Loss of desmin triggers mechanosensitivity and up-regulation of Ankrd1 expression through Akt-NF-κB signaling pathway in smooth muscle cells

Muscle cells, including human airway smooth muscle cells (HASMCs) express ankyrin repeat protein 1 (Ankrd1), a member of ankyrin repeat protein family. Ankrd1 efficiently interacts with the type III intermediate filament desmin. Our earlier study showed that desmin is an intracellular load-bearing protein that influences airway compliance, lung recoil, and airway contractile responsiveness. These results suggest that Ankrd1 and desmin may play important roles on ASMC homeostasis. Here we show that small interfering (si)RNA-mediated knockdown of the desmin gene in HASMCs, recombinant HASMCs (reHASMCs), up-regulates Ankrd1 expression. Moreover, loss of desmin in HASMCs increases the phosphorylation of Akt, inhibitor of κB kinase (IKK)-α, and inhibitor of κB (IκB)-α proteins, leading to NF-κB activation. Treatment of reHASMCs with Akt, IKKα, IκBα, or NF-κB inhibitor inhibits the loss of desmin-induced Ankrd1 up-regulation, suggesting Akt/NF-κB-mediated Ankrd1 regulation. Transfection of reHASMCs with siRNA specific for p50 or p65 corroborates the NF-κB-mediated Ankrd1 regulation. Luciferase reporter assays show that NF-κB directly binds on Ankrd1 promoter and up-regulates Ankrd1 levels. Overall, our data provide a new link between desmin and Ankrd1 regulation, which may be important for ASMC homeostasis.     

Hepatitis B virus X protein induces IKKα nuclear translocation via Akt-dependent phosphorylation to promote the motility of hepatocarcinoma cells

Hepatitis B virus (HBV) X protein (HBx) has been implicated in HBV-associated carcinogenesis through activation of IκB kinase (IKK)/nuclear factor kappa B (NF-κB) signaling pathway. Besides activating NF-κB in the cytoplasm, IKKα was found in the nucleus to regulate gene expression epigenetically in response to various stimuli. However, it is unknown whether nuclear IKKα plays a role in HBx-associated tumor progression. Moreover, the molecular mechanism underlying IKKα nuclear transport also remains to be elucidated. Here, we disclosed HBx as a new inducer of IKKα nuclear transport in hepatoma cells. HBx induced IKKα nuclear transport in an Akt-dependent manner. HBx-activated Akt promoted IKKα nuclear translocation via phosphorylating its threonine-23 (Thr23). In addition, IKKα ubiquitination enhanced by HBx and Akt also contributed to the IKKα accumulation in the nucleus, indicating the involvement of ubiquitination in Akt-increased IKKα nuclear transport in response to HBx. Furthermore, inhibition of IKKα nuclear translocation by mutation of its nuclear localization signal and Thr23 diminished IKKα-dependent cell migration. Taken together, our findings shed light on the molecular mechanism of IKKα nuclear translocation and provide a potential role of nuclear IKKα in HBx-mediated hepatocellular carcinoma (HCC) progression.     

Grape seed procyanidin b2 inhibits human aortic smooth muscle cell proliferation and migration induced by advanced glycation end products

Advanced glycation end product (AGE)-induced vascular smooth muscle cell (VSMC) proliferation is vital to the progression of diabetic vasculopathy. A grape seed procyanidin extract has been reported to possess anti-oxidative and anti-inflammatory properties and to display a significant cardiovascular protective effect, but little is know about the underlying mechanism. The objective of this present study was to determine whether GSPB2 (grape seed procyanidin B2), which is a dimeric procyanidin and more biologically active, could inhibit AGE-induced VSMC proliferation by affecting the production of ubiquitin COOH-terminal hydrolase 1 (UCH-L1), the degradation of IκB-α and nuclear translocation of NF-κB in human aortic smooth muscle cells (HASMCs). Our data show that GSPB2 preincubation markedly inhibited AGE-induced proliferation and migration of HASMCs in a dose-dependent manner and upregulated the protein level of UCH-L1. Further studies revealed that the GSPB2 pretreatment markedly attenuated the degradation of IκB-α and nuclear translocation of NF-κB by modulating ubiquitination of IκB-α in AGE-exposed HASMCs. These results collectively suggest that AGE-induced HASMC proliferation and migration was suppressed by GSPB2 through regulating UCH-L1 and ubiquitination of IκB-α. GSPB2 may therefore have therapeutic potential in preventing and treating vascular complications of diabetes mellitus.     

Grape Seed Procyanidin B2 Inhibits Human Aortic Smooth Muscle Cell Proliferation and Migration Induced by Advanced Glycation End Products

Advanced glycation end product (AGE)-induced vascular smooth muscle cell (VSMC) proliferation is vital to the progression of diabetic vasculopathy. A grape seed procyanidin extract has been reported to possess anti-oxidative and anti-inflammatory properties and to display a significant cardiovascular protective effect, but little is know about the underlying mechanism. The objective of this present study was to determine whether GSPB2 (grape seed procyanidin B2), which is a dimeric procyanidin and more biologically active, could inhibit AGE-induced VSMC proliferation by affecting the production of ubiquitin COOH-terminal hydrolase 1 (UCH-L1), the degradation of IκB-α and nuclear translocation of NF-κB in human aortic smooth muscle cells (HASMCs). Our data show that GSPB2 preincubation markedly inhibited AGE-induced proliferation and migration of HASMCs in a dose-dependent manner and upregulated the protein level of UCH-L1. Further studies revealed that the GSPB2 pretreatment markedly attenuated the degradation of IκB-α and nuclear translocation of NF-κB by modulating ubiquitination of IκB-α in AGE-exposed HASMCs. These results collectively suggest that AGE-induced HASMC proliferation and migration was suppressed by GSPB2 through regulating UCH-L1 and ubiquitination of IκB-α. GSPB2 may therefore have therapeutic potential in preventing and treating vascular complications of diabetes mellitus.     

Docosahexaenoic acid inhibition of inflammation is partially via cross-talk between Nrf2/heme oxygenase 1 and IKK/NF-κB pathways

We examined the underlying mechanisms involved in n-3 docosahexaenoic acid (DHA) inhibition of inflammation in EA.hy926 cells. The present results demonstrated that pretreatment with DHA (50 and 100 μM) inhibited tumor necrosis factor-alpha (TNF-α)-induced intercellular adhesion molecule 1 (ICAM-1) protein, mRNA expression and promoter activity. In addition, TNF-α-stimulated inhibitory kappa B (IκB) kinase (IKK) phosphorylation, IκB phosphorylation and degradation, p65 nuclear translocation, and nuclear factor-κB (NF-κB) and DNA binding activity were attenuated by pretreatment with DHA. DHA triggered early-stage and transient reactive oxygen species (ROS) generation and significantly increased the protein expression of heme oxygenase 1 (HO-1), induced nuclear factor erythroid 2-related factor 2 (Nrf2) translocation to the nucleus and up-regulated antioxidant response element (ARE)-luciferase reporter activity. Moreover, DHA inhibited Nrf2 ubiquitination and proteasome activity. DHA activated Akt, p38 and ERK1/2 phosphorylation, and specific inhibitors of respective pathways attenuated DHA-induced Nrf2 nuclear translocation and HO-1 expression. Transfection with HO-1 siRNA knocked down HO-1 expression and partially reversed the DHA-mediated inhibition of TNF-α-induced p65 nuclear translocation and ICAM-1 expression. Importantly, we show for the first time that HO-1 plays a down-regulatory role in NF-κB nuclear translocation, and inhibition of Nrf2 ubiquitination and proteasome activity are involved in increased cellular Nrf2 level by DHA. In this study, we show that HO-1 plays a down-regulatory role in NF-κB nuclear translocation and that the protective effect of DHA against inflammation is partially via up-regulation of Nrf2-mediated HO-1 expression and inhibition of IKK/NF-κB signaling pathway.     

Beyond NF-κB activation: nuclear functions of IκB kinase α

IκB kinase (IKK) complex, the master kinase for NF-κB activation, contains two kinase subunits, IKKα and IKKβ. In addition to mediating NF-κB signaling by phosphorylating IκB proteins during inflammatory and immune responses, the activation of the IKK complex also responds to various stimuli to regulate diverse functions independently of NF-κB. Although these two kinases share structural and biochemical similarities, different sub-cellular localization and phosphorylation targets between IKKα and IKKβ account for their distinct physiological and pathological roles. While IKKβ is predominantly cytoplasmic, IKKα has been found to shuttle between the cytoplasm and the nucleus. The nuclear-specific roles of IKKα have brought increasing complexity to its biological function. This review highlights major advances in the studies of the nuclear functions of IKKα and the mechanisms of IKKα nuclear translocation. Understanding the nuclear activity is essential for targeting IKKα for therapeutics.     

Disruption of IkappaB kinase (IKK)-mediated RelA serine 536 phosphorylation sensitizes human multiple myeloma cells to histone deacetylase (HDAC) inhibitors

Post-translational modifications of RelA play an important role in regulation of NF-κB activation. We previously demonstrated that in malignant hematopoietic cells, histone deacetylase inhibitors (HDACIs) induced RelA hyperacetylation and NF-κB activation, attenuating lethality. We now present evidence that IκB kinase (IKK) β-mediated RelA Ser-536 phosphorylation plays a significant functional role in promoting RelA acetylation, inducing NF-κB activation, and limiting HDACI lethality in human multiple myeloma (MM) cells. Immunoblot profiling revealed that although basal RelA phosphorylation varied in MM cells, Ser-536 phosphorylation correlated with IKK activity. Exposure to the pan-HDACIs vorinostat or LBH-589 induced phosphorylation of IKKα/β (Ser-180/Ser-181) and RelA (Ser-536) in MM cells, including cells expressing an IκBα "super-repressor," accompanied by increased RelA nuclear translocation, acetylation, DNA binding, and transactivation activity. These events were substantially blocked by either pan-IKK or IKKβ-selective inhibitors, resulting in marked apoptosis. Consistent with these events, inhibitory peptides targeting either the NF-κB essential modulator (NEMO) binding domain for IKK complex formation or RelA phosphorylation sites also significantly increased HDACI lethality. Moreover, IKKβ knockdown by shRNA prevented Ser-536 phosphorylation and significantly enhanced HDACI susceptibility. Finally, introduction of a nonphosphorylatable RelA mutant S536A, which failed to undergo acetylation in response to HDACIs, impaired NF-κB activation and increased cell death. These findings indicate that HDACIs induce Ser-536 phosphorylation of the NF-κB subunit RelA through an IKKβ-dependent mechanism, an action that is functionally involved in activation of the cytoprotective NF-κB signaling cascade primarily through facilitation of RelA acetylation rather than nuclear translocation.     

Coordinate Regulation of TPL-2 and NF-κB Signaling in Macrophages by NF-κB1 p105

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.     

Peptidylarginine deiminase 2 suppresses inhibitory {kappa}B kinase activity in lipopolysaccharide-stimulated RAW 264.7 macrophages

Peptidylarginine deiminases (PADs) are enzymes that convert arginine to citrulline in proteins. In this study, we examined PAD-mediated citrullination and its effect on pro-inflammatory activity in the macrophage cell line RAW 264.7. Citrullination of 45-65-kDa proteins was induced when cells were treated with lipopolysaccharide (LPS; 1 μg/ml). Protein citrullination was suppressed by the intracellular calcium chelator BAPTA/AM (30 μM). LPS treatment up-regulated COX-2 levels in cells. Interestingly, overexpressing PAD2 reduced LPS-mediated COX-2 up-regulation by 50%. PAD2 overexpression also reduced NF-κB activity, determined by NF-κB-driven luciferase activity. The effect of PAD2 on NF-κB activity was further examined by using HEK 293 cells transfected with NF-κB luciferase, IκB β/γ kinase (IKKβ/γ) subunits, and PAD2. IKKβ increased NF-κB activity, but this increase was markedly suppressed when PAD2 was present in cells. IKKβ-mediated NF-κB activation was further enhanced by IKKγ in the presence of calcium ionophore A23187. However, this stimulatory effect of IKKβ/γ was abolished by PAD2. Coimmunoprecipitation of cell lysates showed that IKKγ and PAD2 can coimmunoprecipitate in the presence of the Ca(2+) ionophore. IKKγ coimmunoprecipitated truncation mutants, PAD2(1-385) and PAD2(355-672). The substitution of Gln-358 (a putative ligand for Ca(2+) binding) with an Ala abolished coimmunoprecipitation. Conversely, PAD2 coimmunoprecipitated truncation mutants IKKγ(1-196) and IKKγ(197-419). In other experiments, treating RAW 264.7 cells with LPS induced citrullination in the immunoprecipitates of IKKγ. In vitro citrullination assay showed that incubation of purified PAD2 and IKKγ proteins in the presence of Ca(2+) citrullinated IKKγ. These results demonstrate that PAD2 interacts with IKKγ and suppresses NF-κB activity.     

Anti-osteoclastic effect of caffeic acid phenethyl ester in murine macrophages depends upon the suppression of superoxide anion production through the prevention of an active-Nox1 complex formation

This study investigated the anti-osteoclastic effect of caffeic acid phenethyl ester (CAPE) through suppression of Nox1-mediated superoxide anions production. The multi-nucleated cells were counted and followed by measuring their tartrate-resistant acid phosphatase (TRAP) activity. The superoxide anion production was determined by using fluorescent probe dihydroethidium (DHE). After one day of exposure to the receptor activator of nuclear factor-κB ligand (RANKL), the expression of the proteins involved in superoxide anion production was determined by western blotting. A potent anti-osteoclastic effect of CAPE was observed; the superoxide anion level reached a maximum value after one day of incubation. CAPE attenuated the expression of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) and Rac1, and mitigated the RANKL-induced translocation of p47^phox to the cell membrane. In addition, CAPE suppressed the expression of nuclear factor-kappa B (NF-κB p65), its translocation to the nucleus, and the activation of NF-κB inhibitor (IκBα) and its kinase (IKKβ). Furthermore, CAPE diminished the expression and activation of the c-jun N-terminal kinase (JNK) and the expression of protein-1 activators (AP-1) such as c-Fos and c-Jun. The expression of Nox1 was suppressed by CAPE through the down-regulation of IKKβ/IκBα/NF-κB and JNK/AP-1 signal pathway. This study provides evidence that the anti-osteoclastic effect of CAPE depends upon the attenuated superoxide anion production, which is closely related with interruption of an active Nox1 complex formation due to the attenuated catalytic subunit Nox1 expression resulting from suppression of the IKKβ/IκBα/NF-κB and JNK/AP-1 signaling pathway and the down-regulation of the p47^phox subunit translocation to the cell membrane.     

Induction of vascular cell adhesion molecule-1 expression by IL-4 in human aortic smooth muscle cells is not associated with increased nuclear NF-κB levels

Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) are upregulated in vascular endothelial and smooth muscle cells by cytokines produced at sites of inflammation. The cytokine profile for induction of VCAM-1, however, is different for the two cell types. Tumor necrosis factor-α (TNF-α) induced both VCAM-1 and ICAM-1 expression in human umbilical vein endothelial cells (HUVECs; ED₅₀ ∼ 300 and 30 U/ml, respectively). TNF-α and interleukin-1β (IL-1β) stimulated cell surface ICAM-1 expression, but not VCAM-1 expression, in human aortic smooth muscle cells (HASMCs). Conversely, IL-4 was a potent VCAM-1 inducer in HASMCs (ED₅₀ ∼ 100 pg/ml) but did not induce ICAM-1 expression. Nuclear extracts from IL-4-treated cells were compared with untreated cells for relative nuclear factor-kappa B (NF-κB) levels by using an electrophoretic mobility shift assay and surface plasmon resonance techniques. No significant increase in nuclear NF-κB DNA binding activity was detected in IL-4-treated HASMCs by either method of analysis. IL-1β and TNF-α stimulated nuclear NF-κB levels by about fourfold and fivefold, respectively, in HASMCs. The antioxidant pyrrolidine dithiocarbamate (PDTC) similarly inhibited VCAM-1 upregulation in HASMCs incubated with IL-4 and in HUVECs incubated with TNF-α (IC₅₀s of 25 and 40 μM, respectively). These data suggest that a significant increase in nuclear NF-κB levels is not necessary or sufficient for VCAM-1 upregulation in HASMCs and does not determine the relative sensitivity to inhibition of gene expression by PDTC. J. Cell. Physiol. 180:381–389, 1999. © 1999 Wiley-Liss, Inc.     

NEMO differentially regulates TCR and TNF-α induced NF-κB pathways and has an inhibitory role in TCR-induced NF-κB activation

NF-κB essential modulator (NEMO), the regulatory subunit of the IκB kinase (IKK) complex, is an essential adaptor both for inflammation stimuli and TCR-induced NF-κB activation. However, the exact mechanism of its function has not been fully understood. Here, we report that knockdown of NEMO by RNA interference in Jurkat E6.1 cells enhanced TCR-induced NF-κB report gene activity and IL-2 production by promotion of IκBα degradation and p65 nuclear translocation, whereas inhibited TNF-α and LPS-induced IκBα degradation without influencing the phosphorylation of MAPKs. In human primary T and Jurkat E6.1 cells, both CD3/CD28 and PMA/Ionomycin induced NF-κB activation showed a para-curve correlation with the dosage of small interfering RNA targeting NEMO (siNEMO): the NF-κB report gene activity was increased along with ascending doses of transfected siNEMO and reached the highest activity when knockdown about 70% of NEMO, then turned to decline and gradually be blocked once almost thoroughly knockdown of NEMO. Meanwhile, TNF-α induced NF-κB was always inhibited no matter how much NEMO was knockdown. Subcellular fractionation results suggested that upon CD3/CD28 costimulation, NEMO and IKKβ may not cotranslocate to cytoskeleton fraction as a conventional NEMO/IKK complex with a static stoichiometric ratio, instead the ratio of NEMO: IKKβ continuously shift from high to low. Depletion of NEMO accelerated TCR-induced cytoskeleton translocation of IKKβ. Altogether, this study suggests that NEMO may function as a rheostat exerting a negative action on TCR-induced NF-κB activation and differentially regulates TNF-α and TCR-induced NF-κB pathways.