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• W2070602875 abstract "TAK1 (transforming growth factor-β-activated kinase-1), a MAP3K with considerable sequence similarity to Raf-1 and MEKK-1, has been identified as a transforming growth factor-β/bone morphogenetic protein (BMP)-activated cytosolic component of the MAPK pathways. In this investigation, the molecular interactions between TAK1 and Smad proteins were characterized as well as their influence on BMP-mediated mesenchymal cell differentiation along the osteogenic/chondrogenic pathway. In co-immunoprecipitations we found an interaction of TAK1 with all Smads tested, R-Smads Smads1-5, the co-Smad Smad4, and the inhibitory Smads (I-Smad6 and I-Smad7). Smad interaction with TAK1 takes place through their MH2 domain. This interaction is dependent on the presence of an active kinase domain in TAK1. TAK1 dramatically interferes with R-Smad transactivation in reporter assays and affects subcellular distribution of Smad proteins. Activated TAK1 also interferes with BMP-dependent osteogenic development in murine mesenchymal progenitor cells (C3H10T½). A potential TAK1-mediated apoptosis process could be excluded for these cells. Both synergistic and interfering influences of TAK1 on BMP-mediated Smad-signaling have been reported previously. We suggest that TAK1 is a factor that is involved in the fine-tuning of BMP effects during osteogenic development. TAK1 (transforming growth factor-β-activated kinase-1), a MAP3K with considerable sequence similarity to Raf-1 and MEKK-1, has been identified as a transforming growth factor-β/bone morphogenetic protein (BMP)-activated cytosolic component of the MAPK pathways. In this investigation, the molecular interactions between TAK1 and Smad proteins were characterized as well as their influence on BMP-mediated mesenchymal cell differentiation along the osteogenic/chondrogenic pathway. In co-immunoprecipitations we found an interaction of TAK1 with all Smads tested, R-Smads Smads1-5, the co-Smad Smad4, and the inhibitory Smads (I-Smad6 and I-Smad7). Smad interaction with TAK1 takes place through their MH2 domain. This interaction is dependent on the presence of an active kinase domain in TAK1. TAK1 dramatically interferes with R-Smad transactivation in reporter assays and affects subcellular distribution of Smad proteins. Activated TAK1 also interferes with BMP-dependent osteogenic development in murine mesenchymal progenitor cells (C3H10T½). A potential TAK1-mediated apoptosis process could be excluded for these cells. Both synergistic and interfering influences of TAK1 on BMP-mediated Smad-signaling have been reported previously. We suggest that TAK1 is a factor that is involved in the fine-tuning of BMP effects during osteogenic development. TAK1 (transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF, transforming growth factor; BMP, bone morphogenetic protein; BMPR-IA, BMPR-IB, BMP-receptor type IA and IB; MAP3K, mitogen-activated protein kinase kinase kinase; PMSF, phenylmethylsulfonyl fluoride; RT, reverse transcription SBE, Smad-binding element; wt, wild type; ca, constitutively active; IL-1, interleukin 1; HA, hemagglutinin; ALP, alkaline phosphatase; PBS, phosphate-buffered saline; siRNAs, short interfering RNAs; dn, dominant-negative; HEK, human embryonic kidney; IP, immunoprecipitation; NLS, nuclear localization signal; NES, nuclear export signal; MAP, mitogen-activated protein; CAT, chloramphenicol acetyltransferase; PTH/PTHrP, parathyroid hormone/parathyroid hormone-related peptide. -activated kinase) has initially been identified as a cytosolic component of mitogen-activated protein kinase (MAPK) pathways activated by ligands of the TGF-β/BMP family of secreted factors (1Yamaguchi K. Shirakabe T. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1175) Google Scholar). TAK1 is a MAPK kinase kinase (MAPKKK and MAP3K) that is activated by several cytokines such as IL-1 and tumor necrosis factor-α in addition to TGF-β/BMPs. The protein consists of about 600 amino acids and harbors an N-terminal kinase domain of roughly 300 amino acids that shares about 30% identity with the catalytic domain of other MAP3Ks (e.g. Raf-1, MEKK-1) (1Yamaguchi K. Shirakabe T. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1175) Google Scholar, 2NinomiyaTsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z.D. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1019) Google Scholar, 3Craig R. Larkin A. Mingo A.M. Thuerauf D.J. Andrews C. McDonough P.M. Glembotski C.C. J. Biol. Chem. 2000; 275: 23814-23824Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). TAK1 mediates activation of c-Jun N-terminal kinases, p38 MAPK, and NF-κB pathways (4Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 5Shirakabe K. Yamaguchi K. Shibuya H. Irie K. Matsuda S. Moriguchi T. Gotoh Y. Matsumoto K. Nishida E. J. Biol. Chem. 1997; 272: 8141-8144Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). However, molecular details of the signaling cascade(s) involving TAK1 still remain to be elucidated, especially with regard to the initial steps. During the past few years, the intracellular mechanisms leading to propagation of signaling by TGF-β family members have been elucidated in significant detail. A family of eight intracellular proteins, termed Smad proteins (Smad1 to Smad8), has been identified in vertebrates, and its members transduce signals for TGF-β ligands including the BMPs (reviewed by Attisano and Wrana (6Attisano L. Wrana J.L. Science. 2002; 296: 1646-1647Crossref PubMed Scopus (1134) Google Scholar)). Smad proteins can be classified into three types according to their structure and mechanism of action. The receptor-regulated Smads (R-Smads), Smad1, Smad5, and Smad8, are directly phosphorylated and activated by a BMP type I receptor (IA, IB, and ALK2, which is also an activin receptor (ActR-I)). Smad2 and Smad3 are activated by activin or TGF-β type I receptors (ALK4/ActR-IB and ALK5/TβR-I, respectively) (7Heldin C.H. Miyazono K. Ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3349) Google Scholar, 8Macias-Silva M. Hoodless P.A. Tang S.J. Buchwald M. Wrana J.L. J. Biol. Chem. 1998; 273: 25628-25636Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). Another Smad type, the common mediator Smad (Co-Smad; Smad4), associates with activated R-Smads, although hetero-oligomeric activated R-Smad complexes without Smad4 have also been detected (reviewed in Refs. 9Ten Dijke P. Miyazono K. Heldin C.H. Trends Biochem. Sci. 2000; 25: 64-70Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar and 10Moustakas A. Souchelnytskyi S. Heldin C.H. J. Cell Sci. 2001; 114: 4359-4369Crossref PubMed Google Scholar). The Smad complexes accumulate in the nucleus and participate in the regulation of target genes. This occurs predominantly by binding to other transcription factors and transcriptional regulators (for a review see Moustakas et al. (10Moustakas A. Souchelnytskyi S. Heldin C.H. J. Cell Sci. 2001; 114: 4359-4369Crossref PubMed Google Scholar)). However, direct DNA binding of some Smads via their MH1 domain, or even a combination of both mechanisms, may also operate for many target genes. The third type of Smads interferes with the activation of, and subsequent complex formation by, R-Smads and is termed anti-Smads or inhibitory Smads (I-Smads, Smad6 and Smad7) (11Hayashi H. Abdollah S. Qiu Y.B. Cai J.X. Xu Y.Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone Jr., M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1164) Google Scholar, 12Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (871) Google Scholar, 13Nakao A. Afrakhte M. Morén A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata N. Heldin N.E. Heldin C.H. Ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1564) Google Scholar). Contrasting results on the biological role of TAK1-activated cascades in TGF-β/BMP signaling have been reported. In particular, a synergism for Smad and MAPK signaling cascades has been postulated (14Cocolakis E. Lemay S. Ali S. Lebrun J.J. J. Biol. Chem. 2001; 276: 18430-18436Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 15Uchida K. Suzuki H. Ohashi T. Nitta K. Yumura W. Nihei H. Biochem. Biophys. Res. Commun. 2001; 289: 376-381Crossref PubMed Scopus (33) Google Scholar). For instance, Smad and TAK1 pathways integrate ATF-2 as a common nuclear target in cells stimulated with TGF-β (16Sano Y. Harada J. Tashiro S. GotohMandeville R. Maekawa T. Ishii S. J. Biol. Chem. 1999; 274: 8949-8957Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). TAK1 was reported to restore the ability of P19CL6[noggin] cells to differentiate into cardiac myocytes mimicking the effect of BMP2 upon these cells, whereas forced expression of a dominant-negative form of TAK1 interfered with this induced differentiation (17Monzen K. Hiroi Y. Kudoh S. Akazawa H. Oka T. Takimoto E. Hayashi D. Hosoda T. Kawabata M. Miyazono K. Ishii S. Yazaki Y. Nagai R. Komuro I. J. Cell Biol. 2001; 153: 687-698Crossref PubMed Scopus (128) Google Scholar). TAK1 (co-expressed with its activator protein TAB1) has been proposed to synergize with Smads and to induce ventral mesoderm formation in Xenopus embryos, thereby mimicking BMP activity. This is substantiated by the fact that dominant-negative TAK1 (with a K63W mutation) is able to antagonize the effects of BMPs (18Shibuya H. Iwata H. Masuyama N. Gotoh Y. Yamaguchi K. Irie K. Matsumoto K. Nishida E. Ueno N. EMBO J. 1998; 17: 1019-1028Crossref PubMed Scopus (190) Google Scholar). However, other reports document that the p38 signaling pathway, which can be activated by TAK1, mediates Smad-independent TGF-β responses or even interferes with Smad activity (19Yu L. Hebert M.C. Zhang Y.E. EMBO J. 2002; 21: 3749-3759Crossref PubMed Scopus (588) Google Scholar, 20Sowa H. Kaji H. Yamaguchi T. Sugimoto T. Chihara K. J. Biol. Chem. 2002; 277: 36024-36031Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). A physical interaction between the inhibitory I-Smad6 molecule and TAK1 has also been observed (14Cocolakis E. Lemay S. Ali S. Lebrun J.J. J. Biol. Chem. 2001; 276: 18430-18436Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 21Kimura N. Matsuo R. Shibuya H. Nakashima K. Taga T. J. Biol. Chem. 2000; 275: 17647-17652Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 22Yanagisawa M. Nakashima K. Takeda K. Ochiai W. Takizawa T. Ueno M. Takizawa M. Shibuya H. Taga T. Genes Cells. 2001; 6: 1091-1099Crossref PubMed Scopus (45) Google Scholar, 23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). These observations prompted us to study potential molecular interactions between TAK1 and Smad proteins in more detail. We tested the ability of TAK1 to modify mesenchymal cell differentiation along the osteogenic/chondrogenic pathway in murine mesenchymal stem cells (C3H10T½ cells). This cell line can differentiate into myoblasts, adipocytes, chondrocytes, and osteoblasts (24Taylor S.M. Jones P.A. Cell. 1979; 17: 771-779Abstract Full Text PDF PubMed Scopus (899) Google Scholar, 25Wang E.A. Israel D.I. Kelly S. Luxenberg D.P. Growth Factors. 1993; 9: 57-71Crossref PubMed Scopus (458) Google Scholar, 26Ahrens M. Ankenbauer T. Schröder D. Hollnagel A. Mayer H. Gross G. DNA Cell Biol. 1993; 12: 871-880Crossref PubMed Scopus (311) Google Scholar). It has been documented recently that forced expression of the BMP-dependent signaling protein Smad1, some of its domains, or Smad5 induces osteogenic but not chondrogenic differentiation in C3H10T½ (27Ju W. Hoffmann A. Verschueren K. Tylzanowski P. Kaps C. Gross G. Huylebroeck D. J. Bone Miner. Res. 2000; 15: 1889-1899Crossref PubMed Scopus (53) Google Scholar). Here we demonstrate that TAK1 directly interacts with the MH2 domain of the entire Smad family of signaling mediators and that TAK1 interferes with BMP-mediated signaling cascades that normally lead to osteogenesis in C3H10T½ cells. Generation of TAK1 Expression Plasmids—Based upon the published sequence of murine TAK1 (GenBank™ accession number D76446), a forward primer harboring an EcoRI and reverse primer with a BamHI restriction site (underlined) were generated for cloning of wild-type (wt) TAK1: forward 5′-TATAGAATTCCGCGGGGGATCATGTCGACAGCC and reverse 5′-TATAGGATCCTCATCACTTGTCATCGTCATCCTTGTAGTCTGAAGTGCCTTGTCGTTTCTGCTG. The reverse primer additionally contained a sequence encoding a single FLAG epitope sequence. Expand™ High Fidelity enzyme (Roche Applied Science) was used to perform PCR on cDNA from murine kidney. Agarose gel electrophoresis revealed a single DNA product of about the expected size (1740 bp), which was used for cloning into the eukaryotic expression vector pMT7T3 (26Ahrens M. Ankenbauer T. Schröder D. Hollnagel A. Mayer H. Gross G. DNA Cell Biol. 1993; 12: 871-880Crossref PubMed Scopus (311) Google Scholar). The entire TAK1-cDNA insert was sequenced on both strands (BigDye Terminator Cycle Sequencing Ready Reaction Mix, Applied Biosystems-PerkinElmer Life Sciences) with the ABI Prism 310 capillary sequencer. By using this expression plasmid as template, constitutively active TAK1 (lacking the 22 N-terminal amino acids as described by Yamaguchi et al. (1Yamaguchi K. Shirakabe T. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1175) Google Scholar)) was generated. Additionally, dominant-negative TAK1 (K63W) was generated with standard methods using the QuickChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA). To investigate the tissue-specific expression of murine TAK1 splice variants by PCR, the following primers were used: forward, 5′-CAACTCAGCCACCAGCACAGG, and reverse, 5′-GACTGCGAGCTGGCTTCTCTG. For Co-IP studies, TAK1 variants were re-cloned with the C-terminal FLAG or HA tags into pcDNA3. All constructs were verified by sequencing. Cells and Transfection—Human embryonic kidney 293T cells, murine C2C12 myoblasts, and C3H10T½ progenitor cells and C3H10T½-BMP2 were cultured in high glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The features of C3H10T½-BMP2 cells have been described (28Hollnagel A. Ahrens M. Gross G. J. Bone Miner. Res. 1997; 12: 1993-2004Crossref PubMed Scopus (45) Google Scholar, 29Bächner D. Ahrens M. Schroder D. Hoffmann A. Lauber J. Betat N. Steinert P. Flohe L. Gross G. Dev. Dyn. 1998; 213: 398-411Crossref PubMed Scopus (65) Google Scholar). Recombinant human BMP4 and TGF-β1 were purchased from R&D Systems (Wiesbaden, Germany), and recombinant human IL-1β was from RELIATech GmbH (Braunschweig, Germany). Recombinant human BMP2 from Escherichia coli was prepared as described (30Vallejo L.F. Brokelmann M. Marten S. Trappe S. Cabrera-Crespo J. Hoffmann A. Gross G. Weich H.A. Rinas U. J. Biotechnol. 2002; 94: 185-194Crossref PubMed Scopus (134) Google Scholar). C3H10T½-BMP2 cells were stably transfected with expression plasmids encoding wt TAK1, dominant-negative (dn) or constitutively active (ca) TAK1 in pMT7T3-f1 using DOSPER™ according to the manufacturer's protocol (Roche Applied Science) together with a selection plasmid conferring puromycin resistance (pBSpacΔp, parental C3H10T½ cells) or G418 resistance (pAG60, BMP2-expressing C3H10T½ cells). Individual clones were picked, propagated, and tested for recombinant expression of TAK1 variants by RT-PCR using a vector-specific and a gene-specific primer. Control cell lines (empty expression vector) were established at the same time. Selected cell clones were subcultivated in the presence of puromycin (5 μg/ml) or puromycin/G418 (5 μg/ml + 750 μg/ml), and the selective pressure was maintained during the entire cultivation period. Transient transfections of HEK293T cells and C2C12 were performed using FuGENE 6 as described by the manufacturer (Roche Applied Science). The quantities of transfected DNA were kept constant by adding an appropriate amount of empty vector (31Verschueren K. Remacle J. Collart C. Kraft H. Baker B.S. Tylzanowski P. Nelles L. Wuytens G. Su M.-T. Bodmer R. Smith J.C. Huylebroeck D. J. Biol. Chem. 1999; 274: 20489-20498Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). In Vitro Osteogenic/Chondrogenic Differentiation—Stable cell lines expressing recombinant TAK1wt, TAK1ca, or TAK1dn, parental C3H10T½ cells (C3H10T½wt) and C3H10T½ cells constitutively expressing BMP2 (C3H10T½-BMP2) were plated at a density of 5,000 cells/cm2. After reaching confluence (arbitrarily termed day 0), ascorbic acid (50 μg/ml) and β-glycerophosphate (10 mm) were added as specified by Owen et al. (32Owen T.A. Aronow M. Shalhoub V. Barone L.M. Wilming L. Tassinari M.S. Kennedy M.B. Pockwinse S. Lian J.B. Stein G.S. J. Cell. Physiol. 1990; 143: 420-430Crossref PubMed Scopus (1377) Google Scholar). Recombinant cells cultured in Roux flasks (25 cm2) were harvested at different time points at (day 0) and after (days 4, 7, 10, 13) confluence in TriReagentLS or fixed with 3% paraformaldehyde, respectively. Cell pellets were used for protein analysis by Western blot. Protein was extracted from the cell pellets as described under “Immunoprecipitation and Immunoblotting.” Protein concentration was determined by the Bradford method, and equal amounts (25 μg/lane) of total protein were analyzed by Western blot. Alkaline phosphatase (ALP) activity was determined after fixation of cells with ethanol for 30 min at 4 °C followed by wash with PBS. Enzyme activity in the supernatant was determined spectrophotometrically at 405 nm by measuring the conversion to p-nitrophenol from p-nitrophenyl phosphate in 50 mm Na2CO3 at pH 9.5 and 0.5 mm MgCl2 at 37 °C for 60 min. One unit was defined as the activity catalyzing the hydrolysis of 1 μmol of p-nitrophenyl phosphate/min. Histological Methods and Verification of Cellular Phenotypes—Alkaline phosphatase activity in osteoblasts was visualized by cellular staining with SIGMA FAST 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma) as described in the manufacturer's protocol. Proteoglycan-secreting chondrocytes were identified by staining with Alcian blue at pH 2.5 (Alcian blue 8GS, Roth, Karlsruhe, Germany). RNA Preparation and Semi-quantitative RT-PCR—Total cellular RNA was prepared by TriReagentLS according to the manufacturer's protocol (Molecular Research Center Inc., Ohio, MS). Five μg of total RNA was reverse-transcribed with oligo(dT) primers, and cDNA aliquots were subjected to PCR. RT-PCR was normalized by the transcriptional levels of hypoxanthine-guanine phosphoribosyltransferase (cf. Fig. 8). The primer pairs and PCR conditions used to evaluate osteogenic/chondrogenic differentiation for collagen Ia1, collagen IIa1, osteocalcin, and the PTH/PTHrP receptor have been described (27Ju W. Hoffmann A. Verschueren K. Tylzanowski P. Kaps C. Gross G. Huylebroeck D. J. Bone Miner. Res. 2000; 15: 1889-1899Crossref PubMed Scopus (53) Google Scholar, 33Hoffmann A. Czichos S. Kaps C. Bachner D. Mayer H. Zilberman Y. Turgeman G. Pelled G. Gross G. Gazit D. J. Cell Sci. 2002; 115: 769-781Crossref PubMed Google Scholar). Immunoprecipitation and Immunoblotting—Human embryonic kidney (HEK) 293T transfected with different constructs were harvested 36 h after transfection and lysed in 1% (w/v) Nonidet P-40, 150 mm NaCl, 20 mm Tris, pH 7.5, 2 mm EDTA, 50 mm NaF, 1 mm Na4P2O7, 1 mm PMSF, supplemented with protease inhibitors (Protease Inhibitor Mixture Tablets, Roche Applied Science). IP reactions were performed as described by Verschueren et al. (31Verschueren K. Remacle J. Collart C. Kraft H. Baker B.S. Tylzanowski P. Nelles L. Wuytens G. Su M.-T. Bodmer R. Smith J.C. Huylebroeck D. J. Biol. Chem. 1999; 274: 20489-20498Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar) in the same lysis buffer containing M2-FLAG antibody (Sigma) or mouse monoclonal c-Myc antibody beads (catalog number sc-40AC, Santa Cruz Biotechnology). Endogenous complexes were isolated with TAK1 antibodies (Santa Cruz Biotechnology, catalog number sc-7162), Smad1 antibodies (Upstate Biotechnology, Inc., catalog number 06-653), or HA-antibodies (Santa Cruz Biotechnology, catalog number sc-805) covalently coupled to protein G-Sepharose beads (Seize X Protein G immunoprecipitation kit, Pierce catalog number 45210). Cell extracts and immunoprecipitates were analyzed by immunoblotting using the appropriate antibodies. Phospho-Smad1 (catalog number 9511) as well as p38 and pp38 antibodies were from Cell Signaling Technology (catalog number 9210); Smad2/3 antibodies were purchased from BD Transduction Laboratories (catalog number 610842), and phospho-Smad2 antibodies were a gift from C.-H. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden). GAL4-antibodies were from Santa Cruz Biotechnology (catalog number sc-510). Exposed films were scanned with the Epson scanner 1680 Pro and quantified by analysis on a PC using the public domain NIH Image program “ImageJ” (developed at the National Institutes of Health and available on the Internet at rsb.info.nih.gov/nih-image/). Reporter Assays—Reporter assays using the GAL4 DNA binding domain fused to various forms of Smad proteins were performed as described (34Meersseman G. Verschueren K. Nelles L. Blumenstock C. Kraft H. Wuytens G. Remacle J. Kozak C.A. Tylzanowski P. Niehrs C. Huylebroeck D. Mech. Dev. 1997; 61: 127-140Crossref PubMed Scopus (63) Google Scholar). Briefly, 420 ng of DNA were transfected/well of a 24-well plate seeded with 1 × 105 HEK293T cells the day before transfection. The DNA mix included a plasmid expressing β-galactosidase under the strong constitutive Rous sarcoma virus promoter for normalization purposes and a reporter plasmid encoding firefly luciferase under the control of five GAL4-binding sites upstream of a minimal TATA box (pG5 luc, Promega, Mannheim, Germany) and combinations of effector plasmids. Where appropriate, transfection reactions were supplemented with empty vector. Cells were harvested 24 h after transfection and lysed with a detergent lysis solution (100 mm potassium phosphate, pH 7.8, 0.2% Triton X-100, 0.5 mm dithiothreitol). β-Galactosidase activity was measured with the luminescent β-galactosidase kit II from Clontech and luciferase activity with the luciferase assay system from Promega. All results are expressed as luciferase activity normalized to β-galactosidase values (“relative luciferase activities”). Reporter assays with the Smad1 DNA-binding element SBE-9 (GCCGx9) (35Kusanagi K. Inoue H. Ishidou Y. Mishima H.K. Kawabata M. Miyazono K. Mol. Biol. Cell. 2000; 11: 555-565Crossref PubMed Scopus (155) Google Scholar), cloned into pCAT5 vector (36Boshart M. Kluppel M. Schmidt A. Schütz G. Luckow B. Gene (Amst.). 1992; 110: 129-130Crossref PubMed Scopus (230) Google Scholar), were performed similarly except that cells were lysed in CAT lysis buffer (Roche Applied Science). CAT enzyme was measured with the CAT enzyme-linked immunosorbent assay and β-galactosidase with the chemiluminescent β-galactosidase reporter gene assay (both manufactured by Roche Applied Science). Immunofluorescence—For endogenous immunofluorescence studies with cytokine or growth factor treatments, C3H101/2 or C2C12 cells were seeded onto poly-d-lysine-coated glass chamber slides at 8,500 c/cm2. The following day, cells were switched to serum-free medium for 1 h, then treated with 50 ng/ml IL-1 for 10 min (or solvent as control), and then with 50 ng/ml recombinant human BMP2 for an additional 30 min. For overexpression studies, HEK293T cells were transiently transfected with the indicated constructs as described above. Then cells were fixed with 4% paraformaldehyde in PBS for 10 min, washed twice with PBS, and permeabilized with methanol followed by 0.5% Triton X-100 in PBS. After washing with 0.1% Triton X-100 in PBS (PBT), blocking was performed with 5% fetal calf serum in PBT for 1 h followed by an overnight incubation in blocking buffer containing appropriate primary antibodies. The detection was done with goat anti-mouse Alexa 488 (A-11001, Molecular Probes) antibody for murine primary antibodies at 5 μg/ml or goat anti-rabbit Alexa 568 (A-11011, Molecular Probes) for rabbit primary antibodies or vice versa. Yeast Two-hybrid Assays—Yeast two-hybrid assays were performed according to the Matchmaker GAL4 Two-hybrid System 3 (Clontech). For prey SIP1 and bait Smad1 constructs see Ref. 31Verschueren K. Remacle J. Collart C. Kraft H. Baker B.S. Tylzanowski P. Nelles L. Wuytens G. Su M.-T. Bodmer R. Smith J.C. Huylebroeck D. J. Biol. Chem. 1999; 274: 20489-20498Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar. TAK1 cDNA was cloned as an SfiI-BamHI fragment isolated from the bait vector into pACT2. To generate the ALK2 bait construct, cDNA encoding the cytoplasmic domain of mouse ALK2 was generated by PCR and cloned into pGBT9. Fractionation Procedure—HEK293T cells were harvested and washed twice with ice-cold PBS, and the cytosol fraction was obtained by lysis on ice for 10 min with hypotonic buffer containing 0.5% (w/v) Nonidet P-40, 100 mm NaCl, 10 mm HEPES, pH 7.4, 10% glycerol, 1 mm MgCl2, 1 mm dithiothreitol, 50 mm NaF, 1 mm Na4P2O7, 1 mm PMSF, protease inhibitors (Protease Inhibitor Mixture Tablets, Roche Applied Science). After a low speed centrifugation (4500 rpm, 1 min) nuclear pellets were resuspended in high salt lysis buffer (300 mm NaCl, 10 mm HEPES, pH 7.4, 10% glycerol, 1 mm MgCl2, 1 mm dithiothreitol, 50 mm NaF, 1 mm Na2P2O7, 1 mm PMSF, protease inhibitors (Protease Inhibitor Mixture Tablets, Roche Applied Science)). After 10 min of lysis on ice nuclear extracts were clarified by centrifugation (7500 rpm, 2 min). Equal amounts of protein in cytosolic and nuclear extracts were loaded on SDS gels for Western detection. Experiments with Short Interfering (si) RNAs—The following sequence for TAK1-siRNA was synthesized according to Ref. 37Sakurai H. Suzuki S. Kawasaki N. Nakano H. Okazaki T. Chino A. Doi T. Saiki I. J. Biol. Chem. 2003; 278: 36916-36923Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar: 5′-UGGCGUAUCUUACACUGGA. The scrambled siRNA sequence was 5′-AUUGUCAGACUGACGUCGU. The siRNAs were synthesized by Dharmacon (Lafayette, CO) and delivered in a deprotected, desalted, and annealed form. For transient transfections, 4 μl of Lipofectamine 2000 (1 mg/ml, Invitrogen) were mixed with 200 μl of Opti-MEM (Invitrogen) and incubated for 5 min at room temperature. Additionally, 4 μl of siRNA duplex (20 μm) were mixed with 200 μl of Opti-MEM. Then the Lipofectamine-containing mix was added dropwise to the siRNA-containing mix and incubated at room temperature for another 20 min. Cells were trypsinized, centrifuged, and resuspended at 400,000 cells/ml in serum-free Dulbecco's modified Eagle's medium and were added to the Lipofectamine/siRNA mix. These mixes were seeded immediately into wells of a 6-well dish and incubated for 4 h before addition of medium with serum (20%) overnight. 35,000 cells/ml were reseeded into poly-d-lysine-coated LabTek glass chamber slides (12,000 c/cm2 in 2 well chamber slides, Nunc). After another overnight incubation, cells were starved for 1 h in serum-free Dulbecco's modified Eagle's medium before addition of IL-1 at a final concentration of 50 ng/ml (or solvent as control) for 5 min followed by treatment with 50 ng/ml BMP2 for another 30 min. The cells were then fixed with 4% paraformaldehyde. Tissue Distribution of Murine TAK1 Splice Variants—To investigate both the interactions of TAK1 with Smads and the biological activity for TAK1 in C3H10T1/2 cells, full-length TAK1 cDNA was generated by PCR, starting from total murine kidney RNA, using primers encompassing the N- and C-terminal coding sequences of the protein (see “Experimental Procedures”). At that time, the isolated TAK1 cDNA was an undescribed murine splice variant with a high identity to human TAK1b. Recently, the sequence of this unspliced murine TAK1 variant sequence has been confirmed in an independent study (GenBank™ accession number XM_131329; Fig. 1a). Deletions generated by alternative splicing are located in the C-terminal part of the protein and do not affect the kinase domain (Fig. 1a). Among the nine tissues examined, both the murine TAK1 long and short splice variant mRNAs are expressed in any tissue tested apart from kidney where the long form is prevailing (Fig. 1c). In gut and testis, this long form is also predominantly expressed, whereas in muscle and brain the shorter form is more abundant. The uterus, heart, and liver express both splice variants in similar amounts. In lung tissue, TAK1 mRNA seems not to be expressed at detectable levels. The mRNA of both TAK1 splice variants is also expressed in comparable amounts in C3H10T½ cells (see Fig. 8a, below). The capacity of our cloned longer TAK1 variants to activate the endogenous p38 pathway was investigated by transient overexpression of the wild-type protein (TAK1wt) and two variants, constitutively active TAK1 (TAK1ca) and dominant-negative TAK1 (TAK1dn) in HEK293T cells (Fig. 1b). Here TAK1wt and TAK1ca were considerably more active in activating the p38 pathway than TAK1dn. A basal level of phosphorylated p38 (pp38) in TAK1dn expressing cells is expected because several pathways converge on this p" @default.
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• W2070602875 title "Transforming Growth Factor-β-activated Kinase-1 (TAK1), a MAP3K, Interacts with Smad Proteins and Interferes with Osteogenesis in Murine Mesenchymal Progenitors" @default.
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