Brydon L. Bennett

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 (Ki = 0.19 μM). 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-γ, TNF-α, 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-α 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.

c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis

Mitogen-activated protein kinase (MAPK) cascades are involved in inflammation and tissue destruction in rheumatoid arthritis (RA). In particular, c-Jun N-terminal kinase (JNK) is highly activated in RA fibroblast-like synoviocytes and synovium. However, defining the precise function of this kinase has been difficult because a selective JNK inhibitor has not been available. We now report the use of a novel selective JNK inhibitor and JNK knockout mice to determine the function of JNK in synoviocyte biology and inflammatory arthritis. The novel JNK inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one) completely blocked IL-1--induced accumulation of phospho-Jun and induction of c-Jun transcription in synoviocytes. Furthermore, AP-1 binding and collagenase mRNA accumulation were completely suppressed by SP600125. In contrast, complete inhibition of p38 had no effect, and ERK inhibition had only a modest effect. The essential role of JNK was confirmed in cultured synoviocytes from JNK1 knockout mice and JNK2 knockout mice, each of which had a partial defect in IL-1--induced AP-1 activation and collagenase-3 expression. Administration of SP600125 modestly decreased the rat paw swelling in rat adjuvant-induced arthritis. More striking was the near-complete inhibition of radiographic damage that was associated with decreased AP-1 activity and collagenase-3 gene expression. Therefore, JNK is a critical MAPK pathway for IL-1--induced collagenase gene expression in synoviocytes and in joint arthritis, indicating that JNK is an important therapeutic target for RA.

IκB Kinase (IKK)-Associated Protein 1, a Common Component of the Heterogeneous IKK Complex

ABSTRACT Activation of the transcription factor NF-κB is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IκB. We recently purified a large multiprotein complex, the IκB kinase (IKK) signalsome, which contains two regulated IκB kinases, IKK1 and IKK2, that can each phosphorylate IκBα and IκBβ. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFκB signal transduction cascade in vivo. In this report, we demonstrate reconstitution of IκB 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-κB signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-κB signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-κB activation.

Inhibitor of nuclear factor κB kinase β is a key regulator of synovial inflammation

Objective Inhibitor of nuclear factor κB kinase β (IκB kinase β, or IKKβ) has emerged as a key regulator of the transcription factor nuclear factor κB (NF-κB). Since IKKβ 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 IKKβ wild-type gene (Ad.IKKβ-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.IKKβ-wt or Ad.GFP for studies of IKK activity and NF-κB binding. For studies of the effects of inhibition of IKKβ activity, Lewis rats were immunized with Mycobacterium tuberculosis in mineral oil. The ankle joints were injected on day 12 with an adenoviral construct encoding IKKβ KM (dominant negative, IKKβ-dn) or Ad.GFP. We evaluated paw swelling and NF-κB expression on day 25. Results Intraarticular gene transfer of IKKβ-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 IKKβ-wt–injected ankle joints, coincident with enhanced NF-κB DNA binding activity. Intraarticular gene transfer of IKKβ-dn significantly ameliorated the severity of adjuvant arthritis, accompanied by a significant decrease in NF-κB DNA expression in the joints of Ad.IKKβ-dn–treated animals. Conclusion IKKβ plays a key role in rodent synovial inflammation. Intraarticular gene therapy to inhibit IKKβ activity represents an attractive strategy for the treatment of chronic arthritis.

The NF-κB Cascade Is Important in Bcl-x L Expression and for the Anti-Apoptotic Effects of the CD28 Receptor in Primary Human CD4 + Lymphocytes

We explored the role of the NF-κB pathway in the survival of primary human CD4+ T lymphocytes during CD28 costimulation. Transduction of proliferating CD4+ T cells with a tetracycline-regulated retrovirus encoding for a dominant-interfering, degradation-resistant I-κBα (inhibitor of κBα factor) mutant induced apoptosis. Using DNA arrays, we show that Bcl-xL features as a prominent anti-apoptotic member among a number of early CD28-inducible genes. A 1.2-kb segment of the proximal Bcl-xL promoter, linked to a luciferase reporter, responded to CD3/CD28 stimulation in Jurkat cells. Mutation of an NF-κB site around −840 decreased, while ectopic expression of I-κB kinase-β (IKKβ) enhanced reporter gene activity. Na+-salicylate and cyclopentenone PGs, direct inhibitors of IKKβ, interfered in the activation of the Bcl-xL promoter and induced apoptosis in CD28-costimulated CD4+ T cells. Moreover, salicylate blocked nuclear localization of NF-κB factors that bind to the NF-κB binding site in the Bcl-xL promoter, as well as the expression of Bcl-xL protein. HuT-78, a lymphoblastoid T cell line with constitutive NF-κB activity, contained elevated levels of Bcl-xL protein and, similar to proliferating CD4+ T cells, was resistant to apoptotic stimuli such as anti-Fas and TNF-α. In contrast, the same stimuli readily induced apoptosis in a Jurkat T cell clone with no detectable Bcl-xL expression. Jurkat BMS2 cells also differed from HuT-78 in collapse of mitochondrial membrane potential and superoxide generation in the mitochondrium. Taken together, these data demonstrate that CD3/CD28-induced activation of IKKβ and expression of Bcl-xL promote the survival of primary human CD4+ T lymphocytes.

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-κB/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 IκB cytoplasmic inhibitors, IκBα, IκBβ, and IκBε, by these kinases triggers proteolytic degradation and the release of NF-κB/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 IκBs and the nuclear translocation of NF-κB. In transfection experiments, overexpression of wild type IKK1, a dominant negative mutant IKK1 (K44M), or wild type IKK2 did not affect LPS-induced κB-dependent transcription in monocytic cells. In contrast, a dominant negative mutant of IKK2 inhibited LPS induction of κB-dependent transcription in a dose-dependent manner. These results indicate that LPS induction of κB-dependent gene expression in human monocytic cells requires activation of IKK2.

Differential requirement for c-Jun NH2-terminal kinase in TNFα- and Fas-mediated apoptosis in hepatocytes

The c‐Jun NH2‐terminal kinase (JNK) is involved in the regulation of cell death, but its role in tumor necrosis factor (TNF)‐α‐and Fas‐mediated apoptosis in primary cells is not well defined. In primary rat hepatocytes expressing an IκB superrepressor, the JNK inhibitor SP600125 strongly decreased TNF‐α‐induced cell death, caspase 3 activation, and DNA laddering. In contrast, SP600125 did not rescue mouse hepatocytes from Fas‐induced apoptosis. Apoptosis in mouse hepatocytes, induced by human TNF‐α, was blocked by SP600125, indicating that TNF‐receptor (TNF‐R) 1‐mediated JNK activation is important for TNF‐α‐induced death. However, mouse TNF‐α was more efficient than human TNF‐α in activating JNK and killing mouse hepatocytes, suggesting that TNF‐R1 and TNF‐R2 cooperate in JNK activation and apoptosis. SP600125 rescued actinomycin D‐pretreated hepatocytes and hepatocytes expressing a dominant negative c‐Jun from TNF‐α, indicating that JNK exerts its proapoptotic effect independently of transcription and c‐Jun. SP600125 delayed the mitochondrial permeability transition, inhibited cytochrome c release and prevented bid degradation after TNF‐α, suggesting that JNK‐regulated proapoptotic factors act upstream of the mitochondria. Moreover, overexpression of JNK1 activated a mitochondrial death pathway in hepatocytes, albeit less efficiently than TNF‐α . This study demonstrates that JNK augments TNF‐α‐induced apoptosis in hepatocytes through a signaling pathway that is distinct from the pathway by which it regulates proliferation.

JNK: a new therapeutic target for diabetes

Jun N-terminal kinase (JNK) regulates the transcription factor AP-1, which is implicated in the controlled expression of many genes involved in the immune response. For this reason, drug discovery efforts have focused on the development of JNK inhibitors for chronic inflammatory diseases. However, recent genetic evidence and emerging pharmacological data indicate that activated JNK could be critical in causing diabetes, insulin resistance and obesity. Indeed, if JNK is considered as a stress-activated protein kinase, there appear to be multiple mechanisms through which it might promote diabetes.

NF-κB Regulation by IκB Kinase-2 in Rheumatoid Arthritis Synoviocytes

IκB kinase-1 and IκB kinase-2 (IKK1 and IKK2; also called IKKα and IKKβ, respectively) are part of the signal complex that regulates NF-κB activity in many cell types, including fibroblast-like synoviocytes (FLS). We determined which of these two kinases is responsible for cytokine-induced NF-κB activation in synoviocytes and assessed the functional consequences of IKK1 or IKK2 overexpression and inhibition. FLS were infected with adenovirus constructs encoding either wild-type (wt) IKK1 or IKK2, the dominant negative (dn) mutant of both kinases, or a control construct encoding green fluorescence protein. Analysis of the NF-κB pathway revealed that cytokine-induced IKK activation, IκB degradation, and NF-κB activation was prevented in cells expressing the IKK2 dn mutant, whereas baseline NF-κB activity was increased by IKK2 wt. In addition, synthesis of IL-6 and IL-8, as well as expression of ICAM-1 and collagenase, was only increased by IKK2 wt, and their cytokine-induced production was abrogated by IKK2 dn mutant. However, the IKK1 dn mutant did not inhibit cytokine-mediated activation of NF-κB or any of the functional assays. These data indicate that IKK2 is the key convergence pathway for cytokine-induced NF-κB activation. Furthermore, IKK2 regulates adhesion molecule, matrix metalloproteinase, and cytokine production in FLS.

A Pathogenic Role for c-Jun Amino-Terminal Kinase Signaling in Renal Fibrosis and Tubular Cell Apoptosis

Renal fibrosis and tubular apoptosis are common mechanisms of progressive kidney disease. In vitro studies have implicated the c-Jun amino-terminal kinase (JNK) pathway in these processes. Both of the major JNK isoforms, JNK1 and JNK2, are expressed in the kidney, but their relative contribution to JNK signaling is unknown. This study investigated the role of JNK signaling in renal fibrosis and tubular apoptosis in the unilateral ureteral obstruction model using two different approaches: (1) Mice that were deficient in either JNK1 or JNK2 and (2) a specific inhibitor of all JNK isoforms, CC-401. Western blotting and immunostaining identified a marked increase in JNK signaling in the obstructed kidney, with substantial redundancy between JNK1 and JNK2 isoforms. Administration of CC-401 blocked JNK signaling in the rat obstructed kidney and significantly inhibited renal fibrosis in terms of interstitial myofibroblast accumulation and collagen IV deposition. This effect was attributed to suppression of gene transcription for the profibrotic molecules TGF-beta1 and connective tissue growth factor. CC-401 treatment also significantly reduced tubular apoptosis in the obstructed kidney. Genetic deletion of JNK1 or JNK2 did not protect mice from renal fibrosis in the unilateral ureteral obstruction model, but JNK1 deletion did result in a significant reduction in tubular cell apoptosis. In conclusion, this is the first study to demonstrate that JNK signaling plays a pathogenic role in renal fibrosis and tubular apoptosis. Furthermore, JNK1 plays a nonredundant role in tubular cell apoptosis. These studies identify the JNK pathway as a potential therapeutic target in progressive kidney disease.