Protein ubiquitination regulated by ubiquitin ligases plays important roles in innate immunity. However, key regulators of ubiquitination during innate response and roles of new types of ubiquitination (apart from Lys48- and Lys63-linkage) in control of innate signaling have not been clearly understood. Here we report that F-box only protein Fbxo21, a functionally unknown component of SCF (Skp1–Cul1–F-box protein) complex, facilitates Lys29-linkage and activation of ASK1 , and promotes type I interferon production upon viral infection. Fbxo21 deficiency in mice cells impairs virus-induced Lys29-linkage and activation of ASK1, attenuates c-Jun N-terminal kinase (JNK) and p38 signaling pathway, and decreases the production of proinflammatory cytokines and type I interferon, resulting in reduced antiviral innate response and enhanced virus replication. Therefore Fbxo21 is required for ASK1 activation via Lys29-linkage of ASK1 during antiviral innate response, providing mechanistic insights into non-proteolytic roles of SCF complex in innate immune response.DOI:http://dx.doi.org/10.7554/eLife.14087.001 The innate immune system is the body’s first line of defense against being infected by viruses and other microbes. Upon recognizing a virus, host cells trigger the innate immune response in an effort to eliminate the threat. However, innate immune responses must be carefully controlled because an excessive response can cause inflammation that harms the body.The innate immune response involves a variety of cells and processes that are each activated through a series of communication systems called signaling pathways. While much has been learned about which parts of a virus trigger the innate immune response, it is not clear how the immune response to the virus is controlled.It has been suggested that a process known as ubiquitination could be involved in regulating the activity of signaling pathways that activate the innate immune response. During ubiquitination, enzymes attach a small molecule called ubiquitin to a specific target protein. Ubiquitin often acts as a label that targets a particular protein for destruction. Enzymes called E3 ubiquitin ligases play central roles in identifying specific target proteins for ubiquitination. Some of these enzymes consist of a single protein unit that acts alone, but other E3 ubiquitin ligases are formed by groups (or “complexes”) of several proteins working together.Members of the F-box only protein family are components of some ubiquitin ligase complexes. Here, Yu et al. used a “microarray” technique to assess which F-box only proteins in mice are produced during an immune response to two viruses. The experiments identified an F-box protein called Fbxo21 as a potential candidate for a role in regulating the innate immune response.Additional experiments revealed that Fbxo21 is involved in adding ubiquitin to a specific location on a signaling protein called ASK1, which is known to be crucial for innate immune responses. Instead of targeting ASK1 for destruction, this ubiquitination activates ASK1. Therefore, Yu et al.’s findings demonstrate that Fbxo21 plays an important role in regulating innate immune responses. A future challenge is to investigate exactly how ASK1 is activated by the ubiquitin.DOI:http://dx.doi.org/10.7554/eLife.14087.002 Innate immune system serves as the first line defense of invading microorganisms and endogenous danger signals. Host cells can recognize pathogen-associated molecular patterns (PAMPs) of invading pathogens via pattern recognition receptors (PRRs), initiate signaling pathways to regulate the expression of proinflammatory cytokines and type I interferons (IFN α/β) . Upon viUbiquitin (Ub) modification is one type of the crucial posttranslational modification mechanisms, and has been implicated in regulation of both innate and adaptive immunity . As compC. elegans and drosophila to mammals (C. elegans (NSY-1) and drosophila (DASK1) are important in control of apoptosis or innate immunity  is a mitogen-activated protein kinase (MAPK) kinase kinase that plays pivotal roles in stress and immune responses . ASK1 ca mammals . ASK1 hoimmunity . So ASK1Fbxo21), is required for the production of inflammatory cytokines and type I IFNs upon vesicular stomatitis virus (VSV) and herpes simplex virus 1 (HSV-1) infection. Our study suggests that Fbxo21 facilitates non-proteolytic Ub-modification of ASK1, adding insights into the mechanistic understanding of antiviral innate response.Previously we have investigated the roles of polyubiquitination in the regulation of innate signaling and innate cytokine production . To furtIfnb expression in peritoneal macrophages (PMs) with knockdown of indicated Fbxo molecules , poly (dA:dT) and poly (dG:dC), or infection with VSV and HSV-1 , the type I IFNs-associated IRF3, but not the NF-κB-associated IKKα/β-IκBα  of endogenous Fbxo21 in RAW264.7 cells plus liquid chromatography mass spectrometry (LC-MS) assays. The MS data indicated that Fbxo21 could interact with ASK1 . Using IWe then mapped the domains in Fbxo21 and ASK1 mediating the interaction. After transfection of Fbxo21 with ASK1 (mutants) plasmids  in HEK29Fbxo21).To validate that Fbxo21 is a component of the SCF complex, we examined the endogenous Skp1, Cul1 and Rbx1 in Fbxo21 immune complexes, and found that Skp1, Cul1 and Rbx1 could be coimmunoprecipitated with Fbxo21 . Then weFbxo21 affected the signaling pathways via ASK1. Since SCF complexes usually mediate the Ub-modification and proteasome degradation of substrates  could be linked to ASK1 by Fbxo21 , is required for Lys29-linkage of ASK1.We then analyzed the Ub-modification type of ASK1 by SCFy Fbxo21 . In Fbxonfection . When thnfection , we founnfection , indicatin vitro polyubiquitination assays. In the presence of recombinant GST-Fbxo21, ASK1 and Cul1-Rbx1-Skp1, as well as the NEDD8 conjugation system (neddylation), ASK1 could be efficiently polyubiquitinated; while in the presence of MLN-4924, the polyubiquitination of ASK1 was significantly inhibited  specific for Lys29-linkage of Ub chains have not been identified, we utilized conjugation fraction A that contains predominantly E1 and E2 enzymes instead of defined E2s in the in vitro-/-Fbxo21 RAW264.7 cells reconstituted with Fbxo21 and infected with VSV or HSV-1, we found that wild type ASK1 but not K13-ASK1 could be polyubiquitinated  by CRISPR-Cas9 genome editing , we found that ASK1, but not K13-ASK1 or ASK1-K716A, could rescue the impaired IL-6 and IFNβ production and the inhibited antiviral response  in regulating kinase activation, and suggests that SCF complexes may not only mediate degradation of protein substrates but non-proteolytic regulation of substrates. Considering that ASK1 is a remarkably conserved kinase crucial in defense of infection  and SCFFbxw11 (β-Trcp2) that mediate the processing of NF-κB components and degradation of IκB isoforms  were purchased from Joint Ventures Sipper BK Experimental Animal . All animal experiments were undertaken in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, and with the approval of the Scientific Investigation Board of Second Military Medical University, Shanghai. HEK293, RAW264.7 and L929 cells were obtained from ATCC  and cultured as recommended. Peritoneal macrophages and bone marrow-derived macrophages were obtained, prepared and cultured as described . RecombiMap3k5 or Fbxo21, the siRNA duplexes specific for Map3k5  or Fbxo21 (5’ GCAGAAAGCTGGGTTAGAA 3’) were transfected using the INTERFERin-HTS according to the standard protocol . The nonsense sequence 5’-TTCTCCGAACGTGTCACG-3’ was used as control siRNA.The recombinant vectors encoding mouse ASK1 (GenBank No. NM_008580.4), Fbxo21 (GenBank No. NM_145564.3) and the indicated mutations were constructed as described . Flag-taFbxo21 and Map3k5 were tested, and the target sequences  were mostly efficient in depletion of Fbxo21 and ASK1. Cells with both red and green fluorescence were then sorted by using Gallios Flow Cytometer . Sorted cells were cultured for 3–5 days, and clones propagated from single cell were picked out. The depletion of Fbxo21 or ASK1 was confirmed by both Western blot and DNA sequencing.For the depletion of Fbxo21 or ASK1, pc3-U6-guide RNA-CMV-RED (encoding guiding RNA and red fluorescent protein) and Cas9-IRES-EGFP (encoding Cas9 and green fluorescent protein) plasmids  were cotransfected into RAW264.7 or L929 cells . Four taβ-actin was used as the internal quantitative control.Total cellular RNA was extracted using Trizol reagent , and cDNA was synthesized by using the AMV Reverse Transcriptase kit . Specific primers used for Q-PCR assays and the mRNA quantification methods were as described . β-actinMicroarray expression analysis was performed using a high-density oligonucleotide array  as described , and micELISA kits for mouse IFNβ and IL-6 were from R&D Systems . The concentrations of cytokines in the culture supernatants were determined as described .The AP-1, NF-κB, IRF3 and IFNβ luciferase reporter plasmids were obtained from Panomics of Affymetrix  or as described . LuciferFor propagation of VSV and HSV-1, Vero cells were infected with virus at a MOI of 2, and the viruses produced by one replication cycle were harvested. For purification of virus, the cell cultures were first freeze-thawed twice, and the supernatants were clarified by centrifugation at 3000 ×g for 1 hr, followed by pelleting of the virus by ultracentrifugation at 45,000 ×g for 1 hr. For quantification of virus in infected cells, cells were homogenized in MEM supplemented with 2% FCS just before use. The homogenates were pelleted by centrifugation at 1600 ×g for 30 min, and the supernatants (100 μl) were used for plaque assays on monolayers of Vero cells. The cells were left overnight to settle and were infected by incubation for 1 hr at 37°C with serial dilutions. After 1 hr, 1 ml of medium was added, and the plates were incubated for 2 days. After incubation, the plates were stained with 0.03% methylene blue to allow quantification of plaques. The numbers of plaque formation unit (PFU) were counted, and the results were expressed as PFU/ml of homogenates.Immunoprecipitated Fbxo21 immune complexes were separated on SDS-PAGE gel as described previously . After sThe immunoprecipitations using anti-Fbxo21, anti-Flag or anti-Myc, and the immunoblot assays were performed as described previously .AM Transcription Factor ELISA Kits from Active Motif Inc.  as recommended.Nuclear proteins were extracted by NE-PER Protein Extraction Reagent  according to the manufacturer’s instructions. Protein concentration was determined by the BCA protein assay (Pierce). For the analysis of nuclear transcription factors that could bind with specific DNA sequences, nuclear extracts were examined for c-fos, IRF3 and p65 by using the TransFbxo21 were inserted 3’ and in frame to glutathione S-transferase (GST) coding sequence in pGEX-2T vector . Escherichia coli BL21 (DE3) cells  harboring the recombinant expression vectors were incubated with 1 mM isopropyl β-D-thiogalactopyranoside  at 37°C for 3 hr. For GST fusion protein purification, cells were harvested by centrifugation and suspended in 10 ml PBST containing 2 mM EDTA, 0.1% β-mercaptoethanol, 0.2 mM PMSF, and 5 mM benzamidine. Cells were lysed by passing through a French press twice at 1200 lb/in2. 1 ml of bacterial supernatant was mixed with 100 μl 50% (v/v) glutathione-agarose beads  and incubated for 30 min at 4°C with gentle rotating. The agarose beads were washed four times with 10 ml ice-cold PBST or lysis buffer. The fusion proteins were eluted by 100 μl of 10 mM glutathione in 50 mM Tris (pH 8.0) at 4°C. The purity and quantity of fusion proteins were examined by SDS-PAGE followed by Coomassie blue staining.Mouse wild-type or truncation coding sequences for The GST pull-down assays were conducted as described previously .3VO4 and 1 mM PMSF). Each washed pellet was resuspended in the kinase assay buffer  and supplemented with 10 μM ATP and 0.5 μg of recombinant MKK4 (Abcam) in a total volume of 30 μl at 30°C for 30 min. Then phosphorylated MKK4 was examined by Western blot.For in vitro kinase assays, 100 μg proteins contained in total cell extracts or nuclear extracts were immunoprecipitated with indicated antibodies plus protein A/G beads by gently rocking at 4°C for 2 hr followed by centrifugation at 4°C for 5 min. The pellets were washed with the lysis buffer  Nonidet-P40, 0.5% (wt/vol) sodium deoxycholate, 1% (wt/vol) SDS and proteinase inhibitors) supplemented with 10 mM N-ethylmaleimide, were immunoprecipitated with indicated antibodies and analyzed by immunoblot as described .2 supplemented with 2 mM ATP, 1 μM ubiquitin aldehyde, 1 μg Conjugation Fraction A , 1 μg/μl ubiquitin and derivatives (BostonBiochem), 10 ng/μl recombinant ASK1 (MAP3K5-9515M) and Cul1-Rbx1-Skp1 (CUL1-147H)  and 10 ng/μl GST-Fbxo21 in the presence or absence of Nedd8 Conjugation System  and 5 μM MLN-4924 for 30 min at 37°C. Reaction products were finally processed for Ub immunoblotting.The in vitro analysis of ubiquitination of ASK1 was performed as described  with modAll the experiments were independently repeated at least three times. Dr. X.Z. who is blinded to group design performed the analysis. Results are given as mean ± SE or mean ± SD. Multiple comparisons were done with one-way ANOVA followed by Bonferonni’s multiple comparison test. Statistical significance was determined as p<0.05.Complete microarray data set, GSE72077. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Fbxo21 is required for antiviral innate response" for consideration by eLife. Your article has been reviewed by two peer reviewers, Hidenori Ichijo and Michaela Gack, and the evaluation has been overseen by Ruslan Medzhitov as the Reviewing Editor and Tadatsugu Taniguchi as the Senior Editor.Thank you for submitting your work entitled "Lys29-linkage of ASK1 by SCFThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Fbxo21, the authors conclude that Fbxo21 acts by targeting six lysine residues  in ASK1 for SCF-mediated non-degradative K29-linked ubiquitination. Finally, the authors provide data which show that K29-linked ubiquitination of ASK1 is critical for downstream activation of JNK1/2, p38, and IRF3 and thus cytokine responses to infection with VSV and HSV-1. The authors propose a model in which Fbxo21 targets ASK1 for K29-linked ubiquitination by the SCF complex, which enables ASK1 to activate various innate immune signaling molecules, ultimately resulting in the transcription of proinflammatory cytokines and interferons.In this manuscript, Yu et al. show that the F-box only protein, Fbxo21, is a component of the E3 ubiquitin ligase SCF complex and is critical for the innate immune response to viral infection. A microarray-based screen of Fbxo protein expression levels during viral infection identified Fbxo21 as being potentially involved in innate immune signaling. Using Fbxo21 knockout cells, the authors show that Fbxo21 plays an important role in the activation of innate immune signaling, specifically the production of cytokines and type I interferon, in response to viral infection. Using mass spectrometry analysis, the authors identify the protein kinase ASK1 as an interaction partner of Fbxo21. Through extensive characterization of ASK1 ubiquitination by SCFEssential revisions:1) Although it is convincing that Fbxo21 has an ability to lead Lys29-linked ubiquitination of ASK1 shown in 2) I recommend one additional experiment to strengthen the authors' model that Fbxo21 is a component of the SCF complex during viral infection.The experiments intended to demonstrate that Fbxo21 is part of the SCF complex were performed by overexpressing tagged forms of individual SCF components. Performing an endogenous IP of SCF components and Fbxo21 in uninfected and virus-infected cells would strengthen the authors' point that Fbxo21 is indeed part of the SCF complex during viral infection. Essential revisions:1) Although it is convincing that Fbxo21 has an ability to lead Lys29-linked ubiquitination of ASK1 shown in Fbxo21+/+ or Fbxo21–/– RAW264.7 cells upon virus infection in the presence or absence of MLN-4924 as suggested. Moreover, we also examined the dynamic poly-Ub modification  and dynamic activation of ASK1 in Fbxo21+/+ or Fbxo21–/– RAW264.7 cells. These new data are now presented in Fbxo21 is required for ASK1 activation”.The net effects of Fbxo21 deficiency on Lys29-linkage of ASK1 have not been directly demonstrated because of the lack of Lys29-specific antibody. To address this concern, we examined the phosphorylation and kinase activity of ASK1 in 2) I recommend one additional experiment to strengthen the authors' model that Fbxo21 is a component of the SCF complex during viral infection.The experiments intended to demonstrate that Fbxo21 is part of the SCF complex were performed by overexpressing tagged forms of individual SCF components. Performing an endogenous IP of SCF components and Fbxo21 in uninfected and virus-infected cells would strengthen the authors' point that Fbxo21 is indeed part of the SCF complex during viral infection.As suggested, we performed the assays of endogenous interactions of Fbxo21 with Skp1, Cul1 and Rbx1 in wild type RAW264.7 cells before and after VSV or HSV-1 infection. The data are now presented in


Background. The ability of epithelial cells (ECs) to discriminate between commensal and pathogenic microbes is essential for healthy living. Key to these interactions are mucosal epithelial responses to pathogen-induced damage.Methods. Using reconstituted oral epithelium, we assessed epithelial gene transcriptional responses to Candida albicans infection by microarray. Signal pathway activation was monitored by Western blotting and transcription factor enzyme-linked immunosorbent assay, and the role of these pathways in C. albicans–induced damage protection was determined using chemical inhibitors.Results. Transcript profiling demonstrated early upregulation of epithelial genes involved in immune responses. Many of these genes constituted components of signaling pathways, but only NF-κB, MAPK, and PI3K/Akt pathways were functionally activated. We demonstrate that PI3K/Akt signaling is independent of NF-κB and MAPK signaling and plays a key role in epithelial immune activation and damage protection via mammalian target of rapamycin (mTOR) activation.Conclusions. PI3K/Akt/mTOR signaling may play a critical role in protecting epithelial cells from damage during mucosal fungal infections independent of NF-κB or MAPK signaling. The ability of mucosal surfaces to discriminate commensal from pathogenic organisms is critical in maintaining health. A distinguishing feature of mucosal pathogens over commensals is their ability to cause epithelial damage  and aparInnate immune response activation is mediated by pattern recognition receptors (PRRs) via intracellular signal pathways, including nuclear factor kappa B (NF-κB), phosphoinositide 3 kinase (PI3K), mitogen-activated protein kinase (MAPK) pathways , 3, nuclCandida albicans is a normal microbiota constituent in approximately 50% of individuals but causes mucosal diseases with significant morbidity in immunocompromised hosts , and Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) signaling [4]). There is also significant enrichment in other pathways . Analysis of MetaCore Process Networks enrichment  provides further evidence of immune activation, with Th17-derived cytokines (6.5), ERBB-family signaling (5.3), MIF signaling (3.6), and innate inflammatory response (3.6) among the 10 most enriched processes. Several cell survival processes show enrichment . These data suggest that ECs mount an immediate and robust response to C. albicans infection.At 6 hours, several pathways showed significant enrichment in gene expression Figure A, includA), and analysis of enriched MetaCore Process Networks identifies several DNA damage-associated processes (Base Excision Repair-Nucleotide Excision Repair (BER-NER) repair [8.9], DNA Mismatch Repair (MMR) repair [6.3], DBS repair [4.9], and core processes [3.9]) . Furthermore, there is enrichment of networks involved in cell cycle (DNA production (S phase [4.2]), likely linked with DNA repair processes. Along with enrichment in mitochondrial translation genes (translation in mitochondria [2.1]), these may represent a continuation of earlier apoptotic processes. Other enriched processes suggest changes in intercellular communication  and stress responses (response to hypoxia and oxidative stress [2.2]). Together, these data suggest a general response to an invasive, damage-inducing pathogen.The responses 24 hours postinfection are more heterogeneous, reflecting the variety of stimuli affecting ECs at this time point. The most enriched MetaCore pathways include those involved in metabolism , indicating that these pathways are functional in TR146 cells. STAT3 phosphorylation was not detected up to 2 hours postinfection (data not shown), and STAT1 DNA binding and transcriptional activity showed no increase at 30 minutes or 3 hours postinfection . Finally, although NFAT signaling is associated with myeloid cell responses to C. albicans [3B). Together, these data suggest that IRF3, JAK/STAT, and NFAT signaling play no role in EC responses to C. albicans infection.As well as NF-κB and MAPK signaling , the mic 2 hours ) in mono 2 hours , IRF3 ph 2 hours . To confC. albicans–infected TR146 oral ECs demonstrated increased PDK1 phosphorylation as early as 5 minutes postinfection, peaking at 2 hours  with a matching increase in Akt phosphorylation . We also observed increases in GSK-3β phosphorylation with similar kinetics .Microarray analysis demonstrated several PI3K-activating receptor-ligand interactions , whereas LY294002 additionally reduced G-CSF levels (P < .001). In contrast, IL-1α production was increased, although not significantly , whereas IL-6 production was unaffected. Given that IL-1α production is associated with damage [C. albicans. Therefore, we analyzed 24 hours postinfection supernatant LDH levels with or without inhibition, finding significantly increased LDH release . Inhibition of p38 signaling had no effect on LDH release , indicating that despite mediating discrimination between C. albicans yeast and hyphae via c-Fos [Because PI3K/Akt signaling plays a role in modulating TLR-induced cytokine production in ECs , we inhih damage , these dia c-Fos , p38 plaP < .01), indicating increased cell damage . As with PI3K/Akt inhibition, levels of G-CSF (P < .05) and GM-CSF (P < .001) were significantly reduced . However, unlike Akt inhibition, there was a significant increase in IL-6 production (P < .05). The increased damage in PI3K/Akt/mTOR-inhibited cells was not due to toxic effects of the inhibitors, as treatment with inhibitors alone did not result in a significant increase in LDH release (data not shown).The Akt target, mammalian target of rapamycin (mTOR), plays an important role in cell survival . Therefoe Figure C. As witd Figure D. HoweveC. albicans infection as previously identified [E and F), indicating that NF-κB and MAPK signaling is independent of PI3K/Akt signaling in response to fungal infection.PI3K/Akt signaling can modulate activation of NF-κB and AP-1 TFs , 25. Thuentified . PI3K/AkC. albicans infection and identify PI3K/Akt signaling in addition to MAPK and NF-κB signaling as the major epithelial response pathways against this fungus. PI3K/Akt signaling is independent of NF-κB and MAPK signaling, and although it plays a minor role in inducing EC proinflammatory responses, PI3K/Akt signaling may play a key role in protecting ECs from C. albicans–induced damage.In this study, we describe the global transcriptional response of oral ECs to C. albicans–infected ROE reveals interesting findings. At 6 hours postinfection, several enriched immune response pathways were related to cytokine and other receptor signaling pathways but, notably, neither TLR or other conventional fungal PRR receptor pathways were enriched, supporting our previous findings implying no role for TLRs or other conventional PRRs in early recognition of C. albicans by ECs [C. albicans infections [TREM-1 expression. Thus, this enrichment possibly represents an increase in PRR signaling-associated gene expression. Enrichment of amphoterin and IL-1 signaling pathways suggests involvement of damage-associated molecular patterns in EC responses to C. albicans. This is expected given the stress/damage resulting from tissue invasion. Together, these pathways probably form the core EC response to fungal invasion and damage. The increase in the protease inhibitor SerpinB2 is interesting but should be interpreted with caution as a role has yet to be identified in host infection responses [The transcriptome of s by ECs . In contfections , 27. Altesponses .C. albicans [The 24-hour pathways and networks show enrichment in cell survival, DNA repair, or metabolism, reflecting differing EC priorities during late infection, predominantly targeting apoptosis and cell growth. Along with increases in tissue remodeling genes, this suggests a general damage repair mechanism, maintaining mucosal barrier integrity. The late appearance of apoptosis genes suggests that apoptotic mechanisms are induced at later stages of infection, as previously reported for albicans , 29 and albicans .dusp1) and other members of this family—HVH3 (dusp5) and MKP3 (dusp6). As with MKP1, these proteins negatively regulate MAPK signaling, but with different specificity, acting to dephosphorylate ERK1/2 [C. albicans infection. Other up-regulated MAPK regulators include 2 Sprouty genes, spry2 and spry4, involved in regulating ERK1/2 signaling [trib1 controlling activation of MAPKs by MAP2Ks [Caenorhabditis elegans [In the intermediate infection stage (6 hours), NF-κB–induced genes were up-regulated, which was expected given this pathway's role in EC antifungal responses . Althouge ERK1/2 . Given tignaling , 32, andy MAP2Ks  and invo elegans .In addition to MAPK regulatory factors, we observed up-regulation of several AP-1 proteins, persisting throughout infection, confirming a central role for AP-1 in EC antifungal responses . In partC. albicans infection, most notably EGR1 and EGR3. These proteins are associated with myeloid cell responses to C. albicans [Along with MAPK and NF-κB pathways, other signaling pathways and TFs show upregulation after albicans , 36, plaalbicans , or NFATalbicans , 31. Othalbicans .C. albicans rather than subsequent secreted components. Despite the microarray evidence, only PI3K/Akt signaling via PDK1/Akt appears to be functionally activated alongside MAPK and NF-κB, with no evidence for activation of IRF, STATs, or NFAT. Required for dectin-1 signaling in myeloid cells [C. albicans [C. albicans infection. Given that PI3K/Akt signaling also protects macrophages against fungal-induced killing [The observed discrepancy between microarrays and functional studies reflects the difference between RNA expression and subsequent protein activation, with many signaling pathways showing crossover in target genes. Microarray data represent an expression snapshot, whereas signal activation data from 0–3 hours postinfection represent EC responses to id cells , 36, thealbicans . Inductialbicans  and the albicans , 40, TLRalbicans , and cytalbicans , 41. Heralbicans , 43. In  killing . EvidencCandida albicans–induced endocytosis in ECs is clathrin-dependent, and PI3K/Akt signaling is thought to be crucial for this process [PI3K/Akt signaling may be involved in other EC response mechanisms connected to damage, for example, fungal invasion.  process  and for  process . Interes process , 46, 47,C. albicans infection studies of both monolayers and organotypic models [It is important to note that this work was carried out using the TR146 carcinoma cell line and thus may not accurately reflect findings in a normal host. However, this cell line has been used extensively in c models , 48–50 ac models .C. albicans hyphae [Our data suggest that the role of PI3K/Akt signaling in hyphal discrimination is minimal but may constitute a major damage protection response in ECs. We therefore propose a model whereby (1) p38/c-Fos signaling identifies s hyphae , 17, (2)s hyphae , 17, (3)s hyphae , 17, andSupplementary materials are available at The Journal of Infectious Diseases online . Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.


Drosophila is composed of differentiated functional cells and groups of imaginal tracheoblasts that build the adult trachea during metamorphosis. Air sac primordium cells (ASP) are tracheal imaginal cells that form the dorsal air sacs that supply oxygen to the flight muscles of the Drosophila adult. The ASP emerges from the tracheal branch that connects to the wing disc by the activation of both Bnl-FGF/Btl and EGFR signaling pathways. Together, these pathways promote cell migration and proliferation. In this study we demonstrate that Vein (vn) is the EGF ligand responsible for the activation of the EGFR pathway in the ASP. We also find that the Bnl-FGF/Btl pathway regulates the expression of vn through the transcription factor PointedP2 (PntP2). Furthermore, we show that the FGF target gene escargot (esg) attenuates EGFR signaling at the tip cells of the developing ASP, reducing their mitotic rate to allow proper migration. Altogether, our results reveal a link between Bnl-FGF/Btl and EGFR signaling and provide novel insight into how the crosstalk of these pathways regulates migration and growth.How several signaling pathways are coordinated to generate complex organs through regulation of tissue growth and patterning is a fundamental question in developmental biology. The larval trachea of  Signaling pathways regulate many processes underlying organogenesis, such as patterning, cell division and growth. However, in order to form a fully functional organ, signaling pathways need to be synchronized during development in a very precise way. One process that requires the coordinated orchestration of multiple signaling pathways is Tubulogenesis. This process underlies the formation of organs such as the mammalian lung, kidney, mammary gland or the insect tracheal system. However, the spatial and temporal regulation of these signaling pathways is poorly understood. Here we demonstrate that the fibroblast growth factor (Bnl/FGF) pathway plays a key role in the regulation of epidermal growth factor (EGFR) signaling during the development of the tracheal system of the adult fly.Drosophila metamorphosis the tracheal system undergoes an extensive remodeling to give rise to the new respiratory structures in the adult organism, such as the air sacs of the dorsal thorax. These structures are dilations of the main tracheal trunks that work as air reservoirs in the adult1During While Bnl/FGF signaling controls ASP migration, EGFR regulates cell proliferation and cell survival1vn abrogates the formation of this organ. In addition we find that the transcription factor esg, a FGF target gene, which is highly expressed at the tip cells, attenuates EGF signaling and thus cell proliferation in this region of the ASP. Importantly, this study reveals a link between Bnl/FGF and EGFR pathways during ASP development and provides new insight into how these signaling pathways coordinate to control cell migration and tissue growth.Here we show that the Bnl/FGF pathway coordinates ASP growth by the activation of the EGF ligand Vn expression through the transcription factor Pointed (Pnt). Our genetic analysis reveal that Vn is the main EGF ligand in the stalk cells of the ASP, as silencing of Drosophila has four EGFR ligands: Spitz (Spi), Keren (Krn), Gurken (Grk) and Vn. Vn is produced as a mature secreted protein, while the other three ligands require maturation by the protease Rhomboid (Rho)grk is a specific ligand of EGFR in oogenesisKrn has a minor role as homozygous Krn mutant flies are viableDrosophila EGFR ligands is responsible for EGFR pathway activation in the ASP we disrupted spi and vn gene function in the tracheal system of third instar larvae. This was achieved by the expression of specific RNAi constructs for either gene, using the trachea-specific btlGal4 driver, and controlled temporally through the co-expression of a temperature-sensitive Gal80ts suppressor. The efficacy of the RNAi-lines was validated in the wing disc where a typical EGFR loss of function phenotype can be easily observed  a marker often used to identify dividing cellsvnRNAi throughout the tracheal system using btlGal4 caused a drastic reduction specifically in the ASP, in proliferative cells  were generated in the ASP. Tracheoblasts with over-activated Bnl/FGF signaling expressed higher vn levels and therefore proliferated at a higher rate , Btl/FGFr and EGFR have been implicated in the control of ASP growth and migration along the underlying wing imaginal disc. While Bnl-FGF/Btl signaling is required for directional cell migration, EGFR signaling is needed for cell division and survivalvn, as over-activation of the pathway produces overgrowth by up-regulating vn expression, and such overgrowth is reverted when vn is silenced , UASpntRNAi (#105390) and UASesgRNAi (#9794) from VDRC; UASspiRNAi , UASpntRNAi (HMS01452), UASesgRNAi (HMS00025) from Harvard Transgenic RNAi Project. UASmae, UASp35 and UASpntP2VP16 from Bloomington Stock center; UAS-esgUASvnUASaosUAStor-btlUASDERDNdppGal4 UASGFP (Bloomington) was crossed to UASbnlConditional activation of either RNAi or gene expression in the tracheal system was achieved by using the Gal4/Gal80RNAi #1050 and UASvnGal4, UAS-GFPnlsvnrF264lacZ reporter were used to visualize vn expression. pntHS20pnt1277to visualize pntP1 and pntP2 isoform expression respectively. rholacZ was used to monitor rho gene expression (Bloomington Stock Center).The enhancer trap strain hsflp; btl>y>Gal4; UAStauGFP females were crossed with males carrying different UAS constructs listed above. Embryos were kept at 25 °C until late L2, incubated 1 hour at 37 °C and transferred to 25°C until late L3. Clone size was measured following the same criteria described inFor clonal analysis, The following antibodies and dilutions were used: rat anti-Twist , mouse anti-β-Gal , mouse anti-Hnt (1:200) and anti-Spi ; rabbit anti-PH3 (1:1000) and mouse anti-Cas3  and TRITC- or CY5-conjugated secondary antibodies . Rabbit and mouse secondary antibodies Alexa 555 and rat Alexa 647  were used. 546-labeled Phalloidin was incubated with the tissue for 20 min to visualize cell membrane. Samples were mounted in medium (Vectashield) with DAPI to stain the nucleus and analyzed by confocal microscopy (Leica SP5). Fixation and staining followed standard protocols.How to cite this article: Cruz, J. et al. FGF coordinates air sac development by activation of the EGF ligand Vein through the transcription factor PntP2. Sci. Rep.5, 17806; doi: 10.1038/srep17806 (2015).


Human T-cell Lymphotropic Virus type-1 (HTLV-1) is the causative agent of an aggressive malignancy of CD4+ T lymphocytes. Many evidences have shown that constitutive activation of NF-kB pathway by Tax-1 is crucial for T-cell transformation. We previously demonstrated that CIITA, the master regulator of MHC class II gene transcription, inhibits HTLV-1 replication by blocking the transcription function of the viral transactivator Tax-1. Here we show that CIITA suppresses also Tax-1-mediated activation of the NF-kB pathway. Of note, CIITA interacts with and retains Tax-1 in cytoplasmic fraction and inhibits Tax-1-dependent nuclear translocation of RelA. Moreover, the overexpression of CIITA does not affect Tax-1 interaction with both RelA and IKKg. CIITA acts by suppressing at least the canonical NF-kB pathway, in that it also inhibits activation of NF-kB by Tax-2, which is known to activate NF-kB through the canonical but not the non-canonical pathway. Future studies will be focused to identify the fine mechanisms through which CIITA exerts this inhibitory function. Overall, these findings indicate that CIITA, beside acting as viral restriction factor against HTLV-1 infection, might counteract Tax-1 transforming activity. Thus, assessing the molecular basis of CIITA-mediated Tax-1 inhibition may be important in defining new strategies to control HTLV-1 spreading and oncogenic potential. Supported by: Fondazione Cariplo (2008-2230), AIRC (IG 8862) and Miur PRIN-COFIN 2008. Rawan Abdallah PhD Fellowship is granted by Fondazione Cariplo Bando 2009: Promuovere la formazione di capitale umano di eccellenza: Percorso di eccellenza in tecnologie biomedicali innovative.


CXCR4 and its ligand CXCL12 mediate the homing of progenitor cells in the bone marrow and their recruitment to sites of injury, as well as affect processes such as cell arrest, survival, and angiogenesis. CXCL12 was long thought to be the sole CXCR4 ligand, but more recently the atypical chemokine macrophage migration inhibitory factor (MIF) was identified as an alternative, non-cognate ligand for CXCR4 and shown to mediate chemotaxis and arrest of CXCR4-expressing T-cells. This has complicated the understanding of CXCR4-mediated signaling and associated biological processes. Compared to CXCL12/CXCR4-induced signaling, only few details are known on MIF/CXCR4-mediated signaling and it remains unclear to which extent MIF and CXCL12 reciprocally influence CXCR4 binding and signaling. Furthermore, the atypical chemokine receptor 3 (ACKR3) (previously CXCR7) has added to the complexity of CXCR4 signaling due to its ability to bind CXCL12 and MIF, and to evoke CXCL12- and MIF-triggered signaling independently of CXCR4. Also, extracellular ubiquitin (eUb) and the viral protein gp120 (HIV) have been reported as CXCR4 ligands, whereas viral chemokine vMIP-II (Herpesvirus) and human β3-defensin (HBD-3) have been identified as CXCR4 antagonists. This review will provide insight into the diversity and inter-connections in the CXCR4 receptor/ligand family. We will discuss signaling pathways initiated by binding of CXCL12 vs. MIF to CXCR4, elaborate on how ACKR3 affects CXCR4 signaling, and summarize biological functions of CXCR4 signaling mediated by CXCL12 or MIF. Also, we will discuss eUb and gp120 as alternative ligands for CXCR4, and describe vMIP-II and HBD-3 as antagonists for CXCR4. Detailed insight into biological effects of CXCR4 signaling und underlying mechanisms, including diversity of CXCR4 ligands and inter-connections with other (chemokine) receptors, is clinically important, as the CXCR4 antagonist AMD3100 has been approved as stem cell mobilizer in specific disease settings. The CXC chemokine receptor CXCR4 is well known for its role in the homing of progenitor cells into the bone marrow. Upon injury or stress or CXCR4 blockade, progenitor cell mobilization to the periphery is enhanced . These fHowever, it has become clear that CXCR4-mediated signaling is more complex as initially thought, as also macrophage migration inhibitory factor (MIF) and extracellular ubiquitin (eUb) have been shown to bind CXCR4 and induce intracellular signaling . FurtherHere, we will summarize the structural characteristics of the chemokine receptor CXCR4, its chemokine ligands CXCL12 and MIF, as well as non-canonical and non-host ligands eUb and HIV gp120. We will review and discuss current knowledge on intracellular signaling through CXCR4 and biological effects triggered by each of these CXCR4 ligands, and will elaborate on how ACKR3 modulates classical CXCR4 signaling triggered by CXCL12. Also, we will discuss the antimicrobial protein HBD-3 as well as the viral chemokine vMIP-II as an endogenous, respectively, viral antagonist for CXCR4 , 10, sum3C chemokines where C is a cysteine residue and X any amino acid (aa) residue ..3C chemo residue , 13. His residue . Alterna residue . The ter residue .Box 13C chemokines they are separated by three amino acids , which are structurally homologous to the GPCRs but are unable to couple to G proteins due to a lacking cytosolic DRYLAIV motif, and fail to induce classical chemokine signaling. Because ACKRs efficiently internalize their ligands, they can function, on the one hand, as chemokine decoy receptors and, on the other hand, they concentrate chemokines in hard-to-reach domains  ortholog . UbiquitUpon ligand binding, CXCR4 is internalized by endocytosis and degraded in lysosomes, mediated by a degradation motif (SSLKILSKGK) in its C-terminus and ubiquitination of vicinal lysine residues .in vitro compared to CXCL12-α , which is the best known ligand of CXCR4, i.e., the “cognate” ligand of CXCR4, is highly conserved between mouse and human (>92%), suggesting an evolutionarily important role . Six isoCXCL12-α , 32. AlsCXCL12-α . FurtherCXCL12-α . In compCXCL12-α , 35.The CXCL12 “pro-protein” contains a signal peptide of 21 aa at the CXCL12 N-terminus, which is cleaved off before secretion of the mature, biologically active CXCL12 protein. In the literature, residue numbers of important motifs of CXCL12 are numbered starting from Lys-22 in the pro-protein, now being counted as Lys-1 in the mature protein. The CXCL12 residue numbers mentioned in this manuscript are numbered accordingly.The structure of the mature CXCL12 protein is characterized by a three-stranded β-sheet that is packed against an α-helix Figure . A reporThis “two-site” model has been proposed as a general functional mechanism of chemokines for a long time , 37. Howin vitro , matrix metalloproteinase 2, neutrophil elastase, and cathepsin G , an enzyme that cleaves off the first two residues of CXCL12 , as alsoin vitro experiments by McQuibban et al., alongside the MMP family members MMP 1, 3, 9, 13, and 14  is capable of cleaving the first four N-terminal amino acids of CXCL12, and was shown to impair CXCR4 signaling in the context of recruitment of neural progenitor cells . MMP-2 w, and 14 . Also, n, and 14 . Finally, and 14 .in vivo with higher efficiency compared to CXCL12-α, despite a lower binding affinity to CXCR4  KHLK in position 24–27; with B = basic aa and X = any other aa) in the first strand of the β-sheet of CXCL12 : a CXCL1to CXCR4 . Althougto CXCR4 . On the to CXCR4 . Togethein vitro experiments indicated that CXCL12 may exist in both monomeric and dimeric forms in the extracellular space. Although recent findings support a 1:1 over a 1:2 ligand:receptor stoichiometry for interaction of CXCR4 with both CXCL12 , which are classified by the presence and spacing of their N-terminal cysteine residues  is surrounded by an envelope consisting of a host cell-derived lipid bilayer and virus-encoded glycoproteins. To enter a new target cell, the virus membrane has to be fused with the target cell membrane, a process mediated by the virus glycoprotein gp120 Box  72). Gp. Gp72). env-gene . They arenv-gene , 76. Theenv-gene . Gp41 fuenv-gene . The evoenv-gene . First, env-gene . This bienv-gene , enablinenv-gene . Gp120 benv-gene . The mecenv-gene . MoleculBox 3Gp120 is the envelope protein of the HI-virus, which is responsible for the entry of the virus into cells . Hence, 2+ mobilization from intracellular stores, nor induce chemotaxis of human monocytes  pathway. Also, oligomerization of CXCR4 or CXCL12, as well as interaction of CXCR4 with other signaling molecules affects the outcome of CXCL12/CXCR4 signaling. Each of these aspects will be discussed in the following section.In vitro, such CXCR4 cleavage could be induced by the neutrophil proteases neutrophil elastin and cathepsin G  in the bone marrow, and their mobilization to the periphery in conditions of stress or injury. Homing of HSPCs is mediated by CXCL12 secretion by endothelial cells in the bone marrow sinusoids as well as by bone marrow stromal cells. CXCR4-positive HSPCs flowing through the bloodstream are triggered to firmly adhere to the endothelium of bone marrow sinusoids through CXCL12/CXCR4-induced integrin activation , followehepsin G , althougIn addition to HSPC homeostasis, the CXCL12/CXCR4 axis has been implicated to be involved in progenitor cell survival and proliferation , 92. AlsCxcr4 in mice causes several lethal birth defects, such as defective trafficking of HSPCs from the fetal liver to the embryonic bone marrow, impaired B-lymphopoiesis, impaired vascularization, and abnormal cerebellum development  and phosphatidylinositol-3-kinase (PI3K) pathway activation (2) to diacylglycerol (DAG) and inositol triphosphate (IP3) by phospholipase C (PLC) and subsequent mobilization of Ca2+ ions from intracellular stores  to diacylglycerol (DAG) and inositol triphosphate (IP3) by phospholipase C (PLC) and subsequent mobilization of Ca2+ ions from intracellular stores  production, activating protein kinase A (PKA) and thus the transcription factor cAMP-responsive element-binding protein (CREB). Both Gαi and Gαq are involved in activating PLC and subsequent mobilization of Ca2+ ions from intracellular stores, as well as inhibiting adenylyl cyclase activity and triggering activation of MAP kinases, the phosphatidylinositol-3-kinase (PI3K) pathway, and the NF-κB pathway  is bound to the G protein complex. Upon ligand binding and subsequent activation and conformational change of the GPCR , GDP is r stores , 104. Th pathway .G protein-coupled signaling triggers diverse downstream signaling pathways and hence needs to be tightly regulated. Signaling termination is initiated by hydrolysis of Gα-bound GTP to GDP by an intrinsic GTPase activity of the Gα subunit, with subsequent re-association of the inactive G protein trimer/receptor complex , 109. Thi-specific inhibitor pertussis toxin. However, it is interesting to note that the complex of CXCR4 with JAK2/3 does not dissociate in pertussis toxin-treated cells, as it would normally do. This implies that the Gαi protein could be involved in the recycling of the JAK/STAT receptor complex by uncoupling of JAK2/3 from CXCR4  upon stimulation of T-cells with CXCL12, leading to several cellular outcomes such as ERK activation, increased Caoduction . For exaoduction . Interesoduction .Furthermore, in an intriguing study conducted by Wagner et al., it was shown that in a mouse model of hindlimb ischemia, the administration of a TLR2 blocking antibody had similar pro-angiogenic effects compared with administration of CXCL12 and induced the activation of both ERK1/2 and AKT, the canonical signal transduction pathways of CXCR4. Immunoprecipitation experiments revealed an interaction of TLR2 and CXCR4 in endothelial cells and it was proposed that the effects of the TLR2 blocking antibody were mediated through CXCR4. Indeed, upon CXCR4 knockdown or G protein inhibition, the observed pro-angiogenic effects of the TLR2 blocking antibody were abolished, supporting a role for a functional interaction of CXCR4 with TLR2 in mediating pro-angiogenic effects of TLR2 blocking .Furthermore, recent research implicates a functional interaction of a CXCR4/ACKR3 dimer with the androgen receptor, regulating CXCL12-dependent cellular motility in a prostate tumor cell line . Also, hin vivo , a damage-associated molecular pattern released from damaged cells, was revealed to form a heterocomplex with CXCL12, shifting the efficiency of CXCR4 activation to lower concentrations and mediating inflammatory cell recruitment in vivo .Together, these findings show that the CXCL12/CXCR4 axis is not monogamous, but is connected to a variety of other signaling receptors.Migration inhibitory factor is involved in the progression of diverse acute and chronic inflammatory diseases including septic shock, atherosclerosis, septic lupus erythematosus, bladder pain, and allergic diseases such as eosinophilic esophagitis , 146–149CD74, the surface-expressed form of MHC class-II-associated invariant chain, has been identified as a MIF receptor. In a signaling complex with CD44, MIF/CD74 interaction leads to ERK1/2 and AKT phosphorylation promoting cell survival and proliferation   was first described in 1989 as a putative GPCR cloned from thyroid complementary DNA . FurtherACKR3 is expressed in a variety of tissues, including, for example, embryonic neuronal and heart tissue, hematopoietic cells, and activated endothelium . Its expper se induce G protein signaling upon binding of CXCL12, based on which the previous name CXCR7 was recently renamed into ACKR3 based on its classification in the group of ACKRs that do not signal through G proteins  was also identified as a receptor for the chemokine CXCL11, which was previously believed to exclusively bind to CXCR3 , 224. Thin vitro assay  syndrome was first described in 1990 by Wetzler et al. . WHIM sy in 2003 , 235, co in 2003 . A highlFurthermore, CXCR4 is the chemokine receptor most widely expressed in malignant tumors . It playIn contrast to pathological effects of CXCR4 signaling, CXCR4 also exerts important protective functions in the context of disease. For example, the CXCL12/CXCR4 axis exerts cardioprotective effects after myocardial ischemia by enhancing the incorporation of progenitor cells in the infarcted region and promoting survival of cardiomyocytes , 99, 239clinicaltrials.gov (search term “CXCR4”)  Table .in vitro and in vivo experimental research. For example, TG-0054 has recently been investigated for its therapeutic potential in an animal model of myocardial infarction. It could be shown that the administration of TG-0054 after induction of myocardial infarction leads to a mobilization of mesenchymal stem cells, preventing left ventricular dysfunction and causing a decrease in inflammatory cytokine levels . Additionally, researchers are constantly aiming to improve AMD3100, for example, efforts to increase its anti-HIV properties by functionalizing the phenyl moiety are still ongoing (Since AMD3100 is currently the only FDA-approved CXCR4 antagonist, it is being used in multiple clinical trials (search term “AMD3100” at  ongoing . Mechani ongoing , however ongoing .In the last decade, many new insights in the CXCR4 signaling cascade have been revealed, both in terms of the ligands to which CXCR4 binds, the different signaling pathways that are initiated downstream, the formation of heteromeric receptor complexes involving CXCR4, as well as the interaction of other receptor complexes with the CXCR4 cascade. This will considerably help to understand the functions of CXCR4 and its ligands both in physiological settings as well as in disease. However, there is still a lot of haziness that remains to be clarified. For example, the specific residues of CXCR4 involved in the site 1 and site 2 interaction with CXCL12 vs. MIF are still unknown, as is its affinity to CXCL12 compared to MIF binding in physiological settings, and thus the potential competition between CXCL12 and MIF for CXCR4 binding. Furthermore, both CXCL12 and MIF can interact with ACKR3, and, despite the initial view that ACKR3 merely serves as a chemokine decoy receptor, first reports have now indicated that both MIF- and CXCL12-induced ACKR3 signaling regulate important biological processes, and it can be expected that additional functions of these signaling pathways in both physiological as well as pathological conditions will be revealed. Also, the roles of CXCR4, CXCL12, and MIF in certain physiological or pathophysiological settings may be very complex depending on the cell type, microenvironment, and perhaps potentially inter-individual settings. For example, CXCR4, CXCL12, as well as MIF have been associated with a pro- as well as anti-inflammatory role after myocardial infarction , 239–242The 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.


HTLV-1 is recognized as the aetiological agent of adult T-cell leukemia/lymphoma (ATLL), and HTLV-associated myeolopathy/tropical spastic paraparesis (HAM/TSP), in contrast, HTLV-2 does not cause hematologic disorders and is only sporadically associated with cases of subacute myelopathy. HTLV-1 and HTLV-2 also exhibit distinct cellular tropisms in vivo: HTLV-1 is mainly found in CD4+T lymphocytes, whereas CD8+T-cells are the preferred target for HTLV-2.Human T-cell leukemia viruses type 1 and 2 (HTLV-1 and HTLV-2) share a common genetic organization, expression strategy and ability to infect and immortalize T-cells The articles contributed in this Research Topic are covering all the different aspects that characterize HTLV-1 and HTLV-2, by highlighting differences in their biology that might provide clues to their distinct pathogenic properties.Emphasis was placed on the comparison of mRNA expression (Cavallari et al., The fine tuning of expression of HTLV-1 and HTLV-2 (Cavallari et al., in vivo and in vitro. Ren and Cheng (Given the relevance of Tax in the initial steps of T-cell transformation, their key role as the main viral oncoproteins was discussed in four different articles, each one focusing on a specific aspect of Tax function. Romanelli et al. , highlignd Cheng , discussnd Cheng , discussThe function of the Rex protein was presented by Ciminale et al. . AlthougThe role in viral replication and viral pathogenesis of accessory proteins HTLV-1 p30 and HTLV-2 p28 were highlighted by Anupam et al. . Both p3The functions of other accessory proteins have been presented by Ciminale et al. . HTLV-1 The possible role played by antisense proteins HTLV-1 HBZ and HTLV-2 APH-2 in the establishment of pathologies induced by viral infection has been carefully considered by Barbeau et al. . Unlike Finally the problem of HIV/HTLV co-infection has been discussed by Pilotti et al. . During In conclusion, in this Research Topic, HTLV-1 and HTLV-2 have been thoroughly compared and major differences outlined and discussed. Of particular interest is that HTLV-2 infection, contrary to HTLV-1, is characterized by an oligoclonal distribution in asymptomatic hosts; this indicates that the etiology of malignant transformation by HTLV-1 cannot be uniquely attributed to this phenomenon and further investigation is needed to understand why HTLV-2 is defective in promoting leukemogenesis.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Telomeres shorten with aging and short or damaged telomeres have been implicated in degenerative conditions. We hypothesized that telomere length might be altered in chronic HTLV-1 and -2 infection and could be a marker of HTLV-associated disease and viral dynamics.45 HTLV-1, 45 HTLV-2, and 45 seronegative subjects were selected from the larger HTLV Outcomes Study (HOST) cohort, and stratum-matched on age, sex and race/ethnicity. The telomere-to-single copy gene (T/S) ratio and HTLV-1 and -2 proviral load were measured using real-time PCR on the same PBMC samples. Unpaired t-tests, linear regression and logistic regression were used to test associations.Ln T/S ratio was inversely associated with age among seronegatives (p=.006) but HTLV-1 and -2 subjects did not show an inverse age association. There was no difference in mean T/S ratio between HTLV-1 (1.02), HTLV-2 (1.03) and matched seronegative (0.99) subjects. In HTLV-1 subjects, there was a borderline inverse association (p=0.07) between T/S ratio and log10 proviral load which did not persist after multivariate adjustment (p=0.17). Among HTLV-2 subjects only, Ln T/S ratio was significantly associated (p=0.026) with increased odds of vibration-sensation impairment.We found no evidence for an overall difference in telomere length between HTLV cases and controls but there was a weak association between HTLV-1 proviral load and telomere length. The association between telomere length and impaired vibration sense in the HTLV-2-positive group is intriguing, and suggests avenues for future investigation of previously described neuropathy in that group.


HTLV proviral load in PBMCs is one of the few laboratory parameters used for clinical follow up and assessment of development of symptomatic disease associated with HTLV infection. High levels of proviral load are associated with symptomatic disease  and occurance of higher levels among asymptomatic individuals has been suggested prognostic for development of symptomatic HTLV disease. Commonly, standard PCR methods including standard curves have been used. Droplet Digital PCR (ddPCR) is a new method that enables absolute quantification of DNA and RNA without the use of a standard curve. In ddPCR, the PCR sample is partitioned into 20 0 nanoliter-sized droplets prior to PCR and based on the number of positive droplets and Poisson algorithms the concentration of the sample can be determined. We have established and evaluated an assay for identification, quantification and determination of PVL of HTLV-1 and 2 in PBMC using ddPCR. For determination of PVL, the human gene RPP30 was quantified and used as reference [1]. The samples were extracted using the QIAamp DNA Blood Mini kit on the QIAcube (Qiagen) prior to ddPCR. Sixty HTLV-1 – positive (PVL 0.01%-59.8%) and 12 HTLV-2 – positive samples (PVL 0.02%-11.3%) previously analyzed with real-time PCR at Imperial College, St Mary's Hospital, London (Prof Graham Taylor) as well as 20 negative samples and dilution series of both HTLV-1 and 2 were examined. The results of ddPCR were in concordance with real-time-PCR, however a few weak positive samples were only detected by one of the methods, see Figure 1. The detection levels of the ddPCR were determined to 1-2 copies per PCR reaction for both HTLV-1 and 2. Both the intra- and inter-assay variability of ddPCR was low, 0.97-3.34% and 1.84-6.13% respectively. In conclusion, ddPCR allows for reliable and sensitive determination of PVL for both HTLV-1 and 2.


There is emerging evidence that exocytosis plays an important role in regulating T cell receptor (TCR) signaling. The trafficking molecules involved in lytic granule (LG) secretion in cytotoxic T lymphocytes (CTL) have been well-studied due to the immune disorder known as familial hemophagocytic lymphohistiocytosis (FHLH). However, the knowledge of trafficking machineries regulating the exocytosis of receptors and signaling molecules remains quite limited. In this review, we summarize the reported trafficking molecules involved in the transport of the TCR and downstream signaling molecules to the cell surface. By combining this information with the known knowledge of LG exocytosis and general exocytic trafficking machinery, we attempt to draw a more complete picture of how the TCR signaling network and exocytic trafficking matrix are interconnected to facilitate T cell activation. This also highlights how membrane compartmentalization facilitates the spatiotemporal organization of cellular responses that are essential for immune functions. The key signaling molecules involved in the T Cell Receptor (TCR) signaling network have been well-characterized. T cell signaling is initiated upon TCR engagement by major histocompatibility complex (MHC) molecules bound to peptide antigens (pMHC). Upon TCR engagement, the TCR-associated CD3 dimers are phosphorylated by the kinase Lck on intracellular immunoreceptor tyrosine-based activation motif (ITAM) consensus sites, leading to the recruitment and activation of downstream signaling molecules, such as the adaptor protein Linker for Activated T cells (LAT). Subsequent activation responses including the secretion of lytic granules (LG) target infected or cancer cells for lysis. The formation of a structured interface between a T cell and an antigen-presenting cell (APC), termed the immunological synapse, is critical for the efficient delivery of effector molecules to the APC and intracellular signals in the T cell. The architecture of the immunological synapse was first described in 1998 as a “bull's eye pattern,” consisting of the central supramolecular activation cluster (cSMAC), where signaling molecules such as the TCR accumulated, surrounded by a ring of adhesion molecules known as the peripheral SMAC (pSMAC), with other molecules such as CD45 being excluded and localized in the distal SMAC  and SNX27 are implicated in recycling TCR toward the immunological synapse  and National Health and Medical Research Council of Australia (APP1037320 and APP1059278 to KG and APP1102730 to JR).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.


Altogether, the present data demonstrated for the first time that anti-HTLV-1 gp46 neutralizing antibodies are capable of not only preventing new infection but also eliminating HTLV-1-infected cells in the presence of autologous PBMCs mainly via an antibody-dependent cellular cytotoxicity (ADCC) in vitro. Thus, a vaccine candidate that can elicit or boost anti-gp46 neutralizing antibody response may have a potential for prevention and therapy against HTLV-1 infection.In order to establish a basis for vaccine development against human T cell leukemia virus type-I (HTLV-1), we have evaluated the roles of anti-HTLV-1 neutralizing antibodies using a rat monoclonal antibody (mAb) against HTLV-1 envelope gp46 (LAT-27) and human polyclonal IgG purified from sera of HTLV-1-associated myelopathy (HAM) patients (HAM-IgG). LAT-27 and HAM-IgG completely blocked the HTLV-1-mediated syncytium formation and T-cell transformation in vitro. Interestingly, when these antibodies were added to the cultures of CD8+ T cell-depleted peripheral blood mononuclear cells (PBMCs) from HAM patients, proliferation of Tax-expressing T cells and HTLV-1 p24 production were blocked. In addition, co-culture of HTLV-1-immortalized T cells with autologous PBMCs in the presence of LAT-27 or HAM-IgG, but not F(ab)’2 fragment of LAT-27 or the other non-neutralizing anti-gp46 mAbs, resulted in eradication of Tax-expressing cells and the p24 production. A


Elevation in intracellular cAMP is associated with the control of a number of distinct platelet functions including actin polymerisation, granule secretion, calcium mobilization and integrin activation. Unfortunately, in atherosclerotic disease the protective effects of cAMP are compromised, which may contribute to pathological thrombosis. The cAMP signaling network in platelets is highly complex with the presence of multiple isoforms of adenylyl cyclase (AC), PKA, and phosphodiesterases (PDEs). However, a precise understanding of the relationship between specific AC, PKA, and PDE isoforms, and how individual signaling substrates are targeted to control distinct platelet functions is still lacking. In other cells types, compartmentalisation of cAMP signaling has emerged as a key mechanism to allow precise control of specific cell functions. A-kinase anchoring proteins (AKAPs) play an important role in this spatiotemporal regulation of cAMP signaling networks. Evidence of AKAP-mediated compartmentalisation of cAMP signaling in blood platelets has begun to emerge and is providing new insights into the regulation of platelet function. Dissecting the mechanisms that allow cAMP to control excessive platelet activity without preventing effective haemostasis may unleash the possibility of therapeutic targeting of the pathway to control unwanted platelet activity.Atherothrombosis, the pathology underlying numerous cardiovascular diseases, is a major cause of death globally. Hyperactive blood platelets play a key role in the atherothrombotic process through the release of inflammatory mediators and formation of thrombi. In healthy blood vessels, excessive platelet activation is restricted by endothelial-derived prostacyclin (PGI Simultaneously, thrombin and thrombospondin-1 activate PDE3A, leading to reduced intracellular cAMP . The cAMP signal is conveyed through assembly of macromolecules that constrain cAMP signaling to specific regions in the cell. The formation of multiple cAMP signaling hubs where individual isoforms of PKA interact with anchoring proteins that focus PKA activity on specific substrates, allows the control of specific biological functions in response to distinct stimuli . Early sractions .Examinins Figure . This wa1 Figure . The secs Figure . Finallys Figure . The pre2+ mobilization and increased vasodilator stimulated phosphoprotein (VASP) phosphorylation , AKAP9, AKAP10, AKAP11, AKAP13, moesin, ezrin, Rab32, BIG2 (brefeldin-A-inhibited guanine-nucleotideexchange protein 2), WAVE-1 (Wiskott–Aldrich syndrome protein verprolin homologous 1), MAP2 (microtubuleassociated protein 2), smAKAP  and neurobeachin. Of those fifteen AKAPs we were able to verify the presence of several by immunoblotting techniques. To the best of our knowledge only two functional AKAPs have been identified in platelets, small membrane AKAP (smAKAP) and moesin. SmAKAP was identified in platelets and cardiomyocytes by using chemical proteomics. This AKAP specifically targets PKA-I to cellular membrane through myristoylation and palmitoylation anchors . Verific3R1-IRAG-PDE5 complex that regulate calcium release in response to NO (3R1 through AKAP-dependent mechanisms (unpublished), suggesting the presence of numerous individual PKA-AKAP complexes. Unfortunately, platelet research in this area is hampered by an inability to apply conventional gene silencing or overexpression technologies and therefore relies on the identification of native complexes through protein purification. As protein purification techniques become more sensitive, the ability to identify and characterize native complexes increases and may allow functional relevance to be established. Our data with cell permeable disruptor peptides provide one approach to address this issue. However, these peptides are limited in their specificity and only allow resolution of isoform-specific signaling and functional events. Future lines of investigation could involve the identification of individual PKA-AKAP complexes and the application of a new generation of peptide disruptors that discriminate between individual PKA-AKAP complexes (in vivo. For example, mice heterozygous for Neurobeachin, an established AKAP (The cAMP signaling pathway represents the most potent endogenous system for the control of platelet activation through its ability to modulate multiple platelet functions. However, the molecular mechanisms by which cAMP/PKA signaling controls individual platelet functions are unclear. Fundamental to elucidating this issue is an improved understanding of the organization and spatial resolution of the downstream effectors of cAMP in platelets. Emerging data suggests that compartmentalization of key cyclic nucleotide signaling complexes may control aspects of platelet function as evident by the identification of a localized PKG1β-IPse to NO . The chase to NO , PDE3A aomplexes . An impohed AKAP , demonsthed AKAP . DetaileThe original AKAP hypothesis has now evolved beyond simple anchoring of PKA signaling, with identification of numerous other signaling moieties including phosphatases, PDEs and other kinases coalescing into the complexes . MoreoveZR, AA, and KN designed the experimental work, planned and edited the manuscript; ZR and AA performed the experiments; ZR and KN analyzed the data and wrote 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.


HTLV-1 infects 5 to 10 million people worldwide. Tropical spastic paraparesis / HTLV associated myelopathy (TSP/HAM) is a neurological disease, that impairs functional capacity, daily life activities and quality of life of infected individual. Objective: To evaluate the functional capacity and flexibility of patients infected with HTLV-1. Methods: Sample was comprised of 65 HTLV-1-infected individuals  followed at the referral center for HTLV of Bahiana School of Medicine and Public Health in Salvador, Brazil. Twenty-six uninfected individuals were enrolled as controls. Inclusion criteria were age from 18 to 65 years, HTLV-1 serology (ELISA and Western Blot) positive (HTLV-group) and negative  and for HTLV-1 infected individuals clinical status defined according to the Belem criteria for the HAM/TSP diagnosis. Patients with stroke or trauma history, those coinfected with HIV, HBV, HCV or syphilis were excluded. The functional capacity of all individuals was assessed using the Group of Latin American development for Maturity (GDLAM) protocol: i) 10 Meter Walking Test, ii) time to get up from a chair, iii) time to get up from the prone position and iv) time to get up from a chair and walk around the house. Test performances were expressed in seconds, and used to calculate the GDLAM index. The flexibility was assessed using the sit and reach test, measured in centimeters. Results: The GDLAM index was significantly lower in the uninfected controls (20.80±3.75 seconds) compared to both asymptomatic (26.45±5.99 seconds) and TSP/HAM patients (51.37±14.99 seconds) . Moreover, the flexibility of uninfected controls (29.16 ± 7.52cm) was significantly higher compared to asymptomatic HTLV-1-infected individuals (23.36 ± 7.97cm) and HAM/TSP patients (14.45 ± 7.52cm) . Conclusions: HTLV-1-infeted individuals have an impairment of their functional capacity and flexibility when compared to uninfected individuals, especially patients with TSP/HAM. Interestingly, HTLV-1 asymptomatic individuals also had a decrease of both functional capacity and flexibility, prior to clinically detectable signs of myelopathy.


Despite much effort, identification of modular structures and study of their organizing and functional roles remain a formidable challenge in molecular systems biology, which, however, is essential in reaching a systematic understanding of large-scale cell regulation networks and hence gaining capacity of exerting effective interference to cell activity. Combining graph theoretic methods with available dynamics information, we successfully retrieved multiple feedback modules of three important signaling networks. These feedbacks are structurally arranged in a hierarchical way and dynamically produce layered temporal profiles of output signals. We found that global and local feedbacks act in very different ways and on distinct features of the information flow conveyed by signal transduction but work highly coordinately to implement specific biological functions. The redundancy embodied with multiple signal-relaying channels and feedback controls bestow great robustness and the reaction hubs seated at junctions of different paths announce their paramount importance through exquisite parameter management. The current investigation reveals intriguing general features of the organization of cell signaling networks and their relevance to biological function, which may find interesting applications in analysis, design and control of bio-networks. With the arrival of the post-genomic era, high-throughput experiments start to provide enormous amount of data to enable a systematic understanding of cell regulation mechanism with unprecedented accuracy. However, we are still lacking of powerful analytic tools to probe how sets of proteins function as an effective machine orchestrating intracellular responses to disparate external stimuli. Take the cancer therapy as an example. Albeit beneficial response is seen upon treatment with the EGFR Tyr kinase inhibitors, the lung tumor often revives by finding ways around, such as inducing a PI3K mutation or activating alternative cancerous signaling pathways . To copeThe past decade witnessed much progress on analyzing structures and functions of complex biological networks. Great effort has been devoted to the detection of reaction patterns of diverse gene regulation and metabolic networks . Useful In this paper, we treat the problem from an information and graph theoretic point of view combined with a consideration of dynamical factors. More specifically, we focus on information flow transmitted from external cues to target proteins through forward signaling pipelines adjusted by miscellaneous feedback modules . Here isWe applied our method to quite a few signaling systems among which three are presented here: the G protein coupled receptor (GPCR) mediated calcium spark network –22, the It is commonly believed that cellular multitasking responses to various extracellular stimuli are attributed to complex signaling regulation. The study of information flow in cell signaling helps unravel the relationship between structure and function of these networks. Our strategy is to decompose signaling pathways into information forwarding pipelines and feedback loops, which turns out to be an efficient way to determine signaling modules. We apply our method to three signaling networks: the GPCR signaling networks, the MAPK signaling system and the JAK/STAT signaling pathway. These systems are chosen since they are activated by three different kinds of motifs capturing the majority of signaling stimuli and, in most cases, they coexist in the same cell cross-talking to each other. Moreover, the JAK-STAT signaling networks contains gene regulation. Common rules shared by these systems are very likely to apply to other signaling networks. We find that they all consist of a signal-relaying module which transmits signals quickly from the input to output and several signal-modulating modules which control detailed temporal profile of signals , althougThe paths marked blue in the forward module of each full signaling network in et al is alleviated by the removal of Module 2, which implies that Module 2 is engaged in the quick attenuation of the output. The concentration of (STAT1n*)2 is reduced to about one fourth after we knock out Module 4, which is explained by the fact that the absorbed STAT1c* enters Module 1 and gets back to the forward flow as STAT1c through Module 4. Blocking this channel cuts down output signals. Since Module 1 absorbs (STAT1c*)2 in the cytoplasm but not directly affects the (STAT1n*)2 in the nucleus, the dynamics are more influenced by Module 2 indicating that if different controllers act on the same biochemical pathway, the ones closer to the target have more influence.Unlike previous two systems, the JAK/STAT signaling network involves gene expression regulation. Among the identified four feedback modules, Module 3 has significant influence on the output since removing this module disables the system in bringing down the concentration of (STAT1n*)Functional redundancy or structural degeneracy is closely related to robustness of biological systems  since th2 in the JAK/STAT system 2-GAP-Grb2-Sos are both Shc independent and serve similar functions as shown in On the other hand, multiple signaling paths and crosstalks in the forward module bestow extra robustness to a signaling network. With regard to the inherent complexity of signaling networks, such as multiple input-output pairs and functional diversity of hub proteins, it is not totally out of expectation that different signaling channels exist in the forward module. In the forward module, the MEPU is the most sensitive part in response to extracellular stimuli which can be observed in The clean decomposition of a network enables looking in great detail into biochemical information flows and answering questions like: how reaction chains transduce external stimuli to the target, and how feedbacks efficiently regulate concentration of specific products. Answers to these questions help us identify reaction hubs in signal transduction. The reaction hub is an essential node to transmit information in a signaling network, which also facilitates crosstalks between modules and collect signals from different channels to fine-tune the response to stimuli . Our ana2-GAP, which acts as a reaction hub since three pathways leading to the target protein originate from this compound. It is easy to see that [ERK-PP] is very sensitive to the value of this kinetic parameter. A slight increase or decrease of the parameter affects the concentration of ERK-PP to a large extent. Observably, when the parameter is a quarter of the original value, the production of ERK-PP is dramatically decreased indicating possible existence of a threshold of activation. Analogously, in the GPCR signaling network, the reaction that transforms Gβγ-Gαq-GDP to Gαq-GTP and Gβγ is a reaction hub. And sensitivity analysis 2-GAP-Shc*-Grb2-Sos are also regulated by more than one feedback modules. As depicted in Fig 2-GAP-Shc*-Grb2-Sos are more effective in controlling the output indicating that the Shc dependent and independent pathways are not functionally equivalent. It is also proved in simulation as shown in The signaling skeletons of the three schematic plots  were obtt signal  which hit signal  when theWe apply our decomposition procedure to three typical signaling networks: the GPCR mediated calcium spark network, the EGFR induced MAPK signaling network, and the JAK/STAT signaling network to acquire a quantitative and systematic understanding of the underlying organizing principles. Several common features are recognized that could be ubiquitous in cell signaling since the three signaling networks employ different types of receptors inducing diverse signaling pathways and play distinct yet important roles in living cells. The results show that all three signaling networks are decomposed into one signal forward subgraph and several local or global feedback modules, which brings great convenience and provide new means in analyzing correlation between network structure and their individual function.Feedback modules in cell signaling networks are organized in an intricate yet controlled way. Local feedbacks are usually coupled with fast signaling procedures in the forward module in a straightforward manner. Global feedbacks are structured and behave in much more diverse ways, not only in organizations, but also in functions. From our computation it seems that global feedbacks are closely related to crosstalks since they employ various multi-functional proteins. Although the JAK/STAT pathway we discussed here is not overly complicated, the components in this system closely correlate to different signaling pathways, such as the MAPK signaling, the PI3K signaling and many others . The GPCet al.Local and global feedbacks work together to shape the behavior of the whole signaling network. The simulation results show that dynamical behavior of target proteins is modulated by the detailed control associated to each module. For example, in the GPCR signaling system, Module 2 brings down the signal, but acts during a relatively long time interval. Module 3 suppresses the signal most efficiently; Module 4 and 5 act at a time scale in between. Different time scales of regulations keep the concentration of IP3 at an acceptable level and with a characteristic temporal profile. However, signaling behavior with strong local feedback modules being removed changes much more than when removing global feedbacks, which indicates that local feedback modules are associated with strong regulation and global feedbacks subtly control output signals. It is a good strategy for the strong local negative feedbacks to maintain homeostasis since abnormally high concentration of signaling targets may be toxic to cell and could also be a waste of energy and resources. For extra fine control, multiple long-ranged feedbacks are needed to tenderly trigger or gently inhibit signaling. The cooperation between feedback modules is essential for the robustness of biological systems and acclimates cells to assorted environments and random non-crucial mutations. It also contributes to the functional versatility of signaling networks. For example, in the MAPK system, the local feedbacks turn out to be the oscillation and bistability generators while the global ones prolong the oscillation. In the JAK/STAT signaling, one local and one global feedback are intertwined to adjust the dynamic pattern. On the other hand, such control strategy may as well speed up evolution of signaling networks since new biological functions could emerge from the mutation changing connections between well conserved feedbacks and forward modules or starting new crosstalks between existing feedback controllers. Meanwhile, a mutation that substantially changes local feedback connections is quite unlikely for two reasons: first, the number of involved nodes is small and the probability of random mutation is small; second, any unbalance in the control would significantly decrease the survival rate of such cells. This is probably the reason why many local feedback modules are well conserved during evolution. On the other hand, structure variation affects dynamical behavior of signaling networks in a context-dependent manner. Structures designed as such are utilized efficiently by cells in different tissues to give different responses for diversified signals. For example, different time scales of the ERK-PP oscillation or activation may induce different activities in human breast adenocarcinoma MCF-7 cells or carcinoma SCC-12F cells , 48. Likβγ (node 17). In the MAPK (2-GAP-Grb2-Sos and (EGF-EGFR*)2-GAP-Shc*-Grb2-Sos. In the JAK/STAT signaling, the global feedbacks activate STAT1c to get STAT1c*. As previously discussed, concentration of target proteins is very sensitive to the alteration of kinetic parameters related to the reaction hubs. It seems that feedback modules act on specific points of a network. Since the behavior of the system is greatly controlled by these points, this type of regulation may be the major cooperation mechanism between feedback modules. Our decomposition and simulation analysis also offers an intriguing glimpse of how degeneracy and robustness is achieved in complex cell signaling systems. Cells tend to employ different redundancy strategies throughout the whole network to ensure the robustness of dynamics. Feedback modules acting on same signal forwarding paths are likely to embed functional redundancy, while even those controlling disparate signaling processes may have similar control over output signals. In the forward module, a set of pathways linking the same endpoints often have similar influences on the target protein manifesting their redundancy in relaying signals. Hierarchical feedback modules and functionally redundant structures bestow cells capacity of stabilizing signal transduction processes and coordinate their activities with environmental changes . The Michaelis constant Km is the substrate concentration when the reaction rate is half of the Vmax. In the supplementary tables, such reactions are marked. The kinetic parameters and the initial concentration of chemical species are adopted from the literature [terature , 23, 26.βγ-GDP-Gαq (N21) and Gβγ-GDP-Gαi (N15) are not placed in the higher layer Click here for additional data file.S2 FigThe network model of the MAPK signaling network. The green node is the input and the red one is the output. There are 121 nodes and 313 edges in the whole network.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.


Tumor-induced angiogenesis is the bridge between avascular and vascular tumor growth phases. In tumor-induced angiogenesis, endothelial cells start to migrate and proliferate toward the tumor and build new capillaries toward the tumor. There are two stages for sprout extension during angiogenesis. The first stage is prior to anastomosis, when single sprouts extend. The second stage is after anastomosis when closed flow pathways or loops are formed and blood flows in the closed loops. Prior to anastomosis, biochemical and biomechanical signals from extracellular matrix regulate endothelial cell phenotype; however, after anastomosis, blood flow is the main regulator of endothelial cell phenotype. In this study, the critical signaling pathways of each stage are introduced. A Boolean network model is used to map environmental and flow induced signals to endothelial cell phenotype . Using the Boolean network model, blockade of intracellular signaling molecules of endothelial cell is investigated prior to and after anastomosis and the cell fate is obtained in each case. Activation of apoptotic signal in endothelial cell can prevent the extension of new vessels and may inhibit angiogenesis. It is shown that blockade of a few signaling molecules in endothelial cell activates apoptotic signal that are proposed as antiangiogenic strategies. Cancer is the leading cause of death in North America and develops rapidly in other countries . More thBlood vessels are constructed from ECs which are phenotypically inactive. When, due to some external stimuli like TAFs, EC changes to an active cell, it decides to stay inert, go, and/or grow. EC migration, proliferation, survival, and lumen formation are regulated by signals from extracellular matrix (ECM). Growth factors are the main sources of biochemical signals , 5, whilThe environmental and flow induced signals regulate EC phenotype through activation of signaling cascades inside ECs. Several receptors and dozens of intracellular proteins are involved in signal transduction in the cascades. Each receptor is specialized to receive specific signals. Downstream of the receptor, there are intracellular proteins that transduce signals. Each EC can be considered as a biological system with inputs and outputs. The system inputs are biochemical and biomechanical signals, while the outputs are EC phenotype and lumen formation. Inside an EC, interaction of intracellular signaling molecules regulates the relation between the inputs and outputs.EC migration, proliferation, and lumen formation are essential for successful angiogenesis. Intracellular signaling molecules play a vital role in regulation of the system output, so blocking one or a few signaling molecules changes the EC fate. If blockade of intracellular signaling molecules drives the cell to go for apoptosis, the process of angiogenesis stops. It is also possible to inhibit angiogenesis through inhibition of proliferation, migration, and lumen formation of ECs. Any interruption in or inhibition of angiogenesis reduces tumor growth or may even stop it. This strategy in cancer treatment is called antiangiogenic therapy. Cellular behavior is controlled by signals that EC receives in the cell surface. Integrins, vascular endothelial (VE) Cadherin, and transmembrane receptors  are responsible for receiving EC environmental signals ; howeverMapping of environmental cues (inputs) to specific cell phenotypes (outputs) needs a model that takes into account intracellular molecules interactions and receptor cross talk. Regarding the intracellular signaling molecules behavior, Boolean network can be used to model signal transduction networks. Application of Boolean network in biological and medical modeling dates back to the 1960s when Stuart Kauffman used Boolean network to model genetic regulatory networks . Other tLi et al. used a Boolean model to develop a dynamic model of guard cell abscisic acid signaling . In the In this study, two different stages are considered for vascular development during sprouting angiogenesis. Prior to anastomosis, ECs receive the biochemical and biomechanical signals from ECM. The relation between environmental signals and EC lumen formation has not been considered in the previous models. To build a model for EC phenotype and lumen formation prediction, critical signaling pathways of EC migration, apoptosis, proliferation, and lumen formation are combined. After anastomosis, blood flow induced shear stress is the main regulator of EC phenotype. Different signaling cascades are activated in each stage (before and after anastomosis). Driving ECs to go for apoptosis is possible through blockade of intracellular signaling molecules. Effect of intracellular signaling molecules blockade is investigated for each signaling cascade (before and after anastomosis). Activation of apoptotic signal in ECs inhibits further vascular development and may help to stop tumor growth.The basic step in development of a model for EC phenotype determination is to obtain the signaling cascades and logical rules of interaction (Boolean dependence relations) between intracellular signaling molecules. In the next step, the signaling cascades and the Boolean dependence relations are used in a Boolean network model to construct the network.During single sprout development, biochemical and biomechanical signals from ECM regulate EC phenotype. Bauer et al. proposed a signaling cascade for EC phenotype determination in single sprouts . In addi in vivo and in vitro models, signaling cascade of lumen formation has been investigated [Lumen formation is a key step in vascular morphogenic events. An increasing number of studies try to explain acquirement of lumenal compartments in blood vessels –43. Usinstigated , 44–47. stigated , 48, 49.stigated .ε [ζ. Activation of Src, Pak2, Pak4, Par3, Par6, and PKCζ is required for lumen formation.Downstream of Cdc42 and Rac1, other proteins are activated to transduce signals and modulate cell cytoskeleton. Small GTPase Rac1 activates Pak2. Cdc42 activates Pak2 and Pak4. Pak2 and Pak4 are also activated by protein kinase C (PKC), especially isoform PKCε . Cdc42 a in vivo and in vitro experiments, Davis et al. proposed a signal transduction pathway for EC lumen and tube formation [Based on the availableormation . A schemε. It is assumed that, in the process of sprouting angiogenesis, external signals exist to activate PKCε. MMP establishes vascular guidance tunnel that is essential for tube formation, so the role of MMP is neglected in signaling cascade of lumen formation.In the proposed signaling pathway for lumen formation in To incorporate lumen formation signaling pathway into the phenotype determination signaling pathway, the signaling pathway in The incorporation of signaling pathway of lumen formation into the signaling pathways of cell phenotype determination is performed and the result is presented in In the schematic presented in Three main inputs from the surface receptors of ECs are considered for this network. In the first input, cell-cell contact or Cadherin is representative of VE Cadherin in endothelial cells. The second input is RTK and represents chemical signals from VEGF in the domain. The third one, integrin, is responsible for sensing the amount of attachment of ECs to ECM molecules such as matrix fibers. Activation or deactivation of any receptor and the downstream effectors directly affects cell response. In ECs, the response is cell phenotype, that is, proliferation, apoptosis, and/or motility or lumen formation.Rac and Rho are main agents in cross talk between signaling pathways. Different feedback mechanisms for interplay between Rac and Rho are reported in the literature –55; howeEC function and phenotype are affected by shear stress induced by blood flow. Cultured ECs reorient their longitudinal axis according to the streamlines of the flow. The reorientation will reduce the effective shear stress on ECs . SeveralAt the intracellular scale, experimental studies determine the role of cell surface receptor and intracellular signaling molecules in signaling cascade of shear stress. Integrin is involved in shear stress mechanotransduction  and actiDifferent candidates for shear stress sensors in ECs were introduced; however, as mentioned before, main events in ECs are regulated by RTK, integrin, and VE Cadherin. Based on the available experimental data, a signaling cascade activated by shear stress is proposed here and shown in The signaling cascades in Figures In each case, there are three system inputs, that is, RTK, integrin, and VE Cadherin. Our analyses show that, with and without blood flow, when RTK or integrin signals are not active, the apoptosis is the system output. This is the first general result in activation of apoptotic signal, which requires the blockade of the surface receptors of EC. To investigate the effect of intracellular signaling molecules blockade in cell fate, it is assumed that both integrin and RTK signals are active in the rest of the analysis.Before anastomosis and blood flow start, the main regulator of EC phenotype is biochemical and biomechanical signals from ECM. Targeted inhibition of specific signaling molecules can alter the cell phenotype, thus inhibiting angiogenesis. In this method, using blocking antibodies or technologies to suppress the expression of individual genes, specific signaling molecules are inhibited. The change may make the ECs unable to form lumen, proliferate, and migrate. Ideally, the ECs undergo apoptosis.A Boolean network model is used to analyze the signaling cascade in Each surface receptor and box in The first column in It should be mentioned that the network output (state of nodes 25 to 28 in the last iteration) is independent of initial conditions of intracellular signaling molecules (nodes 4 to 28). This is verified by multiple runs from several initial condition sets, which all converge to a unique set of phenotypes.The state of Rac plays a central role in receptor cross talk in this model. This is verified by activating or deactivating this node, which results in different cell phenotypes . ConsideEC phenotypes alteration due to inhibition of intracellular signaling molecules is presented in The four combinations of Cadherin and Rac are shown in the top row of Blockade of a few molecules like Raf-1 and MEK1 does not lead to a specific result; that is, the cell fate changes, but it cannot help to inhibit angiogenesis. In some cases, blockade of signaling molecules changes the cell fate and makes them unable to go and/or grow. Making the cell unable to migrate, proliferate, or form lumen may help to inhibit angiogenesis.After anastomosis, blood flow starts in the capillaries and the shear stress induced by blood flow is the main regulator of EC phenotype. Similar to the case without blood flow, both integrin and RTK are assumed to be active during analysis; else, the apoptotic signal is produced. The signaling cascade of The structure of Considering each EC as a system with inputs and outputs, specific strategies are tested to inhibit angiogenesis. A Boolean network model of receptor cross talk for cell phenotype determination including lumen formation is used. This is, to our knowledge, the first study that presents a comprehensive model of cell phenotype determination and lumen formation based on environmental cues. Effect of inhibition of each intracellular signaling molecule and possible antiangiogenic effects are investigated in this study. The model predicts that inhibition of a few intracellular signaling molecules can be used to inhibit angiogenesis, thus posing a strategy to achieve the antiangiogenic effects.