Due to the growing importance of cellular signaling mediated by reactive oxygen species (ROS), proteins that are reversibly modulated by these reactant molecules are of high interest. In this context, protein kinases and phosphatases, which act coordinately in the regulation of signal transduction through the phosphorylation and dephosphorylation of target proteins, have been described to be key elements in ROS-mediated signaling events. The major mechanism by which these proteins may be modified by oxidation involves the presence of key redox-sensitive cysteine residues. Protein kinase C (PKC) is involved in a variety of cellular signaling pathways. These proteins have been shown to contain a unique structural feature that is susceptible to oxidative modification. A large number of scientific studies have highlighted the importance of ROS as a second messenger in numerous cellular processes, including cell proliferation, gene expression, adhesion, differentiation, senescence, and apoptosis. In this context, the goal of this review is to discuss the mechanisms by which PKCs are modulated by ROS and how these processes are involved in the cellular response. Phosphorylation, acetylation, ubiquitinylation, and glycosylation are among the most well-known post-translational modifications. The concept of protein oxidation as a post-translational modification has only recently gained acceptance. Protein oxidation occurs as an outcome of a chemical attack by reactive oxygen species (ROS) or reactive nitrogen species (RNS) on susceptible amino acids, such as tyrosine, tryptophan, histidine, lysine, methionine, and cysteine . Indeed,The goal of this review is to discuss the mechanisms by which protein kinase Cs (PKCs) can be modulated by ROS and how these processes are involved in the cellular response. The review will focus on the structural mechanism of action of ROS in the activity of these enzymes, how ROS interact with their target molecules, the regulation of these enzymes by oxidants, and finally the major consequences of this tightly controlled mechanism on cell signaling.2-terminal regulatory domains and COOH-terminal catalytic domains [2+/calmodulin-dependent kinase (CaMK) superfamily. For a complete review of PKD see [The PKC family is composed of serine/threonine protein kinases that are involved in a variety of pathways that regulate cell growth, differentiation, apoptosis, transformation and tumorigenicity. Most cells express more than one isoform, and each type of PKC mediates different cellular events ,9. The v domains . They al domains . However domains . Thus, t domains –14. The  domains ,12. They domains ,15. Figu PKD see ,17.C-terminus. The first residue has been termed the “turn motif” because it is flanked by a proline residue, and the terminal site is called the “hydrophobic motif” because it is flanked by hydrophobic amino acid residues. The autophosphorylation produces a mature and fully phosphorylated enzyme that is able to respond to lipid second messengers [PKC isoforms require serine/threonine phosphorylation for their activation. There are three distinct phosphorylation sites in the catalytic domain; the first and rate-limiting phosphorylation step occurs in the activation loop sequence ,15. Phosssengers ,15,18,202) to generate DAG and inositol trisphosphate (IP3), which subsequently mobilize intracellular calcium [2+ and DAG, PKCs are maintained in an inactive conformation by binding of the PS to the substrate-binding cavity [2+, which binds to the C2 domain. After Ca2+ signaling, the binding of DAG to the C1 domain increases the affinity of PKCs for membrane lipids, inducing conformational changes that lead to a catalytically competent form. Phorbol esters bind to the same site as DAG and mimic its activation [2+ and activated directly by DAG [Phospholipase C (PLC) is an enzyme that, when activated by growth factor receptors, hydrolyzes phosphatidylinositol 4, 5-bisphosphate , hydrogen peroxide (H2O2) and hydroxyl radicals (·OH) [•OH is the most unstable radical, reflecting its limited ability to transmit signals across any significant distance. In addition, O2− and H2O2 can be considered more stable species, and because of this feature, they may be the most favorable ROS to operate as signaling molecules [Reactive oxygen species (ROS) are produced in the natural course of metabolism. They can originate from different sources, including the mitochondrial electron transport chain, xanthine oxidase, myeloperoxidase, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox enzymes), and lipoxygenase ,25. Nox ls (·OH) . These solecules . ROS werolecules ,25.2+ or phospholipids. Cysteine residues are also found in the C-terminal catalytic domain, but they are uncoordinated in a different manner to those in the regulatory domain. Redox modifications at the C-terminal catalytic domain lead to the inactivation of the kinase due to the loss of the free sulfhydryls required for its catalytic activity. Furthermore, the intracellular redox state has been shown to affect the distribution of the PKC isoforms [2O2 this signaling pathway was shown to be dependent of the activation of PKD by two coordinated signaling events: the phosphorylation of Tyr463 mediated by the Src-Abl signaling pathway, which then facilitates the second step, the phosphorylation of the PKD activation loop of Ser738/Ser742 by the Src-PKCδ pathway, leading to an increase in cell survival exposed to oxidative stress [In the PKC structure, two pairs of zinc fingers are found within the regulatory domain. They are sites of DAG and phorbol ester binding. Each zinc finger is formed by a structure that is composed of six cysteine residues and two zinc atoms, as presented in isoforms ,23. Becaisoforms . The aPKisoforms . This chisoforms . PKCδ seisoforms . In HeLae stress ,42. Thise stress . Later te stress . The spe2, such as Nox enzymes, xanthine oxidase and cytochrome P-450, play important roles in cellular metabolism and signaling [In addition to hormones and growth factors, ROS are currently recognized as initiating factors for signaling cascades in the cell, as they are involved in numerous physiological signaling pathways and cellular functions. Thus, sites of ROS production within the cell, such as mitochondria, or enzymes that are capable of one-electron transfer to Oignaling –36.2− production. With respect to complex III, two components were proposed to be auto-oxidizable electron donors to oxygen: semiubiquinone and reduced cytochrome b. The other site in the respiratory chain that is involved in O2− formation is complex I. In this large multi-subunit complex, electron transfer functions at near equilibrium, and thus, O2− production may be linked to both forward electron transport and reverse electron transport. For a complete review, see Rigoulet et al. 2011 [The main superoxide producer in the cell is the respiratory chain, and the primary factor governing mitochondrial ROS generation is the redox state of the respiratory chain ,44. In fal. 2011 .PKCs exist in an inactive form and are activated depending on their subcellular redistribution and the availability of the appropriate substrates after a distinct stimulus ,47. Curr2O2 stimulates its accumulation in the mitochondria, and this fact can be related to apoptosis in different cell types [The presence of PKCs in the mitochondria has long been known, but their functions are not well defined ,49. The ll types –52. On tll types .2− being the major species responsible for the activation of PLC-dependent PKCγ, which is part of a pro-survival pathway to protect the cells from the cytotoxicity of silibinin [In human melanoma cells, mitochondrial ROS were found to have a cytoprotective effect in cells treated with the chemotherapeutic silibinin. In a study performed by Jiang and colleagues , silibinilibinin .2− and Sorbitol, the product resulting from the enzymatic conversion of glucose, can produce fructose by the action of sorbitol dehydrogenase. This reaction increases the ratio of NADH/NAD+, enhancing the oxidized form of triose phosphates with de novo synthesis of DAG. DAG can then activate several PKC isoforms, which can lead to various clinical complications in diabetes [2− to H2O2, results in a decrease in mitochondrial ROS production, which prevents PKC activation and sorbitol accumulation [PKC activation is also involved in cell damage induced by hyperglycemia. Hyperglycemia results in a loss of antioxidant reducing equivalents, which culminates in an overproduction of Odiabetes ,56. In bmulation .2O2 was able to mimic hypoxic responses in normoxic conditions, leading to an increase in PKCɛ activity in pulmonary arteries. Interestingly, H2O2 also significantly activated PKCɛ in mesenteric arteries although not under hypoxic conditions [2O2 as a second messenger, but the effectiveness of the signal triggered by this molecule may also be related to the site of its production, in this case, the mitochondria. In hepatocytes, mitochondrial ROS generation after cell exposure to sodium arsenite (NaAsO2) was shown to induce PKCδ activation, which in turn activates c-Jun N-terminal kinases (JNK) leading to the progression of apoptosis. On the other hand, in the same study, the authors showed that treatment of hepatocytes with taurine was able to inhibit PKCδ and JNK activation, thus avoiding arsenic-induced ROS generation in the liver, preventing apoptosis [Mitochondrial ROS also have an important role in pulmonary vasoconstriction induced by hypoxic conditions. In artery smooth muscle cells that were submitted to an acute hypoxic period, an increase in mitochondrial ROS production was shown, followed by the activation of total PKC and PKCɛ. The experiments performed by Rathore and colleagues  providednditions . These epoptosis . In contpoptosis .N-(4-hydroxyphenyl)retinamide (4HPR), a synthetic retinoid effective in cancer chemoprevention and therapy was shown to increase mitochondrial ROS generation, which in turns activates PKC and MAPK kinases . Downstream of PKC activation is the activation of p38 and ERK, resulting in an enhancement of apoptosis via caspase 9 and caspase 3 pathway [ROS-induced PKC activation also has an important function in the cardioprotective signaling cascade. Sevoflurane, an anesthetic with the ability to protect the myocardium from ischemia and reperfusion injury, triggers a protective signal transduction cascade . The pre pathway .In addition to interactions between receptors and ligands, other events are involved in mitochondrial ROS-induced PKC activation. Several studies implicate that mechanical stretch increases ROS production in endothelial cells –66. In b2− with secondary production of H2O2) in a regulated manner in response to different stimuli, including growth factors, cytokines and calcium signals [b558, which is composed of two subunits, a large glycosylated gp91phox that forms the catalytic subunit and the smaller regulatory subunit p22phox, and a cytosolic component formed by four major proteins, the regulatory subunits p47phox, p67phox, and p40phox, and the small GTPase RAC [b558 to form the active enzyme, a process that is tightly regulated by protein-protein interactions and phosphorylation of the p47phox subunit [Nox enzyme is a complex multi-subunit enzyme that uses NADPH as a substrate to convert molecular oxygen into ROS  (amino acids 363–368) [PKC isoforms are upstream regulators of Nox. The activity of these enzymes is important for the assembly and activation of the Nox1-3 isoforms, which require the phosphorylation-dependent assembly of several cytosolic subunits to be catalytically active; this includes p47phox, which is the best studied of the phosphorylated Nox enzyme components . The C-t363–368) ,77. In r363–368) ,78. Diff363–368) ,80.PKC is not the only protein kinase able to phosphorylate p47phox. PKA ,96, MAPKA growing body of research has shown that the activation of the PKC/Nox signaling complex regulates ROS levels and is involved in various pathophysiological conditions, including neurodegenerative disorders ,106, hum2− generation by Nox enzymes in several types of cells, including phagocytes [PKC has been shown to activate Oagocytes ,71, cardagocytes , aortic agocytes , plateleagocytes , and renagocytes .The interaction between ligand and receptor is crucial to trigger the signaling pathways that involve the PKC and Nox enzymes. The response is different in each cell and involves distinct PKC and Nox isoforms. In vascular smooth muscle cells (VSMC), for example, three isoforms of Nox enzymes are present, Nox1, Nox2, and Nox4. They are thought to have different functions and subcellular localization. It seems that Nox1, when activated, is present on the cell surface and co-localizes with caveolins, while Nox2 can be located intracellularly or in the cell membrane. Nox4 is localized in focal adhesions and in nuclei –122. In PKC can also activate Nox2. In human monocytes and murine macrophages PI3K and PKC pathways are involved in Nox2 stimulation in response to insulin. This mechanism triggers ERK1/2, p38MAPK and NFκB, which culminates in the activation of monocytes and proliferation of macrophages . PKC-dep2O2 in response to various hormone stimuli. Generation of H2O2 by Nox4 after insulin stimulation of differentiated adipocytes was shown to be important in both early and late events in insulin signal transduction, which includes the activation of insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, and the activation of downstream serine kinases and glucose uptake [2O2 and diacylglycerol, through a mechanism dependent of phospholipase A2, but apparently independent of PKC activity. The arachidonic acid generated in this system by phospholipase A2 is responsible for the oxidase activation and the increase of intracellular ROS production [Several studies demonstrate that Nox4 is inducible and can generate He uptake . Similare uptake . Nox4 waoduction , similaroduction .+-channel in the rat cortical collecting duct through the activation of Nox-dependent PKC phosphorylation [2− that is generated primarily by PKC-dependent Nox activation [2− generation in the thick ascending limb of the renal medulla through the activation of PKCα and the stimulation of NADPH oxidase [+–K+ pump [It has been well established that angiotensin II exerts important intra–cellular signaling through ROS generation in many physiological and pathological conditions . In manyrylation . In the tivation . In phys oxidase . This me oxidase . Similar oxidase  and mono oxidase . In myoc–K+ pump . Convers–K+ pump .2+ and PKC activation are critical for AT1 receptor-induced Nox2 activation and ROS production. The authors suggest that this mechanism could play a role in the vascular dysregulation mediated by the central autonomic system [Angiotensin II can also act as key neuromodulator in central autonomic nuclei ,140. Vagc system .O-tetradecanoylphorbol-13-acetate (TPA) or PMA, are also involved in PKC-dependent Nox activation. In human lung adenocarcinoma cells, the tumor-promoting TPA and the tumor necrosis factor-alpha (TNF-α) were implicated in the stimulation of the expression of the mitochondrial manganese-dependent isoform of SOD (MnSOD) mediated by the Nox enzyme pathway. This stimulation is in part regulated by PKCα, which phosphorylates a transcription factor of cAMP-responsive element-binding protein, or by the PKCɛ pathway, which culminates with the activation of forkhead transcription factor 3 (FOXO3) [Phorbol esters, such as 12- (FOXO3) . In a re (FOXO3) , suggest (FOXO3) . PMA inf (FOXO3)  in a cal (FOXO3) . Althoug (FOXO3) ,146,147. (FOXO3) .Several works also highlight the participation of formyl peptide formyl-methionyl-leucyl-phenylalanine (fMLP) in the activation of PKC and Nox enzyme. This participation includes superoxide generation, which in turn stimulates leukocytes, phagocytic cells, and neutrophils ,149,150.Although most biological functions of PKC have been recognized to occur at the plasma membrane or in the cytoplasm, several studies have pointed out the role of PKCs in nuclear functions . Almost The nuclear presence of PKC suggests an involvement of these enzymes in the regulation of DNA replication, RNA synthesis and processing, gene expression, transport between nucleus and cytoplasm, and chromatin structure . MoreoveBeside PKCs, Nox enzymes subunits have been described to be localized in subcellular compartments of different cells . This inshc protein [shc phosphorylation, preserving mitochondrial morphology and reducing apoptosis in cells under oxidative stress [PKC also has been shown to promote the production of endogenous ROS to induce a positive feedback loop . The β i protein . This pr protein . In mouse stress .In myeloid leukemia cells, ROS are not necessary for the translocation of PKCβII from the cytosol to the cell membrane. However, PKCβII was shown to be essential for ROS production induced by the PKC activator TPA. This ROS-dependent mechanism is associated with the stress-activated protein kinase pathway (SAPK). SAPK is associated with the molecular response of myeloid leukemia cells to TPA, through the phosphorylation and activation of c-jun, activating transcription factor 2 (ATF2) and Elk-1, contributing to the induction of the c-jun and Erg-1 early response genes involved in this signaling process . The PKCBecause different PKC isoforms are present in a variety of cells and are involved in a number of signaling pathways, it is difficult to ascribe a common ROS-dependent mechanism for all PKC isotypes. However, it seems that under pathological conditions, a general signaling mechanism is triggered in mitochondrial dysfunction. In this context, an increase in ROS production by mitochondria activates local PKCs, which in turn activate Nox enzymes. Nox enzymes could also activate another group or the same group of PKCs in a feedback mechanism. Consequently, the redox state of the cell becomes imbalanced. Depending on the stimuli, proapoptotic signaling may occur through ROS activation of MAPK, such as SAPK/JNK, ERK1/2 and p38, or antiapoptotic signals ccould be triggered through activation of the NFκB or Akt/ASK1 pathway . Figure


The molecular pathways by which long chain polyunsaturated fatty acids (LCPUFA) influence skeletal health remain elusive. Both LCPUFA and parathyroid hormone type 1 receptor (PTH1R) are known to be involved in bone metabolism while any direct link between the two is yet to be established. Here we report that LCPUFA are capable of direct, PTH1R dependent activation of extracellular ligand-regulated kinases (ERK). From a wide range of fatty acids studied, varying in chain length, saturation, and position of double bonds, eicosapentaenoic (EPA) and docosahexaenoic fatty acids (DHA) caused the highest ERK phosphorylation. Moreover, EPA potentiated the effect of parathyroid hormone (PTH(1–34)) in a superagonistic manner. EPA or DHA dependent ERK phosphorylation was inhibited by the PTH1R antagonist and by knockdown of PTH1R. Inhibition of PTH1R downstream signaling molecules, protein kinases A (PKA) and C (PKC), reduced EPA and DHA dependent ERK phosphorylation indicating that fatty acids predominantly activate G-protein pathway and not the β-arrestin pathway. Using picosecond time-resolved fluorescence microscopy and a genetically engineered PTH1R sensor (PTH-CC), we detected conformational responses to EPA similar to those caused by PTH(1–34). PTH1R antagonist blocked the EPA induced conformational response of the PTH-CC. Competitive binding studies using fluorescence anisotropy technique showed that EPA and DHA competitively bind to and alter the affinity of PTH1 receptor to PTH(1–34) leading to a superagonistic response. Finally, we showed that EPA stimulates protein kinase B (Akt) phosphorylation in a PTH1R-dependent manner and affects the osteoblast survival pathway, by inhibiting glucocorticoid-induced cell death. Our findings demonstrate for the first time that LCPUFAs, EPA and DHA, can activate PTH1R receptor at nanomolar concentrations and consequently provide a putative molecular mechanism for the action of fatty acids in bone. As age increases, the risk of bone loss and fracture increases s-cAMP-PKA q/11-PLC-PKC Parathyroid hormone (PTH) has been shown to play an important role in bone homeostasis It is well documented that lipids play an important role in skeletal biology and bone health In human studies, it has been shown that consuming EPA improved bone quality in elderly female subjects Studies on LCPUFA and CLA (conjugated linoleic acid) in laboratory animals suggest that dietary intakes of different PUFAs and CLA can affect bone remodeling through biosynthesis of prostaglandins and insulin-like growth factors The venous blood concentration of fatty acids is known to vary from 250 µM to 3 mM depending on the nutritional state Fatty acids have been shown to activate certain GPCRs To gain insights into the mechanism by which LCPUFA influence bone cells, we have chosen to primarily study activation of the MAPK/ERK pathway via PTH1R, which is known to have an important role in bone remodeling. In this study we test if LCPUFA can: activate the ERKs via PTH1R, modulate PTH hormone signaling, bind to, cause conformational changes and activate PTH1R. Additionally we aimed to determine if (1) a PTH1R antagonist or knockdown of PTH1R is able block PTH1R mediated activation of EPA; (2) if activation of the ERK cascade by LCPUFA is PKA and PKC-dependent; and (3) if LCPUFA play a similar biological survival role as PTH in osteoblasts by examining effects of LCPUFA on Akt phosphorylation and cell survival.max = pERKmax/ERK) of ∼3.5 and ∼3.0 respectively in HEK293 cells.To examine fatty acid activation of the ERK1/2 cascade via PTH1 receptor, we treated PTH1R transfected Human embryonic kidney 293 (HEK293) and Murine calvarial preosteoblast (MC3T3-E1) cells with distinct fatty acids for 5 minutes as shown on 11, D-Trp12]-PTH-RP (7–34) amide  8,18 Tyr34]PTH (3–34) which was more efficient at inhibition in MC3T3-E1 cells  amide . PhosphoE1 cells .11, D-Trp12]-PTH-RP (7–34) on inhibiting PTH1R activation by EPA, we have performed a Schild analysis of the antagonist induced inhibition of ERK activation. -PTH-RP (7–34) produced a parallel rightward shift in the EPA concentration dependence curve for stimulation of the ERK cascade via PTH1R activation (max) for EPA and did not detectably affect ERK phosphorylation in the absence of the agonist  acts as a competitive antagonist of EPA stimulated PTH1R-dependent ERK phosphorylation over the range of antagonist concentrations tested.To further examine the mechanism of action of antagonist PTH(3–34) amide, blocked the conformational response of the PTH1 receptor to EPA. Although GPCR FRET constructs of this type have been shown to have a reduced efficacy of cognate ligands in stimulating conformational changes due to the insertion of the fluorescing proteins into the native receptor structure To test if EPA induces a PTH1R conformational change, we used the previously constructed and characterized PTH1R FRET sensor, PTH-CC, containing an intramolecular FRET pair that enables detection of conformational activity upon ligand stimulation TMR fluorescent ligand, where a TMR fluorophore was linked to the lysine side chain (at position 13) of PTH(1–34) as described earlier TMR (iK = 4.66±4 nM) was close to that of PTH(1–34) for PTH1 receptor (iK = 2.4±0.1 nM) To further characterize the association between the fatty acids and the PTH1 receptor, we used the PTH(1–34)TMR, at a concentration of 2 nM per well in DPBS was incubated with human PTH1 receptor membrane fractions for four hours at room temperature in the presence of varying concentrations of EPA and DHA. The incubation time we used was based on previous studies in membrane fractions 50 to iK values i values . Both EP11, D-Trp12]-PTH-RP (7–34) antagonist which lead to a significant reduction EPA-induced Akt phosphorylation.To test if LCPUFA similarly to PTH, can modulate biological survival pathway in osteoblasts 11, D-Trp12]-PTH-RP (7–34) antagonist that significantly reduced the fatty acid survival effect.To investigate if EPA-dependent PTH1R activation has a direct biological effect, we investigated cell survival. Treatment of MC3T3 cells with glucocorticoid, dexamethasone (Dex), induces apoptotic cell death as indicated by an increase in trypan blue staining , similarTo confirm that LCPUFA induced ERK phosphorylation is unique for and dependent on the PTH1 receptor in differentiated MC3T3 cells, PTH1R was knocked down by siRNA and the effect of LCPUFA were analyzed. MC3T3s were tranfected with 100 nM siPTH1R or scrambled control siRNA for 96 hours prior to treatment. The cells were then treated with either 10 nM PTH(1–34), 1 µM EPA or 1 µM DHA for five minutes. siPTH1R transfected cells had a significant decrease in ERK phosphorylation when treated with PTH, EPA, or DHA compared to the scrambled siRNA transfected cells .The results presented in this study indicate that LCPUFA, EPA or DHA, can act as an agonist to the PTH1 receptor at nanomolar concentrations. Our data suggest that the mechanism of activation of PTH1R by EPA involves: binding to the PTH1 receptor, inducing a conformational change similar to that of its cognate ligand (PTH(1–34)), and activation of G protein downstream effectors, PKA and PKC, that ultimately lead to ERK phosphorylation.Although our primary focus in this study was EPA, which caused the greatest ERK phosphorylation response via PTH1R, DHA and other LCPUFA, both n-3 and n-6, also caused a significant ERK phosphorylation response with somewhat lower potency. To decipher a possible trend in fatty acid induced ERK activation via PTH1R, we tested fatty acids with different carbon lengths and double bond number. Our data presented in 11, D-Trp12]-PTH-RP (7–34) appears to be a pure antagonist to EPA within the detection limits of the assays used.The selective PTH1R antagonist or knockdown of PTH1R strongly inhibits the response to EPA or DHA ; 5; 8. Ts and Gq pathways and not through β-arrestin pathway.It has been shown that PTH1R can stimulate ERK1/2 through several distinct signal transduction pathways: a G protein dependent pathway mediated by PKA and PKC and a G protein independent pathway mediated through β-arrestin max of fatty acids on ERK1/2 phosphorylation was somewhat lower as compared to PTH(1–34)  , Fig. 2AAlthough in the case of PTH(1–34), intermittent treatment favors bone anabolism and continuous treatment induces the opposite pattern max at higher EPA concentrations (>100 nM)   is due t>100 nM)  leading LCPUFAs had similar biological effects as PTH on the Akt and cell survival pathway The present study reveals the significant effects of fatty acids on PTH1R signaling. In combination, our current findings, numerous previous reports on the importance of PTH signaling on bone homeostasis and the fact that free circulating LCPUFA are present in blood at concentrations used in this study clearly calls for more detailed studies of the role of LCPUFA on PTH pathway both in in-vitro and in-vivo models as it may contribute to development of alternative approaches for treatments of bone diseases such a osteoporosis.iK values  similar to cognate agonist PTH(1–34) (∼2.4 nM). Furthermore, we show that other LCPUFAs are also able to activate PTH1 receptor at concentrations of fatty acids typically found in blood. Analysis of the signaling cascade suggest EPA and DHA actions are mediated through PKA and PKC which are known to be downstream signaling molecules of sG and qG pathways coupled to PTH1 receptor. The Schild analysis and competitive binding studies suggest that EPA and DHA competitively bind to the PTH1 receptor and EPA acts synergistically with PTH(1–34) causing superagonistic response of ERK1/2. The Akt and cell survival analysis suggests that EPA and DHA may affect osteoblast turnover both by directly activating PTH1R and by modulating the potency of endogenous PTH. In combination, these data provide a molecular basis for the involvement of the PTH1 receptor in mediation of LCPUFAs anabolic effects in bone. Furthermore we believe our study is a starting point for more detailed studies of interactions between fatty acids and other types of GPCRs and more detail understanding of known beneficial effects of fatty acids on human health.In conclusion, we have identified novel PTH1 receptor agonists, EPA and DHA, which are able to induce PTH1R response with 11, D-Trp12]-PTH-RP (7–34) Amide and PTH (3–34) Amide was purchased from Phoenix Pharmaceuticals, INC . Human [Lys-13(Nε-5-carboxy-TMR)]PTH(1–34)NH2 [herein termed PTH(1–34)TMR] was synthesized by Selleck Chemicals LLC . Dexamethasone was purchased by Sigma-Aldrich . siRNAs specific for murine PTH1R (1027416) were purchased from Qiagen. Adrenic Acid 22∶4 (n-6) was purchased from Enzo . Docosahexaenoic Acid 22∶6 (n-3) and Eicosapentaenoic Acid 20∶5 (n-3) were obtained from Cayman Chemical . Dihomo-gamma-Linolenic acid 20∶3 (n-6), Docosadienoic Acid 22∶2 , Docosapentaenoic Acid 22∶5 (n-3), Docosapentaenoic Acid 22∶5 (n-6), and Eicosatrienoic Acid 20∶3 (n-3) were purchased from Nu-Chek-Prep . Arachidonic Acid 20∶4 (n-6), Conjugated Linoleic Acid 18∶2 (n-6/7), and Eicosadienoic Acid 20∶2 (n-6), were purchased from Matreya  Linoleic Acid 18∶2 (n-6), Oleic Acid 18∶1 (n-9), and Stearic Acid 18∶0 were purchased from Sigma-Aldrich . GF109203X and H-89 were purchased from Enzo .HEK293  cells were grown in DMEM media  containing 4.5 g/L D-Glucose and transfected using Targefect-293 . MC3T3-E1  cells were cultured in Ascorbic Acid(AA)-free α-modified Eagle's medium  containing 10% FBS. Differentiation was induced by addition of 50 µg/mL AA. Cells were grown to confluence and treated for 10 days in AA-containing medium. Xfect  was used for transfection of MC3T3-E1 cells. Human PTH(1–34) was obtained from Bachem . Human -PTH-RP (7–34) Amide, for 30 minutes prior to fatty acid treatment; MC3T3-E1 cells were treated with PTH1R inhibitor, Bovine PTH(3–34), prior to fatty acid treatment. To further examine downstream signaling, HEK293 cells transfected with PTH1R were incubated with 20 µM of PKA inhibitor, H-89, or 2.5 µM of PKC inhibitor, GF109203X, for 15 minutes prior to fatty acid treatment.To examine fatty acid dependent ERK phosphorylation, both MC3T3-E1 and HEK293 cells transfected with PTH1R were treated with various fatty acids containing 0.1% Ethanol and 3.5×10A control FRET sensor that is localized to the plasma membrane of cells (PM-CC) was constructed as previously described AATKLRETNA-Citrine-SCDTRQQYRKLLKST , replacing PTH1R's GRCDTRQQYRKLLKSTATKLRETNA (underlined are the 2 residues where Citrine is inserted in between), and has cytoplasmic COOH terminus fused to Cerulean at residue Ser495 of PTH1R with insertion of two extra residues encoding Pro-Val to accommodate an Age1 restriction site. The sequences of all constructs were confirmed by sequencing service.PTH-CC was constructed and characterized in our previous study as described FRET measurements in single living cells, 24 hours after being transfected, were performed using multichannel time resolved single photon counting as previously described Both MC3T3-E1 and HEK293 cells were washed with ice cold DPBS, collected, and lysed in lysis buffer containing 50 mM Tris –HCl, 135 mM NaCl, 60 mM n-octyl beta-D-glucopyranoside , protease inhibitor cocktail, and phospahtase inhibitor cocktail  for 30 minutes on ice. SDS sample buffer was added to the lysate and incubated at 95°C for 5 min. Cell extracts were resolved by SDS-PAGE and transferred to polyvinylidine difluoride (PVDF) membranes. Blots were probed with anti-phospho-p44/42 MAPK , anti-p44/42 MAPK , and anti-phopho-Akt . Beta-tubulin (H-235) was from Santa Cruz Biotechnology. Immunoreactive bands were detected with the appropriate horseradish peroxidase-conjugated secondary antibodies  and visualized by enhanced chemiluminescence . The band intensities were measured by densitometry analysis using Image J  and the change in phosphorylated ERK in both cell lines was calculated as the pERK/ERK ratio. The change in phosphorylated Akt was calculated as the pAkt/beta-tubulin ratio.g for 5 minutes. Cell pellet was resuspended in 1 mL DPBS with protease inhibitor cocktail  and 0.5 mm Zirconia/Silica Beads . The sample was homogenized by vortexing at 4°C for 10 minutes. The homogenate was then centrifuged at 1000 g for 5 minutes to remove unbroken cells and larger debris. The supernatant was centrifuged at 50,000 g at 4°C for 60 minutes  and the pellet resuspended in DPBS. Cell membrane fractions were stored at −80°C.Membrane fractions were prepared using confluent monolayers of HEK293 cells transfected with PTH1R in 10-cm plates. Cells were lifted and centrifuged at 1000 G is the calibration factor for the plate reader. Spectral filters, polarizers, and dichroic mirrors were either factory supplied or obtained from CVI Melles Griot . Excitation filter 527 nm±5 nm, emission filter 600 nm±20 nm, and dichroic-2 mirror. The measurements were recorded using 25 excitation flashes with a 40 µs integration time in black flat bottom 96-well plates . In order to have a consistent surface tension and reduce the effect of increased peptide concentration on the meniscus in each well, the plates were blocked with 10% non-fat milk for 2 hours and washed with DPBS prior to start of assay. Total assay volume was 150 µL, obtained by the sequential addition of 80 µL of buffer (DPBS), 5 µL of membrane preparation, 15 µL of competitive displacing ligand, and 50 µL of fluorescent tracer PTH(1–34)TMR .Fluorescence anisotropy was measured on TECAN GENios Pro  according to the equation, Cell death was quantified after combining nonadherent and adherent cells released from the culture dish using trypsin-EDTA. Cells were resuspended in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) prior to analysis. For determination of dead cells by dye exclusion, 0.4% Trypan Blue  was added, and the percentage of cells exhibiting both nuclear and cytoplasmic trypan blue staining was determined using a hemocytometer. A minimum of 100 cells were counted.t-test. A p value of <0.05 was considered to be statistically significant. Calculations of the mean, standard error of the mean,and Student's t-test were performed using Microsoft Excel and OriginPro .Statistical significance was evaluated for differences between groups from at least three independent experiments using Student's x) is for DPTH alone and (Dx)EPA is for DEPA alone that can activate the ERK pathway to the factional effect x. In the numerator, (D)PTH+(D)PTH is a given combination of PTH and EPA that activates the ERK pathway to the same fractional effect x. Concentration values were obtained using data from Synergy was characterized using combination index Figure S1pERK levels in HEK393 and MC3T3 untransfected or transfected with the empty plasmid pcDNA3.1. Data represents mean ± SEM of at least 3 independent experiments.(DOCX)Click here for additional data file.Figure S2TMR as function of PTH(1–34) concentration after 4 hours incubation at room temperature.Fluorescence anisotropy of PTH1R membranes labeled with nM PTH(1–34) Data represents mean ± SEM of at least 15 independent experiments.(DOCX)Click here for additional data file.


In vitro phosphorylation analysis using purified yeast proteins revealed that free Clb5-Cdk1 can create positive feedback by phosphorylating Sic1 that is bound in the inhibitory complex, and that Sic1 inhibits Clb5-Cdk1 with a sub-nanomolar inhibition constant. Our model also predicts that if the G1-phase CDK complex is too efficient at targeting Sic1 for destruction, then G1/S becomes a smooth and readily reversible transition. We propose that the optimal role for the G1-phase CDK in the switch would not be to act as a kinase activity directly responsible for abrupt degradation of CKI, but rather to act as a priming signal that initiates a positive feedback loop driven by emerging free S-phase CDK.The G1/S transition is a crucial decision point in the cell cycle. At G1/S, there is an abrupt switch from a state of high cyclin-dependent kinases (CDK) inhibitor (CKI) levels and low S-phase CDK activity to a state of high S-phase CDK activity and degraded CKI. In budding yeast, this transition is triggered by phosphorylation of the Cdk1 inhibitor Sic1 at multiple sites by G1-phase CDK  and S-phase CDK  complexes. Using mathematical modeling we demonstrate that the mechanistic basis for the abruptness of the G1/S transition is the highly specific phosphorylation of Sic1 by S-phase CDK complex. This switch is generated by a double-negative feedback loop in which S-CDK1 phosphorylates Sic1, thus targeting it for destruction, and thereby liberating further S-CDK1 from the inhibitory Sic1-S-CDK1 complex. Our model predicts that the abruptness of the switch depends upon a strong binding affinity within the Sic1-S-CDK inhibitory complex.  The major cell cycle transitions are triggered by phosphorylation or dephosphorylation of the targets of cyclin-dependent kinases (CDKs)Morgan, . At the SIC1 starts in late mitosis and its protein levels increase until the end of G1 phase, followed by a rapid turnover at the G1/S transition  activation rates, in this study we focus on the analysis of parameters that control the mutual counteraction of CDK and CKI. We used a minimal model containing both G1- and S-phase CDK complexes together with the CKI, which is an inhibitor of the S-phase-CDK. For the model simulations we used our experimentally validated range of inhibition and phosphorylation parameters and realistic concentrations based on experimental data obtained from the literature. The results provide several important predictions about the parameter ranges that allow for bistability of the system, as well as the realistic dynamic paths of the G1/S switch. The conclusions based on these simulations lay out a general framework for understanding the CDK/CKI switches in eukaryotes.in vitro phosphorylation and inhibition assay with purified Sic1 and cyclin-Cdk1 complexes. Figures M value for the phosphorylation of a non-inhibitory version of Sic1 (data not shown). This suggests that Sic1 only inhibits Cln2-Cdk1 as a competitive substrate.One of the important parameters controlling the G1/S switch is the strength of the inhibition of Clb5/6-Cdk1 complexes by Sic1. To quantitatively measure this inhibition we performed an M,ATP = 70 μM, and the value for KM,H1 is above 10 μM (our unpublished data). Therefore, the obtained value of IC50 is a valid estimate of the apparent inhibition constant for substrate phosphorylation at saturating and nearly physiological ATP concentration. However, the experiment presented in Figures i value to be in the range of 100 pM to 1 nM. The exact value of such a constant is difficult to determine using available methods. This extremely tight inhibition complex should exhibit a very low off-rate, which is an important factor in modeling the Sic1 phosphorylation and degradation switch, as discussed below.The experiment presented in Figures in vivo experiments showing that, in the absence of B-type cyclin (Clb) activity, the Clns alone were not able to cause the degradation of Sic1. Thus, Clb-Cdk1 activity is the driving force of Sic1 degradation at the G1/S transition. This underscores the likely importance of a double-negative feedback mechanism whereby Clb5-Cdk1 phosphorylates Sic1, leading to its destruction and thereby releasing further active Clb5-Cdk1 from the inhibitory Sic1-Clb5-Cdk1 complex.The dynamics of the switch are also shaped by the rates of Sic1 phosphorylation by Cln1,2-Cdk1 and Clb5,6-Cdk1. We have previously shown that both Clb5-Cdk1 and Cln2-Cdk1 efficiently phosphorylate the N-terminal part of the truncated non-inhibitory version of Sic1 (Sic1ΔC) . It is partially also the likely reason why the full length Sic1 showed slightly faster phosphorylation rate compared to Sic1ΔC Figures . Indeed,To quantitatively analyze the impact of the inhibition strength and the phosphorylation rates on the abruptness and the degree of irreversibility of the G1/S switch, we constructed a minimal model of Sic1 phosphorylation and degradation Figure , Table 1in vitro, and that multiply phosphorylated forms of Sic1 do not accumulate in vivo after cells are released from an α-factor arrest . Considering that there are about six to seven phosphorylation steps, and only a slight phosphatase counteraction, the net rate constant for the process of Clb5-dependent phosphorylation (kClb5) and degradation of Sic1 was taken to be 0.001 nM−1 × min−1. Although Cln2-Cdk1 showed only a 3–4-fold lower rate for net phosphorylation of Sic1 in the Sic1-Clb5-Cdk1 complex  was initially taken to be 0.00001 nM−1 × min−1, two orders of magnitude lower than the corresponding value for Clb5-Cdk1. The importance of this difference on the properties of the switch will be further analyzed below. Note, as explained above, that these rate constant are the net constants for both phosphorylation and degradation.The values for the phosphorylation rate constants were derived from our experimentally determined rates of the phosphorylation of Sic1 and other specific model substrates by cyclin-Cdk1 complexes , the threshold of Total (Clb5) decreased and the discontinuity of the Clb5-Cdk1 switch was lost. Thus, both tight inhibition and cyclin specificity are important factors for an abrupt transition and a high degree of irreversibility. Interestingly, the effect of Cln2 specificity on bistability was more pronounced compared to the effect of an increase in Ki of the same order.Next, in the diagrams presented in Figure in vivo. On the other hand, Cln2-Cdk1 can efficiently phosphorylate the initial residues in the Sic1 cascade, and this primes Sic1 for efficient subsequent phosphorylation by Clb5-Cdk1, which will finalize the cascade to send Sic1 to degradation , with free Clb5-Cdk1 only starting to appear when the concentration of total Clb5 surpassed the initial concentration of Sic1 at 1000 nM  introduced about a 5–10 min delay in Sic1 degradation after the release of the cells from the G1 arrest . The abruptness of the switch was considerably reduced and the whole transition followed a path of a slow and gradual change . Additionally, recent work by Barberis and colleagues has shown the involvement of Clb5 and also later B-type cyclins in degradation of Sic1 ]. For the inhibition assay twelve reactions with Sic1wt concentrations ranging from 10 to 0.04 nM and two reactions containing no inhibitor were used. As a reference substrate, bovine histone H1 (Upstate) with final concentration 2.5 μM was used. The concentration of kinase complex in the assay was 1.5–2 nM. Before starting the reaction enzyme-inhibitor complex was formed (2 min). Reaction was started by adding H1. Two timepoints, 7 and 14 min, were collected and analyzed. For the phosphorylation assays presented in Figure The general composition of the kinase assay mixture was as follows: 50 mM HEPES pH 7.4, 5 mM MgClM values for Cdk1-dependent phosphorylation of Sic1, velocities were expressed using kcat/KM as a second order rate constant (v0 = kcat/KM[S][E]) instead of the Michaelis–Menten equation. The kinetic schemes of the models are presented in Figure The models were constructed based on mass action kinetics using values of the kinetic constants derived from biochemical experiments. Because Sic1 concentrations in the model were several times below the KThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to assess the presence of a Lipid Transfer Proteins (LTP) in green kiwifruit (Actinidia deliciosa) and to evaluated the allergenic activity of the newly identified protein.Pulp and seeds of green kiwifruit were manually separated and homogenized. Act d 10 was purified from seed extracts by ion exchange chromatography and RP-HPLC, and sequenced by an automatic amino acid sequencer. Act d 10 was evaluated in vivo by skin prick test (SPT) and in vitro by detecting IgE using streptavidin-conjugated CAP . Act d 10 was spotted on a customized ISAC microarray  for evaluating IgE prevalence in a larger cohort of allergic patients.The purification procedure allowed to obtain about 0.4 mg of natural Act d 10 from 1 g of kiwifruit seeds. The primary structure comprises 92 amino acids with a molecular mass of 9,458 Da. Homology by BLAST search in Uniprot database showed Act d 10 displaying the higher identity (56%) with an isoform of Ara h 9. Act d 10 shares 47% of overall sequence identity with Pru p 3. However, higher levels of residue conservation are detected in some regions. Seven patients selected with clinical history of kiwifruit allergy and specific IgE to Pru p 3 were positive to Act d 10 by SPT; the same patients had specific IgE to Act d 10 as detected by the CAP system. 804 randomly selected allergic patients were tested for IgE to Act d 10 and 170 of them (21.14%) were detected positive; 152 of these 170 patients (89.41%) had IgE to Pru p 3.Act d 10 is a new LTP isolated from Actinidia deliciosa, contained in significant amounts in the green kiwi seed. The sequence identity between Act d 10 and other already known allergenic LTPs is not very high, however the significant conservation of some epitope regions suggested potential IgE cross-reactivities.The IgE binding activity of sera from clinically allergic patients has been shown by SPT and direct IgE detection on two different systems. IgE co-recognition of kiwi LTPs and other known LTPs seems to be dependent on the epitope distribution on the different allergens.


The complement component C3a activates human mast cells via its cell surface G protein coupled receptor (GPCR) C3aR. For most GPCRs, agonist-induced receptor phosphorylation leads to receptor desensitization, internalization as well as activation of downstream signaling pathways such as ERK1/2 phosphorylation. Previous studies in transfected COS cells overexpressing G protein coupled receptor kinases (GRKs) demonstrated that GRK2, GRK3, GRK5 and GRK6 participate in agonist-induced C3aR phosphorylation. However, the roles of these GRKs on the regulation of C3aR signaling and mediator release in human mast cells remain unknown.2+ mobilization, attenuated C3aR desensitization, and enhanced degranulation as well as ERK1/2 phosphorylation when compared to shRNA control cells. By contrast, GRK5 or GRK6 knockdown had no effect on C3aR desensitization, but caused a significant decrease in C3a-induced mast cell degranulation. Interestingly, GRK5 or GRK6 knockdown rendered mast cells more responsive to C3a for ERK1/2 phosphorylation.We utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of GRK2, GRK3, GRK5 and GRK6 in human mast cell lines, HMC-1 and LAD2, that endogenously express C3aR. Silencing GRK2 or GRK3 expression caused a more sustained CaThis study demonstrates that GRK2 and GRK3 are involved in C3aR desensitization. Furthermore, it reveals the novel finding that GRK5 and GRK6 promote C3a-induced mast cell degranulation but inhibit ERK1/2 phosphorylation via C3aR desensitization-independent mechanisms. These findings thus reveal a new level of complexity for C3aR regulation by GRKs in human mast cells. Removal of potential phosphorylation sites within the carboxyl terminus of C3aR leads to more robust degranulation when compared to wild-type receptors The complement component C3a plays an important role in innate immunity and also promotes allergic diseases such as bronchial asthma Agonist occupied GPCRs are phosphorylated by a family of protein kinases, collectively known as G protein coupled receptor kinases (GRKs). Of the seven known GRKs, four  are expressed ubiquitously. It is well established that GPCR phosphorylation by GRKs leads to the recruitment of β-arrestin, which results in receptor desensitization and internalization In addition to desensitization, receptor phosphorylation by GRKs leads to the activation of extracellular signal-regulated kinases (ERK1/2) in a β-arrestin-dependent manner. In HEK293 cells, knockdown of GRK5 and GRK6 inhibits angiotensin II and vasopressin-induced β-arrestin-dependent ERK1/2 phosphorylation In the present study, we utilized lentivirus shRNA to knockdown the expression of GRK2, GRK3, GRK5 and GRK6 in human mast cells (HMC-1 and LAD2) that endogenously express functional C3aR. Using this system, we report unexpected findings regarding the roles of GRKs on signaling and mediator release in mast cells via both receptor desensitization-dependent and independent pathways.To determine the roles of GRKs on the regulation of C3aR signaling in human mast cells, we used the Mission shRNA lentiviral system to stably knockdown the expression of GRK2, GRK3, GRK5 or GRK6 in a human mast cell line, HMC-1 cells. Cells were separately transduced with 5 different shRNA constructs targeting different regions of each GRK, and the construct that gave the most efficient knockdown was selected for further study. For control, we used a scrambled shRNA construct purchased from Sigma. Although all 5 constructs reduced the expression of each GRK to variable levels, TRCN0000230149 for GRK2, TRCN0000159482 for GRK3, TRCN0000000842 for GRK5 and TRCN0000001368 for GRK6 were the most efficient in knocking down GRKs in HMC-1 cells. These constructs were therefore used for all subsequent studies in HMC-1 and LAD2 mast cells. Using real time quantitative PCR we found that the mRNA levels of GRK2 and GRK5 were reduced by >90% in HMC-1 cells . However2+ mobilization provides a rapid, sensitive and real-time assay to measure desensitization 2+ spike, which decays rapidly and reaches baseline within ∼3 min 2+ spike, which is followed by a sustained response that remains elevated for an extended period of time. We therefore used Ca2+ mobilization as an assay to determine the effect of GRK2, GRK3, GRK5, GRK6 silencing on C3aR desensitization. C3a caused a transient Ca2+ mobilization in shRNA control cells (2+ response to C3a (100 nM) was similar to shRNA control cells. However, the response remained elevated for longer time period  possess a similar structural organization consisting of an amino terminal domain (185 amino acids), a catalytic domain (270 amino acids) and a carboxyl terminal domain (105 to 230 amino acids). There are, however, important differences in the mechanism via which GRK2/GRK3 and GRK5/GRK6 are localized to the proximity of the receptor to induce receptor phosphorylation 2+ mobilization, greater degranulation and enhanced ERK1/2 phosphorylation when compared to shRNA control cells. The carboxyl terminal tail of C3aR possesses nine potential phosphorylation sites and both GRK2 and GRK3 promote agonist-induced C3aR phosphorylation In the present study, we demonstrated that GRK2, GRK3, GRK5 and GRK6 are expressed in two human mast cell lines, HMC-1 and LAD2. Using shRNA-mediated knockdown of GRKs, we confirmed our previous finding regarding the role of GRK2 on agonist-induced C3aR desensitization in mast cells. In addition, we made the novel observation that GRK3 participates in C3aR desensitization in human mast cells. Thus, silencing the expression of GRK2 or GRK3 resulted in more sustained Ca2+ response but caused substantial inhibition of C3a-induced mast cell degranulation. This suggests that GRK5 and GRK6 provide stimulatory signals for mast cell degranulation. It is noteworthy that GRK5 phosphorylates β-arrestin-1 Previous studies showed that GRK5 and GRK6 promote agonist-induced C3aR phosphorylation in transfected COS cells It is generally accepted that GRK5 and GRK6 promote GPCR-mediated mitogen-activated protein kinase signaling pathways. Thus, siRNA-mediated knockdown of these GRKs inhibit agonist-induced ERK1/2 phosphorylation 4 receptor-mediated ERK1/2 phosphorylation. They also demonstrated that GRK5, but not GRK2, phosphorylates β-arrestin-1 (at Ser412) and that this phosphorylation is required for the inhibition of ERK1/2 activity. We have recently shown that silencing the expression of β-arrestin-1 enhances C3a-induced ERK1/2 phosphorylation via a receptor desensitization-independent pathway 4 receptor, agonist-induced C3aR phosphorylation by GRK5/6 recruits β-arrestin-1 to inhibit C3a-induced ERK1/2 phosphorylation. Tipping et al., Drosophila, Kurtz (Krz) directly binds to and sequesters an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. It is therefore possible that C3a induces β-arrestin-1 phosphorylation via GRK5/GRK6 to promote a complex formation between ERK and β-arrestin-1, leading to the inhibition of ERK1/2 activity. Thus, silencing the expression of GRK5/GRK6 or β-arrestin-1 removes this inhibitory constraint to enhance ERK1/2 phosphorylation. Whether this or other mechanisms participate in the regulation of ERK1/2 activity by GRK5/GRK6 in C3a-activated mast cells remains to be determined.The mechanism by which GRK5/GRK6 inhibit C3a-induced ERK1/2 phosphorylation is not known. Similar to our finding, Barthet et al., In summary, the present study demonstrates that GRK2 and GRK3 participate in C3aR desensitization in human mast cells. It also provides the novel finding that GRK5 and GRK6 promote C3a-induced mast cell degranulation but inhibit ERK1/2 phosphorylation via mechanisms that are independent of receptor desensitization.Mission shRNA bacterial glycerol stocks for GRK subtypes were purchased from Sigma Life Sciences . Indo-1 AM was from Molecular Probes . All tissue culture reagents were purchased from Invitrogen . Anti-GRK 2/3 and anti-GRK5/6 was obtained from Millipore . Anti-human C3aR was obtained from Santa Cruz Biotechnology . Phycoerythrin (PE)-labeled donkey anti-mouse IgG was purchased from eBioscience . All recombinant human cytokines were purchased from Peprotech . Rabbit anti-ERK1/2 and anti-phospho-ERK1/2 antibodies were purchased from Cell Signaling . LightCycler FastStart RNA Master SYBR Green I was obtained from Roche . SuperSignal® West Femto Maximum Sensitivity Substrate and HRP-labeled goat anti-rabbit IgG were from Thermo Scientific . Purified C3a and Phorbol-12 myristate 13-acetate (PMA) were obtained from Advanced Research Technologies  and Calbiochem, (Germany), respectively.HMC-1 cells were cultured in Iscove's modified Dulbecoo's medium (IMDM) supplemented with 10% FCS, glutamine (2 mM), penicillin (100 IU/mL) and streptomycin (100 µg/mL) 6 cells). Eight hours after transduction, the medium was changed and cells were replenished with fresh medium. After a recovery period of 24 h, puromycin (2 µg/ml) was added to select cells with stable virus integration into the genome. Cells were analyzed for GRK knockdown after one week of antibiotic selection.Lentivirus generation was performed according to the manufacture's manual (Sigma). Cell transduction was conducted by mixing 1.5 ml of virus with 3.5 ml of HMC-1 or LAD2 cell  on a Roche LightCycler2.0 instrument. The primers used for real time PCR were: hGAPDH Forward, 5′-GAGTCCACTGGCGTCTTCA-3′ and hGAPDH Reverse, 5′-GGGGTGCTAAGCAGTTGGT -3′ were used for GAPDH. hGRK2 Forward, 5′-ACT TCAGCGTGCATCGCAT-3′ and hGRK2 Reverse, 5′- GCTTTTTGTCCAGGCACTTCAT-3′ were used for GRK2. hGRK3 Forward, 5′-AGCTGTACCTCAGGTGAAGTT-3′ and hGRK3 Reverse, 5′-AGCTTGCTTTGAGAAAGGATGT-3′ were used for GRK3. hGRK5 Forward, 5′-GACCACACAGACGACGACTTC-3′ and hGRK5 Reverse, 5′-CGTTCAGCTCCTTAAAGCATTC-3′ were used for GRK5. hGRK6 Forward, 5′-TAGCGAACACGGTGCTACTC-3′ and hGRK6 Reverse, 5′-GCTGATGTGAGGGAACTGGA-3′ were used for GRK6. The amplification conditions were as follows;, Reverse transcription at 55°C for 10 min. Denaturation at 95°C for 30 s, 40 cycles at 95°C for 10 s, 66°C (69°C for GRK6) for 10 s, and 72°C for 20 s. Melting curve: conditions were; 95°C, 65°C for 15 s, and 95°C (slope 0.1°C/s). Analysis was performed according to ΔΔCt (delta delta Ct) method. Results are expressed as a ratio of GRK to GAPDH.Total RNA was extracted from 1×102+ mobilization was determined as described previously 6) were washed twice with HEPES buffer  containing 1 mg/ml BSA and incubated with 1 µM of Indo-1 for 30 min in dark. Cells were then washed and resuspended in 1.5 ml of the same buffer. The cells were stimulated with 100 nM C3a for 5 min and mobilization of intracellular Ca2+ was monitored with a Hitachi F-2500 fluorospectrophotometer. For desensitization assays, cells were removed from the cuvette, washed three times in cold buffer, resuspended in warm buffer and Ca2+ mobilization to a subsequent exposure of C3a (100 nM) was determined. Desensitization was expressed as percentage decrease in calcium response following second stimulation relative to the first one.Receptor desensitization assay based on Ca6/ml and stimulated with C3a (100 nM) for 1 min or 5 min at 37°C. Cells were washed twice and resuspended in 48 µl of ice-cold FACS buffer (PBS containing 2% FBS). C3aR antibody (2 µL) or Isotype control was added and the cells were incubated on ice for 1 h. After washing twice with cold FACS buffer, cells were resuspended in 48.5 µl of ice-cold FACS buffer. PE-labeled donkey anti-mouse antibody (1.5 µL) was added and the cells were incubated on ice for 1 h. Cells were washed twice and fixed in 300 µl of 2% formaldehyde solution. The samples were acquired and analyzed using FACS Calibur flow cytometer (BD Biosciences).Cells were washed and resuspended in fresh medium at a concentration of 0.5 × 106/ml. Cells were stimulated with C3a (100 nM) or PMA (10 nM) for different time points. Three-fold volume of ice-cold PBS containing 1 mM sodium orthovanadate was added to stop the reaction. Total cell lysates were prepared in RIPA buffer , 5 mM EDTA, 10 mM NaF, 10 mM Na-pyrophosphate and protease inhibitor cocktail) and subsequently analyzed by Western blot using rabbit polyclonal antibodies for phospho-p44/42 MAPK (pERK1/2) and p44/42 MAPK (ERK1/2).shRNA control and GRK knockdown HMC-1 cells were washed twice and resuspended in serum-free IMDM at a concentration of 1×104) were seeded into 96-well plates in a total volume of 50 µl of HEPES buffer containing 1 mg/ml BSA and exposed to different concentrations of C3a . For total β-hexosaminidase release, control cells were lysed in 50 µl of 0.1% Triton X-100. Aliquots (20 µl) of supernatants or cell lysates were incubated with 20 µl of 1 mM p-nitrophenyl-N-acetyl-β-D-glucosamine for 1.5 h at 37°C. The reaction was stopped by adding 250 µl of a 0.1 M Na2CO3/0.1 M NaHCO3 buffer and absorbance was measured at 405 nm LAD2 cells (1.0×10The results are expressed as mean ± S.E.M for the values obtained from multiple experiments. Statistical significance was determined by unpaired two-tailed t test. *, p<0.05; **, p<0.01; ***, p<0.001.


The regulation of mitochondrial dynamics is vital in complex cell types, such as neurons, that transport and localize mitochondria in high energy-demanding cell domains. The Armcx3 gene encodes a mitochondrial-targeted protein (Alex3) that contains several arm-like domains. In a previous study we showed that Alex3 protein regulates mitochondrial aggregation and trafficking. Here we studied the contribution of Wnt proteins to the mitochondrial aggregation and dynamics regulated by Alex3. Overexpression of Alex3 in HEK293 cells caused a marked aggregation of mitochondria, which was attenuated by treatment with several Wnts. We also found that this decrease was caused by Alex3 degradation induced by Wnts. While the Wnt canonical pathway did not alter the pattern of mitochondrial aggregation induced by Alex3, we observed that the Wnt/PKC non-canonical pathway regulated both mitochondrial aggregation and Alex3 protein levels, thereby rendering a mitochondrial phenotype and distribution similar to control patterns. Our data suggest that the Wnt pathway regulates mitochondrial distribution and dynamics through Alex3 protein degradation. Mitochondria are essential organelles for many biological processes, including respiration, energy production and cell viability. Because of the length of neuronal processes (axons and dendrites) and the function of mitochondria in neurotransmission and neural integration, the correct distribution of these organelles is crucial for neuronal function 2+-dependent, and Alex3 was found to control mitochondrial aggregation, dynamics and trafficking in neurons In a previous study we provided evidence that proteins encoded by the Eutherian-specific Armcx gene family localize to mitochondria Arm-containing protein Lost in Epithelial cancers linked to the X chromosome) were initially described as putative tumor-suppressor genes, as their expression is reduced in several epithelial-derived carcinomas, including lung, prostate, colon, and pancreas cancer Members of the Alex protein family -GFP 5′-GGATCCAAGCCTGCGCTGATTTTCGG-3′; for Alex3(1–106)-GFP 5′-GGATCCCATCATCATCATCAGACCA-3′; for Alex3(1–45)-GFP 5′-GGATCCATCACCAGAGCCACCCTCA-3′; for Alex3(1–41)-GFP 5′-GGATCCCCAGAGCCACCCTCAGCCA-3′ and for Alex3(1–30)-GFP 5′-GGATCCCTCAGCCATTTTCTCCTTG-3′). All the constructs generated were sequenced with BigDye-Terminator v3.1 (Applied Biosystems). Mitochondrial-targeted DsRed (MitDsRed) was a gift from Antonio Zorzano (IRB Barcelona). pcDNA-Wnt1 and pcDNA-β-catenin S33 were a gift from E. Batlle (IRB Barcelona). pcDNA-Fz2-HA, pcDNA-Wnt5a, pcDNA-Wnt11 and pcDNA-Dvl2 were a gift from P. Bovolenta .Recombinant mouse Wnt3a was used at 200 or 400 ng/ml and Recombinant mouse Wnt5a at 400 or 800 ng/ml (both from R&D Systems). LiCl and SB216763 (Sigma Aldrich) were used as inhibitors of GSK3β at 10 mM and 10 µM respectively. MG-132  was used as proteasome inhibitor at 10 µM, SP600125 (Sigma-Aldrich) as Jun-kinase inhibitor at 10 µM and KN62 (Bioscience) as CAMKII inhibitor at 25 µM. Cypermetrin (Biogen) was used as Calcineurin inhibitor at 10 µM, Casein kinase II inhibitor I  as CK2 inhibitor at 100 µM, Calphostin C  as PKC inhibitor at 1 µM, BAPTA/AM  as intracellular calcium chelator at 20 µM, and cycloheximide (Sigma-Aldrich) as protein-synthesis inhibitor at 40 µg/ml. These drugs and reagents were used 4 h after transfection. TPA  at 1 µM was used as PKC activator 19 h after transfection. Twenty-four hours after transfection, all the cells were fixed or lysed for immunocytochemistry or Western analysis respectively.2. Upon confluence, cells were trypsinized (0.25% w/v) and plated at the desired density. After two days, cells were transfected using Fugene6 (Roche Diagnostics), following the manufacturer’s instructions, and using a 1∶1 DNA ratio (or as indicated) when two constructs were transfected. Cells were processed as required 24–36 h after transfection.HEK293 cells were used for all the experiments. Cells were cultured in DMEM medium supplemented with 10% Fetal Bovine Serum (FBS), 2 mM glutamine, 120 µg/ml Penicillin and 200 µg/ml Streptomycin and were maintained at 37°C in the presence of 5% COHEK293 cells were fixed in 4% paraformaldehyde. After fixation, they were permeabilized with Triton X-100 in PBS and blocked with blocking buffer (10% FBS (Roche Diagnostics), 0.2 M glycine, 0.1% Triton X-100 and 0.05% Deoxicolic acid in PBS-2% gelatin) for 1 h at room temperature. To label the cells, the following antibodies or dyes were used: rabbit anti-Alex3 (1∶300) HEK293 cells were obtained and lysed in Laemmli Buffer (LB) at 98°C for 5 min. 20 µg of protein for each sample was loaded and run in polyacrylamide gels at 100 V. Transfer to nitrocellulose membranes was performed in 120 mM glycine, 125 mM Tris, 0.1% SDS, and 20% methanol at 35 V o.n. Membranes were then blocked in 5% powder milk in TBS and incubated with primary antibodies anti-Alex3 (1∶2000), anti-GFP , anti-β-catenin , anti-HA . Anti-actin  or β-tubulin  were used as a loading control. Secondary antibodies coupled to HRP were used diluted 1∶2500 in TBS containing 5% powder milk. Labeling was visualized with ECL plus (Amersham Pharmacia Biotech).For quantification of mitochondrial phenotypes, cells were classified as “Normal” (cells with an even distribution of mitochondria forming a dense meshwork), “Aggregated” referred to mitochondrial phenotypes with clustered mitochondria near the perinuclear zone, and “Mild-phenotype” for intermediate phenotypes Fluorodish plates  transfected with Alex3-GFP, MitDsRed or Wnt1 (as above) and filmed 24 h later using a Leica TCS SP2 confocal microscope (Leica Microsystems) equipped with a 63x immersion oil objective. Treatment with TPA was used as described above. All the cultures were kept at 37°C using a heating insert on the microscope stage and an incubating chamber allowing circulation of a controlled CO2 (5%)-air heated mixture for the control of pH. Time-lapse series of image stacks composed of 5 images (512×512 px) were taken every 6 sec over 8 min using Leica Confocal Software (Leica Microsystems). Further image processing and video compilation (7 frames per sec) and edition was done with ImageJ software .HEK293T cells were seeded onto Poly-D-lysine-coated Alex3 is a 379-aa protein containing several regions and motifs, including 6 Arm-like domains conforming a DUF463 domain (aa 110–363), a nuclear localization signal (aa 89–98), and an N-terminal region containing a transmembrane domain (aa 7–29) and a putative outer mitochondrial membrane targeting signal . Gavel aThe Alex3 protein sequence contains 6 Arm-like domains arranged at the C-terminal region . Some prDuring the course of the above experiments, we noted that Alex3 immunofluorescence signals decreased upon treatment with Wnt1 and Fz2 see . To suppWe next addressed whether components of the canonical Wnt pathway, which are known to activate the β-catenin pathway, affected Alex3 protein levels. First, we found that co-transfection of Alex3 with constitutively active β-catenin S33A Finally, incubation with two inhibitors of GSK3β activity, namely LiCl or SB216763, did not prevent Wnt1-induced Alex3 degradation . Taken tWe next tested whether the degradation of Alex3 triggered by Wnt1 was dependent on the proteasome. First, we demonstrate through cycloheximide assays that Alex3 Wnt/PKC-dependent reduction levels is due to protein degradation . We then2+ pathway 2+ pathways, did not reduce the degradation of Alex3 protein caused by Wnt1 incubation . We next tested the impact of additional, non-canonical signaling pathways that are activated by the Wnt cascade, such as the Wnt/Planar Cell Polarity (PCP) Jun kinase pathway and the Wnt/Cacubation .2+ intracellular chelator BAPTA/AM slightly enhanced the Alex3 degradation induced by Wnt1 (Phosphorylation/dephosphorylation often targets proteins to degradation  by Wnt1 . Interes by Wnt1 .We further examined whether the Alex3(1–45)-GFP and Alex3(1–106)-GFP constructs, which lack putative PKC phosphorylation sites , respondIn a previous study we showed that Alex3 overexpression in HEK293 cells leads to mitochondrial aggregation and reduced mitochondrial motility Mitochondrial trafficking and dynamics is essential for cell respiration and thus for cell viability In addition to mitochondrial localization and putative mitochondrial targeting sequences, Alex3 protein contains six Arm-like repeats, arranged in a single DUF463 domain, at the C-terminal region. On the basis of this enrichment in Arm-like domains, we propose that Alex3 functions and thus Alex3-dependent mitochondrial dynamics are regulated by the Wnt/β-catenin signaling cascade. The present data show that overexpression of Alex3 leads to mitochondrial aggregation and decreased mitochondrial dynamics and trafficking. These effects were reversed upon Wnt1 treatment, which led to reduced Alex3 protein levels and concomitantly rendered mitochondrial morphology and dynamics similar to control phenotypes. In our experimental conditions, a similar, though less marked effect, was found with co-transfection with the receptor Fz2 or the Wnt members Wnt5a and Wnt11. Interestingly, neither transfection with a constitutively active β-catenin S33A cDNA or a Dvl2 cDNA, or incubation with GSK3β enzymatic inhibitors (LiCl and SB216763) had effects on Alex3 protein levels or mitochondrial aggregation. These findings suggest that these typical components of the Wnt/β-catenin pathway are not required for the Wnt1-dependent regulation of Alex3-mitochondrial phenotypes.Xenopus embryos In addition to the canonical β-catenin pathway, the Wnt pathway signals through several non-canonical cascades, including the kinases CKII and PKC. Originally, Wnts were classified as canonical (such as Wnt1 or Wnt3a) or non-canonical (Wnt5a or Wnt11) depending or their capacity to induce secondary axis in 2+-dependent effectors CAMKII and Calcineurin do not affect Alex3 protein levels or Alex3-dependent mitochondrial morphology. In contrast, inhibition of CKII was shown to increase Wnt1-dependent Alex3 degradation and, conversely, activation of PKC abolished Wnt1-dependent Alex3 protein levels and mitochondrial morphology and dynamics. These data suggest that these 2 kinases are involved in the regulation of Alex3 protein levels. Although we cannot confirm direct phosphorylation by these kinases, this notion is consistent with the prediction of several CKII and PKC phosphorylation sites in the Alex3 sequence. Moreover, Alex3 fragments lacking these phosphorylation sites are not affected by either Wnt1 transfection or by Calphostin C treatment. Interestingly, up to 5 of these phosphorylation sites fall within the DUF463 domain, which contains six Arm-like domains  The mitochondrial red marker MitoTracker (red) and the mitochondrial green marker MitoGFP (green), which shares the same mitochondrial targeting sequence of MitoDsRed (from the subunit VIII of human cytochrome c oxidase), completely co-localized in HEK293T cells. Alex3-GFP  and Alex3(1–106)-GFP  fully co-localized with both mitochondrial markers, MitoTracker  and MitoDsRed , in HEK293T cells. Scale Bar: 10 µm.(TIF)Click here for additional data file.Figure S2Mitochondrial localization of Alex3 after co-expression of Wnts and Fz2. (A–F) Mitochondrial localization of Alex3 (green) in HEK293AD cells after co-expression with the members of the Wnt/Frizzled signaling pathway used in G–L) High magnifications of boxed areas shown in (A–F). Nuclei were labeled with bisbenzimide (blue). Scale bar: 10 µm.(TIF)Click here for additional data file.Figure S3Alex3 does not colocalize with Frizzled2-HA. Co-expression of Alex3 (green) and Frizzled2-HA (red) in HEK293T cells does not show colocalization. Nuclei are stained in blue (bisbenzimide). Scale Bar: 10 µm.(TIF)Click here for additional data file.Figure S4Recombinant Wnt5a leads to Alex3 degradation and mitochondrial disaggregation in cells.  High-magnification micrographs illustrating that Wnt5a treatment leads to mitochondrial disaggregation. (C) Histogram showing percentage of mitochondrial phenotypes in control and Wnt5a-treated, Alex3-transfected cells. Scale Bar: 20 µm.(TIF)Click here for additional data file.Figure S5Wnt3a treatment does not reverse Alex3 mitochondrial aggregation. Representative Alex3-transfected cells (green) treated with recombinant Wnt3a show mitochondrial aggregate phenotypes. β-catenin in red and nuclei in blue (bisbenzimide). Scale Bar: 10 µm.(TIF)Click here for additional data file.Figure S6Wnt5a and Calphostin C produce Alex3 protein degradation. Overexpressing Alex3 HEK293T cells treated with Wnt5a or Calphostin C and in presence of cycloheximide show a reduction in protein levels compared with cells treated only with cycloheximide.(TIF)Click here for additional data file.Figure S7Alex3-GFP deletion constructs lacking PKC phosphorylation sites do not respond to Wnt1 and Calphostin C. (A) Co-transfection of Wnt1 with Alex3-GFP (1–106) and (1–45) deletion constructs (right panels), which lack PKC phosphorylation sites, does not lead to Alex3 protein degradation, in comparison with co-transfection with full-length Alex3-GFP protein (left panel). (B) Incubation with the PKC inhibitor Calphostin C leads to degradation of full-length Alex3 (left panel) but not of Alex3-GFP (1–106) or (1–45) deletion constructs (right panel). The quantification of Alex3-GFP protein levels is shown at the bottom.(TIF)Click here for additional data file.Video S1Videorecording (7.5 minutes) illustrating mitochondrial dynamics in control cells.(AVI)Click here for additional data file.Video S2Videorecording (7.5 minutes) illustrating mitochondrial dynamics in cells transfected with Alex3-GFP.(AVI)Click here for additional data file.Video S3Videorecording (7.5 minutes) illustrating mitochondrial dynamics in Alex3-GFP/Wnt1.(AVI)Click here for additional data file.Video S4Videorecording (7.5 minutes) illustrating mitochondrial dynamics in Alex3-GFP/Wnt1 cells treated with TPA.(AVI)Click here for additional data file.


In this study, we chemically synthesized three intermediate compounds between VK2 and VK3, namely MK-3, MK-2 and MK-1, and investigated the inhibitory effects of all five compounds on the activity of mammalian pols. Among these compounds, MK-2 was the strongest inhibitor of mammalian pols α, κ and λ, which belong to the B, Y and X families of pols, respectively; whereas VK3 was the strongest inhibitor of human pol γ, an A-family pol. MK-2 potently inhibited the activity of all animal species of pol tested, and its inhibitory effect on pol λ activity was the strongest with an IC50 value of 24.6 μM. However, MK-2 did not affect the activity of plant or prokaryotic pols, or that of other DNA metabolic enzymes such as primase of pol α, RNA polymerase, polynucleotide kinase or deoxyribonuclease I. Because we previously found a positive relationship between pol λ inhibition and anti-inflammatory action, we examined whether these compounds could inhibit inflammatory responses. Among the five compounds tested, MK-2 caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in mouse ear. In addition, in a cell culture system using mouse macrophages, MK-2 displayed the strongest suppression of the production of tumor necrosis factor (TNF)-α induced by lipopolysaccharide (LPS). Moreover, MK-2 was found to inhibit the action of nuclear factor (NF)-κB. In an in vivo mouse model of LPS-evoked acute inflammation, intraperitoneal injection of MK-2 in mice led to suppression of TNF-α production in serum. In conclusion, this study has identified VK2 and VK3 intermediates, such as MK-2, that are promising anti-inflammatory candidates.Previously, we reported that vitamin K This compound inhibited the activity of all of the pols from mammals, and 50% inhibition of the A, B, X and Y families of pols was observed at a dose of 68.8, 27.6–29.1, 24.6–31.0 and 35.3–39.0 μM, respectively; therefore, the strength of the inhibitory effect of MK-2 on mammalian pol families can be ranked as follows: B-family pols = X-family pols > Y-family pols > A-family pol. MK-2 showed the strongest inhibition of pol λ activity among the pols investigated, with an IC50 value of 24.6 μM. This compound also suppressed the activity of the animal pols from fish  and insect (fruit fly) at almost the same concentrations as it inhibited the activity of mammalian pols.Among the five compounds investigated, MK-2 displayed the strongest inhibitory effect on mammalian pols  and was E. coli pol I, Taq pol or T4 pol  pol α or prokaryotic pols, such as r T4 pol . The thrTo test whether MK-2 is an intercalating agent that distorts DNA and subsequently inhibits enzyme activity, we measured the thermal transition of DNA in the presence or absence of this compound. The thermal transition profile of DNA was the same with or without the compound (data not shown). Therefore, the inhibition of pols by MK-2 is not due to DNA distortion, but seems to be due to a direct effect of this compound on the enzymes themselves.2.3.2 and VK3. Application of TPA (0.5 μg) to the mouse ear induced edema, resulting in a 241% increase in the weight of the ear disk 7 h after application. As shown in 3 had a weak effect on the level of inflammation. Therefore, the anti-inflammatory effect of these compounds correlated with their inhibitory effect on mammalian pols including pol λ, which was strongly inhibited by MK-2  and thereby initiates an inflammatory response, resulting in various inflammatory diseases -deoxythymidine 5′-triphosphate (dTTP) (43 Ci/mmol) were purchased from GE Healthcare Bio-Sciences . The oligo(dT)18 DNA primer was customized by Sigma-Aldrich Japan K.K. . LPS was purchased from Sigma-Aldrich. For Western blot analysis, anti-NF-κB p65 antibody, anti-β-actin antibody and horseradish peroxidase-conjugated anti-rabbit IgG antibody  were obtained from Santa Cruz Biotechnology  and Thermo Scientific , respectively. All other reagents were of analytical grade and purchased from Nacalai Tesque Inc. .VK4.2.2 and VK3 derivatives  were chemically synthesized according to a procedure reported by Mayer and Isler with slight modification -dTTP), and incubation was carried out at 37 °C for 60 min, except for Taq pol, which was incubated at 74 °C for 60 min. Activity without the inhibitor was considered to be 100%, and the activity remaining at each concentration of inhibitor was determined relative to this value. One unit of pol activity was defined as the amount of enzyme that catalyzed the incorporation of 1 nmol dNTP (dTTP) into the synthetic DNA template-primer (poly(dA)/oligo(dT)18, A/T = 2/1) in 60 min at 37 °C under normal reaction conditions for each enzyme (scintillation counts: approximately 1 pmol of incorporated radioactive nucleotides = 100 cpm) [The components of the pol assay were poly(dA)/oligo(dT)100 cpm) ,52.4.4.All animal studies were performed according to the guidelines outlined in the “Care and Use of Laboratory Animals” of Kobe University. The animals were anesthetized with pentobarbital before undergoing cervical dislocation. Female 8-week-old C57BL/6 mice that had been bred in-house with free access to food and water were used for all experiments. All of the mice were maintained under a 12-h light/dark cycle and housed at a room temperature of 25 °C.4.5.2 and VK3 intermediates (250 or 500 μg in 20 μL) or 20 μL of acetone as a vehicle control was applied to the inner part of the mouse ear. Thirty minutes after the test compound was applied, a TPA solution (0.5 μg/20 μL of acetone) was applied to the same part of the ear. To the other ear of the same mouse, methanol, followed by TPA solution, was applied as a control. After 7 h, a disk (6 mm diameter) was obtained from the ear and weighed. The inhibitory effect (IE) is presented as a ratio of the increase in weight of the ear disks:The mouse inflammatory test was performed according to Gschwendt’s method . In brie4.6.2–95% air.A mouse macrophage cell line, RAW264.7, was obtained from American Type Culture Collection (ATCC) . The cells were cultured in Eagle’s Minimum Essential Medium (MEM) supplemented with 4.5 g of glucose per liter plus 10% fetal calf serum, 5 mM L-glutamine, 50 units/mL penicillin and 50 units/mL streptomycin. The cells were cultured at 37 °C in standard medium in a humidified atmosphere of 5% CO4 cells/well and incubated for 24 h. The cells were pretreated with the VK2 and VK3 intermediates  for 30 min before the addition of 100 ng/mL of LPS. After stimulation with LPS for 24 h, the cell culture medium was collected to measure the amount of TNF-α secreted. The concentration of TNF-α in the culture medium was quantified by using a commercially available enzyme-linked immunosorbent assay (ELISA) development system  in accordance with the manufacturer’s protocol.RAW264.7 cells were placed in a 12-well plate at 5 × 104.7.5 cells/well were incubated with 10 μM of MK-2 or DMSO (1 μL/mL) as a vehicle control for 30 min, followed by treatment with 100 ng/mL of LPS for 30 min. After treatment, cells were harvested with lysis buffer consisting of 10 mM Hepes (pH 7.9), 10 mM KCl, 1.5 mM MgCl2 and 0.5 mM DTT containing protease inhibitors  and phosphatase inhibitors (10 mM NaF and 1 mM Na3VO4) and stood on ice for 15 min with occasional mixing. The mixture was centrifuged at 1000 × g for 10 min at 4 °C. The precipitate was suspended in extraction buffer consisting of 20 mM Hepes (pH 7.6), 20% (v/v) glycerol, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1.0 mM DTT and 0.1% (v/v) Nonidet P-40 containing the same protease and phosphatase inhibitors. The suspension was then gently mixed on a rotary device for 1 h at 4 °C before being centrifuged at 15,000 × g for 20 min at 4 °C. The resulting supernatant was used as a nuclear extract. The protein concentration was measured by using a bicinchoninic acid (BCA) assay kit in accordance with the manufacturer’s protocol. The nuclear proteins were subjected to Western blotting to evaluate the nuclear translocation of NF-κB.RAW264.7 cells on a 6-well plate at 5 × 104.8.The nuclear proteins (30–50 μg of protein) were boiled in a quarter-volume of sample buffer  and then separated on 10% SDS polyacrylamide gels. Each gel was then electroblotted onto a PVDF membrane. The membrane was blocked with 1% skimmed milk in Tris-buffered saline TBS-T  and probed with anti-NF-κB p65 antibody (1:500) and anti-β-actin antibody (1:5000), before being reacted with horseradish peroxidase (HRP)-conjugated secondary antibody . The protein-antibody complex was visualized with ChemiLumiONE  and detected using an Image Reader . The intensity of each band was analyzed using ImageJ, which was developed at the National Institute of Health.4.9.Mice were intraperitoneally injected with 200 μL of MK-2 dissolved in corn oil at 100 mg/kg body weight (BW), or 200 μL of corn oil as a vehicle control. After 30 min, the mice were intraperitoneally injected with 200 μL of 250 μg/kg BW LPS dissolved in PBS or 200 μL of PBS as a vehicle control. After 1 h, the mice were killed, and blood samples were collected. The blood serum was separated by centrifugation at 15,000 × g for 10 min at 4 °C, and the TNF-α level in the serum was measured by ELISA.4.10.All data are expressed as the means ± SE of at least three independent determinations for each experiment. Statistical significance between each experimental group was analyzed using Student’s t-test, and a level of probability of 0.01 and 0.05 was used as the criterion of significance.5.2 (MK-4) and VK3, potently inhibited the activity of animal pols, especially pol λ. MK-2 also suppressed mouse ear inflammation stimulated by TPA and reduced NF-κB activation and TNF-α production. The molecular mechanism that links the LPS-induced inflammatory response and anti-inflammatory activity in the model of TPA-induced ear edema is unknown. Because activated NF-κB has been observed in a model of TPA-induced ear edema [Our study is the first to demonstrate that MK-2, which is an intermediate between VKar edema , the ant


Human Cytomegalovirus (HCMV) has been implicated in the acceleration of vascular disease and chronic allograft rejection. Recently, the virus has been associated with glioblastoma and other tumors. We have previously shown that the HCMV-encoded chemokine receptor pUS28 mediates smooth muscle cell (SMC) and macrophage motility and this activity has been implicated in the acceleration of vascular disease. pUS28 induced SMC migration involves the activation of the protein tyrosine kinases (PTKs) Src and Focal adhesion kinase as well as the small GTPase RhoA. The PTK Pyk2 has been shown to play a role in cellular migration and formation of cancer, especially glioblastoma. The role of Pyk2 in pUS28 signaling and migration are unknown.In the current study, we examined the involvement of the PTK Pyk2 in pUS28-induced cellular motility. We utilized in vitro migration of SMC to determine the requirements for Pyk2 in pUS28 pro-migratory signaling. We performed biochemical analysis of Pyk2 signaling in response to pUS28 activation to determine the mechanisms involved in pUS28 migration. We performed mass spectrometric analysis of Pyk2 complexes to identify novel Pyk2 binding partners.Expression of a mutant form of Pyk2 lacking the autophosphorylation site (Tyr-402) blocks pUS28-mediated SMC migration in response to CCL5, while the kinase-inactive Pyk2 mutant failed to elicit the same negative effect on migration. pUS28 stimulation with CCL5 results in ligand-dependent and calcium-dependent phosphorylation of Pyk2 Tyr-402 and induced the formation of an active Pyk2 kinase complex containing several novel Pyk2 binding proteins. Expression of the autophosphorylation null mutant Pyk2 F402Y did not abrogate the formation of an active Pyk2 kinase complex, but instead prevented pUS28-mediated activation of RhoA. Additionally, pUS28 activated RhoA via Pyk2 in the U373 glioblastoma cells. Interestingly, the Pyk2 kinase complex in U373 contained several proteins known to participate in glioma tumorigenesis.These findings represent the first demonstration that pUS28 signals through Pyk2 and that this PTK participates in pUS28-mediated cellular motility via activation of RhoA. Furthermore, these results provide a potential mechanistic link between HCMV-pUS28 and glioblastoma cell activation. Human cytomegalovirus (HCMV) is a beta-herpesvirus able to establish a life-long persistent infection after primary infection has been cleared. Although infection is ubiquitous in the human population, persistent HCMV infection is commonly asymptomatic in healthy, immunocompetent individuals. However, HCMV causes significant disease in immunosuppressed patients and, despite effective antiviral therapies, HCMV infection is still a significant problem in congenital disease and in bone marrow transplant recipients. Additionally, HCMV has been associated with long-term diseases including the vascular diseases atherosclerosis, restenosis following angioplasty, and chronic allograft rejection following solid organ transplantation -4. Recen3C-chemokine Fractalkine/CX3CL1 with high affinity in addition to binding a variety of CC-chemokines . Th. Th34]. In order to further characterize the role of Pyk2 in pUS28-mediated cellular migration, we examined phosphorylation of Pyk2 at Y402 in Ad-pUS28 infected mouse fibroblasts lacking FAK (FAK-/-). FAK-/- cells are known to have elevated levels of endogenous Pyk2, partially compensating for the lack of FAK ,47. We hpUS28-expressing FAK-/- stimulated with CCL5 showed sustained phosphorylation of Pyk2 at Y402. Levels of Pyk2 phosphorlyation were ~2-fold higher in the presence of pUS28 than control Ad-Trans infected cells similarly stimulated. CCL5 stimulation had a small but quantifiable effect on Pyk2 phosphorylation in FAK-/- cells infected with Trans only, we believe this activation is attributable to the effects of calcium gradients produced by the addition of media upon stimulation Figure . Pyk2 kiin vitro kinase assays on Pyk2 immunoprecipitation reactions. For these experiments we returned to examining Pyk2 signaling in the context of SMC. However, in human SMC endogenous production of pUS28 ligands obscures signaling activation [in vitro kinase assay. The Pyk2 kinase activity was analyzed via SDS-PAGE and autoradiography. pUS28 signaling promoted the formation of a large kinase complex involving Pyk2 and a number of other interacting phosphoproteins. This protein complex begins to form by 2.5 min after the addition of CCL5 peaking at 10 min post ligand treatment  patients, a clear mechanistic link between HCMV and GBM tumorigenesis has not been established ,46,57. HWe tested the participation of Pyk2 in the pUS28-mediated activation of RhoA in this system. Similar to results obtained in FAK-/- fibroblasts, pUS28-specific activation of RhoA is readily observed in U373. pUS28-expressing U373 showed 49% activation of RhoA at 5 min post addition of CCL5. Adenovirus expression of WT Pyk2 resulted in similar activation of RhoA, but altered the timing of RhoA activation such that the peak of RhoA activation was seen at 2.5 min post addition of CCL5. Overexpression of the Pyk2 F402Y mutant prevents pUS28-mediated activation of RhoA resulting in less than 1% activation of RhoA throughout the timecourse of stimulation Figure .In order to compare pUS28-mediated activation of Pyk2 in U373 to that seen in RSMC, we performed proteomics analysis of Pyk2 complexes in U373 Figure . In contin vivo including brain, hematopoetic cells, endothelial cells, SMC and fibroblasts. Interestingly, many of these cell types are capable of undergoing migration events in response to various external stimuli including integrin, growth factor, hormone and chemokine-mediated signals. There is very little consensus in the literature regarding the participation of Pyk2 in such migration events. Indeed, requirements for Pyk2 signaling, and downstream effectors of Pyk2 activation are highly cell type and signal type-specific [In this paper, we examined the ability of the HCMV-encoded chemokine receptor pUS28 to activate Pyk2 and determined the role of Pyk2 in pUS28 mediated SMC migration Figure . Pyk2 exMigration of vascular SMC is induced by a variety of stimuli including growth factors, cytokines, extracellular matrix (ECM) components and peptide hormones. Pro-migratory signaling in SMC is best understood in the context of platelet-derived growth factor (PDGF) binding through receptor tyrosine kinases PDGFR-α and β. PDGF-mediated migration in SMC is characterized by Src-dependent activation of FAK, RhoA mediated activation of ROCK and subsequent phosphorylation and inhibition of myosin light chain phosphatase as well as activation of various other effector molecules via ERK, p38MAPK and JNK MAPK families . AngioteThe results presented herein represent the first example of a chemokine-mediated pro-migratory signaling cascade in VSMC involving Pyk2. We have previously demonstrated that chemokine binding to the viral chemokine receptor pUS28 causes SMC migration via a Src-mediated activation of FAK and ERK , and theIn this paper, we explore the contribution of pUS28 signaling to Pyk2 in migration processes associated with HCMV-mediated vascular disease. However, HCMV has been implicated in other disease processes in which the induction of cellular migration may play a role. In particular, HCMV infection has been associated with several human malignancies including prostate and colon cancers as well as glioblastoma multiforme (GBM) . GBM is pUS28 signaling has recently been reported to produce a transformed phenotype in transfected NIH-3T3 cells characterized by increased proliferative capacity, and increased expression of the pro-angiogenic marker VEGF. In addition, the study demonstrated that WT HCMV infection of a human glioblastoma cell line (U373) induces VEGF expression while a pUS28 deletion mutant of HCMV fails to induce VEGF upregulation in glioblastoma cells  implicatin vitro and in vivo. In these studies, Pyk2 is demonstrated to promote migration and invasion of glioma cells while FAK controls their proliferation. The authors demonstrate that the migratory capacity of individual glioma cell lines can be correlated to the relative levels of Pyk2 expression and activation. Perhaps the most compelling evidence for the role of Pyk2 and FAK in glioma tumor progression utilized an in vivo xenograft model of glioblastoma in mice. In this study, Pyk2 deficient cells were observed to invade only locally and survival was significantly increased when mice were engrafted with Pyk2 or FAK deficient glioma cells compared to mice engrafted with wild-type glioma cells [In addition to its role in vascular remodelling, Pyk2 has been identified as a pro-metastatic and pro-angiogenic molecule in several cancer models including small cell lung cancer ,80, prosma cells . Our curma cells ,84. Pyk2ma cells . TherefoWe observed the formation of a cell type specific Pyk2 complex in response to pUS28 signaling in U373 glioblastoma cells. This complex included several proteins previously implicated in glioma tumorigenesis, but never before identified as Pyk2 binding partners. Our data suggests at least two novel mechanisms for the participation of Pyk2 and pUS28 in glioma tumorigenesis. First, we identified TCF-4 and AF-17 binding to Pyk2 in response to pUS28 signaling. Both proteins are members of the Wnt/β-catenin signaling pathway that has recently been linked to glioma progression ,66. IndeThese findings refine our understanding of the pro-migratory signaling pathways activated by pUS28 in SMC and establish Pyk2 as an attractive target for the treatment of CMV-mediated vascular disease because it acts early in the pUS28 pro-migratory signaling cascade and its expression is restricted to a few cell types, reducing the opportunities for off-target effects. Further, these results suggest a role for HCMV pUS28 in pathologies associated with aberrant activation of Pyk2 including some malignancies.In this report, we determined the involvement of Pyk2 in pUS28 signaling and cellular migration Figure . We founThe authors declare that they have no competing interests.JEV participated in the design of the study, carried out all signaling and immunofluorescence assays, prepared samples for proteomics analysis and drafted the manuscript. SV coordinated the mass spectrometry and participated in MS data analysis. RM developed the RhoA activation assay. PS performed SMC migration assays and helped to edit the manuscript. LPT prepared samples for mass spectrometry and participated in data analysis, JIS participated in MS data analysis, DNS conceived of the study, participated in its design and coordination and helped to draft and edit the manuscript. All authors read and approved the final manuscript.Table S1: Mass Spectrometry Data for Pyk2 Complexes in RSMC. For each Pyk2 associated protein, spectral hits are shown for each unique peptide over the timecourse of stimulation. Total spectral hits per peptide are shown to the right of the timecourse for each condition. Total peptides and spectral hits for each timepoint are shown below the list of peptides for each protein.Click here for fileTable S2: Mass Spectrometry Data for Pyk2 Complexes in U373. For each Pyk2 associated protein, spectral hits are shown for each unique peptide over the timecourse of stimulation. Total spectral hits per peptide are shown to the right of the timecourse for each condition. Total peptides and spectral hits for each timepoint are shown below the list of peptides for each protein.Click here for file