Chemical Genetics Define the Roles of p38α and p38β in Acute and Chronic Inflammation
2007; Elsevier BV; Volume: 282; Issue: 48 Linguagem: Inglês
10.1074/jbc.m704236200
ISSN1083-351X
AutoresStephen J. O’Keefe, John S. Mudgett, Susan Cupo, Janey N. Parsons, Nicole Chartrain, Catherine E. Fitzgerald, Shiow-Ling Chen, Karen Lowitz, Cordelia Rasa, Denise M. Visco, Silvi Luell, Ester Carballo‐Jane, Karen Owens, Dennis M. Zaller,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe p38 MAP kinase signal transduction pathway is an important regulator of proinflammatory cytokine production and inflammation. Defining the roles of the various p38 family members, specifically p38α and p38β, in these processes has been difficult. Here we use a chemical genetics approach using knock-in mice in which either p38α or p38β kinase has been rendered resistant to the effects of specific inhibitors along with p38β knock-out mice to dissect the biological function of these specific kinase isoforms. Mice harboring a T106M mutation in p38α are resistant to pharmacological inhibition of LPS-induced TNF production and collagen antibody-induced arthritis, indicating that p38β activity is not required for acute or chronic inflammatory responses. LPS-induced TNF production, however, is still completely sensitive to p38 inhibitors in mice with a T106M point mutation in p38β. Similarly, p38β knock-out mice respond normally to inflammatory stimuli. These results demonstrate conclusively that specific inhibition of the p38α isoform is necessary and sufficient for anti-inflammatory efficacy in vivo. The p38 MAP kinase signal transduction pathway is an important regulator of proinflammatory cytokine production and inflammation. Defining the roles of the various p38 family members, specifically p38α and p38β, in these processes has been difficult. Here we use a chemical genetics approach using knock-in mice in which either p38α or p38β kinase has been rendered resistant to the effects of specific inhibitors along with p38β knock-out mice to dissect the biological function of these specific kinase isoforms. Mice harboring a T106M mutation in p38α are resistant to pharmacological inhibition of LPS-induced TNF production and collagen antibody-induced arthritis, indicating that p38β activity is not required for acute or chronic inflammatory responses. LPS-induced TNF production, however, is still completely sensitive to p38 inhibitors in mice with a T106M point mutation in p38β. Similarly, p38β knock-out mice respond normally to inflammatory stimuli. These results demonstrate conclusively that specific inhibition of the p38α isoform is necessary and sufficient for anti-inflammatory efficacy in vivo. The ubiquitous involvement of protein kinases in cellular signal transduction pathways makes them attractive targets for pharmacologic intervention. Low molecular weight kinase inhibitors enable titratable control of signaling pathways. However, because of the high degree of structural conservation within protein kinases, the generation of truly specific inhibitors is very challenging (1Fischer P.M. Curr. Med. Chem. 2004; 11: 1563-1583Crossref PubMed Scopus (127) Google Scholar, 2Vieth M. Sutherland J.J. Robertson D.H. Campbell R.M. Drug Discov. Today. 2005; 10: 839-846Crossref PubMed Scopus (160) Google Scholar). In addition, tools are not readily available for comprehensive assessment of inhibitor specificity across the large family of >500 kinases in mammalian species (3Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6259) Google Scholar, 4Caenepeel S. Charydczak G. Sudarsanam S. Hunter T. Manning G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11707-11712Crossref PubMed Scopus (249) Google Scholar). Genetic techniques, such as gene knock-outs and RNA interference, offer alternative ways to study the biological function of kinases. Nevertheless, interpretations of data generated using genetic techniques are not always straightforward because of the potential for compensation within highly adaptable signal transduction pathways. The phenotypes associated with genetic deletion of a kinase can be quite distinct from that associated with pharmacologic inhibition (5Jaeschke A. Karasarides M. Ventura J.J. Ehrhardt A. Zhang C. Flavell R.A. Shokat K.M. Davis R.J. Mol. Cell. 2006; 23: 899-911Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). One way to overcome these difficulties is through chemical genetics, which combines specific genetic mutations and pharmacological techniques to manipulate the selectivity of low molecular weight inhibitors. Chemical genetic approaches in which kinases are mutated to enable the binding of specific inhibitors have been described (5Jaeschke A. Karasarides M. Ventura J.J. Ehrhardt A. Zhang C. Flavell R.A. Shokat K.M. Davis R.J. Mol. Cell. 2006; 23: 899-911Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 6Bishop A.C. Buzko O. Shokat K.M. Trends Cell Biol. 2001; 11: 167-172Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 7Crews C.M. Splittgerber U. Trends Biochem. Sci. 1999; 24: 317-320Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In this report, we describe an alternative approach in which genetic knock-in mice are used to render p38α or p38β kinase resistant to the effects of specific inhibitors. Comparisons of the effects of in vivo administration of p38 inhibitors to wild-type versus drug-resistant knock-in mice provide powerful tools to dissect the biological function of these specific kinase isoforms. The p38 subfamily of MAP 2The abbreviations used are: MAP, mitogen-activated protein; CAIA, collagen antibody-induced arthritis; Me2SO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; HPLC, high performance liquid chromatography; HRP, horseradish peroxidase; neo, neomycin resistance; TK, thymidine kinase; TNF, tumor necrosis factor; GST, glutathione S-transferase. kinases consists of 4 members, p38α, p38β, p38γ, and p38δ, which share high sequence homology and a signature TGY phosphorylation motif in the kinase activation loop (8Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2885) Google Scholar). p38α MAP kinase was originally identified as an enzyme that was phosphorylated and activated in LPS-stimulated monocytes and was subsequently shown to be an important mediator of TNFα and IL-1 signaling (9Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3138) Google Scholar, 10Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2413) Google Scholar). The function of the various p38 family members has been investigated using a variety of techniques, including overexpression of wild-type and mutant kinases (11Lechner C. Zahalka M.A. Giot J.F. Moller N.P. Ullrich A. Proc. Natl. Acad. Sci. U. S. 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Ther. 1999; 82: 389-397Crossref PubMed Scopus (334) Google Scholar). The majority of p38 inhibitors that have been described are active against both p38α and p38β with minimal activity against p38γ and p38δ (18Hynes Jr., J. Leftheri K. Curr. Top. Med. Chem. 2005; 5: 967-985Crossref PubMed Scopus (65) Google Scholar). These dual p38α/β inhibitors have potent anti-inflammatory activity in preclinical models, and several compounds have advanced into the early stages of clinical development (16Kumar S. Boehm J. Lee J.C. Nat. Rev. Drug Discov. 2003; 2: 717-726Crossref PubMed Scopus (1071) Google Scholar). The dual specificity of p38 kinase inhibitors for both p38α and p38β does not allow the use of these compounds as definitive tools to study the specific functions of the individual p38 isoforms. The deletion of the p38α gene in mice was reported by four separate groups and, in all cases, was associated with embryonic lethality (19Adams R.H. Porras A. Alonso G. Jones M. Vintersten K. Panelli S. Valladares A. Perez L. Klein R. Nebreda A.R. Mol. Cell. 2000; 6: 109-116Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, 20Allen M. Svensson L. Roach M. Hambor J. McNeish J. Gabel C.A. J. Exp. Med. 2000; 191: 859-870Crossref PubMed Scopus (252) Google Scholar, 21Mudgett J.S. Ding J. Guh-Siesel L. Chartrain N.A. Yang L. Gopal S. Shen M.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10454-10459Crossref PubMed Scopus (329) Google Scholar, 22Tamura K. Sudo T. Senftleben U. Dadak A.M. Johnson R. Karin M. Cell. 2000; 102: 221-231Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). These studies revealed that p38α is required for placental development (19Adams R.H. Porras A. Alonso G. Jones M. Vintersten K. Panelli S. Valladares A. Perez L. Klein R. Nebreda A.R. Mol. Cell. 2000; 6: 109-116Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, 20Allen M. Svensson L. Roach M. Hambor J. McNeish J. Gabel C.A. J. Exp. Med. 2000; 191: 859-870Crossref PubMed Scopus (252) Google Scholar, 21Mudgett J.S. Ding J. Guh-Siesel L. Chartrain N.A. Yang L. Gopal S. Shen M.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10454-10459Crossref PubMed Scopus (329) Google Scholar, 22Tamura K. Sudo T. Senftleben U. Dadak A.M. Johnson R. Karin M. Cell. 2000; 102: 221-231Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar) and may play a role in the expression of erythropoietin during early development (22Tamura K. Sudo T. Senftleben U. Dadak A.M. Johnson R. Karin M. Cell. 2000; 102: 221-231Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). While tetraploid rescue of the placental defect in p38α knockouts enabled survival of the embryos (19Adams R.H. Porras A. Alonso G. Jones M. Vintersten K. Panelli S. Valladares A. Perez L. Klein R. Nebreda A.R. Mol. Cell. 2000; 6: 109-116Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar), there is little information available on the phenotype of adult p38α knock-out mice. The deletion of the p38β gene in mice was not associated with any known phenotype (23Beardmore V.A. Hinton H.J. Eftychi C. Apostolaki M. Armaka M. Darragh J. McIlrath J. Carr J.M. Armit L.J. Clacher C. Malone L. Kollias G. Arthur J.S. Mol. Cell Biol. 2005; 25: 10454-10464Crossref PubMed Scopus (202) Google Scholar). Mouse embryonic fibroblast cells from p38β knock-out mice displayed normal signaling in response to stress stimuli. p38β knock-out mice had normal in vivo responses to LPS and still showed pathological responses to the overexpression of TNFα. However, it is difficult to draw firm conclusions from the results obtained with p38β knockout mice because of the potential for compensation between different p38 isoforms. This type of compensation was previously described in p38γ knock-out mice in which phosphorylation of SAP97 was insensitive to a dual p38α/β inhibitor, SB203850, in wild-type cells but became sensitive to this compound in p38γ knock-out cells (24Sabio G. Arthur J.S. Kuma Y. Peggie M. Carr J. Murray-Tait V. Centeno F. Goedert M. Morrice N.A. Cuenda A. EMBO J. 2005; 24: 1134-1145Crossref PubMed Scopus (195) Google Scholar). To generate better animal models to study the function of these p38 isoforms, we generated knock-in mice with point mutations in Thr106 of p38α or p38β. These mutations do not affect the kinetic properties of the kinases but render them resistant to certain classes of p38 inhibitors. We used these knock-in mice to demonstrate conclusively that specific inhibition of the p38α isoform is necessary and sufficient for antiinflammatory efficacy in vivo. Antibodies—The murine monoclonal antibody against p38β and the goat anti-mouse HRP-conjugated antibody were from Invitrogen (Zymed Laboratories Inc.). The rabbit polyclonal antibody against p38α was from Cell Signaling Technology. Donkey anti-rabbit HRP-conjugated antibody was from Santa Cruz Biotechnology. The Arthrogen monoclonal antibody mixture was from Chemicon/Millipore. Compounds—The synthesis of MRK 4g and MRK 48 has been described (29Bao J. Hunt J.A. Miao S. Rupprecht K.M. Stelmach J.E. Liu L. Ruzek R.D. Sinclair P.J. Pivnichny J.V. Hop C.E. Kumar S. Zaller D.M. Shoop W.L. O'Neill E.A. O'Keefe S.J. Thompson C.M. Cubbon R.M. Wang R. Zhang W.X. Thompson J.E. Doherty J.B. Bioorg. Med. Chem. Lett. 2006; 16: 64-68Crossref PubMed Scopus (11) Google Scholar, 30Liverton N.J. Butcher J.W. Claiborne C.F. Claremon D.A. Libby B.E. Nguyen K.T. Pitzenberger S.M. Selnick H.G. Smith G.R. Tebben A. Vacca J.P. Varga S.L. Agarwal L. Dancheck K. Forsyth A.J. Fletcher D.S. Frantz B. Hanlon W.A. Harper C.F. Hofsess S.J. Kostura M. Lin J. Luell S. O'Neill E.A. Orevillo C.J. Pang M. Parsons J. Rolando A. Sahly Y. Visco D.M. O'Keefe S.J. J. Med. Chem. 1999; 42: 2180-2190Crossref PubMed Scopus (207) Google Scholar). Compounds were dissolved in Me2SO for the in vitro experiments. Mice—The Merck Institutional Animal Care and Use Committee approved all animal procedures and were performed in accordance with institutional policy and National Institutes of Health guidelines governing the humane treatment of vertebrate animals. 129/SvEv mice were obtained from Taconic Farms, Inc. (Germantown, NY). Mice were housed at up to ten mice per cage in a temperature- and humidity-controlled room (21 °C, 50%) with a 12 h light/dark cycle (lights on at 0700) either in microisolator cages in a specific pathogen-free barrier facility or in a conventional facility. The mice had access to a standard rodent laboratory diet (Harland Teklad Laboratory Rodent Diet 7012) and reverse-osmosis water ad libitum. Sentinel animals were evaluated quarterly as part of a health surveillance program and were determined to be specific pathogen-free by Charles River Laboratory Assessment Plus profile (Wilmington, MA), endo- and ectoparasite examinations, and gross necropsy. In the LPS-induced TNF production experiments, male mice 12–15 weeks of age were used. In the collagen antibody-induced arthritis (CAIA) experiments, female mice 11–15 weeks of age were used. All mice used for the in vivo experiments were on a 129S6 background. Cloning and Expression of p38 Proteins—Proteins were expressed, purified, and activated as described previously (47LoGrasso P.V. Frantz B. Rolando A.M. O'Keefe S.J. Hermes J.D. O'Neill E.A. Biochemistry. 1997; 36: 10422-10427Crossref PubMed Scopus (102) Google Scholar). Kinase Assays and Km Determinations—Kinase assays were performed as previously described using 2 μm GST-ATF2 as the protein substrate (47LoGrasso P.V. Frantz B. Rolando A.M. O'Keefe S.J. Hermes J.D. O'Neill E.A. Biochemistry. 1997; 36: 10422-10427Crossref PubMed Scopus (102) Google Scholar). The ATP Km was determined by measuring the rate at various ATP concentrations. The resulting data were fit using Grafit software version 4.3 (Erithacus Software). Construction Targeting Vectors and Transfection of ES Cells—Portions of a murine p38β cDNA were used as hybridization probes to screen a mouse genomic library, and several phage clones were isolated that contained fragments of the p38β genomic sequence. One phage clone contained the entire p38β genomic sequence on a single 12.2-kb EcoRI fragment. The p38β allele was confirmed by DNA sequencing. To generate a MAPK11 (p38β)-null allele, a targeting vector consisting of a 5.4-kb 5′ arm of homology preceded by a thymidine kinase cassette, a neomycin resistance cassette and a 3-kb 3′ arm of homology was constructed as follows: The 5′ arm of the targeting vector was created by inserting a 5.4 BamHI fragment of the MAPK11 cosmid clone into the BglII site of pKO Scrambler NKTV-1901 to generate pKOp38βRIKpn. The 3′ arm was constructed from a KpnI-EcoRI cosmid fragment containing exons 10–12 inserted into the KpnI-EcoRI sites of pKO Scrambler NTKV-1902. A KpnI fragment of the MAPK11 cosmid containing part of exon 7 and all of exons 8 and 9 was ligated into the KpnI site of pKOp38βRIKpn, and clones in the correct orientation were selected. NotI-XhoI fragments were ligated together to generate the completed targeting vector. To generate a Mapk11 allele containing a T106M mutation in exon 4, a targeting vector consisting of a 4.2 kb 5′ arm of homology preceded by a thymidine kinase cassette, a neomycin resistance cassette and a 5kb 3′ arm of homology was constructed as follows: A 333-bp Sse83871 fragment of the murine Mapk11 gene containing exon 4 and the T106M mutation (ACG → ATG) was generated by site-directed mutagenesis and confirmed by sequence analysis. The 333-bp Sse83871 fragment containing the T-M mutation replaced the corresponding wild-type sequence in a 9.2-kb p38β genomic EcoRI fragment obtained from a cosmid clone. XhoI linkers were added to the mutated 9.2-kb genomic fragment, and it was subcloned into the XhoI site of pKI-TK3988, a modified pKOScramber-NTKV-1901 vector in which the neo cassette and the unique NotI site of the parental vector were removed. To introduce the neo cassette into the p38β genomic segment, a NotI site was introduced into a BsmBI-BspEI fragment from the intron between exons 1 and 2 using PCR mutagenesis. Finally, a 1.8-kb neomycin cassette flanked by loxP sites obtained from pBS246neoTK was inserted at the NotI site of the BsmBI-BspEI fragment, and the resulting product was used to replace the corresponding BsmBI-BspEI fragment in the p38β genomic sequence. To introduce a T106M mutation into exon 4 of the Mapk14 gene, a targeting vector consisting of a 4.2-kb long arm of homology, a neomycin resistance cassette and a 2.3-kb short arm of homology was constructed as follows: A 6.5-kb HindIII genomic fragment containing exon 4 of the murine Mapk14 gene (21Mudgett J.S. Ding J. Guh-Siesel L. Chartrain N.A. Yang L. Gopal S. Shen M.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10454-10459Crossref PubMed Scopus (329) Google Scholar) was isolated and subcloned into pBS-KS. To mutate the fragment, a 1.8-kb KpnI-HindIII genomic fragment was created using PCR to insert the T106M mutation (ACC → ATG) just downstream of the 5′-terminal KpnI site. The corresponding fragment in the original subclone was replaced with the mutated fragment and confirmed by sequence analysis. The resulting mutated 6.5-kb genomic fragment was subcloned into the HindIII site of pBS-KS. Last, a 1.8-kb neomycin cassette flanked by loxP sites obtained from pBS246neoTK was inserted at the PsiI site of Mapk14 located 0.74-kb upstream of the mutation to generate a 4.2-kb 5′ long arm and a 2.3-kb 3′ short arm. The targeting vectors were linearized and electroporated into the CMTI-1 ES line (obtained from CMTI, Inc.) and seeded on irradiated murine fibroblast feeder cells. The clones were selected in medium containing 300 μg/ml G418 for 3 days following electroporation and 150 μg/ml for routine culture. G418-resistant ES colonies were picked and placed into individual wells of a 96-well tissue culture plate. Genomic DNA was prepared from ES cells, and the various clones were screened by Southern blot analysis using DNA probes as described in the figure legends. Positive clones were identified and cultured in vitro to obtain adequate cell numbers for blastocyst injections. Positive ES clones that had the correct karyotype were used for injection into C57BL/6 blastocysts. Coat color chimeras were mated to C57BL/6 females to determine if germline transmission was achieved. PCR was used to genotype mice using DNA isolated from tail snips. For genotyping p38β-null mice, the following set of PCR primers were used: 5′-CCACCCACCTCCACCCCAGAAGTTACTTAGACATT-3′, 5′-CCTTCCCAGCCTCTGAGCCCAGAAA-3′, 5′-GATCCTCCCTTAGGAGACCCCTTTGAGTGGACAA-3′, 5′-AATCCTTCCTGTGAGCCTGGGGAGG-3′. The PCR reaction produces a 1004-bp product for the wild-type allele and a 749-bp product for the null allele. A second 2.5-kb product may be generated from the wild-type gene with these primers but not under the conditions used. For genotyping p38β(T106M) knock-in mice, the following set of PCR primers were used: 5′-CCACCCACCTCCACCCCAGAAGTTACTTAGACATT-3′, 5′-GATCCTCCCTTAGGAGACCCCTTTGAGTGGACAA-3′, 5′-TCACGAGGCCCTTTCGTCTTCAAGAATTCAAGTT-3′. The PCR reaction produces a 1000-bp product for the wild-type allele and a 714- and a 1200-bp product for the T106M allele. For genotyping p38α(T106M) knock-in mice, the following set of PCR primers were used: 5′-TCCTTCCTTTAAGGGAGAGCAGGATAGTACTCAT-3′, 5′-CGAGGCTAGAGCGGCCATCAAGCTTAGGATC-3′, 5′-TCAGCTTCTGGCACTTCACGATGTTGTTCA-3′. The PCR reaction produces a 1200-bp product for the wild-type allele and a 941-bp product for the T106M allele. RPA Assay—Tissue samples were homogenized in 1 ml of TRIzol reagent per 50–100 mg of tissue using a Polytron homogenizer, and RNA was isolated following the manufacturer's instructions. A 307-bp p38β(T106M) probe containing a single mismatch to the p38β(T106M) sequence and two mismatches to the wild-type sequence was generated by PCR and cloned into the KpnI/PstI sites of pGEM3Z. A 250-bp p38α (T106M) probe that was an exact match for the mutant sequence was generated in a similar fashion. Ribonuclease protection assays were carried out using the RPA III™ Ribonuclease Protection Assay kit (Ambion) following the manufacturer's instructions. Western Blot Analysis—Soluble protein extracts were run on 4–20% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk overnight. The blocked membranes were incubated with anti-p38α or p38β antibodies (1:1000 dilution) for 1 h at room temperature with gentle agitation followed by three washes (10 min each) with buffer containing 10 mm Tris, pH 7.5, 50 mm NaCl, and 0.1% Tween 20 and then incubated with HRP-conjugated anti-rabbit or anti-mouse antibodies for 1 h at room temperature. Blots were developed using Western chemiluminescent reagent (PerkinElmer Life Sciences) and exposed to x-ray film. Peritoneal Exudate Cell Preparation and LPS-induced TNF Assay—Peritoneal exudate cells (PECs) were harvested from 2 to 3 mice of each genotype matched for age and gender 3 days after intraperitoneal inoculation of 1 ml of 8% Brewer thioglycollate medium (Sigma). Mice were euthanized and the abdomens flushed with 10 ml of phosphate-buffered saline plus heparin. Cells were collected from the peritoneal lavage fluid by centrifugation for 10 min at 1200 rpm, and resuspended at 5.56 × 105/ml in RPMI supplemented with 10% fetal calf serum (Hyclone), 100 units/ml penicillin, 100 units/ml streptomycin, 0.3 mg/ml l-glutamine (Invitrogen), 1 mm sodium pyruvate (Invitrogen), 1× non-essential amino acids (Invitrogen), and 10 μm 2-mercaptoethanol (BioRad). The PEC suspension (180 μl) was dispensed into wells of a 96-well tissue culture plate and 2 μl of MRK 4g dissolved in Me2SO was added to the cell suspension. The cells were stimulated with 20 μl of a1 μg/ml solution of LPS (Salmonella minnesota Re 595, Sigma) and incubated overnight at 37 °C in a humidified atmosphere of 5% CO2. The supernatant was harvested, and the TNFα concentration was determined by ELISA. LPS Challenge—12-week-old mice were dosed with a p38 inhibitor (p.o., 3 mg/kg in 0.5% methylcellulose) or vehicle 2.5 h prior to injection of 10 μg/mouse LPS (Escherichia coli Serotype 0111:B4, Sigma) and 800 mg/kg d-galactosamine (Sigma) in saline. Animals were euthanized 90 min later, and plasma TNFα was measured by ELISA. Plasma compound levels were measured by HPLC. Collagen Antibody Arthritis Induction in Mice—Female mice were weighed and placed into vehicle or drug treatment groups on day 0. Arthritis-inducing monoclonal antibody mixture (Chemicon International, ECM 1400) was injected intravenously into the caudal portion of each tail following the manufacturer's protocol for arthritis induction in C57BL/6 mice. Briefly, 0.8 ml of the monoclonal antibody mixture was injected on day 0. On day 3 postinoculation, mice were each given an intraperitoneal injection with LPS. Vehicle or compound was given once daily by oral gavage beginning 24 h post-LPS injections. Limbs were scored daily using the following system: 0, normal; 1, one or two swollen digits; 2, swelling of more than two digits as well as swelling of the entire carpus/tarsus; 3, deformity; 4, rigidity and immobility of the joints. The lowest possible score was 0 for an asymptomatic mouse, and the highest possible score was 16, which would indicate all four limbs were rigid. Mutation of Thr106 to Methionine Does Not Affect the ATP Km but Does Decrease Inhibitor Potency—A variety of studies have demonstrated the important role of Thr106 within the ATP binding site of p38α in determining sensitivity to pyridinyl imidazole inhibitors (25Gum R.J. McLaughlin M.M. Kumar S. Wang Z. Bower M.J. Lee J.C. Adams J.L. Livi G.P. Goldsmith E.J. Young P.R. J. Biol. Chem. 1998; 273: 15605-15610Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 26Wilson K.P. McCaffrey P.G. Hsiao K. Pazhanisamy S. Galullo V. Bemis G.W. Fitzgibbon M.J. Caron P.R. Murcko M.A. Su M.S. Chem. Biol. 1997; 4: 423-431Abstract Full Text PDF PubMed Scopus (282) Google Scholar, 27Eyers P.A. Craxton M. Morrice N. Cohen P. Goedert M. Chem. Biol. 1998; 5: 321-328Abstract Full Text PDF PubMed Scopus (280) Google Scholar, 28Lisnock J. Tebben A. Frantz B. O'Neill E.A. Croft G. O'Keefe S.J. Li B. Hacker C. de Laszlo S. Smith A. Libby B. Liverton N. Hermes J. LoGrasso P. Biochemistry. 1998; 37: 16573-16581Crossref PubMed Scopus (112) Google Scholar). We expressed mutant p38α and p38β proteins containing T106M mutations in E. coli and determined the effect of the mutation on the ATP Km and the IC50 of two structurally distinct p38α/β inhibitors, MRK 4g and MRK 48 (Fig. 1 and Refs. 29Bao J. Hunt J.A. Miao S. Rupprecht K.M. Stelmach J.E. Liu L. Ruzek R.D. Sinclair P.J. Pivnichny J.V. Hop C.E. Kumar S. Zaller D.M. Shoop W.L. O'Neill E.A. O'Keefe S.J. Thompson C.M. Cubbon R.M. Wang R. Zhang W.X. Thompson J.E. Doherty J.B. Bioorg. Med. Chem. Lett. 2006; 16: 64-68Crossref PubMed Scopus (11) Google Scholar, 30Liverton N.J. Butcher J.W. Claiborne C.F. Claremon D.A. Libby B.E. Nguyen K.T. Pitzenberger S.M. Selnick H.G. Smith G.R. Tebben A. Vacca J.P. Varga S.L. Agarwal L. Dancheck K. Forsyth A.J. Fletcher D.S. Frantz B. Hanlon W.A. Harper C.F. Hofsess S.J. Kostura M. Lin J. Luell S. O'Neill E.A. Orevillo C.J. Pang M. Parsons J. Rolando A. Sahly Y. Visco D.M. O'Keefe S.J. J. Med. Chem. 1999; 42: 2180-2190Crossref PubMed Scopus (207) Google Scholar). As shown in Table 1, the mutations, even though within the ATP binding site, had no significant effect on the binding of ATP compared with the wild-type proteins. Nevertheless, the mutation of Thr106 to Met dramatically decreased the potency of the two tested inhibitors 200–1000-fold. Both of these compounds are highly selective for p38α and β with at least 500-fold decrease in potency against other kinases that have been tested (29Bao J. Hunt J.A. Miao S. Rupprecht K.M. Stelmach J.E. Liu L. Ruzek R.D. Sinclair P.J. Pivnichny J.V. Hop C.E. Kumar S. Zaller D.M. Shoop W.L. O'Neill E.A. O'Keefe S.J. Thompson C.M. Cubbon R.M. Wang R. Zhang W.X. Thompson J.E. Doherty J.B. Bioorg. Med. Chem. Lett. 2006; 16: 64-68Crossref PubMed Scopus (11) Google Scholar, 30Liverton N.J. Butcher J.W. Claiborne C.F. Claremon D.A. Libby B.E. Nguyen K.T. Pitzenberger S.M. Selnick H.G. Smith G.R. Tebben A. Vacca J.P. Varga S.L. Agarwal L. Dancheck K. Forsyth A.J. Fletcher D.S. Frantz B. Hanlon W.A. Harper C.F. Hofsess S.J. Kostura M. Lin J. Luell S. O'Neill E.A. Orevillo C.J. Pang M. Parsons J. Rolando A. Sahly Y. Visco D.M. O'Keefe S.J. J. Med. Chem. 1999; 42: 2180-2190Crossref PubMed Scopus (207) Google Scholar). Expression of such mutant proteins in animals would essentially make these dual inhibitors specific for the remaining wild-type kinase and allow us to probe the role of each kinase in vivo pharmacologically. The use of such a pharmacological approach in the presence of a complete complement of normally active kinases would eliminate any possible developmental or other type of compensation.TABLE 1Comparison of kinetic parameters for wild-type and mutant enzymesWild-type(α)αT106MWild-type(β)βT106MVmax (arbitrary units)19 ± 0.158 ± 0.289 ± 1465 ± 9ATP Km (μm)128 ± 2.472 ± 4.869 ± 2535 ± 13MRK 48 IC50 (nm)0.08 ± 0.03 (n = 4)28 ± 12 (n = 5)0.25 ± 0.1 (n = 5)260 ± 30 (n = 5)MRK 4g IC50 (nm)0.1 (n = 1)34 (n = 1)0.1 (n = 1) Open table in a new tab Generation of p38α(T106M)-, p38β(T106M)-, and p38β-null Mice—The p38α and β loci were targeted by homologous recombination in murine ES cells. To generate p38β-null mice, we performed a targeted deletion of exons 1–7 in one of the p38β alleles in ES cells (Fig. 2A). Positive clones (Fig. 2B) were used to generate chimeric mice that successfully transmitted the null allele to their offspring. The genotypes of the mice were determined by PCR (Fig. 2C). The p38β–/– mice were fertile with no apparent phenotype in agreement with the observations of Beardmore et al. (23Beardmore V.A. Hinton H.J. Eftychi C. Apostolaki M. Armaka M. Darragh J. McIlrath J. Carr J.M. Armit L.J. Clacher C. Malone L. Kollias G. Arthur J.S. Mol. Cell Biol. 2005; 25: 10454-10464Crossref PubMed Scopus (202) Google Scholar). For the generation of mice expressing p38β(T106M),
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