A pathogenic role for JNK signaling in experimental anti-GBM glomerulonephritis.

Activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway is involved in the immune response; however, little is known of its role in immune-induced renal injury. In this study, we examine JNK signaling in the rat anti-glomerular basement membrane (GBM) disease model using CC-401, a specific JNK inhibitor. Animals were given CC-401, vehicle alone or no treatment starting before anti-GBM serum injection and continued treatment until killing. In acute disease, CC-401 blocked JNK signaling and reduced proteinuria in the first 24 h. The transient neutrophil influx seen at 3 h of disease was not affected, however. Continued CC-401 treatment suppressed glomerular and tubulointerstitial damage usually seen at 14 days. The protective effect may be due to modulation of macrophage activation, as CC-401 had no effect upon glomerular macrophage infiltration at day 14 despite the suppression of glomerular lesions and a marked reduction in renal tumor necrosis factor-alpha and inducible nitric oxide synthase messenger RNA levels. Treatment with CC-401 had no apparent effect on T cell or humoral immune responses. These studies suggest that JNK signaling promotes renal injury in acute and progressive rat anti-GBM disease. JNK inhibitors may be a novel therapeutic approach for the treatment of human glomerulonephritis.

SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase.

Jun N-terminal kinase (JNK) is a stress-activated protein kinase that can be induced by inflammatory cytokines, bacterial endotoxin, osmotic shock, UV radiation, and hypoxia. We report the identification of an anthrapyrazolone series with significant inhibition of JNK1, -2, and -3 (K(i) = 0.19 microM). SP600125 is a reversible ATP-competitive inhibitor with >20-fold selectivity vs. a range of kinases and enzymes tested. In cells, SP600125 dose dependently inhibited the phosphorylation of c-Jun, the expression of inflammatory genes COX-2, IL-2, IFN-gamma, TNF-alpha, and prevented the activation and differentiation of primary human CD4 cell cultures. In animal studies, SP600125 blocked (bacterial) lipopolysaccharide-induced expression of tumor necrosis factor-alpha and inhibited anti-CD3-induced apoptosis of CD4(+) CD8(+) thymocytes. Our study supports targeting JNK as an important strategy in inflammatory disease, apoptotic cell death, and cancer.

TAK1/JNK and p38 have opposite effects on rat hepatic stellate cells.

After liver injury, hepatic stellate cells (HSCs) undergo a process of activation with expression of smooth muscle alpha-actin (alpha-SMA), an increased proliferation rate, and a dramatic increase in synthesis of type I collagen. The intracellular signaling mechanisms of activation and perpetuation of the activated phenotype in HSCs are largely unknown. In this study the role of the stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38, were evaluated in primary cultures of rat HSCs. The effect of JNK was assessed by using an adenovirus expressing a dominant negative form of transforming growth factor beta (TGF-beta)-activated kinase 1 (TAK1) (Ad5dnTAK1) and a new selective pharmacologic inhibitor SP600125. The effect of p38 was assessed with the selective pharmacologic inhibitor SB203580. These kinases were inhibited starting either in quiescent HSCs (culture day 1) or in activated HSCs (culture day 5). Although blocking TAK1/JNK and p38 decreased the expression of alpha-SMA protein in early stages of HSC activation, no effect was observed when TAK1/JNK or p38 were inhibited in activated HSCs. JNK inhibition increased and p38 inhibition decreased collagen alpha1(I) mRNA level as measured by RNase protection assays, with maximal effects observed in early stages of HSC activation. Furthermore, TAK1/JNK inhibition decreased HSC proliferation, whereas p38 inhibition led to an increased proliferation rate of HSCs, independently of its activation status. These results show novel roles for the TAK1/JNK pathway and p38 during HSC activation in culture. Despite similar activators of TAK1/JNK and p38, their functions in HSCs are distinct and opposed.

Inhibitor of nuclear factor kappaB kinase beta is a key regulator of synovial inflammation.

OBJECTIVE: Inhibitor of nuclear factor kappaB kinase beta (IkappaB kinase beta, or IKKbeta) has emerged as a key regulator of the transcription factor nuclear factor kappaB (NF-kappaB). Since IKKbeta could have both pro- and antiinflammatory activity, we examined whether its constitutive activation was sufficient to cause a chronic inflammatory disease such as rheumatoid arthritis. METHODS: Normal Lewis rats were evaluated for paw swelling by plethysmometry and histologic assessment after intraarticular injection of an adenoviral construct encoding the IKKbeta wild-type gene (Ad.IKKbeta-wt); controls received an adenoviral construct encoding green fluorescent protein (Ad.GFP). The rats were killed after 7 days. Additionally, rats were killed 48 hours after intraarticular injection of Ad.IKKbeta-wt or Ad.GFP for studies of IKK activity and NF-kappaB binding. For studies of the effects of inhibition of IKKbeta activity, Lewis rats were immunized with Mycobacterium tuberculosis in mineral oil. The ankle joints were injected on day 12 with an adenoviral construct encoding IKKbeta K-->M (dominant negative, IKKbeta-dn) or Ad.GFP. We evaluated paw swelling and NF-kappaB expression on day 25. RESULTS: Intraarticular gene transfer of IKKbeta-wt into the joints of normal rats resulted in significant paw swelling and histologic evidence of synovial inflammation. Increased IKK activity was detectable in the IKKbeta-wt-injected ankle joints, coincident with enhanced NF-kappaB DNA binding activity. Intraarticular gene transfer of IKKbeta-dn significantly ameliorated the severity of adjuvant arthritis, accompanied by a significant decrease in NF-kappaB DNA expression in the joints of Ad.IKKbeta-dn-treated animals. CONCLUSION: IKKbeta plays a key role in rodent synovial inflammation. Intraarticular gene therapy to inhibit IKKbeta activity represents an attractive strategy for the treatment of chronic arthritis.

NF-kappaB stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-alpha- and Fas-mediated apoptosis.

BACKGROUND AND AIMS: Hepatocyte apoptosis is induced by tumor necrosis factor alpha (TNF-alpha) and Fas ligand. Although nuclear factor-kappaB (NF-kappaB) activation protects hepatocytes from TNF-alpha-mediated apoptosis, the NF-kappaB responsive genes that protect hepatocytes are unknown. Our aim was to study the role of NF-kappaB activation and inducible nitric oxide synthases (iNOSs) in TNF-alpha- and Fas-mediated apoptosis in hepatocytes. METHODS: Primary cultures of hepatocytes from wild-type and iNOS knockout mice were treated with TNF-alpha, the Fas agonistic antibody Jo2, a nitric oxide (NO) donor (S-nitroso-N-acetylpenicillamine), an NO inhibitor (N(G)-methyl-L-arginine acetate), and/or adenovirus-expressing NF-kappaB inhibitors. RESULTS: The IkappaB superrepressor and a dominant-negative form of IkappaB kinase beta (IKKbeta) inhibited NF-kappaB binding activity by TNF-alpha or Jo2 and sensitized hepatocytes to TNF-alpha- and Jo2-mediated apoptosis. TNF-alpha and Jo2 induced iNOS messenger RNA and protein levels through the induction of NF-kappaB. S-nitroso-N-acetylpenicillamine inhibited Bid cleavage, the mitochondrial permeability transition, cytochrome c release, and caspase-8 and -3 activity, and reduced TNF-alpha- and Fas-mediated death in hepatocytes expressing IkappaB superrepressor. N(G)-methyl-L-arginine acetate partially sensitized hepatocytes to TNF-alpha- and Fas-mediated cell killing. TNF-alpha alone or Jo2 alone induced moderate cell death in hepatocytes from iNOS(-)/(-) mice. CONCLUSIONS: NO protects hepatocytes from TNF-alpha- and Fas-mediated apoptosis. Endogenous iNOS, which is activated by NF-kappaB via IKKbeta, provides partial protection from apoptosis.

Differential role of I kappa B kinase 1 and 2 in primary rat hepatocytes.

Nuclear factor-kappa B (NF-kappa B) protects hepatocytes from undergoing apoptosis during embryonic development and during liver regeneration. Activation of NF-kappa B is mediated through phosphorylation of its inhibitor, I kappa B, by a kinase complex that contains 2 I kappa B kinases. We analyzed the differential role of I kappa B kinase 1 (IKK1) and I kappa B kinase 2 (IKK2) in tumor necrosis factor alpha (TNF-alpha)- and interleukin-1 beta (IL-1 beta)-mediated NF-kappa B activation in primary rat hepatocytes. Maximal induction of IKK activity was observed 5 minutes after TNF-alpha and 15 minutes after IL-1 beta treatment, and activated IKK was able to phosphorylate GST-I kappa B (1-54) and GST-p65 (354-551), but not a GST-p65 (354-551) substrate with a serine-to-alanine substitution at position 536. Infection with an adenovirus containing catalytically inactive IKK2K44M (Ad5IKK2dn) completely blocked both TNF-alpha- and IL-1 beta-induced GST-I kappa B and GST-p65 phosphorylation, I kappa B degradation, and NF-kappa B DNA binding. Adenovirally transduced, catalytically inactive IKK1K44M (Ad5IKK1dn) reduced IKK activity and NF-kappa B DNA binding only slightly. Accordingly, Ad5IKK2dn induced apoptosis in 75% (+/-6%) of hepatocytes after 12 hours of TNF-alpha, which was accompanied by activation of caspases 3 and 8, nuclear fragmentation, and DNA laddering. In contrast, Ad5IKK1dn led to 21% (+/-2%) apoptosis in TNF-alpha-treated hepatocytes after 12 hours and comparatively low activity of caspases 3 and 8. Furthermore, Ad5IKK2dn completely blocked the induction of inducible nitric oxide synthase (iNOS), whereas Ad5IKK1dn had no influence on the expression of iNOS. Thus, IKK2 is the main mediator for cytokine-induced NF-kappa B activation in primary hepatocytes and protects against TNF-alpha-induced apoptosis, whereas IKK1 kinase activity is not required for NF-kappa B activation.

Expression and regulation of inducible IkappaB kinase (IKK-i) in human fibroblast-like synoviocytes.

IkappaB kinase (IKK) plays a key role in the regulation of nuclear factor kappaB (NF-kappaB). We previously demonstrated the expression of two kinases, IKK1 and IKK2, in fibroblast-like synoviocytes (FLS) and determined their functional consequences for inflammatory gene expression in vitro and in vivo. Recently, a novel inducible IkappaB kinase has been described, namely, IKK-i or IKK-epsilon, which is functionally and structurally distinct from constitutively expressed IKK1 and IKK2. Therefore, we investigated the expression and regulation of this novel kinase in FLS from patients with rheumatoid arthritis and osteoarthritis. Interestingly, constitutive gene expression and protein expression were observed in all cell lines examined. TNFalpha stimulation for 24 h increased IKK-i expression 7.2 +/- 1.8-fold in FLS (P < 0.02). IL-1 also significantly increased IKK-i gene expression. Time course experiments demonstrated that IKK-i gene expression increased within 3 h of TNFalpha stimulation and persisted for at least 24 h. Dose-response studies showed that as little as 1 ng/ml of TNFalpha increased IKK-i gene expression. Constitutive IKK-1 gene expression was also noted in rheumatoid arthritis, osteoarthritis, and normal synovium. This is the first report demonstrating constitutive expression and cytokine regulation of this novel kinase in primary human synovial cells.

NF-kappa B regulation by I kappa B kinase in primary fibroblast-like synoviocytes.

NF-kappa B is a key regulator of inflammatory gene transcription and is activated in the rheumatoid arthritis (RA) synovium. In resting cells, NF-kappa B is retained as an inactive cytoplasmic complex by its inhibitor, I kappa B. Phosphorylation of I kappa B targets it for proteolytic degradation, thereby releasing NF-kappa B for nuclear translocation. Recently, two related I kappa B kinases (IKK-1 and IKK-2) were identified in immortalized cell lines that regulate NF-kappa B activation by initiating I kappa B degradation. To determine whether IKK regulates NF-kappa B in primary cells isolated from a site of human disease, we characterized IKK in cultured fibroblast-like synoviocytes (FLS) isolated from synovium of patients with RA or osteoarthritis. Immunoreactive IKK protein was found to be abundant in both RA and osteoarthritis FLS by Western blot analysis. Northern blot analysis showed that IKK-1 and IKK-2 genes were constitutively expressed in all FLS lines. IKK function in FLS extracts was determined by measuring phosphorylation of recombinant I kappa B in vitro. IKK activity in both RA and osteoarthritis FLS was strongly induced by TNF-alpha and IL-1 in a concentration-dependent manner. Activity was significantly increased within 10 min of stimulation and declined to near basal levels within 80 min. Activation of IKK in FLS was accompanied by phosphorylation and degradation of endogenous I kappa B alpha as determined by Western blot analysis. Concomitant activation and nuclear translocation of NF-kappa B was documented by EMSA and immunohistochemistry. Transfection with a dominant negative IKK-2 mutant prevented TNF-alpha-mediated NF-kappa B nuclear translocation, whereas a dominant negative IKK-1 mutant had no effect. This is the first demonstration that IKK-2 is a pivotal regulator of NF-kappa B in primary human cells.

IkappaB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.

Activation of the transcription factor NF-kappaB is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IkappaB. We recently purified a large multiprotein complex, the IkappaB kinase (IKK) signalsome, which contains two regulated IkappaB kinases, IKK1 and IKK2, that can each phosphorylate IkappaBalpha and IkappaBbeta. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFkappaB signal transduction cascade in vivo. In this report, we demonstrate reconstitution of IkappaB kinase activity in vitro by using purified recombinant IKK1 and IKK2. Recombinant IKK1 or IKK2 forms homo- or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells we identified two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, which display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, we set out to identify IKK-associated proteins. To this end, we purified and cloned a novel component common to both complexes, named IKK-associated protein 1 (IKKAP1). In vitro, IKKAP1 associated specifically with IKK2 but not IKK1. Functional analyses revealed that binding to IKK2 requires sequences contained within the N-terminal domain of IKKAP1. Mutant versions of IKKAP1, which either lack the N-terminal IKK2-binding domain or contain only the IKK2-binding domain, disrupt the NF-kappaB signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-kappaB signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-kappaB activation.

Role of IKK1 and IKK2 in lipopolysaccharide signaling in human monocytic cells.

Mononuclear phagocytes play a major role in immune and inflammatory responses. Bacterial lipopolysaccharide (LPS) induces monocytes to express a variety of genes by activating the NF-kappaB/Rel transcription factor family. Recently, we have reported that the tumor necrosis factor and interleukin 1 signaling pathways activate two kinases, IKK1 and IKK2. Phosphorylation of the IkappaB cytoplasmic inhibitors, IkappaBalpha, IkappaBbeta, and IkappaBepsilon, by these kinases triggers proteolytic degradation and the release of NF-kappaB/Rel proteins into the nucleus. At present, the role of the IKKs in LPS signaling has not been investigated. Here, we report that LPS induces IKK activity in human monocytes and THP-1 monocytic cells. The kinetics of activation of kinase activity in monocytic cells are relatively slow with maximal activity observed at 60 min, which coincides with the degradation of IkappaBs and the nuclear translocation of NF-kappaB. In transfection experiments, overexpression of wild type IKK1, a dominant negative mutant IKK1 (K44M), or wild type IKK2 did not affect LPS-induced kappaB-dependent transcription in monocytic cells. In contrast, a dominant negative mutant of IKK2 inhibited LPS induction of kappaB-dependent transcription in a dose-dependent manner. These results indicate that LPS induction of kappaB-dependent gene expression in human monocytic cells requires activation of IKK2.

Selective inhibition of TNF-alpha induced cell adhesion molecule gene expression by tanapox virus.

Poxviruses encode virulence factors that have been identified as proteins that are secreted from infected host cells. Some of these secretory proteins impede host immune defences. We have previously demonstrated that tanapox virus (TPV) infected cells secrete an early 38 kDa glycopeptide that binds to human (h) interferon-gamma, hIL-2, and hIL-5. We now show an additional activity in the supernatant from TPV infected cells that down-regulates the expression of tumour necrosis factor-alpha (TNF-alpha) induced cell adhesion molecule gene expression. This activity was not detected in mock infected cells. Enzyme linked immunosorbent assays (ELISA) on primary human endothelial cells, show the induction of E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) following TNF-alpha or IL-1 beta treatment, as expected. Supernatant from TPV infected cells significantly decreased the TNF-alpha but not IL-1 beta-induced expression of these molecules. Mobility shift assays and Northern blot analyses further show that the supernatant from TPV infected cells inhibited TNF-alpha-induced activation of the nuclear transcription factor-kappa B (NF-kappa B) and transcriptional activation of the E-selectin, VCAM-1 and ICAM-1 genes. Based on TNF-alpha affinity chromatography, this activity appears to be associated with a 38 kDa glycopeptide.

Glucose transport in a murine mammary epithelial cell line.

The glucose transport systems of the COMMA-D cell line (a murine mammary epithelial cell line) were examined using 2-deoxyglucose as substrate. The kinetics and inhibition studies with other sugars including xylose suggested that the transport system had properties of both GLUT-1 and Glut-3. Subsequent analysis of mRNA transcripts using cDNAs for GLUT-1 to 4 showed that only GLUT-1 was expressed in the COMMA-D cells. The results highlight the fact that kinetic and substrate specificity are not sufficient, by themselves, for the identification and characterisation of GLUT isoforms in cultured cells.

Interleukin-4 suppression of tumor necrosis factor alpha-stimulated E-selectin gene transcription is mediated by STAT6 antagonism of NF-kappaB.

Interleukin-4 (IL-4), an immunoregulatory cytokine secreted from activated T-helper 2 lymphocytes, eosinophils, and mast cells, stimulates the expression of a number of immune system genes via activation of the transcription factor, STAT6. However, IL-4 can concomitantly suppress the expression of other immune-related gene products, including kappa light chain, FcgammaRI, IL-8, and E-selectin. We demonstrate that IL-4 activates STAT6 in human vascular endothelial cells and that two STAT6 binding sites are present in the promoter of the E-selectin gene. IL-4-induced STAT6 binding does not activate E-selectin transcription but instead suppresses tumor necrosis factor alpha-induced expression of the E-selectin gene. STAT6 was found to compete for binding to a region in the E-selectin gene promoter containing overlapping STAT6 and NF-kappaB binding sites, effectively acting as an antagonist of NF-kappaB binding and transcriptional activation. This novel mechanism for IL-4-mediated inhibition of inflammatory gene expression provides an example of a STAT factor acting as a transcriptional repressor rather than an activator.

Identification of signal-induced IkappaB-alpha kinases in human endothelial cells.

Activation of the nuclear transcription factor-kappaB is an early event in endothelial activation. NF-kappaB activation is regulated by the inducible phosphorylation and subsequent degradation of the inhibitory subunit IkappaB-alpha. We identified two discrete kinases of approximately 36 and 41 kDa in the cytoplasm of human umbilical vein endothelial cells that specifically bind to and phosphorylate the IkappaB-alpha subunit. IkappaB-alpha kinase activity is transiently elevated following treatment with either tumor necrosis factor alpha, interleukin-1beta, or bacterial lipopolysaccharides and precedes activation of either mitogen-activated kinase or Jun kinase. Furthermore, activation of the IkappaB-alpha kinases precedes both the appearance of hyperphosphorylated IkappaB-alpha and its subsequent degradation, as well as the translocation of NF-kappaB to the nucleus. Deletion mutagenesis of the IkappaB-alpha polypeptide revealed that these kinases bind in or around the ankyrin repeat domains and phosphorylate residues within the C terminus. These kinases, however, were not identical to casein kinase II and displayed a pharmacologic profile distinct from other known kinases. These kinases may represent components of a signal transduction pathway regulating IkappaB-alpha levels in vascular endothelium.

Interaction between adrenergic and peptide stimulation in the rat pineal: pituitary adenylate cyclase-activating peptide.

The 27 amino acid peptide, pituitary adenylate cyclase-activating polypeptide (PACAP-27), and its 38 amino acid analogue, PACAP-38, stimulate serotonin-N-acetyltransferase (NAT) activity and N-acetylserotonin (NAS) and melatonin content of pineal glands from adult rats. Maximal stimulation of rat pineal NAT by PACAP-38 is not increased further significantly by concurrent stimulation with the two related peptides, vasoactive intestinal polypeptide (VIP) and/or peptide N-terminal histidine C-terminal isoleucine (PHI). Isoproterenol was a more potent inducer of NAT activity than any of these peptides alone or in combination. PACAP-38 also stimulates melatonin production by chicken pineal cells in culture as does VIP. Stimulation by both was not greater than after either alone. Prior stimulation of rat pineal NAT activity with VIP, PHI, or PACAP-38 reduces the magnitude of subsequent stimulation with PACAP-38 or forskolin. Concurrent stimulation of alpha-receptors or treatment with active phorbol ester augments rat pineal response to PACAP-38 stimulation just as it increases the response to VIP, PHI, and beta-receptor stimulation. Pineals from newborn rats respond to PACAP-38 with an increase in NAT activity and the increase is augmented by concomitant alpha 1-adrenergic stimulation. The putative PACAP inhibitor PACAP (6-38) and the putative VIP inhibitor (Ac-Tyr,D-Phe)-GRF 1-29 amide, in 100-1,000-fold excess, did not affect the stimulatory activity of any of the peptides. Pineal melatonin concentration parallels changes in pineal NAT activity.

Commonalities in vasoactive intestinal peptide and peptide N-terminal histidine C-terminal isoleucine stimulation of N-acetyltransferase activity in the rat pineal.

Exposure of adult rat pineal glands in organ culture to the polypeptides vasoactive intestinal polypeptide (VIP), and peptide N-terminal histidine C-terminal isoleucine (PHI) increases pineal serotonin N-acetyltransferase (NAT) activity and melatonin synthesis. The following research results are taken to indicate that VIP and PHI share common components of the NAT induction system: (1) The effects of the two peptides are additive at concentrations of 10 nM VIP and 100 nM PHI but not at higher peptide concentrations. (2) Pineals from newborns also respond to PHI with a dose dependent increase in NAT activity. NAT responses are additive at the same concentrations as seen with the adult pineals. (3) Light exposure affects the sensitivity of pineals to VIP and PHI stimulation in a similar manner; pineals taken after 3 hr of light are much less sensitive to PHI or VIP than those taken after 13 hr of light. (4) Pineals exposed for 48 hr to either PHI or VIP have a reduced NAT response to either agonist, which is reversible by culture in agonist-free media. (5) Neither VIP nor PHI stimulation of NAT activity is affected by concentrations of the VIP antagonists (N-Ac-Tyr1,D-Phe2)-GRF(1-29)-NH2 (NAcTDGRF), L-8-K, VIP-Neurotensin Hybrid (VIPNET), or (4Cl-D-Phe6, Leu17)-VIP (4C1PLVIP), which affect VIP binding or function in other tissues.