Anthony M. Manning

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.

Multiple signals converging on NF-κB

Abstract The recent identification of molecular components of the signal transduction pathway regulating activation of nuclear factor-κB (NF-κB) in response to cytokines such as tumor necrosis factor α and interleukin-1β allows the evaluation of how other diverse stimuli impinge on the NF-κB activation pathway. These studies suggest a basis for specificity in activation of specific Rel-related family members and the genetic responses they promote.

Identification of the receptor component of the IκBα-ubiquitin ligase

NF-κB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IκBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin–proteasome system to eliminate inhibitory regulators. Here we isolate the pIκBα–ubiquitin ligase (pIκBα-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-κB inhibitor pIκBα. Taking advantage of its high affinity to pIκBα, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIκBα degradation motif as an F-box/WD-domainprotein belonging to a recently distinguished family of β-TrCP/Slimb proteins. This component, which we denote E3RSIκB (pIκBα-E3 receptor subunit), binds specifically to pIκBα and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RSIκB, which tightly binds pIκBα but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIκBα and consequently NF-κB activation. E3RSIκB represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.

NF-κB as a primary regulator of the stress response

A myriad of unrelated exogenous or endogenous agents that represent a threat to the organism are capable of inducing NF-κB activity, including viral infection, bacterial lipids, DNA damage, oxidative stress and chemotherapuetic agents. Likewise, NF-κB regulates the expression of an equally diverse array of cellular genes. These findings are indicative of the widespread significance of NF-κB as a mediator of cellular stress. Remarkably, the NF-κB pathway displays the capacity to activate, in a cell- and stimulus-specific manner, only a subset of the total repertoire of NF-κB-responsive genes. The seemingly promiscuous nature of NF-κB activation poses a regulatory quagmire as to how specificity is achieved at the level of gene expression. The review will summarize recent findings and explore how they further our understanding of the mechanism by which stimulus-specific activation of NF-κB is achieved in response to cellular stress.

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.

Identification of the receptor component of the IkappaBalpha-ubiquitin ligase.

NF-κB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IκBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin–proteasome system to eliminate inhibitory regulators. Here we isolate the pIκBα–ubiquitin ligase (pIκBα-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-κB inhibitor pIκBα. Taking advantage of its high affinity to pIκBα, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIκBα degradation motif as an F-box/WD-domainprotein belonging to a recently distinguished family of β-TrCP/Slimb proteins. This component, which we denote E3RSIκB (pIκBα-E3 receptor subunit), binds specifically to pIκBα and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RSIκB, which tightly binds pIκBα but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIκBα and consequently NF-κB activation. E3RSIκB represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.

Intercellular adhesion molecule 1 (ICAM-1) expression and its role in neutrophil-induced ischemia-reperfusion injury in rat liver.

The potential role of intercellular adhesion molecule‐1 (ICAM‐1) in the pathogenesis of reperfusion injury was investigated in male Fischer rats subjected to 45 min of hepatic ischemia and 24 h of reperfusion. ICAM‐1 mRNA levels increased during ischemia in the ischemic liver lobes; however, during reperfusion mRNA levels increased in both the ischemic and nonischemic lobes. Immunohistochemical evaluation indicated ICAM‐1 expression only on sinusoidal lining cells in controls; ischemia‐reperfusion enhanced ICAM‐1 expression in the sinusoids and induced some expression on hepatocytes. The monoclonal anti–ICAM‐1 antibody 1A29, but not an immunoglobulin G control antibody, administered at 1 h and 8 h of reperfusion (2 mg/kg) significantly attenuated liver injury as indicated by 51% lower plasma alanine aminotransferase activities and 32–36% less hepatic necrosis at 24 h without affecting reactive oxygen formation by Kupffer cells and hepatic neutrophils. Although 1A29 reduced neutrophil extravasation in a glycogen peritonitis by 60%, the antibody had no significant effect on hepatic neutrophil infiltration during reperfusion. These data suggest that ICAM‐1 plays a significant role during the neutrophil‐dependent injury phase after hepatic ischemia and reperfusion and therefore blocking this adhesion molecule may have therapeutic potential against postischemic acute liver failure. J. Leukoc. Biol. 57: 368–374; 1995.

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 temporal profiles of ICAM-1 protein and mRNA expression after transient MCA occlusion in the rat

Leukocytes may contribute to ischemic cell damage. ICAM-1 expression on endothelial cells facilitates the migration of leukocytes into tissue. Therefore, we measured the temporal profiles of ICAM-1 mRNA and protein in rat brain after transient (1 or 2 h) of middle cerebral artery (MCA) occlusion. Male Wistar rats (n = 86) were subjected to 1 or 2 h MCA of occlusion, or 2 h of MCA occlusion followed by reperfusion for a variety of durations ranging from 1 h to 1 week. 10 additional control animals were employed. ICAM-1 mRNA and protein were measured during ischemia and reperfusion, and immunohistochemical methods were used to identify specific cell types expressing ICAM-1. ICAM-1 mRNA was detected 1 h after the onset of ischemia. mRNA maximized at 10 h of reperfusion and persisted out to 1 week of reperfusion. ICAM-1 significantly increased in microvascular endothelial cells at 2 h of reperfusion, maximized at 46 h and persisted out to 1 week of reperfusion (P < 0.05). ICAM-1 mRNA and protein are present in ischemic brain early after the onset of ischemia and reperfusion, respectively. These data provide support for the role of ICAM-1 in mediating leukocyte-endothelial adhesion after transient MCA occlusion in the rat.

E-Selectin in Focal Cerebral Ischemia and Reperfusion in the Rat

The selectin family of glycoproteins facilitates the early phase of polymorphonuclear leukocyte adhesion to the endothelial cell and, thus, may promote ischemic cell damage. To evaluate E-selectin in the pathogenesis of focal cerebral ischemia and reperfusion injury, we cloned rat E-selectin cDNA and measured the temporal profiles E-selectin mRNA (Northern blot) and protein (immunohistochemistry) during (1 h of ischemia) and after (up to 1 week) transient (2 h) middle cerebral artery (MCA) occlusion in the male Wistar rat. We also tested the effect on these rats of administration of CY-1503, an analog of sialyl Lewisx (SLex), on ischemia cell damage. mRNA for E-selectin was first detected in the ischemic hemisphere at 2 h of reperfusion and persisted to 46 h of reperfusion. E-selectin (protein) was localized to microvessels within the ischemic lesion at 0 h of reperfusion and persisted to 70 h of reperfusion. Treatment of the ischemic animals with CY-1503 (50 mg/kg) (n = 8) significantly reduced infarct volume by 42% (p < 0.05) and significantly reduced myeloperoxidase immunoreactive cells in the ischemic lesion by 60% (p < 0.05). These findings provide the first direct evidence for the involvement of E-selectin in transient MCA occlusion in rats and suggest that the E-selectin may facilitate neutrophil adhesion and subsequent cerebral ischemic cell damage.