J. Telliez - Academia.edu (original) (raw)

Papers by J. Telliez

Research paper thumbnail of Jak3 deficiency blocks innate lymphoid cell development

Mucosal Immunology, 2018

Loss-of-function mutations in the tyrosine kinase JAK3 cause autosomal recessive severe combined ... more Loss-of-function mutations in the tyrosine kinase JAK3 cause autosomal recessive severe combined immunodeficiency (SCID). Defects in this form of SCID are restricted to the immune system, which led to the development of immunosuppressive JAK inhibitors. We find that the B6.Cg-Nr1d1 tm1Ven /LazJ mouse line purchased from Jackson Laboratories harbors a spontaneous mutation in Jak3, generating a SCID phenotype and an inability to generate antigen-independent professional cytokine-producing innate lymphoid cells (ILCs). Mechanistically, Jak3 deficiency blocks ILC differentiation in the bone marrow at the ILC precursor and the pre-NK cell progenitor. We further demonstrate that the pan-JAK inhibitor tofacitinib and the specific JAK3 inhibitor PF-06651600 impair the ability of human intraepithelial ILC1 (iILC1) to produce IFN-c, without affecting ILC3 production of IL-22. Both inhibitors impaired the proliferation of iILC1 and ILC3 and differentiation of human ILC in vitro. Tofacitinib is currently approved for the treatment of moderate-to-severely active rheumatoid arthritis. Both tofacitinib and PF-06651600 are currently in clinical trials for several other immune-mediated conditions. Our data suggest that therapeutic inhibition of JAK may also impact ILCs and, to some extent, underlie clinical efficacy.

Research paper thumbnail of The NMR Solution Structure of the RIP Death Domain and Characterization of the Interaction with TRADD

Research paper thumbnail of These include

cooperatively to facilitate activation by calcium. domains of the exchange factor Ras-GRF act The... more cooperatively to facilitate activation by calcium. domains of the exchange factor Ras-GRF act The N-terminal pleckstrin, coiled-coil, and IQ

Research paper thumbnail of Jak3 deficiency blocks innate lymphoid cell development

Mucosal immunology, Jan 17, 2017

Loss-of-function mutations in the tyrosine kinase JAK3 cause autosomal recessive severe combined ... more Loss-of-function mutations in the tyrosine kinase JAK3 cause autosomal recessive severe combined immunodeficiency (SCID). Defects in this form of SCID are restricted to the immune system, which led to the development of immunosuppressive JAK inhibitors. We find that the B6.Cg-Nr1d1(tm1Ven)/LazJ mouse line purchased from Jackson Laboratories harbors a spontaneous mutation in Jak3, generating a SCID phenotype and an inability to generate antigen-independent professional cytokine-producing innate lymphoid cells (ILCs). Mechanistically, Jak3 deficiency blocks ILC differentiation in the bone marrow at the ILC precursor and the pre-NK cell progenitor. We further demonstrate that the pan-JAK inhibitor tofacitinib and the specific JAK3 inhibitor PF-06651600 impair the ability of human intraepithelial ILC1 (iILC1) to produce IFN-γ, without affecting ILC3 production of IL-22. Both inhibitors impaired the proliferation of iILC1 and ILC3 and differentiation of human ILC in vitro. Tofacitinib is...

Research paper thumbnail of OP0155 Development of A JAK3 Specific Inhibitor Clinical Candidate: Functional Differentiation of JAK3 Selective Inhibition over PAN-JAK or JAK1 Selective Inhibition

Annals of the Rheumatic Diseases, 2016

The support of the Danish arthritis association "Gigtforeningen" has made it possible to fund our... more The support of the Danish arthritis association "Gigtforeningen" has made it possible to fund our youth projects without having to go fundraising ourselves, which we are very grateful for.

Research paper thumbnail of Serum, AP-1 and Ets-1 stimulate the human ets-1 promoter

Nucleic Acids Research, 1992

The ets-1 proto-oncogene codes for a transcription factor. In order to understand how ets-1 Is re... more The ets-1 proto-oncogene codes for a transcription factor. In order to understand how ets-1 Is regulated, we have cloned its promoter. We show that the promoter Is inducible by serum and expression of c-Fos and c-Jun, and it is positively auto-regulated by its gene product. A 50 base-pair sequence is sufficient to confer c-Fos + c-Jun and c-Ets-1 responsiveness to a heterologous promoter. This element contains two AP1 and one Ets-1 like motifs. Striking, AP-1 and Ets-1 motifs are found in oncogene responsive units (ORU's) of other promoters, suggesting that combining these motifs is a common mechanism for generating mttogen responsive transcription elements.

Research paper thumbnail of Structural analysis of the mouse c-HA-ras gene promoter

Molecular Carcinogenesis, 1991

Previous studies have demonstrated that the mouse c-Harvey ras proto-oncogene (c-Ha-ras) promoter... more Previous studies have demonstrated that the mouse c-Harvey ras proto-oncogene (c-Ha-ras) promoter sequences are GC rich and contain several potential transcription factor SP1 binding sites. We investigated the endonuclease hypersensitivity of this region in nuclei in vitro and whole mouse tissues in vivo and identified a very strong, ubiquitous hypersensitive site covering the proximal promoter sequences. Footprint protection studies using nuclear extracts from various cell types including fibroblasts, erythroid cells, and both normal and transformed epithelial cells revealed a consistent protein-binding pattern. Five protein binding sites were observed, four of which correlated with potential SP1 binding sites. Competition experiments using an oligonucleotide corresponding to a consensus SP1 binding site confirmed that these sequences were indeed bound by the SP1 (or SP1-like) trans-acting factor. In addition, no differences were observed between the footprint patterns obtained using extracts from cells of different lineages or between normal and transformed epithelial cells carrying activated ras genes. The controlling elements responsible for differential c-Ha-ras transcription between cell types or at different stages of carcinogenesis therefore probably lie in other regions of the gene.

Research paper thumbnail of Mutational analysis and NMR studies of the death domain of the tumor necrosis factor receptor-1

Journal of Molecular Biology, 2000

Tumor necrosis factor receptor-1 (TNFR-1) death domain (DD) is the intracellular functional domai... more Tumor necrosis factor receptor-1 (TNFR-1) death domain (DD) is the intracellular functional domain responsible for the receptor signaling activities. To understand the transduction mechanism of TNFR-1 signaling we performed structural and functional analysis of the TNFR-DD. The secondary structure of the TNFR-DD shows that it consists of six anti-parallel a-helices. The determination of the topological fold and an extensive mutagenesis analysis revealed that there are two opposite faces that are involved in self-association and interaction with the TRADD death domain. Interestingly, the same critical residues in TNFR-DD are involved in both interactions. There is a good correlation between the binding activities of the mutant proteins and their cytotoxic activities. These results provide important insight into the molecular interactions mediating TNFR-DD self-association and subsequent recruitment of TRADD in the signaling activity of TNFR-1.

Research paper thumbnail of The N-terminal pleckstrin, coiled-coil, and IQ domains of the exchange factor Ras-GRF act cooperatively to facilitate activation by calcium

Molecular and Cellular Biology, 1996

We have recently shown that the neuronal exchange factor p140 Ras-GRF becomes activated in vivo i... more We have recently shown that the neuronal exchange factor p140 Ras-GRF becomes activated in vivo in response to elevated calcium levels [C. L. Farnsworth, N. W. Freshney, L. B. Rosen, A. Ghosh, M. E. Greenberg, and L. A. Feig, Nature (London) 376:524-527, 1995]. Activation is mediated by calcium-induced calmodulin binding to an IQ domain near the N terminus of Ras-GRF. Here we show that the adjacent N-terminal pleckstrin homology (PH), coiled-coil, and IQ domains function cooperatively to allow Ras-GRF activation. Deletion of the N-terminal PH domain redistributes a large percentage of Ras-GRF from the particulate to the cytosolic fraction of cells and renders the protein insensitive to calcium stimulation. A similar cellular distribution and biological activity are observed when only the core catalytic domain is expressed. Although the PH domain is necessary for particulate association of Ras-GRF, it is not sufficient for targeting the core catalytic domain to this cellular location...

Research paper thumbnail of OP0147 Lack of Differentiation of Janus Kinase Inhibitors in Rheumatoid Arthritis Based on Janus Kinase Pharmacology and Clinically Meaningful Concentrations

Annals of the Rheumatic Diseases, 2014

Research paper thumbnail of Regulatory elements in the first intron of the mouse ha-ras gene

Molecular Carcinogenesis, 1995

The Ha-ras gene is one of the three oncogenes (Ha-ras, Ki-ras, and N-ras) of the ras superfamily ... more The Ha-ras gene is one of the three oncogenes (Ha-ras, Ki-ras, and N-ras) of the ras superfamily of small G proteins. The p21ras proteins encoded by the ras genes are key proteins involved in the transduction of signals from membrane receptor-tyrosine kinases to downstream targets. The ras genes seem to play a ubiquitous role in the control of cell proliferation and cell differentiation. At the same time, ras genes may perform specific differentiated functions in certain cell types. Little is known about the regulation of expression of the Ha-ras gene. The first intron of the Ha-ras gene has been reported to be highly conserved between human and rodent. We investigated the role that this intron may play in the regulation of expression of Ha-ras. The promoter region of the Ha-ras gene exhibits characteristics of a housekeeping gene. Deletion analysis shows the existence of an enhancer-type element in the 5' region of the first intron (intron 0). DNase 1 footprinting experiments reveal five sites that interact with nuclear proteins from fibroblast and epithelial cell lines. Deletion and site-directed mutagenesis of three of these sites show that two are involved in a positive effect and one in a negative effect on the regulation of expression of the mouse Ha-ras gene.

Research paper thumbnail of Solution Structure of the N-Terminal Domain of the TNFR1 Associated Protein, Tradd

Research paper thumbnail of Hippocampus-dependent learning and memory is impaired in mice lacking the Ras-guanine-nucleotide releasing factor 1 (Ras-GRF1)

Neuropharmacology, 2001

Previous results have suggested that the Ras signaling pathway is involved in learning and memory... more Previous results have suggested that the Ras signaling pathway is involved in learning and memory. Ras is activated by nucleotide exchange factors, such as the calmodulin-activated guanine-nucleotide releasing factor 1 (Ras-GRF1). To test whether Ras-GRF1 is required for learning and memory, we inactivated the Ras-GRF1 gene in mice. These mutants performed normally in a rota-rod motor coordination task, and in two amygdala-dependent tasks (inhibitory avoidance and contextual conditioning). In contrast the mutants were impaired in three hippocampus-dependent learning tasks: contextual discrimination, the social transmission of food preferences, and the hidden-platform version of the Morris water maze. These studies indicate that Ras-GRF1 plays a role in hippocampal-dependent learning and memory. 

Research paper thumbnail of Stress induced gene expression: a direct role for MAPKAP kinases in transcriptional activation of immediate early genes

Nucleic Acids Research, 2011

Immediate early gene (IEG) expression is coordinated by multiple MAP kinase signaling pathways in... more Immediate early gene (IEG) expression is coordinated by multiple MAP kinase signaling pathways in a signal specific manner. Stress-activated p38a MAP kinase is implicated in transcriptional regulation of IEGs via MSK-mediated CREB phosphorylation. The protein kinases downstream to p38, MAPKAP kinase (MK) 2 and MK3 have been identified to regulate gene expression at the posttranscriptional levels of mRNA stability and translation. Here, we analyzed stress-induced IEG expression in MK2/3-deficient cells. Ablation of MKs causes a decrease of p38a level and p38-dependent IEG expression. Unexpectedly, restoration of p38a does not rescue the full-range IEG response. Instead, the catalytic activity of MKs is necessary for the major transcriptional activation of IEGs. By transcriptomics, we identified MK2-regulated genes and recognized the serum response element (SRE) as a common promoter element. We show that stress-induced phosphorylation of serum response factor (SRF) at serine residue 103 is significantly reduced and that induc

Research paper thumbnail of Solution Structure of N-TRADD and Characterization of the Interaction of N-TRADD and C-TRAF2, a Key Step in the TNFR1 Signaling Pathway

Molecular Cell, 2000

recruited to the TNFR1 in a TNF-dependent manner. TRADD contains two functionally separate domain... more recruited to the TNFR1 in a TNF-dependent manner. TRADD contains two functionally separate domains, which allow the protein to couple to at least two distinct signaling pathways (Hsu et al., 1996). The C-terminal region of the protein (aa 196-301) contains a death do-Dé siré e H. main that mediates the interaction between TRADD and the death domains of TNFR1, FADD, and RIP (Boldin et Genetics Institute Wyeth Research al., 1995; Chinnaiyan et al., 1995; Stanger et al., 1995). The recruitment of FADD initiates the activation of the 87 Cambridge Park Drive Cambridge, Massachusetts 02140 caspase cascade, which eventually leads to apoptosis. The N-terminal region of TRADD (N-TRADD) spanning from residues 1-169 appears to be a novel domain since a BLAST (Altschul et al., 1997) search did not identify Summary any sequence homology to known proteins. N-TRADD is responsible for the binding of TRAF2, a TNFR-associ-TRADD is a multifunctional signaling adaptor protein ated factor (Hsu et al., 1996). This interaction is mediated that is recruited to TNFR1 upon ligand binding. The through the C-terminal region of TRAF2 (aa 348-501), C-terminal of TRADD comprises the "death domain" termed C-TRAF2. The interaction of N-TRADD with that is responsible for association of TNFR1 and other C-TRAF2 initiates TRAF2-mediated signaling processes death domain-containing proteins such as FADD and central to the cellular inflammatory response, such as RIP. The N-terminal domain (N-TRADD) promotes the JNK and NF-B activation (Rothe et al., 1995; Cao et recruitment of TRAF2 to TNFR1 by binding to the al., 1996; Reinhard et al., 1997; Song et al., 1997). This C-terminal of TRAF2, leading to the activation of JNK/ crucial role of N-TRADD in TNF signaling is supported AP1 and NF-B. The solution structure of N-TRADD by the observation that the expression of N-TRADD (aa was determined, revealing a novel protein fold. A com-1-194) can inhibit TNF-mediated NF-B and JNK activabination of NMR, BIAcore, and mutagenesis experition in a dominant-negative manner (Kieser et al., 1999). ments was used to help identify the site of interaction In order to understand how N-TRADD may interact of N-TRADD with C-TRAF2, providing a framework for with the adaptor protein TRAF2, we have determined future attempts to selectively inhibit the TNF signaling the three-dimensional structure of N-TRADD (1-169) by pathways. NMR spectroscopy. The solution structure of N-TRADD consists of five ␣ helices and four ␤ strands, arranged Introduction in a unique fashion. Using the structure, together with site-directed mutagenesis, we have identified a region Tumor necrosis factor (TNF) is a proinflammatory cytoof N-TRADD that interacts with C-TRAF2. This informakine that is involved in a variety of biological activities, tion, in addition to the recently published structures of through its binding to two distinct cell surface receptors, C-TRAF2 (McWhirter et al., 1999; Park et al., 1999), TNFR1 and TNFR2 (Rothe et al., 1992; Tartaglia and allows us to gain an insight into the interaction of Goeddel, 1992; Baker and Reddy, 1998). Both TNF re-N-TRADD and C-TRAF2. ceptors are part of the larger TNF receptor superfamily (Smith et al., 1994; Grell, 1995), which includes CD27, CD30, CD40, and Fas antigen, among others. These Results and Discussion receptors share no obvious sequence similarities in the cytoplasmic domain, with the exception of TNFR1 and Structure Determination The structure of N-TRADD was determined from 2402 Fas, which each have an ‫08ف‬ amino acid "death domain" (DD) at the C-terminal with ‫%82ف‬ sequence iden-NMR-derived restraints obtained using uniformly 15 Nand 15 N/ 13 C-labeled protein, with double and triple reso-tity. These death domains can induce apoptosis by mediating self-association of both TNFR1 and Fas upon nance NMR experiments. N-TRADD was soluble to ‫1ف‬ mM, but high concentrations of dithiothreitol (20 mM) ligand binding to each receptor, a critical event to trigger downstream signaling pathways by recruiting and acti-were required, to prevent aggregation of the protein. Under these conditions, the sample was stable for 6-8 vating receptor-associated effector molecules (Tartaglia and Goeddel, 1992; Boldin et al., 1995). Recently, many weeks. The structural statistics and root-mean-square deviations are in Table 1, and the superposition of the of these downstream signaling proteins were identified and shown to contain a DD, which mediates the interac-ensemble of 25 structures is shown in Figure 1B. The atomic root-mean-square deviation about the mean co-tion with the receptor through a DD-DD interaction. TRADD, one of the earlier TNFR1 adaptor proteins ordinate for residues 14-161 is 0.56 Å for the backbone atoms and 1.01 Å for all atoms. For secondary structure identified (Hsu et al., 1995), is a 34 kDa protein that is elements only, the rmsd is 0.46 Å for backbone atoms and 0.92 Å for all atoms. The N-terminal residues 1-10 ‡ To whom correspondence should be addressed (e-mail: dtsao@ and C-terminal residues 162-169 are disordered. The genetics.com [D. H. H. T.], llin@genetics.com [L. L. L]). § These authors contributed equally to this work.

Research paper thumbnail of Distinct Cellular Functions of MK2

Molecular and Cellular Biology, 2002

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is activated upon stress... more Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is activated upon stress by p38 MAPK␣ and -␤, 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.

Research paper thumbnail of 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

Molecular and Cellular Biology, 2007

Deletion of the MK2 gene in mice resulted in an impaired inflammatory response although MK3, whic... more 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.

Research paper thumbnail of MAPKAP Kinase 2-Deficient Mice Are Resistant to Collagen-Induced Arthritis

The Journal of Immunology, 2006

TNF-␣ is a pleiotropic cytokine considered a primary mediator of immune regulation and inflammato... more TNF-␣ 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-␣ 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-␣ 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.

Research paper thumbnail of Engagement of Tumor Necrosis Factor (TNF) Receptor 1 Leads to ATF-2- and p38 Mitogen-activated Protein Kinase-dependent TNF-alpha Gene Expression

Journal of Biological Chemistry, 1999

Engagement of the tumor necrosis factor-alpha (TNF-alpha) receptors by the TNF-alpha ligand resul... more Engagement of the tumor necrosis factor-alpha (TNF-alpha) receptors by the TNF-alpha ligand results in the rapid induction of TNF-alpha gene expression. The study presented here shows that autoregulation of TNF-alpha 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-alpha 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-activating death 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-alpha gene expression. Thus, the TNF-alpha CRE site is the target of TNFR1 stimulation and mediates the autoregulation of TNF-alpha gene transcription.

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

Journal of Biological Chemistry, 2007

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine that controls the initiatio... more Tumor necrosis factor alpha (TNFalpha) 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 TNFalpha, interleukin (IL)-1beta, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFalpha 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-1beta-induced TNFalpha 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 E(2), 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 TNFalpha 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.

Research paper thumbnail of Jak3 deficiency blocks innate lymphoid cell development

Mucosal Immunology, 2018

Loss-of-function mutations in the tyrosine kinase JAK3 cause autosomal recessive severe combined ... more Loss-of-function mutations in the tyrosine kinase JAK3 cause autosomal recessive severe combined immunodeficiency (SCID). Defects in this form of SCID are restricted to the immune system, which led to the development of immunosuppressive JAK inhibitors. We find that the B6.Cg-Nr1d1 tm1Ven /LazJ mouse line purchased from Jackson Laboratories harbors a spontaneous mutation in Jak3, generating a SCID phenotype and an inability to generate antigen-independent professional cytokine-producing innate lymphoid cells (ILCs). Mechanistically, Jak3 deficiency blocks ILC differentiation in the bone marrow at the ILC precursor and the pre-NK cell progenitor. We further demonstrate that the pan-JAK inhibitor tofacitinib and the specific JAK3 inhibitor PF-06651600 impair the ability of human intraepithelial ILC1 (iILC1) to produce IFN-c, without affecting ILC3 production of IL-22. Both inhibitors impaired the proliferation of iILC1 and ILC3 and differentiation of human ILC in vitro. Tofacitinib is currently approved for the treatment of moderate-to-severely active rheumatoid arthritis. Both tofacitinib and PF-06651600 are currently in clinical trials for several other immune-mediated conditions. Our data suggest that therapeutic inhibition of JAK may also impact ILCs and, to some extent, underlie clinical efficacy.

Research paper thumbnail of The NMR Solution Structure of the RIP Death Domain and Characterization of the Interaction with TRADD

Research paper thumbnail of These include

cooperatively to facilitate activation by calcium. domains of the exchange factor Ras-GRF act The... more cooperatively to facilitate activation by calcium. domains of the exchange factor Ras-GRF act The N-terminal pleckstrin, coiled-coil, and IQ

Research paper thumbnail of Jak3 deficiency blocks innate lymphoid cell development

Mucosal immunology, Jan 17, 2017

Loss-of-function mutations in the tyrosine kinase JAK3 cause autosomal recessive severe combined ... more Loss-of-function mutations in the tyrosine kinase JAK3 cause autosomal recessive severe combined immunodeficiency (SCID). Defects in this form of SCID are restricted to the immune system, which led to the development of immunosuppressive JAK inhibitors. We find that the B6.Cg-Nr1d1(tm1Ven)/LazJ mouse line purchased from Jackson Laboratories harbors a spontaneous mutation in Jak3, generating a SCID phenotype and an inability to generate antigen-independent professional cytokine-producing innate lymphoid cells (ILCs). Mechanistically, Jak3 deficiency blocks ILC differentiation in the bone marrow at the ILC precursor and the pre-NK cell progenitor. We further demonstrate that the pan-JAK inhibitor tofacitinib and the specific JAK3 inhibitor PF-06651600 impair the ability of human intraepithelial ILC1 (iILC1) to produce IFN-γ, without affecting ILC3 production of IL-22. Both inhibitors impaired the proliferation of iILC1 and ILC3 and differentiation of human ILC in vitro. Tofacitinib is...

Research paper thumbnail of OP0155 Development of A JAK3 Specific Inhibitor Clinical Candidate: Functional Differentiation of JAK3 Selective Inhibition over PAN-JAK or JAK1 Selective Inhibition

Annals of the Rheumatic Diseases, 2016

The support of the Danish arthritis association "Gigtforeningen" has made it possible to fund our... more The support of the Danish arthritis association "Gigtforeningen" has made it possible to fund our youth projects without having to go fundraising ourselves, which we are very grateful for.

Research paper thumbnail of Serum, AP-1 and Ets-1 stimulate the human ets-1 promoter

Nucleic Acids Research, 1992

The ets-1 proto-oncogene codes for a transcription factor. In order to understand how ets-1 Is re... more The ets-1 proto-oncogene codes for a transcription factor. In order to understand how ets-1 Is regulated, we have cloned its promoter. We show that the promoter Is inducible by serum and expression of c-Fos and c-Jun, and it is positively auto-regulated by its gene product. A 50 base-pair sequence is sufficient to confer c-Fos + c-Jun and c-Ets-1 responsiveness to a heterologous promoter. This element contains two AP1 and one Ets-1 like motifs. Striking, AP-1 and Ets-1 motifs are found in oncogene responsive units (ORU's) of other promoters, suggesting that combining these motifs is a common mechanism for generating mttogen responsive transcription elements.

Research paper thumbnail of Structural analysis of the mouse c-HA-ras gene promoter

Molecular Carcinogenesis, 1991

Previous studies have demonstrated that the mouse c-Harvey ras proto-oncogene (c-Ha-ras) promoter... more Previous studies have demonstrated that the mouse c-Harvey ras proto-oncogene (c-Ha-ras) promoter sequences are GC rich and contain several potential transcription factor SP1 binding sites. We investigated the endonuclease hypersensitivity of this region in nuclei in vitro and whole mouse tissues in vivo and identified a very strong, ubiquitous hypersensitive site covering the proximal promoter sequences. Footprint protection studies using nuclear extracts from various cell types including fibroblasts, erythroid cells, and both normal and transformed epithelial cells revealed a consistent protein-binding pattern. Five protein binding sites were observed, four of which correlated with potential SP1 binding sites. Competition experiments using an oligonucleotide corresponding to a consensus SP1 binding site confirmed that these sequences were indeed bound by the SP1 (or SP1-like) trans-acting factor. In addition, no differences were observed between the footprint patterns obtained using extracts from cells of different lineages or between normal and transformed epithelial cells carrying activated ras genes. The controlling elements responsible for differential c-Ha-ras transcription between cell types or at different stages of carcinogenesis therefore probably lie in other regions of the gene.

Research paper thumbnail of Mutational analysis and NMR studies of the death domain of the tumor necrosis factor receptor-1

Journal of Molecular Biology, 2000

Tumor necrosis factor receptor-1 (TNFR-1) death domain (DD) is the intracellular functional domai... more Tumor necrosis factor receptor-1 (TNFR-1) death domain (DD) is the intracellular functional domain responsible for the receptor signaling activities. To understand the transduction mechanism of TNFR-1 signaling we performed structural and functional analysis of the TNFR-DD. The secondary structure of the TNFR-DD shows that it consists of six anti-parallel a-helices. The determination of the topological fold and an extensive mutagenesis analysis revealed that there are two opposite faces that are involved in self-association and interaction with the TRADD death domain. Interestingly, the same critical residues in TNFR-DD are involved in both interactions. There is a good correlation between the binding activities of the mutant proteins and their cytotoxic activities. These results provide important insight into the molecular interactions mediating TNFR-DD self-association and subsequent recruitment of TRADD in the signaling activity of TNFR-1.

Research paper thumbnail of The N-terminal pleckstrin, coiled-coil, and IQ domains of the exchange factor Ras-GRF act cooperatively to facilitate activation by calcium

Molecular and Cellular Biology, 1996

We have recently shown that the neuronal exchange factor p140 Ras-GRF becomes activated in vivo i... more We have recently shown that the neuronal exchange factor p140 Ras-GRF becomes activated in vivo in response to elevated calcium levels [C. L. Farnsworth, N. W. Freshney, L. B. Rosen, A. Ghosh, M. E. Greenberg, and L. A. Feig, Nature (London) 376:524-527, 1995]. Activation is mediated by calcium-induced calmodulin binding to an IQ domain near the N terminus of Ras-GRF. Here we show that the adjacent N-terminal pleckstrin homology (PH), coiled-coil, and IQ domains function cooperatively to allow Ras-GRF activation. Deletion of the N-terminal PH domain redistributes a large percentage of Ras-GRF from the particulate to the cytosolic fraction of cells and renders the protein insensitive to calcium stimulation. A similar cellular distribution and biological activity are observed when only the core catalytic domain is expressed. Although the PH domain is necessary for particulate association of Ras-GRF, it is not sufficient for targeting the core catalytic domain to this cellular location...

Research paper thumbnail of OP0147 Lack of Differentiation of Janus Kinase Inhibitors in Rheumatoid Arthritis Based on Janus Kinase Pharmacology and Clinically Meaningful Concentrations

Annals of the Rheumatic Diseases, 2014

Research paper thumbnail of Regulatory elements in the first intron of the mouse ha-ras gene

Molecular Carcinogenesis, 1995

The Ha-ras gene is one of the three oncogenes (Ha-ras, Ki-ras, and N-ras) of the ras superfamily ... more The Ha-ras gene is one of the three oncogenes (Ha-ras, Ki-ras, and N-ras) of the ras superfamily of small G proteins. The p21ras proteins encoded by the ras genes are key proteins involved in the transduction of signals from membrane receptor-tyrosine kinases to downstream targets. The ras genes seem to play a ubiquitous role in the control of cell proliferation and cell differentiation. At the same time, ras genes may perform specific differentiated functions in certain cell types. Little is known about the regulation of expression of the Ha-ras gene. The first intron of the Ha-ras gene has been reported to be highly conserved between human and rodent. We investigated the role that this intron may play in the regulation of expression of Ha-ras. The promoter region of the Ha-ras gene exhibits characteristics of a housekeeping gene. Deletion analysis shows the existence of an enhancer-type element in the 5' region of the first intron (intron 0). DNase 1 footprinting experiments reveal five sites that interact with nuclear proteins from fibroblast and epithelial cell lines. Deletion and site-directed mutagenesis of three of these sites show that two are involved in a positive effect and one in a negative effect on the regulation of expression of the mouse Ha-ras gene.

Research paper thumbnail of Solution Structure of the N-Terminal Domain of the TNFR1 Associated Protein, Tradd

Research paper thumbnail of Hippocampus-dependent learning and memory is impaired in mice lacking the Ras-guanine-nucleotide releasing factor 1 (Ras-GRF1)

Neuropharmacology, 2001

Previous results have suggested that the Ras signaling pathway is involved in learning and memory... more Previous results have suggested that the Ras signaling pathway is involved in learning and memory. Ras is activated by nucleotide exchange factors, such as the calmodulin-activated guanine-nucleotide releasing factor 1 (Ras-GRF1). To test whether Ras-GRF1 is required for learning and memory, we inactivated the Ras-GRF1 gene in mice. These mutants performed normally in a rota-rod motor coordination task, and in two amygdala-dependent tasks (inhibitory avoidance and contextual conditioning). In contrast the mutants were impaired in three hippocampus-dependent learning tasks: contextual discrimination, the social transmission of food preferences, and the hidden-platform version of the Morris water maze. These studies indicate that Ras-GRF1 plays a role in hippocampal-dependent learning and memory. 

Research paper thumbnail of Stress induced gene expression: a direct role for MAPKAP kinases in transcriptional activation of immediate early genes

Nucleic Acids Research, 2011

Immediate early gene (IEG) expression is coordinated by multiple MAP kinase signaling pathways in... more Immediate early gene (IEG) expression is coordinated by multiple MAP kinase signaling pathways in a signal specific manner. Stress-activated p38a MAP kinase is implicated in transcriptional regulation of IEGs via MSK-mediated CREB phosphorylation. The protein kinases downstream to p38, MAPKAP kinase (MK) 2 and MK3 have been identified to regulate gene expression at the posttranscriptional levels of mRNA stability and translation. Here, we analyzed stress-induced IEG expression in MK2/3-deficient cells. Ablation of MKs causes a decrease of p38a level and p38-dependent IEG expression. Unexpectedly, restoration of p38a does not rescue the full-range IEG response. Instead, the catalytic activity of MKs is necessary for the major transcriptional activation of IEGs. By transcriptomics, we identified MK2-regulated genes and recognized the serum response element (SRE) as a common promoter element. We show that stress-induced phosphorylation of serum response factor (SRF) at serine residue 103 is significantly reduced and that induc

Research paper thumbnail of Solution Structure of N-TRADD and Characterization of the Interaction of N-TRADD and C-TRAF2, a Key Step in the TNFR1 Signaling Pathway

Molecular Cell, 2000

recruited to the TNFR1 in a TNF-dependent manner. TRADD contains two functionally separate domain... more recruited to the TNFR1 in a TNF-dependent manner. TRADD contains two functionally separate domains, which allow the protein to couple to at least two distinct signaling pathways (Hsu et al., 1996). The C-terminal region of the protein (aa 196-301) contains a death do-Dé siré e H. main that mediates the interaction between TRADD and the death domains of TNFR1, FADD, and RIP (Boldin et Genetics Institute Wyeth Research al., 1995; Chinnaiyan et al., 1995; Stanger et al., 1995). The recruitment of FADD initiates the activation of the 87 Cambridge Park Drive Cambridge, Massachusetts 02140 caspase cascade, which eventually leads to apoptosis. The N-terminal region of TRADD (N-TRADD) spanning from residues 1-169 appears to be a novel domain since a BLAST (Altschul et al., 1997) search did not identify Summary any sequence homology to known proteins. N-TRADD is responsible for the binding of TRAF2, a TNFR-associ-TRADD is a multifunctional signaling adaptor protein ated factor (Hsu et al., 1996). This interaction is mediated that is recruited to TNFR1 upon ligand binding. The through the C-terminal region of TRAF2 (aa 348-501), C-terminal of TRADD comprises the "death domain" termed C-TRAF2. The interaction of N-TRADD with that is responsible for association of TNFR1 and other C-TRAF2 initiates TRAF2-mediated signaling processes death domain-containing proteins such as FADD and central to the cellular inflammatory response, such as RIP. The N-terminal domain (N-TRADD) promotes the JNK and NF-B activation (Rothe et al., 1995; Cao et recruitment of TRAF2 to TNFR1 by binding to the al., 1996; Reinhard et al., 1997; Song et al., 1997). This C-terminal of TRAF2, leading to the activation of JNK/ crucial role of N-TRADD in TNF signaling is supported AP1 and NF-B. The solution structure of N-TRADD by the observation that the expression of N-TRADD (aa was determined, revealing a novel protein fold. A com-1-194) can inhibit TNF-mediated NF-B and JNK activabination of NMR, BIAcore, and mutagenesis experition in a dominant-negative manner (Kieser et al., 1999). ments was used to help identify the site of interaction In order to understand how N-TRADD may interact of N-TRADD with C-TRAF2, providing a framework for with the adaptor protein TRAF2, we have determined future attempts to selectively inhibit the TNF signaling the three-dimensional structure of N-TRADD (1-169) by pathways. NMR spectroscopy. The solution structure of N-TRADD consists of five ␣ helices and four ␤ strands, arranged Introduction in a unique fashion. Using the structure, together with site-directed mutagenesis, we have identified a region Tumor necrosis factor (TNF) is a proinflammatory cytoof N-TRADD that interacts with C-TRAF2. This informakine that is involved in a variety of biological activities, tion, in addition to the recently published structures of through its binding to two distinct cell surface receptors, C-TRAF2 (McWhirter et al., 1999; Park et al., 1999), TNFR1 and TNFR2 (Rothe et al., 1992; Tartaglia and allows us to gain an insight into the interaction of Goeddel, 1992; Baker and Reddy, 1998). Both TNF re-N-TRADD and C-TRAF2. ceptors are part of the larger TNF receptor superfamily (Smith et al., 1994; Grell, 1995), which includes CD27, CD30, CD40, and Fas antigen, among others. These Results and Discussion receptors share no obvious sequence similarities in the cytoplasmic domain, with the exception of TNFR1 and Structure Determination The structure of N-TRADD was determined from 2402 Fas, which each have an ‫08ف‬ amino acid "death domain" (DD) at the C-terminal with ‫%82ف‬ sequence iden-NMR-derived restraints obtained using uniformly 15 Nand 15 N/ 13 C-labeled protein, with double and triple reso-tity. These death domains can induce apoptosis by mediating self-association of both TNFR1 and Fas upon nance NMR experiments. N-TRADD was soluble to ‫1ف‬ mM, but high concentrations of dithiothreitol (20 mM) ligand binding to each receptor, a critical event to trigger downstream signaling pathways by recruiting and acti-were required, to prevent aggregation of the protein. Under these conditions, the sample was stable for 6-8 vating receptor-associated effector molecules (Tartaglia and Goeddel, 1992; Boldin et al., 1995). Recently, many weeks. The structural statistics and root-mean-square deviations are in Table 1, and the superposition of the of these downstream signaling proteins were identified and shown to contain a DD, which mediates the interac-ensemble of 25 structures is shown in Figure 1B. The atomic root-mean-square deviation about the mean co-tion with the receptor through a DD-DD interaction. TRADD, one of the earlier TNFR1 adaptor proteins ordinate for residues 14-161 is 0.56 Å for the backbone atoms and 1.01 Å for all atoms. For secondary structure identified (Hsu et al., 1995), is a 34 kDa protein that is elements only, the rmsd is 0.46 Å for backbone atoms and 0.92 Å for all atoms. The N-terminal residues 1-10 ‡ To whom correspondence should be addressed (e-mail: dtsao@ and C-terminal residues 162-169 are disordered. The genetics.com [D. H. H. T.], llin@genetics.com [L. L. L]). § These authors contributed equally to this work.

Research paper thumbnail of Distinct Cellular Functions of MK2

Molecular and Cellular Biology, 2002

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is activated upon stress... more Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is activated upon stress by p38 MAPK␣ and -␤, 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.

Research paper thumbnail of 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

Molecular and Cellular Biology, 2007

Deletion of the MK2 gene in mice resulted in an impaired inflammatory response although MK3, whic... more 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.

Research paper thumbnail of MAPKAP Kinase 2-Deficient Mice Are Resistant to Collagen-Induced Arthritis

The Journal of Immunology, 2006

TNF-␣ is a pleiotropic cytokine considered a primary mediator of immune regulation and inflammato... more TNF-␣ 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-␣ 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-␣ 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.

Research paper thumbnail of Engagement of Tumor Necrosis Factor (TNF) Receptor 1 Leads to ATF-2- and p38 Mitogen-activated Protein Kinase-dependent TNF-alpha Gene Expression

Journal of Biological Chemistry, 1999

Engagement of the tumor necrosis factor-alpha (TNF-alpha) receptors by the TNF-alpha ligand resul... more Engagement of the tumor necrosis factor-alpha (TNF-alpha) receptors by the TNF-alpha ligand results in the rapid induction of TNF-alpha gene expression. The study presented here shows that autoregulation of TNF-alpha 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-alpha 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-activating death 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-alpha gene expression. Thus, the TNF-alpha CRE site is the target of TNFR1 stimulation and mediates the autoregulation of TNF-alpha gene transcription.

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

Journal of Biological Chemistry, 2007

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine that controls the initiatio... more Tumor necrosis factor alpha (TNFalpha) 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 TNFalpha, interleukin (IL)-1beta, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFalpha 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-1beta-induced TNFalpha 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 E(2), 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 TNFalpha 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.