Negative regulation of TGF-β signaling in development

The TGF-β superfamily members have important roles in controlling patterning and tissue formation in both invertebrates and vertebrates. Two types of signal transducers, receptors and Smads, mediate the signaling to regulate expression of their target genes. Despite of the relatively simple signal transduction pathway, many modulators have been found to contribute to a tight regulation of this pathway in a variety of mechanisms. This article reviews the negative regulation of TGF-β signaling with focus on its roles in vertebrate development.

Negative regulation of NLRP3 infl ammasome signaling

Infl ammasomes are multiprotein complexes that serve as a platform for caspase-1 activation and interleukin-1β(IL-1β)maturation as well as pyroptosis.Though a number of infl ammasomes have been described,the NLRP3 inflammasome is the most extensively studied.NLRP3 inflammasome is triggered by a variety of stimuli,including infection,tissue damage and metabolic dysregulation,and then activated through an integrated cellular signal.Many regulatory mechanisms have been identifi ed to attenuate NLRP3 infl ammasome signaling at multiple steps.Here,we review the developments in the negative regulation of NLRP3 inflammasome that protect host from inflammatory damage.

Negative regulation of resistance protein-mediated immunity by master transcription factors SARD_1 and CBP60g

Summary Salicylic acid （SA） is an essential defence hormone in plants. Upon pathogen infection, induced biosynthesis of SA is mediated by Isochorismate synthase 1 （ICS1）, whose gene transcription is controlled mainly through two redundant transcription factors, SAR Deficient 1 （SARD0 and Calmodulin- binding protein 6o-like g （CBP60g）.

A novel short transcript isoform of chicken IRF7 negatively regulates interferon-βproduction

Type I interferon(IFN-I)provides an important first line to protect avian species against pathogens invasion.IFN regulatory factor 7(IRF7)has been identified as the most important regulator for both DNA and RNA virus-induced IFN-I production in chickens.Although four splicing variants of IRF7 have been identified in mammals,it is still unclear whether alternative splicing patterns and the function of IRF7 isoform(s)exist in chickens.In this study,we reported a novel short transcript isoform of chicken IRF7(chIRF7),termed chIRF7-iso,which contained an intact N-terminal DNA-binding domain(DBD)and 14 amino acids different from chIRF7 in the C-terminal.Overexpression of chIRF7 in chicken leghorn male hepatocellular(LMH)cells activated the IFN-βpromoter and significantly inhibited Newcastle disease virus(NDV)and fowl adenovirus serotype 4(FAdV-4)replication.Conversely,overexpression of chIRF7-iso blocked the IFN-βpromoter activity and was favorable for NDV and FAdV-4 replication in vitro.Collectively,our results confirm that a novel chIRF7 isoform-mediated negative regulates IFN-βproduction,which will contribute to understanding the role of chIRF7 in innate antiviral response in chicken.

Long noncoding RNA negative regulator of antiviral response contributes to pancreatic ductal adenocarcinoma progression via targeting miR-299-3p

BACKGROUND Pancreatic ductal cancer(PDAC)has high malignancy and poor prognosis.Long noncoding RNAs(lncRNAs)are associated with high levels of malignancy,including PDAC.However,the biological and clinical significance of negative regulator of antiviral response(NRAV)in PDAC is unclear.AIM To study the regulatory role of lncRNA NRAV in PDAC.METHODS GEPIA analyzed lncRNA NRAV and miRNA(miR-299-3p)expression levels in PDAC tissues and measured them in PDAC cells by quantitative measurements in real time.The specific role of NRAV and miR-299-3p in cell proliferation and transfer potential was evaluated by cell formation analysis,Cell Counting Kit-8 and Transwell analysis.The relationship between NRAV and miR-299-3p was studied by predictive bioinformatics,RNA immunoassay,and fluorescence enzyme analysis.In vivo experiments included transplantation of simulated tumor cells under naked mice.RESULTS The expression level of lncRNA NRAV was higher in both tumor tissues and cell lines of PDAC and was negatively associated with the clinical survival of PDAC patients.Functionally,overexpression of NRAV promoted cell proliferation and metastasis of PDAC cells,while knockdown of NRAV reversed these effects.Finally,NRAV was performed as a molecular sponge of miR-299-3p.Moreover,overexpression of miR-299-3p could reverse the promoting effects of NRAV on cell proliferation and metastasis of PDAC cells.CONCLUSION NRAV facilitates progression of PDAC as a molecular sponge of miR-299-3p and may be a potential molecular marker for diagnosis and treatment of PDAC.

Dominating expression of negative regulatory factors downmodulates major histocompatibility complex Class-Ⅱexpression on dendritic cells in chronic hepatitis C infection

AIM: To elucidate the molecular mechanisms leading to development of functionally impaired dendritic cells(DCs) in chronic hepatitis C(CHC) patients infected with genotype 3 virus.METHODS: This prospective study was conducted on the cohorts of CHC individuals identified as responders or non-responders to antiviral therapy. Myeloid DCs were isolated from the peripheral blood of each subject using CD1c(BDCA1)+ DC isolation Kit. Monocytes from healthy donor were cultured with DC growth factors such as IL-4 and GM-CSF either in the presence or absence of hepatitis C virus(HCV) viral proteins followed by LPS stimulation. Phenotyping was done by flowcytometry and gene expression profiling was evaluated by real-time PCR.RESULTS: Non-responders [sustained virological response(SVR)-ve] to conventional antiviral therapy had significantly higher expression of genes associated with interferon responsive element such as IDO1 and PD-L1(6-fold) and negative regulators of JAK-STAT pathway such as SOCS(6-fold) as compared to responders(SVR+ve) to antiviral therapy. The downregulated genes in non-responders included factors involved in antigen processing and presentation mainly belonging to major histocompatibility complex(MHC) Class-Ⅱ family as HLA-DP, HLA-DQ(2-fold) and superoxide dismutase(2-fold). Cells grown in the presence of HCV viral proteins had genes downregulated for factors involved in innate response, interferon signaling, DC maturation and co-stimulatory signaling to T-cells, while the genes for cytokine signaling and Toll-like receptors(4-fold) were upregulated as compared to cells grown in absence of viral proteins.CONCLUSION: Underexpressed MHC class-Ⅱ genes and upregulated negative regulators in non-responders indicate diminished capacity to present antigen and may constitute mechanism of functionally defective state of DCs.

The43,000Growth - associated Protein Functions as a Negative Growth Regulator in Glioma.

Previous molecular analyses of human astrocytomas have identified many genetic changes associated with astrocy-toma formation and progression.In an effort to identify novel gene expression changes associated with astrocytomaformation,which might reveal new potential targets for glioma therapeutic drug design,we used the B8-RAS-transgenic mouse astrocytoma model.Using multiplex gene expression profiling,we found that

NRL3 Interacts with OsK4 to Regulate Heading Date in Rice

NRL3 is essential for the growth and development of rice leaves.In this study,we found that the loss function of NRL3 also delayed heading date under natural long daylight and short daylight conditions.The yeast two-hybrid and the bimolecular fluorescence complementation proved that NRL3interacts with OsK4,a Snf1-related kinase.OsK4 localized to the nucleus and expressed in various rice tissues.The rhythmic expression pattern of Os K4 was similar to NRL3 under long daylight and short daylight conditions.Knock-out mutants of Os K4 exhibited early heading under long daylight conditions,indicating that it acts as a negative regulator of heading date in rice.Interestingly,the OsK4 mutant under the nrl3 mutant background rescued the late heading phenotype of nrl3 under long daylight conditions,suggesting that Os K4 functions downstream of NRL3.Moreover,both NRL3 and Os K4 controlled heading date through regulating the expression of Hd3a and RFT1 genes.These findings shed light on the heading date regulation in rice and provide a sound theoretical base to improve regional adaptability of rice.

A Study on the Molecular Switch of Gene Expression of the Mouse Heart Nuclear DNA Fragments

It is observed by in situ stain that LDH (1 5) ...nNAD + can probably enter the nucleopore and can be bound bound specifically with the genes that encode them. During the in vitro expression, the dilution of heart nuclear DNA fragments could enhance the expression activity of LDH/DNA and the amount of expressed LDH (1 5) is in proportion to the amount of dissociable LDH (1 5) on the LDH/DNA. With the integration of 14C Leu to the proteins, it is also observed that the addition of LDH (1 5) ...nNAD + can suppress the in vitro expression activity of LDH/DNA. AFM observation shows that the regulation sequence at the both ends of active genes may be bound with such active factors as proteins encoded by the genes which probably is the main molecular switch of gene expression and regulation we have been always searching for. Our work shows the prospective application of the combination of AFM and isotope labeling in the research of biological reaction.

Synergistic and feedback signaling mechanisms in the regulation of inflammation in respiratory infections

Pneumonia, the most typical and frequent lower respiratory tract infection （LRTI）, is a leading cause of health problems in the United States. Bacteria represent the most prevailing cause of pneumonia in both children and adults. Although pneumonia with a single bacterial infection is common, a significant portion of patients with pneumonia is polymicrobial. This infection is often complexed with other physiological factors such as cytokines and growth factors. Nontypeable Haemophilus influenzae （NTHi） is the most frequently recovered Gram-negative bacterial pathogen in the respiratory system and induces strong inflammatory responses. NTHi also synergizes with other respiratory pathogens, such as Streptococcus pneumoniae and respiratory viruses and pro-inflammatory cytokines, such as tumor necrosis factor-alpha （TNF-α）. It is noteworthy that NTHi not only synergizes with growth factors such as transforming growth factor-beta （TGF-β）, but also utilizes growth factor receptors such as TGF-β receptor and epidermal growth factor receptor （EGFR）, to enhance inflammatory responses. Although appropriate inflammation is a protective response against invading pathogens, an uncontrolled inflammatory response is often detrimental to the host. Thus, inflammation must be tightly regulated. The human immune system has evolved strategies for controlling overactive inflammatory response. One such important mechanism is via regulation of negative feedback regulators for inflammation. CYLD, a multifunctional deubiquitinase, was originally reported as a tumor suppressor, but was recently identified as a negative regulator for nuclear factor-kappa B （NF-κB） signaling. It is induced by NTHi and TNF-α via a NF-κB-dependent mechanism, thereby serving as an inducible negative feedback regulator for tightly controlling inflammation in NTHi infection.

MicroRNA negatively regulates NF-κB-mediated immune responses by targeting NOD1 in the teleost fish Miichthys miiuy

Inflammation is a self-protection mechanism that can be triggered when innate immune cells detect infection.Eradication of pathogen infection requires appropriate immune and inflammatory responses,but excessive inflammatory responses can cause uncontrolled inflammation,autoimmune diseases,or pathogen dissemination.Mounting evidence has shown that microRNAs(miRNAs)in mammals act as important and versatile regulators of innate immunity and inflammation.However,miRNAmediated regulation networks are largely unknown in inflammatory responses in lower vertebrates.Here,miR-144 and miR-217 are identified as negative regulators in teleost inflammatory responses.We find that Vibrio harveyi and lipopolysaccharide(LPS)treatment significantly upregulate the expression of fish miR-144 and miR-217.Upregulated miR-144 and miR-217 suppress LPS-induced inflammatory cytokine expression by targeting nucleotide-binding oligomerization domain-containing protein 1(NOD1),thereby avoiding excessive inflammatory responses.In addition,miR-144 and miR-217 regulate inflammatory responses through NOD1-induced nuclear factor kappa(NF-κB)signaling pathways.These findings demonstrate that miR-144 and miR-217 play regulatory roles in inflammatory responses by modulating the NOD1-induced NF-κB signaling pathway.

ISG60 Negatively Regulates Cell Antiviral Responses by Disrupting the VISA-Associated Complexes

Viral infection activates the transcription factors NF-KB and interferon regulatory factor 3 （IRF3）, which collaborate to induce type I interferons （IFNs） and initiate host innate antiviral response. IFN-stimulated gene 56 （ISG56） induced by type I IFNs is a negative regulator of cellular antiviral response. In this study, we identified ISG60 as an ISG56-associated protein by biochemical purification and mass spectrometry analysis. Overex- pression of ISG60 inhibited Sendai virus-induced activation of NF-KB and IRF3. Coimmunoprecipitation assays indicated that ISG60 interacted with MDA5 and VISA, two important signaling proteins participating in virus-triggered production of type I IFNs. Furthermore, ISG60 disrupted the interaction of VISA with MDA5 or RIG-I. These results indicate that ISG60 is a negative regulator of virus-triggered type I IFNs induction.

CPC, a Single-Repeat R3 MYB, Is a Negative Regulator of Anthocyanin Biosynthesis in Arabidopsis

Single-repeat R3 MYB transcription factors like CPC （CAPRICE） are known to play roles in developmental processes such as root hair differentiation and trichome initiation. However, none of the six Arabidopsis single-repeat R3 MYB members has been reported to regulate flavonoid biosynthesis. We show here that CPC is a negative regulator of anthocyanin biosynthesis. In the process of using CPC to test GAL4-dependent driver lines, we observed a repression of anthocyanin synthesis upon GAL4-mediated CPC overexpression. We demonstrated that this is not due to an increase in nutrient uptake because of more root hairs. Rather, CPC expression level tightly controls anthocyanin accumulation. Microarray analysis on the whole genome showed that, of 37 000 features tested, 85 genes are repressed greater than three-fold by CPC overexpression. Of these 85, seven are late anthocyanin biosynthesis genes. Also, anthocyanin synthesis genes were shown to be down-regulated in 35S：：CPC overexpression plants. Transient expression results suggest that CPC competes with the R2R3-MYB transcription factor PAP1/2, which is an activator of anthocyanin biosynthesis genes. This report adds anthocyanin biosynthesis to the set of programs that are under CPC control, indicating that this regulator is not only for developmental programs （e.g. root hairs, trichomes）, but can influence anthocyanin pigment synthesis.

ROP11 GTPase is a Negative Regulator of Multiple ABA Responses in Arabidopsis

The phytohormone abscisic acid （ABA） plays crucial roles in plant development and plant responses to environmental stresses. Although ABA receptors and a minimal set of core molecular components have recently been discovered, understanding of the ABA signaling pathway is still far from complete. In this work, we characterized the function of ROP11, a member of the plant-specific ROP small GTPases family, in the ABA signaling process. ROP11 is preferentially expressed in guard cells in all plant organs with stomata. Expression of a constitutively active ROP11 （CA-ROP11） suppresses ABA-mediated responses, whereas reduced expression of ROP11 or expression of its dominant-negative form （DN-ROP11） causes the opposite phenotypes. The affected ABA-mediated responses by ROP11 include seed germination, seedling growth, stomatal closure, induction of ABA-responsive genes, as well as plant response to drought stress. Furthermore, we showed that ROP11 and its closest-related family member, ROP10, act in parallel in mediating these responses. ABA treatment does not affect ROP11 transcription and protein abundance; however, it causes the accumulation of CA-ROP11 in the nucleus. These results demonstrated that ROP11 is a negative regulator of multiple ABA responses in Arabidopsis.

A Dual-Function Transcription Factor, AtYY1, Is a Novel Negative Regulator of the Arabidopsis ABA Response Network

Abscisic acid （ABA） plays crucial roles in plant growth and development, as well as in response to various environmental stresses. To date, many regulatory genes involved in the ABA response network have been identified; however, their roles have remained to be fully elucidated. In this study, we iden- tified AtYY1, an Arabidopsis homolog of the mammalian C2H2 zinc-finger transcription factor Yin Yang 1 （YY1）, as a novel negative regulator of the ABA response. AtYY1 is a dual-function transcription factor with both repression and activation domains. The expression of AtYY1 was induced by ABA and stress conditions including high salt and dehydration. The yyl mutant was more sensitive to ABA and NaCI than the wild-type, while overexpressing AtYY1 plants were less sensitive. AtYY1 loss also enhanced ABA-induced stomatal closing and drought resistance. Moreover, AtYYI can bind the ABA REPRESSOR1 （ABR1） promoter and directly upregulate ABR1 expression, as well as negatively regulate ABA- and saR-responsive gene expression. Additional analysis indicated that ABA INSENSITIVE4 （ABI4） might positively regulate AtYY1 expression and that ABR1 can antagonize this regulation. Our findings provide direct evidence that AtYY1 is a novel negative regulator of the ABA response network and that the ABI4-AtYY1-ABR1 regulatory pathway may fine-tune ABA-responsive gene expression in Arabidopsis.

The Salicylic Acid Receptor NPR3 Is a Negative Regulator of the Transcriptional Defense Response during Early Flower Development in Arabidopsis

Arabidopsis NON-EXPRESSOR OF PR1 （NPR1） is a transcription co-activator that plays a central role in regulating the transcriptional response to plant pathogens. The NPR family consists of NPR1 and five NPRl-like genes. The NPR1 paralog NPR3 has recently been shown to function as a receptor of the plant hormone salicylic acid and to mediate proteosomal degradation of NPR1. The function of NPR3 protein during early flower development was revealed through a detailed molecular-genetic analysis including promoter transcriptional fusion analysis, phenotype characterization of npr3-3 mutants/overexpressors, and whole-plant fitness analysis. The physical interaction between NPR3 and NPR1/ TGA2 was explored using bimolecular fluorescence complementation analysis in onion epidermal cells, Here, we show that NPR3 expression was strongest in the petals and sepals of developing flowers and declined after flower opening. Consistently with this observation, an npr3 knockout mutant displayed enhanced resistance to Pseudomonas syringae infection of immature flowers, but not leaves. Developing npr3 flowers exhibited increased levels of basal and induced PR1 transcript accumulation. However, the npr3 mutant showed lower fitness compared to Col-0 in the absence of pathogen. Moreover, NPR3 was shown to interact with NPR1 and TGA2 in vivo. Our data suggest that NPR3 is a negative regulator of defense responses during early flower development and it may function through the association with both NPR1 and TGA2.

Interferon regulatory factor 3-CL,an isoform of IRF3,antagonizes activity of IRF3

Interferon regulatory factor 3(IRF3),one member of the IRF family,plays a central role in induction of type I interferon(IFN)and regulation of apoptosis.Controlled activity of IRF3 is essential for its functions.During reverse transcription(RT)-PCR to clone the full-length open reading frame(ORF)of IRF3,we cloned a full-length ORF encoding an isoform of IRF3,termed as IRF3-CL,and has a unique carboxyl-terminus of 125 amino acids.IRF3-CL is ubiquitously expressed in distinct cell lines.Overexpression of IRF3-CL inhibits Sendai virus(SeV)-triggered induction of IFN-β and SeV-induced and inhibitor of NF-kB kinase-e(IKKε)-mediated nuclear translocation of IRF3.When IKKε is overexpressed,IRF3-CL is associated with IRF3.These results suggest that IRF3-CL,the alternative splicing isoform of IRF-3,may function as a negative regulator of IRF3.

Programmed Death Ligand-1 on Microglia Regulates Th1 Differentiation via Nitric Oxide in Experimental Autoimmune Encephalomyelitis

Microglia are considered to be potential anti- gen-presenting cells and have the ability to present antigen under pathological conditions. Nevertheless, whether and how microglia are involved in immune regulation are lar- gely unknown. Here, we investigated the suppressive activity of microglia during experimental autoimmune encephalomyelitis （EAE） induced by myelin oligodendro- cyte glycoprotein, with the goal of understanding their role in regulating the T cell reaction. Using flow cytometric analysis, we found that microglia were characterized by increased cell number and up-regulated programmed death ligand-1 （PD-L1） at the peak phase of EAE. Meanwhile, both the CD4＋ T cells and microglia that infiltrated the central nervous system expressed higher levels of PD1, the receptor for PD-L1, accompanied by a decline of Thl cells. In an ex vivo co-culture system, microglia from EAE mice inhibited the proliferation of antigen-specific CD4＋ T cells and the differentiation of Thl cells, and this was significantly inhibited by PD-L 1 blockade. Further, microglia suppressed Thl cells via nitric oxide （NO）, the production of which was dependent on PD-L1. Thus, these data suggest a scenario in which microglia are involved in the regulation of EAE by suppressing Thl-cell differenti- ation via the PD-L1-NO pathway.