Toll-like receptors (TLRs) are a family of pattern recognition receptors that shape the innate immune system by identifying pathogen-associated molecular patterns and host-derived damage-associated molecular patterns. TLRs are widely expressed on both immune cells and non-immune cells, including hematopoietic stem and progenitor cells, effector immune cell populations, and endothelial cells. In addition to their well-known role in the innate immune response to acute infection or injury, accumulating evidence supports a role for TLRs in the development of hematopoietic and other malignancies. Several hematopoietic disorders, including lymphoproliferative disorders and myelodysplastic syndromes, which possess a high risk of transformation to leukemia, have been linked to aberrant TLR signaling. Furthermore, activation of TLRs leads to the induction of a number of proinflammatory cytokines and chemokines, which can promote tumorigenesis by driving cell proliferation and migration and providing a favorable microenvironment for tumor cells. Beyond hematopoietic malignancies, the upregulation of a number of TLRs has been linked to promoting tumor cell survival, proliferation, and metastasis in a variety of cancers, including those of the colon, breast, and lung. This review focuses on the contribution of TLRs to hematopoietic malignancies, highlighting the known direct and indirect effects of TLR signaling on tumor cells and their microenvironment. In addition, the utility of TLR agonists and antagonists as potential therapeutics in the treatment of hematopoietic malignancies is discussed. Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that play a central role in innate immunity. TLRs are expressed on a wide variety of effector immune and stromal cell types, as well as hematopoietic stem and progenitor cells (HSPCs) –3, and sThis review focuses on the contribution of TLR signaling to impaired hematopoiesis and hematopoietic malignancy. We will explore the role of cell-autonomous aberrant TLR signaling to loss of normal HSPC function and leukemogenesis, as well as the non-cell-autonomous effects of TLR signaling that may contribute to disease pathogenesis. Finally, we will discuss the utility of TLRs and their downstream effectors as therapeutic targets in hematopoietic neoplasms.A total of 12 TLR family members have been described in mice, and 10 in humans . In addiSignaling through TLRs requires the recruitment of intracellular adaptor proteins, with all TLRs, except TLR3, using the myeloid differentiation primary response gene 88 (MyD88). Following recruitment of MyD88 to activated TLRs, this adaptor complexes with the serine–threonine kinase interleukin-1 receptor-associated kinase 4 (IRAK4) , which iToll-like receptor3 functions independently of MyD88, utilizing instead the adaptor protein TIR-domain-containing adaptor-inducing interferon-B (TRIF) . TRIF, uin vivo treatment of mice with the TLR4 agonist LPS, for example, leads to HSC cycling and a promotion of myeloid differentiation with a loss of HSC repopulating activity in transplantation experiments  is associated with enhanced HSC repopulating activity . Both mouse and human HSCs express TLRs –3, and a-renewal . Conversactivity , 42. Thiin vitro studies showing that incubation of HSPCs with TLR agonists induces cell cycling and myeloid differentiation  by endothelial cells ) subtype of MDS, for example, loss of two micro-RNAs, miR-145 and miR-146a, which normally inhibit the TLR signaling intermediates TIR-domain-containing adaptor protein (TIRAP that associates with MyD88 and assists in the formation of the Myddosome) and TRAF6, leads to inappropriate activation of TLR signaling  describein vitro, further supporting a role for this receptor in the pathogenesis of MDS. They proposed a model whereby augmented TLR2 signaling leads to activation of the histone demethylase JMJD3, thus stimulating the abnormal epigenetic activation of multiple innate immunity genes, including IL-8, and promoting ineffective hematopoiesis. Based on these data, a phase I/II study of an anti-TLR2 antibody (OPN-305) is underway for second-line treatment of lower-risk MDS patients .In addition to the data supporting a role for enhanced TLR signaling in del (5q) MDS, numerous studies have shown increased expression of TLRs and TLR pathway intermediates in patients with all types of MDS. For example, Maratheftis and colleagues showed increased TLR4 expression in the CD34+ cells of patients with MDS in a small series of 21 cases, and this expression was associated with increased apoptosis . Velegra3CSK4, versus the TLR2/6 agonist, PAM2CSK4, led to distinct activation kinetics of the NF-κB and PI3K pathways. Furthermore, only PAM3CSK4 sensitized ALL cells to vincristine-mediated toxicity. Further studies are needed to determine whether there are heterodimer-specific effects of TLR2 stimulation on MDS cells, as this could have implications for the development of targeted therapies.Interestingly, while expression of TLR2 itself in the above study correlated with low-risk disease by the International Prognostic Scoring System and longer survival, higher expression of its binding partner, TLR6, was associated conversely with higher-risk disease and increased bone marrow blasts . This fiIn addition to the TLRs themselves, the expression of numerous downstream signaling mediators is increased in patients with MDS, as well. For example, increased expression of the TLR signaling adaptor, MyD88, was found in 40% of individuals in a cohort of 64 patients with MDS, and high expression correlated with shorter survival . SimilarRps14, which has been linked to the severe macrocytic anemia observed in del (5q) MDS patients, to activation of the calcium binding proteins, S100A8 and S100A9. The authors found that S100A8, acting upstream of TLR4 and TNFα, contributed to a p53-dependent erythroid-differentiation defect. Further studies are needed to comprehensively define the different bone marrow populations in which aberrant TLR signaling occurs in MDS and to determine the relative contribution of cell-autonomous versus cell non-autonomous signaling to the disease pathogenesis.Thus, multiple studies have linked increased TLR signaling to MDS; however, the specific contribution of this signaling to disease progression is not clear. The existing data suggest that aberrant TLR signaling promotes ineffective hematopoiesis and likely contributes to the cytopenias characteristic of the disease. However, it is not at all clear whether this aberrant signaling promotes the transformation of MDS to acute leukemia. Further studies will be necessary to determine if TLR expression and signaling in premalignant MDS HSCs changes upon transformation, and whether TLR stimulation contributes to the acquisition of clonal dominance in this disease. In addition, it is not clear to what extent enhanced TLR signaling outside of the CD34+ population contributes to MDS pathogenesis. Accumulating evidence supports a role for the bone marrow microenvironment in the evolution of MDS, with numerous alterations in the bone marrow stromal and hematopoietic populations and deregulation of inflammatory cytokine secretion contributing to ineffective hematopoiesis. For example, myeloid-derived suppressor cells (MDSCs), an immature myeloid population with potent T-cell suppressive capabilities, are expanded in the bone marrow of MDS patients , 58. MDSin vitro and ex vivo with the TLR2/1 agonist, PAM3CSK4, and found that agonist treatment led to expansion, increased leukemic cell survival, and a shift toward differentiation. Similarly, in a study of mRNA expression of TLRs in myeloid leukemia cell lines, Okamoto and colleagues found that 10 TLRs were expressed in the majority of leukemic cell lines assessed , Rybka and colleagues found that the mRNA expression of TLR2 and TLR4 was significantly higher in the bone marrow of patients with myelomonocytic and monoblastic acute leukemia compared to those with other types of AML. Furthermore, the expression of these receptors was also higher in patients with no response to induction therapy compared to those who achieved complete remission, and the overall survival of patients with higher TLR2 and TLR4 expression was significantly shorter than that of patients with lower expression . One of assessed . The autin vitro in the absence of exogenous TLR ligands. However, this proliferation was dependent upon intact signaling by the RNA/DNA-sensing TLRs, as it was inhibited by the 3dUnc93b1 mutation  , which ultimately promotes enhanced IRAK1 phosphorylation and activation of NF-κB and JAK–STAT3 signaling with production of proinflammatory cytokines and promotion of cell survival . Finally, and 9) , 69, TLRin vitro .via autocrine secretion of interleukin-6 (IL-6)  , protected CLL cells from spontaneous apoptosis. Finally, single nucleotide polymorphisms (SNPs) in several TLRs have been associated with lymphoma risk (In addition to the activating MyD88 (L265P) mutation, the expression of TLR signaling pathway components is increased in lymphoid malignancies. For example, TLRs have been found to be more highly expressed in the plasma cells obtained from multiple myeloma patients as compared to healthy control donors . Additio6 (IL-6) –73. In ar (VEGF) . A studyr (VEGF) . Furtheroma risk –79.Apcmin/+ mice . Furthermore, TLR2 is expressed on breast epithelia, and TLR2 agonist treatment of luminal and basal breast epithelial cells promotes in vitro colony growth. Conversely, TLR2 loss inhibited the growth of breast cancer cells both in vitro and in vivo. Another recent study showed that STAT3-mediated upregulation of TLR2 expression on gastric epithelial cells promoted cell proliferation and survival, and inhibition of TLR2 suppressed gastric tumor formation  suppresses NF-κB signaling and induces WM cell death. Ibrutinib is a BTK inhibitor that was recently approved for the treatment of WM following a prospective, multicenter trial that showed its efficacy with high response rates and improved progression-free survival in previously treated patients  is currently underway for the second-line treatment of lower-risk MDS patients based on preclinical studies showing improved erythroid colony formation upon TLR2 inhibition in MDS CD34+ cells patients , 86.in vitro terminal differentiation and inhibits proliferation of AML cell lines and primary patient samples in a TLR8–MyD88–p38-dependent fashion. Furthermore, R848 significantly inhibited AML tumor growth in an AML NSG mouse xenograft model. In human myeloma cell lines, stimulation with polyinosinic–polycytidylic acid (poly I:C), an agonist of TLR3, led to increased apoptosis and induced activation of the NFκB pathway and type I IFN secretion, thereby inhibiting cell growth. Poly I:C also led to TLR3 upregulation, type I IFN induction, and increased apoptosis in primary AML cells . Furthermore, while multiple different TLRs have been shown to have elevated expression in various tumors, it is not clear to what extent these different receptors may confer unique signals leading to the promotion of tumor development . Furthermore, the downstream mediators of TLR signaling that regulate HSC cycling, differentiation, and function, and that ultimately therefore may promote leukemogenesis, are not well known. And finally, the contribution of cell-autonomous versus cell non-autonomous TLR signaling to hematopoietic malignancies is not well understood. Although the expression and signaling of TLRs is known to be increased on the CD34+ cells of MDS patients, for example, as well as the leukemia cells in various myeloid and lymphoid cancers, it is not known whether other cell types within the bone marrow microenvironment  also display aberrant TLR signaling, and what role this non-autonomous signaling may play in the pathogenesis of the disease.via promotion of an antitumor immune response. A more defined understanding of the mechanism through which TLRs influence hematopoietic neoplasms is therefore necessary to better delineate the utility of TLR agonists versus antagonists as novel therapeutic options and balance the potential benefits of reducing hyperactivated TLR signaling on hematopoiesis with the risks of suppressing the immune response against the tumor cells as well as infections , there are multiple studies conversely showing an antitumor effect of TLR stimulation, with TLR agonists either directly affecting cell growth and chemosensitivity or indirectly supporting tumor cell death fections .Based on the studies discussed within, it has become increasingly apparent that the effects of TLR signaling on tumor growth and progression requires careful consideration of numerous factors, including the TLR in question, unique downstream effectors, the influence of the tumor microenvironment, and the tumor type. Future studies aimed at gaining a comprehensive mechanistic understanding of the role of TLR signaling in lymphoproliferative disorders, MDS, and other hematopoietic neoplasms are required to more fully appreciate the influence of this signaling on disease pathogenesis and determine the appropriate utility of TLR agonists and antagonists as therapeutic agents.DM, SB, and LS wrote and edited the manuscript.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.


UA inhibited endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production in HUVECs, increased intracellular HMGB1 expression and extracellular HMGB1 secretion, and upregulated RAGE expression. UA also activated NF-κB and increased the level of inflammatory cytokines. Blocking RAGE significantly suppressed the upregulation of RAGE and HMGB1 and prevented the increase in DNA binding activity of NF-κB and the levels of inflammatory cytokines. It also blocked the decrease in eNOS expression and NO production induced by UA. Our results suggest that high concentrations of UA cause endothelial dysfunction via the HMGB1/RAGE signaling pathway. Uric acid (UA) is a risk factor for endothelial dysfunction, a process in which inflammation may play an important role. UA increases high mobility group box chromosomal protein 1 (HMGB1) expression and extracellular release in endothelial cells. HMGB1 is an inflammatory cytokine that interacts with the receptor for advanced glycation end products (RAGE), inducing an oxidative stress and inflammatory response, which leads to endothelial dysfunction. In this study, human umbilical vein endothelial cells (HUVECs) were incubated with a high concentration of UA (20 mg/dL) after which endothelial function and the expression of HMGB1, RAGE, nuclear factor kappa B (NF- Uric acid (UA), a final breakdown product of purine metabolism, is degraded by urate oxidase to allantoin and freely excreted in the urine in most mammals . HoweverThe receptor for advanced glycation end products (RAGE), a transmembrane multiligand receptor of the immunoglobulin superfamily, has been implicated in many chronic diseases , 20, incα. HMGB1 participates in the pathogenesis of systemic inflammation after the early mediator response has resolved [ HMGB1 from human umbilical vein endothelial cells (HUVECs) [κB) which leads to proinflammation. In this study, we investigated whether the HMGB1/RAGE signaling pathway contributes to endothelial dysfunction induced by UA.As a high affinity ligand of RAGE, HMGB1 is a recently discovered important extracellular mediator in systemic inflammation . HMGB1 iresolved . HMGB1 cresolved . High co(HUVECs) . Extrace2 culture bottles and propagated in Dulbecco's modified eagle medium with 1.0 g/L glucose supplemented with 10% heat-inactivated fetal bovine serum. HUVECs were incubated at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide. The medium was refreshed every 2 to 3 days.HUVECs were obtained from China Center for Type Culture Collection. Uric acid crystallites  were dissolved in 1 N NaOH solution. HUVECs were cultured on gelatin-coated 25 cmμg/mL) for 2 h before UA (20 mg/dL) stimulation. For controls, HUVECs were cultured with control medium, a normal goat IgG control (5 μg/mL), or medium with a control antibody (5 μg/mL) and UA (20 mg/dL).HUVECs were cultured with 20 mg/dL UA solution for different times . Inhibition of the interaction between HMGB1 and RAGE was evaluated by using a specific RAGE blocking antibody . HUVECs were cultured with anti-RAGE antibody  according to the manufacturer's protocol. Fluorogenic quantitative PCR (FQ-PCR) was performed using the StepOne™ Real-Time PCR System . Primers were designed and synthesized by Shanghai Sangon Biotech . There were five pairs of primers. The primer sequences for HMGB1 were 5′-GGGATGGCAAAGTTTTTCCCTTTA-3′ and 5′-CACTAACCCTGCTGTTCGCT-3′. For RAGE, the primer sequences were 5′-GCTTGGAAGGTCCTGTCTCC-3′ and 5′-CACGGACTCGGTAGTTGGAC-3′. For intercellular adhesion molecule- (ICAM-) 1, the primer sequences were 5′-CACAGTCACCTATGGCAACG-3′ and 5′-GTGTCTCCTGGCTCTGGTTC-3′. For vascular adhesion molecule- (VCAM-) 1, the primer sequences were 5′-AGTTGAAGGATGCGGGAGTA-3′ and 5′-GTGTCTCCTGGCTCTGGTTC-3′. The mRNA expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference.Total RNA was extracted from HUVECs using the TRIzol reagent  according to the manufacturer's instructions. The RNA integrity was checked by nucleic acid agarose gel electrophoresis, and the RNA concentration was determined by ultraviolet spectrophotometer. Briefly, 1 κB was examined in nuclear and plasma protein samples by western blotting. Nuclear protein and cytoplasm protein fractions were obtained using a nuclear plasma protein extraction kit  following the manufacturer's instructions. The protein samples were dissolved in Laemmli buffer, boiled for 10 min at 100°C, and equal amount of proteins was separated on 10% SDS-PAGE gels . The membrane was blocked in 5% wt/vol skim milk for 1 hour after the membrane was transferred to a nitrocellulose membrane (Cell Scientific). The proteins were incubated with primary antibodies and followed with secondary antibodies. Polyclonal rabbit antibodies against HMGB1 , RAGE (Abcam Inc.), NF-κB p65 , eNOS (Abcam Inc.), ICAM-1 , VCAM-1 , proliferating cell nuclear antigen , and a monoclonal mouse antibody to β-actin (Affbiotech.) were used as primary antibodies according to the manufacturers' protocol (including dilutions). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies  were used. The proteins were detected using enhanced chemiluminescence reagents . The images were captured through Gel Doc XR system  and analyzed using Image Lab software (vision 4.0). Anti-β-actin antibody was used as the loading control for total proteins and cytoplasm protein, and the relative protein levels of nuclear protein were calculated as densitometric ratios to PCNA.RAGE, endothelial nitric oxide synthase (eNOS), ICAM-1, and VCAM-1 were examined in cell samples by western blotting. The extracellular HMGB1 was concentrated using cellulose 3,000 molecular weight cut-off (MWCO) concentrating centrifugal filter units  prior to western blotting. NF-Nitric oxide (NO) levels in cell culture supernatants were measured with Griess reagent by the nitrate reductase method using a nitric oxide assay kit  according to the manufacturer's protocol.α and IL-6 were measured in duplicate using ELISA kits according to the manufacturer's instructions .Immunoreactive TNF-t-test or one-way ANOVA followed by post hoc Dunnett's test, as appropriate. All statistical analyses were two-sided, and a P value of less than 0.05 was considered statistically significant. Statistical analyses were carried out using SPSS version 17 .Data are expressed as means ± standard deviation (SD). Differences among groups were analyzed by two-tailed Student's P < 0.05) (P < 0.05)  , as was  < 0.05) . These rTo examine whether a high concentration of UA can upregulate the expression of RAGE and HMGB1, we detected the mRNA and protein expression of RAGE and HMGB1 by FQ-PCR and western blotting assay in HUVECs treated with 20 mg/dL UA. When the HUVECs were stimulated with 20 mg/dL UA, the mRNA expression of RAGE and HMGB1 significantly increased in a time-dependent manner . At the κB, the expression of NF-κB p65 in the cytoplasm and nucleus of HUVECs was detected by western blotting assays. After treatment with 20 mg/dL UA, we observed a decrease in cytoplasmic NF-κB  to neutralize RAGE. Treatment of HUVECs with anti-RAGE antibody for 24 h significantly blocked the decrease in eNOS expression and NO production induced by 20 mg/dL UA Figures . These rκB and inflammation signals in endothelial cells induced by UA were mediated by RAGE, we evaluated the effects of anti-RAGE antibody on the expression of RAGE, HMGB1, NF-κB, ICAM-1, and VCAM-1 and the release IL-6 and TNF-α from HUVECs. Treatment of HUVECs with anti-RAGE antibody for 24 h significantly suppressed the upregulation of RAGE, HMGB1, ICAM-1, and VCAM-1  and adhesion molecules (ICAM-1 and VCAM-1). These results demonstrate that high concentrations of UA can induce inflammatory responses and result in endothelial dysfunction. However, the exact mechanisms have not been described yet.Hyperuricemia not only is the etiological factor of gout , 31, but−/− mice showed increased plaque area as well as increased expression of RAGE and its ligands, but diabetic RAGE−/−/apoE−/− mice showed significantly reduced plaque accumulation and expression of RAGE and its ligands [κB in HUVECs and further promotes the production and release of proinflammatory mediates and contributes to the amplification of the inflammatory response and finally induces endothelial dysfunction [κB signaling in endothelial cells. We also found that blocking (by anti-RAGE antibody) RAGE significantly suppressed the upregulation of RAGE and HMGB1 and inhibited the increase in DNA binding activity of NF-κB and the levels of inflammatory cytokines (IL-6 and TNF-α) and adhesion molecules (ICAM-1 and VCAM-1) induced by a high concentration of UA. At the same time, blockage of RAGE significantly prevented the decrease in the eNOS expression and the NO production induced by a high concentration of UA. These results imply that UA induces endothelial dysfunction by stimulating the HMGB1/RAGE signaling pathway (There is a large body of evidence to suggest that the RAGE-ligands axis has an important role in the pathogenesis of atherosclerosis. RAGE is a multiligand receptor of the immunoglobulin superfamily, which has been implicated in many chronic inflammation diseases, including atherosclerosis . Previou ligands . The stu ligands , 42. As  ligands . Extrace ligands . RAGE acfunction . Treatmefunction , which ifunction . Interacfunction , 47. In  pathway .In conclusion, the present study indicated that high concentrations of UA induce endothelial dysfunction by the HMGB1/RAGE signaling pathway. These results provide new insight into the mechanisms of UA-induced endothelial dysfunction and atherosclerosis.


DrNOD2) and RIG-I (DrRIG-I) in innate immune responses. Luciferase assays were conducted to determine the activation of NF-κB and interferon signaling. Morpholino-mediated knockdown and mRNA-mediated rescue were performed to further confirm the regulatory roles between DrNOD2 and DrRIG-I. Results showed that DrNOD2 and DrRIG-I shared conserved structural hallmarks with their mammalian counterparts, and activated DrRIG-I signaling can induce DrNOD2 production. Surprisingly, DrNOD2-initiated signaling can also induce DrRIG-I expression, indicating that a mutual regulatory mechanism may exist between them. Studies conducted using HEK293T cells and zebrafish embryos showed that DrRIG-I could negatively regulate DrNOD2-activated NF-κB signaling, and DrNOD2 could inhibit DrRIG-I-induced IFN signaling. Moreover, knocking down DrRIG-I expression by morpholino could enhance DrNOD2-initiated NF-κB activation, and vice versa, which could be rescued by their corresponding mRNAs. Results revealed a mutual feedback regulatory mechanism underlying NOD2 and RIG-I signaling pathways in teleosts. This mechanism reflects the coordination between cytosolic antibacterial and antiviral PRRs in the complex network of innate immunity.Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and retinoic acid-inducible gene I (RIG-I) are two important cytosolic pattern recognition receptors (PRRs) in the recognition of pathogen-associated molecular patterns (PAMPs), initiating innate antibacterial and antiviral signaling pathways. However, the relationship between these PRRs, especially in teleost fish models, is rarely reported. In this article, we describe the mutual regulation of zebrafish NOD2 ( Host defense against pathogen infection is initiated by the recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs). Well-recognized PRRs include toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors (CLRs), and the family of DNA sensors ,3,4,5,6.NOD2 is a member of the NLR family and a general cytoplasmic sensor for most bacteria ,10,11. SRIG-I is an essential cytoplasmic receptor for recognizing various RNA viruses . This PRAlthough NOD2 and RIG-I are important in innate antibacterial/viral processes in the hosts, studies on the correlation between the NOD2 and RIG-I signaling pathways are limited. Previous studies showed that NOD2 could be significantly induced by IFN-β produced through the activation of RIG-I signaling pathway upon viral stimulation ,20. HoweDrNOD2 and DrRIG-I were highly conserved and similar to their mammalian counterparts [DrRIG-I and DrNOD2 could induce each other, and a close negative mutual regulation between these molecules was observed. Our results provide insights into the coordination of cytosolic antibacterial and antiviral PRRs. This information can improve the understanding on the functional modulation among innate signaling pathways in the complex innate immunity network from an evolutionary perspective.Teleosts were used in several studies to investigate the structures, signaling pathways, and antipathogen functions of NOD2 and RIG-I ,33,34,35terparts ,37,38. TDrRIG-I and DrNOD2 with their mammalian counterparts to investigate the mutual regulation of NOD2 and RIG-I signaling pathways using zebrafish. We used SMART and Pfam databases to refer the protein domains and found that DrRIG-I and DrNOD2 possessed all the functional domains shared by their Homo sapiens counterparts  was used, instead of wild-type DrRIG-I, because the latter remains in an auto-inhibited state without ligand stimulation [DrNOD2, a mutant without the LRR domain (DrNOD2 (ΔLRR)) was also used to activate the NOD2 signaling. Our results showed that DrNOD2 was induced at 6 and 12 h post injection (hpi) of DrRIG-I (CARD) and reached its highest level at 6 h. Surprisingly, we also observed the induction of DrRIG-I when DrNOD2 signaling was activated at 24 hpi (DrRIG-I could be observed as early as 12 hpi in the DrNOD2 (ΔLRR)-injected group  alone led to a robust NF-κB activation in the HEK293T cells (DrRIG-I or DrRIG-I-CARD was co-injected (DrRIG-I (ΔCARD) was also used as negative control, and this mutant could not inhibit the DrNOD2- and DrNOD2 (ΔLRR)-activated NF-κB signaling -mediated NF-κB activation and TNF-α expression. Good rescue was achieved with the simultaneous injection of DrRIG-I mRNA .To prove our hypothesis, we determined the function of 3T cells A and zeb3T cells B. Activainjected A,B. The ignaling B. The Drobserved C. Co-admG-I mRNA D,E. MDP- embryos F. These DrNOD2 in DrRIG-I-induced IFN signaling were also examined. DrRIG-I (CARD) overexpression induced the ISG15 expression in the HEK293T cells (DrNOD2 or DrNOD2 (ΔLRR) was co-injected (DrNOD2 (ΔCARD) could not inhibit the DrRIG-I (CARD)-initiated Mx activation (DrNOD2 by MO (DrNOD2 mRNA decreased the level of activation (DrNOD2 or DrNOD2 (ΔLRR) was co-injected in zebrafish embryos .The roles of 3T cells A and Mx 3T cells B. ISG15 injected A,B. The tivation B. HighlyD2 by MO C resultetivation D,E. Furt embryos F. These RIG-I and NOD2 are two of the most important cytosolic PRRs participating in the recognition of viral and bacterial invasion in mammals. In the present study, we used zebrafish as an attractive model organism to investigate the NOD2- and RIG-I-mediated immunology. The zebrafish was selected because of its conserved structural and functional characteristics compared with its mammalian counterparts.DrNOD2 expression and that NOD2 signaling activation could induce DrRIG-I expression. These observations suggest a feedback regulatory mechanism between the two signaling pathways. Further evaluation showed that the overexpression of DrRIG-I or DrRIG-I-CARD in the HEK293T cells and zebrafish embryos could significantly inhibit DrNOD2 and DrNOD2 (ΔLRR)-activated NF-κB signaling. Similarly, administration of DrNOD2 or DrNOD2 (ΔLRR) in cells and embryos could significantly suppress DrRIG-I (CARD)-induced IFN signaling. Further knockdown and rescue experiments also confirmed this proposition. Therefore, negative feedback and mutual regulation exist between DrNOD2 and DrRIG-I signaling pathways. Moreover, the results showed the physiological significance of this mutual feedback regulation. DrRIG-I could negatively regulate MDP-initiated NOD2 signaling, and DrNOD2 could negatively regulate LMW poly I:C initiated RIG-I signaling. NOD2 was recently confirmed to interact with RIG-I in a zebrafish cell line (ZF4) by co-immunoprecipitation analysis [DrNOD2 and DrRIG-I signaling pathways may have resulted from the sequestering of the CARDs of DrRIG-I and DrNOD2 away from their adaptors, MAVS and RIPK2, respectively.We used the zebrafish model to demonstrate that RIG-I signaling activation could induce analysis , and we analysis . These dDrNOD2 and DrRIG-I could be induced by their corresponding signaling pathway and negative regulate the signaling. We believe that this feedback might be of great biological significance in innate antibacterial or antiviral immunity. For example, when the host NOD2/RIG-I signaling is activated via bacterial/viral infection, a feedback regulation might contribute to maintain the homeostasis of immune responses. This characteristic prevents excessive immune reactions. Meanwhile, the induced NOD2 or RIG-I would alert the host, leading to a much faster and stronger immune response against a secondary bacterial or viral invasion. Thus, dysfunction in this feedback regulation may result in various pathological disorders. Secondary bacterial infection commonly develops after viral infections, and this process is accompanied by bursts of inflammatory responses [Therefore, esponses . In addiesponses . We infeDanio rerio) weighing 0.5–1 g and measuring 1–2 cm in length were purchased from the National Zebrafish Resources of China. The fish were kept in tanks with recirculating water at 28 °C and fed daily with commercial pellets at 0.7% of their body weight. The fish were acclimatized and evaluated for overall fish health at least two weeks before the experiments.Wild-type AB zebrafish  between the EcoRI and XhoI sites to construct the eukaryotic expression vector (pCMV-DrNOD2). The LRR motif-deleted mutant (ΔLRR) construct (pCMV-DrNOD2 (ΔLRR)) and the CARD motif-deleted mutant (ΔCARD) construct (pCMV-DrNOD2 (ΔCARD)) were cloned and inserted into the pCMV-HA between the sites of EcoRI and XhoI. The CARD motif-deleted mutant (ΔCARD) construct of DrRIG-I was cloned and inserted into the pcDNA6 between the sites of BamHI and EcoRI. The NF-κB luciferase vector was purchased from Clontech , while the pRL-TK vector was obtained from Promega . Human ISG15 and zebrafish Mx promoters (hISG15-pro-luc and DrMx-pro-luc), as well as the RIG-I expression plasmids pcDNA6-DrRIG-I and pcDNA6-DrRIG-I (CARD), were constructed in our previous study [The open-reading frame (ORF) of us study ,45. All DrRIG-I and splice junction MO of DrNOD2 were designed, synthesized by Gene Tools , and dissolved in water (2 mM). The MO sequences used were as follows: DrRIG-I MO, 5′-GATTCTCCTTCTCCAGCTCGTACAT-3′; and DrNOD2 MO, 5′-ACCTGCCAAAAATCCAACATGGTTA-3′. The 5′UTR sequence (complement to the MO sequence) of DrRIG was amplified with DrRIG-I F1 and R1 primers (DrRIG-I MO. Along with DrRIG-I MO or standard control MO (4 ng/embryo), the constructed vector was injected into one-cell stage embryos (100 pg/embryo). The embryos were collected at 24 hpi, and the GFP fluorescence was visualized through an Olympus MVX10 MacroView. The splice inhibition efficiency of DrNOD2 MO was examined through RT-PCR. The one-cell stage embryos of zebrafish were injected with DrNOD2 MO (4 or 6 ng/embryo) or standard control MO (6 ng/embryo). The embryos were collected at 24 hpi for RNA isolation and cDNA reverse transcription. A forward primer in exon 1 and reverse primer in exon 2 were used to detect the deletion of exon 2  according to the manual and then solubilized in DEPC water.The translation-blocking MOs of  primers , and thef exon 2 . Capped v/v) FBS , penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 °C in 5% CO2. Cells were seeded into six-well plates to allow growth until 70–90% confluence on the day of transfection. Transient transfection and cell lysate preparation were performed as previously described [HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% (escribed .HEK293T cells or zebrafish embryos were transfected (1 µg/mL) or injected (100 pg/embryo) with relative stimulant plasmids and NF-κB/ISG15/Mx luciferase reporter vectors. The pRL-TK renilla luciferase reporter plasmid was used as internal control. An empty control plasmid was then added to ensure same amounts of the total DNA. Subsequently, the cells and embryos were lysed at 24 hpi, and dual-luciferase reporter assay was performed as described previously . LuciferDrRIG-I (CARD) or pCMV-DrNOD2 or pCMV-DrNOD2 (ΔLRR) (100 pg/embryo) was injected into one-cell stage embryos. The mRNA levels of NOD2 or RIG-I relative to β-actin were examined at 6, 12, and 24 hpi using RT-PCR to determine whether RIG-I-initiated signaling induced NOD2 production and vice versa. The empty plasmid injection group was set as control. RT-PCR was conducted with the following parameters: (1) 40 cycles of amplification at 95 °C for 30 s and 60 °C for 20 s; (2) melting curve analysis at 95 °C for 5 s, 65 °C for 15 s, and 95 °C for 15 s; and (3) cooling at 40 °C for 30 s. Relative gene expression was calculated using the 2Ct−∆∆ method with NOD2/RIG-I, which were initially normalized against β-actin. The primers used are shown in pcDNA6-DrRIG-I (CARD) (Myc tag) and pCMV-DrNOD2 (HA tag) were transfected into HEK293T cells. At 48h post transfection, cells were lysed with cold lysis buffer ) containing protease inhibitor mixture  for 30 min at 4 °C. lysates were centrifuged for 15 min at 14,000 rpm and the supernatants were incubated with mouse anti-HA Ab  at 4 °C overnight and then incubated with protein A-agarose beads (Roche) for 4 h. The obtained samples were subjected to Western blot assays using rabbit anti-c-Myc tag and HRP-conjugated goat anti-rabbit IgG Ab (Abcam) antibodies, and visualized with ECL reagents as described before.pcDNA6-DrNOD2 and DrRIG-I was examined in the HEK293T cells and zebrafish embryos. The role of DrRIG-I in the DrNOD2-activated NF-κB signaling was analyzed by administering pCMV-DrNOD2 or pCMV-DrNOD2 (ΔLRR), alone or together, with pcDNA6-DrRIG-I, pcDNA6-DrRIG-I (CARD), or pcDNA6-DrRIG-I (ΔCARD) and the NF-κB luciferase reporter plasmid and internal control plasmid pRL-TK to the HEK293T cells or one-cell stage zebrafish embryos. The CARD deletion mutant was used as the negative control. The role of DrNOD2 in DrRIG-I-induced IFN signaling was analyzed by administering pcDNA6-DrRIG-I (CARD), alone or together, with pCMV-DrNOD2, pCMV-DrNOD2 (ΔLRR), or pCMV-DrNOD2 (ΔCARD). The IFN luciferase reporter plasmid (hISG15-pro-luc in HEK293T and DrMx-pro-luc in embryo) and control plasmid pRL-TK were also administered. The CARD deletion mutant was used as a negative control. The cells/embryos were harvested and lysed at 24 h post transfection/injection for dual-luciferase reporter assay.The mutual regulation between DrRIG-I in DrNOD2-initiated pathways and DrNOD2 in DrRIG-I-initiated pathways. For the former, one-cell stage embryos were injected with pCMV-DrNOD2/pCMV-DrNOD2 (ΔLRR) (100 pg/embryo) and DrRIG-I MO (4 ng/embryo), or together with capped DrRIG-I mRNA (100 pg/embryo). For the latter, one-cell stage embryos were injected with pcDNA6-DrRIG-I (CARD) (100 pg/embryo) and DrNOD2 MO (4 ng/embryo) or together with capped DrNOD2 (100 pg/embryo) mRNA. The embryos in each group were harvested at 24 hpi and lysed for dual-luciferase reporter assay. Furthermore, qRT-PCR was conducted to examine the relative expression levels of TNFα  and Mx .Knockdown and rescue experiments were conducted to further confirm the roles of DrRIG-I and DrNOD2 by evaluating the role of DrRIG-I in MDP-initiated NOD2 signaling and DrNOD2 in LMW poly I:C-initiated RIG-I signaling. For DrRIG-I, one-cell stage embryos were administered with 2 nL (1 µg/μL) of MDP, alone or together, with pcDNA6-DrRIG-I or pcDNA6-DrRIG-I (CARD) (100 pg/embryo), as well as NF-κB luciferase reporter plasmids. The embryos were collected at 24 hpi, and luciferase assays were conducted to examine the NF-κB activation level. For DrNOD2, one-cell stage embryos were administered with 4 nL (1 µg/μL) of LMW poly I:C alone or together with pCMV-DrNOD2/pCMV-DrNOD2 (ΔLRR) (100 pg/embryo), as well as Mx luciferase reporter plasmids. The embryos were collected at 24 hpi, and luciferase assays were conducted to examine Mx activation level.We further confirmed the physiological significance of the mutual negative regulatory roles between t-test for paired samples. Statistical significance were considered at * p < 0.05 and ** p < 0.01.Data from three independent experiments were expressed as mean ± SD. Groups were compared statistically using Student’s


In the present study, we examined the effects of central administration of Urotensin II on blood pressure, heart rate, and baroreceptor heart rate reflexes in conscious normotensive rabbits. Preliminary operations were undertaken to implant a balloon cuff on the inferior vena cava for baroreflex assessments and to implant cannula into the lateral and fourth ventricle. After 2 weeks of recovery cumulative dose response curves to Urotensin II  given into the ventricles, or Ringer's solution as a vehicle were performed on separate days. Injections were given each hour and baroreflex assessments were made 30 min after each administration. Analysis of the dose response curves to Urotensin II compared to vehicle administered into the lateral or fourth ventricle, indicated little change to blood pressure or heart rate. Analysis of the time course to the highest dose over a 30 min period revealed a small (−5 mmHg) depressor response maximal at 10 min when injected into the fourth ventricle but no effect when injected into the lateral ventricle. Baroreflex assessments made at each dose showed that there was no change in baroreflex sensitivity but that an increase in the upper plateau was observed when Urotensin was injected into the lateral ventricle and a tendency for a reduced lower heart rate plateau was observed after fourth ventricle administration. Clonidine administration in the fourth ventricle decreased blood pressure and heart rate, thus confirming catheter patency. In conclusion, our findings suggest that Urotensin II in the forebrain and brainstem may play a role in modulating cardiac sympathetic and vagal baroreflexes but only during large acute changes in blood pressure. In vivo studies show marked species differences in the actions of intravenously administered Urotensin II. In the rat, intravenous Urotensin II elicits a depressor response due to mesenteric and hindquarter vasodilatation associated with tachycardia   after induction with intravenous administration of propofol (10 mg/kg).During the first procedure, a small perivascular silastic balloon was implanted around the intrathoracic inferior vena cava through a right thoracotamy while the rabbit was artificially ventilated. The exterior extremity of the tubing attached to the balloon was sutured under the skin between the scapulae. The lungs were reinflated, air removed from the chest cavity, the ribs drawn tightly together and the muscle and skin sutured.Two weeks after the initial surgery, the six rabbits underwent a second surgical procedure. An intracerebroventricular catheter  was implanted into the lateral ventricle  as described previously , L-phenylephrine HCl , and clonidine HCl . The doses of drugs were expressed in μg of the base. Drugs administered into the lateral ventricle or fourth ventricle were dissolved in Ringer's  solution while drugs given i.v. were dissolved in normal saline .Experiments were conducted in conscious rabbits held in a standard single rabbit holding box. Arterial pressure was measured from the central ear artery which was catheterized transcutaneously  and HR was derived by a rate-meter triggered from the arterial pulse. Mean arterial pressure (MAP), HR and respiration rate were digitized and averaged over 2 s periods by computer using the LabVIEW programming language . Throughout the experiment, cardiovascular parameters were recorded on a six-channel polygraph .The first experiment was conducted at least 14 days after implantation of the fourth ventricle catheter. Each rabbit underwent three experiments, each separated by 2 week recovery, during which a dose response curve to either Urotensin II into the lateral ventricle, Urotensin II into the fourth ventricle or a control experiment was performed which consisted of administering the same volumes of Ringer's solution . For each experiment, there was an initial 30 min acclimatization period and a 30 min period during which baseline baroreflex parameters were obtained in duplicate. Dose-response curves were performed using a range of five increasing doses of Urotensin II administered in 25 μL. The doses were 10 ng (7 pmol), 100 ng (72 pmol), 1 μg (0.7 nmol), 10 μg (7 nmol), and 100 μg (72 nmol) and thus covered 4 orders of magnitude. The dose range was based on a previous publication  or mean difference ± standard error of the difference (SED). For all parameters, a repeated measure ANOVA was performed on the change (delta) from control values. The between treatment sums of squares was partitioned into two non-orthogonal contrasts comparing each treatment with vehicle. In addition, the between doses sums of squares was partitioned into a linear trend (indicating a dose response relationship) and the non-linear component. A Bonferroni adjustment of the t statistic was made to account for the multiple testing and lessen the likelihood of a type 1 error.There were no differences in MAP or HR with Urotensin II injection into the lateral ventricle, into the fourth ventricle and control experiment over time .F1, 70 linear trend = 12, P < 0.01). The analysis of variance showed no difference in the effect of Urotensin on HR compared to vehicle administration  had no effect on blood pressure at any time within the 30 min period examined Figure . By contP < 0.01, Figure F1, 70 linear trend = 5.3, P < 0.05, Figure Baroreflex assessments were made by using the rapid ramp method before and 30 min after each dose of Urotensin or vehicle. The blood pressure set-point (BP50) and the baroreflex sensitivity (average gain) did not change over the course of the experiment with repeated assessments during either the Urotensin II or vehicle administration protocols Figure . HoweverP < 0.05, Figure P = 0.07, Figure The lower HR plateau was reduced after 1 μg Urotensin II into the fourth ventricle compared to the control  we did observe there a significant increase in the upper HR plateau of the curve compared to the control curves (To confirm the patency of the fourth ventricular catheter clonidine was administered at the end of the last experiment in all 6 animals. Administration of 5 μg/kg of clonidine into the fourth ventricle elicited a rapid reduction in HR and blood pressure (Figure The main findings of the present study were that Urotensin II administered into the fourth or lateral ventricle of conscious rabbits differentially affected the maximum and minimum plateaus of the baroreceptor HR reflex with few other changes observed to arterial pressure or HR. We observed a small depressor response to 100 μg Urotensin II administered into the fourth ventricle but the small increases in HR were not related to a specific effect of Urotensin II. The lateral ventricle administration increased the upper plateau of the HR curves which is where the sympathetic activity is maximally activated during hypotension. By contrast, the fourth ventricular administration inhibited the lower plateau which is predominately the vagal end of the curve. Taken together these results suggest that central administration of Urotensin II modifies autonomic control of heart rate but only when these reflex systems are responding close to maximal changes in blood pressure. Interestingly, the forebrain action of Urotensin II appears to facilitate the maximum cardiac sympathetic activity while the hindbrain action appears to be to inhibit the maximum cardiac vagal activity. The combined action would be to shift the curve upward.To our knowledge, this is the first report of a central effect of Urotensin II on autonomic reflexes in conscious rabbits. It has been shown that autoradiographic labeling of brain slices indicate the presence of urotensin II binding sites in the lateral septum, bed nucleus of the stria terminalis, medial amygdaloid nucleus, anteroventral thalamus, anterior pretectal nucleus, pedunculopontine tegmental nucleus, pontine nuclei, geniculate nuclei, parabigeminal nucleus, dorsal endopiriform nucleus, and cerebellar cortex (Jégou et al., Previous studies examining the central cardiovascular actions of Urotensin II have been performed so far only with rats (Lin et al., In the present study we used the human form of Urotensin II but across phyla from fishes to primates there is a great deal of homology of the key cyclic region of the peptide which is responsible for its biological action (Coulouarn et al., KL, YS, KJ, SB, and GH contributed to the analysis of the data and writing of the manuscript. KL and SB conducted animal experiments.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 Hippo signaling pathway plays a crucial role in cell proliferation, apoptosis, differentiation, and development. Major effectors of the Hippo signaling pathway include the transcriptional co-activators Yes-associated protein 1 (YAP) and WW domain-containing transcription regulator protein 1 (TAZ). The transcriptional activities of YAP and TAZ are affected by interactions with proteins from many diverse signaling pathways as well as responses to the external environment. High YAP and TAZ activity has been observed in many cancer types, and functional dysregulation of Hippo signaling enhances the oncogenic properties of YAP and TAZ and promotes cancer development. Many biological elements, including mechanical strain on the cell, cell polarity/adhesion molecules, other signaling pathways , and cellular metabolic status, can promote oncogenesis through synergistic association with components of the Hippo signaling pathway. Here, we review the signaling networks that interact with the Hippo signaling pathway and discuss the potential of using drugs that inhibit YAP and TAZ activity for cancer therapy. Drosophila and Mst1/2, Sav1, Lats1/2, and Mob1a/b in mammals) inhibit the activation of transcriptional co-activators Yorkie (Yki), YAP, and TAZ  gene in  defects , 2. Foll defects , 4, Hipp defects –9, and M defects  genes reosophila . SubsequDrosophila, Scalloped (Sd) binding to Yki increases the transcriptional activity of Sd [in vivo [Because Yki, YAP, and TAZ cannot bind to DNA, they need to bind to another transcription factor that interacts with DNA directly. In ty of Sd . Sd belo[in vivo . While e[in vivo , 14, 15.[in vivo , 17.in vivo, S127 and S381 are regarded as the critical sites related to increased oncogenic activity. S127 phosphorylation results in 14-3-3 binding and cytoplasmic sequestration. S381 phosphorylation induces the subsequent phosphorylation of S384 and S387 by CK1δ/ε. These phosphorylated phosphodegrons recruit the SCFbeta-TrCP E3 ubiquitin ligase, which ultimately leads to YAP degradation [In mammals, the five consensus HXRXXS motifs in YAP  are phosphorylated by Lats kinase. Although all of these are phosphorylated radation .Although the immediate mechanism of regulation of YAP and TAZ by Hippo core components has been demonstrated, several proteins have recently been found to regulate Hippo core components in less direct fashion, bypassing the linear kinase modules to activate YAP and TAZ. These studies have shown that mechanical cues, cell polarity/adhesion molecules, other signaling pathways, and cellular metabolic status can regulate YAP and TAZ activity.The growth of normal cells in culture systems halts when they physically encounter other cells. YAP is inhibited by cell-cell contact  through Hippo signaling pathway activation, and overexpression of YAP can overcome cell-contact inhibition and promote cell proliferation .Drosophila, extra F-actin formation through the loss of capping proteins led to overgrowth in imaginal discs through the inhibition of Hippo signaling activity [The rigidity and cell geometry of the extracellular matrix (ECM) regulate YAP and TAZ activity Figure . While cactivity , 23. In activity .12/13-protein-coupled receptors. In contrast, glucagon and epinephrine, which stimulate Gαs-protein-coupled receptors, activated Lats1/2 [Rho family small guanosine triphosphatases (GTPases) play a key role in actin cytoskeleton organization Figure . The spe Lats1/2 , 26. Dep Lats1/2 .Drosophila. Spectrin downregulation led to the phosphorylation of regulatory light chain of myosin II by Rho-associated protein kinase, resulting in the generation of contractile tension and Yki activation [Spectrin is a cytoskeletal protein found on the intracellular side of the plasma membrane and involved in cytoskeletal tension. Spectrin acts as a tumor suppressor and upstream regulator of the Hippo signaling pathway in tivation , 29.Drosophila, Ajuba LIM protein binds to α-catenin and Wts at adherens junctions under conditions of high cytoskeletal tension and then inhibits Wts activity [Mechanical force appears to change cytoskeletal organization in a context-dependent manner and to modulate YAP activity. In activity . Mechaniactivity .Drosophila, Merlin (Mer), Expanded (Ex), Kibra, and Crumbs interact with the cytoskeleton at the apical domain of the cell. The FERM domain proteins Mer and Ex control proliferation and apoptosis through Hippo signaling activity [Drosophila [Drosophila and Mst in mammals [Drosophila.In activity . The WW osophila –35  family proteins inhibit YAP and TAZ activity via direct binding at tight junctions independently of YAP and TAZ phosphorylation status –45 Figu. α-CatenAlthough several lines of evidence indicate that the modulation of cell polarity/adhesion molecules influences Hippo core components and YAP and TAZ activity, their relationship may be mostly indirect. Additional studies are required to define the link between cell polarity/adhesion molecules and the Hippo signaling pathway.The Hippo signaling pathway is integrated upstream or downstream of other biological processes. The prominent signaling pathways related to the Hippo signaling pathway are GPCR, epidermal growth factor (EGF), bone morphogenetic protein (BMP)/transforming growth factor β (TGFβ), Wnt, and Notch signaling pathways Figure  and 2C.12/13 stimulates YAP and TAZ activity by inhibiting Lats1/2 kinase [s activates Lats1/2 kinase activity [s, activates protein kinase A (PKA). Activated PKA activates Lats kinase by inhibition of Rho GTPases [q/11 can activate protein kinase C (PKC), which can either activate or inhibit YAP activity, dependent on the isoforms of PKC [GPCR signaling has been implicated in almost every aspect of the physiological regulation of Hippo signaling Figure . As ment2 kinase , 51, andactivity . Cyclic  GTPases , 52. Gαqs of PKC .Treatment with an EGF ligand induces PI3-kinase (PI3K)–phosphoinositide-dependent kinase (PDK1) axis activation and inhibits Hippo core components independently of AKT Figure . In confTGFβ and BMP signaling acts as a ligand-induced transcriptional activator or repressor of responsive target genes that act through the SMAD (Sma and Mad-related family) protein complex  Figure . TGFβ stHippo and Wnt signaling reciprocally regulate each other's activity through a variety of mechanisms Figure . First, YAP/TEAD complex directly regulates the transcription of Jag1  and NotcIn response to energy deprivation, cell growth and proliferation are inhibited, and YAP and TAZ activity decreases Figure . Energy Under hypoxic conditions, hypoxia-inducible factor 1 (HIF1) is rapidly accumulated, and the transcriptional activity of HIF1 is elevated in response to changes in available oxygen in the cellular environment. The hypoxia-induced HIF1 transcriptional target genes include TAZ and ubiquitin ligase SIAH1/2, which is required for the ubiquitination and degradation of Lats2 kinase , 86 Fig. YAP alsβγ protein and Rho GTPase activity though geranylgeranylation, enhancing YAP and TAZ activity [The mevalonate pathway produces numerous essential metabolic biomolecules, such as cholesterol, steroid hormones, and vitamin K from acetyl-coenzyme A (CoA) . An inhiactivity , 89. Mevactivity .Drosophila, and Cyclin D and BclXL expression levels are correlated with YAP expression levels in human colorectal cancers [Elevated YAP and TAZ activity induce hyperplasia, dysplasia, and tumors in a number of mouse models. YAP-inducible transgenic mice show irregular expansion in liver tissue and undifferentiated intestinal progenitor cells . YAP ind cancers , 92. YAP cancers . Thus, Y cancers , 95.Deletion of Hippo core component genes can also cause hyperplasia or expansion of specific cell types. In the liver-specific Lats1/2-knockout mouse, liver is filled with highly proliferated immature biliary epithelial cells . DeletioThe correlation between Hippo signaling and cancer is not the same in all tissues or cells. For example, the epidermis of mice with skin-specific deletion of Mst1/2 or Lats1/2 shows no abnormality and YAP-S127 phosphorylation is not increased . Also, nAlthough the dysregulation of YAP has been reported in many types of cancer, little is known about the contributions of mutations of Hippo signaling pathway genes. Data from the Catalogue of Somatic Mutation in Cancer (COSMIC) census show that Hippo pathway genes, with the exception of NF2 and TAZ, are not cancer-related genes . AlthougAs discussed above, the Hippo signaling pathway is interconnected with other biological processes that promote tumor formation and development. YAP and TAZ activity is high in many cancer types, which suggests that Hippo signaling components are potential targets for cancer therapy. The Hippo core kinases Mst1/2 and Lats1/2 are the most attractive therapeutic targets. However, Mst1/2 and Lats1/2 kinases are tumor suppressive, and their activity is low in many cancer types, unlike most oncogenic kinases, such as Src, Abl, Raf, and Akt, which are activated by mutations and involved in tumorigenesis . TherefoRecently, YAP was identified as a key survival input that mediates resistance by acting parallel to other known pathways in cancer progression. YAP silencing enhanced the response to RAF and MEK inhibitor in a wide spectrum of BRAF-mutated cancer cell lines, and patients who have BRAF-mutated tumors with lower YAP expression respond better to treatment with RAF and MEK inhibitor , 116. Inin vitro [in vitro and in vivo [The direct inhibition of YAP and TAZ oncoproteins is also a therapeutic target for anti-cancer treatment. Heterozygous and homozygous deletion of YAP dramatically suppresses tumorigenesis in LKB1-, Mob1-, and NF2-deficient liver , 63, 120in vitro . Lentiviin vitro . The TDU in vivo . FurtherIn high-throughput screening, Verteporfin, a small molecule that belongs to the porphyrin family, was identified as an inhibitor of YAP-TEAD interactions Table . Treatmein vivo. Activation of YAP and TAZ may be triggered by other biological processes that are vulnerable to oncogenic mutations and increases in the transcription of genes that are related to oncogenic processes. Understanding the synergistic effects between the Hippo signaling pathway and other biological processes that result in tumorigenesis could facilitate the development of new anti-cancer drugs.Hippo core components and YAP and TAZ activity are regulated by a variety of mechanisms. A number of studies have reported that the Hippo signaling pathway is associated with cellular mechanical strain, cell polarity/adhesion molecules, other signaling pathways , and cellular metabolic status. Dysregulation of Hippo signaling generally results in aberrant YAP and TAZ transcriptional activity that enhances their oncogenic properties. High YAP and TAZ activity has been detected in many types of cancer, but genetic mutations of Hippo core components, YAP, or TAZ are rarely found. This implies that aberrant activation of the Hippo signaling pathway may be insufficient for cancer formation


We have reported that integrins crosstalk with growth factors through direct binding to growth factors , neuregulin-1, fractalkine) and subsequent ternary complex formation with cognate receptor . IGF1 and IGF2 are overexpressed in cancer and major therapeutic targets. We previously reported that IGF1 binds to integrins ανβ3 and α6β4, and the R36E/R37E mutant in the C-domain of IGF1 is defective integrin binding and signaling functions of IGF1, and acts as an antagonist of IGF1R. We studied if integrins play a role in the signaling functions of IGF2, another member of the IGF family. Here we describe that IGF2 specifically binds to integrins ανβ3 and α6β4, and induced proliferation of CHO cells (IGF1R+) that express ανβ3 or α6β4 (β3- or α6β4-CHO cells). Arg residues to Glu at positions 24, 34, 37 and/or 38 in or close to the C-domain of IGF2 play a critical role in binding to integrins and signaling functions. The R24E/R37E/R38E, R34E/R37E/R38E, and R24E/R34E/R37E/R38E mutants were defective in integrin binding and IGF2 signaling. These mutants suppressed proliferation induced by WT IGF2, suggesting that they are dominant-negative antagonists of IGF1R. These results suggest that IGF2 also requires integrin binding for signaling functions, and the IGF2 mutants that cannot bind to integrins act as antagonists of IGF1R. The present study defines the role of the C-domain in integrin binding and signaling. Integrins are transmembrane receptor heterodimers formed by α and β chains. Integrins bind to extracellular matrix ligands , cell surface ligands  and soluble ligands including several growth factors . We haveIGF1 and IGF2 are homologues and share 67% sequence identity , and areWe studied the role of integrins in IGF2 signaling through IGF1R. We show that IGF2 binds to integrins ανβ3 and α6β4, and induces intracellular signaling in an integrin-dependent manner in CHO cells (IGF1R+) that overexpress these integrins. We located the integrin-binding site of IGF2 in the C-domain that connects B and A domains, suggesting that the C-domain plays a critical role in IGF signaling. The IGF2 mutants defective in integrin binding were defective in signaling and acted as antagonists of IGF1R, indicating that the IGF2 signaling also fits well with the ternary complex model.We studied if soluble integrin αvβ3 binds to immobilized IGF2 in an ELISA-type binding assay. We found that soluble αvβ3 bound to IGF2 in a dose dependent manner, but did not bind to wells coated only with BSA . We studWe studied if IGF2-induced intracellular signaling requires αvβ3 in β3-CHO cells. All the experiments were performed in anchorage-independent conditions (using polyHEMA coated plates) to reduce signals generated by cell-extracellular matrix interaction. IGF2 induced proliferation of β3-CHO cells in a dose dependent manner to a much higher extent than in CHO cells . Cell prOur results so far suggest that αvβ3 binds to IGF2 and is involved in IGF1R signaling. To further study the role of ανβ3 in IGF2 signaling, we generated IGF2 mutants that are defective in integrin binding. We previously reported that Arg36 and Arg37 in the C-domain of IGF1 are critical for integrin-binding . Based oWe studied the signaling functions of the R24E/R37E/R38E, R34E/R37E/R38E, and R24E/R34E/R37E/R38E mutants using β3-CHO cells in polyHEMA-coated plates. R24E/R37E/R38E, R34E/R37E/R38E, and R24E/R34E/R37E/R38E were defective in inducing activation of IGF1R, AKT, and ERK1/2  and cellOur previous studies suggested that α6β4, another integrin that is overexpressed in cancer cells, is critically involved in IGF1/IGF1R signaling through direct binding to IGF1 [We studied if the IGF2 mutants bind to the immobilized soluble IGF1R ectodomain in ELISA-type assays. We detected the binding of WT and three IGF2 mutants to IGF1R. R24E/R37E/R38E bound to IGF1R to the similar extent to that of WT IGF2, but the binding of R34E/R37E/R38E and R24E/R34E/R37E/R38E was weaker than that of WT IGF2 . We showIn the present study, we establish that IGF2 specifically binds to ανβ3 and induced proliferation of β3-CHO cells and cyclic RGDfV suppressed the cell proliferation induced by IGF2. This suggests that the binding of IGF2 to integrin ανβ3 plays a role in IGF2/IGF1R signaling, as in the case of IGF1 , and IGFThe biological role of the C-domains of IGF1 and IGF2 has not been established. In our previous studies, IGF1 binds to integrins ανβ3, amino acid residues of IGF1 critical for integrin binding were localized in the C-domain (Arg residues at positions 36 and 37) , which aWe used the integrin-binding defective IGF2 mutants  to define the role of integrins in IGF2 signaling. The IGF2 mutants were defective in activating IGF1R and in inducing intracellular signaling and cell proliferation in β3-CHO cells. These findings suggest that IGF2 requires integrin binding to induce signaling through IGF1R, as in the case of IGF1 . We prevChinese hamster ovary (CHO), and MBA-MB-231 human breast cancer cells were obtained from American Type Culture Collection (ATCC). CHO cells expressing human integrin β1 (β1-CHO) or human integrin β3 (β3-CHO) were described . CHO celAYRPSETLCGGELLVDTLQFVCGDRGFYFSRPASSRVSRRSRGIVEECCFRSCDLLALLETYCATPAKSE. Protein was synthesized as insoluble protein by isopropyl β-d-thiogalactoside (IPTG) induction in Escherichia coli BL21. The C-terminal His tag of the protein was used to purify the protein with nickel-nitrilotriacetic acid affinity chromatography under denaturing conditions (in 8 M urea). The nickel-nitrilotriacetic acid resin was washed with 1% Triton X-114 before eluting the bound protein to eliminate endotoxin. Purified proteins were refolded in vitro following the protocols (“Isolation of proteins from inclusion bodies” available from the Björkman laboratory). In brief, the purified proteins were eluted in 8 M urea. After elution, the proteins were diluted into refolding buffer  on ice. The dilution was kept for 16 hrs at 4°C with a slow stirring movement. Then the proteins were concentrated by ultrafiltration. Around 2 milligrams of purified proteins were obtained from 1 liter of bacterial culture. WT IGF2 protein concentration was determined by measuring A280.A cDNA fragment encoding WT IGF2 was amplified by PCR with synthetic oligonucleotides 5-gtggtgctcgagctcggacttggcgggggtagc-3 and 5-ccgacgcatccatggctgcttaccgccccagtgag-3 using human placenta cDNA library as a template. The PCR fragment was digested with NcoI and XhoI and subcloned into the NcoI/XhoI site of PET28a. The expression construct encodes IGF2 (residues 322–523) with a His6 tag at the C terminus, 2 Poly(2-hydroxyethyl methacrylate) (poly-HEMA) following the protocol as described [2 atmosphere. The cells were collected and plated in poly-HEMA plate for starvation for 4 hrs in DMEM without FBS. Serum-starved cells were treated with WT IGF2 or IGF2 mutants . After treatment, cells were collected by spinning the samples at 3.5 rpm and then solubilized using lysis buffer  and 1 mM Na3VO4). BSA assay (Thermo Scientific) was performed to determine the protein concentration to each sample. The cell lysates were analyzed by western blotting using different antibodies. The HRP-conjugated anti-IgG antibody, Supersignal West Pico and Femto (Thermo Scientific) were used to detect IgG. Fuji LAS 4000 mini luminescent image analyzer and Multi Gauge V3.0 software  were used for analysis of the images.The anchorage-independent conditions were used to perform all the signaling assays. Wells of microtiter plates were coated with 1.2 mg/cmescribed . Cells w4 cells/well) were serum-starved overnight in serum free media (DMEM) and incubated with proteins (WT or IGF2 mutant) for 24 hrs in 96 wells plate coated with 1.2 mg/cm2 of poly-HEMA following the protocol as described [Cells  at 1 μg/ml in PBS for 1 h at 37°C, and the remaining protein-binding sites were block by incubating with 1 mg/ml BSA for 1 h at room temperature. WT and mutant IGF2 (2.5 μg/50 μl in PBS) were added to the wells and incubated in PBS/0.05% Tween 20 at room temperature for 1 hr. After washing with PBS/0.05% Tween 20, wells were incubated with anti-5His antibody conjugated with HRP (Qiagen), then peroxidase substrate.Cell adhesion assays  and bind


FAS -670 polymorphism in an interferon (IFN)-regulated STAT1-binding site has been associated to both ATL and HAM/TSP susceptibility. Fashi T stem cell memory (Tscm) cells have been identified as the hierarchical apex of ATL, but have not been investigated in HAM/TSP. In addition, both FAS and STAT1 have been identified in an IFN-inducible HAM/TSP gene signature, but its pathobiological significance remains unclear. We comprehensively explored Fas expression (protein/mRNA) and function in lymphocyte activation, apoptosis, proliferation, and transcriptome, in PBMC from a total of 47 HAM/TSP patients, 40 asymptomatic HTLV-1-infected individuals (AC), and 58 HTLV-1 -uninfected healthy controls. Fas surface expression followed a two-step increase from HC to AC and from AC to HAM/TSP. In HAM/TSP, Fas levels correlated positively to lymphocyte activation markers, but negatively to age of onset, linking Fashi cells to earlier, more aggressive disease. Surprisingly, increased lymphocyte Fas expression in HAM/TSP was linked to decreased apoptosis and increased lymphoproliferation upon in vitro culture, but not to proviral load. This Fashi phenotype is HAM/TSP-specific, since both ex vivo and in vitro Fas expression was increased as compared to multiple sclerosis (MS), another neuroinflammatory disorder. To elucidate the molecular mechanism underlying non-apoptotic Fas signaling in HAM/TSP, we combined transcriptome analysis with functional assays, i.e., blocking vs. triggering Fas receptor in vitro with antagonist and agonist-, anti-Fas mAb, respectively. Treatment with agonist anti-Fas mAb restored apoptosis, indicating biased, but not defective Fas signaling in HAM/TSP. In silico analysis revealed biased Fas signaling toward proliferation and inflammation, driven by RelA/NF-κB. Correlation of Fas transcript levels with proliferation (but not apoptosis) was confirmed in HAM/TSP ex vivo transcriptomes. In conclusion, we demonstrated a two-step increase in Fas expression, revealing a unique Fashi lymphocyte phenotype in HAM/TSP, distinguishable from MS. Non-apoptotic Fas signaling might fuel HAM/TSP pathogenesis, through increased lymphoproliferation, inflammation, and early age of onset.Human T-cell lymphotropic virus (HTLV)-1 was the first human retrovirus to be associated to cancer, namely adult T-cell leukemia (ATL), but its pathogenesis remains enigmatic, since only a minority of infected individuals develops either ATL or the neuroinflammatory disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A functional  A two-step increase in cell death receptor Fas occurs upon HTLV-1 infection and disease progression.Unexpectedly, higher Fas level was linked to decreased cell death, increased lymphocyte proliferation/activation, and early disease onset.FAS (but not FASL) as a part of an interferon (IFN)-regulated gene signature in HAM/TSP patients (FAS -670 gene polymorphism is associated to both ATL (Human T-cell lymphotropic virus 1 (HTLV-1) is an exogenous human retrovirus infecting 5–10 million people worldwide, mostly in HTLV-1 endemic regions , 3. Thirpatients . In addiboth ATL  and HAM/both ATL  disease ex vivo, in vitro, and in silico experimental approach, while patient information and sample use is summarized in Table A flow chart diagram Figure  providesn = 47, 66.0% female, mean age 50.2 ± 11.5 years, mean disease duration 5.6 ± 4.0 years (range 0.8–14 years), EDSS range 3–7 (mean 5.1 ± 1.2)] were recruited from three endemic regions  following written informed consent. Age- and gender-matched HTLV-1-infected asymptomatic carriers  and uninfected healthy controls  from the same endemic regions were included in the study. The study was approved by the Ethics Committees of University of Sao Paulo and FIOCRUZ-Bahia in Brazil and Universidad Peruana Cayetano Heredia in Lima, Peru. Diagnosis of HAM/TSP was according to WHO criteria , and 10% heat-inactivated fetal calf serum .PBMC isolated from 5–10 ml of heparinized venous blood by Ficoll–Hypaque density gradient centrifugation (Sigma-Aldrich) were washed twice with PBS and were plated in 24-well tissue culture plates  at 4 × 10in vitro culture. Proviral load  in HAM/TSP patients and AC was quantified as published (Human T-cell lymphotropic virus-1 matrix protein p19 was quantified in cell-free supernatant of HAM/TSP patients’ PBMC and AC and HC using RetroTek HTLV-1/2 p19 Antigen ELISA kit (ZeptoMetrix) after 48 h of ublished , 31.3 in PBS (+20% human serum to block Fc receptors), and incubated for 30 min on ice with mAbs specific for CD3, CD4, CD8, CD80, CD86, CD95/Fas, and HLA-DR and corresponding isotype controls (BD Biosciences). For total Fas surface quantification and apoptosis, a minimum of 100,000 events/sample were stained and acquired with FACSort and FACSCanto II flow cytometers (BD Biosciences) and analyzed using CellQuest and Diva software, respectively.For phenotypic analysis, PBMC were resuspended at a density of 200,000 cells in 50 µl of 1% BSA, 0.1% NaN3H]-thymidine incorporation and flow cytometry -thymidine incorporation in HAM/TSP patients. Surprisingly, we found that Fas expression positively correlates to spontaneous lymphoproliferation in vitro  lymphocytes ex vivo in HAM/TSP patients, stained immediately after PBMC isolation, without in vitro culture. As shown in Figure hi (99.2 ± 0.8%), as compared to non-proliferating lymphocytes .We quantified o Figure A, which escribed  sensitivin vitro with anti-Fas mAb (agonist or antagonist) or anti-CD3 mAb as a positive control. No decrease in spontaneous apoptosis was observed upon treatment with antagonist anti-Fas mAb, confirming our hypothesis of inactive Fas-FasL signaling in vitro in HAM/TSP. Interestingly, treatment with agonist anti-Fas mAb resulted in significantly increased apoptosis , similar to treatment with anti-CD3 mAb  , while treatment with antagonist anti-Fas mAb downregulated 38 genes and upregulated 18 genes . Thus, triggering Fas signaling effects a broader gene spectrum than inhibiting it. This was also evident from Ingenuity® pathway analysis (IPA), since no biological functions were significantly associated with antagonist anti-Fas mAb treatment, whereas treatment with agonist anti-Fas mAb resulted in 22 significantly associated biological functions (5% FDR-adjusted and a stringent cut-off of at least five enriched molecules per pathway) . The top 10 biological functions activated by agonist anti-Fas mAb , highlight cellular migration, especially of myeloid cells. In addition, IPA network analysis . Again, blocking Fas signaling did not yield any significantly enriched upstream regulators .Considering the significant correlation between s Figure B of Fas-ex vivo global gene expression profile in PBMC of HAM/TSP patients (n = 6). Using transcriptome-wide correlation, 4,554 genes significantly correlated to Fas transcript levels , after stringent FDR-correction for multiple testing. Using annotated ingenuity pathways, we found a significant enrichment for proliferation-related genes . However, apoptosis, as defined by IPA, was not enriched amongst the ex vivo Fas-correlating genes .Finally, we used a pathway-based data mining approach, to test our hypothesis of biased Fas signaling, and to possibly extend our findings by including additional pro- and anti-apoptotic genes . For this purpose, we explored possible interactions of Fas mRNA within the ex vivo, in vitro, and systems analysis of Fas expression with functional apoptosis and proliferation assays, thereby providing an all-inclusive approach of the biological and clinical relevance of Fas signaling in HAM/TSP. We observed a two-step increase in ex vivo Fas expression: first, a greater percentage of Fas+ lymphocytes upon HTLV-1 infection and second, a strong increase in expression of the death receptor at the single-cell level upon HAM/TSP disease progression. In addition, for the first time, we demonstrate that Fas expression correlates negatively to apoptosis and age of onset, but positively to immune activation and lymphoproliferation.In this study, we combined ex vivo and in vitro Fas levels negatively correlated to in vitro apoptosis  significantly correlating to Fas mRNA level, but are not enriched for apoptotic pathways. Taken together, our data indicate that the death receptor is fully functional in HAM/TSP, and not in a dormant state, but skewed towards other biological pathways. Similar to our observation in HAM/TSP, increased Fas   . Interesng sets) . Furtherng sets)  or envirng sets)  linked, t of PVL .ex vivo, in vitro, in silico approach identifies biased pro-inflammatory and proliferative Fas signaling in HAM/TSP, revealing possible novel therapeutic targets.In conclusion, our results suggest that defective Fas-mediated apoptosis is linked to early disease onset and might be an additional factor in HAM/TSP pathogenesis, independent of PVL. Triggering Fas signaling, rather than inhibiting it, induces a specific gene set with a central role for NFκB pro-survival signaling. Thus, our integrated JVW designed research; SMM, FEL, TD, Ricardo K, DD, GS-S, GL, and JVW performed research; SVS, DFN, JV, and AMV contributed to data analysis; FEL, Ramon K, CA, MT, EG, DB, RL, and BGC provided patient samples; SMM and JVW analyzed data and wrote the paper.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 reviewer MM and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Recent investigations showed single associations between uric acid levels, functional parameters, exacerbations and mortality in COPD patients. The aim of this study was to describe the role of uric acid within the network of multiple relationships between function, exacerbation and comorbidities.We used baseline data from the German COPD cohort COSYCONET which were evaluated by standard multiple regression analyses as well as path analysis to quantify the network of relations between parameters, particularly uric acid.1, reduced 6-MWD, higher burden of exacerbations (GOLD criteria) and cardiovascular comorbidities, in addition to risk factors such as BMI and packyears. These associations remained significant after taking into account their multiple interdependences. Compared to uric acid levels the diagnosis of hyperuricemia and its medication played a minor role.Data from 1966 patients were analyzed. Uric acid was significantly associated with reduced FEVWithin the limits of a cross-sectional approach, our results strongly suggest that uric acid is a biomarker of high impact in COPD and plays a genuine role for relevant outcomes such as physical capacity and exacerbations. These findings suggest that more attention should be paid to uric acid in the evaluation of COPD disease status. Recent investigations showed an increased mortality in COPD patients with elevated uric acid (UA) levels , 2 and dCOPD and Systemic Consequences-Comorbidities Network).Based on this the aim of this study was to identify the role of the biomarker UA for spirometric parameters, 6-min walk distance, exacerbation rate and cardiovascular comorbidities in COPD while taking into account the fact that these outcome measures are related to each other. The hypothesis was that UA has direct effects on these measures that cannot be explained by their mutual relationships and common risk factors. Such information could be helpful to understand pathophysiological mechanisms and the responses to therapy. For this purpose we used data from the German COPD cohort study COSYCONET  [1), forced vital capacity (FVC) and CAT. Moreover, valid data on body-mass index (BMI), packyears of smoking, 6-min walk distance (6-MWD), serum levels of UA and creatinine, as well as hyperuricemia-related medication were required. The exacerbation risk for the ABCD grouping was based on the 12-month history of exacerbations of all severities, including hospitalization, as described by GOLD  [From D (2017)  based onD (2017) ) were inmission) . This remission) . The COS1 and FVC, each in % predicted, as well as a binary exacerbation category. The categorization of low and high exacerbations was based on the GOLD groups A-D asdefined in GOLD 2017, and we collapsed the A-D groups into binary subgroups i.e. “low” comprising groups A and B, and “high” comprising groups C and D [1 and FVC were taken from the Global Lung Initiative (GLI) [The study protocol and panel of assessments have been described previously . The dia C and D . PredictData are presented in the tables as numbers or mean values and standard deviations (SD). Comparisons between the three groups  were performed by univariate analysis of variance (ANOVA), or by chi-square-tests in the case of categorical variables. The relationships between variables were assessed using multiple linear regression analysis in case of continuous outcome variables, and binary logistic regression analysis for the exacerbation variable. The distributions of packyears and creatinine were right-skewed and therefore transformed to reduce a potential bias in the numerical estimates of associations. Approximately symmetric distributions were achieved by taking the square root of the variable “packyears”.p ≥ 0.05. All other statistical analyses were performed by the package SPSS version 24 . P-values < 0.05 were considered as statistically significant.The results demonstrated multiple relationships between the variables that could only partially be addressed by conventional regression analysis. There are statistical methods to describe complex networks and especially to differentiate between direct and indirect (i.e. mediated) effects. A common approach is path analysis  which hap < 0.001, ANOVA). Post-hoc tests revealed pairwise significant differences between patients without the diagnosis of hyperuricemia, those with specific therapy, and those with diagnosis of HU without specific therapy (p < 0.05 each). This underlines that the diagnosis of hyperuricemia is not congruent with elevation of the biomarker uric acid. In addition we stratified UA levels according to the GOLD A-D groups .The transformed values of packyears (see methods) were used for all subsequent analyses. To properly describe the associations between variables in the presence of multiple correlations we followed a stepwise approach, using linear and logistic regression analyses which were performed using the complete data set ; FVC % predicted on age (p = 0.010); 6-MWD on gender, age and BMI (p < 0.05 each); creatinine on gender, age, BMI and packyears (p < 0.05 each); and UA on gender, age, BMI and packyears (p < 0.05 each). When FEV1% predicted was introduced as an additional predictor of 6-MWD, this association was also significant (p < 0.001). The count of cardiovascular comorbidities depended on gender, age and BMI (p < 0.001 each) but not packyears. Moreover, a logistic regression analysis revealed that the exacerbation category depended, among risk factors, only on age (p = 0.005).First we determined the associations with the risk factors age, BMI, gender and packyears which were taken as predictors in multiple linear regression analyses. FEV1% predicted and FVC % predicted were dependent on UA (p ≤ 0.003), as well as 6-MWD on UA (p < 0.001). In addition, the cardiovascular comorbidity count was associated with UA (p < 0.001), as well as the binary exacerbation category . Gender was significant (p < 0.05 each) in all of these analyses except for FEV1% predicted.Second, we determined the associations with UA as predictor of lung function and physical capacity in linear regression analyses, taking into account the risk factors. FEV1% predicted, 6-MWD (p < 0.001 each) and cardiovascular comorbidities (p = 0.023) were significant predictors, whereas FVC % predicted was far from significance (p = 0.592). The same result was obtained using the cardiovascular comorbidity count as depended variable (p < 0.001 each).Third, the association between the binary exacerbation category and the functional parameters was analysed using multiple logistic regression, again with gender as covariate. FEVThe results described above revealed multiple relationships between risk factors, functional variables, exacerbations and biomarkers. To delineate this network of interdependences in greater detail we used the approach of path analysis.1, posing a problem due to collinearity. Moreover, the effects of gender were taken into account by using gender-adjusted values for all variables; the only exception was the binary exacerbation category for which no meaningful adjustment could be introduced. Therefore gender was implicit in the path analyses and disappeared as an explicit predictor. In designing the model we used the results of the regression analyses as well as pathophysiological considerations.FVC was omitted from these analyses as it was highly correlated with FEV1% predicted and 6-MWD. In the third step we incorporated results for the binary exacerbation category and the count of cardiovascular comorbidities as given in “Exacerbation category versus function and cardiovascular comorbidities”. Only associations identified as statistically significant were kept in the final model. It should be noted, that even in the presence of multiple other pathways UA had independent effects on FEV1 (p = 0.005), 6-MWD (p < 0.001) and cardiovascular comorbidity count (p < 0.001), whereby UA levels were influenced by age, BMI and packyears. Except for one link (between UA and exacerbation category) all relationships identified as statistically significant in the regression analyses remained significant in the comprehensive model. This consistency, together with the good fit of the data, indicated the validity of the model which we found. The chi-square value of fit with generalized least squares estimation was 12.306 with 8 degrees of freedom and a p-value of 0.138, the corresponding CFI 0.996, and the RMSEA 0.017. The standardized regression and correlation coefficients of the final path analysis model are shown in Table The final model integrated risk factors, UA, functional indices, cardiovascular comorbidities and exacerbations into a comprehensive network that was compatible with the results of the conventional regression analyses. In a first step we used the results of the regression analyses that can be found under “Dependence of functional parameters on risk factors”. Additionally, we introduced correlations between the predictors BMI and packyears, and age and packyears, as these were suggested by the data. In a second step we used the result given under “Associations with uric acid”, to describe the additional relationships between UA, FEVFor the purpose of illustration the final model is shown in two parts, the first describing the relationships to risk factors Fig. , and then = 1966). As indicated in Table n = 1610); the model of Figs. p = 0.028), underlining the importance of UA per se. This link had been only at the border of statistical significance (p = 0.063) when using the whole study population.All analyses described above were performed in the total population of patients n = 966. As i1, lower physical capacity in terms of 6-MWD and a greater number of cardiovascular comorbidities. Both FEV1 and 6-MWD mediated an indirect effect of UA on exacerbations as defined by GOLD 2017. In the absence of hyperuricemia-specific medication there was even a direct link from UA to exacerbations, probably due to the fact that untreated patients had higher uric acid levels  for mortality, exacerbations and lung function in COPD , 2. The 1 in lung healthy subjects [1 in COPD patients [1 to be linked to UA. In parallel to findings in patients with pulmonary hypertension [The observed association of lung function with UA is in line with previous results regarding FVC and FEVsubjects , or FEV1patients . In our rtension , we obsertension , as wellrtension . Both fi1, which is a common finding in COPD [1, pointing towards greater airflow limitation. Taken together, this observation again underlines that UA itself has effects that are explained by common risk factors like BMI.UA levels are known to be influenced by a variety of factors, among them overweight, age and male gender. These risk factors were also significant predictors in our data. In the path analysis gender was implicit and taken into account by the use of appropriately adjusted values. As excretion rate has an effect on UA levels , we tent in COPD . At the Thus, patients with hyperuricemia showed different ranges of UA depending on the presence of specific medication as illustrated in Fig. 1, it was also directly associated with 6-MWD. We do not know whether this could be partially due persistent motoric impairment from gout arthritis even in the absence of acute episodes.Beyond lung function and physical capacity, the rate and severity of exacerbations are determinants of the prognosis in COPD , 23. We 1. The independent effect of UA on exacerbations was statistically significant only when excluding patients with hyperuricemia-specific medication. This seems understandable due to the reduction in the range of variation of UA compared to patients with hyperuricemia without specific UA-lowering medication. These observations support the hypothesis that UA influences the risk/severity of COPD exacerbations. Taking into account the different paths linking UA to exacerbations, our findings suggested that an increase in UA level by 2 mg/dl was linked to a shift by about 5% from the low to the high exacerbation category. A potential pathophysiological link could be endothelin-1 which is known to be elevated in hyperuricemia, but also in asthma and COPD exacerbations [In the path analysis model exacerbations were dependent on FEVrbations . The assrbations  which inrbations . Taken tObvious limitations of the study are its cross-sectional and non-interventional design. Furthermore, we did not have data on the history of clinical manifestations of gout, in terms of acute episodes. Patients with the physician-based diagnosis of hyperuricemia are potentially more health-conscious and may therefore have a better physical capacity and prognosis, which could influence our results. Moreover, there was no information on the specific medical reasons why patients received hyperuricemia-specific treatment. Also, there was no information on the duration of the hyperuricemia-specific treatment. This, however, appeared to be of minor importance, since the major results were independent of excluding or including these patients. Long-term follow-up data could reveal whether the relationships observed in a cross-sectional analysis are also reflected in the relationships between the changes of parameters within the follow-up period. The strength of our study was its large sample size which allowed the use of sophisticated statistical methods with the aim to disentangle the role of UA from other influencing factors. Moreover we could rely on comprehensive, high quality data of lung function and patients’ clinical characteristics.Within the limits of a cross-sectional approach, our results strongly suggest that uric acid is not only a useful biomarker for single entities in COPD but also plays a central role in this disease. It is simultaneously associated with determinants of worse COPD prognosis including airway obstruction, cardiovascular comorbidities, exacerbations and physical capacity, irrespective of common risk factors. This suggests that more attention should be paid to the levels of uric acid in the evaluation of COPD disease status. The described role of uric acid should be substantiated in future targeted intervention trials.