Distinct role of calmodulin and calmodulin-dependent protein kinase-II in lipopolysaccharide and tumor necrosis factor-alpha-mediated suppression of apoptosis and antiapoptotic c-IAP2 gene expression in human monocytic cells

Exposure of phagocytic cells to bacterial endotoxin (lipopolysaccharide; LPS) or inflammatory cytokines confers antiapoptotic survival signals; however, in the absence of the appropriate stimulus, monocytes are programmed to undergo apoptosis. Macrophage survival may thus influence inflammatory and immune responses and susceptibility to microbial pathogens. Herein, we demonstrate that LPS and the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), enhance monocytic cell survival through the induction of the antiapoptotic c-IAP2 gene in a human promonocytic THP-1 cell line. We also investigated the role of upstream signaling molecules including the mitogen-activated protein kinases, phosphatidylinositol 3-kinase, and the calcium signaling pathways in the regulation of c-IAP2 expression and eventual survival of monocytic cells. Our results suggest that LPS and TNF-alpha-induced c-IAP2 expression was regulated by calmodulin (CaM) through the activation of calmodulin-dependent protein kinase-II (CaMKII). In addition, CaM and CaMKII regulated c-IAP2 expression in LPSand TNF-alpha-stimulated cells through NF-kappaB activation. Moreover, the CaM/CaMKII pathway also regulated LPS- and TNF-alpha-mediated inhibition of apoptosis in these cells. Taken together, these results suggest that LPS- and TNF-alpha-induced c-IAP2 expression and its associated antiapoptotic survival signals in THP-1 cells are regulated selectively by CaM/CaMKII through NF-kappaB activation.     

Berberine suppresses neuroinflammatory responses through AMP‐activated protein kinase activation in BV‐2 microglia

The AMPK cascade is a sensor of cellular energy change, which monitors the AMP/ATP ratio to regulate cellular metabolism by restoring ATP levels, but its regulation of neuroinflammation mechanism remains unclear. Berberine, one of the major constituents of Chinese herb Rhizoma coptidis, has been shown to improve several metabolic disorders, such as obesity and type II diabetes. However, the effect of berberine on neuroinflammatory responses in microglia are poorly understood. This study shows that berberine represses proinflammatory responses through AMP‐activated protein kinase (AMPK) activation in BV‐2 microglia. Our findings also demonstrate that berberine significantly down‐regulates LPS‐ or interferon (IFN)‐γ‐induced nitric oxide synthase (iNOS) and cyclo‐oxygenase‐2 (COX‐2) expression in BV‐2 microglia cells. Berberine also inhibited LPS‐ or IFN‐γ‐induced nitric oxide production. In addition, berberine effectively inhibited proinflammatory cytokines such as TNF‐α, IL‐1β, and IL‐6 expression. On the other hand, upon various inflammatory stimulus including LPS and IFN‐γ, berberine suppressed the phosphorylated of ERK but not p38 and JNK in BV‐2 microglia. AMPK activation is catalyzed by upstream kinases such as LKB1 and Ca2+/calmodulin‐dependent protein kinase kinase‐II (CaMKK II). Moreover, berberine induced LKB1 (Ser428), CaMKII (Thr286), and AMPK (Thr172) phosphorylation, but not AMPK (Ser485). Furthermore, the inhibitory effect of berberine on iNOS and COX‐2 expression was abolished by AMPK inhibition via Compound C, an AMPK inhibitor. Berberine‐suppressed ERK phosphorylation was also reversed by Compound C treatment. Our data demonstrate that berberine significantly induces AMPK signaling pathways activation, which is involved in anti‐neuroinflammation. J. Cell. Biochem. 110: 697–705, 2010. © 2010 Wiley‐Liss, Inc.     

Increases in calmodulin abundance and stabilization of activated inducible nitric oxide synthase mediate bacterial killing in RAW 264.7 macrophages

The rapid activation of macrophages in response to bacterial antigens is central to the innate immune system that permits the recognition and killing of pathogens to limit infection. To understand regulatory mechanisms underlying macrophage activation, we have investigated changes in the abundance of calmodulin (CaM) and iNOS in response to the bacterial cell wall component lipopolysaccharide (LPS) using RAW 264.7 macrophages. Critical to these measurements was the ability to differentiate free iNOS from the CaM-bound (active) form of iNOS associated with nitric oxide generation. We observe a rapid 2-fold increase in CaM abundance during the first 30 min that is blocked by inhibition of either NFkappaB nuclear translocation or protein synthesis. A similar 2-fold increase in the abundance of the complex between CaM and iNOS is observed with the same time dependence. In contrast, there are no detectable increases in the CaM-free (i.e., inactive) form of iNOS within the first 2 h; it remains at a very low abundance during the initial phase of macrophage activation. Increasing cellular CaM levels in stably transfected macrophages results in a corresponding increase in the abundance of the CaM/iNOS complex that promotes effective bacterial killing following infection by Salmonella typhimurium. Thus, LPS-dependent increases in CaM abundance function in the stabilization and activation of iNOS on the rapid time scale associated with macrophage activation and bacterial killing. These results explain how CaM and iNOS coordinately function to form a stable complex that is part of a rapid host response that functions within the first 30 min following bacterial infection to upregulate the innate immune system involving macrophage activation.     

Involvement of Syk kinase in TNF-induced nitric oxide production by airway epithelial cells

We have recently found that Syk is widely expressed in lung epithelial cells (EC) and participates in beta1 integrin signaling. In this study, we assessed the role of Syk in regulation of NO production. Stimulation of human bronchial EC line HS-24 by TNF caused an increased expression of inducible nitric oxide synthase (iNOS). Inhibition of Syk using siRNA or piceatannol down-regulated the iNOS expression and reduced NO production. This effect occurred in EC simultaneously stimulated via beta1 integrins, suggesting that TNF and beta1 integrins provide co-stimulatory signals. Inhibition of Syk down-regulated TNF-induced p38 and p44/42 MAPK phosphorylation and nuclear translocation of p65 NF-kappaB. Thus, TNF-induced activation of pro-inflammatory signaling in EC leading to enhanced expression of iNOS and NO production was dependent on Syk. Syk-mediated signaling regulates NO production at least partly via activating the MAPK cascade. Understanding the role of Syk in airway EC may help in developing new therapeutic tools for inflammatory lung disorders.     

iNOS signaling interacts with COX-2 pathway in colonic fibroblasts

COX-2 and iNOS are two major inflammatory mediators implicated in colorectal inflammation and cancer. Previously, the role of colorectal fibroblasts involved in regulation of COX-2 and iNOS expression was largely ignored. In addition, the combined interaction of COX-2 and iNOS signalings and their significance in the progression of colorectal inflammation and cancer within the fibroblasts have received little investigation. To address those issues, we investigated the role of colonic fibroblasts in the regulation of COX-2 and iNOS gene expression, and explored possible mechanisms of interaction between COX-2 and iNOS signalings using a colonic CCD-18Co fibroblast line and LPS, a potential stimulator of COX-2 and iNOS. Our results clearly demonstrated that LPS activated COX-2 gene expression and enhanced PGE(2) production, stimulated iNOS gene expression and promoted NO production in the fibroblasts. Interestingly, activation of COX-2 signaling by LPS was not involved in activation of iNOS signaling, while activation of iNOS signaling by LPS contributed in part to activation of COX-2 signaling. Further analysis indicated that PKC plays a major role in the activation and interaction of COX-2 and iNOS signalings induced by LPS in the fibroblasts.     

Innate immune responses to TREM-1 activation: overlap, divergence, and positive and negative cross-talk with bacterial lipopolysaccharide

TREM-1 (triggering receptor expressed on myeloid cells-1) is an orphan immunoreceptor expressed on monocytes, macrophages, and neutrophils. TREM-1 associates with and signals via the adapter protein DAP12/TYROBP, which contains an ITAM. TREM-1 activation by receptor cross-linking has been shown to be proinflammatory and to amplify some cellular responses to TLR ligands such as bacterial LPS. To investigate the cellular consequences of TREM-1 activation, we have characterized global gene expression changes in human monocytes in response to TREM-1 cross-linking in comparison to and combined with LPS. Both TREM-1 activation and LPS up-regulate chemokines, cytokines, matrix metalloproteases, and PTGS/COX2, consistent with a core inflammatory response. However, other immunomodulatory factors are selectively induced, including SPP1 and CSF1 (i.e., M-CSF) by TREM-1 activation and IL-23 and CSF3 (i.e., G-CSF) by LPS. Additionally, cross-talk between TREM-1 activation and LPS occurs on multiple levels. Although synergy in GM-CSF protein production is reflected in commensurate mRNA abundance, comparable synergy in IL-1beta protein production is not. TREM-1 activation also attenuates the induction of some LPS target genes, including those that encode IL-12 cytokine family subunits. Where tested, positive TREM-1 outputs are greatly reduced by the PI3K inhibitor wortmannin, whereas this attenuation is largely PI3K independent. These experiments provide a detailed analysis of the cellular consequences of TREM-1 activation and highlight the complexity in signal integration between ITAM- and TLR-mediated signaling.     

Tumor Necrosis Factor (TNF) Receptor-associated Factor (TRAF)-interacting Protein (TRIP) Negatively Regulates the TRAF2 Ubiquitin-dependent Pathway by Suppressing the TRAF2-Sphingosine 1-Phosphate (S1P) Interaction

The signaling pathway downstream of TNF receptor (TNFR) is involved in the induction of a wide range of cellular processes, including cell proliferation, activation, differentiation, and apoptosis. TNFR-associated factor 2 (TRAF2) is a key adaptor molecule in TNFR signaling complexes that promotes downstream signaling cascades, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase activation. TRAF-interacting protein (TRIP) is a known cellular binding partner of TRAF2 and inhibits TNF-induced NF-κB activation. Recent findings that TRIP plays a multifunctional role in antiviral response, cell proliferation, apoptosis, and embryonic development have increased our interest in exploring how TRIP can affect the TNFR-signaling pathway on a molecular level. In our current study, we demonstrated that TRIP is negatively involved in the TNF-induced inflammatory response through the down-regulation of proinflammatory cytokine production. Here, we demonstrated that the TRAF2-TRIP interaction inhibits Lys⁶³-linked TRAF2 ubiquitination by inhibiting TRAF2 E3 ubiquitin (Ub) ligase activity. The TRAF2-TRIP interaction inhibited the binding of sphingosine 1-phosphate, which is a cofactor of TRAF2 E3 Ub ligase, to the TRAF2 RING domain. Finally, we demonstrated that TRIP functions as a negative regulator of proinflammatory cytokine production by inhibiting TNF-induced NF-κB activation. These results indicate that TRIP is an important cellular regulator of the TNF-induced inflammatory response.     

Nitric oxide suppression of cellular proliferation depends on cationic amino acid transporter activity in cytokine-stimulated pulmonary endothelial cells

Inducible nitric oxide (NO) synthase (iNOS) is a stress response protein upregulated in inflammatory conditions, and NO may suppress cellular proliferation. We hypothesized that preventing L-arginine (L-arg) uptake in endothelial cells would prevent lipopolysaccharide/tumor necrosis factor-α (LPS/TNF)-induced, NO-mediated suppression of cellular proliferation. Bovine pulmonary arterial endothelial cells (bPAEC) were treated with LPS/TNF or vehicle (control), and either 10 mM L-leucine [L-leu; a competitive inhibitor of L-arg uptake by the cationic amino acid transporter (CAT)] or its vehicle. In parallel experiments, iNOS or arginase II were overexpressed in bPAEC using an adenoviral vector (AdiNOS or AdArgII, respectively). LPS/TNF treatment increased the expression of iNOS, arginase II, CAT-1, and CAT-2 mRNA in bPAEC, resulting in greater NO and urea production than in control bPAEC, which was prevented by L-leu. LPS/TNF treatment resulted in fewer viable cells than in controls, and LPS/TNF-stimulated bPAEC treated with L-leu had more viable cells than LPS/TNF treatment alone. LPS/TNF treatment resulted in cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase expression, which was attenuated by L-leu. AdiNOS reduced viable cell number, and treatment of AdiNOS transfected bPAEC with L-leu preserved cell number. AdArgII increased viable cell number, and treatment of AdArgII transfected bPAEC with L-leu prevented the increase in cell number. These data demonstrate that iNOS expression in pulmonary endothelial cells leads to decreased cellular proliferation, which can be attenuated by preventing cellular L-arg uptake. We speculate that CAT activity may represent a novel therapeutic target in inflammatory lung diseases characterized by NO overproduction.     

The 15-deoxy-delta12,14-prostaglandin J2 inhibits the inflammatory response in primary rat astrocytes via down-regulating multiple steps in phosphatidylinositol 3-kinase-Akt-NF-kappaB-p300 pathway independent of peroxisome proliferator-activated receptor gamma

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), such as 15-deoxy-12,14-PGJ2 (15d-PGJ2), have been proposed as a new class of anti-inflammatory compounds because 15d-PGJ2 was able to inhibit the induction of inflammatory response genes such as inducible NO synthase (iNOS) and TNF (TNF-alpha) in a PPAR-dependent manner in various cell types. In primary astrocytes, the anti-inflammatory effects (inhibition of TNF-alpha, IL-1beta, IL-6, and iNOS gene expression) of 15d-PGJ2 are observed to be independent of PPARgamma. Overexpression (wild-type and dominant-negative forms) of PPARgamma and its antagonist (GW9662) did not alter the 15d-PGJ2-induced inhibition of LPS/IFN-gamma-mediated iNOS and NF-kappaB activation. The 15d-PGJ2 inhibited the inflammatory response by inhibiting IkappaB kinase activity, which leads to the inhibition of degradation of IkappaB and nuclear translocation of p65, thereby regulating the NF-kappaB pathway. Moreover, 15d-PGJ2 also inhibited the LPS/IFN-gamma-induced PI3K-Akt pathway. The 15d-PGJ2 inhibited the recruitment of p300 by NF-kappaB (p65) and down-regulated the p300-mediated induction of iNOS and NF-kappaB luciferase reporter activity. Coexpression of constitutive active Akt and PI3K (p110) reversed the 15d-PGJ2-mediated inhibition of p300-induced iNOS and NF-kappaB luciferase activity. This study demonstrates that 15d-PGJ2 suppresses inflammatory response by inhibiting NF-kappaB signaling at multiple steps as well as by inhibiting the PI3K/Akt pathway independent of PPARgamma in primary astrocytes.     

Kruppel-like factor 4 is a mediator of proinflammatory signaling in macrophages

Activation of macrophages is important in chronic inflammatory disease states such as atherosclerosis. Proinflammatory cytokines such as interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), or tumor necrosis factor-alpha can promote macrophage activation. Conversely, anti-inflammatory factors such as transforming growth factor-beta1 (TGF-beta1) can decrease proinflammatory activation. The molecular mediators regulating the balance of these opposing effectors remain incompletely understood. Herein, we identify Kruppel-like factor 4 (KLF4) as being markedly induced in response to IFN-gamma, LPS, or tumor necrosis factor-alpha and decreased by TGF-beta1 in macrophages. Overexpression of KLF4 in J774a macrophages induced the macrophage activation marker inducible nitric-oxide synthase and inhibited the TGF-beta1 and Smad3 target gene plasminogen activator inhibitor-1 (PAI-1). Conversely, KLF4 knockdown markedly attenuated the ability of IFN-gamma, LPS, or IFN-gamma plus LPS to induce the iNOS promoter, whereas it augmented macrophage responsiveness to TGF-beta1 and Smad3 signaling. The KLF4 induction of the iNOS promoter is mediated by two KLF DNA-binding sites at -95 and -212 bp, and mutation of these sites diminished induction by IFN-gamma and LPS. We further provide evidence that KLF4 interacts with the NF-kappaB family member p65 (RelA) to cooperatively induce the iNOS promoter. In contrast, KLF4 inhibited the TGF-beta1/Smad3 induction of the PAI-1 promoter independent of KLF4 DNA binding through a novel antagonistic competition with Smad3 for the C terminus of the coactivator p300/CBP. These findings support an important role for KLF4 as a regulator of key signaling pathways that control macrophage activation.     

TNF-α、IL-1β及LPS对血管平滑肌细胞一氧化氮合酶基因表达的影响及作用机制研究

通过ＲＮＡ印迹分析和亚硝酸盐含量测定检查ＴＮＦ－α、ＩＬ－１β和ＬＰＳ对大鼠血管平滑肌细胞（ＶＳＭＣ）诱导型一氧化氮合酶（ｉＮＯＳ）基因表达及ＮＯ生成的影响．结果表明,ＴＮＦ－α、ＩＬ－１β和ＬＰＳ均能显著诱导ＶＳＭＣｉＮＯＳ基因表达和促进ＮＯ生成,其作用强度与浓度和作用时间有关;双因素（ＴＮＦ－α＋ＬＰＳ,ＬＰＳ＋ＩＬ－１β）对诱导ｉＮＯＳ基因表达及ＮＯ生成产生协同作用．ＰｏｌｙｍｙｘｉｎＢ和地塞米松可部分抑制ＴＮＦ－α对ｉＮＯＳ基因表达的诱导作用及ＮＯ生成     

IFN-gamma + LPS induction of iNOS is modulated by ERK, JNK/SAPK, and p38(mapk) in a mouse macrophage cell line

Nitric oxide (NO.) produced by inducible nitric oxide synthase (iNOS) mediates a number of important physiological and pathophysiological processes. The objective of this investigation was to examine the role of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS and NO. by interferon-gamma (IFN-gamma) + lipopolysaccharide (LPS) in macrophages using specific inhibitors and dominant inhibitory mutant proteins of the MAPK pathways. The signaling pathway utilized by IFN-gamma in iNOS induction is well elucidated. To study signaling pathways that are restricted to the LPS-signaling arm, we used a subclone of the parental RAW 264.7 cell line that is unresponsive to IFN-gamma alone with respect to iNOS induction. In this RAW 264.7gammaNO(-) subclone, IFN-gamma and LPS are nevertheless required for synergistic activation of the iNOS promoter. We found that extracellular signal-regulated kinase (ERK) augmented and p38(mapk) inhibited IFN-gamma + LPS induction of iNOS. Dominant-negative MAPK kinase-4 inhibited iNOS promoter activation by IFN-gamma + LPS, also implicating the c-Jun NH(2)-terminal kinase (JNK) pathway in mediating iNOS induction. Inhibition of the ERK pathway markedly reduced IFN-gamma + LPS-induced tumor necrosis factor-alpha protein expression, providing a possible mechanism by which ERK augments iNOS expression. The inhibitory effect of p38(mapk) appears more complex and may be due to the ability of p38(mapk) to inhibit LPS-induced JNK activation. These results indicate that the MAPKs are important regulators of iNOS-NO. expression by IFN-gamma + LPS.