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Cardiac fibrosis and chronic inflammation are common complications in type 2 diabetes mellitus (T2D). Since nucleotide oligomerization-binding domain 1 (NOD1), an innate immune receptor, is involved in the pathogenesis of insulin resistance and diabetes outcomes, we sought to investigate its involvement in cardiac fibrosis. Here, we show that selective staining of cardiac fibroblasts from T2D (db/db;db) mice exhibits up-regulation and activation of the NOD1 pathway, resulting in enhanced NF-κB and TGF-β signalling. Activation of the TGF-β pathway in cardiac fibroblasts from db mice was prevented after inhibition of NF-κB with BAY-11-7082 (BAY). Moreover, fibrosis progression in db mice was also prevented by BAY treatment. Enhanced TGF-β signalling and cardiac fibrosis of db mice was dependent, at least in part, on the sequential activation of NOD1 and NF-κB since treatment of db mice with a selective NOD1 agonist induced activation of the TGF-β pathway, but co-administration of a NOD1 agonist plus BAY, or a NOD1 inhibitor prevented the NOD1-induced fibrosis. Therefore, NOD1 is involved in cardiac fibrosis associated with diabetes, and establishes a new mechanism for the development of heart fibrosis linked to T2D., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

The effect of acanthoic acid analogs on the response to proinflammatory challenge was investigated. Some pimarane diterpenes are known activators of the LXRαβ nuclear receptors, but we show here that they also exert a rapid, potent, and selective activation of the p110γ and p110δ subunits of PI3K. Combination of these effects results in an important attenuation of the global transcriptional response to LPS in macrophages. PI3K/Akt activation leads to inhibition of the LPS-dependent stimulation of IKK/NF-κB and p38 and ERK MAPKs. Macrophages from LXRαβ-deficient mice exhibited an inhibition of these pathways similar to the corresponding wild-type cells. Silencing or inhibition of p110γ/δ suppressed the effect of these diterpenes (DTPs) on IKK/NF-κB and MAPKs signaling. Taken together, these data show a multitarget anti-inflammatory mechanism by these DTPs including a selective activation of PI3K isoenzymes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The ability of neonatal and adult cardiomyocytes to activate the nuclear factor (NF)-kappaB pathway in response to lipopolysaccharide and interleukin-1beta challenge has been investigated and compared with that of peritoneal macrophages. The activation of the IkappaB kinase and the phosphorylation and degradation of IkappaBalpha and IkappaBbeta was much lower in adult cardiomyocytes than in the neonatal counterparts and macrophages. This restricted activation of the NF-kappaB pathway resulted in a significant reduction in the time of nuclear activation of NF-kappaB, as deduced by electrophoretic mobility shift assays and in the transcription of target genes, such as IkappaBalpha, cyclooxygenase-2 (COX-2) and nitric-oxide synthase-2 (NOS-2). Studies on chromatin immunoprecipitation showed binding of NF-kappaB proteins to the regulatory kappaB sites identified in the promoters of the IkappaBalpha, COX-2, and NOS-2 genes in macrophages and, to a lower extent, in neonatal cardiomyocytes. The binding to these kappaB sites in adult cardiomyocytes was observed only in the IkappaBalpha promoter and was minimal or absent in the COX-2 and NOS-2 promoters, respectively, suggesting a restricted activation of NF-kappaB-regulated genes in these cells. These data indicate that the function of the NF-kappaB pathway in adult cardiomyocytes is limited in time, which results in the expression of a reduced number of genes and provides a functional explanation for the absence of NOS-2 inducibility in these cells under proinflammatory conditions.

Objectives and Background: The expression of inflammatory genes in the heart plays an important role in organ dysfunction. However, the contribution of cardiomyocytes to this process, and in particular to the synthesis of high concentrations of nitric oxide and prostaglandins, has not been analyzed in detail. For this reason, cultured isolated cardiomyocytes were used to evaluate the response to pro-inflammatory stimuli., Methods and Results: Isolated cultured foetal, neonatal, and adult rat cardiomyocytes were stimulated with lipopolysaccharide and cytokines, and the expression of nitric oxide synthase-2 (NOS-2) and cyclooxygenase-2 (COX-2) in these cells was investigated. Only cultured foetal cardiomyocytes expressed NOS-2 and COX-2 under these conditions, whereas the neonatal counterparts required various days in culture to gain this response. Analysis of the NF-kappaB signalling pathway showed an impaired activation of IkappaB kinase in response to lipopolysaccharide and cytokines in cells maintained in culture for 1 day. These data were confirmed by DNA microarray analysis. However, other early signalling pathways, such as the p38 and Erk MAPKs, were not affected by the time in culture., Conclusions: Neonatal and adult cardiomyocytes are resistant to the expression of pro-inflammatory genes due to impairment in the activation of IkappaB kinase, a process that might contribute to preventing rapid organ dysfunction in the course of various inflammatory pathologies, such as septic shock and myocarditis.

The effect of rofecoxib, a selective cyclooxygenase-2 inhibitor, on inflammatory signaling has been investigated in elicited murine peritoneal macrophages. Macrophages treated with 10 microM rofecoxib exhibited an important inhibition in the early activation of nuclear factor kappa B (NF-kappa B) and the mitogen-activated protein kinase p38, the extracellular-regulated kinase p44, and the c-Jun N-terminal kinase. Moreover, this drug decreased the protein levels of nitric-oxide synthase-2 and cyclooxygenase-2 in lipopolysaccharide (LPS)-treated macrophages. Rofecoxib delayed and attenuated NF-kappa B activation, which impaired significantly the expression of kappa B-dependent genes. This drug and related coxibs did not affect cell viability and protected against LPS-induced apoptosis through the impairment of the inflammatory response. These data show an additional anti-inflammatory mechanism of selective cyclooxygenase-2 inhibitors through the attenuation of macrophage activation.

Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) transiently activates protein kinase C zeta (PKC zeta) and Jun N-terminal kinase (JNK) through a phosphoinositide-3-kinase (PI3-kinase)-dependent pathway. Incubation of LPS-treated cells with the cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)) promoted a sustained activation of PKC zeta and JNK and inhibited I kappa B kinase (IKK) and NF-kappa B activity. Accordingly, 15dPGJ(2) induced an imbalance between JNK and IKK activities by increasing the former signaling pathway and inhibiting the latter signaling pathway. Under these conditions, apoptosis was significantly enhanced; this response was very dependent on PKC zeta and JNK activation. The effect of 15dPGJ(2) on PKC zeta activity observed in LPS-activated macrophages was not dependent on a direct action of this prostaglandin on the enzyme but was due to the activation of a step upstream of PI3-kinase. Moreover, LPS promoted the redistribution of activated PKC zeta from the cytosol to the nucleus, a process that was enhanced by treatment of the cells with 15dPGJ(2) that favored a persistent and broader distribution of PKC zeta in the nucleus. These results indicate that 15dPGJ(2) and other cyclopentenone prostaglandins, through the sustained activation of PKC zeta, might contribute significantly to the process of resolution of inflammation by promoting apoptosis of activated macrophages.

Stimulation of fetal hepatocytes with proinflammatory cytokines and lipopolysaccharide promotes the expression of cyclooxygenase-2 (COX-2) and nitric oxide synthase-2 (NOS-2), whereas the hepatoma cell line HepG2 exhibits a behavior similar to that described for adult hepatocytes and only expresses NOS-2. The effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on the inflammatory onset was analyzed in these cells since in addition to the inhibition of cyclooxygenase activity, these drugs interfere with other signaling pathways related with the inflammatory response. Inhibition of nuclear factor kappaB (NF-kappaB) activation by aspirin and salicylate has been described in many cells. However, incubation of hepatic cells with salicylate, aspirin, indomethacin, ibuprofen, or 5,5-dimethyl-3(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone (DFU), a fluorinated derivative of rofecoxib, failed to impair IkappaB kinase activity, the processing of NF-kappaB, and the expression of NF-kappaB-dependent genes, such as NOS-2. Moreover, selective COX-2 inhibitors did not promote apoptosis in hepatocytes under inflammatory conditions, suggesting that prostaglandins are not required to maintain cell viability. In conclusion, these data indicate that hepatocytes are not sensitive to NF-kappaB inhibition by NSAIDs and that these drugs, especially the COX-2 selective inhibitors, do not alter cell viability.

Recently isolated trophoblasts express nitric oxide synthase 2 (NOS-2) and cyclooxygenase 2 (COX-2), decreasing the levels of the corresponding mRNAs when the cells were maintained in culture. The sustained expression of COX-2 and NOS-2 in trophoblasts was dependent on the activation of nuclear factor kappaB (NF-kappaB) since proteasome inhibitors and antioxidants that abrogated NF-kappaB activity suppressed the induction of both genes. The time-dependent fall of the mRNA levels of NOS-2 and COX-2 paralleled the inhibition of NF-kappaB, determined by electrophoretic mobility shift assays, and the increase of the IkappaBalpha and IkappaBbeta inhibitory proteins. Isolated trophoblasts synthesized reactive oxygen intermediates (ROI), a process impaired after culturing the cells, and that might be involved in the NF-kappaB activation process. Moreover, treatment of recently isolated cells with ROI scavengers suppressed the expression of COX-2 and NOS-2. Challenge of trophoblasts with interleukin-1beta up-regulated the expression of both proteins, an effect that was potentiated by lipopolysaccharide. These results indicate that the physiological expression of NOS-2 and COX-2 in trophoblasts involves a sustained activation of NF-kappaB which inhibition abrogates the inducibility of both genes.

A concerted activation of transcription factors involved in the transactivation of type II NO synthase (iNOS) gene occurred after partial hepatectomy (PH), resulting in the transient expression of iNOS. The corresponding mRNA and protein levels of iNOS reached a maximum at 4 h and 8 h post-PH respectively. This induction was preceded by an early and transient activation of nuclear factor kappaB (NF-kappaB). Analysis of the kappaB inhibitory (I) proteins showed an important role for IkappaBalpha in the process of NF-kappaB activation, whereas the contribution of IkappaBbeta was less evident. Interferon regulatory factor 1, which has been described as an important activator of iNOS expression, was up-regulated after PH but failed to bind to the corresponding DNA binding sequences of the iNOS promoter. The transcriptional control of iNOS after PH, was compared with the events associated with the hepatic expression of this enzyme in animals challenged with lipopolysaccharide, showing a differential pattern of transcription-factor activation and IkappaB degradation between both models. Transfection of hepatoma cell lines with iNOS promoter constructs, followed by stimulation with post-PH sera, revealed the requirement of NF-kappaB activation for iNOS expression. These data suggest that there is an important role for the restricted NF-kappaB activation in the temporal pattern of iNOS expression in regenerating liver.

Lipopolysaccharide (LPS) administration to mice elicited the activation of nuclear factor kappaB (NF-kappaB) in several tissues including liver and macrophages. Maximal activation was observed 1 h after treatment but declined at 3 and 6 h. The levels of IkappaBalpha and IkappaBbeta were analyzed during this period in an attempt to correlate NF-kappaB activity with IkappaB resynthesis. Degradation of IkappaBalpha was very rapid and was followed by recovery 1 h after LPS administration. IkappaBbeta degradation, which has been associated with persistent NF-kappaB activation, was complete at 1 h. However, a rapid recovery of IkappaBbeta in these tissues was observed at 3 h in parallel with the abrogation of NF-kappaB activity. Immunolocalization of newly synthesized IkappaBbeta by confocal microscopy revealed its preferential accumulation in the cytosol. Analysis of IkappaBbeta by Western blot using high resolution polyacrylamide gel electrophoresis showed the presence of two bands in cytosolic extracts of LPS-treated macrophages at 3 h, but only one band with the same mobility as the control was detected at 6 h. Moreover, treatment of extracts of resynthesized IkappaBbeta with alkaline phosphatase resulted in the accumulation of the protein of slightly higher electrophoretic mobility, indicating the prevalence of a rapid phosphorylation of the newly synthesized IkappaBbeta. At the mRNA level, up-regulation of IkappaBbeta was observed in macrophages stimulated for 1 h with LPS. When the effect of pro-inflammatory cytokines was investigated, tumor necrosis factor alpha, but not interleukin-1 or interferon-gamma, promoted an important degradation of IkappaBbeta followed by an increase in the mRNA at 1 h. These results suggest the existence of LPS- and tumor necrosis factor alpha- specific pathways involved in a rapid IkappaBbeta degradation and resynthesis and might explain the transient period of activation of NF-kappaB in these tissues upon stimulation with these factors. This rapid control of NF-kappaB function may contribute to the attenuation of the inflammatory response of these cells.

Incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS), S-[2,3-bis(palmitoyloxy)-(2-R, S)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys4 (TPP), a synthetic lipopeptide present in bacterial cell wall lipoproteins, or with phorbol 12,13-dibutyrate (PDBu) induced an increase in nitric oxide synthesis through the expression of type II nitric oxide synthase (iNOS). Transfection of hepatocytes with a HindII fragment corresponding to the promoter region of the murine iNOS gene (from nucleotide -1588 to +165) resulted in the expression of the reporter gene when cells were stimulated with these factors. The transcription factors activated by these stimuli involved an increase in the nuclear content of proteins that bind to kappaB, AP-1, GAS, and SIE sequences. Inhibition of NF-kappaB activation with pyrrolidine dithiocarbamate eliminated the expression of iNOS in hepatocytes stimulated with LPS, TPP, or PDBu. In addition to this, transfection of hepatocytes with promoter mutants in which a sequential 2-base pair change within the kappaB sites was introduced (position -971 to -961 and -85 to -75, respectively), resulted in approximately 17 and 35%, respectively, of the activity of the naive promoter. Simultaneous mutation of both kappaB sites abolished the promoter activity. Analysis of the proteins involved in kappaB binding showed the presence of p50/p65 dimers in the nuclei of activated cells at the time that an important decrease of IkappaB-alpha was observed soon after cell stimulation with LPS, TPP, or PDBu. However, only LPS was able to decrease the amount of IkappaB-beta. These results suggest that LPS, TPP, and PDBu, although activating different signal transduction pathways, use a common mechanism mediating iNOS expression in cultured hepatocytes.

The effect of acanthoic acid analogs on the response to proinflammatory challenge was investigated. Some pimarane diterpenes are known activators of the LXRαβ nuclear receptors, but we show here that they also exert a rapid, potent, and selective activation of the p110γ and p110δ subunits of PI3K. Combination of these effects results in an important attenuation of the global transcriptional response to LPS in macrophages. PI3K/Akt activation leads to inhibition of the LPS-dependent stimulation of IKK/NF-κB and p38 and ERK MAPKs. Macrophages from LXRαβ-deficient mice exhibited an inhibition of these pathways similar to the corresponding wild-type cells. Silencing or inhibition of p110γ/δ suppressed the effect of these diterpenes (DTPs) on IKK/NF-κB and MAPKs signaling. Taken together, these data show a multitarget anti-inflammatory mechanism by these DTPs including a selective activation of PI3K isoenzymes.