The transforming growth factor β (TGFβ) superfamily of signal transduction molecules plays crucial roles in the regulation of cell behavior. TGFβ regulates gene transcription through Smad proteins and signals via non-Smad pathways. The TGFβ pathway is strictly regulated, and perturbations lead to tumorigenesis. Several pathway components are known to be targeted for proteasomal degradation via ubiquitination by E3 ligases. Smurfs are well known negative regulators of TGFβ, which function as E3 ligases recruited by adaptors such as I-Smads. TGFβ signaling can also be enhanced by E3 ligases, such as Arkadia, that target repressors for degradation. It is becoming clear that E3 ligases often target multiple pathways, thereby acting as mediators of signaling cross-talk. Regulation via ubiquitination involves a complex network of E3 ligases, adaptor proteins, and deubiquitinating enzymes (DUBs), the last-mentioned acting by removing ubiquitin from its targets. Interestingly, also non-degradative ubiquitin modifications are known to play important roles in TGFβ signaling. Ubiquitin modifications thus play a key role in TGFβ signal transduction, and in this review we provide an overview of known players, focusing on recent advances. One of the major regulators of cell communication in all multicellular organisms is transforming growth factor β (TGFβ). TGFβ is the prototypic family member of 33 secreted, structurally related human cytokines. They are involved in the regulation of cell proliferation, differentiation, apoptosis, and motility of diverse cell types. Whereas practically all human cell types respond to TGFβ, the actions of certain family members are more cell type-selective. The importance of proper TGFβ signaling is highlighted by the observation that perturbed TGFβ signaling results in tumorigenesis, and many different mutations or other alterations in TGFβ signaling components have been identified in human cancers. Intriguingly, TGFβ plays a dual role in cancer development and progression. During the early stages of tumorigenesis, TGFβ acts as a tumor-suppressor by inhibiting proliferation. Later in cancer progression, TGFβ has pro-angiogenic and immunosuppressive effects, and it promotes metastasis by inducing epithelial to mesenchymal transition (EMT). Cancer cells that manage to evade the anti-proliferative effects of TGFβ and simultaneously maintain the tumor-promoting effects benefit from this distorted signaling. The wide variation in cellular responses to TGFβ demonstrates the complexity of the intracellular signaling pathways. By studying TGFβ signaling and its cross-talk to other pathways, we gain insight into the regulation of cell behavior and consequently in the mechanisms underlying cancer development. In this review we will focus on the regulation of TGFβ signaling by the ubiquitin system. We will discuss several mechanisms by which TGFβ pathway components are targeted for degradation as a way of negative regulation. Yet TGFβ signaling can also be enhanced via the degradation of negative regulators, and ubiquitination even plays a crucial part downstream of TGFβ in various cross-talk pathways. Furthermore, the roles of deubiquitination and non-degradative ubiquitination will be discussed. Finally, we will touch upon the opportunities these regulatory mechanisms give us for pharmacological intervention.The TGFβ family of cytokines consists of many members including bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), and activins. TGFβ ligands signal through receptors and Smads and regulate target gene transcription . The difSince TGFβ signaling is important for a wide variety of cell functions, it must itself be tightly regulated. It cannot simply be regarded as an on/off switch, because both signal strength and duration are important factors affecting the cellular response outcome. The cellular response to TGFβ is highly dependent on the expression levels of receptors , Smads, Ubiquitination is a post-translational modification of proteins, which can affect their stability, activity, and cellular localization. Ubiquitin (Ub) is a small, 8.5 kDa, protein that is conjugated onto target proteins via its C-terminal glycine Gly76) residue. Ub is produced in a precursor state of linear chains of Ub moieties or fused to ribosomal proteins. The activity of deubiquitinating enzymes (DUBs) is required to produce free Ub, either by processing precursor Ub or by recycling Ub by removing it from its targets . The act residue.E3 ligases display both substrate and chain specificity. While one E3 ligase may preferentially target its substrates for degradation via K48 Ub chains, another may regulate the localization of its targets via mono-ubiquitination. Directly opposing the conjugating function of E3 ligases are the deubiquitinating enzymes (DUBs). DUBs are proteases that remove Ub moieties from their targets. In case of the K48 Ub chain-mediated proteasomal degradation pathway, DUBs remove the Ub chain and stabilize the protein. DUBs also show substrate and chain specificity and therefore represent another layer of regulation of the ubiquitin system.TGFβ induces the expression of various genes, among which are negative regulators, such as I-Smads  and SmurSmurfs also regulate the canonical TGFβ pathway at the level of Smad signaling. Smurf1 was shown to ubiquitinate Smad1 and Smad5 , while SFbw1a, and this is dependent on TGFβ  is a transcriptional repressor of TGFβ signaling. Phosphorylated TGIF1 was found to be targeted for degradation by Fbxw7. Fbxw7 is the substrate recognition component of a ubiquitin ligase complex which was found to target proteins such as cyclin E, c-Myc, Notch, and c-Jun for degradation. Fbxw7 stimulates TGFβ signaling by inducing the degradation of TGIF1 .Another E3 ligase involved in TGFβ regulation is WWP2. The full-length WWP2 (WWP2-FL) was shown to ubiquitinate both Smad2/3 and Smad7. Interestingly, the WWP2-N isoform stimulates the degradation of Smad2/3, yet the WWP-C isoform and WWP2-FL preferentially ubiquitinate Smad7 after TGFβ stimulation. These findings imply that depending on the specific isoforms expressed, TGFβ signaling can be either activated or inhibited .TGFβ exerts its effects via Smad-dependent and independent pathways. Some of the downstream functions of TGFβ signaling are dependent on the ubiquitin system. TGFβ was recently found to induce the ubiquitination and subsequent proteasomal degradation of Krüppel-like factor 4 (KLF4). KLF4 is a transcription factor involved in the regulation of core cell functions such as proliferation, differentiation, and apoptosis, and it has been implicated in carcinogenesis. TGFβ signaling induces the ubiquitination of KLF4 by Cdh1/APC, and this pathway is important for TGFβ-mediated transcription regulation .Besides its function in regulating protein levels of TGFβ signaling components, Smurf1 has also been shown to function downstream of TGFβ as a regulator of RhoA signaling . TGFβ isSmurfs also mediate TGFβ anti-inflammatory signals together with Smad6 by targeting MyD88 for degradation . Smurf1 DUBs remove Ub chains or mono-ubiquitin modifications from target proteins, thereby counteracting the function of E3 ligases. They show specificity for the type of Ub modification and the substrate, yet generally they are less specific than E3 ligases. Some DUBs have been identified to target components of the TGFβ pathway. One such DUB is UCH37, which was shown to bind to Smad7 and deubiquitinate TβRI . It stabTwo other DUBs that have been implicated in TGFβ signal transduction are AMSH, associated molecule with the SH3 (Src homology 3) domain of SA  , and AMSAs previously discussed, not all forms of ubiquitination lead to proteasomal degradation of the target protein. Other types of Ub modifications, such as mono-ubiquitination or K11 and K63 chains, serve to alter the activation state of the protein, its subcellular localization, or its ability to form protein–protein interactions. In the TGFβ signaling pathway various examples of these types of post-translational modifications have been identified, which demonstrate the biological importance of this ‘alternative side’ of the ubiquitin system.One of the first steps in the canonical TGFβ pathway is the phosphorylation of R-Smads by the TβRI. The efficiency of this activation step was shown to be modulated via ubiquitination. Athophin 1-interacting protein 4 (AIP4) or Itch was shown to ubiquitinate Smad2 and thereby promote the phosphorylation of Smad2 by TβRI. This results in an enhancement of TGFβ signaling . E3 ligain vivo during embryonic development  p300/CBP to the chromatin to promote transcription. The acetylation of histones is thought to increase their affinity for proteins such as Ectodermin/TIF1γ, an E3 ligase. Bound to chromatin, Ectodermin/TIF1γ is activated to mono-ubiquitinate Smad4 at K519, and this disrupts the association of Smad4 with phospho-Smad2 . Mono-ubelopment .Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a ubiquitin E3 ligase, which preferentially conjugates K63 Ub chains onto its substrates. TRAF6 interacts with TβRI and is activated via autoubiquitination, induced by TGFβ ligand binding to the receptors. Active TRAF6 ubiquitinates and thereby activates TGFβ-associated kinase (TAK1), which is important for the TGFβ-induced activation of the p38 MAP kinase pathway  77). TA. TA77). in vivo is dependent on other proteins to act as adaptors or activators, and the specificity of E3 ligases is also dependent on the E2 enzyme providing the Ub moiety  onto TGFβ signaling components have also been found to be important. Both receptors and Smads have been shown to be SUMOylated, affecting their function . DiffereAs disruptions in TGFβ signal transduction are implicated in a wide variety of cancers and the ubiquitin system is important for the regulation of this pathway, it does not come as a surprise that in several cancers dysregulations of E3 ligases such as Smurfs and Arkadia have been found . In variThe ubiquitin system can be targeted pharmacologically at different levels. The only drug now being used in the clinic is bortezomib, a general proteasome inhibitor. This drug has cytotoxic effects due to the non-specific inhibition of protein degradation. It is prescribed for multiple myeloma and mantle cell lymphoma, and more proteasome inhibitors are currently under investigation as anti-cancer drugs. Yet the ubiquitin system has the potential of providing us with specific drug targets . Small m


The porous PtCu NCs were composed of amounts of PtCu nanograins with an average particle size of 2.9 nm. The electrocatalytic performance of the PtCu NC electrode towards H2O2 was studied by cyclic voltammetry (CV) and chronoamperometry. The prepared PtCu NC electrode exhibited excellent electrocatalytic activity towards H2O2, with a wide liner range from 5 μM to 22.25 mM, a relatively high sensitivity of 295.3 μA mM-1 cm-2, and a low detection limit of 5 μM (S/N = 3). The hollow porous nanostructure has potential applications in biosensors.Cubic PtCu nanocages (NCs) were successfully synthesized through a redox reaction using cuprous oxide (Cu The detection limit of H2O2 was found to be 5 μM (S/N = 3) with a relatively high sensitivity of 295.3 μA mM-1 cm-2. The sensitivity and linear range obtained at PtCu NC electrode are better than those for HRP immobilized at multiwalled carbon nanotube/alumina-coated silica nanocomposite modified glassy carbon electrode  3-/4- (A), n is the number of electrons transferred (n = 1), D is the diffusion coefficient (0.76 × 10-5 cm2 s-1), v is the scan rate (V s-1), and C is the concentration of K3Fe(CN)6 (5 mM). The calculated value of A is 0.83 cm2 for the PtCu NC electrode, which is 11.75 times of the bare GCE.where 2O as the template. The PtCu NC electrode exhibited excellent electrocatalytic activity towards H2O2. The observed detection limit and sensitivity for PtCu NC electrode was 5 μM and 295.3 μA mM-1 cm-2, respectively, with a wide linear range from 5 μM to 22.25 mM. On the basis of our research, the PtCu NC electrode has potential applications for the design of hydrogen peroxide sensor.Cubic PtCu NCs were successfully synthesized using CuThe authors declare that they have no competing interests.LT designed the experiment and wrote the paper. XZ and WH prepared the solution and the modified electrode. BL carried out the synthesis of PtCu nanocage. YL did the electrochemical measurements. All authors read and approved the final manuscript.


Since 1989, our Institute provides HTLV-1 screening to the relatives of newly diagnosed HTLV-1 cases.We report HTLV-1 infection and associated diseases in twin pairs in which both siblings were tested at ages older than 3.We identified six pairs of twins. Among four monozygotic pairs, 3 were male; median age at HTLV-1 screening was 19. Two pairs were HTLV-1-negative, one positive, and one discordant. The mother of the concordant positive pair was tested 32 years after twins’ delivery because of strongyloidiasis, and developed Adult-T-cell- leukemia-lymphoma (ATLL). Length of breastfeeding was reported the same for both twins, but not recalled. At age 35 one developed ATLL, the other remained asymptomatic. In the discordant monozygotic pair, length of breastfeeding was not clarified for the negative sibling, and six months for the HTLV-1 positive one; the latter developed crusted scabies at age 15. In the concordant negative pairs, born to mothers who later developed HAM/TSP, each twin was breastfed for less than three months. In the case of dizygotic twins, four female, both pairs were HTLV-1-discordant. In one pair, whose mother was HTLV-1-positive, twins were equally breastfed for six months. In the other pair, only the positive sibling received breastfeeding, for two years. Both positive siblings presented HAM/TSP. According to medical interviews, no other risk factors for HTLV-1-adult transmission were present in these twins.These findings suggest the interplay of genetic and non genetic factors, including the confirmed role of breastfeeding, on HTLV-1 mother-to-child transmission and HTLV-1-associated diseases.


HTLV-1 carriers and the patients with adult T-cell leukemia (ATL) frequently suffer from the opportunistic infections. Although it has been known that HTLV-1 infection evokes cell-mediated immune deficiency, its mechanism remains unknown. HTLV-1 bZIP factor (HBZ) is encoded in the minus strand of HTLV-1, which is constitutively expressed and involved in the proliferation of HTLV-1-infected cells. In this study, we investigated the role of HBZ in immunodeficiency in a mice model. HBZ transgenic (HBZ-Tg) mice were infected with HSV-2 and Listeria monocytogenes and evaluated for cellular immunity to those pathogens. The clearance of both pathogens was impaired in HBZ-Tg mice compared with the non-transgenic littermate. In the both infection, production of IFN-gamma in CD4+ T cells was significantly reduced in HBZ-Tg mice. In addition, ectopic expression of HBZ in primary human CD4+ T cells impaired IFN-gamma production in vitro. As the molecular mechanisms, we found that HBZ suppressed transcription of IFNG promoter. Our results suggest that HBZ plays a crucial role for cellular immunodeficiency in HTLV-1-infected subjects.


PCR-based testing has been widely utilized to assess the prevalence of Xenotropic Murine Leukemia Virus-related Virus (XMRV) in prostate cancer and chronic fatigue syndrome patients. An alternative approach, screening for antibodies elicited by XMRV infection, represents a more desirable option for large-scale epidemiologic studies. In this study, blood donor and retrovirus-infected populations were screened for serologic evidence of XMRV infection.Plasma from 1000 US blood donors, 100 HIV-1 infected Cameroonians, 486 HTLV-I infected Japanese, and 156 Japanese HTLV-uninfected controls were screened for antibodies to XMRV gp70 and p15E using recombinant-based chemiluminescence immunoassays (CMIAs). CMIA reactive samples were further evaluated by Western Blot (WB) and real-time RT-PCR for XMRV pol and env sequences.Of US donors, 0.8% (8/1000) were CMIA reactive: 1 p15E and 3 of 7 gp70 reactive samples WB confirmed yielding a 0.4% seroreactive rate. No HIV-1 infected specimens were reactive. Of the Japanese samples, 1/156 uninfected (0.6%) and 20/486 HTLV-infected samples (4.1%) had detectable p15E antibodies by CMIA and WB. Eight additional HTLV-infected samples were gp70 reactive; 4 of 486 (0.8%) were gp70 reactive in WB. No XMRV pol or env sequences were detected in the seroreactives.XMRV seroprevalence ranged from 0 - 0.6% in US blood donors, HIV-1 infected and HTLV uninfected subjects. Notably, 4.1% of Japanese HTLV-I infected individuals were p15E reactive. Inspection of sequence homology between HTLV and XMRV revealed a high level of conservation within the immunodominant region of HTLV gp21 suggesting increased seroreactivity is due to cross-reactive antibodies.


A recent review paper, published online on April 24 2012 in Frontiers in Psychology, argues that antidepressants interfere with the normal homeostatic response of the body and cause more harm than good  improve both mental and physical health outcomes (Katon et al., Finally, the argument that evidence from randomized control trials that show little difference between antidepressants and placebos proves that antidepressants are not effective is a simplistic interpretation of the data. Recent work has demonstrated that these trials show small mean differences between the two groups because they lump responders together with non-responders. When looking at subgroups of responders, there are large differences between antidepressants and placebo (Thase et al.,


Cellular signal transduction pathways are highly interconnected and form a complex network where many signaling molecules interact with each other in a complicated manner -2. The c


Novel technological advances have improved our understanding of how cyclic nucleotides are able to convey signals faithfully between cellular compartments. Phosphodiesterases play a crucial role in shaping these signals in health and disease. The concept of compartmentalization is guiding the search for therapies that have the potential to offer greater efficacy and tolerability compared with current treatments. In 1958, Sutherland and Rall  identifiThe phosphodiesterase (PDE) family of enzymes catalyze the hydrolytic degradation of cAMP and cGMP. PDEs are encoded by 21 genes from which are derived 11 different families, designated PDE 1–11 according to their structure, kinetics, substrate specificity, and regulatory mechanisms . As the In an attempt to improve tolerability, drugs have been developed that target individual families of PDE. In particular, the PDE4 family of enzymes has been implicated in the pathogenesis of airway diseases such as COPD and asthma. PDE4 exclusively hydrolyses cAMP and is encoded by four separate genes that give rise to subfamilies A to D . In the Inhibition of PDE5 by sildenafil or tadalafil has proved clinically useful for augmenting vasodilatation for the treatment of erectile dysfunction. Similar effects of the PDE5 inhibitors on the pulmonary circulation have also been exploited for the treatment of pulmonary arterial hypertension . DespiteThe apparent lack of specificity of second messengers was a concern from early in the development of the field. Sutherland's initial observations of cAMP signaling being involved in multiple cellular functions encountered significant opposition from his contemporaries: How could such a seemingly ubiquitous signaling molecule mediate such a large variety of specific effects within cells? Indeed, in 1971, Rall  contempl1) increased cAMP accumulation and PKA activity in the soluble fraction only. At the time of the experiments, cyclic AMP concentration ([cAMP]) was determined by an assay that takes advantage of the favorable binding of PKA to acidified cAMP  A and G, respectively. Other effectors include ion channels and, in the case of cAMP, the “exchange protein directly activated by cAMP” or EPAC . PKA comCyclic GMP (cGMP) was first identified as a second messenger in photo-transduction in the retina . It is sThe development of fluorescent reporters of specific components of nucleotide signaling, such as those that make use of the FRET phenomenon, have permitted the direct visualization of the limited diffusion of cyclic nucleotides that characterizes subcellular microdomains. Restricted domains of cAMP that are dependent on the activity of PDEs have been shown in the ventricular cardiomyocytes of neonatal rats in response to β-adrenergic receptor stimulation. Using a FRET biosensor, a striated pattern of [cAMP] consistent with the sarcomeric structure of cardiomyocytes was visualized, which was subsequently abolished upon inhibition of PDEs. This observation indicates an important role for these enzymes in limiting the spatial propagation of the signal . Using cEach PDE may be classified according to its substrate specificity . PDE4, 72-terminal portions of PDE4A5 has identified multiple regions of the protein that target the isoform to the cell membrane  to an inhibitory variant (Gi), thus forming a feedback loop. However, Baillie et al.  is found mainly within the cytoplasm, whereas the majority of type II PKA  is associated with specific membranous cellular structures . The loc2+-dependent ATPase (SERCA) pump to replenish the SR with Ca2+ during myocyte relaxation  at the sarcoplasmic reticulum (SR), was shown to affect the ability of the sarco-/endoplasmic reticulum Calaxation .In the heart, the small heat-shock protein Hsp20 has been associated with protection against pathological hypertrophy, apoptosis, and ischemia-reperfusion injury , the pat1) phase before DNA synthesis, whereas displacement of PDE4D3 from the centrosome, causing a local increase in [cAMP], leads to cell-cycle arrest at prophase of mitosis. Another example of the role played by localized PDEs has been described in a cancer cell model. The D5 isoform of PDE4 has been shown to interact with focal adhesion kinase (FAK) and the receptor for activated C-kinase (RAK). Displacement of the PDE isoform from the PDE4D5/RAK/FAK complex leads to a significantly less invasive phenotype  and its downstream effectors PDEs and PKG. The role of cGMP as a second messenger was first identified in the highly organized signal transduction pathway of vertebrate photo-transduction in the retina that is dependent on cGMP acting directly on ion channels. Absorption of a photon of light by the photoreceptor rhodopsin in turn activates the G-protein transducin that subsequently stimulates PDE6 to catabolize cGMP to GMP and leads to closure of CNG channels responsible for the depolarizing “dark” current . PDE6, a−/− cells , derived from the nitric oxide synthase 3 (NOS3) isoform, is a potent stimulator of soluble guanylyl cyclase (sGC). In the failing myocardium, PDE5 expression is elevated compared with the normal heart . Immunoc/− cells . If cGMP/− cells . This bi/− cells . Therefo/− cells . Such a /− cells . These dIn addition to anchoring PKA isoenzymes, AKAPs aid the assembly of multi-protein signaling complexes that include other proteins such as PDEs, protein kinases, and protein phosphatases . In this1-adrenoceptor stimulation results in greater cAMP synthesis in the PKA-RII compartment than the RI compartment, which leads to phosphorylation of modulators of cardiac contractility such as the L-type calcium channel (LTCC), PLB, and troponin I (TnI)  concentrations  . Howevertenuated . Stimulatrations . In oppotrations . Through3-adrenceptors exert a functionally antagonistic effect to β1/2-adrenoceptor signaling , which localizes nNOS to the sarcolemma, exhibit displacement of nNOS activity to the cytosol. This fails to drive PDE2-mediated cAMP hydrolysis, resulting in an elevation in [cAMP] (This signaling cross talk provides a mechanism to explain how βignaling  in that n [cAMP] . Anothern [cAMP] . The subn [cAMP] .1 and PGI2 are important mediators of platelet function that stimulate cAMP production (Cross talk between the cAMP and cGMP signaling pathways through PDEs 2 and 3 has important physiological functions in tissues other than the heart. Platelets are enucleate, granulocytic fragments of megakaryocytes that are important mediators of thrombus formation and wound repair. NO is an important modulator of platelet function in thrombogenesis through actions on adhesion and recruitment at the site of injury . NO, foroduction .Compartmentalization, the structural and functional restriction of signaling domains, allows distinct pools of cyclic nucleotide to interact with particular effectors. PDEs are key modulators of cyclic nucleotide signaling and hence represent attractive therapeutic targets for cardiovascular, respiratory , neurodeFrom the early discoveries relating to cyclic nucleotide signaling over 60 years ago, significant advances in our understanding of cyclic nucleotide signaling have been achieved. With our increasing understanding of the localized nature of cyclic nucleotide signaling, specific targeted therapy to subcellular domains represents not only a challenge but an opportunity to improve both therapeutic tolerability and efficacy.


MDO) orchestrates the development of the thalamus by releasing secreted signaling molecules such as Shh.A fundamental requirement for development of diverse brain regions is the function of local organizers at morphological boundaries. These organizers are restricted groups of cells that secrete signaling molecules, which in turn regulate the fate of the adjacent neural tissue. The thalamus is located in the caudal diencephalon and is the central relay station between the sense organs and higher brain areas. The mid-diencephalic organizer (MD organizer in zebrafish. Wnt signaling induces the MDO in a narrow time window of 4 hours - between 10 and 14 hours post fertilization. Loss of Wnt3 and Wnt3a prevents induction of the MDO, a phenotype also observed upon blockage of canonical Wnt signaling per se. Pharmaceutical activation of the canonical Wnt pathways in Wnt3/Wnt3a compound morphant embryos is able to restore the lack of the MDO. After blockage of Wnt signaling or knock-down of Wnt3/Wnt3a we find an increase of apoptotic cells specifically within the organizer primordium. Consistently, blockage of apoptosis restores the thalamus organizer MDO in Wnt deficient embryos.Here we show that canonical Wnt signaling in the caudal forebrain is required for the formation of the Shh-secreting MDO and consequently for the development of the major relay station of the brain - the thalamus. We propose that Wnt ligands are necessary to maintain the primordial tissue of the organizer during somitogenesis by suppressing Tp53-mediated apoptosis.We have identified canonical Wnt signaling as a novel pathway, that is required for proper formation of the  The anatomical ZLI border zone contains a small cell population, which releases signaling molecules. This signaling center orchestrates thalamus development by controlled release of the morphogen Sonic hedgehog and thus, we termed it the middiencephalic organizer . Lack otebrates -6. Thus (MHB) organizer [MDO establishment has to be determined.A further important diffusible, external cue during neural development is Wnt signaling. Patterning of the vertebrate anterior neural tube requires the function of this pathway at multiple stages . Canonicrganizer ,11. Severganizer . Despiterganizer ,14. Recerganizer . HoweverMDO. By a Morpholino-based knock-down approach we identified Wnt3 and Wnt3a as the responsible ligands and hence are required to maintain the primordium of the MDO. Lack of canonical Wnt signaling per se or knock-down of Wnt3/Wnt3a leads similarly to an increase of apoptosis specifically within the organizer primordium. Consistently, blockage of Tp53-mediated apoptosis is able to rescue the MDO. Furthermore, abrogation of the repressive factors Fezf2 and Irx1b leads to restoration of the organizer. In summary, we propose that canonical Wnt signaling triggered by Wnt3/Wnt3a is necessary to suppress Tp53-mediated apoptosis and thus maintain the organizer tissue during development.Here we show that blockage of the canonical Wnt signaling pathway leads to malformation of the wnt8b expression is restricted by the Shh-dependent transcription factor FoxG1 [MDO, we treated embryos with a specific Wnt signaling antagonist, the tankyrase inhibitor IWR1 [Shh in the central part of the MDO, whereas expression at the dorsal tip remained . The tha Figure . Thus, wwnt3 MO leads to a severe down-regulation of Wnt3 protein by Western blot analysis -based knock-down approach . Injectis Figure . Brain ms Figure . This suwnt3/wnt3a MO) led to a strong decrease of the expression of the pan-MDO marker shh in the central part of the MDO at 24 and 36 hpf , we find that the expression of the paralogue shh-b is absent in the organizer in compound morphant embryos  adjacent to the MDO in wnt3/wnt3a morphant embryos  misspecification of thalamic cells - both due to a lack of Wnt signaling.The foregoing descriptions notwithstanding, our knowledge of the requirement for Wnt signaling for the formation of the thalamus , and thefezf2  were maintained at 28°C on a 14 h light/10 h dark cycle [Shh::RFP [Nkx2.2::GFP and masterblind mutant line carrying a mutation in axin1 [Breeding zebrafish , 0.25 mM wnt3a MO [irx1b MO [fezf2 MO [Tp53 MO [Transient knock-down of gene expression was performed as described in . We usedwnt3a MO , irx1b Mirx1b MO , fezf2 Mfezf2 MO , and Tp5[Tp53 MO . The injin-vivo, we used BIO [MDO of wild-type embryos at the 10 hpf. HS-Dkk1-GFP DNA [To manipulate Wnt signaling used BIO ; used BIO . The bea-GFP DNA  was injein situ hybridizations were performed as described in [wnt3 (formerly known as wnt3l) and wnt3a [shh (shh-a) [shh-b [ptc1 [axin2 [lhx5 [irx1b [otx1l and otx2 [neurog1 [fezf2 [Prior to staining, embryos were fixed in 4% paraformaldehyde/PBS at 4°C overnight for further analysis. Whole-mount mRNA ribed in . The expnd wnt3a , shh (sh (shh-a) ), shh-b ) [shh-b , ptc1 [6-b [ptc1 , axin2 [1 [axin2 , lhx5 [6n2 [lhx5 , irx1b [5 [irx1b , otx1l aand otx2 , neurog1[neurog1 , fezf2 [1 [fezf2 .SDS-PAGE/Western blot analysis was performed with polyclonal antibodies to detect Wnt3  and Wnt3a  and a monoclonal antibody against PCNA  as loading control, respectively.Prior to imaging, embryos were de-yolked, dissected and mounted in 70% (v/v) glycerol/PBS on slides with cover slips. Images were taken on an Olympus SZX16 microscope equipped with a DP71 digital camera by using the imaging software Cell A. For confocal analysis, embryos were embedded for live imaging in 1.5% low-melting-point agarose (Sigma) dissolved in 1× Ringer's solution containing 0.016% tricaine at 48 hpf. Confocal images stacks were obtained using the Leica TCS SP5 confocal laser-scanning microscope. We collected a series of optical planes (z-stacks) to reconstruct the imaged area. Rendering the volume in three dimensions provided a view of the image stack at different angles. The step size for the z-stack was usually 1 to 2 μm and was chosen upon calculation of the theoretical z-resolution of the 40× objective. Images were further processed using Imaris 6 .Cer: Cerebellum; cFb: Caudal forebrain; DMSO: Dimethyl sulfoxide; Eth: Epithlamus; HyTh: Hypothalamus; Mb: Midbrain; MDO: Mid-diencephalic organizer; MHB: Midbrain-hindbrain boundary; PBS: Phosphate-buffered saline; PTh: Pre-thalamus; PTh-p: Pre-thalamus primordium; Tel: Telencephalon; Th: Thalamus; Th-p: Thalamus primordium; ZLI: Zona limitans intrathalamica.The authors declare that they have no competing interests.BM, SW and JP carried out the molecular genetic studies displayed in this manuscript. QC carried out the immunoassays. SS, CH and GD conceived the study, and participated in its design and coordination. SS guided the project and wrote the manuscript. All authors read and approved the final manuscript.


Both p30 and p28 regulate viral gene expression at the post-transcriptional level whereas p30 can also function at the transcriptional level. Recently, several reports have implicated p30 and p28 in multiple cellular processes, which provide novel insight into HTLV spread and survival and ultimately pathogenesis. In this review we summarize and compare what is known about p30 and p28, highlighting their roles in viral replication and viral pathogenesis.Extensive studies of human T-cell leukemia virus (HTLV)-1 and HTLV-2 over the last three decades have provided detailed knowledge on viral transformation, host–viral interactions and pathogenesis. HTLV-1 is the etiological agent of adult T cell leukemia and multiple neurodegenerative and inflammatory diseases while HTLV-2 disease association remains elusive, with few infected individuals displaying neurodegenerative diseases similar to HTLV-1. The HTLV group of oncoretroviruses has a genome that encodes structural and enzymatic proteins Gag, Pro, and Env, regulatory proteins Tax and Rex, and several accessory proteins from the pX region. Of these proteins, HTLV-1 p30 and HTLV-2 p28 are encoded by the open reading frame II of the pX region. Like most other accessory proteins, p30 and p28 are dispensable for  Human T-cell leukemia virus (HTLV) are complex deltaretroviruses, with HTLV-1 and HTLV-2 causing the most prevalent worldwide infections. HTLV-1 infects approximately 25 million people worldwide and is endemic in Japan, Africa, South America, Iran, and the Caribbean basin . In contenv gene and 3′ long terminal repeat (LTR) encodes four open reading frames (ORF I to IV) with a potential for encoding several proteins , CREB and AP-1 to the Tax response element (TRE) in the LTR region . Tax driderstood . Studiesormation . Rex binormation . p30 andormation  and in cormation , albeit + T-cells in infected patients with HTLV-1 (symptomatic and asymptomatic) against p30, demonstrate the importance of HTLV-1 accessory proteins in viral persistence and ultimately in the viral life cycle  and p28 (HTLV-2) are required for efficient viral replication and persistence Figure 1 for summary of key p30 domains). Structurally, p30 contains a bipartite nucleolar/nuclear localization signal (NoRS/NLS), which is comprised of two arginine rich domains  transcriptional complex . FurtherIn order to further characterize the role of p30 in cellular transcription, various groups have performed microarray analysis. The initial microarray analysis showed that p30 altered expression of cellular genes involved in apoptosis, cell cycle regulation, cell adhesion, transcription, and T-cell signaling and activation. It was found that p30 selectively activates genes involved in T cell signaling/activation and also enhances transcription mediated by NFAT, NF-κB, and AP-1 when stimulated . The abitax/rex mRNA in the nucleus. The expression of p30 results in decreased levels of tax/rex mRNA specifically: this effect was not observed with other viral mRNAs. p30 binds to the second splice junction of tax/rex mRNA, which then is retained in the nucleus to repress tax expression and, in turn, viral gene expression  and Cyclins that phosphorylate key molecules allowing cell cycle progression. Different studies have shown that p30 also influences cell cycle progression. The G2/M checkpoint is regulated by a tight balance of phosphorylation and dephosphorylation events, where Cdc25c plays a crucial role in the onset of mitosis . p30 actWaf expression. In contrast to p30, the expression of p28 had no effect on S phase entry and p28 was incapable of binding to Cyclin E  are highly toxic to cells and are associated with developmental, immunological, neurological disorders, and various cancers . Anupam pression . In contpression . The distax/rex mRNA is not known; however, the mass spectrometry analyses identified interactions between p28 and hnRNP H1 and hnRNP F. Conversely, p30 does not have an interaction with hnRNP H1 was also identified. NIP30 is predicted to bind DNA binding/EF hand/Leucine zipper protein (NEFA), which has been shown to localize to the Golgi complex. Preliminary characterization of NIP30 indicated that it is a nuclear protein . It has been shown that PRMT5 can regulate transcription and also is involved in mRNA processing, mainly splicing . Moreovein vivo models demonstrate a more complex viral regulatory mechanism that allows the virus to infect and persist in the host. HTLVs encode various proteins that affect multiple signaling nodes -1 and HTLV-2 are related retroviruses that have different pathological outcomes. While HTLV-1 infection is linked to cancer, inflammatory and neurodegenerative diseases, HTLV-2 is minimally pathogenic. The differences in the viral oncoprotein Tax-1 and Tax-2 are in part responsible for the distinct outcomes of infection with these viruses. However the absence of Tax expression in most ATL patients and the requirement for the accessory genes in HTLV persistence in ng nodes  thereby Comparison of the roles of p30 and p28 has provided further insight into HTLV biology. Further studies are needed to determine their distinct roles in viral latency, low penetrance and ultimately the differential pathological outcomes of HTLV-1 and HTLV-2.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.


Bone morphogenetic proteins (BMPs) are important extracellular cytokines that play critical roles in embryogenesis and tissue homeostasis. BMPs signal via transmembrane type I and type II serine/threonine kinase receptors and intracellular Smad effector proteins. BMP signaling is precisely regulated and perturbation of BMP signaling is connected to multiple diseases, including musculoskeletal diseases. In this review, we will summarize the recent progress in elucidation of BMP signal transduction, how overactive BMP signaling is involved in the pathogenesis of heterotopic ossification and Duchenne muscular dystrophy, and discuss possible therapeutic strategies for treatment of these diseases. Bone morphogenetic proteins (BMPs) were first discovered and described by Marshall Urist as secreted proteins that guide proliferation and differentiation of mesenchymal cells of muscle into bone cells , 2. NowaMore than 20 BMPs have been identified and characterized. Although BMPs were initially identified for their ability to induce bone formation , not allBMPs are morphogens and can induce different cell fates at different concentrations , 7. TheyDrosophila homologues of BMP2 and BMP7, were shown to induce bone formation in mammals [Drosophila embryos [BMPs are structurally related cytokines that are found in all multi-cellular organisms. The crystal structure confirmed that the monomers of BMP7 and BMP2 share a common scaffold , 16. Fun mammals  and huma embryos . Mature  embryos . Most cu embryos .Like other members in the TGFβ family, BMPs signal across the plasma membrane by interacting and inducing complexes composed of type I and type II receptors that are endowed with intrinsic serine/threonine kinase activity Fig. . In mammBoth type I and type II receptors are required for signal transduction . The typUpon formation and subsequent activation of a BMP ligand-receptor complex, the activated type I receptors phosphorylate receptor regulated Smad proteins (R-Smads) at their two C-terminal serine residues. ALK1, -2, -3, and -6 mediate the phosphorylation of R-Smad1, -5, and -8. The phosphorylated R-Smads can form complexes with the common mediated Smad (Co-Smad), Smad4, and translocate into the nucleus. In the nucleus, this Smad complex binds the DNA and in collaboration with co-activators and repressors and other transcription factors regulates the expression of specific genes .Recently, the nuclear localized Smads were found to promote also miRNA maturation . TGFβ anXenopus embryos [In addition to Smad proteins, BMPs are also able to transduce signals via Smad-independent signaling pathways, for example via ERK, p38, and JNK MAP kinases, small GTPases, and PI3K–Akt/PKPB pathways. BMPs can also activate TGFβ-activated kinase-1 (TAK1), a member of the MAP kinase kinase kinase family , which m embryos , however embryos . Recentl embryos –44. TherGiven the important role BMP signaling is playing in a wide variety of biological processes, it has to be tightly regulated. This is achieved by both positive and negative regulation, which occurs at each step of the BMP/Smad signaling pathway. The expression, localization, and activation of BMP ligands, receptors, and Smads are intricately regulated, and this also involves the crosstalk with other signaling pathways , 39. ForNumerous secreted proteins have been identified as BMP antagonists. BMP antagonists can directly bind to BMPs and thereby prohibit BMPs from binding to their receptors. All of these BMP antagonists have a cysteine-knot structure. Based on the size of cysteine-knots, the BMP antagonists can be divided into three subfamilies: the DAN subfamily (eight-membered ring) including USAG-1 and Sclerostin, the twisted gastrulation (Tsg) subfamily (nine-membered ring), and chordin and Noggin (ten-membered ring) , 46. DetXenopus BAMBI and its mammalian homolog Nma have been identified as pseudo-receptors that contain extracellular domains structurally related to that of type I receptors. They lack the intracellular kinase domain, and as a consequence function as repressors of BMP signaling to prevent the formation of active receptor complexes [Currently a number of transmembrane and membrane-anchored proteins have been characterized as co-receptors or type III receptors, and function as modulators for TGFβ/BMP signaling. The repulsive guidance molecules (RGM) family, comprised of RGMa , RGMb , RGMc , and RGMd (currently only found in fish ) form thomplexes , 68.The inhibitory Smads (I-Smad) comprise Smad6 and Smad7, which serve as inhibitors for the Smad signaling pathways. Smad6 mainly targets BMP signaling while Smad7 represses both TGFβ and BMP signaling –71. SmadThe ubiquitin system also actively participates in the regulation of BMP signaling. Smad7 can recruit Smurf E3 ubiquitin ligases and mediate the turnover of activated type I receptors , 75. In Smad anchor for receptor activation (SARA) protein can enhance TGFβ signaling by recruiting and presenting non-phosphorylated R-Smads to active membrane type I receptors . RecentlSince the BMP signaling is transduced by phosphorylated C-terminal R-Smads, phosphatases for R-Smads could function as repressor for BMP signaling. The phosphatase proteins, small C-terminal domain phosphatase (SCP1/2) and protein phosphatase magnesium-dependent 1A (PPM1A), can efficiently dephosphorylate the C-terminal domain of Smad1 in the nucleus and attenuate BMP signaling , 82. RecIn the nucleus, the activated Smad complexes interact with other transcription factors to control gene expression. Histone deacetylases (HDACs), chromatin modulators, function as negative regulators for BMP signaling. c-Ski and Twist-1 are well-studied negative regulators for BMP signaling, which interact with Smad4 and recruit HDACs to the Smad complex thereby repressing its transcriptional activity and antagonize BMP signaling. Both c-Ski and Twist-1 can inhibit BMP-induced osteoblast differentiation –88.Formation of the ligand-receptor complex can initiate endocytosis of active ligands and receptors. Endocytosis-mediated internalization of receptors cannot only control receptor density, thus modulating signaling activity, but is also required for signal transduction in some situations , 90.It was proposed that BMP type I receptors internalization is mediated by clathrin-mediated endocytosis, which is required for continuation of Smad signaling . EndofinIn the previous section, we have discussed various negative regulatory mechanisms for BMP signaling. Multiple negative regulators have been demonstrated to be direct target genes of BMP signaling. For instance, Noggin/Chordin and I-Smads are all well established as direct BMP target genes –96. EndoIn the above sections, we have summarized current research results on BMP signaling. BMPs were originally discovered as bone inducers and repressors of myogenesis , 4, 97. Heterotopic ossification (HO) is defined as bone formation at aberrant locations outside the skeleton; mature bone tissue can be found in the soft tissue where bone normally does not exist. The presence of HO might cause joint stiffness, limited range of motion, swelling and pain, and can even result in severe functional limitations . HO was Fibrodysplasia ossificans progressiva (FOP) has an incidence of 1 in 2 million. Patients develop progressive heterotopic ossification (HO) in the soft tissues either as a result of trauma or spontaneously. Children born with FOP appear normal at birth apart from deformed great toes . Before In 1997, Shafritz reported that BMP4 is overexpressed in lymphoblastoid cells and lesional cells of FOP patients . The BMPThe mutated ALK2 in FOP patients that leads to elevated BMP signaling plays a pivotal role in ectopic bone formation in the FOP patients. However, transgenic mice with global postnatal expression of constitutively activated (CA)-ALK2 (induced without inflammation) do not develop ectopic bone. CA-ALK2 in combination with local inflammation mediated by adenoviral infection induced bone formation in skeletal muscle, joint fusion, and functional impairment . MoreoveFor a long time, osteoprogenitor cells for ectopic bone were considered to be the mesenchymal stem cells residing in the skeletal muscles which have the potential to differentiate into multiple mesenchymal lineages , 123–126Elevated BMP signaling due to a defect in the auto-regulatory feedback loop was already confirmed in the lymphocytes from FOP patients . LymphocMutated ALK2 from FOP patients can directly convert mature endothelial cells into multi-potent mesenchymal cells, and sensitize mesenchymal cells to BMP-induced osteoblast differentiation , 127, thAs mentioned before, FOP is a very rare genetic disease characterized by progressive heterotopic ossification (HO) induced by an activated ALK2 mutant. The most common form of HO is the acquired form, which is either induced by trauma or linked to damage in the nervous system . Post-traumatic HO is caused by injuries at soft tissue at any site. The most common post-traumatic HO is observed after severe long bone fracture or in the hip after total hip arthroplasty . More thAs mentioned, inflammatory conditions have been reported to be important for the progression of the disease in FOP , 145. LiBMPs may mediate the induction of acquired HO. In the valve where HO was identified, BMP2 and BMP4 were found to be expressed by myofibroblasts and preosteoblasts in areas adjacent to B- and T-lymphocyte infiltrations . Moreove+ progenitor cells are discovered as major osteoprogenitor cells that respond to an inflammatory stimulation and further differentiate into heterotopic bones in BMP2 and BMP4-induced HO in the skeletal muscle [+ cells residing in the interstitium of skeletal muscle and other tissues, displaying multi-potent ability to differentiate into mesoderm linage cells including osteoblasts and adipocytes [+ cells from the endothelium, it is this Tie2+ cell population of non-endothelium origin that are responsive to BMP2-induced HO in the skeletal muscle [+ multipotent mesenchymal cells might be converted from endothelial cells, or whether the niche holding these cells could facilitate transition of endothelial cells into multipotent mesenchymal cells in vivo. In addition to Tie2+ cells, mrSCs also contributed to the HO induced by BMP2 and BMP9 in the damaged muscle [Tie2l muscle . Tie2 isl muscle , 138. Inipocytes . Insteadl muscle . Medici l muscle  discoverd muscle . In addid muscle .+ cells as osteoprogenitor cells responsive to BMP2 to induce bone formation in skeletal muscle [Interestingly, in an in vitro study, BMP9 appeared to be more potent for inducing differentiation of mrSCs into osteoblasts than BMP2 . In vivol muscle . FurtherDifferent from FOP patients, in whom the ectopic bone is impossible to be removed by surgical operation, the only effective treatment of symptomatic established HO is surgical resection of ectopic bone tissue. To prevent the possible relapse of HO, it is prudent to avoid soft-tissue trauma in the operation room. Gentle handling of tissues includes complete wound lavage and removal of all bone debris and reaming was suggested to decrease the risk of HO after surgery , 154. RaIn damaged regenerating muscle, BMPs, such as BMP2, BMP4, and BMP9, can potently induce bone formation . Therefo+, MyoD−), which reside between the basement membrane and sarcolemma of individual muscle fibers, are activated; activated satellite cells  proliferate and subsequently either differentiate into myoblasts , which initiate myogenic differentiation  and fuse to repair damaged fibers or form nascent muscle fibers. Part of the activated satellite cells convert back to quiescent satellite cells  thereby self-renewing the satellite cell pool [Muscle regeneration is comprised of three steps: upon muscle damage, quiescent muscle stem cells or satellite cells (characterized by Pax7ell pool . Togetheell pool . In addiell pool . The relell pool –166.Upon muscle damage, ALK3 expression is elevated in the activated satellite cells, and Smad1/5/8 phosphorylation is detected in the nucleus of the activated satellite cells, implying the activation of the BMP pathway . In contActivation of BMP signaling is necessary for maintenance of activated satellite cells in the damaged muscle. However, BMP2, BMP4, and BMP9 have been demonstrated to induce ectopic bone formation in the damaged muscle. Therefore one might be prudent when using BMP inhibitors to repress HO occurrence in skeletal muscle as they may disturb the muscle regeneration. One option to overcome this complication could be to use the inhibitors that could specifically target the osteoprogenitor cells for ectopic bone cells. Another possibility is to inhibit the activity of receptors that are involved in the HO process, but not in the muscle regeneration process. Up to now, ALK3 is the only BMP type I receptor discovered to be involved in the muscle regeneration process  and it iBMP signaling is important in balancing the satellite cell proliferation and differentiation program. Dysregulated BMP signaling might be linked with progression of muscle diseases. DMD disease is one of the diseases in which elevated BMP signaling in the satellite cells might exacerbate the disease .DMD gene [DMD disease is a recessive X-linked form of muscular dystrophy that results in muscle degeneration. The disease is caused by mutations in the DMD gene , encodinDMD gene , 171. DuDMD gene . In addiDMD gene . FurtherDMD gene , 174. MoDMD gene .To find the underlying molecular mechanism for the inefficient differentiation of DMD myoblasts, Sterrenburg and colleagues performed a microarray assay to compare expression profiles in DMD myoblasts and healthy myoblasts . BMP4 exMuscle fibrosis is a prominent pathological symptom in DMD patients. The TGFβ signaling pathway has already been established as a key factor involved in fibrosis in DMD patients . Multipl+ cells residing between skeletal muscle and endothelium was established as multipotent cells, and could differentiate into adipocytes in vitro [As mentioned above, adipose tissue also replaces muscle fibers in dystrophic muscle of DMD patients . A groupin vitro . BMP4 isin vitro . Therefomdx mice with the BMP antagonist Noggin [To validate whether BMP signaling indeed contributes to the progression of DMD disease, we administrated dystrophin-deficient t Noggin . We obset Noggin , but couIn this review, we have summarized the current progress of research on HO and DMD diseases that are related to elevated BMP signaling. The critical involvement of overactive BMP signaling in ectopic bone formation in HO patients is well established. Whether deregulated BMP signaling also contributes to DMD pathology by repressing muscle regeneration needs more investigation. Inhibition of excessive BMP signaling might be a promising therapeutic approach for treatment of these diseases, especially in FOP patient and DMD patients, in which the surgical treatment is impossible.BMPs have been well established as crucial cytokines that control multiple biological phenomena, either during embryonic development or to control postnatal tissue homeostasis. Therefore anti-BMP treatment in the above-mentioned diseases should be considered with care to prevent the possible on-target side-effects and dose-dependent effects of such treatments should be determined in animal models. The BMP type I receptor kinase inhibitors dorsomorphin and LDN-193189 have shown to inhibit at high dose both BMP and TGFβ activity. The BMP antagonist Noggin is not so stable in vivo and was shown not to be able to inhibit BMP6 and BMP9 . The sol


Drosophila melanogaster ortholog Shifted (Shf) binds Hh and extends the range of Hh activity in the developing D. melanogaster wing. Shf activity is thought to depend on reinforcing interactions between Hh and glypican HSPGs. Using zebrafish embryos and the heterologous system provided by D. melanogaster wing, we report on the contribution of glypican HSPGs to the Wnt-inhibiting activity of zebrafish Wif1 and on the protein domains responsible for the differences in Wif1 and Shf specificity. We show that Wif1 strengthens interactions between Wnt and glypicans, modulating the biphasic action of glypicans towards Wnt inhibition; conversely, glypicans and the glypican-binding “EGF-like” domains of Wif1 are required for Wif1's full Wnt-inhibiting activity. Chimeric constructs between Wif1 and Shf were used to investigate their specificities for Wnt and Hh signaling. Full Wnt inhibition required the “WIF” domain of Wif1, and the HSPG-binding EGF-like domains of either Wif1 or Shf. Full promotion of Hh signaling requires both the EGF-like domains of Shf and the WIF domains of either Wif1 or Shf. That the Wif1 WIF domain can increase the Hh promoting activity of Shf's EGF domains suggests it is capable of interacting with Hh. In fact, full-length Wif1 affected distribution and signaling of Hh in D. melanogaster, albeit weakly, suggesting a possible role for Wif1 as a modulator of vertebrate Hh signaling.Proper assignment of cellular fates relies on correct interpretation of Wnt and Hedgehog (Hh) signals. Members of the Wnt Inhibitory Factor-1 (WIF1) family are secreted modulators of these extracellular signaling pathways. Vertebrate WIF1 binds Wnts and inhibits their signaling, but its  Drosophilamelanogaster, the secreted Shifted protein has been shown to bind Hh and to increase Hh signaling, likely by reinforcing interactions between Hh and cell surface proteins of the glypican family. We provide evidence that the vertebrate homolog of Shifted, Wnt Inhibitory Factor-1 (Wif1), inhibits Wnt activity by a similar mechanism, reinforcing interactions between Wnts and glypicans in a manner that sequesters Wnts from their receptors. We also examine the structural basis for the specificities of Wif1 and Shifted for Wnt and Hh signaling, respectively, and provide evidence that Wif1, although a potent inhibitor of Wnt activity, influences D. melanogaster Hh signaling.In developing organisms, cells choose between alternative fates in order to make appropriately patterned tissues, and misregulation of those choices can underlie both developmental defects and cancers. Cells often make these decisions because of signals received from neighboring cells, such as those mediated by the secreted signaling proteins of the Wnt and Hedgehog (Hh) families. While signaling can be regulated by the levels of signaling or receptor proteins expressed by cells, another level of control is exerted by proteins that bind signaling proteins outside of cells and either inhibit or promote the signaling process. In the fruitfly  Drosophila melanogaster WIF1 homolog Shifted  binds Hh and promotes Hh signaling D. melanogaster WIF1 family proteins.The extracellular space provides an important milieu for the regulation of signaling by Wnt and Hedgehog (Hh) morphogens. Several factors are known that bind secreted Wnts or Hhs and regulate either their extracellular levels, their movement through tissues, or their access to receptors. Members of the Wnt Inhibitory Factor-1 (WIF1) family of secreted proteins are unusual, however, because they can impact either the Wnt or Hh pathways. Vertebrate WIF1 binds Wnts and inhibits Wnt signaling D. melanogaster Wnt Wingless  and, in gain-of-function assays, WIF1 inhibits vertebrate Wnt and D. melanogaster Wg signaling wif1 in zebrafish (Danio rerio) embryos results in shortening along the anterior-posterior axis, defective somites and increased canonical Wnt signaling in the developing swimbladder Wif1 in mice does not lead to obvious developmental defects, it does increase the growth of radiation-induced osteosarcomas WIF1 is also epigenetically silenced in many tumors that have heightened Wnt signaling, and addition of exogenous WIF1 to such tumors reduces Wnt signaling, slows tumor growth and increases apoptosis Human WIF1 (NCBI Gene ID: 11197) binds vertebrate Wnts and the D. melanogaster member of the WIF1 family but, unlike vertebrate WIF1, Shf cannot inhibit Wg signaling. Instead, Shf binds Hh (NCBI Gene ID: 42737), and loss of Shf reduces both the accumulation of extracellular Hh and the range of Hh signaling in the D. melanogaster wing disc D. melanogaster glypicans, Dally (NCBI Gene ID: 39013) and Dally-like protein , or blocking synthesis of their HS glycosaminoglycan sidechains, reduces the extracellular accumulation of extracellular Shf dally and dlp or HS synthesis in D. melanogaster wing discs mimics the loss of Shf, similarly reducing the accumulation of extracellular Hh and the range of Hh signaling in wing discs Shf is the only D. melanogaster and vertebrate glypicans to promote or inhibit Wnt signaling is “biphasic”, depending in part on their concentration; low levels promote and high levels inhibit While binding has been demonstrated between HS and vertebrate WIF1 D. melanogaster, and the early embryo of zebrafish, the inhibitory activity of the zebrafish WIF1 homolog  is greatly facilitated by its ability to act as a bridge between Wnts and glypicans. In this sense, Wif1 can bias the biphasic activity of glypicans, increasing their ability to inhibit Wnt signaling.We will provide evidence that in at least two contexts, the exogenous assay provided by the wing disc of We have also examined the structural basis for this interaction. All WIF1 family members (including Shf) are composed of two distinct regions. At the N-terminal end is the Wnt-binding ‘WIF’ domain D. melanogaster Hh, suggesting that vertebrate WIF1 proteins have the potential to regulate vertebrate Hh signaling.Finally, given the similarities between Wif1 and Shf, what controls their pathway specificity, and does vertebrate Wif1 have any overlapping activity in the promotion of Hh activity? To answer these questions we swapped domains between Wif1 and Shf. Our results show that the ‘EGF-like’ domains, but not the ‘WIF’ domains, of Wif1 and Shf are largely interchangeable for the inhibition of Wnt signaling. The ‘EGF-like’ domains are not, however, interchangeable for the promotion of Hh signaling, while the ‘WIF’ domains are. We will also show that Wif1 can affect the accumulation and, weakly, the activity of D. melanogaster. Three D. melanogaster Wnt family members, Wg, Wnt4 (NCBI Gene ID: 34007) and Wnt6 (NCBI Gene ID: 34010), are co-expressed in a narrow stripe of cells along the prospective wing margin in mid-to-late third instar wing discs, but Wnt4 and Wnt6 are not known to affect wing margin development UAS-wif1 with the wing blade driver nubbin-Gal4  produced adult wing phenotypes indicative of reduced Wg activity  by inhibiting Wg signaling. Our initial results argue against this, since wif1 expression did not obviously affect anti-Dlp staining, except by narrowing the zone with low Dlp levels near the wing margin S10, a constitutively active, Wnt-independent form of the D. melanogaster β-Catenin Arm (NCBI Gene ID: 31151) dlp transcription at high levels using UAS-dlp. To bypass the deformation of wing tissues expected from widespread ArmS10 expression, we used the Gal4 Flpout technique to generate clonal clusters of cells misexpressing S10UAS-arm, either alone or in combination with UAS-dlp and/or UAS-wif1. First, we found that ex-Wg levels were much higher within clones expressing S10UAS-arm, UAS-wif1 and UAS-dlp than in clones expressing only S10UAS-arm and UAS-dlp . nub-Gal4-driven expression of pVal-UAS-wif1 did not cause any margin scalloping or wing blade reduction on its own, but did so when co-misexpressed with UAS-dlp  Wnt receptor (UAS-Dfz2-GPI) that binds ex-Wg but cannot transduce Wg signal nub-Gal4-driven UAS-Dfz2-GPI expression were not strengthened by co-expression of UAS-dlp  reporter line wif1 injection also inhibited posterior development , which is similar to Dally and Dlp and interacts with zebrafish Wnts and Wnt-binding proteins elopment , phenocowif1 and gpc4. Control embryos injected with low (5 pg/nL) doses of gpc4 message were morphologically indistinguishable from wild-type individuals and showed normal levels of canonical TOP:dGFP expression in the hindbrain  or high (60 pg/nL) levels of wif1 message, as indicated by an increase in both the fraction of short-tailed embryos and the severity of the defects . To bypass the variability in transgene transcription frequently caused by different genomic insertion sites, we used pVal-UAS-wif1 and pVal-UAS-wif1ΔEGF constructs that integrate into a single, pre-selected genomic location in vitro by D. melanogaster S2 cells at equal levels  in wing discs nub-Gal4-driven expression of wif11-EGFshfUAS-WIF did not extend ex-Wg as far proximally as UAS-wif1  shf-EGFwif1UAS-WIF, had no effect on Wg signaling , and to the Hh receptor Patched , measure low- and high-threshold Hh responses, respectively knot , a high-threshold Hh target shf adult wings the spacing between L3 and L4 is greatly reduced, and in wing discs the normally broader domains of anti-CiAct and anti-Ptc staining regress to thin stripes shf is also required for the extracellular accumulation and movement of Hh: when GFP-tagged Hh (Hh-GFP) is expressed in dorsal cells using ap-Gal4, it accumulates in adjacent ventral cells . Nonetheless, expression of UAS-wif1 using ap-Gal4 significantly increased the ventral accumulation of dorsally expressed Hh-GFP in wing discs mutant for the null allele of shf (x33shf) .We were unable to test the effects of Wif1 on L3–L4 spacing in the adult (shfx33) . This acpression . In someAct staining induced by Wif1 was not an indirect effect caused by reduced Wg signaling, we reduced Wg signaling in shf discs using UAS-Dfz2-GPI. This did not alter anti-CiAct staining . WIF1 binds both Wnts, largely through its ‘WIF’ domain D. melanogaster Wif1 homolog Shf, which binds Hh and glypicans and stabilizes Hh on cell surfaces We have shown that, in the model system provided by the developing D melanogaster glypicans Dlp and Dally increase the effectiveness of Wif1 in developing wings, while the zebrafish glypican Glp4  increases the effectiveness of Wif1 in zebrafish embryos. Previous studies suggest that human WIF1 expression reduces the receptor-mediated endocytosis of Wg in the wing disc But while the stabilization of Hh-glypican interactions by Shf is accompanied by an increase in Hh signaling, our evidence shows that the increase in Wnt-glypican binding caused by WIF1 does not increase signaling. In fact, the glypican interactions strongly potentiate Wif1's Wnt-inhibiting activity: the D. melanogaster Wif1 ortholog Shf; Shf accumulation in wing discs is sensitive to glypican and HS levels, but that sensitivity is lost after a missense mutation in Shf's third ‘EGF-like’ domain In vitro evidence suggests that WIF1 binds the HS sidechains of HSPGs through its ‘EGF-like’ domain D. melanogaster and zebrafish embryos, and that removing the ‘EGF-like’ domains reduced the synergism between Wif1 and Dlp in wing discs, and between Wif1 and the zebrafish glypican Gpc4 in zebrafish embryos. A recent study also found that removing the ‘EGF-like’ domains reduced human WIF1's effectiveness in an in vitro assay; however, the ‘EGF-like’ domains were also reported to reinforce the binding of Wnts to the WIF domain We also found that loss of the ‘EGF-like’ domains greatly reduces Wif1's effectiveness in both One consequence of the interaction between Wif1 and glypicans is that it alters the effects of glypicans on signaling. Increasing the levels of Dlp or Dally in the presence of WIF1 synergistically decreases Wg signaling, and reducing the levels of endogenous Dlp or Dally increases Wg signaling. The latter result is particularly telling, because in the absence of WIF1 removing endogenous Dally reduces Wg signaling in the wing wg-expressing cells of the wing margin, but stimulates signaling in proximal cells distant from the wing margin This underscores the complexity of the role glypicans play in regulating signaling. In some settings the effects of the glypicans on Wnt are known to be biphasic. While endogenous Dally weakly stimulates Wg signaling in the wing disc wnt11 mRNA less effective at rescuing zygotic wnt11 mutants Vertebrate glypicans such as Gpc4 can be similarly biphasic. Removing Gpc4 inhibits non-canonical Wnt signaling in zebrafish embryos, indicating a positive role in signaling, and while overexpression of Gpc4 does not inhibit signaling on its own, at high levels it makes A mathematical model using a different cell surface ligand-binding protein, the BMP-binding protein Cv-2, provides one way of explaining such biphasic effects The ability of glypicans to interact with other Wnt-binding molecules provides another way of altering the biphasic activity of glypicans. Since Wif1 increases the amount of Wnt binding to the glypican, this should increase the glypican's effective concentration, biasing its biphasic activity towards inhibition. Thus, the presence or absence of proteins that bind both glypicans and Wnts may provide an explanation for some of context-specific activities of vertebrate glypicans.D. melanogaster WIF1 homolog Shf contains both ‘WIF’ and ‘EGF-like’ domains, it does not inhibit Wg signaling; instead, it increases the levels or range of Hh signaling The ‘WIF’ domain of WIF1 does not bind HS sidechains, but is sufficient for Wnt binding; the ‘EGF-like’ domains show only weak binding to Wnts on their own, but appear to strengthen Wnt binding to the ‘WIF’ domain UAS-shf did not alleviate margin defects caused by expression of UAS-wg RNAi, even though UAS-Dfz2-GPI and UAS-wg RNAi show a very comparable impact on Wg activity. Alternatively, Shf's effect on Wnt signaling might be due to interactions with the Wnt4 or Wnt6 expressed along the wing margin, which may have redundant roles in wing margin development Surprisingly, Shf did show a weak ability to improve Wg signaling in sensitized backgrounds expressing either Wif1 or the dominant negative DFz2-GPI construct. While we have never detected any obvious effect of Shf on ex-Wg levels, it may weakly interact with Wg in a manner that reduces the levels bound to Wif1 or DFz2-GPI and increases the levels available for the Wg receptors. Consistent with this interpretation, shf mutant wing discs. The Hh-GFP accumulation is abnormal, however, appearing more punctuate than in normal wing discs, perhaps accounting for its ability to reduce the expression of Hh targets.The situation with Hh signaling is more complex. First, vertebrate WIF1's are not known to regulate vertebrate Hh signaling, but we found that zebrafish Wif1 can weakly affect the reduced movement or accumulation of Hh normally observed in Wif1−EGFShf construct almost fully rescues loss of shf function, something not observed after expression of the Shf ‘EGF-like’ domains alone. Together, these data suggest that the ‘WIF’ domains of both Shf and zebrafish Wif1 are capable of interacting with Hh. Like Wnts, Hh is palmitoylated Wif1–EGFShf construct made using zebrafish ‘WIF’ domains, a similar construct made using the ‘WIF’ domain from human WIF1 does not rescue loss of shf function .Placing WIF domain of zebrafish Wif1 in the context of Shf's ‘EGF-like’ domains in a chimeric WIFWif1-EGFShf differ only slightly in their ability to inhibit Wnt signaling and interact genetically with Dlp. We therefore favor the alternative hypothesis that Shf's ‘EGF-like’ domains contribute to Hh signaling through a mechanism independent of glypican binding. While the Shf ‘EGF-like’ domains alone (ShfΔWIF) cannot increase Hh signaling, we have found that they can increase the levels of extracellular Hh , suggesting that they contribute to Hh binding, much as the ‘EGF-like’ domains of WIF1 do to Wnt binding The Shf ‘EGF-like’ domains are necessary to confer a Shf-like level of Hh-promoting activity to the ‘WIF’ domains of zebrafish Wif1. The Hh-promoting activity of Wif1's ‘WIF’ domain is increased by placing it in the context of Shf's ‘EGF-like’ domains, and the low Hh-promoting activity of Shf's ‘WIF’ domain is not changed by placing it in the context of Wif1's ‘EGF-like’ domains. It is unlikely that the ‘EGF-like’ domains of Shf and Wif1 differ significantly in their HSPG-binding activities, since Wif1 and WIFD. melanogaster, an important question is whether it can also do so in vertebrates. Because of its strong effects on Wnt signaling, vertebrate WIF1 family proteins have rarely been assayed for their effects on other pathways, so a weak modulation of one of the vertebrate Hhs remains a possibility.Since Wif1 can alter Hh distribution and, more weakly, signaling in Animals were handled in accordance with guidelines set forth by NIH and IACUC. Our animal use protocols were approved by the University of Wisconsin and Tufts University Institutional Animal Care and Use CommitteespUAS-wif1 and pVal-UAS-wif1 were generated by PCR from full-length wif1 template pVal-wif1ΔEGF terminates at R177; both the pVal inserted constructs also contain a C-terminal V5 epitope. Constructs were expressed in S2 cells as described 282:Q180 (WIFShf-EGFWif1) and T174:C261 (WIFzWIF1-EGFShf). WIFShf-EGFWif1 also contains a V5 epitope N-terminal to the WIF domain. The identically tagged full-length Shf localized and functioned like endogenous Shf protein . Most transgenes were subcloned into pUAST, but for the comparison between Wif1 and Wif1ΔEGF they were subcloned into pValium1 and intergrated into identical attP2 genomic sites D. melanogaster embryos by Injection Services, Inc. .gpc4 open reading frame was generously provided by L. Solnica-Krezel. mRNA was prepared in vitro from linearized plasmids using mMessage mMachine Kit (Ambion), purified and kept at −80 C° in frozen aliquots. Before injection, freshly thawed mRNA was diluted to stock concentrations depicted in Tg(TOP:dGFP) fish were crossed with wild-type and their F1 progeny were injected with the chosen mRNAs as described above. To measure GFP fluorescence 30 hour old embryos were mounted in low melting point agarose on coverslips and photographed using an EM-CCD camera (Photometrix) under constant exposure settings. Calculations of GFP intensity were performed in ImageJ and presented in arbitrary units and compared to the levels of the wild-type controls that were set at 100% after normalization.x33shf2shf80dally and A187dlpap-Gal4, dpp-Gal4, en-Gal4, or nub-Gal4  +>Gal4 UAS-GFPy,w, hs-Flp; Act>y and a 60 minute 37°C heat shock at three days after egg laying. In addition to the UAS lines generated above, we used: UAS-dallyUAS-dlpUAS-wif1, UAS-shf, UAS-shfΔEGF, UAS-shfΔWIFS10UAS-armUAS-Dfz2-GPIUAS-hh-GFPUAS-dlp RNAi (Vienna Drosophila RNAi Center 10299) and UAS-wg RNAi (Transgenic RNAi Project JF01480).Flies were maintained at 25°C. Mutant analyses used Act (1∶20) Late third instar discs were dissected in ice cold PBS and fixed in EM-grade 4% formaldehyde/PBS for 30 minutes at 4°C, rinsed in PBS containing 0.2% Triton X-100, and incubated with primary antibodies overnight at 4°C. Primary antibodies and their working dilutions were: mouse anti-Wg  Extracellular Wg was detected by incubating discs with anti-Wg (1∶3) for 30–45 minutes at 4°C in Shields and Sang M3 culture medium, briefly rinsing in PBS, fixing for 30 min in 4% formaldehyde/PBS and staining with secondary antisera in PBS Figure S1nub-Gal4-driven expression of UAS-wg RNAi in the prospective wing blade (B). Knockdown of wg expression causes loss of wing margin bristles, loss of L1 (arrow) and scalloping of the wing margin. Anterior is up and distal is to the right.Reductions in Wg signaling disrupt wing margin development. Comparison of wild type adult wing (A) featuring dense array of anterior sensory bristles along longitudinal vein 1 , and wing resulting from (PDF)Click here for additional data file.Figure S2nub-Gal4, UAS-wif1 wing almost completely lacks anterior bristles and exhibits moderate notching of the wing margin. (C) nub-Gal4, UAS-Dfz2-GPI wings lack all margin bristles, and shows extreme notching of the wing margin and reduced wing size. (D). nub-Gal4, UAS-Dfz2-GPI, UAS-wif1 wings showed further reduction of wing size than with nub-Gal4 UAS-Dfz2-GFP alone, indicating that nub-Gal4, UAS-Dfz2-GFP are responsive to further reductions in Wnt/Wg signaling. (E) nub-Gal4, UAS-Dfz2-GPI UAS-wif1 showed similar effects as nub-Gal4 UAS-Dfz2-GFP alone, showing that expression of Dlp does not enhance of the effects of DFz2-GPI expression.Expressing DFz2-GPI with Dlp or Wif1. (A) Wild-type wing with normal wing margin and margin bristles. (B) (PDF)Click here for additional data file.Figure S3pVal-wif1 or pVal-wif1ΔEGF genomic insertions produced additive reductions in bristle number. Due to slight variability in wing size, stout bristle number was normalized to the length of the sampling area defined in Arbitrary Units (A.U.) The sampling area began at the distal point where L2 intercepts the wing margin .Effects of Wif1 variants and Dlp on stout bristle differentiation. Expression of Dlp together with moderately driven (PDF)Click here for additional data file.Figure S4Tg(TOP:dGFP) reporter, showing GFP expression in the dorsal midbrain (arrow), anterior to the midbrain-hindbrain boundary. (B) Injection of 40 pg/nL of wif1 mRNA reduces GFP expression (arrow). (C) Injection of 5 pg/nL of kny mRNA does not change GFP expression. (D) GFP intensities were measured in living embryos and their statistically significant differences were verified by two-tailed Mann-Whitney U test. Statistically significant differences in GFP intensity were measured. No differences of statistical significance from were observed between wild-type and kny-injected embryos.Wif1 expression in zebrafish embryos leads to decreased expression of a β-catenin-regulated reporter.  DIC (A) and fluorescence (A′) images of embryos carrying the (PDF)Click here for additional data file.Figure S5D. melanogaster glypican Dally require normal ‘EGF-like’ domains.  Changes in anti-Shf staining (red) in wild type discs (WT) containing dally mutant clones (A) or overexpressing dally (B). Anti-Shf staining is reduced (asterisk) in a dally mutant clone (marked by the absence of GFP in green) in the anterior compartment (A). Dorsal, ap-Gal4-driven expression of UAS-dally  stabilizes Shf .  In 2shf, discs, anti-Shf staining is not reduced in an anterior dally mutant clone , or increased by dorsal, ap-gal4 driven UAS-dally expression . Shf2 protein contains a missense mutation in Shf's third “EGF-like’ domain.Shf interactions with the (PDF)Click here for additional data file.Figure S6Drosophila S2 cells. Constructs were tagged with V5 epitope at their C-termini , which is fully stable since it is secreted at levels virtually equal to the levels of the full length Wif1 (lane B).Full length and EGF-depleted Wif1 are secreted by -termini . Respect(PDF)Click here for additional data file.Figure S7UAS-wif1 or UAS-Dfz2-GPI expressing wings.  UAS-shf reduces notching defects in UAS-wif1 expressing wings, restoring L1 and some anterior bristles.  UAS-shf increases wing growth in UAS-Dfz2-GPI-expressing wings. Wings expressing UAS-shf alone are indistinguishable from wild-type Shf partially alleviates Wg signaling defects in (PDF)Click here for additional data file.Figure S8Act accumulation in shf mutants. (A) Strong inhibition of Wg signaling in a x33shf/Y wing disc, using nub-gal4- driven UAS-Dfz2-GPI, did not alter the width of the domain with strong accumulation of CiAct. Compare to anti-CiAct staining in shf mutants in nub-gal4-driven expression of UAS-wif1 in a x33shf/Y wing disc caused a broader, less intense domain of CiAct.The effects of Wg signaling on Ci(PDF)Click here for additional data file.Figure S9shf nulls (x33shf). The image in the upper panel shows areas sampled for the measurements, and the chart shows the GFP intensities in arbitrary units. Values obtained for the Posterior-Ventral quadrant (P–V) were normalized, and expressed as the percentage of signal intensity compared to the Anterior Ventral quadrant (A–V) where the Hh-GFP signal is at background levels. Graph shows averaged values and error bars show standard deviation. Differences of statistical significance were determined using the two-tailed Mann-Whitney U Test. In shf discs, Hh-GFP diffusion into the ventral compartment was significantly enhanced (asterisks) by Wif1 (p = 0.023) or WIFWif1-EGFShf (p<0.001), but not significantly enhanced by WIFShf-EGFWif1 (p = 0.153).Effects of Wif1 and chimeric Wif1:Shf proteins on Hh-GFP levels. Constructs were expressed in (PDF)Click here for additional data file.


GJA5, investigate the potential functional role of these polymorphisms, and determine their allelic frequencies. The prevalence of nonsynonymous Cx40 mutations in blood and atrial tissue was also compared to mutation frequencies reported in prior studies.The atrial gap junction protein connexin-40 (Cx40) has been implicated to play an important role in atrial conduction and development of atrial fibrillation (AF). However, the frequency of Cx40 mutations in AF populations and their impact on Cx40 expression remains unclear. In this study, we sought to identify polymorphisms in the Cx40 gene GJA5 coding and 3′ UTR regions in blood samples from 91 lone AF subjects and 67 atrial tissue-derived samples from a lone cohort, a mixed AF cohort, and several transplant donors. Reporter gene transfection and tissue allelic expression imbalance assays were used to assess the effects of a common insertion/deletion polymorphism on Cx40 mRNA stability and expression.We conducted direct sequencing of the ′ UTR SNP. A previously noted 25 bp insertion/deletion polymorphism in the 3′ UTR was found to be common  but had no effect on Cx40 mRNA stability and expression. The observed prevalence of nonsynonymous Cx40 mutations in atrial tissues derived from lone AF subjects differed significantly (p = 0.03) from a prior atrial tissue study reporting a high mutation frequency in a group of highly selected young lone AF subjects.We identified one novel synonymous SNP in blood-derived DNA from a lone AF subject. In atrial tissue-derived DNA from lone and mixed AF subjects, we observed one novel nonsynonymous SNP, one rare previously reported synonymous SNP, and one novel 3′ UTR does not appear to play a role in modulating Cx40 mRNA levels.Our results suggest that Cx40 coding SNPs are uncommon in AF populations, although rare mutations in this gene may certainly lead to AF pathogenesis. Furthermore, a common insertion/deletion polymorphism in the Cx40 3 GJA5 encodes two alternative transcripts, Cx40A and Cx40B, comprised of first exons 1A or 1B and a shared coding second exon, respectively  to determine the cDNA allelic expression ratio, normalized to the gDNA ratio, at the rs1043806 position as previously described [Screening of 67 atrial derived gDNA samples for the 25 bp insertion/deletion polymorphism was carried out using the PCR primers ATTCCTCGGAGTAGTGGTGAGATGG (upstream) and ACACCCTAGCAGAAGGAAAGGTTGC (downstream), which amplify a 131 bp region in the gov/LPG) . To measgov/LPG) . This isgov/LPG) ,17. A Cxescribed .Table Cx40 variants in a large cohort of subjects of European ancestry, we conducted direct sequencing of the Cx40 coding region in blood- and atrial tissue-derived gDNA samples from patients diagnosed with lone or mixed AF, as well as a small number of transplant donors. Detected SNPs were determined to be novel if they have not been previously reported in public SNP databases, including those available from dbSNP [Cx40 coding SNP was identified (339G>C)  Figure A. The no) Figure . Sequenc) Figure  yielded GJA5 by sequencing the proximal 1,435 bp of the GJA5 3′ UTR region in atrial tissue genomic DNA samples from 48 patients diagnosed with AF, a subset of the 67 subjects described in Table ′ UTR SNP rs1692141, located 53 bp downstream of the GJA5 stop codon with a minor allele frequency of 0.237 in our samples.We conducted a search for novel regulatory variants of GJA5 3′ UTR insertion/deletion was conducted with the atrial tissue samples, examining eight SNP genotypes situated within the GJA5 region. The SNP genotypes tested consisted of the common Cx40B promoter variant rs10465885, a Cx40A promoter SNP (rs11552588) and five other contiguous GJA5 SNPs from the Illumina Hap550 BeadChip [GJA5 3′ UTR, was used as the indicator SNP in an expression imbalance assay, as described below. Analysis of linkage disequilibrium (LD) relationships using Haploview v.4.1 (Broad Institute) determined that the insertion/deletion polymorphism is in partial LD with rs10465885, with r2 = 0.81  that has been shown to decrease mRNA stability when present in the 3′UTR [GJA5 3′UTR construct demonstrated a 54% reduction in luciferase activity compared to cells transfected with the unmodified reporter  between the frequency of missense Cx40 alleles in our 91 lone AF blood samples  and the frequency observed in 218 lone AF blood samples by Yang and colleagues  [Cx40 missense allele frequencies in our 34 lone AF tissues (average age at surgery 54 years), in which we detected a single nonsynonymous variant , and the frequency observed by Gollob et al. in 15 highly selected and young (average age of onset 45 years) lone AF tissue donors, in which four novel heterozygous Cx40 missense mutations were found  [Fisher’s exact probability test was used to compare the frequencies of nonsynonymous variant , 0.69%) . HoweverGJA5 coding region in 91 lone AF blood samples and 67 atrial tissue samples  from individuals of predominantly European ancestry, identifying a novel nonsynonymous SNP (339G>C), a novel synonymous SNP (951T>C), and a previously reported synonymous SNP (rs2232191). In the 3′ UTR we identified a single novel variant (*22G>A). We also determined that a 25 bp insertion/deletion polymorphism in the GJA5 3′ UTR is common and in partial LD with the Cx40 transcript B promoter SNP rs10465885, which is associated with early onset lone AF and strongly correlated with Cx40B and total Cx40 expression [Cx40A allelic expression imbalance assays indicated that the insertion/deletion is not directly associated with changes in Cx40 expression. The modest and inconsistent 1.39-fold effect observed for the insertion/deletion in the 8 subjects assayed for Cx40A allelic expression imbalance was much weaker than the consistent 3.3-fold effect that rs10465885 has on allelic expression imbalance of the Cx40B transcript [We conducted direct sequencing of the pression . Howeveranscript . These aGJA5 gene to be associated with AF at the conservative genome-wide significance threshold [GJA5 promoter variant that both alters Cx40 expression levels and is weakly associated with AF [Cx40 coding variants have observed very different rare allele frequencies. In fifteen young, idiopathic AF patients of Western European descent, Gollob et al. identified one germ line and three somatic nonsynonymous variants in the Cx40 coding region using DNA derived from atrial tissue samples and blood [Cx40 genetic variants using blood-derived DNA, Yang and colleagues reported the discovery of three nonsynonymous Cx40 variants in 218 lone AF patients of Chinese descent, and no novel variants in 200 control subjects [Cx40 variants in blood-derived DNA samples from 91 lone AF subjects, which was not statistically different from the frequency observed by Yang. Combined with our finding of no Cx40 nonsynonymous variants in 30 mixed AF atrial tissue samples, these results suggest that Cx40 coding SNPs are uncommon in lone and secondary AF patient populations.The most recent and largest AF genome-wide association study has not identified common variants in or near the hreshold . However with AF . Since nnd blood . In contsubjects . Our stuGJA5 variants discovered in atrial tissue were of germline or somatic origin. We also did not have a large cohort of donor atrial tissue available that could be used to compare the frequency of rare variants in AF vs. control atria. Finally, our transfection study was performed in HL-1 cells, an atrial-derived, immortalized mouse cell line that demonstrates some atrial-like properties [′UTR insertion/deletion polymorphism, which we observed to have no effect on reporter activity, could still modify gene expression in vivo in the human atrium.The current study has several limitations. First, all samples were predominantly derived from patients of European ancestry and the sample size was limited to 91 lone AF blood-derived DNA samples and 67 atrial tissue samples, of which only 34 were lone AF subjects. Second, we did not possess paired blood and tissue samples, and thus were unable to determine whether the rare operties . It is tGJA5 gene. We determined that a 25 bp insertion-deletion polymorphism is common in the GJA5 3′ UTR, but we could detect no functional role for this variation. Our study suggests that aggregations of different rare nonsynonymous GJA5 genetic variants are not commonly observed among AF patients. Although rare, GJA5 variants that alter Cx40 function may be directly causal of AF.In 158 atria- and blood-derived DNA samples from subjects with AF, we observed only one nonsynonymous rare genetic variant in the Cx40: Connexin-40; AF: Atrial fibrillation; SNP: Single Nucleotide Polymorphism; LD: Linkage Disequilibrium.The authors declare that they have no competing interests.′ UTR regions, allelic expression imbalance analysis, statistical analysis of Cx40 missense allele frequencies, and drafted the manuscript. RCW carried out sequencing of the GJA5 3′ UTR and characterization of the insertion/deletion polymorphism, including the PCR screen, haplotype analysis and functional assays, and assisted in drafting the manuscript. DRW participated in the design of the study and provided blood and atrial tissue samples. MKC participated in the design of the study, performed chart reviews and provided clinical characteristics of patient samples. JDS conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.GDT conducted direct sequencing of the GJA5 coding and proximal 3This work was supported by NIH/ National Heart, Lung and Blood Institute grant HL090620 and the supplementary American Recovery and Reinvestment Act (ARRA) grant HL090620-01A1S2 to Drs. Chung, Barnard, Smith, and Van Wagoner. It was also supported in part by NIH/National Center for Research Resources (NCRR) Case Western Reserve University/Cleveland Clinic CTSA UL1-RR024989 ; the Heart and Vascular Institute, Department of Cardiovascular Medicine, Cleveland Clinic (Chung); Leducq Foundation grant 07-CVD 03 ; and the Atrial Fibrillation Innovation Center, State of Ohio . Robert Wirka was a Howard Hughes Medical Institute Medical Research Training Fellow. Dr. Tchou was partially supported by a Case Western Reserve University training grant in Cardiovascular Research from the NIH (T32HL1053381).The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/13/102/prepub


Ursolic acid (UA), a pentacyclic triterpenoid widely found in medicinal herbs and fruits, has been reported to possess a wide range of beneficial properties including anti-hyperglycemia, anti-obesity, and anti-cancer. However, the molecular mechanisms underlying the action of UA remain largely unknown. Here we show that UA inhibits leucine-induced activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway in C2C12 myotubes. The UA-mediated inhibition of mTORC1 is independent of Akt, tuberous sclerosis complex 1/2 (TSC1/2), and Ras homolog enriched in brain (Rheb), suggesting that UA negatively regulates mTORC1 signaling by targeting at a site downstream of these mTOR regulators. UA treatment had no effect on the interaction between mTOR and its activator Raptor or inhibitor Deptor, but suppressed the binding of RagB to Raptor and inhibited leucine-induced mTOR lysosomal localization. Taken together, our study identifies UA as a direct negative regulator of the mTORC1 signaling pathway and suggests a novel mechanism by which UA exerts its beneficial function. Ursolic acid (UA) is a triterpene compound derived from certain traditional medicinal plants The mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that plays important roles in the regulation of protein and lipid synthesis, autophagy, as well as mitochondrial metabolism and biogenesis While the mTORC1 signaling pathway has been well established as a key component to promote growth and accelerate cancer, it also plays an important role in regulating metabolism In the current study, we investigated the effect of UA on mTOR signaling, we found that UA inhibits leucine-stimulated mTOR activation in C2C12 myotubes by inhibiting mTOR from targeting to lysosome, uncovering a novel mechanism underlying the negative regulation of the mTORC1 signaling pathway by UA, a small molecule that possess a wide range of beneficial properties including anti-hyperglycemia, anti-obesity, and anti-cancer.Plasmids encoding FLAG-tagged wild-type and Q99L mutant of RagB, the lentiviral vector pWPI, the lentiviral envelope plasmids pMD2.G and psPAX2 were obtained from Addgene. The plasmids encoding wild-type and S16H mutant of Rheb were gifts of Dr. Bo Xiao (Johns Hopkins University School of Medicine) and were described previously −/− and wild-type mouse embryonic fibroblasts (MEFs)  were grown in DMEM (Gibco 11995-065) supplemented with 10% new born calf serum and 1% penicillin/streptomycin C2C12 cells (American Type Culture Collection) were cultured in DMEM (ATCC 30-2002) supplemented with 10% newborn calf serum and 1% penicillin/streptomycin. TSC1/2wt, RagBQ99L, Rhebwt, or RhebS16H. The medium was changed to growth medium eight hours post transfection and collected after 48 hours. The collected medium was filtered via a 0.45-µm filter and stored at −80°C until needed. For cell infection, C2C12 cells were incubated with the lentiviral medium in the presence of 8 µg/ml polybrene for 72 hours. Cells were visually scored for GFP intensity. Control cells were infected with lentiviruses encoding only GFP. No notable difference in proliferation and behave was detected between infected and uninfected parental cells.cDNAs encoding wild-type RagB, Q99L mutant of RagB, wild-type Rheb, S16H mutant of Rheb were subcloned into the lentiviral vector pWPI. Lentiviral particles were produced by transfecting 293T cells with the pMD2.G plasmid, the psPAX2 plasmid, and the pWPI plasmid encoding green fluorescent protein (GFP) vector, RagBCells were rinsed once with ice-cold PBS and lysed in ice-cold lysis buffer . Cell lysates were centrifuged at 13,000 rpm for 10 minutes and supernatants were collected. For IP experiments, primary antibodies were added to the lysates and incubated with rotation overnight at 4°C. After addition of 30 µl of 50% slurry of the protein A-sepharose, the incubation was continued for 2 more hours. Immunoprecipitates were washed three times with ice-cold lysis buffer containing 150 mM NaCl. Immunoprecipitated proteins were detected by Western blot using 8%–12% SDS-PAGE as described The in-cell cross-linking experiments were performed according to the procedure described previously Cells were grown on round cover slips in 12-well plates to 60–80% confluence. Cells were serum starved, treated with or without leucine and/or UA, rinsed with PBS, and fixed with 4% paraformaldehyde for 5 minutes at room temperature. The coverslips were rinsed twice with PBS and permealized with 0.2% Triton X-100 in PBS for 15 minutes. After rinsing two more times with PBS, the coverslips were blocked for one hour in blocking buffer (0.25% BSA in PBS) and incubated with primary antibody overnight at 4°C. After rinsing with blocking buffer, cells were incubated with secondary antibodies for one hour at room temperature. The coverslips were mounted on glass slides using glycerol and the fluorescence images were detected by a Zeiss LSM780 NLO fluorescence microscope with a 63 x objective.P values of *<0.05, **<0.01, and ***<0.001.Quantification of protein phosphorylation and total levels was performed by analyzing Western blots using the NIH Image J program. Statistical analysis of the data was performed using the analysis of variance (ANOVA). Data were presented as mean ± S.E.M. Statistical significance was set at To determine the potential effect of UA on mTOR activity, differentiated C2C12 myotubes were treated with UA at different doses, followed with insulin or leucine treatment. Insulin treatment greatly stimulated the phosphorylation of S6K, 4EBP1, and Akt, and this phosphorylation was suppressed by UA treatment in a dose-dependent manner . InsulinBranched-chain amino acids such as leucine have been found to activate mTOR complex 1 (mTORC1) via inhibition of TSC1/TSC2 S16H. S6K phosphorylation was enhanced in cells overexpressing RhebWT and RhebS16H and the increase was augmented by treatment of the cells with leucine (The small GTPase Rheb has been shown to be necessary for amino acid-dependent mTORC1 activation  leucine . While p leucine , suggestTo determine the potential involvement of the negative regulator Deptor in UA-induced inhibition of the mTORC1 signaling, we examined the effect of UA on leucine-stimulated S6K phosphorylation in C2C12 cells in which the expression levels of Deptor were suppressed by RNAi. As expected, mTORC1 signaling was increased in Deptor-suppressed cells, as demonstrated by increased basal S6K and 4EBP-1 phosphorylation . LeucineQ99L were transiently expressed in C2C12 cells. Overexpression of RagB per se had no significant effect on S6K phosphorylation but potentiated leucine-stimulated S6K phosphorylation (Q99L, on the other hand, significantly promoted basal S6K phosphorylation, which was not further enhanced by leucine treatment (Q99L (The small GTPase Rag has been shown to be critical for leucine-stimulated mTORC1 activation rylation . Overexpreatment . Treatment (Q99L , suggestnt (Q99L . Similarnt (Q99L . Howevernt (Q99L .To further elucidate the mechanism by which UA inhibits mTORC1 signaling, we examined RagB-mediated recruitment of mTORC1 to lysosome, which is critical for leucine-stimulated mTORC1 activation UA has been shown to exert many beneficial effects such as anti-diabetes –induced activation of mTORC1 C2C12 myotubes possess morphological, biochemical and metabolic properties of the isolated skeletal muscle How UA disrupts the binding of RagB to Raptor remains unknown. One possibility may be that UA directly interacts with RagB or mTORC1 components, leading to a conformational change of these proteins and thus impairs their interaction. Since the interaction between mTOR with the Rag proteins is critical for mTOR localization to the lysosome in response to amino acid stimulation In summary, we have demonstrated for the first time that UA inhibits leucine-stimulated mTOR signaling in C2C12 cells. In addition, we show that UA inhibits mTORC1 signaling by disrupting the interaction between RagB-mTORC1. As aberrant activation of the mTORC1 signaling pathway is associated with impaired energy homeostasis and increased incidents of human tumors/cancers