Lymphotoxin-alpha 1 beta 2 and LIGHT induce classical and noncanonical NF-kappa B-dependent proinflammatory gene expression in vascular endothelial cells

Activation of the classical and noncanonical NF-kappaB pathways by ligation of the lymphotoxin (LT)-beta receptor (LTbetaR) plays a crucial role in lymphoid organogenesis and in the generation of ectopic lymphoid tissue at sites of chronic inflammation. Within these microenvironments, LTbetaR signaling regulates the phenotype of the specialized high endothelial cells. However, the direct effects of LTbetaR ligation on endothelial cells remain unclear. We therefore questioned whether LTbetaR ligation could directly activate endothelial cells and regulate classical and noncanonical NF-kappaB-dependent gene expression. We demonstrate that the LTbetaR ligands LIGHT and LTalpha1beta2 activate both NF-kappaB pathways in HUVECs and human dermal microvascular endothelial cells (HDMEC). Classical pathway activation was less robust than TNF-induced signaling; however, only LIGHT and LTalpha1beta2 and not TNF activated the noncanonical pathway. LIGHT and LTalpha1beta2 induced the expression of classical NF-kappaB-dependent genes in HUVEC, including those encoding the adhesion molecules E-selectin, ICAM-1, and VCAM-1. Consistent with this stimulation, LTbetaR ligation up-regulated T cell adhesion to HUVEC. Furthermore, the homeostatic chemokine CXCL12 was up-regulated by LIGHT and LTalpha1beta2 but not TNF in both HUVEC and HDMEC. Using HUVEC retrovirally transduced with dominant negative IkappaB kinase alpha, we demonstrate that CXCL12 expression is regulated by the noncanonical pathway in endothelial cells. Our findings therefore demonstrate that LTbetaR ligation regulates gene expression in endothelial cells via both NF-kappaB pathways and we identify CXCL12 as a bona fide noncanonical NF-kappaB-regulated gene in these cells.     

A small molecule ubiquitination inhibitor blocks NF-kappa B-dependent cytokine expression in cells and rats

A small molecule inhibitor of NF-kappaB-dependent cytokine expression was discovered that blocked tumor necrosis factor (TNF) alpha-induced IkappaB(alpha) degradation in MM6 cells but not the degradation of beta-catenin in Jurkat cells. Ro106-9920 blocked lipopolysaccharide (LPS)-dependent expression of TNFalpha, interleukin-1beta, and interleukin-6 in fresh human peripheral blood mononuclear cells with IC(50) values below 1 microm. Ro106-9920 also blocked TNFalpha production in a dose-dependent manner following oral administration in two acute models of inflammation (air pouch and LPS challenge). Ro106-9920 was observed to inhibit an ubiquitination activity that does not require betaTRCP but associates with IkappaB(alpha) and will ubiquitinate IkappaB(alpha) S32E,S36E (IkappaB(alpha)(ee)) specifically at lysine 21 or 22. Ro106-9920 was identified in a cell-free system as a time-dependent inhibitor of IkappaB(alpha)(ee) ubiquitination with an IC(50) value of 2.3 +/- 0.09 microm. The ubiquitin E3 ligase activity is inhibited by cysteine-alkylating reagents, supported by E2UBCH7, and requires cIAP2 or a cIAP2-associated protein for activity. These activities are inconsistent with what has been reported for SCF(betaTRCP), the putative E3 for IkappaB(alpha) ubiquitination. Ro106-9920 was observed to be selective for IkappaB(alpha)(ee) ubiquitination over the ubiquitin-activating enzyme (E1), E2UBCH7, nonspecific ubiquitination of cellular proteins, and 97 other molecular targets. We propose that Ro106-9920 selectively inhibits an uncharacterized but essential ubiquitination activity associated with LPS- and TNFalpha-induced IkappaB(alpha) degradation and NF-kappaB activation.     

Curcumin prevents alcohol-induced liver disease in rats by inhibiting the expression of NF-kappa B-dependent genes

Induction of NF-kappaB-mediated gene expression has been implicated in the pathogenesis of alcoholic liver disease (ALD). Curcumin, a phenolic antioxidant, inhibits the activation of NF-kappaB. We determined whether treatment with curcumin would prevent experimental ALD and elucidated the underlying mechanism. Four groups of rats (6 rats/group) were treated by intragastric infusion for 4 wk. One group received fish oil plus ethanol (FE); a second group received fish oil plus dextrose (FD). The third and fourth groups received FE or FD supplemented with 75 mg. kg(-1). day(-1) of curcumin. Liver samples were analyzed for histopathology, lipid peroxidation, NF-kappaB binding, TNF-alpha, IL-12, monocyte chemotactic protein-1, macrophage inflammatory protein-2, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nitrotyrosine. Rats fed FE developed fatty liver, necrosis, and inflammation, which was accompanied by activation of NF-kappaB and the induction of cytokines, chemokines, COX-2, iNOS, and nitrotyrosine formation. Treatment with curcumin prevented both the pathological and biochemical changes induced by alcohol. Because endotoxin and the Kupffer cell are implicated in the pathogenesis of ALD, we investigated whether curcumin suppressed the stimulatory effects of endotoxin in isolated Kupffer cells. Curcumin blocked endotoxin-mediated activation of NF-kappaB and suppressed the expression of cytokines, chemokines, COX-2, and iNOS in Kupffer cells. Thus curcumin prevents experimental ALD, in part by suppressing induction of NF-kappaB-dependent genes.     

Osteoprotegerin is an alpha vbeta 3-induced, NF-kappa B-dependent survival factor for endothelial cells

Osteopontin protects endothelial cells from apoptosis induced by growth factor withdrawal. This interaction is mediated by the alpha(v)beta(3) integrin and is NF-kappaB-dependent (Scatena, M., Almeida, M., Chaisson, M. L., Fausto, N., Nicosia, R. F., and Giachelli, C. M. (1998) J. Cell Biol. 141, 1083-1093). In the present study we used differential cloning to identify osteopontin-induced, NF-kappaB-dependent genes in endothelial cells. One of the genes identified in this screen was osteoprotegerin, a member of the tumor necrosis factor receptor superfamily. By Northern and Western blot analysis, osteoprotegerin mRNA and protein levels were very low in endothelial cells plated on the non-integrin cell attachment factor, poly-d-lysine. In contrast, osteoprotegerin mRNA and protein levels were induced 5-7-fold following alpha(v)beta(3) ligation by osteopontin. Osteoprotegerin induction by osteopontin was time-dependent and observed as early as 3 h following treatment. NF-kappaB inactivation achieved by over expression of an IkappaB super repressor in endothelial cells completely inhibited osteoprotegerin induction by osteopontin. Finally, purified osteoprotegerin protected endothelial cells with inactive NF-kappaB from apoptosis induced by growth factor deprivation. These data suggest that alpha(v)beta(3)-mediated endothelial survival depends on osteoprotegerin induction by NF-kappaB and indicate a new function for osteoprotegerin in endothelial cells.     

Biliary obstruction selectively expands and activates liver myeloid dendritic cells

Obstructive jaundice is associated with immunologic derangements and hepatic inflammation and fibrosis. Because dendritic cells (DCs) play a major role in immune regulation, we hypothesized that the immunosuppression associated with jaundice may result from the functional impairment of liver DCs. We found that bile duct ligation (BDL) in mice expanded the myeloid subtype of liver DCs from 20 to 80% of total DCs and increased their absolute number by >15-fold. Liver myeloid DCs following BDL, but not sham laparotomy, had increased Ag uptake in vivo, high IL-6 secretion in response to LPS, and enhanced ability to activate T cells. The effects of BDL were specific to liver DCs, as spleen DCs were not affected. Expansion of liver myeloid DCs depended on Gr-1(+) cells, and we implicated monocyte chemotactic protein-1 as a potential mediator. Thus, obstructive jaundice selectively expands liver myeloid DCs that are highly functional and unlikely to be involved with impaired host immune responses.     

TLR2 expression in astrocytes is induced by TNF-alpha- and NF-kappa B-dependent pathways

Astrocytes participate in CNS innate immune responses as evident by their ability to produce a wide array of inflammatory mediators upon exposure to diverse stimuli. Although we have established that astrocytes use TLR2 to signal inflammatory mediator production in response to Staphylococcus aureus, a common etiological agent of CNS infections, the signal transduction pathways triggered by this pathogen and how TLR2 expression is regulated remain undefined. Three disparate inhibitors that block distinct steps in the NF-kappaB pathway, namely SC-514, BAY 11-7082, and caffeic acid phenethyl ester, attenuated NO, TNF-alpha, and CXCL2 release from S. aureus-activated astrocytes. Among these proinflammatory mediators, autocrine/paracrine TNF-alpha was pivotal for augmenting TLR2 expression, since receptor levels were not elevated in astrocytes isolated from TNF-alpha knockout mice upon bacterial exposure. Since TLR2 is critical for signaling astrocytic cytokine production in response to S. aureus, we evaluated the effect of TNF-alpha loss on proinflammatory mediator release. Interestingly, among the molecules assayed, only NO production was significantly attenuated in TNF-alpha knockout astrocytes compared with wild-type cells. Similar results were obtained following LPS treatment, suggesting that TNF-alpha is an important regulator of astrocytic TLR2 expression and NO release in response to diverse microbial stimuli. In addition, NF-kappaB inhibitors attenuated TNF-alpha-induced TLR2 expression in astrocytes. Overall, this study suggests that two important anti-bacterial effector molecules, TLR2 and NO, are regulated, in part, by NF-kappaB-dependent autocrine/paracrine effects of TNF-alpha in astrocytes.     

Short-term hyperhomocysteinemia-induced oxidative stress activates retinal glial cells and increases vascular endothelial growth factor expression in rat retina

Hyperhomocysteinemia is associated with an increase in the incidence of vascular diseases, including retinal vascular diseases. We examined the effects of high plasma levels of homocysteine on retinal glial cells and vascular endothelial growth factor (VEGF) expression. Male Sprague-Dawley rats were fed either a 3.0 g/kg homocystine diet or a control diet for 2 week. The homocystine-diet group had higher plasma levels of homocysteine and thiobarbituric acid reactive substances (TBARSs) and lower plasma levels of folate, retinol, alpha-tocopherol, and retinal expression of CuZn superoxide dismutase (SOD) than the controls. The rats fed the homocystine-diet showed an increase in vimentin, glial fibrillary acidic protein (GFAP), and VEGF immunoreactivity in the retina as compared to the controls. The increase in vimentin immunoreactivity in the hyperhomocysteinemic rats was correlated with changes in GFAP immunoreactivity in astrocytes within the ganglion cell layer. We found for the first time that short-term hyperhomocysteinemia-induced oxidative stress activates retinal glial cells and increases VEGF expression in the retina.     

Shear stress activates Tie2 receptor tyrosine kinase in human endothelial cells

The receptor tyrosine kinase (RTK) Tie2 is expressed predominantly on endothelial cells. Tie2 is critical for vasculogenesis during development and could be important for maintaining endothelial cell survival and integrity in adult blood vessels. Although most RTKs are activated by shear stress in the absence of ligand activation, the effect of shear stress on Tie2 is unknown. Therefore, we examined the effect of shear stress on Tie2 phosphorylation in primary cultured endothelial cells. Interestingly, shear stress (20 dyne/cm(2)) produced a rapid, marked, and sustained Tie2 phosphorylation, while it produced a rapid but slight and transient phosphorylation of insulin receptor and VEGF receptor 2 (Flk1). In addition, Tie2 phosphorylation in response to shear stress was velocity-dependent, while phosphorylation of insulin receptor and Flk1 was not. Shear stress also produced Akt phosphorylation in a time-, velocity-, and PI 3-kinase-dependent manner. Accordingly, shear stress suppressed serum deprivation-induced endothelial cell apoptosis. Taken together, our results indicated that activation of Tie2/PI 3-kinase/Akt in response to shear stress could be an important signaling cascade for maintaining endothelial survival and integrity in blood vessels.     

Transfection of human endothelial cells with HIV-1 tat gene activates NF-kappa B and enhances monocyte adhesion

Human immunodeficiency virus (HIV)-1 Tat released from HIV-1-infected monocytes is believed to enter other cells via an integrin-facilitated pathway, resulting in altered gene expression. Indeed, exogenous Tat protein can increase cell adhesion molecule gene expression in human endothelial cells. Signaling pathways initiated by Tat in endothelial cells are not known. We evaluated the ability of endogenous tat to stimulate monocyte adhesion via activation of nuclear factor-kappaB (NF-kappaB) within human umbilical vein endothelial cells. Transfection with pcTat, but not control vector DNA, increased NF-kappaB binding activity, NF-kappaB luciferase reporter activity, and monocyte adhesion. pcTat also increased kappaB-dependent HIV-1-LTR-CAT reporter activity 28-fold compared with a 3-fold increase produced by transfection with an equivalent amount of pcTax (from human leukemia virus). The pcTat-induced increase in pNF-kappaB-Luc activity and monocyte adhesion to endothelial cells was blocked by cotransfection with dominant-negative mutant IkappaBalpha and by incubation with 10 mM aspirin. We conclude that monocyte adhesion to human endothelial cells stimulated by pcTat is mediated via an NF-kappaB-dependent mechanism. Furthermore, inhibition studies using aspirin suggest that pcTat-stimulated NF-kappaB activation and monocyte adhesion occur via a redox-sensitive mechanism.     

PGE1 analog alprostadil induces VEGF and eNOS expression in endothelial cells

Endothelial nitric oxide synthase (eNOS), VEGF, and hypoxia-inducible factor 1-alpha (HIF-1alpha) are important regulators of endothelial function, which plays a role in the pathophysiology of heart failure (HF). PGE1 analog treatment in patients with HF elicits beneficial hemodynamic effects, but the precise mechanisms have not been investigated. We have investigated the effects of the PGE1 analog alprostadil on eNOS, VEGF, and HIF-1alpha expression in human umbilical vein endothelial cells (HUVEC) using RT-PCR and immunoblotting under normoxic and hypoxic conditions. In addition, we studied protein expression by immunohistochemical staining in explanted hearts from patients with end-stage HF, treated or untreated with systemic alprostadil. Alprostadil causes an upregulation of eNOS and VEGF protein and mRNA expression in HUVEC and decreases HIF-1alpha. Hypoxia potently increased eNOS, VEGF, and HIF-1alpha synthesis. The alprostadil-induced upregulation of eNOS and VEGF was prevented by inhibition of MAPKs with PD-98056 or U-0126. Consistently, the expression of eNOS and VEGF was increased, and HIF-1alpha was reduced in failing hearts treated with alprostadil. The potent effects of alprostadil on endothelial VEGF and eNOS synthesis may be useful for patients with HF where endothelial dysfunction is involved in the disease process.     

Chunghyuldan activates NOS mRNA expression and suppresses VCAM-1 mRNA expression in human endothelial cells

Chunghyuldan (CHD), a combinatorial drug that has antihyperlipidemic and anti-inflammatory activities, has been shown to improve arterial stiffness and inhibit stroke recurrence in clinical study. To understand the molecular basis of CHD's clinical effects, we explored its effect on cell proliferation and expression of nitric oxide synthase (NOS) and vascular cell adhesion molecule (VCAM-1) in human umbilical vein endothelial cells (HUVECs). Cell number counting and [3H]thymidine incorporation assay demonstrated that nontoxic doses of CHD have an inhibitory effect on DNA synthesis and suppress cell cycle progression of HUVECs. CHD treatment led to a marked induction of NO production through up-regulation of NOS mRNA expression in a dose- and time-dependent manner, whereas it suppressed VCAM-1 expression. CHD inhibition of VCAM-1 expression was totally blocked by pretreatment with the NO synthesis inhibitor L-NMMA, whereas pretreatment with the NO donor DETA-NO further decreased VCAM-1 level in CHD-treated HUVECs, indicating that VCAM-1 regulation by CHD is mediated through increased NO synthesis by CHD. In addition, TNF-alpha-mediated VCAM-1 activation was substantially impeded by CHD treatment. Collectively, our data suggest that anti-inflammatory or anti-hyperlipidemic effects of CHD might be associated with its ability to activate NO production and suppress VCAM-1 expression in human endothelial cells.     

Integrin-linked kinase regulates inducible nitric oxide synthase and cyclooxygenase-2 expression in an NF-kappa B-dependent manner.

Nitric oxide (NO) and prostaglandins are produced as a result of the stimulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2, respectively, in response to cytokines or lipopolysaccharide (LPS). We demonstrate that the activity of integrin-linked kinase (ILK) is stimulated by LPS activation in J774 macrophages. Inhibition of ILK activity by dominant-negative ILK or a highly selective small molecule ILK inhibitor, in epithelial cells or LPS-stimulated J774 cells and murine macrophages, resulted in inhibition of iNOS expression and NO synthesis. LPS stimulates the phosphorylation of IkappaB on Ser-32 and promotes its degradation. Inhibition of ILK suppressed this LPS-stimulated IkappaB phosphorylation and degradation. Similarly, ILK inhibition suppressed the LPS-stimulated iNOS promoter activity. Mutation of the NF-kappaB sites in the iNOS promoter abolished LPS- and ILK-mediated regulation of iNOS promoter activity. Overexpression of ILK-stimulated NF-kappaB activity and inhibition of ILK or protein kinase B (PKB/Akt) suppressed this activation. We conclude that ILK can regulate NO production in macrophages by regulating iNOS expression through a pathway involving PKB/Akt and NF-kappaB. Furthermore, we also demonstrate that ILK activity is required for LPS stimulated cyclooxygenase-2 expression in murine and human macrophages. These findings implicate ILK as a potential target for anti-inflammatory applications.