A new 9,11-secosterol from the soft coral Sinularia granosa

Chemical investigations on the EtOAc-soluble fractions from the EtOH extract of Formosa soft coral afforded a new 9,11-secosteroid, 8αH-3β,11-dihydroxy-5α,6α-expoxy-24-methylene-9,11-secocholestan-9-one (1), along with one known steroid 3β,11-dihydroxy-5β,6β-expoxy-24-methylene-9,11-secocholestan-9-one (2) from Sinularia granosa. The structure of the new metabolite was elucidated on the basis of extensive spectroscopic analysis and by comparison of their NMR data with the known compounds, including 2. Both 1 and 2 were shown to significantly inhibit the accumulation of the pro-inflammatory inducible nitric oxide synthase protein, and 1 also was found to effectively reduce the level of cyclooxygenase-2 protein, in lipopolysaccharide-stimulated RAW264.7 macrophage cells at 10 μM. Furthermore, cytotoxic activity of 1 and 2 toward a limited panel of cancer cell lines was also discovered.     

A novel mechanism of action of chemically modified tetracyclines: inhibition of COX-2-mediated prostaglandin E2 production.

Tetracyclines (doxycycline and minocycline) inhibit inducible NO synthase expression and augment cyclooxygenase (COX)-2 expression and PGE2 production. In contrast, chemically modified tetracyclines (CMTs), such as CMT-3 and -8 (but not CMT-1, -2, and -5), that lack antimicrobial activity, inhibit both NO and PGE2 production in LPS-stimulated murine macrophages, bovine chondrocytes, and human osteoarthritis-affected cartilage, which spontaneously produces NO and PGE2 in ex vivo conditions. Furthermore, CMT-3 augments COX-2 protein expression but inhibits net PGE2 accumulation. This coincides with the ability of CMT-3 and -8 to inhibit COX-2 enzyme activity in vitro. The action of CMTs is distinct from that observed with tetracyclines because 1) CMT-3-mediated inhibition of PGE2 production coincides with modification of COX-2 protein, which is distinct from the nonglycosylated COX-2 protein generated in the presence of tunicamycin, as observed by Western blot analysis and 2) CMT-3 and -8 have no significant effect on COX-2 mRNA accumulation. In contrast, CMT-3 and -8 do not inhibit COX-1 expression in A549 human epithelial cells at the level of protein and mRNA accumulation or modification of COX-1 protein. CMT-3 and -8 inhibit the sp. act. of COX-2 (but not COX-1) in cell-free extracts. These results demonstrate differential action of CMT-3 (Metastat) on COX-1 and -2 expression, which is distinct from other tetracyclines.     

Interleukin-1beta differentially regulates beta2 adrenoreceptor and prostaglandin E2-mediated cAMP accumulation and chloride efflux from Calu-3 bronchial epithelial cells. Role of receptor changes, adenylyl cyclase, cyclo-oxygenase 2, and protein kinase A

Here we tested the effect of interleukin-1beta, a pro-inflammatory cytokine, on cAMP accumulation and chloride efflux in Calu-3 airway epithelial cells in response to ligands binding to adenylyl cyclase-coupled receptors such as the beta2 adrenoreceptor and EP prostanoid receptors. Interleukin-1beta significantly increased isoprenaline-induced cAMP accumulation by increasing beta2 adrenoreceptor numbers via a protein kinase A-dependent mechanism. In contrast, interleukin-1beta significantly impaired prostaglandin E2-induced cAMP accumulation by induction of cyclo-oxygenase-2, prostaglandin E2 production, and a resulting down-regulation of adenylyl cyclase. The cAMP changes were all mirrored by alterations in chloride efflux assessed using the fluorescent chloride probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide with interleukin-1beta increasing chloride efflux in response to isoprenaline and reducing the response to prostaglandin E2. Studies with glibenclamide confirmed that chloride efflux was via the cystic fibrosis transmembrane conductance regulator. Calu-3 expresses EP4 receptors, but not EP2, and receptor expression is reduced by interleukin-1beta. Collectively, these results provide mechanistic insight into how interleukin-1beta can differentially regulate cAMP generation and chloride efflux in response to different adenylyl cyclase-coupled ligands in the same cell. These findings have important implications for diseases involving inflammation and abnormal ion flux such as cystic fibrosis.     

Kruppel-Like Factor 2-Mediated Suppression of MicroRNA-155 Reduces the Proinflammatory Activation of Macrophages

Objective

Recent evidence indicates that significant interactions exist between Kruppel-like factor 2 (KLF2) and microRNAs (miRNAs) in endothelial cells. Because KLF2 is known to exert anti-inflammatory effects and inhibit the pro-inflammatory activation of monocytes, we sought to identify how inflammation-associated miR-155 is regulated by KLF2 in macrophages.

Approach and Results

Peritoneal macrophages from wild-type (WT) C57Bl/6 mice were transfected with either recombinant adenovirus vector expressing KLF2 (Ad-KLF2) or siRNA targeting KLF2 (KLF2-siRNA) for 24 h–48 h, then stimulated with oxidized low-density lipoproteins (ox-LDL, 50 μg/mL) for 24 h. Quantitative real-time polymerase chain reaction showed that KLF2 markedly reduced the expression of miR-155 in quiescent/ox-LDL-stimulated macrophages. We also found that the increased expression of miR-155, monocyte chemoattractant protein (MCP-1) and interleukin (IL)-6 and the decreased expression of the suppressor of cytokine signaling (SOCS)-1 and IL-10 in ox-LDL-treated macrophages were significantly suppressed by KLF2. Most importantly, over-expression of miR-155 could partly reverse the suppressive effects of KLF2 on the inflammatory response of macrophages. Conversely, the suppression of miR-155 in KLF2 knockdown macrophages significantly overcame the pro-inflammatory properties associated with KLF2 knockdown. Finally, Ad-KLF2 significantly attenuated the diet-induced formation of atherosclerotic lesions in apolipoprotein E-deficient (apoE^-/-) mice, which was associated with a significantly reduced expression of miR-155 and its relative inflammatory cytokine genes in the aortic arch and in macrophages.

Conclusion

KLF2-mediated suppression of miR-155 reduced the inflammatory response of macrophages.     

Effects of specific alpha-adrenoceptive agents on extraneuronal uptake (uptake2) of isoproterenol in perfused rat heart

Effects of specific alpha-adrenoceptive agents (alpha 1-agonist, alpha 1-antagonist, alpha 2-agonist and alpha 2-antagonist) on the extraneuronal accumulation of 3H-isoproterenol in the perfused rat heart were examined. The extraneuronal accumulation of 3H-isoproterenol in the hearts perfused with 3H-isoproterenol (10(-6)M) under COMT inhibition by tropolone (10(-4)M) was about 6 times higher than that of intact COMT. The increase in the accumulation by COMT inhibition was regarded as 100% and the effects of specific alpha-adrenoceptive agents on the accumulation was evaluated. alpha 1-agonists, methoxamine and phenylephrine, did not affect the accumulation. alpha 1-antagonists, prazosin, bunazosin and YM-12617, significantly decreased the accumulation of 3H-isoproterenol and these IC50 values were 2 x 10(-6)M, 3.5 x 10(-6)M and 2.3 x 10(-5)M, respectively. alpha 2-agonists, clonidine and guanabenz, significantly reduced the accumulation and these IC50 values were 3.4 x 10(-5)M and 2.9 x 10(-7)M, respectively. The alpha 2-antagonist, yohimbine, did not affect the accumulation. The present experiments clearly demonstrated that the tested alpha 1-antagonists and alpha 2-agonists inhibited uptake2 in rat heart but the tested alpha 1-agonists and an alpha 2-antagonist did not inhibit it.     

Forskolin inhibits 5-hydroxytryptamine-induced phosphoinositide hydrolysis and Ca+2 Mobilisation in canine cultured aorta smooth muscle cells.

The effect of forskolin on 5-hydroxytryptamine (5-HT)-induced inositol phosphate (IP) and Ca2+ mobilisation was investigated in canine cultured aorta smooth muscle cells (ASMCs). Pretreatment of ASMCs with forskolin attenuated 5-HT-induced IP accumulation and Ca2+ mobilisation in a time- and concentration-dependent manner. The half-maximal effects (pEC50) of forskolin to attenuate IP and Ca2+ responses to 5-HT occurred at concentrations of 6.28 and 6.64, respectively. Pretreatment of ASMCs with cholera toxin caused a similar inhibition on 5-HT-induced responses. Even after treatment with forskolin for 24 h, the 5-HT-induced responses were still inhibited. The inhibitory effect of forskolin resulted from both a depression of the maximal response and a shift to the right of the concentration-effect curves of 5-HT in these responses. The water-soluble forskolin analogue L-858051 [7-deacetyl-7beta-(gamma-N-methylpiperazino)-butyryl forskolin] significantly inhibited the 5-HT-stimulated IP accumulation. In contrast, the addition of 1,9-dideoxy forskolin, an inactive forskolin analogue, had little effect on IP response. Moreover, SQ-22536 [9-(tetrahydro-2-furanyl)-9-H-purin-6-amine], an inhibitor of adenylate cyclase, and both H-89 [N-(2-aminoethyl)-5-iosquinolinesulphonamide] and HA-1004 [N-(2-guanidinoethyl)-5-iosquinolinesulphonamide], inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit the 5-HT-stimulated accumulation of IP in ASMCs. These results indicate that activation of cAMP/PKA might inhibit the 5-HT-stimulated IP accumulation and consequently reduce Ca2+ mobilisation, or inhibit both responses independently.     

BH(4) (tetrahydrobiopterin)-dependent activation, but not the expression, of inducible NOS (nitric oxide synthase)-2 in proinflammatory cytokine-stimulated, cultured normal human astrocytes is mediated by MEK-ERK kinases

Nitric oxide (NO) from astrocytes is one of the signalers used by the brain's extensive glial-neuronal-vascular network, but its excessive production by pro-inflammatory cytokine-stimulated glial cells can be cytodestructive. Here, we show how three pro-inflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma) together stimulated the activation, but not the prior expression, of NOS-2 protein via a mechanism involving MEK-ERKs protein kinases in astrocytes from adult human cerebral temporal cortex. The cytokines triggered a transient burst of p38 MAPK activity and the production of NOS-2 mRNA which were followed by bursts of MEK-ERK activities, synthesis of the NOS-2 co-factor tetrahydrobiopterin (BH(4)), a build-up of NOS-2 protein and from it active NOS-2 enzyme. Selectively inhibiting MEK1/MEK2, but not the earlier burst of p38 MAPK activity, with a brief exposure to U0126 between 24 and 24.5 h after adding the cytokine triad affected neither NOS-2 expression nor NOS-2 protein accumulation but stopped BH(4) synthesis and the assembly of the NOS-2 protein into active NOS-2 enzyme. The complete blockage of active NOS-2 production by the brief exposure to U0126 was bypassed by simply adding BH(4) to the culture medium. Therefore, this cytokine triad triggered two completely separable, tandem operating mechanisms in normal human astrocytes, the first being NOS-2 gene expression and accumulation of NOS-2 protein and the second being the synthesis of the BH(4) factor needed to dimerize the NOS-2 protein into active, NO-making NOS-2 enzyme.     

Calcitonin-induced increase in phosphate accumulation in LLC-PK1 cells probably through protein kinase C activation

To assess the role of protein kinase C and cAMP on the calcitonin-induced alteration of phosphate accumulation by renal tubular cells, the effects of phorbol esters, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and DBcAMP on the phosphate accumulation in LLC-PK1 cells were investigated. Calcitonin stimulated phosphate accumulation with a concomitant increase in cAMP production. Phorbol esters and 1-oleoyl-2-acetyl-glycerol, activators of protein kinase C, also stimulated the phosphate accumulation. Furthermore, H-7, an inhibitor of protein kinase C, inhibited a calcitonin-induced increase in phosphate accumulation significantly. Although DBcAMP by itself did not increase the phosphate accumulation, it enhanced the stimulatory effect of 12-0-tetradecanoyl phorbol-13-acetate on the phosphate accumulation. Accordingly, protein kinase C as well as cAMP might be involved in the calcitonin-induced increase in phosphate accumulation in LLC-PK1 cells.     

IL-10 fails to inhibit the production of IL-18 in response to inflammatory stimuli

IL-10 is an inhibitor of the production of pro-inflammatory cytokines such as IL-12 and IL-1beta, but it is not known whether it can inhibit the production of IL-18. Therefore, a variety of in vivo and in vitro models were used to determine whether IL-10 is an inhibitor of IL-18 production. Infection of IL-10-/- mice with Toxoplasma gondii results in increased levels of IL-12 in the serum and in recall responses compared to wild type (WT) mice. Surprisingly, although infection resulted in increased levels of IL-18 in serum, there were no differences between WT and IL-10-/- mice. Moreover, splenocytes from infected WT and IL-10-/- mice produced similar levels of IL-18 and addition of exogenous IL-10 did not inhibit their production of IL-18. To address whether endogenous IL-18 inhibitors were masking increased IL-18 production in the IL-10-/- mice, expression of IL-18 binding protein was examined using RT-PCR. Although infection leads to increased expression of IL-18BP mRNA, no difference was seen between WT and IL-10-/- mice. In addition, splenocytes from IL-10-/- mice produced elevated levels of nitric oxide (NO) compared to WT mice, and NO has been shown to inhibit activity of interleukin-1 converting enzyme (ICE), which is required for IL-18 production. However, the addition of an inhibitor of NO production did not alter the levels of IL-18 produced. Finally, analysis of the levels of cytokine mRNA of macrophages stimulated with LPS and IFN-gamma revealed that although IL-10 is a potent inhibitor of IL-12 mRNA accumulation, it did not inhibit IL-1beta or IL-18. Together, these data indicate that IL-10 is not an inhibitor of the production of IL-18.     

Inhibition of dicarboxylic anion transport by fluorescein isothiocyanate in skeletal sarcoplasmic reticulum.

It has been demonstrated previously that dicarboxylic anions are cotransported during ATP-dependent Ca2+ transport by skeletal muscle sarcoplasmic reticulum (SR) membranes, and that anion cotransport stimulates Ca2+ transport. In the current study, we present evidence that dicarboxylic anion cotransport and Ca2+ transport are kinetically distinct in SR, but both functions are mediated by the CaATPase protein. Preincubation of SR with 40 microM fluorescein isothiocyanate (FITC) (pH 7.0) inhibited essentially all of the Ca2+ ATPase activity, as well as active oxalate-supported and oxalate-independent 45Ca2+ accumulation. The addition of 1 mM beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) to the preincubation media fully protected the dicarboxylic anion-independent Ca2+ ATPase activity and the oxalate-independent active 45Ca2+ accumulation from the inhibitory effects of FITC; however, the ATP-associated [14C]oxalate accumulation, the oxalate-dependent 45Ca2+ accumulation, and the oxalate- and maleate-dependent stimulation of Ca2+ ATPase activity were not protected by AMP-PCP. Thus, the dicarboxylic anion accumulation and the stimulation of Ca2+ uptake by dicarboxylic anions could be functionally separated from the ATP-dependent, anion-independent Ca2+ translocation. FITC bound exclusively to the 100-kDa (CaATPase) and 92-kDa (phosphorylase) proteins in the SR membranes and to purified CaATPase in sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 1 mM AMP-PCP inhibited 50-55% of the FITC fluorescence on the 100-kDa protein, but did not significantly alter fluorescence on the 92-kDa protein. Two-dimensional gel analysis demonstrated a single 100-kDa protein in longitudinal SR membranes. FITC appears to inhibit ATP-dependent Ca2+ transport, and dicarboxylic anion translocation through interaction at separate domains of the CaATPase protein.     

Increased cellular free cholesterol in macrophage-specific Abca1 knock-out mice enhances pro-inflammatory response of macrophages

Macrophage-specific Abca1 knock-out (Abca1(-)(M)(/-)(M)) mice were generated to determine the role of macrophage ABCA1 expression in plasma lipoprotein concentrations and the innate immune response of macrophages. Plasma lipid and lipoprotein concentrations in chow-fed Abca1(-)(M)(/-)(M) and wild-type (WT) mice were indistinguishable. Compared with WT macrophages, Abca1(-)(M)(/-)(M) macrophages had a >95% reduction in ABCA1 protein, failed to efflux lipid to apoA-I, and had a significant increase in free cholesterol (FC) and membrane lipid rafts without induction of endoplasmic reticulum stress. Lipopolysaccharide (LPS)-treated Abca1(-)(M)(/-)(M) macrophages exhibited enhanced expression of pro-inflammatory cytokines and increased activation of the NF-kappaB and MAPK pathways, which could be diminished by silencing MyD88 or by chemical inhibition of NF-kappaB or MAPK. In vivo LPS injection also resulted in a higher pro-inflammatory response in Abca1(-)(M)(/-)(M) mice compared with WT mice. Furthermore, cholesterol depletion of macrophages with methyl-beta-cyclodextrin normalized FC content between the two genotypes and their response to LPS; cholesterol repletion of macrophages resulted in increased cellular FC accumulation and enhanced cellular response to LPS. Our results suggest that macrophage ABCA1 expression may protect against atherosclerosis by facilitating the net removal of excess lipid from macrophages and dampening pro-inflammatory MyD88-dependent signaling pathways by reduction of cell membrane FC and lipid raft content.     

Knockout of toll-like receptor-4 attenuates the pro-inflammatory state of diabetes

Type 1 diabetes (T1DM) is associated with increased vascular complications and is a pro-inflammatory state. Recent findings have shown increased TLR2 and 4 expression, signaling, ligands, and functional activation in T1DM subjects compared to controls and further accentuated in T1DM with microvascular complications. Thus, the aim of this study was to examine if genetic deficiency of TLR4 attenuates the increased inflammation associated with T1DM using the streptozotocin-induced diabetic mouse model. C57BL/6 and TLR4(-/-) mice were obtained and studied in the native state and following induction of diabetes using streptozotocin. Diabetic (WT+STZ) mice had increased expression of both TLR2 and TLR4, while TLR4(-/-) STZ mice had increased expression only of TLR2, but not TLR4 compared to the non-diabetic mice TLR2 expression was significantly increased with STZ-induced diabetes and was unaffected by knockout of TLR4. Also, levels of MyD88, IRAK-1 protein phosphorylation, Trif, IRF3, and NF-κB activity were significantly reduced in TLR4(-/-) +STZ mice compared to the WT+STZ mice. WT+STZ mice exhibited significantly increased levels of serum and macrophage IL-1β, IL-6, KC/IL-8, IP-10, MCP-1, IFN beta and TNF-α compared to WT mice and this was significantly attenuated in TLR4(-/-) +STZ mice (P<0.01). Thus, TLR4 contributes to the pro-inflammatory state and TLR4KO attenuates inflammation in diabetes.     

An acidic pH environment increases cell death and pro-inflammatory cytokine release in osteoblasts: the involvement of BAX inhibitor-1

BAX Inhibitor-1 (BI-1), a transmembrane protein on the endoplasmic reticulum, has been studied previously in various physio/pathological conditions, but not in bone cells. In this study, using the MG63 osteoblast cell line and osteoblasts differentiated from stem cells, the role of BI-1 was studied. First, expression of BI-1 was confirmed in osteoblasts, as well as osteoclasts, in mouse tibiae bone immunohistochemistry. For evaluation of a recently published property of BI-1, an acidic pH-dependent Ca2? channel-like effect in osteoblasts, acidic pH-associated cell death, and pro-inflammatory cytokine release were examined. In MG63 osteoblasts, acidic pH induced a pH-dependent increase in cell death and ER stress, as determined by elevated expression of GRP78, CHOP, phospho-eIF2α, IRE-1α, spliced XBP-1, and phospho-JNK. In osteoblasts, mitochondrial Ca2? also showed a strong pH-dependent increase. BI-1 knock-down using siRNA protected cells against acidic pH, regulating mitochondrial Ca2? accumulation, possibly via the acidic pH-dependent Ca2? channel-like effect of BI-1. BI-1 knock-down also resulted in inhibition of acidic pH-induced release of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. In addition, bone marrow stem cells were differentiated into human osteoblasts, which showed increased expression of BI-1 mRNA and protein. In differentiated primary human osteoblasts, acidic pH-associated cell death, mitochondrial Ca2? accumulation, and pro-inflammatory cytokine release were more significant than in non-differentiated stem cells. In summary, endogenous expression of BI-1 is associated with acidic pH-induced Ca2? release, cell death, and pro-inflammatory cytokine release in human osteoblasts.     

Artemisinin attenuates lipopolysaccharide-stimulated proinflammatory responses by inhibiting NF-κB pathway in microglia cells

Microglial activation plays an important role in neuroinflammation, which contributes to neuronal damage, and inhibition of microglial activation may have therapeutic benefits that could alleviate the progression of neurodegeneration. Recent studies have indicated that the antimalarial agent artemisinin has the ability to inhibit NF-κB activation. In this study, the inhibitory effects of artemisinin on the production of proinflammatory mediators were investigated in lipopolysaccharide (LPS)-stimulated primary microglia. Our results show that artemisinin significantly inhibited LPS-induced production of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1) and nitric oxide (NO). Artemisinin significantly decreased both the mRNA and the protein levels of these pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS) and increased the protein levels of IκB-α, which forms a cytoplasmic inactive complex with the p65-p50 heterodimeric complex. Artemisinin treatment significantly inhibited basal and LPS-induced migration of BV-2 microglia. Electrophoretic mobility shift assays revealed increased NF-κB binding activity in LPS-stimulated primary microglia, and this increase could be prevented by artemisinin. The inhibitory effects of artemisinin on LPS-stimulated microglia were blocked after IκB-α was silenced with IκB-α siRNA. Our results suggest that artemisinin is able to inhibit neuroinflammation by interfering with NF-κB signaling. The data provide direct evidence of the potential application of artemisinin for the treatment of neuroinflammatory diseases.     

Human immunodeficiency virus type-1 Tat protein induces nuclear factor (NF)-κB activation and oxidative stress in microglial cultures by independent mechanisms

We have extended our previous findings and shown that human immunodeficiency virus Tat protein, in addition to nitric oxide (NO), stimulated rat microglial cultures to release pro-inflammatory cytokine interleukin-1beta and tumour necrosis factor-alpha in a nuclear factor (NF)-kappaB-dependent manner. At the same time, Tat stimulated the accumulation of free radicals, as indicated by the increased levels of isoprostane 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)), a reliable marker of lipid peroxidation and oxidative stress, by a mechanism unrelated to NF-kappaB activation. The presence of free radical scavengers abrogated Tat-induced 8-epi-PGF(2alpha) accumulation without affecting NO and cytokine production. Consistently, Tat-induced IkappaBalpha degradation - an index of NF-kappaB activation - was not affected by free radical scavengers, but was prevented by an NF-kappaB-specific inhibitor. Our observations indicate that NF-kappaB plays a key role in Tat-dependent microglial activation, and that oxidative stress and NF-kappaB activation induced by Tat occur by independent mechanisms.