Lih-g Lin

Cytosolic phospholipase A2.

To summarize the regulation of cPLA2, we have proposed a model for the activation of cPLA2 based both on our previous studies (Clark et al., 1991; Lin et al., 1993) and the work of many others (Fig. 5). In this model, cPLA2 is tightly regulated by multiple pathways, including those that control Ca2+ concentration, phosphorylation states and cPLA2 protein levels, to exert both rapid and prolonged effects on cellular processes, such as inflammation. cPLA2 is rapidly activated by increased intracellular Ca2+ concentration and phosphorylation by MAP kinase. When cells are stimulated with a ligand for a receptor, such as ATP or PDGF, PLC is activated via either a G protein-dependent or -independent process, leading to the production of diacylglycerol (DAG) and inositol triphosphate (IP3). The rise in these intracellular messengers cause the activation of PKC and mobilization of intracellular Ca2+. Alternatively, the increase in intracellular Ca2+ can result from a Ca2+ influx. Increased Ca2+ acts through the CaLB domain to cause translocation of cPLA2 from the cytosol to the membrane where its substrate, phospholipid, is localized. This step is essential for the activation of cPLA2 and may account for the partial activation of cPLA2 in the absence of phosphorylation. MAP kinase activation can occur through both PKC-dependent and -independent mechanisms (Cobb et al., 1991; Posada and Cooper, 1992; Qiu and Leslie, 1994). In many cases, this pathway is also G protein-dependent. Activated MAP kinase phosphorylates cPLA2 at Ser-505, causing increased enzymatic activity of cPLA2, which is realized only upon translocation of cPLA2 to the membrane. Therefore, full activation of cPLA2 requires both increased cytosolic Ca2+ and cPLA2 phosphorylation at Ser-505. In a more delayed response, cPLA2 activity in the cells can be controlled by changes in its expression levels, such as in response to inflammatory cytokines and certain growth factors. Thus the expression level of cPLA2 is regulated by both transcriptional and post-transcriptional mechanisms.

Distinct cellular functions of MK2.

ABSTRACT Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is activated upon stress by p38 MAPKα and -β, which bind to a basic docking motif in the C terminus of MK2 and which subsequently phosphorylate its regulatory sites. As a result of activation MK2 is exported from the nucleus to the cytoplasm and cotransports active p38 MAPK to this compartment. Here we show that the amount of p38 MAPK is significantly reduced in cells and tissues lacking MK2, indicating a stabilizing effect of MK2 for p38. Using a murine knockout model, we have previously shown that elimination of MK2 leads to a dramatic reduction of tumor necrosis factor (TNF) production in response to lipopolysaccharide. To further elucidate the role of MK2 in p38 MAPK stabilization and in TNF biosynthesis, we analyzed the ability of two MK2 isoforms and several MK2 mutants to restore both p38 MAPK protein levels and TNF biosynthesis in macrophages. We show that MK2 stabilizes p38 MAPK through its C terminus and that MK2 catalytic activity does not contribute to this stabilization. Importantly, we demonstrate that stabilizing p38 MAPK does not restore TNF biosynthesis. TNF biosynthesis is only restored with MK2 catalytic activity. We further show that, in MK2-deficient macrophages, formation of filopodia in response to extracellular stimuli is reduced. In addition, migration of MK2-deficient mouse embryonic fibroblasts (MEFs) and smooth muscle cells on fibronectin is dramatically reduced. Interestingly, reintroducing catalytic MK2 activity into MEFs alone is not sufficient to revert the migratory phenotype of these cells. In addition to catalytic activity, the proline-rich N-terminal region is necessary for rescuing the migratory phenotype. These data indicate that catalytic activity of MK2 is required for both cytokine production and cell migration. However, the proline-rich MK2 N terminus provides a distinct role restricted to cell migration.

The Mitogen-Activated Protein Kinase (MAPK)-Activated Protein Kinases MK2 and MK3 Cooperate in Stimulation of Tumor Necrosis Factor Biosynthesis and Stabilization of p38 MAPK

ABSTRACT MK2 and MK3 represent protein kinases downstream of p38 mitogen-activated protein kinase (MAPK). Deletion of the MK2 gene in mice resulted in an impaired inflammatory response although MK3, which displays extensive structural similarities and identical functional properties in vitro, is still present. Here, we analyze tumor necrosis factor (TNF) production and expression of p38 MAPK and tristetraprolin (TTP) in MK3-deficient mice and demonstrate that there are no significant differences with wild-type animals. We show that in vivo MK2 and MK3 are expressed and activated in parallel. However, the level of activity of MK2 is always significantly higher than that of MK3. Accordingly, we hypothesized that MK3 could have significant effects only in an MK2-free background and generated MK2/MK3 double-knockout mice. Unexpectedly, these mice are viable and show no obvious defects due to loss of compensation between MK2 and MK3. However, there is a further reduction of TNF production and expression of p38 and TTP in double-knockout mice compared to MK2-deficient mice. This finding, together with the observation that ectopically expressed MK3 can rescue MK2 deficiency similarly to MK2, indicates that both kinases share the same physiological function in vivo but are expressed to different levels.

MAPKAP Kinase 2-Deficient Mice Are Resistant to Collagen-Induced Arthritis

TNF-α is a pleiotropic cytokine considered a primary mediator of immune regulation and inflammatory response and has been shown to play a central role in rheumatoid arthritis (RA). MAPKAP kinase 2 (MK2) is a serine/threonine kinase that is regulated through direct phosphorylation by p38 MAPK, and has been shown to be an essential component in the inflammatory response that regulates the biosynthesis of TNF-α at a posttranscriptional level. The murine model of collagen-induced arthritis (CIA) is an established disease model to study pathogenic mechanisms relevant to RA. In this study, we report that deletion of the MK2 gene in DBA/1LacJ mice confers protection against CIA. Interestingly, the MK2 heterozygous mutants display an intermediate level of protection when compared with homozygous mutant and wild-type littermates. We show that MK2−/− and MK2+/− mice exhibit decreased disease incidence and severity in the CIA disease model and reduced TNF-α and IL-6 serum levels following LPS/d-Gal treatment compared with wild-type mice. Additionally, we show that levels of IL-6 mRNA in paws of mice with CIA correlate with the disease status. These findings suggest that an MK2 inhibitor could be of great therapeutic value to treat inflammatory diseases like RA.

Innate Immune Responses to TREM-1 Activation: Overlap, Divergence, and Positive and Negative Cross-Talk with Bacterial Lipopolysaccharide

TREM-1 (triggering receptor expressed on myeloid cells-1) is an orphan immunoreceptor expressed on monocytes, macrophages, and neutrophils. TREM-1 associates with and signals via the adapter protein DAP12/TYROBP, which contains an ITAM. TREM-1 activation by receptor cross-linking has been shown to be proinflammatory and to amplify some cellular responses to TLR ligands such as bacterial LPS. To investigate the cellular consequences of TREM-1 activation, we have characterized global gene expression changes in human monocytes in response to TREM-1 cross-linking in comparison to and combined with LPS. Both TREM-1 activation and LPS up-regulate chemokines, cytokines, matrix metalloproteases, and PTGS/COX2, consistent with a core inflammatory response. However, other immunomodulatory factors are selectively induced, including SPP1 and CSF1 (i.e., M-CSF) by TREM-1 activation and IL-23 and CSF3 (i.e., G-CSF) by LPS. Additionally, cross-talk between TREM-1 activation and LPS occurs on multiple levels. Although synergy in GM-CSF protein production is reflected in commensurate mRNA abundance, comparable synergy in IL-1β protein production is not. TREM-1 activation also attenuates the induction of some LPS target genes, including those that encode IL-12 cytokine family subunits. Where tested, positive TREM-1 outputs are greatly reduced by the PI3K inhibitor wortmannin, whereas this attenuation is largely PI3K independent. These experiments provide a detailed analysis of the cellular consequences of TREM-1 activation and highlight the complexity in signal integration between ITAM- and TLR-mediated signaling.

Engagement of tumor necrosis factor (TNF) receptor 1 leads to ATF-2- and p38 mitogen-activated protein kinase-dependent TNF-alpha gene expression.

Engagement of the tumor necrosis factor-α (TNF-α) receptors by the TNF-α ligand results in the rapid induction of TNF-α gene expression. The study presented here shows that autoregulation of TNF-α gene transcription by selective signaling through tumor necrosis factor receptor 1 (TNFR1) requires p38 mitogen-activated protein (MAP) kinase activity and the binding of the transcription factors ATF-2 and Jun to the TNF-α cAMP-response element (CRE) promoter element. Consistent with these findings, TNFR1 engagement results in increased p38 MAP kinase activity and p38-dependent phosphorylation of ATF-2. Furthermore, overexpression of MADD (MAP kinase-activatingdeath domain protein), an adapter protein that binds to the death domain of TNFR1 and activates MAP kinase cascades, results in CRE-dependent induction of TNF-α gene expression. Thus, the TNF-α CRE site is the target of TNFR1 stimulation and mediates the autoregulation of TNF-α gene transcription.

Catalytically active MAP KAP kinase 2 structures in complex with staurosporine and ADP reveal differences with the autoinhibited enzyme

MAP KAP kinase 2 (MK2), a Ser/Thr kinase, plays a crucial role in the inflammatory process. We have determined the crystal structures of a catalytically active C-terminal deletion form of human MK2, residues 41-364, in complex with staurosporine at 2.7 A and with ADP at 3.2 A, revealing overall structural similarity with other Ser/Thr kinases. Kinetic analysis reveals that the K(m) for ATP is very similar for MK2 41-364 and p38-activated MK2 41-400. Conversely, the catalytic rate and binding for peptide substrate are dramatically reduced in MK2 41-364. However, phosphorylation of MK2 41-364 by p38 restores the V(max) and K(m) for peptide substrate to values comparable to those seen in p38-activated MK2 41-400, suggesting a mechanism for regulation of enzyme activity.

Pharmacologic Inhibition of Tpl2 Blocks Inflammatory Responses in Primary Human Monocytes, Synoviocytes, and Blood

Tumor necrosis factor α (TNFα) is a pro-inflammatory cytokine that controls the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Tpl2 is a MAPKKK in the MAPK (i.e. ERK) pathway, and the Tpl2-MEK-ERK signaling pathway is activated by the pro-inflammatory mediators TNFα, interleukin (IL)-1β, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFα expression. Thus, pharmacologic inhibition of Tpl2 should be a valid approach to therapeutic intervention in the pathogenesis of rheumatoid arthritis and other inflammatory diseases in humans. We have developed a series of highly selective and potent Tpl2 inhibitors, and in the present study we have used these inhibitors to demonstrate that the catalytic activity of Tpl2 is required for the LPS-induced activation of MEK and ERK in primary human monocytes. These inhibitors selectively target Tpl2 in these cells, and they block LPS- and IL-1β-induced TNFα production in both primary human monocytes and human blood. In rheumatoid arthritis fibroblast-like synoviocytes these inhibitors block ERK activation, cyclooxygenase-2 expression, and the production of IL-6, IL-8, and prostaglandin E2, and the matrix metalloproteinases MMP-1 and MMP-3. Taken together, our results show that inhibition of Tpl2 in primary human cell types can decrease the production of TNFα and other pro-inflammatory mediators during inflammatory events, and they further support the notion that Tpl2 is an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases.

Selective inhibitors of tumor progression loci-2 (Tpl2) kinase with potent inhibition of TNF-α production in human whole blood

Tpl2 (cot/MAP3K8) is an upstream kinase of MEK in the ERK pathway. It plays an important role in Tumor Necrosis Factor-alpha (TNF-alpha) production and signaling. We have discovered that 8-halo-4-(3-chloro-4-fluoro-phenylamino)-6-[(1H-[1,2,3]triazol-4-ylmethyl)-amino]-quinoline-3-carbonitriles (4) are potent inhibitors of this enzyme. In order to improve the inhibition of TNF-alpha production in LPS-stimulated human blood, a series of analogs with a variety of substitutions around the triazole moiety were studied. We found that a cyclic amine group appended to the triazole ring could considerably enhance potency, aqueous solubility, and cell membrane permeability. Optimization of these cyclic amine groups led to the identification of 8-chloro-4-(3-chloro-4-fluorophenylamino)-6-((1-(1-ethylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methylamino)quinoline-3-carbonitrile (34). In a LPS-stimulated rat inflammation model, compound 34 showed good efficacy in inhibiting TNF-alpha production.

Proteomic characterization of the dynamic KSR-2 interactome, a signaling scaffold complex in MAPK pathway

KSR-1 is a scaffold protein that is essential for Ras-induced activation of the highly conserved RAF-MEK-ERK kinase module. Previously, we identified a close homolog of KSR-1, called KSR-2, through structural homology-based data mining. In order to further understand the role of KSR-2 in MAPK signaling, we undertook a functional proteomics approach to elucidate the dynamic composition of the KSR-2 functional complex in HEK-293 cells under conditions with and without TNF-alpha stimulation. We found nearly 100 proteins that were potentially associated with KSR-2 complex and 43 proteins that were likely recruited to the super molecular complex after TNF-alpha treatment. Our results indicate that KSR-2 may act as a scaffold protein similar as KSR-1 to mediate the MAPK core (RAF-MEK-ERK) signaling but with a distinct RAF isoform specificity, namely KSR-2 may only mediate the A-RAF signaling while KSR-1 is responsible for transducing signals only from c-RAF. In addition, KSR-2 may be involved in the activation of many MAPK downstream signaling molecules such as p38 MAPK, IKAP, AIF, and proteins involved in ubiquitin-proteasome, apoptosis, cell cycle control, and DNA synthesis and repair pathways, as well as mediating crosstalks between MAPK and several other signaling pathways, including PI3K and insulin signaling. While interactions with these molecules are not known for KSR-1, its reasonable to hypothesize that KSR-1 may also play a similar role in mediating these downstream signaling pathways.