Lufen Chang

Mammalian MAP kinase signalling cascades

Mitogen-activated protein kinases (MAPKs) are important signal transducing enzymes, unique to eukaryotes, that are involved in many facets of cellular regulation. Initial research concentrated on defining the components and organization of MAPK signalling cascades, but recent studies have begun to shed light on the physiological functions of these cascades in the control of gene expression, cell proliferation and programmed cell death.

A central role for JNK in obesity and insulin resistance

Obesity is closely associated with insulin resistance and establishes the leading risk factor for type 2 diabetes mellitus, yet the molecular mechanisms of this association are poorly understood. The c-Jun amino-terminal kinases (JNKs) can interfere with insulin action in cultured cells and are activated by inflammatory cytokines and free fatty acids, molecules that have been implicated in the development of type 2 diabetes. Here we show that JNK activity is abnormally elevated in obesity. Furthermore, an absence of JNK1 results in decreased adiposity, significantly improved insulin sensitivity and enhanced insulin receptor signalling capacity in two different models of mouse obesity. Thus, JNK is a crucial mediator of obesity and insulin resistance and a potential target for therapeutics.

Reactive Oxygen Species Promote TNFα-Induced Death and Sustained JNK Activation by Inhibiting MAP Kinase Phosphatases

TNFalpha is a pleiotropic cytokine that induces either cell proliferation or cell death. Inhibition of NF-kappaB activation increases susceptibility to TNFalpha-induced death, concurrent with sustained JNK activation, an important contributor to the death response. Sustained JNK activation in NF-kappaB-deficient cells was suggested to depend on reactive oxygen species (ROS), but how ROS affect JNK activation was unclear. We now show that TNFalpha-induced ROS, whose accumulation is suppressed by mitochondrial superoxide dismutase, cause oxidation and inhibition of JNK-inactivating phosphatases by converting their catalytic cysteine to sulfenic acid. This results in sustained JNK activation, which is required for cytochrome c release and caspase 3 cleavage, as well as necrotic cell death. Treatment of cells or experimental animals with an antioxidant prevents H(2)O(2) accumulation, JNK phosphatase oxidation, sustained JNK activity, and both forms of cell death. Antioxidant treatment also prevents TNFalpha-mediated fulminant liver failure without affecting liver regeneration.

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.

JNK2 and IKKβ Are Required for Activating the Innate Response to Viral Infection

Viral infection or double-stranded (ds) RNA induce interferons (IFN) and other cytokines. Transcription factors mediating IFN induction are known, but the signaling pathways that regulate them are less clear. We now describe two such pathways. The first pathway leading to NF-kappaB depends on the dsRNA-responsive protein kinase (PKR), which in turn activates IKB kinase (IKK) through the IKKbeta subunit. The second viral-and dsRNA-responsive pathway is PKR independent and involves Jun kinase (JNK) activation leading to stimulation of AP-1. Both IKKbeta and JNK2 are essential for efficient induction of type I IFN and other cytokines in response to viral infection or dsRNA. This study establishes a general role for these kinases in activation of innate immune responses.

Defective neural tube morphogenesis and altered apoptosis in the absence of both JNK1 and JNK2

Mice lacking both c-Jun-NH(2)-terminal kinases (JNK1 and JNK2) were generated to define their roles in development. Jnk1/jnk2 double mutant fetuses die around embryonic day 11 (E11) and were found to display an open neural tube (exencephaly) at the hindbrain level with reduced apoptosis in the hindbrain neuroepithelium at E9.25. In contrast, a dramatic increase in cell death was observed one day later at E10.5 in both the hindbrain and forebrain regions. Moreover, about 25% of jnk1-/-jnk2+/- fetuses display exencephaly probably due to reduced levels of JNK proteins, whereas jnk1+/-jnk2-/- mice are viable. These results assign both pro- and anti-apoptotic functions for JNK1 and JNK2 in the development of the fetal brain.

JNK1 Is Required for Maintenance of Neuronal Microtubules and Controls Phosphorylation of Microtubule-Associated Proteins

Microtubules (MTs) play an important role in elaboration and maintenance of axonal and dendritic processes. MT dynamics are modulated by MT-associated proteins (MAPs), whose activities are regulated by protein phosphorylation. We found that a member of the c-Jun NH(2)-terminal protein kinase (JNK) subgroup of MAP kinases, JNK1, is involved in regulation of MT dynamics in neuronal cells. Jnk1(-/-) mice exhibit disrupted anterior commissure tract formation and a progressive loss of MTs within axons and dendrites. MAP2 and MAP1B polypeptides are hypophosphorylated in Jnk1(-/-) brains, resulting in compromised ability to bind MTs and promote their assembly. These results suggest that JNK1 is required for maintaining the cytoskeletal integrity of neuronal cells and is a critical regulator of MAP activity and MT assembly.

IKKβ Is Required for Prevention of Apoptosis Mediated by Cell-Bound but Not by Circulating TNFα

Abstract IκB kinase β (IKKβ) is required for NF-κB activation and suppression of TNFα-mediated liver apoptosis. To investigate how IKKβ suppresses apoptosis, we generated hepatocyte-specific Ikkβ knockout mice, Ikkβ Δhep , which exhibit little residual NF- κB activity but are healthy with normal liver function. Unexpectedly, Ikkβ Δhep mice are slightly more sensitive than controls to LPS-induced liver apoptosis but are highly susceptible to liver destruction following concanavalin A (ConA)-induced T cell activation. Unlike LPS, a potent inducer of circulating TNFα, ConA exerts cytotoxic effects through cell-bound TNFα, which activates type 1 and 2 TNF receptors (TNFR). While TNFR2 does not contribute to NF-κB activation, it is important for ConA-induced JNK activation, which is augmented by the absence of IKKβ. Using JNK-deficient mice we show that JNK is required for ConA-induced liver damage. Thus, the antiapoptotic function of IKKβ, which is most critical in situations that involve cell-bound TNFα, is mediated partially through attenuation of JNK activity.

Essential Roles of Receptor-Interacting Protein and TRAF2 in Oxidative Stress-Induced Cell Death

ABSTRACT Oxidative stress and reactive oxygen species (ROS) can elicit and modulate various physiological and pathological processes, including cell death. However, the mechanisms controlling ROS-induced cell death are largely unknown. Data from this study suggest that receptor-interacting protein (RIP) and tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2), two key effector molecules of TNF signaling, are essential for ROS-induced cell death. We found that RIP−/− or TRAF2−/− mouse embryonic fibroblasts (MEF) are resistant to ROS-induced cell death when compared to wild-type cells, and reconstitution of RIP and TRAF2 gene expression in their respective deficient MEF cells restored their sensitivity to H2O2-induced cell death. We also found that RIP and TRAF2 form a complex upon H2O2 exposure, but without the participation of TNFR1. The colocalization of RIP with a membrane lipid raft marker revealed a possible role of lipid rafts in the transduction of cell death signal initiated by H2O2. Finally, our results demonstrate that activation of c-Jun NH2-terminal kinase 1 is a critical event downstream of RIP and TRAF2 in mediating ROS-induced cell death. Therefore, our study uncovers a novel signaling pathway regulating oxidative stress-induced cell death.

c-Jun N-terminal kinase activation is required for the inhibition of neovascularization by thrombospondin-1

Thrombospondin-1 (TSP-1) is a potent inhibitor of angiogenesis that acts directly on endothelial cells via the CD36 surface receptor molecule to halt their migration, proliferation, and morphogenesis in vitro and to block neovascularization in vivo. Here we show that inhibitory signals elicited by TSP-1 did not alter the ability of inducers of angiogenesis to activate p42 and p44 mitogen-activated protein kinase (MAPK). Rather, TSP-1 induced a rapid and transient activation of c-Jun N-terminal kinases (JNK). JNK activation by TSP-1 required engagement of CD36, as it was blocked by antagonistic CD36 antibodies and stimulated by short anti-angiogenic peptides derived from TSP-1 that act exclusively via CD36. TSP-1 inhibition of corneal neovascularization induced by bFGF was severely impaired in mice null for JNK-1, pointing to a critical role for this stress-activated kinase in the inhibition of neovascularization by TSP-1.