The Hedgehog (Hh) pathway is a signaling cascade that plays a crucial role in many fundamental processes, including embryonic development and tissue homeostasis. Moreover, emerging evidence has suggested that aberrant activation of Hh is associated with neoplastic transformations, malignant tumors, and drug resistance of a multitude of cancers. At the molecular level, it has been shown that Hh signaling drives the progression of cancers by regulating cancer cell proliferation, malignancy, metastasis, and the expansion of cancer stem cells (CSCs). Thus, a comprehensive understanding of Hh signaling during tumorigenesis and development of chemoresistance is necessary in order to identify potential therapeutic strategies to target various human cancers and their relapse. In this review, we discuss the molecular basis of the Hh signaling pathway and its abnormal activation in several types of human cancers. We also highlight the clinical development of Hh signaling inhibitors for cancer therapy as well as CSC-targeted therapy. Drosophila melanogaster (D. melanogaster) [The Hedgehog (Hh) signaling pathway was first discovered in 1980 by Nusslein-Volhard and Wieschaus in a large-scale genetic screening to find mutations that affect larval body segment development in the fruit fly, ogaster)  that plaogaster) ,4,5. Howogaster) ,11 as weogaster) ,16,17,18ogaster) , liver [ogaster) , prostatogaster) , and blaogaster) , and thiDrosophila and other higher organisms, the mechanisms of the Hh signaling pathway are highly conserved [Drosophila), (3) a G-protein-coupled receptor (GPCR)-like receptor Smoothened (SMO in humans and Smo in mice/Drosophila), and (4) three transcription factors  named from the correlation of GLI1 and glioma [Even though there are several important differences between onserved . In humad glioma .Hh proteins are one of the secreted signaling protein families that can be diffused and establish gradients in target tissues after secretion, and thus are responsible for cell interactions during embryonic development . Hh protD. melanogaster of all the discovered Hh ligands [In mammals, there are three Hh gene family members: Sonic hedgehog (SHH), Indian hedgehog (IHH), and Desert hedgehog (DHH). They are all considered to be similar in their physiological effects, but their roles in development are different in many organ systems due to the diversely regulated patterns of expression . Of all  ligands . DHH exp ligands  and Leyd ligands  in the t ligands , where i ligands .Patched gene have been demonstrated in several diseases such as basal cell nevus syndrome (BCNS), nevoid basal cell carcinoma syndrome, sporadic basal cell carcinomas, and medulloblastomas [The Hh/SHH receptor is PTCH ,61, a 12lastomas ,66,67.SMO is a seven-pass integral membrane protein that is a member of the Frizzled (FzD) class of G-protein-coupled receptors (GPCRs) and functions as a positive regulator of the Hh signaling pathway because of its physical characteristics and position in Hh signaling by acting downstream of or in parallel to Patched . SMO hasGli gene is located at chromosome 12 (q13 to q14.3) and was identified by Vogelstein in 1987 because of its gene amplification of more than 50-fold in glioblastoma multiforme (GBM) and its derived cell line [The human ell line . In mammell line . These fell line . After bell line .DrosophilaCi is more closely functionally related to mammalian GLI2 and GLI3, as they can activate transcription and undergo proteolysis to generate both activating and repressing domains, whereas GLI1 cannot be modified post-translationally and thus only plays a role as a transcriptional activator of the Hh signaling pathway [Among the three GLI family members, the  pathway . GLI1 ha pathway  by bindi pathway . On the  pathway . Mechani pathway ,83,84.Drosophila) [In the absence of an Hh ligand, PTCH1, a 12-pass transmembrane receptor, localizes at the base of the primary cilium (PC), a subcellular membrane extension that is important for the distribution and function of most of the components in this pathway. PTCH1 then destabilizes SMO by repressing its ciliary accumulation and consequently inhibits SMO activity, which is an essential step for activating this pathway in mammals (but not in sophila) . The fulsophila) . The phosophila) . The trusophila) .PTCH1 [In the canonical Hh signaling pathway, Hh regulates the GLI family of transcription factors. Upon Hh binding to PTCH1, PTCH1 leaves the cilia by internalizing together with its ligand SHH, and both proteins are degraded in lysosomes . This biPTCH1 . A schemHowever, emerging evidence over the past few years has indicated that Hh proteins can also signal through GLI-independent mechanisms, the so-called “noncanonical” Hh signaling . In thisThe SMO-independent noncanonical signaling pathway, which operates via PTCH and three Hh proteins , can inhibit the activation of caspase-3 and promote cell survival through inactivating the PTCH1 pro-apoptotic activity in an SMO-independent manner . SurprisIn the noncanonical signaling pathway, which works through functions or activities of SMO, it has been indicated that via the rapid stimulation of Rac1 and RhoA small GTPases by SMO, heterotrimeric Gi Proteins are required for SHH-induced fibroblast migration in human endothelial cells . AdditioAccumulating evidence suggests that aberrant activation of the Hh signaling pathway by deregulation of any component within may be involved in the development and progression of cancers and diseases. In various cancer types, three types of proposed mechanisms of uncontrolled activation of Hh signaling are explained below, as summarized in Patched gene [The first evidence of a link between aberrant Hh signaling and cancer is the rare condition Gorlin syndrome , which is caused by the activating mutation in the hed gene . Gorlin hed gene ,109. Canhed gene ,110, thehed gene ,112, thehed gene .PTCH1 gene are observed in invasive transitional cell carcinoma of the bladder [The tumorigenesis is ligand independent among all these cases, in which the pathway is constitutively activated in the absence of the ligand through mutations in components of the Hh pathway, including activating mutations in the SMO and inactivating mutations in the PTCH1 or Sufu. In Gorlin syndrome, a PTCH1 mutation on chromosome 9 was found . Interes bladder , esophag bladder , and tri bladder .Ptch1 gene [Sufu gene also develop a skin phenotype similar to that in Gorlin syndrome such as basaloid changes and jaw keratocysts [These observations have been confirmed by experiments in a variety of preclinical and clinical models. As heritable mutations in BCNS patients and a somatic mutation in a sporadic BCC were found in the ch1 gene , Ptch1 hch1 gene ,124. In ch1 gene . Moreoveatocysts .Besides activation via the mutations in components of the Hh pathway without ligands, Hh signaling could also be ligand-dependent autocrine/juxtacrine-activated, in which the Hh ligand is profusely released and taken up, activating the same or surrounding tumor cells. In addition to the overexpression of the Hh ligand, other tumors display a high level of PTCH1 and GLI expression.As the Hh ligand is known to be widely expressed in the foregut endoderm and involved in the regulation of branching morphogenesis in the mammalian lung , Hh signIn addition to autocrine/juxtacrine activation of the Hh signaling pathway, the Hh pathway can be activated through a paracrine manner in stromal cells as well. In this type, Hh ligands released by cancer cells will bind to the PTCH1 receptor and switch on the Hh signaling in stromal cells. These stromal cells will secrete paracrine growth signals such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), interleukin-6 (IL-6), Wnt, Platelet-derived growth factor (PDGF), and Bone morphogenetic proteins (BMP) to induce the growth of the tumor . The parMoreover, this type of Hh signaling pathway could also be in a reverse paracrine manner, in which the cancer cells take up the Hh ligands released by stromal cells and promote their growth. For example, Hh ligands released by bone-marrow, nodal, and splenic stroma could activate Hh signaling, which plays an important role in the survival of B and plasma cell malignancies . MoreoveAs previously mentioned, while a controlled Hh signaling pathway contributes to numerous biological processes throughout embryonic development of normal tissues and organs as well as to tissue repair and tissue homeostasis, uncontrolled activation of the pathway may promote tumorigenesis , such asBasal cell carcinoma (BCC) is the first group of cancers where the oncogenic potential of deregulated Hh signaling was identified. The pioneer study by Hanh et al. and Jonhson et al. indicated that patients with Gorlin’s syndrome had a strong tendency to develop BCC ,108. It Cyclopamine, an Hh signaling inhibitor, is the first drug that has been used to treat BCC . Since t+CD45− cells have been identified as a CSC population in BCC. This population was resistant to chemotherapy and expressed a high level of multidrug resistance protein 1 (MDR1), whose expression gradually increased upon drug exposure. CD200+ BCC also expressed a high level of GLI1 and relied on GLI1 for their survival, but not GLI2 [+CD45− cells were able to initiate BCC tumor growth with identical histology to the parental cells and showed the upregulation of SHH signaling genes. Inhibiting Hh signaling by Smo antagonists was successful in eradicating BCC CSCs. Thus, this suggested a hypothesis that dual therapy using available anti CD200+ neutralizing antibody in combination with Smo antagonists would be a novel strategy for a complete inhibition of BCC progression [CD200not GLI2 ,201. In gression .The Hedgehog (Hh) signaling pathway is essential for cell growth as well as the development of gastrointestinal tracts . AccumulBCL2 and CCND1, which resulted in decreased proliferarion of colon cancer cells [It has been reported that Hh signaling interacts with other signaling pathways, such as Wnt/b-catenin and PI3K pathways in several cancers, including in colon cancer . Dual taer cells .+ was identified as a marker of colon CSCs, and Hh-GLI1 signaling was also reported to play important roles in colon CSC survival and expansion [CD133xpansion . Treatmexpansion . A studyxpansion . Noncanoxpansion ,212. Trexpansion .Aberrant activation of Hh signaling has been observed in the several types of breast cancers. As we discussed earlier, Hh signaling alteration in cancer arises due to reasons such as overexpression of Hh ligands, which results in the activation of either autocrine or paracrine signaling, loss of function of the receptor, somatic mutation of Hh components such as GLI1 and PTCH1, and the overexpression of the Hh components . In breaHh inhibitor sonidegib was used in the treatment of several cancers and has been used clinically to treat breast cancer patients, either independently or in a combination with docetaxel . Cyclopa+/CD24−/Lin− is a subpopulation of breast CSCs that has the ability to produce large numbers of tumors having self-renewal function. CSCs expressing CD44+/CD24−/Lin− have increased PTCH1, GLI1, and GLI2 mRNA expression [CD44pression ,226. P63pression . In addipression ,228. Hh pression ,165.Pancreatic ductal adenocarcinoma (PDAC) is thought to be one of the leading causes of cancer death with 5-year survival rates of 1 to 5% . It has Hh signaling in pancreatic cancer belongs to type III ligand-dependent signaling . Among tVarious studies have shown the ability of cyclopamine to induce apoptosis in a variety of tumor cell lines and to inhibit pancreatic cancer ,232. Cyc+CD24+ESA+ cells were identified as a population of pancreatic CSC [In pancreatic cancer, CD44atic CSC . Hh signatic CSC . Kim et atic CSC . Li et aatic CSC . Inhibitatic CSC . Baicaleatic CSC . Sanguinatic CSC . Gemcitaatic CSC . Treatmeatic CSC ,172. In atic CSC . Taken tMedulloblastoma, an aggressive childhood tumor of cerebellar origin, is a malignancy exhibiting a well-recognized reliance on aberrant Hh signaling. The Hh pathway of medulloblastoma belongs to the ligand-independent type I Hh signaling . Abnorma+ cells expressed higher levels of Shh target genes, such as GLI1 and CYCLIN-D1, than that with CD15− cells [CD15 has been identified as a marker for medulloblastoma CSCs. Medulloblastoma with CD155− cells ,175. The5− cells . When th5− cells .Bcl2 [Hh pathway activity has a critical role in the maintenance of normal and neoplastic stem cells in the hematopoietic system and correlates with the chemotherapy-resistant phenotype of myeloid leukemia cells and disease recurrence ,176,178.Bcl2 .+ chronic phase (CP) CML cells compared to in normal CD34+ hematopoietic cells [In a chronic myelogenous leukemia (CML) study, Chen et al. reported that a loss of Smoothened (SMO) damaged the renewal of hematopoietic stem cells and impaired induction of CML by the BCR-ABL1 oncoprotein . Moreoveic cells . Tyrosinic cells . Cyclopaic cells ,179. Theic cells .+ leukemic cells [+ leukemia cells. Inhibition of Hh signaling with Smoothened antagonist cyclopamine, endogenous Hh inhibitor Hh-interacting protein, or anti-Hh neutralizing antibody induced apoptosis of CD34+ leukemic cells, although these CD34+ cell lines have a resistance to cytarabine (Ara-C) [+ leukemic cells. Ara-C treatment in combination with cyclopamine significantly enhanced the sensitivity to Ara-C, which resulted in the complete abrogation of bulk leukemia cells [In acute myeloid leukemia (AML), activation of Hh signaling has been shown in several human AML cells, especially primary CD34ic cells . Not onlic cells . A high ic cells . Moreove (Ara-C) . Furtheria cells . PF-0444ia cells . Recentlia cells .In lymphoblastic leukemia, Hh pathway components are upregulated in precursor B-ALL, and SMO inhibitor has been reported to reduce self-renewal of ALL cells in vitro and in vivo ,184. MorThe Hh pathway is evolutionarily conserved and essential for the regulation of normal development and differentiation in invertebrates and vertebrates. However, aberrant Hh pathway activation by either mutation or ligand overexpression is closely correlated with many types of human cancers. Thus, targeting the Hh pathway may provide therapeutic options for the treatment of numerous types of human cancers. Effective inhibition of the Hh signaling pathway can be obtained through several approaches, such as using anti-Hh antibodies to inhibit receptor activation or small-molecule antagonists that inhibit downstream Hh signaling, such as SMO and GLI1. Synthetic Hh antagonists are now available, and some are undergoing clinical evaluation . In this


Patients with advanced gastric cancer usually have a poor prognosis and limited therapeutic options. Overcoming this challenge requires novel targets and effective drugs. The Hedgehog (Hh) signaling pathway plays a crucial role in the development of the gastrointestinal tract and maintenance of the physiologic function of the stomach. Aberrantly activated Hh signaling is implicated in carcinogenesis as well as maintenance of cancer stem cells. Somatic mutations in the components of Hh signaling (PTCH1 and SMO) have been shown to be a major cause of basal cell carcinoma, and dozens of Hh inhibitors have been developed. To date, two inhibitors (GDC-0449 and LDE225) have been approved by the U.S. Food and Drug Administration to treat basal cell carcinoma and medulloblastoma. Here, we review the role of the Hh signaling in the carcinogenesis and progression of gastric cancer and summarize recent findings on Hh inhibitors in gastric cancer. Hedgehog signaling is often aberrantly activated and plays an important role during inflammation and carcinogenesis of gastric epithelial cells. Further study of the precise mechanisms of Hh signaling in this disease is needed for the validation of therapeutic targets and evaluation of the clinical utility of Hh inhibitors for gastric cancer. Gastric cancer (GC) is highly heterogeneous, largely because of the complex molecular mechanisms of its carcinogenesis  –3. ConseThe Hh signaling pathway is crucial in embryonic development and tissue homeostasis –7. Over In this review, we will focus on the role of the Hh signaling pathway in GC and CSCs and discuss the therapeutic potential of Hh inhibitors for GC.The central components of the Hh signaling pathway are three Hh ligands , the transmembrane receptor Patched1 (PTCH1), the G-protein-coupled receptor Smoothened (SMO), the negative regulator Suppressor-of-fused (SUFU), and the transcription factors GLI1, GLI2, and GLI3 . In geneGLIs play a central role in Hh signaling because abnormal activation of Hh signaling, whether due to mutations of pathway components or ligand-dependent or -independent mechanisms, triggers the downstream effector GLIs. Among the three GLIs, GLI1 acts exclusively as a transcription activator, while GLI2 and GLI3 act either as transcriptional activators or truncated transcriptional repressors . Indeed,Broadly, the canonical mechanisms linking abnormal activation of the Hh signaling pathway to cancer can be ligand dependent or ligand independent. The best-known example of the ligand-independent mechanism is in BCC . SomaticOn the other hand, the ligand-dependent mechanism has been observed in some gastrointestinal adenocarcinomas, e.g., pancreatic cancer and colon cancer . This meHh signaling has been implicated as a critical factor in gastric gland organogenesis and differentiation during embryonic development. Although all three Hh ligands activate identical signaling cascades by binding to PTCH1, the distribution of these ligands exhibits tissue specificity. SHH and IHH are most highly expressed in the gastrointestinal tract , while DHelicobacter pylori infection, expression of SHH is downregulated in inflamed tissues [In precancerous lesions in the stomach, the balance of this “paracrine” regulation is broken, which results in metaplastic transformation and growth of the fibrous tissue, proceeding to carcinogenesis. Interestingly, in the chronic inflammatory setting, e.g.,  tissues , 51, mai tissues . However tissues . These o tissues . Further tissues . Thus, ePrevious studies demonstrated that the overactivity of Hh signaling is a common molecular event in GC and that this abnormal activity is blocked by Hh inhibitors  and Hh antibodies , 56. In Abundant evidence indicates that Hh signaling is involved in the maintenance of CSCs in many cancers –20. CompTo date, the molecular characterization and functional relevance of CSCs in solid tumors are not well understood. Nevertheless, the close relationship between Hh signaling and CSCs raises the possibility of the combination of an Hh inhibitor and standard chemotherapy to improve antitumor efficacy. To achieve the goal, the precise molecular mechanisms of CSCs with Hh signaling need to be further investigated. Meanwhile, from a technical viewpoint, at least two challenges need to be resolved. First is the identification of reliable biomarkers to distinguish CSCs and to predict benefit from therapy. The baseline expression of Hh ligands in tumor tissue appears not to provide a positive association between clinical benefit and high activation of Hh signaling from treatment with Hh inhibition . InsteadConsidering the critical role of the Hh pathway in carcinogenesis, targeting of Hh signaling has attracted substantial interest. So far, dozens of small-molecule inhibitors of Hh have been developed, and two (GDC-0449 and LDE225) have been approved by the FDA for treating BCC and medulloblastoma , 16, butThe anti-SHH monoclonal antibody 5E1 blocks Hh signaling by binding at the SHH pseudo-active site groove . 5E1 hasCyclopamine is the first small-molecule inhibitor of Hh signaling, extracted from corn lilies, and it inactivates SMO by directly binding to its heptahelical bundle , 81. CycGDC-0449 (vismodegib), which acts by binding to the extracellular domain of SMO and antagonizing Hh signaling, has shown promising anti-tumor activity in advanced BCC and became the first Hh signaling inhibitor approved by the FDA in 2012 for the treatment of metastases and locally advanced BCC , 16. AltOther SMO inhibitors, including LDE225 (sonidegib), PF-04449913 (glasdegib), itraconazole, LEQ506, BMS-833923, LY2940680, and vitamin D3, have been effective against a variety of human cancers –90, and Since the GLI1 transcription factor is the final effector of the Hh signaling cascade, targeting GLI1 may yield more efficient antitumor activity than other targets have thus far. In 2007, two small-molecule GLI1 inhibitors (GANT58 and GANT61) were identified through cell-based screening . GANT61 Yan and colleagues treated GC cell lines with cyclopamine and GANT61 and found that both inhibitors repressed cell growth . AnotherBesides direct GLI1 inhibitors, indirect GLI1 inhibitors have also recently emerged as candidates for suppressing Hh-dependent tumors. JQ1, the bromodomain and extra-terminal domain (BET) protein inhibitor, specifically impacts GLI1 transcription . BET proThe Hh signaling pathway has long been thought to play a crucial role in embryonic development, tissue homeostasis, carcinogenesis, and maintenance of CSCs. Since the predominant role of Hh signaling in BCC has been verified, leading to the development and marketing of vismodegib, efforts are underway to exploit this pathway to treat other cancers. However, so far, the results from clinical trials targeting Hh signaling in a number of cancers, such as GC, colorectal cancer, ovarian cancer, and pancreatic cancer, have been inconsistent with expectations , 99, 100The reasonable way to overcome this problem is the identification of precise predictive biomarkers and optimized combination strategies, such as an Hh signaling inhibitor combined with standard chemotherapy or other targeted therapy. In one study of Hh signaling in medulloblastoma, Shou and colleagues validated a five-gene signature that identifies Hh pathway activation and patients most likely to respond to therapy targeting the Hh signaling pathway . AlthougHh signaling is often aberrantly activated and plays an important role in the inflammation and carcinogenesis of gastric epithelial cells. However, the clinical utility of Hh inhibitors for GC should be further evaluated through more well-designed clinical trials. In addition, given the complexity of Hh signaling and the heterogeneity of GC, the precise mechanisms of Hh signaling need to be studied further for the validation of therapeutic targets and ideal biomarkers.


C. elegans mediates its response to both osmotic stress and starvation. We report that in response to osmotic stress the cytosolic sulfotransferase SSU-1 antagonizes insulin-like signaling and promotes developmental arrest. Both SSU-1 and the DAF-16 FOXO transcription factor, which is activated when insulin signaling is low, are needed to drive specific responses to reduced insulin-like signaling. We demonstrate that SSU-1 functions in a single pair of sensory neurons to control intercellular signaling via the nuclear hormone receptor NHR-1 and promote both the specific transcriptional response to osmotic stress and altered lysophosphatidylcholine metabolism. Our results show the requirement of a sulfotransferase–nuclear hormone receptor neurohormonal signaling pathway for some but not all consequences of reduced insulin-like signaling.Insulin and insulin-like signaling regulates a broad spectrum of growth and metabolic responses to a variety of internal and environmental stimuli. For example, the inhibition of insulin-like signaling in  C. elegans response to many environmental stressors, but how distinct outcomes are achieved is unknown. The authors show that the cytosolic sulfotransferase SSU-1 controls neurohormonal signaling via NHR-1 to specify the animals’ osmotic stress response.Reduced insulin-like signaling is required for  This response is independent of plasma insulin concentration1, indicating that the consequences of insulin signaling are distinct in different physiological contexts. Similarly, the consequences of insulin signaling can change during development. For example, injection of the insulin-like peptide Bombyxin-II into hemolymph of larvae but not into hemolymph of adults of the silk moth Bombyx mori results in increased conversion of trehalose into glucose4.Changes in insulin and insulin-like growth factor signaling can elicit distinct responses in different cellular or developmental contexts. For example, obese humans exhibit a transcriptional response to insulin in adipose tissue that differs from that of non-obese individualsAlthough the specific physiological responses to changes in insulin signaling vary among animals, the general ability to respond to insulin signaling and to modify the specificity of responses to insulin signaling as a consequence of changing environmental and developmental stimuli is conserved among metazoa. An understanding of the molecular mechanisms that govern the sensitivity and specificity of insulin signaling in different contexts is essential for the understanding of animal development and physiology and likely to prove important for the understanding of pathologies that result from abnormalities in insulin signaling, such as obesity and type 2 diabetes.C. elegans arrests development immediately after hatching in response to osmotic stress and that this developmental arrest involves the inhibition of insulin-like signaling and subsequent activation of the FOXO transcription factor DAF-165. Earlier studies had showed that C. elegans also arrests development immediately after hatching in response to starvation and that this developmental arrest also involves the inhibition of insulin-like signaling and activation of DAF-166. Despite these similarities in the timing and regulation of developmental arrest in response to osmotic stress and starvation, we found that these two developmental arrests are physiologically distinct5. Specifically, (a) developmental arrest in response to osmotic stress results in animals that are immobile and do not respond to touch, whereas animals arrested in response to starvation remain mobile and responsive, and (b) most genes that exhibit changes in expression in animals arrested in response to osmotic stress do not exhibit changes in expression in animals arrested in response to starvation and vice versa5. These results indicate that the inhibition of insulin-like signaling in C. elegans can elicit distinct physiological responses to starvation and osmotic stress. The mechanistic basis of these different responses has been unknown. Here we demonstrate that the cytoplasmic sulfotransferase SSU-1 is required in the ASJ sensory neurons for developmental arrest in response to osmotic stress, but not in response to starvation, and functions by controlling neurohormonal signaling via the nuclear hormone receptor NHR-1 to antagonize insulin-like signaling.We previously reported that ssu-1  resulted in approximately 40% of animals failing to arrest development in response to 500 mM NaCl  caused animals to fail to arrest development in response to osmotic stress . A nonsense allele of the cytosolic sulfotransferase gene aCl Fig. . Similaress Fig. . To testion Fig. . By contion Fig. , consist7. To determine if SSU-1 functions in the ASJ sensory neurons to regulate developmental arrest in response to osmotic stress, we expressed a rescuing transgene of ssu-1( + ) under the control of the ASJ-specific promoter trx-1 in ssu-1(-) animals8. Expression of SSU-1 in the ASJ sensory neurons restored developmental arrest in response to osmotic stress   and pregnenolonest) Fig. . For exaU-1 Fig. . We confter Fig. . GFP waster Fig. . We notesod-5::gfp in response to osmotic stress. We identified five alleles of the nuclear hormone receptor gene nhr-1   failed to arrest development in response to osmotic stress, and (2) failed to express 42) Fig. .nhr-1 acts cell-autonomously by testing for rescue of the loss of sod-5::GFP expression in nhr-1 mutants. We expressed a copy of the wild-type nhr-1 gene using either the endogenous nhr-1 promoter or any of a variety of cell-type-specific promoters, but we were unable to observe rescue, possibly because of toxic consequences of nhr-1 overexpression (see Supplementary Discussion). Besides arresting development in response to food deprivation or high osmolarity, C. elegans also arrests its development in response to harsh environmental conditions, entering an alternative third-larval stage known as the dauer larva13. Like the other developmental arrests, dauer arrest is regulated by insulin-like signaling13. Interestingly, we found that sod-5::GFP was expressed in dauer-arrested animals and that this expression required NHR-1, as in animals that arrest in response to osmotic stress  or strong (500 mM NaCl) osmotic stress. Consistent with our previous findings, nearly 100% of daf-2 mutant embryos arrested development at 300 mM NaCl    Fig. . None ofssu-1 and daf-16 mutant embryos did not produce more glycerol than wild-type embryos in response to osmotic stress  in wild-type animals in response to osmotic stress, including glycerol, and 12 polar metabolites that decreased in abundance more than twofold  in wild-type animals in response to osmotic stress, including several TCA-cycle intermediates , or specific unsaturated LPC (18:1) into standard NGM Petri plates. We placed wild-type embryos on these plates and observed that in all cases embryos developed to adulthood normally. However, animals fed either mixed LPCs from egg yolk or saturated LPC (18:0), but not unsaturated LPC (18:1), generated exclusively dead/arrested embryos Fig. . These dC. elegans, we discovered that an SSU-1 sufotransferase—NHR-1 nuclear hormone receptor signaling pathway acts in concert with an insulin-like signaling pathway that includes the insulin-like peptide INS-31, the insulin-like receptor DAF-21, and the FOXO transcription factor DAF-165 to drive gene expression in response to osmotic stress  neurons, can control responses to insulin signaling, including alterations in gene expression in distant tissues16. However, neither neuronal control of nor cellular responses to insulin signaling have previously been linked to cytosolic sulfotransferase-controlled hormonal signaling. Given the strong conservation of the insulin signaling pathway as a regulator of growth and metabolism throughout metazoan , we propose that neuronal cytosolic sulfotransferase activity functions broadly in neurohormonal signaling in mammals and that abnormalities in such signaling underlie a variety of human pathologies known to be caused by abnormalities in insulin signaling, such as obesity and type 2 diabetes.More generally, numerous studies have established that the central nervous system plays an important role in regulating insulin sensitivity and specificityC. elegans strains were cultured and maintained at 20 °C unless noted otherwise17. The Bristol strain N2 was the wild-type strain. Mutations used were::daf-16LGI:daf-2(e1370)LGIIIssu-1, zIs356 , ayIs7 [hlh-8::GFP fusion + dpy-20( + )]LGIV: :nhr-1LGX:wuIs57 Unknown linkage:nEx2685 , nEx2722 , nEx2720 , nEx2719Extrachromosomal arrays2O, sodium hypochlorite (Sigma), 5 M KOH) was added to dissolve the animals. Worms were centrifuged after 1.5–2 min in the hypochlorite solution and fresh solution was added. Total time in the hypochlorite solution was 7–10 min. Embryos were washed three times in S-basal buffer (including 0.1% ethanol and 5 ng/µL cholesterol) before final suspension in 5 mL S-basal at a density of 1 worm/µL. Embryos were cultured in a 16 mm glass tube on a tissue culture roller drum at approximately 25 rpm and 21–22 °C. For the M-cell division assay during starvation, the larvae were starved for 7 days before an average of 150 larvae per replicate were examined on a 5% Noble agar slide on a compound fluorescent microscope.Mixed-stage cultures on 10 cm NGM plates were washed from the plates using S-basal and centrifuged. A hypochlorite solution . Image acquisition settings were calibrated to minimize the number of saturated pixels and were kept constant throughout the experiment.Embryos were placed onto Petri plates containing 500 mM NaCl in NGM agar seeded with Approximately 200 developing eggs from mothers grown at 50 mM NaCl (unless otherwise noted) were collected and placed on standard NGM Petri plates containing varying concentrations of NaCl for 48 h. After 48 h, animals that remained immobile and were not feeding were scored as arrested. Mobile animals that were feeding were scored as developing. Percent failing to arrest was defined as the percent of animals mobile and feeding  and includes L1-stage larvae.ssu-1  approximately 500 L4 stage wild-type animals were incubated with 20 µL of ethyl methanesulfonate (EMS) (Sigma) in 4 mL of M9 for 4 h at 20 °C, and approximately 20,000 F3 generation embryos were placed onto Petri plates containing 500 mM NaCl in NGM agar and screened for mutants that hatched and were mobile. In the screen that identified the alleles of nhr-1, approximately 20,000 L4 stage animals expressing SOD-5::GFP (wuIs57) were incubated with 20 µL of EMS in 4 mL of M9 for 4 h at 20 °C, and approximately 200,000 F3 generation embryos were placed onto Petri plates containing 500 mM NaCl in NGM agar and screened for mutants that hatched and were mobile.Two screens were performed to identify mutants that failed to arrest development in response to osmotic stress. In the screen that identified ssu-1 DNA fragment with synthetic introns replacing endogenous introns (endogenous introns are repetitive and could not be synthesized) was obtained from Integrated DNA Technologies using their custom gene-synthesis service. The ssu-1 fragment was amplified with a 3' 5xGly linker using appropriate primers. The pJDM169 vector containing 1.1 kb of the trx-1 promoter sequence upstream of the trx-1 start codon, mCherry and an unc-54 3'UTR was obtained from J. Meisel18. The ssu-1 DNA fragment with 5xGly linker was cloned into the pJDM169 using Infusion HD (Clontech) cloning to generate the plasmid pVD100 that contains Ptrx-1::ssu-1(cDNA) – 5xGly – mCherry::unc-54 3'UTR.A synthetic g at 4 °C. After centrifugation the polar and lipid layers were separated and dried using a SpeedVac concentrator. Liquid chromatography and mass spectrometry were performed as described19.Approximately 100 µL of embryos were collected by bleaching and placed on standard NGM agar plates. After 3 h embryos were collected in M9, pelleted, and frozen. 100 µL of frozen embryos were resuspended in 600 µL methanol and lysed using a BeadBug microtube homogenizer (Sigma) and 0.5 mm Zirconium beads. After lysis, 300 µL of water and 400 µL of cholorform were added to each sample, and samples were vortexed for 1 min at 4 °C and centrifuged for 10 min at 15,000× ssu-1(fc73), and daf-16(m26) embryos were placed on standard NGM plates. After 3 h embryos were collected in M9, lysed using a BeadBug microtube homogenizer (Sigma) and 0.5 mm Zirconium beads (Sigma), and RNA was extracted using the RNeasy Mini kit (QIAGEN). RNA integrity and concentration were assessed using a Fragment Analyzer , and libraries were prepared using a Illumina NeoPrep RNAseq kit. Libraries were loaded for paired-end sequencing using the Illumina NextSeq500. Seventy-five-nucleotide paired-end sequencing reads were mapped against the C. elegans genome assembly ce10 using RSEM v. 1.2.15, with Bowtie v. 1.0.1 for read alignment 21. Expected read counts per gene were retrieved and, after rounding counts to the nearest integer, used to perform differential gene expression analysis with DESeq2 in the R v. 3.2.3 statistical environment. Sequencing library size was estimated for each library to account for differences in sequencing depth and complexity among libraries, as well as gene-specific count dispersion parameters. Gene identifiers were obtained from Wormbase version WS235.Wild-type, For Fig. R09G11.2c was amplified from C. elegans wild-type cDNA and used in all vectors generated for tissue-specific expression of nhr-1 cDNA. Infusion HD (Clontech) cloning was used to clone individual promoter fragments with nhr-1 cDNA, 5xGly linker, mCherry and tbb-2 3'UTR to generate plasmids containing the generic sequence Promoter::nhr-1 cDNA – 5xGly – mCherry::tbb-2 3'UTR. The promoter fragments of the genes ges-1 (in pVD104), unc-54 (in pVD105), and rab-3 (in pVD106) used for tissue-specific expression of nhr-1 cDNA contain 2.9, 1.9, 1.4 and 1.3 kb, respectively, of sequence upstream of each of these genes’ start codons.nEx2685 was generated by injecting pVD100 at 20 μg/mL and the co-injection marker plasmid pQZ22 at 50 μg/mL into ssu-1(fc73); wuIs57 animals. nEx2719, nEx2720, and nEx2722 were generated by injecting pVD104, pVD105, and pVD106, respectively, at 20 μg/mL along with the co-injection marker plasmid pQZ22 at 50 μg/mL for each transgene into wuIs57; nhr-1(n6242) animals.Extrachromosomal arrays were generated by injecting the corresponding plasmid and co-injection marker into the gonad of one-day old adults at the specified concentrations. Animals in the F1 progeny carrying the resulting extrachromosomal arrays were individually picked onto separate Petri plates and examined for transmission to progeny. Plates with progeny carrying transmitted arrays were maintained as independent transgenic lines. E. coli OP50. Wild-type embryos were placed on plates containing either ethanol alone or LPCs in ethanol. Animals were allowed to grow for 72 h at room temperature. After 72 h, embryos were collected from adult animals and placed on new petri plates containing LPCs for 24 h, and the fraction of embryos hatched was counted.Mixed LPCs from egg yolk (Sigma L4129), LPC (18:0) (Sigma L2131), and LPC (18:1) (Sigma L1881) were resuspended in ethanol and added to standard NGM Petri plates at a final concentration of 1 mM. These plates were then seeded with t-tests were used to compare all samples that reflect percentages of populations or populations of animals. For Fig. p-value was calculated using a normal approximation to the hypergeometric probability, as in http://nemates.org/MA/progs/representation.stats.html. No statistical method was used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Statistics source data available as a Source Data file.Two-tail Supplementary InformationSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3Description of Additional Supplementary FilesSource DataTransparent Peer Review File


High serum uric acid (UA) has been reported to be associated with left ventricular (LV) dysfunction; however, the relationship between UA and plasma B-type natriuretic peptide (BNP), a sensitive biomarker of heart failure, is still unclear. This study investigated their relationship to provide an accurate assessment of high UA. The study patients consisted of 3,077 subjects who underwent cardiac catheterization because of various cardiovascular disorders. Since the explanatory factors of multiple regression analysis were mostly confounding with each other, subgroup analysis was performed by quartering the study population using the respective risk factors and by covariance structure analysis. This analysis revealed that UA was almost always well associated with a reduced LV ejection fraction (LVEF), but generally not with BNP. UA was significantly associated with BNP in lean aged females, but not in obese adolescent males, although LVEF was significantly reduced in response to a high UA in both groups. A high UA is a direct risk factor for cardiac dysfunction from the perspective of BNP; however, augmentation of BNP in response to a high UA would likely be restricted among obese adolescent males. On the other hand, the observed LV systolic dysfunction, such as LVEF, reflects a high UA on an almost constant basis. Additionally, high serum UA is associated with the presence of hypertension, diabetes, and metabolic syndrome8. High serum UA levels also predict an increase in morbidity and mortality in patients with heart failure13. Recently, we reported that high UA is causally associated with left ventricular (LV) systolic dysfunction and a reduced LV ejection fraction (LVEF) in patients with ischemic heart disease. Importantly, the effect of high UA on LV dysfunction is exerted not only through an atherosclerotic process in the coronary arteries (cardiac ischemia) but also directly, as represented by a possible cause-and-effect relationship14.High serum uric acid (UA) has recently been discussed not only as a gout culprit but also as a cause of cardiovascular disorders. According to previous studies, serum UA levels predict the progression of chronic kidney disease and the development of stroke19. The plasma level of BNP is a highly reliable surrogate maker of heart failure. Measurement of plasma BNP is recommended as a reliable diagnostic method of heart failure in general practice and emergency medical care21. It is thus conceivable that plasma BNP should be potentially linked to high serum UA and associated LV dysfunction. There have been few reports of a close linkage between serum UA and plasma BNP in heart failure. Instead, there has been a negative report in which UA did not significantly add to the prognostic utility of BNP22. Although a high serum UA level is associated with heart failure, more studies are required to confirm the role of high serum UA in heart failure in consideration of the observed LV dysfunction and plasma BNP.B-type natriuretic peptide (BNP) is a natriuretic peptide that is widely used for diagnosis and predicting prognosis in heart failure24. Additionally, serum UA is associated with the same factors25. To explore the precise effects of high UA on plasma BNP and the observed LV dysfunction, advanced statistical procedures are warranted.Statistical analysis would be of assistance in this study; however, analyses are highly intractable for precise study because plasma BNP levels are associated with many confounding factors, including ageing, gender distinction, body mass index (BMI), LV dysfunction, and renal dysfunctionIn this study, we examined the effect of high serum UA on plasma BNP and observed LV dysfunction in patients with cardiovascular diseases who underwent cardiac catheterization in our institution. We performed a step-by-step statistical analysis to examine the possible action of high serum UA on high plasma BNP and observed LV dysfunction, taking into account possible associated factors such as age, gender distinction, BMI and renal function.Table P < 0.001). There appears to be a close linkage among serum UA, plasma BNP and LVEF.Figure P < 0.001). Age was positively associated with LVEF (b-1) and Log BNP (b-2) but negatively associated with high UA . BMI was negatively associated with Log BNP (c-2) but positively associated with high UA (c-3) and LVEF . Additionally, Cr was positively associated with Log BNP (d-2) and serum UA (d-3) but negatively associated with LVEF . These analyses suggested that surrounding factors such as ageing, BMI and Cr would seemingly modulate the authentic relationship among serum UA, plasma BNP and LVEF.Several potential risk factors, which were listed as candidates based on previous information, were included in the analysis: gender, age, BMI and creatinine (Cr). To examine a possible interruption of a possible linkage of serum UA to plasma BNP and LVEF by these surrounding factors, a single regression analysis was performed , except for male gender. Next, multiple linear regression analysis was adapted for the determination of Log BNP, as shown in Table P < 0.001); however, UA did not affect the Log BNP. These results suggested that high UA clearly represented a direct risk for a reduction in LVEF, even under the influence of surrounding factors. On the other hand, the association between high UA and plasma BNP was low or unrecognizable.Next, to examine the contribution of serum UA and the surrounding factors to LVEF, we adopted a multiple linear regression analysis, as shown in Table These respective multivariate analyses would still be inadequate for correctly interpreting the risk factors because the factors are expected to confound each other Fig. . We thouP < 0.001). Additionally, there was a significant linkage from LVEF to Log BNP (P < 0.001). On the other hand, in the male group (path model A-2), the analysis revealed that UA was significantly linked to LVEF (P < 0.001) but not to Log BNP (P = 0.423), although there was a significant linkage from LVEF to Log BNP (P < 0.001). These results showed that high UA had a negative impact on LVEF in different genders, but it had a positive impact on Log BNP only in females.Path models A-1 and A-2 in Fig. P < 0.001) but not to Log BNP (P = 0.221). Path model B-2 showed that serum UA showed a significant link to LVEF (P < 0.001) and an inverse link to Log BNP (not a positive link) (P = 0.015). Path model B-3 showed that serum UA had a significant link to LVEF (P < 0.001) and Log BNP (P = 0.002). Additionally, path model B-4 showed that serum UA had a significant link to LVEF (P < 0.001) and Log BNP (P = 0.005). These results showed that high UA had a significant impact on LVEF at any age and on Log BNP only in the elderly population (over 67 years).Path model B-1, B-2, B-3 and B-4 in Fig. P < 0.001) and Log BNP (P = 0.010). Path model C-2 showed that serum UA showed a significant link to LVEF (P < 0.001) but not to Log BNP (P = 0.828). Path model C-3 showed that serum UA had a significant link to LVEF (P < 0.001) but not to Log BNP (P = 0.532). Additionally, path model C-4 showed that serum UA had a significant link to LVEF (P < 0.001) but not to Log BNP (P = 0.461). These results showed that high UA had a significant impact on LVEF at any stage of BMI, but it had an effect on Log BNP only in the lean body mass population (BMI ≤ 21.8).Path models C-1, C-2, C-3 and C-4 in Fig. P = 0.303) and was significantly associated with Log BNP (not a positive link) (P = 0.008). Path model D-3 showed that serum UA was significantly associated with LVEF (P < 0.001) but not with Log BNP (P = 0.858). Additionally, path model D-4 showed that serum UA was significantly associated with LVEF (P < 0.001) but not with Log BNP (P = 0.857).Path models D-1, D-2, D-3 and D-4 in Fig. P < 0.001) among lean aged females. On the other hand, there was a close linkage between serum UA and LVEF  but never between serum UA and Log BNP  among obese, adolescent males.Summarizing the results of the covariance structure analysis among all path models  high serum UA was sharply associated with reduced LVEF and that 2) plasma BNP authentically responded to high serum UA, but only under the appropriate conditions, such as among lean aged females but never among obese, adolescent males.28. High XO-induced ROS production would be significant throughout the whole body, but the precise mechanism in the local heart has not been well clarified. However, it has recently been reported that cardiac function is reduced in accordance with energy deficiency, which is linked to possible cardiac UA production and XO activity31. If XO enzymatic activity is sufficiently upregulated for a local increase in oxidative stress in the heart, BNP production may also be activated under such circumstances32. As outlined above, high serum UA, high plasma BNP and low LVEF would be mutually related in heart failure by oxidative stress, which is probably activated not only throughout the whole body but also locally in the heart.As observed above, we reaffirmed that high serum UA is a risk factor for cardiac dysfunction, which can be evaluated not only by LVEF but also by plasma BNP, even under some limitations. Although the molecular mechanisms underlying the unfavorable effects of high serum UA on the heart have not been precisely examined, potential mechanisms are as follows. The UA level is elevated with subsequent upregulation of the xanthine oxidase (XO) enzymatic pathway, which accounts for the production of reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate (NADPH)33. On the other hand, free fatty acid suppresses the production of BNP35. These pathological actions would lead to a close linkage between low fat content and high plasma BNP or between high fat content and low plasma BNP. However, it is quite natural that high body mass would be substantially linked to high UA, suggesting that adipose tissue or FFA has different effects on serum UA (raising) and plasma BNP (lowering). Thus, the authentic correlation between serum UA and plasma BNP becomes less visible in obesity.The possible reasons or mechanisms for blunting the close linkage between serum UA and plasma BNP under a given set of conditions are as follows. First, we briefly describe the possible reasons for the beclouded linkage between serum UA and plasma BNP from the perspective of obesity. The molecular mechanisms have just started to be examined; however, it has recently been reported that natriuretic peptide reduces body mass by improving the biological function of adipocytes and accelerating fat burning24. Ageing then likely has different effects on serum UA (raising) and plasma BNP (lowering); thus, the authentic correlation between serum UA and plasma BNP becomes less apparent in the adolescent population.Second, it is well accepted that serum UA is higher in youth because of increased metabolic activity, high physical activity and a high level of food intake without a relationship to heart failure status. Additionally, the plasma BNP level would generally be low in the young population compared with the aged population38 and with plasma BNP via a wide variety of mechanisms41.Third, the strong linkage was absolute between serum UA and plasma BNP in females; however, the linkage disappeared in males. It is difficult to proceed with a discussion of the precise mechanism(s). Sex steroid hormones may be associated with serum UAFinally, a close linkage between serum UA and plasma BNP was hardly observed in any stage of renal dysfunction. The reasons or mechanisms are unknown; however, other risk factors including ageing, male gender and obesity, which are highly correlated with renal dysfunction, would have substantially affected the values of UA and BNP in diverse ways. Importantly, the close correlation between serum UA and reduced LVEF was mostly recognizable in advanced stages of renal dysfunction in this study.14. However, this study clearly demonstrated that the adverse effect of high serum UA on heart failure status would likely be underestimated only by measuring plasma BNP, which must be dealt with carefully in clinical practice. It is important to search for factors or clinical parameters that are strongly associated with serum UA. For example, oxidative stress markers may be conceivable. Additionally, the direct measurement of XO activity in the circulation enters the picture. On the other hand, the harmful effects of high serum UA are almost always demonstrated by the observed cardiac function such as the LVEF. In a further study, we should investigate whether high serum UA is more likely linked to patients with heart failure with an reduced ejection fraction (HFrEF) or to those with heart failure with a preserved ejection fraction (HFpEF).The direct effect of high serum UA on cardiac damage would be definite based on the results of the current and our previous studyThis was a cross-sectional study, and regarding the underlying diseases of the patients in this study, most had ischemic heart disease (84.2%). Further analysis is warranted to determine the adverse effect of high UA prospectively in a large number and various kinds of patients with heart failure. Additionally, in the present study, we did not directly examine the biological interaction between BNP and UA. Therefore, there may be a limitation to determining whether UA affects BNP or whether BNP affects UA as a definitive conclusion. A precise biological analysis should be conducted in the future.High serum UA is a direct risk factor for cardiac dysfunction from the perspective of plasma BNP; however, augmentation of plasma BNP in response to high serum UA would likely be restricted among obese adolescent males. On the other hand, the observed LV systolic dysfunction such as LVEF sharply reflects high serum UA on an almost constant basis.The study patients consisted of 3,077 subjects with any cause who were consecutively admitted to our institutions from 2012 to 2016. All patients underwent cardiac catheterization for an evaluation of respective cardiac disorders. We excluded patients receiving hemodialysis because cardiac function was largely changed by artificial volume control. The ethics committee of The Jikei University School of Medicine approved the study protocol (24-355[7121]), and we complied with our institution’s routine ethical regulations. Informed consent was obtained from each patient, and all clinical investigations were conducted in accordance with the principles expressed in the Declaration of Helsinki. According to our routine ethical regulations, we posted a notice about the study design and contact information at a public location in our institution.24. The definitions of the diseases were as follows: In brief, ischemic heart disease (IHD) was diagnosed by symptoms, electrocardiography results, blood sampling, and the morphology of the coronary arteries. The patients with IHD included those with clinically stable IHD. Organic stenosis was defined as ≥75% occlusion of the coronary arteries on coronary angiography. The patients with coronary spastic angina were included in the IHD group if the disease activity was stable, and a provocation test was planned during hospitalization. Valvular diseases included heart failure caused by moderate valvular disease and patients who were scheduled for surgery. Arrhythmia included the need for catheter ablation, an implantable cardioverter-defibrillator, cardiac resynchronization therapy, and patients with a pacemaker or syncope. Cardiomyopathy was defined when patients were diagnosed before admission and underwent treatment or if they were diagnosed after admission (excluding cases of ischemic cardiomyopathy). Infectious heart disease included pericarditis, myocarditis, and infectious endocarditis. BMI was calculated by the square of the height divided by the weight on admission.Baseline diseases, hypertension, diabetes mellitus, and dyslipidemia were defined as previously describedLVEF was obtained from the left ventriculography (LVG) trace during the end-systolic and end-diastolic phases. The contrast LVG images were acquired at a frame rate of 30 frames per second in the right anterior oblique 30-degree projection. The LVEF was calculated from single-plane cineangiograms by means of the area-length formula using a semi-automated trace method with QAngio XA version 7.1 .We collected data for plasma BNP and other levels during cardiac catheterization. Whole blood (5 mL) was collected in tubes containing sodium ethylenediaminetetraacetic acid (EDTA) (1 mg/mL blood). Plasma BNP was measured with a rapid enzyme-linked immunosorbent assay (non-extracted) using an antibody against human BNP . Serum biochemical analyses, including UA and Cr, were performed in a central laboratory in our hospital during the study.t-test and/or the Mann–Whitney U test where appropriate. After confirming the normality of distribution of the BNP variable by using a formal test, to achieve a normal distribution, the BNP value was log-transformed to (Log) BNP before conducting the analysis. Multiple regression analysis was performed when multiple values were compared. All statistical analyses were performed using SPSS Statistics version 23.0 .Continuous variables are expressed as the means ± standard deviation (SD) or medians with the interquartile range [IQR]. Categorical variables are expressed as the percentages of overall and/or each sub-group and were compared using a chi-square test as appropriate. Comparisons between two data sets of continuous variables were performed using Pearson’s product-moment correlation coefficient analysis, and significant differences between them were analyzed using a P values < 0.05. The implementation procedures for the covariance structure analysis have been described previously47. The paths between the variables were drawn from the independent to the dependent variables with directional arrows that were used for every regression model . A two-way arrow between two variables indicated a correlation between these variables. For every regression, the total variance in the dependent variable was theorized to be caused either by the independent variables in the model or by extraneous variables (e). Each path had a coefficient that indicated the standardized coefficient of the regressing independent variable on the dependent variable of the relevant path. The indirect effect was determined by multiplying the path coefficients of the intervening variables. The causality model defined certain hierarchical regression models that compare clinical factors in LVEF and plasma BNP. The corresponding author had full access to all the data in the study and takes responsibility for both its integrity and the data analysis.Path analysis based on covariance structure analysis was used to investigate the relationship among clinical factors in this study population and, in particular, to identify probable causal effects in heart failure. Path analysis was performed using IBM SPSS AMOS version 23 . The obtained structural equation models were tested and confirmed at the significance level of Supplementary Tables S1-S4


CARD11 functions as a key signaling scaffold that controls antigen-induced lymphocyte activation during the adaptive immune response. Somatic mutations in CARD11 are frequently found in Non-Hodgkin lymphoma, and at least three classes of germline CARD11 mutations have been described as the basis for primary immunodeficiency. In this review, we summarize our current understanding of how CARD11 signals, how its activity is regulated, and how mutations bypass normal regulation to cause disease. Our understanding of the immune system has benefited greatly from the study of the molecular circuitry in lymphocytes that governs antigen recognition and the cellular and systemic response to pathogens. This circuitry is elaborate, highly regulated, sensitive, and specific. As the study of immune cell signal transduction has highlighted networks of molecules that translate antigen sensing into lymphocyte action, it has also revealed how the dysregulation of signaling machinery can precipitate disease, including immunodeficiencies, autoimmunity syndromes, leukemia, and lymphoma.CARD11 is a fascinating, multi-domain scaffold protein that plays a key role as a signaling hub during the adaptive immune response. Underscoring its importance, the CARD11 gene is extremely intolerant to loss-of-function (LOF) mutation or genetic variation in the human population . Since iCARD11 can be thought of as a string of protein-protein interaction domains, each of which presents surfaces for the binding and regulation of interaction targets Figure . The proin trans.Like many signaling proteins that promote cellular proliferation and activation and whose signaling could be dangerous if dysregulated, CARD11 contains an internal autoinhibitory domain that keeps CARD11 inactive in the absence of receptor triggering –11. Thisin trans that actively prevents cooperative RE action.Antigen receptor engagement by antigen in the appropriate context leads to a cascade of signaling events upstream of CARD11 that have been extensively reviewed . At presUpon activation, the protein interaction surfaces of CARD11 become accessible for binding to a variety of proteins Figure . Bcl10 wIn addition to the intramolecular interactions mediated by the REs in the ID, two other CARD11 regions have been shown to mediate CARD11-CARD11 interactions. The Coiled-coil domain, predicted to form four discontinuous regions with coiled-coil character, mediates assembly of CARD11 into an oligomer of undetermined stoichiometry. The SH3-GUK domain tandem has also been shown to participate in modular inter- and intramolecular binding interactions that appear to be required for higher order clustering of CARD11 visible via microscopy .The transient recruitment of cofactors to CARD11 ultimately leads to the activation of the IKK complex in the canonical NF-κB activation pathway. As it assembles cofactors into complexes, CARD11 orchestrates ubiquitinylation and phosphorylation events that somehow work together to promote IKK kinase action on inhibitory IκB proteins that tether NF-κB in the cytoplasm. The phosphorylation of IκBs promotes their ubiquitinylation and degradation by the proteasome, which allows NF-κB to stably translocate to the nucleus to bind target genes.n-Bcl10 , the levels of LinUbn-Bcl10 produced by each variant correlates with the degree of NF-κB activation it achieves  to conjugate Bcl10 (substrate) with linear ubiquitin chains, to produce LinUbn-Bcl10 . LinUbn- of IKKγ  in an in of IKKγ . LinUbn-achieves . Signaliof LUBAC .n(K63)-Bcl10. This modification, mediated by cIAPs, has been shown to be a prerequisite for linear ubiquitinylation of Bcl10 in the context of chronic BCR signaling -Bcl10, and Ubn(K63)-MALT1 rapidly disappear as the result of the action of A20  have been described so far that result from germline mutations in the CARD11 gene, (1) CARD11 deficiency, (2) BENTA disease, and (3) Immunodeficiency with atopy.Pneumocystis jirovecii infections as infants, displayed normal T and B cell counts but hypogammaglobulinemia, deficits in mature or differentiated B and T cells (CD4 and CD8), reduced Treg numbers, and defective B and T cell activation in vitro. The homozygous mutations found in these patients include a deletion of exon 21 that results in a lack of detectable CARD11 expression , caused by heterozygous GOF CARD11 mutations, has been described in 16 patients so far beginning in 2012 –90. BENTin vitro, consistent with a poor T cell response to prior antigen exposure. Patients also display elevated serum IgE levels but low-to-normal levels of other Ig classes. CARD11 mutations that cause Immunodeficiency with atopy have been found in the CARD , Coiled-coil , and GUK (R975W)  Figure , while R) Figure .A thorough understanding of how CARD11 is kept inactive prior to signaling, and how CARD11 converts to an open, active scaffold will require determination of the three-dimensional structure of CARD11 in “closed” and “open” states. Structural studies so far have solved the structure of the CARD domain of CARD11 and have modeled how the CARD11 CARD can nucleate the formation of Bcl10 filaments , 93, 94.It remains unclear precisely how CARD11 signaling results in the activation of IKK kinase activity. The generation of ubiquitinylated Bcl10, MALT1, and IKKγ species is thought to induce a network of interacting proteins, in which ubiquitin chains are recognized by specific domains within signaling cofactors. This web of intermolecular binding can induce the proximity and clustering of IKK complexes, but how kinase activity is induced under physiological conditions, and whether other requisite components have yet to be discovered are unclear. It should also be explored whether CARD11 simply recruits enzymes  to their substrates or whether CARD11 binding plays a more active role in allosteric regulation of catalytic activity or substrate competency.Although it is clear that the four REs within the ID function cooperatively to keep CARD11 inactive prior to signaling, it remains unclear why the protein has evolved this unique array of redundant repressive elements. RE redundancy does prevent unwanted GOF mutations from occurring in the ID, but it does not prevent their occurrence in the CARD, LATCH, and Coiled-coil. It is possible that the REs determine the kinetics of CARD11 “activation” during signaling, or the kinetics by which CARD11 returns to the basal inactive state following signaling, but further studies are needed to test these hypotheses.The discovery of patients with germline CARD11 LOF and GOF mutations provides exciting opportunities for obtaining insight into the molecular mechanisms of CARD11 signaling and the cellular interplay of immune cell subtypes affected by CARD11 dysfunction. It remains unclear why some CARD11 LOF mutations are dominant negative and manifest disease when heterozygous, while other CARD11 LOF mutations manifest disease only when homozygous. In addition, it is not firmly established whether all disease-associated CARD11 alleles affect signaling to mTOR and JNK, or which dysregulated pathways downstream of CARD11 are responsible for which disease manifestations. Also unknown is whether modifier genes in the patients studied have influenced their presentation, since only a small number of patients have been identified so far. It will be interesting to see whether additional CARD11 alleles will be discovered in the human population, leading to variable phenotypes of immunodeficiency and atopy.Several studies have implicated a role for CARD11 in pathways distinct from antigen receptors, including those emanating from activating NK cell receptors –97, OX40All authors listed have made 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.


OXPHOS) via the five (CI‐CV) mitochondrial respiratory chain (MRC) complexes. The sea squirt alternative oxidase (AOX) is able to bypass the distal part of the MRC and was shown to alleviate the consequences of CIII and CIV defects in several cellular and Drosophila models. In this issue of EMBO Molecular Medicine, Rajendran et al (AOX is capable to extend lifespan and prevent heart failure in a CIII deficient mouse model, raising the possibility of future human AOX bypass treatment.Mitochondrial diseases are a diverse group of inborn disorders affecting cellular energy production by oxidative phosphorylation (an et al  demonstr Q also subsequently receives electrons from additional pathways ; electrons from reduced Q  are transferred via CIII, cytochrome c and CIV (cytochrome c oxidase) to the final electron acceptor oxygen, forming water. Simultaneously, protons are translocated across the IM by CI, CIII and CIV, creating an electrochemical gradient (proton‐motive force), which is utilized by CV (ATP synthase) to generate ATP. This oxygen‐requiring process, termed oxidative phosphorylation (OXPHOS), provides the vast majority of the cell's energy requirements. Under normal conditions, a small fraction of the electrons escape the MRC and form oxygen free radicals (ROS), which may participate in cell signalling  and translation system, present in all enucleated cells. They are involved in numerous cellular pathways , but their main function is to provide cellular energy (ATP) via the mitochondrial respiratory chain (MRC), which is composed of ~89 proteins in five multimeric complexes  embedded in the mitochondrial inner membrane (IM). CI and CII transfer electrons from NADH and FADHMitochondrial diseases affecting the OXPHOS are a heterogeneous group of prevalent genetic disorders transmitted either by maternal inheritance due to mutations in mtDNA genes  or by Mendelian inheritance due to mutations in nuclear genes encoding the remaining MRC subunits or one of the numerous assembly, transcription, translation and replication factors needed for mtDNA and OXPHOS maintenance. The clinical manifestations are extremely variable, can occur at any age and involve several organs, mainly highly energy‐dependent, such as brain, heart, muscle, liver and kidneys. OXPHOS dysfunction is also implicated in common neurodegenerative disorders including Parkinson's disease (PD) and Alzheimer's disease (AD). The cellular consequences of impaired OXPHOS are complex and include energy‐deficit oxidative stress by overproduction of ROS, accumulation of toxic metabolites and metabolic derangement . Indeed, allotropic expression of the single peptide AOX from a sea squirt, Ciona intestinalis, conferred cyanide resistance and restored electron flow to CIV‐deficient cells in culture  part of the electron transport by alternative oxidase (AOX) Fig C. This pEMBO Molecular Medicine, Rajendran and colleagues provide the first proof of concept that AOX bypass alleviates the pathological manifestations and expands lifespan in a relevant genetic mouse model of a human mitochondrial disorder (Rajendran et al, BCS1L, encoding a CIII assembly factor (Fernandez‐Vizarra & Zeviani, In this issue of + deficit by relieving the accumulation of stalled electrons, albeit at the expense of less efficient energy production. An additional advantage of AOX is that the enzyme becomes active only when needed, i.e., when a significant amount of QH2 accumulates, enabling fine‐tuning according to specific tissue demands. Whether AOX or other alternative enzyme bypass therapies are feasible in humans is presently “unchartered territory”, but will most probably be explored in the near future.Although restoration of the specific mutation by viral transfer or gene editing would theoretically be a more precise approach, AOX could provide a universal treatment strategy for all mutations affecting CIII and CIV, and possibly other diseases involving OXPHOS. The precise mechanism of AOX action has not yet been fully elucidated, but could include reducing oxidative and cellular stresses, and preventing NADThe author declares that he has no conflict of interest.


AT1 angiotensin receptor plays important physiological and pathophysiological roles in the cardiovascular system. Renin-angiotensin system represents a target system for drugs acting at different levels. The main effects of ATR1 stimulation involve activation of Gq proteins and subsequent IP3, DAG, and calcium signaling. It has become evident in recent years that besides the well-known G protein pathways, AT1R also activates a parallel signaling pathway through β-arrestins. β-arrestins were originally described as proteins that desensitize G protein-coupled receptors, but they can also mediate receptor internalization and G protein-independent signaling. AT1R is one of the most studied receptors, which was used to unravel the newly recognized β-arrestin-mediated pathways. β-arrestin-mediated signaling has become one of the most studied topics in recent years in molecular pharmacology and the modulation of these pathways of the AT1R might offer new therapeutic opportunities in the near future. In this paper, we review the recent advances in the field of β-arrestin signaling of the AT1R, emphasizing its role in cardiovascular regulation and heart failure. AngII is the main effector of the renin-angiotensin system (RAS), which has pleiotropic effects in the cardiovascular system and salt-water balance regulation. As such, the RAS is an important target in the treatment of various cardiovascular diseases. AT1R blockers (ARBs) and angiotensin-converting enzyme inhibitors are widely used for the treatment of hypertension. Moreover, ARBs have beneficial effects beyond lowering the blood pressure, by preventing cardiovascular organ injuries . Howeverchannels , 4.Following their activation, GPCRs usually undergo G protein kinase (GRK) dependent phosphorylation on serine and threonine residues on their C-terminal tails. This phosphorylation leads to binding of β-arrestin proteins, which shut down G protein activation and target the receptors toward internalization. During the last two decades it became increasingly evident that, in addition to G proteins, β-arrestins can also mediate signaling events. β-arrestin proteins serve as scaffold proteins, bringing together the players of different protein kinase cascades, and instead of turning it off, they switch the signaling toward different routes . Like maThe binding of agonists to G protein-coupled receptors initiate the “classical,” G protein-mediated pathway, which results in the production of second messenger molecules. Termination of signaling has a key role in the regulation of the kinetics of receptor function. Mechanisms regulating receptor function include several consecutive or parallel processes, such as desensitization, internalization into intracellular vesicles, and degradation of the receptors. Receptor internalization is regulated by phosphorylation by GPCR kinases, which promotes the β-arrestin binding leading to desensitization, internalization and altered signaling of GPCRs . The rolLigand binding to a plasma membrane receptor can initiate several parallel signal transduction pathways leading to various responses in the cell. Ligands of the plasma membrane receptors were originally classified as agonists and antagonists (or more recently inverse agonists). However, several ligands are capable of selectively initiating one or more of distinct signal transduction pathways coupled to one receptor, which phenomenon has been referred to as “biased agonism” or “functional selectivity.” It has been revealed that the biased agonism is an important feature of several members of the GPCR superfamily, and it is proposed that the biased agonists can serve as new therapeutic agents . Some ofEarly studies had identified mutant receptors of the highly conserved Asp125Arg126Tyr127 sequence of AT1R , which are not able to couple to G proteins, but still capable to internalize and bind β-arrestins . Using tβ-arrestin-mediated mechanisms can be considered as the second wave of signal transduction events resulting in alternative outcomes. Moreover, the agonist binding of AT1R can lead to direct association with wide spectra of cytoplasmic signaling proteins, such as AT1R-associated protein (ATRAP), SHP-2, JAK2, β-arrestins, and phospholipase Cγ, which explains a plethora of AT1R generated signaling mechanisms, including JAK/STAT pathway and MAPK cascade activation –54. The One such selectively activated pathway of particular interest is the β-arrestin-dependent signaling. β-arrestin-mediated signaling events include activation of Src tyrosine kinases extracellular signal-regulated kinase 1/2 (ERK 1/2), c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK) cascades, Akt and p38 mitogen-activated protein kinases –59.Besides the observation of Erk1/2 activation through β-arrestins, many other cellular functions have been discovered to be regulated through GPCRs in a non-canonical G protein-independent way. These pathways include JNK3, p38, and Akt protein kinase regulation , some ofSince the AngII is a vasopressor hormone, its production and effects are very important in the development of numerous cardiovascular diseases such as hypertension, atherosclerosis, and cardiac hypertrophy. Indeed, AT1R blockers and inhibitors of AngII production (ACE inhibitors) are now extensively used in the treatment of hypertension and other cardiovascular diseases , 70. SpeBoth AT1R-activation of G protein-dependent and G protein-independent mechanisms contribute to the development of cardiac hypertrophy. It was demonstrated that heart-specific overexpression of different AT1R mutants can lead to hypertrophy, moreover, the mutant AT1R, which mediates only G protein-independent signaling mechanisms, caused greater cardiac hypertrophy but less apoptosis and fibrosis than overexpression of wild-type AT1R . ContrarIn the development of pathological cardiac hypertrophy, mechanical stress is thought to be one of the most important factors. Parallel with the discoveries of the β-arrestin-dependent signaling through AT1R, AT1R receptor was also found to be involved in stretch-induced signaling pathways in various cell types. First, it has been discovered, that in cardiomyocytes, stretch-induced Erk1/2 activation was dependent on AT1Rs –80. SincInterestingly, mechanical activation of AT1R caused increased affinity toward β-arrestin biased ligand TRV 120023 , suggestThe involvement of AT1R- β-arrestin interplay in cardiac function has become more evident with the discovery of the beneficial effects of biased AT1R ligands on the heart contractility. RAS is typically activated in patients with HF, with increased circulating AngII levels. Anti-RAS drugs, like AT1R blockers and ACE inhibitors, have been long proven beneficial in the treatment of HF. It seems that the pathological actions of AT1R on vasoconstriction and heart remodeling are mediated through Gq-protein activation. On the contrary, studies have suggested that TRV120023 and TRV120027, a β-arrestin biased ligand of AT1R, decrease blood pressure similarly to AT1R blockers, but unlike those drugs, it improves cardiac performance, preserves cardiac stroke volume, decreases systemic vascular resistance, improves cardiac output while preserving renal functions in animal models , 98.2+ sensitization, in immature mouse cardiomyocytes, TRV120027 activates CaV 1.2 Ca2+ channels as well through Src-family tyrosine kinases and casein kinase 2 in a β-arrestin-dependent manner . Although the AT2R does not bind β-arrestins upon Ang II stimulation , since AT1R mutants used in the studies and biased AT1R ligands failed to activate the usual Gq-protein signaling with IP3 and DAG production and consequent Caen AngII . In addien AngII . Howeveren AngII , raisingen AngII . They foen AngII . These den AngII . The avaen AngII , 127. Soen AngII . When noWe have come a long way in the recent almost 20 years since the interaction of AT1R and β-arrestin has been discovered. The interaction of these two proteins itself had a huge effect in understanding how the GPCRs function, and how are the signaling pathways regulated beside the classical G protein activation. We are at the doorstep of therapeutic exploitation of this system, and although the first attempt to cross it was not successful, recent results suggest that this was probably not our last chance. Recent advances in the field also show that there might be still a lot learn about β-arrestin-dependent signaling, and AT1R might further serve as a good model for such experiments.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.


Neuropeptide W (NPW) and neuropeptide B (NPB) are two structurally and functionally related regulatory peptides, which are highly expressed in several brain regions and, additionally, in some peripheral tissues. Nevertheless, their distributions in the tissues are not similar. They act on target tissues via two subtypes of G protein-coupled receptors which are designated as NPBWR1 (GPR7) and NPBWR2 (GPR8), respectively, and possess different binding affinities. NPB activates NPBWR1, whereas NPW stimulates both the receptors with similar potency. Both of these peptides takes a part in the central regulation of neuroendocrine axes, feeding behavior, energy homeostasis, cardiovascular functions, circadian rhythm, pain sensation, modulation of inflammatory pain, and emotions. Over the past few years, studies have shown that NPB is also involved in sleep regulation. On the contrary, NPW participates in regulation of vascular myogenic tone, inhibits gastric tension sensitive vagal afferents and insulin secretion. Also, expression of NPW in the stomach is regulated by feeding. Abovementioned findings clearly demonstrate the functional diversity among NPW versus NPB signaling systems. In this review, signal transduction pathways of NPW/NPB are critically evaluated and observed together with mapping of expression of their signaling systems. Neuropeptide B (NPB) and neuropeptide W (NPW) are peptides with structural and functional similarities. Both are endogenous ligands for G protein-coupled neuropeptide B/W receptors 1 (NPBWR1) and NPBWR2, respectively, and constitute for NPB/NPW signaling systems, which are responsible for regulation of several physiological processes.Neuropeptide B was independently identified and purified by three different groups from bovine hypothalamus . At the Figure 1; Neuropeptide W is present in two isoforms with lengths of 23 (NPW23) and 30 (NPW30) amino acids. Both of them are produced from a common precursor peptide, prepro-NPW by proteolytic cleavage of two pairs of arginine residues at position 24–25 and 31—2 . The namFigure 1; Neuropeptide B is produced by proteolytic processing from a precursor, prepro-protein called prepro-NPB. In humans, the final peptide sequence of NPB is either shorter (NPB23) or extended (NPB29) which consist of 23 amino acid residues or 29 amino acid residues, respectively . In non-Figure 2. Shorter forms of NPW and NPB display similarity at C-terminal secondary structures, while the N-terminal region is more crucial for receptor binding and interaction  including the hippocampus, hypothalamus, and amygdala  and periaqueductal gray, and in a lesser amount also in the ventral tegmental area, and EW but fail to detect it in the brain cortex  with highest expression in the cerebellum (Tables 4), while the highest expression was found in the hypothalamus  and GPR8 . At thatThe two receptors are functionally coupled to Gi proteins, and their stimulation leads to various intracellular responses in different cell types but also within the cells of the same type. Activation of the receptors may affect the intracellular cAMP level, when the decrease was observed in Chinese hamster ovary cells , whereasIn human, NPBWR1 and NPBWR2 share a 70% nucleotide and a 64% amino acid homology with each other. Additionally, they show about 40% amino acid homology with somatostatin and opioid receptors; however, they do not bind somatostatin. NPBWR1 has a low affinity for non-selective opioid ligands . In mammThese findings suggest that mainly NPB signaling system is not well preserved during vertebrate evolution, which may be reflected also in different function of this system in various species. The involvement of various intracellular signaling pathways in response to activation of NPBWR1 or NPBWR2 in different cell types suggests tissue-specific functions of the signaling system.In the pig, the expression patterns of NPBWR1 and NPWBR2 are largely similar to each other and also to expression of their ligands . Lower cIn the mouse nervous system, strong NPBWR1 mRNA expression was found in the amygdala, suprachiasmatic nucleus, paraventricular nucleus, dorsomedial and ventromedial hypothalamic nucleus, periventricular nucleus, insular cortex, zona incerta, anterior part of the dorsal thalamic nucleus, superior colliculus, nucleus of the solitary tract, and dorsal horn of the spinal cord . NPW-binThe highest expression of NPB mRNA and the highest amount of NPB-binding sites was found in the amygdala, ventral tuberomamillary nucleus, suprachiasmatic nucleus, and hippocampus, the sites where also dense NPB and/or NPW-IR nerve fibers network is present suggesting functional transmission of NPB/NPW signals to target cells.In human peripheral tissues, NPBWR1 and NPBWR2 mRNAs were identified in the testis, lung, large intestine, and skin. NPBWR1 mRNA was additionally detected also in the prostate and trachea, while NPBWR2 mRNA in the adrenal gland, spleen, and lymph node . In the In fact, a high number of specific receptors in different peripheral tissues indicate the importance of the NPB/W signaling system in these parts of the body. Especially those with a high degree of expression of these receptors should undergo detailed functional studies targeted for the mapping of their functions here.A wide distribution of NPB/NPW signaling systems within the CNS suggests several important regulatory functions. Several pharmacological studies indicate their involvement in the regulation of feeding activity, central regulation of neuroendocrine functions, pain sensation, energy homeostasis, autonomic regulation, activation of stress axis, and involvement in the emotions, anxiety, and fear . It is iRegulation of feeding and energy metabolism appears to be primary function of the NPB/NPW signaling system, which is complex and include regulation of energy intake as well as endocrine functions involved in metabolism. NPB/NPW involvement occurs at both central and peripheral levels. Within the CNS, NPB- and NPW-containing neurons were observed in hypothalamic areas essential for feeding behavior and energy metabolism. Chronic intracerebroventricular application of NPW led to reduced food intake and weight loss in rats . SeveralBy contrast, Hypothalamic paraventricular nucleus neurons are known to project directly to the sympathetic preganglionic neurons in the spinal cord and control sympathetic outflow . IntraceNeuropeptide W expressing neurons localized in the midbrain send projections to the central nucleus of amygdala and bed nucleus of the stria terminalis, which are important for emotive reactions, and express NPBWR1 mRNA . This suThe NPB/NPW signaling system is also present in several brain regions linked to pain processing, which is suggestive of a role in modulation of pain transmission. NPB gene knockout mice respond differently to specific types of pain, where they exhibit hyperalgesia in response to acute inflammatory pain, but not thermal or chemical pain . IntrathInvolvement of NPB/NPW signaling system in the regulation of circadian rhythm is indicated by presence of NPW-binding sites in suprachiasmatic nucleus  and NPBWIt is difficult to review the central and peripheral effects of these peptides, as their peripheral actions often involved activity in the CNS. In the peripheral tissues, NPW/NPB signaling system regulates vascular smooth muscle tone, endocrine glands activity, metabolism of adipocytes, and stomach activity .An important source of plasma NPW is likely from gastric G cells, which produce and release the peptide according to the level of satiety. Lower production occurs during fasting in rats, which is increased after refeeding suggesting important role of NPW in feeding regulation in peripheral level in addition to central regulation . Li et aTable 5.As it has been mentioned above, the NPB/NPW signaling system is expressed in the hypothalamus and several endocrine glands, including pancreatic islets, thyroid and parathyroid gland, adrenal gland, ovary, and testis . This suIn vitro experiments show that both peptides are able to stimulate basal glucocorticoid, corticosterone, or aldosterone secretion from dispersed or cultured adrenocortical cells depending on species . NPY is expressed in similar parts of the CNS as NPB and NPW . AdditioCurrently, there is limited information concerning to function of NPB and NPW during development. The NPB/W signaling system was described over/during past two decades, and since then many studies have been devoted to it. Their location was also studied thoroughly and confirmed in the CNS; however, the information related to distribution in peripheral organs and tissues is still sporadic. Although the expression of mRNAs of individual members of this signaling system has been demonstrated in most of the organs, their exact localization at the cellular level is still unknown in many cases. Moreover, its localization and function in endocrine glands have been studied in more detail, with results showing the involvement of this system in a number of regulatory mechanisms. Nevertheless, so far, there has not been any satisfactorily explanation regarding the presence of this signaling system in other organs and tissues.Concerning to cardiovascular system, strong evidence exists suggesting involvement of NPB/NPW signaling system in regulation of blood pressure on the central as well as peripheral level. Information about tissue localization and function of this system within the heart needs to be elucidated. Expression of the signaling system members’ mRNA has already been demonstrated in the heart of several species .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.