibits a functional KP/KISS1R signaling system on D4 of pregnancy. Thus, the goal of this study was to determine whether a functional KP/KISS1R signaling system exists in the mouse uterus on D4 of pregnancy.Expression of kisspeptin (protein) and Kiss1 null uterus expresses functional KISS1R molecules capable of mediating the effects of kisspeptin.Since kisspeptin/KISS1R signaling triggers the phosphorylation of the mitogen-activated protein kinases p38 and ERK1/2, through immunohistochemical analyses, we determined whether exogenously administered kisspeptin could trigger p38 and ERK1/2 phosphorylation in the uterus on D4 of pregnancy. The results clearly demonstrated that kisspeptin could and that its effects were mediated via KISS1R. Additionally, the robust kisspeptin-triggered response was observed in the pregnant uterus only. Finally, it was demonstrated that on D4 of pregnancy the These results lead us to conclude that on D4 of pregnancy, the mouse uterus expresses a functional KP/KISS1R signaling system strengthening the possibility that this signaling system regulates embryo implantation. These findings strengthen the rationale for determining whether such a functional system exists in the uterus of the human female and if so, what role it might play in human pregnancy. It is now well-established that hypothalamic kisspeptins (KPs), signaling via their cognate receptor KISS1R, are potent triggers of gonadotropin-releasing hormone (GnRH) secretion and mice bearing a deletion of the gene encoding KISS1R in the GnRH neuron display hypogonadotropic hypogonadism and infertility . TherefoKiss1r (mRNA) are highly expressed in the mouse uterus on the day of implantation  suggesting a role for this signaling system in regulating embryo implantation [Kiss1. These mice are infertile and it was assumed that their infertility was due to a lack of hypothalamic KP/KISS1R signaling and the resulting reduction in gonadotropins, estradiol and progesterone [Kiss1 null uterus leading us to suggest that it was a lack of uterine and not hypothalamic KP/KISS1R signaling that triggered implantation failure. Despite this important suggestion, we did not actually demonstrate the presence of a functional KP/KISS1R signaling system in the uterus on D4 of pregnancy. Such demonstration would have greatly strengthened our suggestion.We have previously reported that KP (protein) and esterone . HoweverThus, the goal of this study was to determine whether on D4 of pregnancy, the day of embryo implantation, a functional KP/KISS1R signaling system exists in the mouse uterus. It was previously reported that in various cell types, the KP/KISS1R signaling triggers mitogen-activated protein kinase (MAPK) phosphorylation. Specifically, it was demonstrated that KP treatment of KISS1R-expressing cells triggers the phosphorylation of the MAPKs p38 and ERK1/2 –12 but nKiss1 null females  were maImmediately after drug or saline treatment, uteri were removed and simultaneously processed for immunohistochemistry using standard techniques  and 5 μmResults showed that, compared to PBS-treated pregnant mice Fig.  and b, uWhen these studies were conducted in non-pregnant mice, relative to PBS-treated mice Fig. , uteri fWhen compared to PBS-treated pregnant mice Fig. , uteri fConsistent with data obtained in KISS1R-expressing Chinese hamster ovary cells , relativIt was reported that in addition to KISS1R, KP can signal via the NPFF1/GPR147 and NPFF2/GPR74 receptors . Therefon = 2), similar observations were made with pERK1/2 levels at the luminal epithelium and again, consistent with earlier experiments, KP54 had no effect on JNK/SAPK phosphorylated levels both in the presence and absence of P234 pre-treatment (n = 2) (data not shown).As expected, based on earlier findings Fig. -e resultKiss1−/− females [Kiss1r mRNA was still expressed in the Kiss1−/− uterus, this expression did not result in the formation of functional receptor molecules thus precluding the ability of KP to signal and rescue the implantation defect. To test the latter possibility, Kiss1−/− uteri on D4 of pregnancy were assayed for phosphorylated p38 following KP54 treatment. Results showed that relative to PBS-treated pregnant mice , similar observations were made with pERK1/2 levels at the luminal epithelium and again, consistent with earlier experiments, KP54 had no effect on JNK/SAPK phosphorylated levels (n = 2) (data not shown). Together, these results confirm the presence of functional KISS1R molecules coupled to p38 and ERK1/2 phosphorylation on D4 of pregnancy in the Kiss1−/− uterus suggesting that failure of exogenous KP to rescue the implantation defect, as reported by Calder et al. [In a previous study we failed to rescue the implantation defect through the add-back of KP to pregnant  females . We spec females . Anotherice Fig. , uteri fice Fig. . Based oThe goal of this study was to determine whether on D4 of pregnancy, the day of embryo implantation, a functional KP/KISS1R signaling system exists in the mouse uterus. Our data clearly reveal that one exists and this major finding further strengthens the possibility that this signaling system regulates uterine functions in early pregnancy. Because expression, as determined by phosphorylated p38 and ERK1/2 levels was localized to the uterine epithelia, it is possible that KP/KISS1R signaling regulates embryo implantation. For example, through p38 it might regulate the availability (production and/or secretion) of glandular products essential for implantation, or through both p38 and ERK1/2 might regulate embryo implantation at the uterine epithelium. It is also possible that through paracrine signaling, epithelial KP/KISS1R signaling prepares stromal cells for decidualization in the event of successful implantation , 16.


Fibrosis is defined as an excessive accumulation of extracellular matrix components that lead to the destruction of organ architecture and impairment of organ function. Moreover, fibrosis is an intricate process attributable to a variety of interlaced fibrogenic signals and intrinsic mechanisms of activation of myofibroblasts. Being the dominant matrix-producing cells in organ fibrosis, myofibroblasts may be differentiated from various types of precursor cells. Identification of the signal pathways that play a key role in the pathogenesis of fibrotic diseases may suggest potential therapeutic targets. Here, we emphasize several intracellular signaling pathways that control the activation of myofibroblasts and matrix production. Fibrosis, characterized by excessive accumulation of extracellular matrix (ECM) in and around inflamed or damaged tissue, leads to tissue destruction,permanent scarring, and organ malfunction ••. AlthoFibrogenesis is a dynamic and progressive process in which nonresolving inflammation following a persistent injury sets the stage for fibrosis and triggers the activation of matrix-producing myofibroblasts differentiated from a variety of precursor cell types through different mechanisms, including activation of resident fibroblasts and pericytes, phenotypic transition of epithelial and endothelial cells, phenotypic modulation of vascular smooth muscle cells, and recruitment of circulating multipotent monocytes and fibrocytes ••. The mcis-acting elements in the regulatory regions of its target genes , which binds to hamartin (TSC1). TSC1 contributes stability and prolongs the half-life of TSC2 within this complex, and TSC2 becomes phosphorylated and thereby inactivated. TSC2 functions as a GTPase-activating protein (GAP) specifically for Ras homolog enriched in brain (Rheb) , 35. RhePI3K also acts as a branch point in response to TGF-β, leading to activation of PAK2/c-Abl. The latter not only stimulates collagen gene expression in normal fibroblasts, but also induces fibroblast proliferation, thereby increasing the number of myofibroblast precursors . PAK2/c-Rho GTPases, a subfamily of small GTP-binding proteins belonging to the Ras superfamily, modulate the actin cytoskeleton. Their activity is regulated by Rho guanine nucleotide exchange factors. The latter can interact directly with Rho proteins, allowing exchange of GDP for GTP ••. Rho iMultiple target genes for MRTF/SRF are known drivers of fibrosis, including CTGF, α-SMA, and collagen , 47. SRFWnt proteins deliver their signal across the plasma membrane by interacting with Fizzled receptors and coreceptors, members of the LDL receptor-related protein 5/6. Once Wnts bind to their receptors/coreceptors, they initiate a chain of downstream signaling events implicating Disheveled, axin, adenomatosis polyposis coli (APC), casein kinase 1 (CK-1), and glycogen synthase kinase 3β (GSK-3β), leading to dephosphorylation of β-catenin •. EscapiWnt/β-catenin is an evolutionarily conserved cellular signaling system that plays an essential role in a diverse array of biologic processes, such as organogenesis, tissue homeostasis, and the pathogenesis of many human diseases •. The coWith regard to cellular targets, several in vitro studies show that activation of Wnt/β-catenin signaling enhances proliferation, migration, and matrix production in human dermal fibroblasts, suggesting a key role for this signaling pathway in fibroproliferation and differentiation of fibroblasts into myofibroblasts , 53. In Increasing evidence exists regarding crosstalk between Wnt/β-catenin and TGF-β. Wnt/β-catenin signaling can upregulate expression of TGF-β1 , 63, andcis-acting elements regulates the expression of hedgehog target genes [Hedgehog signaling is a highly conserved signaling pathway that orchestrates multiple aspects of embryogenesis, development, and tissue remodeling in a wide spectrum of systems , 71. Hedet genes . Gli1 anet genes , 75. Diret genes •, 76.Among the three vertebrate hedgehogs, sonic hedgehog (Shh) is the best characterized and most widely studied. Shh signaling has been implicated in the regulation of injury repair and wound healing after tissue damage •, 77•. SShh signaling elicits its action by regulating the transcription of its target genes •, 76. ThNotch proteins are single-pass transmembrane receptors with conserved expression among animal species during evolution. Their principal function is the regulation of many developmental processes, including proliferation, differentiation, and apoptosis. Mammals possess four different Notch receptors, referred to as Notch1–4. The Notch receptor consists of an extracellular domain, which is involved in ligand binding, and an intracellular domain that works in signal transduction. Notch ligands also are single-pass transmembrane proteins named Jagged (Jag1 and 2) and Delta  , 82. ActDuring the past few years, activation of Notch signaling has shown fibrogenic effects in a wide spectrum of diseases, including systemic sclerosis (SSc) •, 86, scActivated Notch1 and an elevated level of NICD are found in the lesional skin of SSc patients and in the skin and lungs of mouse models with SSc. Blocking the release of NICD with a γ-secretase inhibitor or treating SSc mice with Notch siRNA may reduce the collagen content in the skin and lungs •, 86, 90Activation of Notch signaling is associated with abnormal differentiation of respiratory epithelial cells in progressive IPF or secondary pulmonary fibrosis as observed in SSc. Hes-1, a Notch target gene, is upregulated in lung mucus cells from patients with chronic obstructive pulmonary disease, idiopathic pulmonary arterial hypertension, and IPF . ActivatThe participation of Notch in chronic kidney disease (CKD) has been studied in detail. Elevated levels of Notch ligands and receptors are detected in several glomerular diseases and tubulointerstitial fibrosis , 88, 89.Abundant studies demonstrate that integrin signaling plays a critical role in the production and assembly of matrix proteins. Integrins are a family of heterodimeric transmembrane receptors containing α and β subunits. Integrins integrate signals between cells and their extracellular environment by connecting the cytoskeleton to the ECM. Because they have no enzymatic or actin-binding activity, integrins transmit their signals by activating their downstream effector kinases, focal adhesion kinase (FAK), and integrin-linked kinase (ILK) •, 96.ILK can integrate and mediate several different intracellular signals associated with fibrogenesis, mainly because it is both a scaffolding protein and a serine/threonine protein kinase. Ectopic expression of ILK directly phosphorylates GSK-3β, then the inactivation of GSK-3β leads to stabilization of Snail1 and β-catenin. Snail1 regulates EMT and fibroblast motility, whereas β-catenin directly governs the transcription of fibronectin and PAI-1. On the other hand, as a scaffolding protein, ILK assembles a multicomponent protein complex that contains ILK, PINCH, and parvin , 97. TheGiven the diversity of known fibrogenic mediators, the intracellular signal cascades involved might be immensely complex, with almost immeasurable crosstalk and feedback. In essence, the activation of major fibrogenic signaling mentioned earlier is characteristic of various fibrotic diseases. During the past decade, our understanding of the profibrotic mechanisms of these signaling pathways has evolved and improved considerably. It seems intriguing and meaningful to identify some of the converging molecular machineries that integrate various signal inputs and control the transcriptional program for myofibroblast activation and matrix production. Because several strategies to target key profibrotic mediators and signaling pathways to block the process of fibrosis are effective in different animal models and clinical trials ••, 101••


Medulloblastoma (MB), a primitive neuroectomal tumor of the cerebellum, is the most common malignant pediatric brain tumor. The cause of MB is largely unknown, but aberrant activation of Hedgehog (Hh) pathway is responsible for ~30% of MB. Despite aggressive treatment with surgical resection, radiation and chemotherapy, 70%–80% of pediatric medulloblastoma cases can be controlled, but most treated patients suffer devastating side effects. Therefore, developing a new effective treatment strategy is urgently needed. Hh signaling controls transcription of target genes by regulating activities of the three Glioma-associated oncogene (Gli1-3) transcription factors. In this review, we will focus on current clinical treatment options of MB and discuss mechanisms of drug resistance. In addition, we will describe current known molecular pathways which crosstalk with the Hedgehog pathway both in the context of medulloblastoma and non-medulloblastoma cancer development. Finally, we will introduce post-translational modifications that modulate Gli1 activity and summarize the positive and negative regulations of the Hh/Gli1 pathway. Towards developing novel combination therapies for medulloblastoma treatment, current information on interacting pathways and direct regulation of Hh signaling should prove critical. Medulloblastoma (MB) is a rapidly-growing, highly invasive tumor arising from embryonal cells in the cerebellum. It is the most common malignant brain tumor in pediatric patients, accounting for about 20% of all brain tumors in children ,2. The mThe World Health Organization classifies medulloblastoma into subtypes based on histological traits: (1) desmoplastic or nodular; (2) medulloblastoma with extensive nodularity; (3) large-cell variant; and (4) anaplastic medulloblastoma . HoweverHowever, as will be described in further detail in this review, current therapies targeting SHH signaling demonstrate several limitations. Towards exploring new venues of drug development for SHH signaling, this review will describe limitations with current therapies, pathways interacting with SHH signaling in MB, as well as pathways interacting with SHH in other cancer types. Finally, the review will discuss points of direct regulation of SHH signaling by different kinases and provide critical insight for drug-resistant in MB treatments.Drosophila, it has been shown that heparin sulfate glycoproteins called glypicans play a role in the transport of Hh ligand [Hedgehog (Hh) signaling is mediated by a group of morphogen ligands: sonic hedgehog (SHH), Desert hedgehog (DHH) and Indian hedgehog (IHH). These are synthesized as precursor proteins that are then processed into two fragments, namely an amino-terminal peptide and a carboxy-terminal peptide. The amino-terminal peptide is responsible for Hh signaling ,14,15. Bh ligand . These gPTCH1) which is localized to the base of primary cilium, releasing its inhibition of Smoothened (SMO), a protein responsible for activating the downstream Hh pathway. Once SMO is activated, it binds to SUFU and induces nuclear translocation of Hh pathway transcription regulators, Gli1 (activator), Gli2 (activator) and Gli3 (repressor) [Gli1, Ptch1, cyclin D, and myc involved in cell survival, proliferation and differentiation [Hh signaling requires intact primary cilium, a microtubule-containing organelle that extends from the surface of nearly all cells in mammalian tissues. Responding to mechanical and chemosensation, primary cilium are localization points for signaling receptors, ion channels and transporters. Acting at the primary cilium, Hh morphogens play essential roles in embryogenesis, cell proliferation and tissue development, and stem cell maintenance . Specifipressor) ,20. Gli1ntiation ,22. LateSmoothened mutations were detected in sporadic basal cell carcinomas and primary neuroectodermal tumors, and activating mutations in Smoothened lead to cerebellar dysplasia and tumors [Smoothened developed medulloblastoma-like cerebellar tumors by five months of age; this incidence increased to over 80% in mice homozygous for constitutively active Smoothened. Mice harboring homozygous constitutively active Smoothened also demonstrated metastasis of cerebellar tumors to the leptomeningeal membranes of the brain [PTCH were found in a subset of sporadic medulloblastomas [PTCH is sufficient to drive medulloblastoma, as the majority of PTCH hemizygous mice with medulloblastoma was found to contain the wild-type sequence in the second PTCH allele [SUFU [During cerebellar development , granule neuron progenitor (GNPs) cells in the outermost layer of the cerebellum  undergo massive proliferation through SHH signaling . In one d tumors ,26,27. Ihe brain . Other clastomas . In addiH allele . A subsele [SUFU .Gli1 alleles resulted in a decrease in spontaneous medulloblastoma formation in PTCH heterozygote mice [Activity of downstream Gli transcriptional activity is closely linked with tumorigenesis. In one study, inactivation of both ote mice . Howeverote mice . The relPTCH1 mutations in approximately equal frequencies across all age groups. On the other hand, SUFU mutations were found enriched in infant SHH-MB, while SMO mutations were enriched in adults. Mutations in TP53 were found in nearly 50% of the 4–17-year-old age group samples [A recent genome sequencing study suggests that infant (≤3 years old), pediatric (4–17 years old) and adult SHH-medulloblastomas are genetically distinct. Sequencing of infant, children and adult medulloblastomas found  samples . These rFor medulloblastoma, the first treatment option is usually surgery to resect as much of the tumor as possible. This is followed by radiation of the brain and spine and/or chemotherapy . Standar+/− mice [+/− mice [Use of the steroidal alkaloid cyclopamine, which targets SMO to suppress Hh signaling, launched the idea of modulating the Hh signaling pathway as a therapeutic ,39,40. C+/− mice . Cyclopa+/− mice . Unfortu+/− mice .vs. 5.8 months) [More SMO inhibitors are currently being evaluated, including vismodegib GDC-0449), sonidegib (LDE-225), BMS-833923, PF-04449913 and LY2940680, in clinical trials in advanced cancers 9, sonide. Vismode months) . Likewis months) .GANT61 and HPI-1 are potential therapeutics for MB that target the Hh signaling pathway; both target Gli1 and Gli2 ,52,53. Let al. 2014 argue that patients with mutations downstream of SMO such as those with GLI2 and NMYC mutations generally would not respond to SMO antagonists. Patients with genetic aberrations upstream of SMO, such as PTCH1 and SHH amplifications, respond to SMO antagonists and include pediatric patients. Therefore, exploration of SMO inhibitors in the treatment of pediatric medulloblastoma is productive, especially when used in combination therapies. Identification of pathways cross-talking with SHH, along with an understanding of pathways to treatment resistance, will aid the development of more efficacious SHH targeting therapeutics.In the context of SHH-MB, Kool ABCG2, an ATP-binding cassette (ABC) transporter associated with stem cell behavior and drug resistance, was found to be elevated. Treatment of surviving cells from certain patients with ABC transporter inhibitors verapamil and reserpine sensitized the cells to radiation [A number of genes have been linked to treatment resistance. In one study, medulloblastoma cells surviving radiation were analyzed for the expression of genes for stem cell behavior, which is linked to the ability of a subset of tumor cells to drive tumor formation or initiate metastasis. adiation . High leadiation . Thus, iThe role of cancer testis antigens (CTA), which are highly elevated across different cancers including melanoma and breast cancer, in modulating chemotherapy resistance has been investigated in the context of medulloblastoma. Expression of members of the CTA family, such as the MAGE and GAGE proteins, were correlated with chemotherapy resistance, and inhibition of these genes sensitized MB cells to agents such as cisplatin and etoposide .Smoothened was discovered to contribute to resistance to the Smo-targeting drug GDC-0449, through a deficiency in drug binding [PTCH1 mutation [Acquired mutations in components of the SHH pathway will also drive resistance to therapies targeting SHH signaling. In a patient with metastatic medulloblastoma, an acquired G-to-C missense mutation at position 1697 (aspartic acid to histidine) in  binding . Whole emutation .SHH amplifications, PTCH1 and SMO mutations) in general may respond more favorably to SMO inhibitors, while those with mutations downstream (GLI2 mutations) may be more resistant to treatment [In light of recent literature, several considerations are important for future therapeutic development for SHH medulloblastoma. The first consideration is the variable genetic backgrounds of SHH-MB in infants, children and adults. For example, patients with mutations upstream in the SHH pathway  MBs, the role of p53 in MB should be examined closely. p53 status is important in the prognosis of patients with SHH-medulloblastoma, as well as disease incidence. A cohort study suggested that TP53 status plays a critical role in survival status of patients, with five-year survival rates differing significantly between 41% and 81%, respectively, for SHH-medulloblastoma patients with and without TP53 mutations [PTCH, incidence of medulloblastoma was 14%; this incidence increases to >95% in the presence of p53 loss, and this increase is specific for p53 loss. However, no significant change in incidence was observed for heterozygous mice carrying a mutation in either APC or p19ARF [utations . In micer p19ARF . Finallyr p19ARF . These rcAMP. The role of cAMP in a variety of brain tumors has been established. Furman et al. found that higher cAMP levels correlated with lower grade brain tumors, while lower adenylate cyclase and cAMP levels correlated with higher malignancy [et al. generated foci of decreased cAMP using phosphodiesterase-4A1 in mice cortex and found the majority of mice developed cortical growth with tumor morphology [et al. found that the phosphorylation by cAMP-dependent PKA results in Gli2 degradation [lignancy . In a nerphology . Possiblradation . These rGNAS gene, encoding the G protein Gαs, is sufficient to initiate SHH medulloblastoma. Downstream Gαs signaling mediated through cAMP levels suppresses SHH signaling, and mice harboring the GNAS mutation demonstrate decreased tumor proliferation when cAMP levels are elevated [Further linking cAMP to SHH-mediated MB, another group of researchers found that ablation of the elevated . TherefoAtoh1 and Boc. Defects in different components of Hh signaling during cerebellar development trigger formation of medulloblastoma. For example, following SHH-induced proliferation, the GNPs exit the cell cycle and migrate inwards the cerebellum where they differentiate into granule neurons in the internal granule layer (IGL). Important in this process is the transcription factor Atoh1, whose expression is restricted to proliferating GNPs and absent in IGL neurons. SHH signaling appears to stabilize Atoh1 protein levels by preventing its proteasomal degradation through a phosphor-dependent mechanism. Abnormal constitutive SHH signaling may lead to Atoh1 overexpression, subsequently transforming GNPs into MB cells [MB cells . AnotherMB cells . Both AtTGF-β. Microarray analyses of medulloblastoma samples from patients with primary and recurrent and metastatic tumors was performed, and TGF-β was found to be significantly differentially expressed. With primary tumors, positive nuclear staining of SMAD3, representing activation of TGF-β pathway, correlated with longer survival [survival . In contsurvival . TherefoSmo and Gli1 mRNA expression were downregulated while Ptch was unaffected. Finally, overexpression of Smad5 was shown to decrease the percentage of SHH-induced proliferating cells in culture. These results suggest that the BMP2-Smad5 axis, specifically its activation, may be an important targeting point that allow a bypass to SHH-induced proliferation [Bone morphogenic proteins, which belong to the TGF-β superfamily of morphogens, play a role in controlling SHH-mediated cell proliferation. Specifically, BMP2 and BMP4 have been shown in primary culture of proliferating granule cell precursors to arrest SHH-induced proliferation, thus allowing the cellular differentiation. Moreover, upon addition of BMP2 to SHH-stimulated cultures, feration .CXC12 and CXCR4. The chemokine CXC12 and its receptor CXCR4 play a synergistic role with SHH signaling in medulloblastoma. CXC12 plays an important role during granular cell development. Deletion of CXCR4 or CXC12 resulted in migration of granule cell precursors away from the EGL [cyclin D1, while CXCR4 antagonism, resulted in decreased cyclin D1 expression and also blocked maximal MB tumor growth. Finally, SHH activation was shown to trigger CXCR4 localization to the cell surface [ the EGL . In addi the EGL . Co-acti surface . These rbFGF. Basic FGF (bFGF) signaling appears to have an inhibitory role on SHH-induced proliferation. Indeed, co-incubation of SHH with bFGF eliminated the Shh-induced proliferation [Gli1, Nmyc, and cyclin D1 [Gli1 transcription. Activation of bFGF is thus another attractive point of co-targeting in SHH-induced MB.feration . Consistferation . In addiyclin D1 . TherefoSurvivin. Survivin regulates cell cycle progression and inhibits apoptosis [poptosis ; Brun anpoptosis . How suret al. identified that Gli1 and Gli3 were MAPK substrates. They can be phosphorylated by JNK1/2 and ERK2, these results provided a direct connection between MAP kinase and hedgehog signaling [The crosstalk between RAS/RAF/MEK and Hh signaling has been demonstrated in several cancer models. In melanoma cells, oncogenic Ras activity, dominant active MEK1 and dominant active AKT1 all increased the subcellular nuclear localization of exogenous Gli1 . Similarignaling .A growing number of studies demonstrate the influence of other signaling pathways on Hh signaling in breast cancers. For example, one study demonstrated cooperative regulation at the transcriptional and epigenetic level leading to SHH overexpression . The autThe balance between cellular apoptosis and autophagy may be crucial in certain SHH-mediated tumors. This balance was found important for hepatocellular carcinoma (HCC) pathology and is mediated in part by Hh signaling. Treatment of cultured human hepatocellular carcinoma tissue with recombinant SHH or other Hh pathway agonists such as purmorphamine prevented the activation of autophagy. Inhibition of Hh signaling with GANT61, an inhibitor of Gli1 and Gli2, induced autophagy. This induction was associated with upregulation of Bnip3, a protein which mediates displacement of anti-apoptotic protein Bcl-2 from autophagy protein beclin-1. As Bcl-2 inhibits beclin-1 mediated autophagy, the inhibition of Hh signaling and subsequent upregulation of Bnip3 leads to upregulation of apoptosis over autophagy. Indeed, the study found that the inhibition of Hh signaling increased HCC apoptosis and decreased cell viability, suggesting that the balance between apoptosis and autophagy as mediated by Hh signaling is important in HCC pathology .Additionally, in hepatocellular carcinoma, protein kinase C delta (PKCδ), which generally slows proliferation and induces cell cycle arrest of various cell lines, is found to suppress Hh signaling. PKCδ prevents GLI1 nuclear localization and inhibits GLI1 transcriptional activity, leading to suppression of Hh signaling .i.e., catabolic vs. anabolic reactions. The crosstalk between PKM2 and Hh signaling suggests an overlap of glucose metabolism with the Hh pathway [et al., 2014. Overexpression of AMP kinase led to a decrease in Gli1 expression, while the knockdown of AMPK increased Gli1 expression [A number of studies implicate the role of glucose metabolism and energy-sensing and Hh pathway in progressing HCC. Overexpression of pyruvate kinase isoenzyme 2 (PKM2) in HCC cells resulted in upregulation of Gli1 transcription, while shRNA-mediated downregulation of PKM2 resulted in downregulation of Gli1 transcription. Notably, PKM2 in tumor cells determines the fate of glucose use,  pathway . Finallypression . SuppresThe activity of Gli1 is crucial determined by its protein stability and localization. Several kinases were known to modulate Gli1 protein stability and localization. Protein kinase A (PKA) retains Gli1 in the cytoplasm and suppresses Gli1 transcriptional activity by phosphorylating it on Thr-374 . In addiRibosomal protein S6 kinase 1 (S6K1) phosphorylates Gli1 on Ser-84, leading to GLI1 activation . PI3K/AKPreviously we found that activation of AMPK reduces Gli1 protein levels and stability, thus blocking SHH-induced transcriptional activity. AMPK phosphorylates Gli1 at Ser-102 and 408 and Thr-1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This in turn leads to increased Gli1 protein stability, transcriptional activity, and oncogenic potency . The kin(1)Clinically, the identification and assessment of novel Gli inhibitors for Hh-mediated cancers should be evaluated in the context of medulloblastoma.(2)versus pediatric MB, assessment of novel MB treatments should be conducted with stratification of patient groups in terms of age.Clinically, given the unique genetic backgrounds of adult (3)Understanding of the resistance that arises from treatment of Hh-mediated cancers should be assessed in the context of Hh-mediated MB.(4)Non-transcriptional mechanisms by which SHH signaling mediates tumorigenesis in MB should be further studied.(5)Pathways such as the RAS/RAF/MEK, NF-κB, autophagy, and glucose sensing signaling have been shown to modulate SHH signaling. The effects of the crosstalk of these pathways on MB tumorigenesis should be further studied.(6)Gli1 represents a downstream signaling point of SHH driving SHH-mediated MB, and knowledge about the regulation points on Gli1 will prove essential to developing therapeutics targeting this transcription factor.Recent literature on the genetic diversity of MB suggests that adult, pediatric and infant MB have unique genetic signatures. Towards developing novel therapeutics for pediatric MB, an understanding of these unique genetic backgrounds is crucial. In addition, successful treatment of SHH MB will benefit from an understanding and targeting of downstream SHH signaling and pathways that cross-talk with SHH. To summarize, venues for continuing and further research include: