linearization-of-the-bradford-protein-assay

Linearization of the Bradford Protein Assay

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. ...

cAMP-response element-binding protein (CREB)-binding protein (CBP) is a general transcriptional co-activator that mediates interactions between transcription factors and the basal transcription machinery. To obtain insights into the mechanism by which the KIX domain of CBP can recognize the transactivation domains of many different transcription factors, we have used NMR and biochemical analyses to study the interactions of KIX with the transactivation domain from the constitutive activator c-Myb and with the kinase-inducible transactivation domain (KID) from CREB. NMR chemical shift mapping shows that both activation domains bind to the same surface of KIX. In the unbound state, both the phosphorylated KID and c-Myb activation domains are only partly structured, and binding to KIX is coupled with folding to form an amphipathic helix. Helix-destabilizing mutations significantly impair binding, whereas mutations that increase the intrinsic secondary structure content of the free phosphorylated KID peptide have only a small influence on binding affinity. Low affinity but specific binding of unphosphorylated KID to KIX was measured by ITC and was also observed in Western blot assays and by a fluorescence resonance energy transfer experiment in living cells. The large increase in the affinity for phosphorylated KID is due to favorable intermolecular interactions involving the phosphate moiety. After induction by phosphorylation, CREB is able to compete effectively with other transcriptional activators for binding to CBP.

Roles of phosphorylation and helix propensity in the binding of the KIX domain of CREB-binding protein by constitutive (c-Myb) and inducible (CREB) activators.

Rollen von Phosphorylierung und Helix Neigung in der Bindung des KIX-Domain von CREB-bindenden Protein, das durch konstitutive (c-Myb) und induzierbarer (CREB) Aktivatoren.

The interactions between cAMP-response element-binding protein (CREB)-binding protein (CBP) and gene-specific transcription factors play an important role in activation of transcription from numerous genes. Cooperative interactions between CBP and multiple transcriptional activators may provide a mechanism for synergistic increases in transcriptional activation. Here we report the characterization of ternary complexes formed by the KIX domain of CBP and the transactivation domain of the trithorax group protein mixed lineage leukemia protein (MLL), together with either the phosphorylated kinase-inducible domain (pKID) of CREB or the activation domain from c-Myb. Isothermal titration calorimetry experiments show that KIX in complex with the MLL activation domain binds the c-Myb activation domain and pKID domain with 2-fold higher affinity than does the KIX domain alone. Thus, the activation domains of Myb and MLL or of pKID and MLL bind cooperatively to KIX. The thermodynamics of these interactions imply different mechanisms of binding cooperativity for the two ternary complexes; the KIX.MLL.pKID complex is stabilized by entropy increases, whereas the enhancement of Myb binding in the presence of the MLL activation domain is due to more favorable enthalpy. NMR experiments show that the MLL-binding site on KIX is distinct from the surface that binds the pKID and c-Myb activation domains. Our data indicate that KIX can directly mediate cooperative interactions between pairs of transcriptional regulatory proteins. In the case of MLL and c-Myb, both proteins are involved in proliferation of hematopoietic cells and leukemogenesis, and synergistic interactions mediated by CBP may play a functional role.

Cooperativity in transcription factor binding to the coactivator CREB-binding protein (CBP). The mixed lineage leukemia protein (MLL) activation domain binds to an allosteric site on the KIX domain.

Die Kooperativit&#xE4;t der Transkriptionsfaktor-Bindung an den Coaktivator CREB-bindenden Protein (CBP). Das gemischte Linie Leuk&#xE4;mie Protein (MLL)-Aktivierungsdom&#xE4;ne bindet an eine allosterische Bindungsstelle auf der KIX-Dom&#xE4;ne.

The hematopoietic transcription factor c-Myb activates transcription of target genes through direct interactions with the KIX domain of the co-activator CBP. The solution structure of the KIX domain in complex with the activation domain of c-Myb reveals a helical structure very similar to that adopted by KIX in complex with the phosphorylated kinase inducible domain (pKID) of CREB. While pKID contains two helices, alphaA and alphaB, which interact with KIX, the structure of bound c-Myb reveals a single bent amphipathic helix that binds in the same hydrophobic groove as the alphaB helix of pKID. The affinity of c-Myb for KIX is lower than that of pKID, and relies more heavily on optimal interactions of the single helix of c-Myb with residues in the hydrophobic groove. In particular, a deep hydrophobic pocket in KIX accounts for more than half the interactions with c-Myb observed by NMR. A bend in the alpha-helix of c-Myb enables a critical leucine side-chain to penetrate into this pocket more deeply than the equivalent leucine residue of pKID. The components that mediate the higher affinity of pKID for KIX, i.e. the phosphate group and the alphaA helix, are absent from c-Myb. Results from isothermal titration calorimetry, together with the structural data, point to a key difference between the two complexes in optimal pH for binding, as a result of differential pH-dependent interactions with histidine residues of KIX. These results explain the structural and thermodynamic basis for the observed constitutive versus inducible activation properties of c-Myb and CREB.

Solution structure of the KIX domain of CBP bound to the transactivation domain of c-Myb.

L&#xF6;sung Struktur der KIX-Dom&#xE4;ne des CBP der Transaktivierungsdom&#xE4;ne von c-Myb gebunden.

Phosphorylation of the cAMP response element binding protein (CREB) at Ser-133 in response to hormonal stimuli triggers cellular gene expression via the recruitment of the histone acetylase coactivator paralogs CREB binding protein (CBP) and p300 to the promoter. The NMR structure of the CREB:CBP complex, using relevant interaction domains called KID and KIX, respectively, reveals a shallow hydrophobic groove on the surface of KIX that accommodates an amphipathic helix in phospho (Ser-133) KID. Using an NMR-based screening approach on a preselected small-molecule library, we identified several compounds that bind to different surfaces on KIX. One of these, KG-501 (2-naphthol-AS-E-phosphate), targeted a surface distal to the CREB binding groove that includes Arg-600, a residue that is required for the CREB:CBP interaction. When added to live cells, KG-501 disrupted the CREB: CBP complex and attenuated target gene induction in response to cAMP agonist. These results demonstrate the ability of small molecules to interfere with second-messenger signaling cascades by inhibiting specific protein-protein interactions in the nucleus.

Identification of small-molecule antagonists that inhibit an activator: coactivator interaction.

Identifizierung von kleinen Molek&#xFC;len Antagonisten, die einen Aktivator hemmen: Coaktivator Interaktion.

Tight regulation of the production of the key pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) is essential for the prevention of chronic inflammatory diseases. In vivo administration of a synthetic phospholipid, named hereafter phospho-ceramide analogue-1 (PCERA-1), was previously found to suppress lipopolysaccharide (LPS)-induced TNF-alpha blood levels. We therefore investigated the in vitro anti-inflammatory effects of PCERA-1. Here, we show that extracellular PCERA-1 potently suppresses production of the pro-inflammatory cytokine TNF-alpha in RAW264.7 macrophages, and in addition, independently and reciprocally regulates the production of the anti-inflammatory cytokine interleukin-10 (IL-10). Specificity is demonstrated by the inability of the phospholipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) to perform these activities. Similar TNF-alpha suppression and IL-10 induction by PCERA-1 were observed in macrophages when activated by Toll-like receptor 4 (TLR4), TLR2 and TLR7 agonists. Regulation of cytokine production is demonstrated at the mRNA and protein levels. Finally, we show that, while PCERA-1 does not block activation of nuclear factor (NF)-kappaB and mitogen-activated protein kinases by LPS, it elevates the intracellular cAMP level. In conclusion, the anti-inflammatory activity of PCERA-1 seems to be mediated by a cell membrane receptor, upstream of cAMP production, and eventually TNF-alpha suppression and IL-10 induction. Thus, identification of the PCERA-1 receptor may provide new pharmacological means to block inflammation.

A ceramide-1-phosphate analogue, PCERA-1, simultaneously suppresses tumour necrosis factor-alpha and induces interleukin-10 production in activated macrophages.

Enge Verordnung der Produktion von der wichtigsten Pro-inflammatorische Zytokin Tumor Nekrose Faktor-Alpha (TNF-Alpha) ist entscheidend f&uuml;r die Vermeidung von chronisch entz&uuml;ndlichen Erkrankungen. In-vivo Verwaltung von einer synthetischen Phospholipid, benannt nachstehend Phospho-Ceramid analog-1 (PCERA-1), wurde bereits festgestellt, dass Lipopolysaccharid (LPS) unterdr&uuml;cken-induzierte TNF-Alpha-Blutspiegel. Wir untersuchten deshalb die in-vitro-entz&uuml;ndungshemmenden Wirkungen von PCERA-1. Hier zeigen wir, dass extrazellul&auml;re PCERA-1 stark unterdr&uuml;ckt die Produktion von der Pro-inflammatorische Zytokin TNF-Alpha in RAW264.7 Makrophagen und dar&uuml;ber hinaus, unabh&auml;ngig und gegenseitig die Produktion von entz&uuml;ndungshemmenden Zytokin Interleukin-10 (IL-10 reguliert). Besonderheit zeigt sich durch die Unf&auml;higkeit der Phospholipide Ceramid-1-Phosphat (C1P), Sphingosin-1-Phosphat (S1P) und Lysophosphatidic S&auml;ure (LPA) diese Aktivit&auml;ten durchf&uuml;hren. &Auml;hnliche Unterdr&uuml;ckung von TNF-Alpha und IL-10 Induktion von PCERA-1 wurden in Makrophagen aktiviert durch Toll-Like Receptor 4 (TLR4), TLR2 und TLR7 Agonisten beobachtet. Regulierung der Zytokin-Produktion zeigt sich auf der mRNA und Protein. Schlie&szlig;lich zeigen wir, dass w&auml;hrend PCERA-1 Aktivierung des nuklearen Faktor (NF) nicht blockiert-KappaB und Mitogen-aktivierte Proteinkinasen von LPS, es erh&ouml;ht den intrazellul&auml;ren cAMP-Ebene. Zusammenfassend scheint die entz&uuml;ndungshemmende Aktivit&auml;t des PCERA-1 durch eine Zellmembran-Rezeptor vermittelt upstream cAMP-Produktion, und schlie&szlig;lich Unterdr&uuml;ckung der TNF-Alpha und IL-10 Induktion. Identifizierung von PCERA-1-Rezeptor kann somit neue pharmakologische Mittel, um die Entz&uuml;ndung zu blockieren bieten.

Ein Ceramid-1-Phosphat-Analog, PCERA-1, gleichzeitig unterdr&uuml;ckt die Tumor-Nekrose-Faktor-Alpha und Interleukin-10 Produktion in aktivierten Makrophagen induziert.

Phospho-ceramide analog-1 (PCERA-1) has been described as a potent in vivo suppressor of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha), and thus as a putative drug for the treatment of inflammatory diseases. However, the in vivo cell target of PCERA-1 has not been identified, and its in vivo effect on secretion of other relevant cytokines has not been reported. We have previously shown that PCERA-1 suppresses lipopolysaccharide (LPS)-induced TNFalpha production in RAW264.7 macrophages in vitro. We therefore hypothesized that PCERA-1 targets TNFalpha production by primary macrophages. In this study we thus investigated the effect of PCERA-1 on LPS-induced release of TNFalpha, interleukin (IL)-10 and IL-12p40, in vivo, and ex vivo. We found that PCERA-1 suppressed production of the pro-inflammatory cytokines, TNFalpha and IL-12p40, and increased production of the anti-inflammatory cytokine, IL-10, in LPS-challenged mice, and in primary peritoneal macrophages as well as bone marrow-derived macrophages (BMDM) stimulated with LPS and interferon (IFN)-gamma. These activities of PCERA-1 were independent of each other. In contrast, PCREA-1 only slightly affected TNFalpha production in the whole blood assay, where LPS-induced cytokines are mainly produced by monocytes. Moreover, isolated blood monocytes were inert to PCERA-1, but acquired responsiveness to PCERA-1 upon macrophage colony stimulating factor (M-CSF)-induced differentiation into macrophages. Pharmacokinetic analysis in mice showed that while the volume of distribution of PCERA-1 is low, the drug was rapidly exchanged between the peritoneum and the systemic circulation. Together, these results suggest that sensitivity to PCERA-1 increases upon differentiation of blood monocytes into tissue macrophages, and imply a mechanistic role for peritoneal macrophages in the in vivo anti-inflammatory activity of PCERA-1. Finally, we show that the mechanism of activity of PCERA-1 and prostaglandin E2 (PGE2) is distinct, and that PCERA-1 signaling is not mediated by EP2, a PGE2 receptor which is also activated by oxidized phospholipids. The independent and reciprocal modulation of production of TNFalpha and IL-12p40, vs. IL-10, suggests that PCERA-1 may be a candidate drug for the treatment of inflammation-linked diseases.

Modulation of TNFalpha, IL-10 and IL-12p40 levels by a ceramide-1-phosphate analog, PCERA-1, in vivo and ex vivo in primary macrophages.

Phospho-Ceramid Analog-1 (PCERA-1) wurde beschrieben als ein potenter in Vivo Entst&ouml;rer der Pro-inflammatorische Zytokin Tumornekrosefaktor Alpha (TNFalpha), und damit als vermeintliche Medikament zur Behandlung von entz&uuml;ndlichen Erkrankungen. Jedoch in-vivo Zelle vorbei PCERA-1 wurde nicht erkannt, und seine in-vivo Wirkung auf die Sekretion von anderen relevanten Zytokine wurde nicht berichtet. Wir haben bereits gezeigt, dass PCERA-1 Lipopolysaccharid (LPS) unterdr&uuml;ckt-induzierte TNFalpha-Produktion in in-vitro-RAW264.7 Makrophagen. Wir daher die Hypothese, dass PCERA-1-Ziele-TNFalpha-Produktion von prim&auml;ren Makrophagen. In dieser Studie untersuchten wir somit die Wirkung der PCERA-1 auf LPS-induzierte Freisetzung von TNFalpha, Interleukin (IL) -10 und IL - 12 p 40, in vivo und ex Vivo. Wir fanden, dass PCERA-1 Produktion der Pro-inflammatorische Zytokine, TNFalpha und IL - 12 p 40 und erh&ouml;hte Produktion von der Anti-inflammatory Cytokine unterdr&uuml;ckt IL-10, LPS gehandicapte M&auml;usen und in prim&auml;ren peritonealen Makrophagen sowie Knochenmark abgeleiteten Makrophagen (BMDM) stimuliert mit LPS und Interferon (IFN)-Gamma. Diese T&auml;tigkeit der PCERA-1 wurden unabh&auml;ngig voneinander. PCREA-1 betroffen dagegen nur leicht TNFalpha-Produktion in der Vollblut-Assay, in denen LPS-induzierte Zytokine vor allem von Monozyten hergestellt werden. Dar&uuml;ber hinaus isoliert Blut Monozyten wurden inert bis PCERA-1, sondern erwarb Reaktionsf&auml;higkeit zu PCERA-1 auf Makrophagen-Kolonie-anregende Faktor (M-CSF)-induzierte Differenzierung in Makrophagen. Pharmakokinetischen Analyse an M&auml;usen zeigten, dass w&auml;hrend das Volumen der Verteilung von PCERA-1 niedrig ist, das Medikament rasch zwischen Peritoneum und den K&ouml;rperkreislauf ausgetauscht wurde. Diese Ergebnisse zusammen, schlagen die Empfindlichkeit gegen&uuml;ber PCERA-1 erh&ouml;ht nach Differenzierung des Blut-Monozyten in Gewebe Makrophagen und implizieren eine mechanistische Rolle f&uuml;r peritonealen Makrophagen in der in-vivo entz&uuml;ndungshemmende Aktivit&auml;t des PCERA-1. Schlie&szlig;lich zeigen wir, dass der Mechanismus der Aktivit&auml;t des PCERA-1 und Prostaglandin E2 (PGE2) unterscheidet, und dieser PCERA-1-Signalisierung von EP2, ein PGE2-Rezeptor, der auch durch oxidierte Phospholipide aktiviert wird nicht vermittelt wird. Die unabh&auml;ngige und gegenseitige Beeinflussung der Produktion von TNFalpha und IL - 12 p 40, vs. IL-10, legt nahe, dass PCERA-1 ein Kandidat-Medikament zur Behandlung von Krankheiten mit Entz&uuml;ndung verbunden sein kann.

TNFalpha, IL-10 und IL - 12 p 40-Level durch ein Ceramid-1-Phosphat-Analog-Modulation, PCERA-1, in Vivo und Ex Vivo in prim&auml;ren Makrophagen.

Expression of the anti-inflammatory cytokine IL-10 can be induced either by TLR agonists such as lipopolysaccharide (LPS), or by various endogenous stimuli, in particular those acting via a cAMP-dependent signaling pathway. We have previously reported that the synthetic phospho-ceramide analogue-1 (PCERA-1) increases cAMP level and subsequently down-regulates production of TNFalpha and up-regulates production of IL-10 in LPS-stimulated macrophages. The objective of this study was to determine the mechanism of activity of PCERA-1 and the role of cAMP in LPS-induced IL-10 production. We show here that PCERA-1 induces IL-10 production in synergism with various TLR agonists in mouse RAW264.7 macrophages. Cooperativity is evident both at the mRNA and protein levels. IL-10 production by LPS and PCERA-1 is mediated by the cAMP pathway and by the p38 MAP kinase. Phosphorylation of p38 is cooperatively accomplished by LPS and PCERA-1 or other cAMP inducers. Furthermore, the activity of PCERA-1 can be partially mimicked by a cell-permeable analog of cAMP, and blocked by the protein kinase A (PKA) inhibitor H89. Finally, in the absence of PCERA-1, the residual IL-10 induction by LPS depends on the basal cAMP level as it can be largely elevated by the phosphodiesterase (PDE)-4 inhibitor rolipram. Our results thus indicate that IL-10 induction by LPS critically depends on basal cAMP level, and that a co-stimulus by a TLR agonist and a cAMP-elevating agent results in synergistic PKA-dependent and p38-dependent IL-10 production.

Synergistic IL-10 induction by LPS and the ceramide-1-phosphate analog PCERA-1 is mediated by the cAMP and p38 MAP kinase pathways.

Ausdruck der Anti-inflammatory Cytokine IL-10 l&auml;sst induzierte TLR Agonisten wie Lipopolysaccharid (LPS), oder durch verschiedene endogene Reize, insbesondere diejenigen, die &uuml;ber ein Lager-abh&auml;ngigen Signalweg. Wir haben bereits berichtet, dass die synthetischen Phospho-Ceramid analog-1 (PCERA-1) cAMP erh&ouml;ht und anschlie&szlig;end nach unten-TNFalpha reguliert und oben-die Produktion von IL-10 in LPS stimuliert Makrophagen reguliert. Das Ziel dieser Studie war den Mechanismus der Aktivit&auml;t des PCERA-1 und die Rolle des Lagers in LPS-induzierte IL-10 Produktion festzustellen. Hier zeigen wir, dass PCERA-1, IL-10 Produktion in Synergismus mit verschiedenen TLR Agonisten in Maus RAW264.7 Makrophagen induziert. Kooperativit&auml;t ist offensichtlich auf der mRNA und Protein. IL-10 Produktion von LPS und PCERA-1 ist durch das cAMP Weg und die p38 MAP Kinase vermittelt. Phosphorylierung von p38 erfolgt kooperativ durch LPS und PCERA-1 oder anderen cAMP Induktoren. Dar&uuml;ber hinaus kann die Aktivit&auml;t des PCERA-1 teilweise nachgeahmt durch eine Zelle-durchl&auml;ssigen Analog des Lagers und der Protein-Kinase A (PKA) Hemmer H89 geblockt. Schlie&szlig;lich h&auml;ngt keine PCERA-1, die verbleibende IL-10-Induktion von LPS die basale cAMP-Ebene wie es weitgehend mit der Phosphodiesterase (PDE)-4-Hemmer Rolipram angehoben werden. Unsere Ergebnisse zeigen somit, dass IL-10 Induktion von LPS kritisch basale cAMP Ebene abh&auml;ngt und eine Co-stimulus durch eine TLR-Agonist und ein Lager-erhebend-Agent synergistische PKA-abh&auml;ngige und p38-abh&auml;ngige IL-10-Produktion f&uuml;hrt.

Synergistische IL-10 Induktion von LPS und die analoge Ceramid-1-Phosphat-PCERA-1 ist durch das cAMP und p38 MAP Kinase Wege vermittelt.

Ceramide-1-phosphate (C1P) is known as a second messenger regulating a multitude of processes including cell growth, apoptosis and inflammation. Exciting recent findings now suggest that C1P can stimulate macrophages migration in an extra-cellular manner via a G protein-coupled receptor (GPCR). Interestingly, a synthetic C1P analog, named phospho-ceramide analogue-1 (PCERA-1), was recently described as a potent in-vivo anti-inflammatory agent, and was suggested to act on macrophages in an extra-cellular manner via a GPCR. Here we summarize and compare the receptor-mediated as well as receptor-independent activities of natural C1P and its synthetic analog. We also provide experimental data in support of distinct C1P and PCERA-1 receptors.

Distinct receptor-mediated activities in macrophages for natural ceramide-1-phosphate (C1P) and for phospho-ceramide analogue-1 (PCERA-1).

Ceramid-1-Phosphat (C1P) bekannt als einen zweiten boten, die Regulierung einer Vielzahl von Prozessen einschlie&szlig;lich Zellwachstum, Apoptose und Entz&uuml;ndungen. Aufregende neue Entdeckungen zufolge nun C1P Makrophagen Migration in einer extrazellul&auml;ren Weise &uuml;ber ein G-Protein-gekoppelter Rezeptor (GPCR) anregen k&ouml;nnen. Interessanterweise eine synthetische C1P analog, benannt Phospho-Ceramid analog-1 (PCERA-1), wurde vor kurzem als in Vivo potente entz&uuml;ndungshemmende Agent beschrieben und wurde angeregt, auf Makrophagen extrazellul&auml;ren &uuml;ber eine GPCR handeln. Hier wir zusammenfassen und vergleichen die Rezeptor-vermittelte als auch Rezeptor-unabh&auml;ngigen Aktivit&auml;ten der nat&uuml;rlichen C1P und seine synthetische Entsprechung. Wir bieten auch experimentelle Daten zur Unterst&uuml;tzung der unterschiedlichen C1P und PCERA-1-Rezeptoren.

Verschiedene Rezeptor-vermittelten Aktivit&auml;ten in Makrophagen f&uuml;r nat&uuml;rliche Ceramid-1-Phosphat (C1P) und Phospho-Ceramid analog-1 (PCERA-1).

The synthetic phospho-ceramide analogue-1 (PCERA-1) down-regulates production of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) and up-regulates production of the anti-inflammatory cytokine interleukin-10 (IL-10) in lipopolysaccharide (LPS) -stimulated macrophages. We have previously reported that PCERA-1 increases cyclic adenosine monophosphate (cAMP) levels. The objective of this study was to delineate the signalling pathway leading from PCERA-1 via cAMP to modulation of TNF-alpha and IL-10 production. We show here that PCERA-1 elevates intra-cellular cAMP level in a guanosine triphosphate-dependent manner in RAW264.7 macrophages. The cell-permeable dibutyryl cAMP was able to mimic the effects of PCERA-1 on cytokine production, whereas 8-chloro-phenylthio-methyladenosine-cAMP, which specifically activates the exchange protein directly activated by cAMP (EPAC) but not protein kinase A (PKA), failed to mimic PCERA-1 activities. Consistently, the PKA inhibitor H89 efficiently blocked PCERA-1-driven cytokine modulation as well as PCERA-1-stimulated phosphorylation of cAMP response element binding protein (CREB) on Ser-133. Finally, PCERA-1 activated cAMP-responsive transcription of a luciferase reporter, in synergism with the phosphodiesterase (PDE)-4 inhibitor rolipram. Our results suggest that PCERA-1 activates a G(s) protein-coupled receptor, leading to elevation of cAMP, which acts via the PKA-CREB pathway to promote TNF-alpha suppression and IL-10 induction in LPS-stimulated macrophages. Identification of the PCERA-1 receptor is expected to set up a new target for development of novel anti-inflammatory drugs.

The ceramide-1-phosphate analogue PCERA-1 modulates tumour necrosis factor-alpha and interleukin-10 production in macrophages via the cAMP-PKA-CREB pathway in a GTP-dependent manner.

Die synthetischen Phospho-Ceramid analog-1 (PCERA-1) nach unten-reguliert die Pro-inflammatorische Zytokin Tumor-Nekrose-Faktor-Alpha (TNF-Alpha) und oben-reguliert die entz&uuml;ndungshemmenden Zytokin Interleukin-10 (IL-10) in Lipopolysaccharid (LPS) - Makrophagen stimuliert. Wir haben bereits berichtet, dass PCERA-1 Zyklisches Adenosinmonophosphat (cAMP) Ebenen erh&ouml;ht. Ziel dieser Studie war es, Signalgebung Weg f&uuml;hrenden von PCERA-1 &uuml;ber cAMP zur Modulation von TNF-Alpha und IL-10 Produktion abzugrenzen. Wir zeigen hier, dass PCERA-1 intra-cellular-cAMP-Ebene auf Guanosintriphosphat-abh&auml;ngigen Weise in RAW264.7 Makrophagen erhebt. Zelle-durchl&auml;ssigen Dibutyryl Lager war in der Lage, die Auswirkungen der PCERA-1 auf Cytokine Produktion, zu imitieren, w&auml;hrend 8-Chloro-Phenylthio-Methyladenosine-cAMP, die speziell das Austausch-Protein aktiviert direkt vom Lager (EPAC) aber nicht Proteinkinase A (PKA) aktiviert, beim PCERA-1-Aktivit&auml;ten zu imitieren Fehler. Konsequent, blockiert die PKA-Inhibitor H89 effizient PCERA-1-gesteuerte Cytokine Modulation sowie PCERA-1-stimuliert die Phosphorylierung von cAMP Response Element verbindliches Protein (CREB) auf Ser-133. PCERA-1 aktiviert schlie&szlig;lich Lager-responsive Transkription der Luciferase Reporter, in Synergismus mit der Phosphodiesterase (PDE)-4-Hemmer Rolipram. Unsere Ergebnisse legen nahe, dass PCERA-1 aktiviert einen G(s)-Protein-gekoppelter Rezeptor f&uuml;hrt zu Erh&ouml;hung von cAMP, die &uuml;ber die PKA-CREB-Bahn zur F&ouml;rderung der Unterdr&uuml;ckung von TNF-Alpha und IL-10 Induktion in LPS stimuliert Makrophagen fungiert. Identifizierung von PCERA-1-Rezeptor wird voraussichtlich ein neues Ziel f&uuml;r die Entwicklung von neuartigen Antiphlogistika einrichten.

Der Ceramid-1-Phosphat-analogen PCERA-1 moduliert Tumor-Nekrose-Faktor-Alpha und Interleukin-10 Produktion in Makrophagen &uuml;ber die cAMP-PKA-CREB-Bahn, GTP-abh&auml;ngigen Weise.

The role of CREB in LPS signaling is controversial. The objective of this study was to evaluate the effect of LPS on phosphorylation and transcriptional activation of CREB, in comparison to isoproterenol, a beta-adrenergic receptor agonist. We show here that LPS elevates intra-cellular cAMP level in RAW264.7 macrophages, with slower kinetics and lower magnitude than isoproterenol. The two agents stimulated CREB phosphorylation on Ser-133 to a similar extent, but with a different mechanism; rapid and mostly PKA-mediated for isoproterenol; slow and MSK1-mediated for LPS. Interestingly, LPS-stimulated phosphorylation of CREB did not result in transcriptional activation of a CRE-regulated luciferase reporter, in contrast to stimulation by isoproterenol. Furthermore, inhibitors of p38 and MSK1, but not PKA, completely blocked the production of IL-10 and TNFalpha in LPS-stimulated macrophages. Distinctively, the PKA inhibitor H89 blocked the suppressive effect of isoproterenol on TNFalpha production, as well as its stimulatory effect on IL-10 induction, in LPS-stimulated macrophages. Likewise, while over-expression of dominant negative CREB had no effect on LPS-stimulated TNFalpha production, it blocked the suppressive effect of isoproterenol on TNFalpha production in the LPS-stimulated macrophages. Our results thus indicate that PKA-mediated phosphorylation of CREB promotes TNFalpha suppression and IL-10 induction, whereas the same phosphorylation event initiated by LPS and mediated by MSK1 is non-functional for transcriptional modulation.

Role of CREB in modulation of TNFalpha and IL-10 expression in LPS-stimulated RAW264.7 macrophages.

Die Rolle des CREB in LPS Signalisierung ist umstritten. Das Ziel dieser Studie war es, den Effekt der LPS auf Phosphorylierung und transkriptionelle Aktivierung von CREB, im Vergleich zu Isoproterenol, ein adrenergenen Rezeptor-Agonisten zu bewerten. Wir zeigen hier, dass LPS hebt intra-cellular cAMP Ebene in RAW264.7 Makrophagen, mit langsameren Kinetik und geringeren Haushaltsproblemen als Isoproterenol. Die beiden Agenten stimuliert CREB Phosphorylierung auf Ser-133 &auml;hnlichen Umfang, aber mit einen anderen Mechanismus; schnelle und vor allem PKA-vermittelte f&uuml;r Isoproterenol; langsam und MSK1-vermittelte f&uuml;r LPS. Interessanterweise LPS stimuliert Phosphorylierung von CREB nicht transkriptionelle Aktivierung des CRE-regulierten Luciferase Reporter, im Gegensatz zur Stimulation durch Isoproterenol f&uuml;hren. Dar&uuml;ber hinaus blockiert Inhibitoren der p38 und MSK1, aber nicht PKA, v&ouml;llig die Produktion von IL-10 und TNFalpha in LPS stimuliert Makrophagen. Markant, blockiert die PKA-Inhibitor H89 die unterdr&uuml;ckende Wirkung von Isoproterenol auf TNFalpha-Produktion, sowie seine stimulierende Wirkung auf IL-10 Induktion, in LPS stimuliert Makrophagen. Ebenso w&auml;hrend &Uuml;berexpression des dominierenden negativen CREB keinen Einfluss auf LPS stimuliert TNFalpha-Produktion hatte, blockiert es die unterdr&uuml;ckende Wirkung von Isoproterenol auf TNFalpha-Produktion in der LPS stimuliert Makrophagen. Unsere Ergebnisse zeigen damit, dass die PKA-vermittelte Phosphorylierung von CREB f&ouml;rdert TNFalpha Unterdr&uuml;ckung und IL-10 Induktion, wogegen das gleiche Ereignis der Phosphorylierung von LPS initiiert und durch vermittelt MSK1 nicht-funktionale transkriptionelle Modulation.

Rolle des CREB Modulation des TNFalpha und IL-10 Ausdruck in RAW264.7 LPS stimuliert Makrophagen.

The Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002 (LY2), has been previously reported to inhibit nuclear factor ÎºB (NFÎºB) activity, in a PI3K-independent mechanism. The goals of the current research were to determine the specificity of LY2 regarding NFÎºB subunits, and to identify relevant modulation of cytokine expression in LPS-stimulated macrophages. We found that LY2 specifically diminished the level of p50, but not p65, NFÎºB in the nucleus of LPS-stimulated mouse RAW264.7 macrophages and human THP-1 monocytes. This activity of LY2 was mimicked by its PI3K-inert analog LY303511 (LY3), but not by another PI3K inhibitor - wortmannin. We further show that LY2 inhibited LPS-induced IL-10 expression by RAW264.7 macrophages, in a PI3K-independent mechanism. Moreover, using a deletion mutant of an IL-10 promoter reporter gene we demonstrate that the activity of the NFÎºB enhancer site at the IL-10 promoter is regulated by LY2 in a PI3K-independent manner. Finally, both LY2 and LY3 elevated TNFÎ± production in the LPS tolerant state which is regulated by p50 NFÎºB homodimers, but not before tolerance development. The effects of LY2 and LY3 on p50 translocation and on cytokine production in LPS-stimulated macrophages are thus consistent with specific PI3K-independent inhibition of p50 NFÎºB homodimer activity by LY2.

The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 modulates cytokine expression in macrophages via p50 nuclear factor ÎºB inhibition, in a PI3K-independent mechanism.

Der Phosphatidylinositol-3-Kinase (PI3K) Hemmer, LY294002 (LY2), wurde zuvor berichtet nuklearer Faktor &kappa;B (NF&kappa;B) Aktivit&auml;t in einen PI3K-unabh&auml;ngigen Mechanismus zu hemmen. Die Ziele der aktuellen Forschung waren die Besonderheit des LY2 bez&uuml;glich NF&kappa;B-Untereinheiten zu bestimmen und entsprechende Modulation der Cytokine Ausdruck in LPS stimuliert Makrophagen zu identifizieren. Wir fanden, dass LY2 speziell das Niveau der p50, aber nicht p65, NF&kappa;B in den Zellkern von LPS stimuliert Maus RAW264.7 Makrophagen und menschlichen THP-1 Monocyten verringert. Diese Aktivit&auml;t des LY2 wurde durch seine PI3K inerte analoge LY303511 nachgeahmt (LY3), aber nicht von einem anderen PI3K Inhibitor - Wortmannin. Wir zeigen weiter, dass LY2 LPS-induzierte IL-10 Ausdruck durch RAW264.7 Makrophagen in einen PI3K-unabh&auml;ngigen Mechanismus gehemmt. Dar&uuml;ber hinaus zeigen eine L&ouml;schung-Mutante eine IL-10 Projekttr&auml;ger Reporter-gen wir, dass die Aktivit&auml;t der NF&kappa;B-Vergr&ouml;&szlig;erer-Website unter der IL-10-Projekttr&auml;ger PI3K-unabh&auml;ngig von LY2 geregelt ist. Schlie&szlig;lich erh&ouml;hte LY2 und LY3 TNF&alpha;-Produktion in der LPS-tolerant-Staat, der von p50 NF&kappa;B Homodimers, jedoch nicht vor Toleranz Entwicklung geregelt ist. Die Auswirkungen von LY2 und LY3 auf p50 Translokation und Cytokine Produktion in LPS stimuliert Makrophagen somit an den spezifischen PI3K-unabh&auml;ngige Hemmung der p50 NF&kappa;B Homodimer Aktivit&auml;t von LY2 entsprechen.

Der Phosphatidylinositol-3-Kinase (PI3K) Hemmer LY294002 moduliert Cytokine Ausdruck in Makrophagen &uuml;ber p50 nuklearer Faktor &kappa;B Hemmung, in einen PI3K-unabh&auml;ngigen Mechanismus.

Macrophages, pivotal cells in onset and progression of inflammation, can benefit from sub-cellular drug targeting to the molecular loci of drug action, whether cell membrane or cell interior. Postulating manipulation of liposome size and surface properties can provide sub-cellular targeting, we studied: thermodynamics of liposome-macrophage binding; liposome cellular localizations; liposome safety including pro-inflammatory cytokine production. We aimed at extending the body of knowledge on interactions of regular unilamellar (RL-ULV) and multilamellar (RL-MLV) liposomes with macrophages. We investigated, for the first time, the interactions of hyaluronan (HA) surface-modified liposomes (HA-ULV and HA-MLV) with macrophages, with respect to multiple equilibria binding combined with cellular localization. Macrophages bound all four liposome types, substantially-favoring the two MLV species over the two ULV species, and internalizing only RL-MLV. Three macrophage-internalization inhibitors (2-deoxyglucose, LY294002 and Wortmannin) reduced RL-MLV internalization but not binding affinity nor binding capacity. Both MLV types were not detrimental to cell proliferation, nor did they elicit TNF-Î± production in resting and in LPS-activated macrophages. Moreover, a 24-hour exposure of LPS-activated macrophages to HA-MLV reduced TNF-Î± production by 40%, indicating potential for anti-inflammatory activity. In conclusion RL-MLV and HA-MLV are the liposomes of choice for delivering anti-inflammatory drugs to the macrophage surface or its interior, according to the loci of drug action.

Hyaluronan-modified and regular multilamellar liposomes provide sub-cellular targeting to macrophages, without eliciting a pro-inflammatory response.

Makrophagen, zentrale Zellen im Ausbruch und Verlauf einer Entz&uuml;ndung, profitieren von subzellulare Droge-targeting die molekulare Loci der ARZNEIMITTELWIRKUNG, ob Zelle Membrane oder Handy-Interieur. Postulierte Manipulation von Liposomen Gr&ouml;&szlig;e und Oberfl&auml;cheneigenschaften subzellulare Ausrichtung bieten kann, die wir untersucht: Thermodynamik der Liposomen-Makrophagen-Bindung; Liposomen zellul&auml;re Lokalisierungen; Liposomen Sicherheit auch Pro-inflammatorische Zytokin-Produktion. Wir abzielt erweitern den K&ouml;rper des Wissens auf Wechselwirkungen von regelm&auml;&szlig;igen Unilamellar (RL-ULV) und multilamellar (RL-MLV) Liposomen mit Makrophagen. Wir untersuchten erstmals, die Interaktionen von Hyalurons&auml;ure (HA) Oberfl&auml;che-modifizierten Liposomen (HA-ULV-Prozessor und HA-MLV) mit Makrophagen, in Bezug auf multiple Gleichgewichte Bindung kombiniert mit zellul&auml;ren Lokalisation. Makrophagen gebunden alle vier Liposomen-Typen, erheblich-beg&uuml;nstigt die beiden MLV-Arten &uuml;ber die beiden ULV-Arten und verinnerlichen nur RL-MLV. Drei Makrophage-Internalisierung Inhibitoren (2-Deoxyglucose, LY294002 und Wortmannin) reduziert RL-MLV Internalisierung aber nicht verbindliche Affinit&auml;t noch Bindungskapazit&auml;t. Beide MLV-Typen waren nicht nachteilig auf die Zellproliferation, noch haben sie entlocken TNF-&alpha;-Produktion in Ruhe und LPS aktivierten Makrophagen. Dar&uuml;ber hinaus reduziert eine 24-Stunden-Exposition von LPS aktivierte Makrophagen zu HA-MLV TNF-&alpha;-Produktion um 40 %, angibt, Potenzial f&uuml;r entz&uuml;ndungshemmende Aktivit&auml;t. Abschlie&szlig;end sind RL-MLV und HA-MLV die Liposomen Wahl f&uuml;r die Lieferung von entz&uuml;ndungshemmende Medikamente f&uuml;r die Makrophagen-Oberfl&auml;che oder das Innere nach den Loci der ARZNEIMITTELWIRKUNG.

Hyaluronan-modifizierte und regelm&auml;&szlig;ige multilamellar Liposomen bieten subzellulare Zielgruppenadressierung auf Makrophagen, ohne eine Pro-inflammatorische Reaktion hervorrufen.

Article	Year
Roles of phosphorylation and helix propensity in the binding of the KIX domain of CREB-binding protein by constitutive (c-Myb) and inducible (CREB) activators. The Journal of biological chemistry\| PubMed ID: 12196545	2002
Cooperativity in transcription factor binding to the coactivator CREB-binding protein (CBP). The mixed lineage leukemia protein (MLL) activation domain binds to an allosteric site on the KIX... The Journal of biological chemistry\| PubMed ID: 12205094	2002
Solution structure of the KIX domain of CBP bound to the transactivation domain of c-Myb. Journal of molecular biology\| PubMed ID: 15019774	2004
Identification of small-molecule antagonists that inhibit an activator: coactivator interaction. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 15585582	2004
A ceramide-1-phosphate analogue, PCERA-1, simultaneously suppresses tumour necrosis factor-alpha and induces interleukin-10 production in activated macrophages. Immunology\| PubMed ID: 18793216	2009
Modulation of TNFalpha, IL-10 and IL-12p40 levels by a ceramide-1-phosphate analog, PCERA-1, in vivo and ex vivo in primary macrophages. Immunology letters\| PubMed ID: 19185589	2009
Synergistic IL-10 induction by LPS and the ceramide-1-phosphate analog PCERA-1 is mediated by the cAMP and p38 MAP kinase pathways. Molecular immunology\| PubMed ID: 19362373	2009
Distinct receptor-mediated activities in macrophages for natural ceramide-1-phosphate (C1P) and for phospho-ceramide analogue-1 (PCERA-1). Molecular and cellular endocrinology\| PubMed ID: 19467294	2010
The ceramide-1-phosphate analogue PCERA-1 modulates tumour necrosis factor-alpha and interleukin-10 production in macrophages via the cAMP-PKA-CREB pathway in a GTP-dependent manner. Immunology\| PubMed ID: 19922425	2010
Role of CREB in modulation of TNFalpha and IL-10 expression in LPS-stimulated RAW264.7 macrophages. Molecular immunology\| PubMed ID: 20303596	2010
Immunology letters\| PubMed ID: 21040745	2011
The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 modulates cytokine expression in macrophages via p50 nuclear factor ÎºB inhibition, in a PI3K-independent mechanism. Biochemical pharmacology\| PubMed ID: 22005520	2012
Hyaluronan-modified and regular multilamellar liposomes provide sub-cellular targeting to macrophages, without eliciting a pro-inflammatory response. Journal of controlled release : official journal of the Controlled Release Society\| PubMed ID: 22019559	2011

Tsaffrir Zor

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