TGF- -activated kinase 1 and TAK1-binding protein 1 cooperate to mediate TGF- 1 - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 09:40 |只看该作者 |倒序浏览 |打印

作者：Sung Il Kim, Joon Hyeok Kwak, Mareena Zachariah, Yanjuan He, Lin Wang, and Mary E. Choi作者单位：Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
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      【摘要】
   We have previously demonstrated that transforming growth factor- 1 (TGF- 1 ) rapidly activates the mitogen-activated protein kinase kinase 3 (MKK3)-p38 MAPK signaling cascade, leading to the induction of type I collagen synthesis in mouse glomerular mesangial cells (Wang L, Ma R, Flavell RA, Choi ME. J Biol Chem 277: 47257-47262, 2002). In the present study, we investigated the functional role of upstream TGF- -activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1) in the TGF- 1 signaling cascade. Rapid activation of endogenous TAK1 activity by TGF- 1 was observed in mouse mesangial cells. Transient overexpression of TAK1 with TAB1 enhanced the activation of MKK3 and p38 MAPK with or without TGF- 1 stimulation, whereas a dominant-negative mutant of TAK1 (TAK1DN) suppressed TGF- 1 -induced activation of MKK3 and p38 MAPK. Moreover, constitutive expression of TAK1DN reduced steady-state protein levels of MKK3 and p38 MAPK as well as MKK3 phosphorylation. Increased p38 MAPK activity by ectopic expression of either TAB1 or wild-type p38 MAPK resulted in enhanced TGF- 1 -induced type I collagen expression. In contrast, constitutive expression of TAK1DN inhibited collagen induction. Taken together, our data indicate that TAK1 and TAB1 play a pivotal role as upstream signal transducers activating the MKK3-p38 MAPK signaling cascade that leads to the induction of type I collagen expression by TGF- 1. In addition, our findings also suggest that TAK1 has a novel function in regulation of the steady-state protein levels of MKK3 and p38 MAPK.
      【关键词】 mouse mesangial cell TGF signaling stable transfection dominant negative mutant of TAK
   TRANSFORMING GROWTH FACTOR - 1 (TGF- 1 ) is a potent inducer of extracellular matrix (ECM) synthesis, and increased TGF- 1 expression has been observed in kidneys from both patients and experimental animals with progressive glomerular fibrosis ( 4, 17 ). Among the various ECM proteins whose expression is induced by TGF-, collagens are major components and play a central role in the development of fibrotic lesions ( 3 ). Although types I, III, IV, and V collagen can be produced by mesangial cells in culture, the major isoform of collagen elaborated is type I ( 10, 16 ). Despite intensive investigations, the signaling pathways mediating TGF- 1 -induced ECM synthesis are still incompletely understood. Recent studies note the involvement of both the Smad and mitogen-activated protein kinase (MAPK) signaling pathways in TGF- 1 -induced production and accumulation of ECM proteins ( 22, 33, 34 ). The phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway has also been implicated in TGF- 1 -induced type I collagen expression ( 21, 23, 28 ). We have previously reported that TGF- 1 -induced type I collagen expression in glomerular mesangial cells requires the activation of the mitogen-activated protein kinase kinase 3 (MKK3)-p38 MAPK signaling cascade ( 6, 37 ). However, mediators farther upstream regulating TGF- 1 -induced MKK3-p38 MAPK activation and type I collagen expression have not yet been identified.- y# d7 h4 K' X& {

TGF- -activated kinase 1 (TAK1), as implied by its name, was originally identified as a member of TGF- -activated MAPK kinase kinase (MKKK) ( 38 ). Recent investigations have indicated that TAK1 is involved in distinct signaling pathways induced by various stimuli, including environmental stress ( 30 ), inflammatory cytokines ( 14 ), and LPS ( 12 ) as well as TGF- 1 ( 29, 38 ). Phosphorylation of two threonine residues (Thr-184 and Thr-187) and one serine residue (Ser-192) in the activation loop of TAK1 protein is required for its activation ( 11, 24, 31 ). Once TAK1 is activated, it has the capability to activate several downstream cell signaling cascades, including the MKK4/7-c-Jun NH 2 -terminal kinase (JNK) cascade, MKK3/6-p38 MAPK cascade, and nuclear factor B (NF- B)-inducing kinase (NIK)-IkB kinase (IKK) cascade that regulates the NF- B activation ( 26 ).
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TAK1 has been consistently shown to be associated with TAK1-binding protein 1 (TAB1), which regulates TAK1 kinase activity ( 14, 29 ). The COOH-terminal 68 amino acids of TAB1, containing a serine- and threonine-rich region, are sufficient for binding to TAK1 ( 19 ). TAB1 also possesses a binding domain for p38 MAPK near the NH 2 -terminal region of the TAK1 binding domain ( 7, 8 ). The interaction can be readily observed when TAB1 and p38 MAPK, but not other p38 MAPK isoforms, are overexpressed together ( 8 ). There are possibilities that the interaction of TAB1 with p38 MAPK occurs endogenously. For instance, in HEK293 cells, stimulation with TNF- shows that endogenous TAB1 indeed interacted with p38 MAPK ( 7 ). Moreover, another isoform, TAB1, in which the TAK1 binding domain has been removed by alternate RNA splicing, has been found in various types of cells. The absence of a TAK1 binding domain in TAB1 facilitates the interaction of TAB1 with p38 MAPK that leads to autophosphorylation of p38 MAPK ( 8 ). However, it is not clear whether such interaction of endogenous TAB1 with p38 MAPK occurs in all cell types in general, or whether it is cell specific.
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In the present study, we show that TGF- 1 rapidly activates endogenous TAK1 in mouse mesangial cells. Blockade of TAK1 signaling by a dominant-negative mutant of TAK1 (TAK1DN) resulted in suppression of TGF- 1 -induced activation of MKK3 and p38 MAPK, while overexpression of TAK1 with TAB1 enhanced activation of MKK3 and p38 MAPK. Moreover, constitutive expression of TAK1DN reduced steady-state protein levels of MKK3 and p38 MAPK as well as MKK3 phosphorylation. Increased p38 MAPK activity by ectopic expression of either TAB1 or wild-type p38 MAPK enhanced TGF- 1 -induced type I collagen expression, whereas TAK1DN inhibited the collagen induction. Thus TAK1 and TAB1 are upstream signal transducers activating the MKK3-p38 MAPK signaling cascade and induction of type I collagen by TGF- 1. In addition, our findings suggest that TAK1 has a novel function in regulation of the steady-state protein levels of MKK3 and p38 MAPK.
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MATERIALS AND METHODS; @# p- ]4 Q% E6 Z. P2 d& c

Reagents. Recombinant human TGF- 1 (rhTGF- 1 ) was purchased from R&D Systems (Minneapolis, MN). Hemaglutinin (HA; Y-11), -actin, TAK1, and TAB1 polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies against p38 MAPK, phospho-p38 MAPK, MKK3, and phospho-MKK3/6 were obtained from Cell Signaling Technologies (Beverly, MA). Anti-type 1 collagen antibodies and monoclonal anti-c-Myc antibodies (9E10) were obtained from Calbiochem (San Diego, CA) and Sigma, respectively. Geneticin (G418 sulfate), SeeBlue Plus2-prestained standards, protein size marker, and Lipofectamine Plus reagent were obtained from Invitrogen (Rockville, MD).
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Murine mesangial cell culture. Mouse glomerular mesangial cells were isolated and characterized as previously described ( 37 ). Primary cultures of mouse mesangial cells were established and maintained in RPMI 1640 medium (Mediatech) supplemented with 15% FBS (Hyclone), insulin (10 µg/ml), 100 U/ml penicillin, and 100 µg/ml streptomycin and incubated in a humidified atmosphere of 5% CO 2 -95% air at 37°C. Using this technique, we have been successful in establishing homogeneous cultures of glomerular mesangial cells that immunostain for anti-vimentin (Dako) and anti-myosin antibodies (Zymed Laboratories), and negative staining for cytokeratin (Roche Molecular Biochemicals) and von Willebrand's factor (Dianova) as well as negative fluorescent acetylated LDL uptake (Biomedical Technologies). Cells between passages 7 and 16 were used for the experiments. To evaluate a TGF- 1 effect, cells grown to subconfluence were rendered quiescent in medium containing 0.5% FBS for 16 h followed by treatment with 2 ng/ml of TGF- 1 for the indicated time periods.

Expression constructs. HA-tagged expression constructs for wild-type TAK1 (pCMV-HA-TAK1) and constitutively active mutant of TAK1 (pCMV-HA-TAK1CA), Myc- or Flag-tagged TAB1 (pCS2Myc-TAB1 and pCMVF-TAB1, respectively), and pQE-31-MKK6 for the bacterial expression and purification of His 6 -tagged MKK6 protein were kindly provided by K. Matsumoto (Nagoya University) ( 14 ). For transient and stable transfection experiments, HA-TAK1 and HA-TAK1CA were recloned into pcDNA3.1 vector. Dominant-negative mutant of TAK1 (TAK1DN), the same as the kinase-deficient TAK1 K63W, was obtained by PCR-based site-directed mutagenesis and cloned into pcDNA3.1 vector with an HA-tag. Expression constructs for Flag-tagged wild-type p38 MAPK (pcMV-Flag-p38 ) and dominant-negative mutant of p38 MAPK (pcMV-Flag-p38 DN) were gifts from J. Han (The Scripps Research Institute). First-strand cDNA of mouse MKK3b was synthesized by Moloney murine leukemia virus reverse transcriptase with oligonucleotide dT 12 according to the manufacturer's instructions and then amplified by PCR using a mouse MKK3b-specific PCR primer set (forward primer: 5'-GGGGTCCTGGGATCTGAATCCTCTCC-3' and reverse primer: 5'-GTCCATGGCTTTGGATCC GTCCCCAAGTAT-3') synthesized according to the sequence from GenBank (accession No. NM_008928 ). PCR products were ligated with pcDNA3.1/TOPO vector (Invitrogen), and correct clones were confirmed by DNA sequencing of the inserted MKK3b cDNA and its surrounding region, and named pcDNA-V5-MKK3.
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Transient transfection of mouse mesangial cells. Transfection of mouse mesangial cells was carried out using Lipofectamine Plus reagent. Cells grown to 60% confluence on either 100- or 60-mm dishes were washed with PBS and transfected with 2 (for 100-mm dish) or 0.6 µg (for 60-mm dish) of respective plasmids for 4 h under serum-free condition. Total amounts of DNA were adjusted with empty vector pcDNA3.1. Control cells were transfected with empty vector pcDNA3.1 alone. Following transfection, the cells were washed with PBS and incubated in RPMI 1640 medium supplemented with 15% FBS for 24 h before each experiment.
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Characterization of stably transfected mouse mesangial cells. To obtain stably transfected mouse mesangial cells expressing respective wild-type TAK1, dominant-negative mutant of TAK1, and constitutively active mutant of TAK1, cells grown to 60% confluence on 100-mm dishes were transfected with 2 µg of pcDNA3.1-based expression vectors harboring HA-TAK1, HA-TAK1DN, and HA-TAK1CA, respectively. For controls, cells were transfected with empty vector pcDNA3.1 using the same procedure. Following transfection, cells were incubated in RPMI 1640 medium supplemented with 15% FBS for 24 h. For the selection of stably transfected clones, the cells were treated with 600 µg/ml Geneticin (G418 sulfate) in RPMI medium supplemented with 15% FBS, and the medium was changed every 3-4 days. G418-resistant colonies emerging 10 days after transfection were subcloned using ring cylinders, amplified, and maintained in RPMI medium containing 15% FBS, 300 µg/ml of G418, 100 U/ml penicillin, and 100 µg/ml streptomycin. Stable transfectant clones expressing each of the HA-tagged TAK1 wild-type and mutant proteins were identified by immunoprecipitation with anti-TAK1 antibodies and Western blotting with anti-HA antibodies.

Western blot analysis, immunoprecipitation, and TAK1 kinase assay. The cells were washed with ice-cold PBS and lysed in lysis buffer containing 1% Nonidet P-40, 20 mM Tris (pH 8.0), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na 3 VO 4, and 1 mM NaF. The cell lysates were passed through 21-gauge needles several times and then centrifuged for 15 min at 14,000 g at 4°C. The protein concentration was determined by using a BCA protein assay reagent kit (Pierce). For Western blotting, protein samples (100 µg) were subjected to 10 or 7.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk for 1 h and then incubated with primary antibodies overnight on a rocker at 4°C. The membranes were washed three times (15 min/each) with TTBS buffer (10 mM Tris, pH 7.5, 50 mM NaCl, and 0.1% Tween 20) and then incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min at room temperature. The target protein bands were detected with LumiGLO (New England Biolabs). The expression levels of type I collagen were quantitated by using Image J software (Research Services Branch, National Institutes of Health). Statistical significance of the experimental data from three independent experiments was determined by the Student's t -test for paired data. P values
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In the case of immunoprecipitation experiments, 200-500 µg of cell lysates were reacted with 2 µg of anti-TAK1 antibodies or anti-c-Myc antibodies for 30 min at 4°C followed by precipitation with 20 µl of protein A/G-Sepharose for 2 h at 4°C. The resultant immunoprecipitates were washed three times with lysis buffer and subjected to Western blotting. For the immunocomplex kinase assay to assess TAK1 activity, immunoprecipitates with anti-TAK1 antibodies were washed one more time with kinase buffer (20 mM Tris·HCl, pH 7.4, 10 mM MgCl 2 ) and resuspended in the same kinase buffer containing 20 µg of bacterially expressed His 6 -MKK6 as a substrate, obtained from Escherichia coli TOP 10 F' (Invitrogen) harboring pQE-31-MKK6 (K. Matsumoto) ( 14 ) and purified with a Ni-NTA spin column (Qiagen). The kinase reaction was initiated by the addition of 250 µM ATP. After 15 min of incubation at 25°C, the reactions were terminated by the addition of SDS sample buffer and boiled for 5 min. Samples were fractionated by 10% SDS-PAGE followed by Western blotting with anti-phospho-MKK3/6 antibodies or anti-His 6 antibodies. All of the experiments were repeated at least three times with essentially the same results, and representative blots are shown.
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Northern blot analysis. For the preparation of cDNA probes, DNA fragments of human p38 MAPK cDNA and mouse MKK3b cDNA, 0.3 and 0.4 Kb in length, respectively, were amplified by PCR using respective Flag-p38 and V5-MMK3 as a template DNA. The specific primer sets for human p38 MAPK cDNA and mouse MKK3b (for human p38 MAPK: forward 5'-TGGCTGTCGACCTACTGGA GA-3', reverse: 5'-AACCAGGTGCTCAGGACTCCA-3'; for mouse MKK3: forward 5'-GCCTTACATGGCCCCTGAGA-3', reverse: 5'-CCTATGAATCCTCTCCCAGG AT-3') were synthesized according to the sequences from GenBank (NM_008928  for mouse MKK3b and NM_011951  for human p38 MAPK). The human pro- 1 (I) collagen cDNA probe used was previously described ( 37 ). Total RNA was isolated with TRIzol (Invitrogen) according to the manufacturer's instructions and size-fractionated (20 µg/lane) on a 1% agarose-2% formaldehyde gel in 20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA (pH 7.2). Fractionated RNA was transferred and ultraviolet cross-linked to nylon membranes (Gene Screen Plus, DuPont). The membranes were prehybridized for 2 h in Church Gilbert's hybridization buffer (Quality Biological) and then hybridized overnight in the same solution containing the appropriate 32 P-labeled probe at 65°C. The membranes were then washed twice in solution containing 0.5% bovine serum albumin, 5% SDS, 40 mM phosphate buffer (pH 7.0), and 1 mM EDTA (pH 8.0) for 30 min each at 65°C, followed by 15-min washes with a solution containing 1% SDS, 40 mM phosphate buffer (pH 7.0), and 1 mM EDTA (pH 8.0) at 65°C. The membranes were exposed to Kodak X-AR film. Similar results were obtained from three independent experiments, and representative blots are shown. To control for the relative equivalence of RNA loading, the same membranes were hybridized with a 32 P-labeled oligonucleotide probe corresponding to 18S rRNA as previously described ( 37 )./ m2 n+ E& I3 G4 G

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Activation of endogenous TAK1 by TGF- 1 in mouse mesangial cells. We first determined whether endogenous TAK1 is activated by TGF- 1 in cultured mouse mesangial cells. The levels of TAK1 protein expression in cell lysates obtained from mouse mesangial cells treated with exogenous TGF- 1 (2 ng/ml) for varying time periods were determined by Western blot analyses using anti-TAK1 antibodies. As shown in Fig. 1 A, exposure to TGF- 1 caused TAK1 to migrate more slowly ( top arrow) on SDS-PAGE. The slower migrating bands represent phosphorylated forms of endogenous TAK1, since treatment with phosphatase has been shown to eliminate these bands ( 11, 24 ). Immunoblotting with anti- -tubulin antibodies was performed for a loading control. We next confirmed increased endogenous TAK1 kinase activities in these cell lysates obtained from mouse mesangial cells treated with exogenous TGF- 1 (2 ng/ml) for the indicated time periods ( Fig. 1 B ). TAK1 kinase activity was determined by an immunecomplex kinase assay using bacterially expressed MKK6 protein as a substrate. As shown in Fig. 1 B, increases in TGF- 1 -induced TAK1 activity were observed after TGF- 1 stimulation in a time-dependent manner. Relative equivalent amounts of His 6 -MKK6 substrate used in each reaction were confirmed by immunoblotting with anti-His 6 antibodies. Thus these results indicate that TGF- 1 rapidly activates endogenous TAK1 in mouse mesangial cells.  K, z- D, s9 j" f, S1 S

Fig. 1. Transforming growth factor- 1 (TGF- 1 ) activates endogenous TGF- -activated kinase 1 (TAK1) in mouse mesangial cells. A : Western blot analysis. Mouse mesangial cells grown in RPMI 1640 medium supplemented with 15% FBS to subconfluence were rendered quiescent in the same medium containing 0.5% FBS for 16 h followed by treatment with exogenous TGF- 1 (2 ng/ml) for the indicated time periods. Cell lysates were then subjected to immunoblotting with anti-TAK1 antibodies ( top ). Immunoblotting with anti- -tubulin antibodies was performed for loading control ( bottom ). B : immunecomplex kinase assay. Cell lysates isolated from mouse mesangial cells treated with exogenous TGF- 1 (2 ng/ml) for the indicated time periods were subjected to immunoprecipitation with anti-TAK1 antibodies ( lanes 2-5 ) or normal mouse IgG ( lane 1 ) as negative control (NC), followed by immunecomplex kinase assay for TAK1 as described in MATERIALS AND METHODS. Following kinase reaction, phospho-MKK6 produced was analyzed by Western blotting with anti-phospho-MKK3/6 antibodies ( top ). Relative equivalent amounts of His-MKK6 substrate used in each reaction were confirmed by Western blotting with anti-His 6 antibodies ( bottom ).
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TAK1 is an upstream activator of the MKK3-p38 MAPK signaling pathway in mouse mesangial cells. It has been reported that TAK1 can activate MKK4, MKK3, and MKK6 ( 13, 30 ), and we have previously demonstrated that MKK3 functions as a critical component of the TGF- 1 signaling pathway and that its activation is required for subsequent p38 MAPK activation and collagen stimulation by TGF- 1 in mouse mesangial cells ( 37 ). Here, we further investigated whether TAK1 serves as an upstream activator of TGF- 1 -induced MKK3-p38 MAPK signaling cascade in mouse mesangial cells. We examined the effects of overexpression of either HA-TAK1 with TAB1 or HA-TAK1DN by transient transfection of mouse mesangial cells. Cells transfected with empty vector pcDNA3.1 alone served as controls. As shown in Fig. 2, transient transfection of HA-TAK1DN partly abrogated the TGF- 1 -induced phosphorylation of both MKK3 and p38 MAPK ( lane 4 ) compared with control pcDNA3.1 transfectants ( lane 2 ) as determined by Western blot analyses using anti-phospho-MKK3 and anti-phospho-p38 MAPK antibodies. Conversely, coexpression of TAK1 and TAB1 resulted in further enhanced phosphorylation of both MMK3 and p38 MAPK in the presence or even in the absence of TGF- 1 stimulation ( lanes 5 and 6 ). Relative equivalence of protein loading was confirmed by immunoblotting the same membranes with the corresponding anti-MKK3 and anti-p38 MAPK antibodies. The transiently expressed HA-TAK1, HA-TAK1DN, and Flag-TAB1 proteins were detected by corresponding anti-HA or anti-Flag antibodies. With transient transfection experiments, the suppression of TGF- 1 -induced MKK3-p38 MAPK activation by TAK1DN was partial. To further clarify whether TAK1 serves as the predominant upstream activator of TGF- 1 -induced MKK3-p38 MAPK signaling cascade in mouse mesangial cells, we next examined the effects of constitutive expression of TAK1DN. Stably transfected mouse mesangial cells with respective HA-TAK1DN, HA-TAK1CA, or empty vector pcDNA3.1 were isolated and verified by immunoprecipitation of cell lysates with anti-TAK1 antibodies followed by immunoblotting with anti-HA antibodies. As shown in Fig. 3 A, representative clones of stably transfected mesangial cells with HA-TAK1DN (TAK1DN#7) or HA-TAK1CA (TAK1CA#25) expressed corresponding HA-tagged TAK1DN or TAK1CA when immunoblotted with anti-HA antibodies, whereas no HA-tagged proteins were detected in the control empty vector pcDNA3.1-transfected cells, as expected. The more slowly migrating bands on SDS-PAGE detected in TAK1CA#25 cells represent phosphorylated forms of TAK1, as previously described ( 11, 24 ), while only nonphosphorylated forms of TAK1 are detected in TAK1DN#7 cells. Total cell lysates obtained from mouse mesangial cells transiently transfected with HA-TAK1 and Flag-TAB1, which results in TAK1 activation, served as positive controls for TAK1 ( Fig. 3 A, lane 1 ) and accordingly, the more slowly migrating band corresponding to phosphorylated TAK1 ( top band) and nonphosphorylated TAK1 ( bottom band) are detected. We next determined the effects of constitutive expression of TAK1DN on TGF- 1-induced MKK3-p38 MAPK activation. Total cell lysates from stably transfected mesangial cells with HA-TAK1DN (TAK1DN#7) and control empty vector pcDNA3.1-transfected cells (pcDNA) treated with or without exogenous TGF- 1 (2 ng/ml) were subjected to Western blotting with anti-phospho-MKK3 and anti-phospho-p38 MAPK antibodies. As shown in Fig. 3 B, exposure to TGF- 1 stimulated the phosphorylation of both MKK3 and p38 MAPK in the control empty vector-transfected cells (pcDNA), as anticipated. Conversely, in TAK1DN#7 cells, activation of both MKK3 and p38 MAPK by TGF- 1 was strongly inhibited. Remarkably, basal levels of phosphorylated forms of MKK3 and p38 MAPK were also decreased, as was total protein expression of MKK3 and p38 MAPK. The reduction of MKK3 and p38 MAPK protein levels was not due to alterations of their steady-state mRNA levels, as evidenced by Northern blot analyses for MKK3 and p38 MAPK mRNA ( Fig. 3 C ).
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Fig. 2. Effects of ectopic expression of TAK1 and dominant-negative mutant of TAK1 (TAK1DN) on MKK3-p38 MAPK signaling pathway in mouse mesangial cells. Mouse mesangial cells transiently transfected with pcDNA-based expression vectors for HA-TAK1, HA-TAK1DN, and Flag-TAB1, as indicated, were cultured in medium supplemented with 15% FBS for 24 h and then rendered quiescent in medium containing 0.5% FBS for another 16 h, before treatment with exogenous TGF- 1 (2 ng/ml) for 20 min. Subsequently, cell lysates were isolated and subjected to Western blotting with corresponding antibodies against phospho-MKK3, MKK3, phospho-p38, and p38 MAPK. Anti-HA and anti-Flag antibodies were used to detect expression of transfected HA-TAK1, HA-TAK1DN, and Flag- TAK1-binding protein 1 (TAB1).

Fig. 3. Constitutive expression of TAK1DN inhibits TGF- 1 -induced MKK3-p38 MAPK signaling pathway in mouse mesangial cells. Mouse mesangial cells stably transfected with TAK1DN, constitutively active mutant of TAK1 (TAK1CA), and empty vector pcDNA3.1 were confirmed by immunoprecipitation (IP) with anti-TAK1 antibodies followed by Western blotting (WB) with anti-HA antibodies. While the stable transfectant of TAK1CA (TAK1CA#25) expressed phosphorylated and nonphosphorylated forms of TAK1 proteins, the TAK1-DN (TAK1DN#7) clone expressed only nonphosphorylated TAK1 protein. Empty vector (pcDNA)-transfected cells did not express any detectable proteins with anti-HA antibodies. Cell lysates obtained from mouse mesangial cells transiently transfected with HA-TAK1 and TAB1 were used as a positive control (PC) for TAK1 protein. B : effects of constitutive expression of TAK1DN on MKK3 and p38 MAPK activation by TGF- 1. Stably transfected control (pcDNA) and TAK1-DN (TAK1DN#7) cells were grown to subconfluence and rendered quiescent in medium supplemented with 0.5% FBS for 16 h followed by TGF- 1 (2 ng/ml) treatment for 20 min. Cell lysates were subjected to Western blotting with corresponding antibodies against phospho-MKK3, MKK3, phospho-p38, and p38 MAPK. Anti-GAPDH antibodies were used to demonstrate relative equivalence of protein loading. C : effects of constitutive expression of TAK1DN on steady-state mRNA levels of MKK3 and p38 MAPK. Total RNA was isolated from stably transfected pcDNA and TAK1DN#7 cells grown to subconfluence in medium containing 15% FBS or rendered quiescent in medium with 0.5% FBS for 16 h. Northern blot analysis was undertaken as described in MATERIALS AND METHODS for detection of steady-state mRNA levels of MKK3 and p38 MAPK. 18S rRNA signals indicate relative equivalence of RNA loading.

TAB1 alone can activate p38 MAPK. It has been reported that TAB1 contains a p38 MAPK binding domain and is capable of direct interaction with p38 MAPK, but not other isoforms of p38 MAPKs, to induce autophosphorylation of p38 MAPK ( 7, 8 ). To determine whether TAB1-directed p38 MAPK activation occurs in mouse mesangial cells, cells cotransfected with Myc-TAB1 and Flag-p38 MAPK were cultured and cell lysates were obtained. Immunoprecipitation experiments demonstrated the interaction of TAB1 with p38 MAPK, and TAB1-bound p38 MAPK was phosphorylated, independently of TGF- 1 stimulation ( Fig. 4 A ). Activation of p38 MAPK mediated by TAB1 was similar to that induced by TGF- 1 in control cells ( Fig. 4 B, lanes 3 and 2, respectively) but less potently than that induced by coexpression of TAK1 and TAB1 ( Fig. 4 B, lane 4 ).! ~4 s* `: I- L0 S5 n* L" d" U0 c

Fig. 4. Ectopic expression of TAB1 activates p38 MAPK in mouse mesangial cells. A : interaction of TAB1 with p38 MAPK in mouse mesangial cells. Myc-TAB1 was expressed ectopically alone or with Flag-p38 MAPK. Phosphorylated p38 MAPK bound to TAB1 was analyzed through immunoprecipitation (IP) with anti-Myc antibodies followed by Western blotting (WB) with anti-phospho-p38 MAPK antibodies. Total p38 was evaluated with Western blotting with anti-p38 MAPK antibodies. B : TAB1-mediated activation of p38 MAPK. Flag-p38 MAPK was expressed ectopically alone or with Flag-TAB1 or with Flag-TAB1 plus HA-TAK1. Levels of p38 MAPK phosphorylation was examined by Western blotting with anti-phospho-p38 MAPK antibodies and total p38 with anti-p38 MAPK antibodies.1 T* W- @  s6 \8 i, _4 K7 o- f

Ectopic expression of either p38 MAPK or TAB1 enhances TGF- 1 -induced type I collagen expression. Our previous report showed that in mouse mkk3 -/- mesangial cells, TGF- 1 stimulation failed to activate p38 MAPK and to induce type I collagen expression ( 37 ). To specifically assess the role of the activation of p38 MAPK in TGF- 1 -induced type I collagen expression, mouse mesangial cells were transiently transfected with either Flag-p38 or dominant-negative mutant p38 (Flag-p38 DN). The ectopic expression of p38 markedly enhanced TGF- 1 -stimulated type I collagen expression ( Fig. 5 A, lanes 5 and 6 ), while p38 DN abrogated the collagen response ( Fig. 5 A, lanes 3 and 4 ). Given that ectopic expression of TAB1 alone can activate p38 MAPK in mouse mesangial cells, we also examined the effects of TAB1 expression on the collagen response. Like p38 MAPK expression, the ectopic expression of TAB1 resulted in the enhancement of TGF- 1 -stimulated type I collagen protein ( Fig. 6, A and B ) and corresponding pro- 1 (I) collagen mRNA expression ( Fig. 6 C ).
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Fig. 5. Ectopic expression of p38 MAPK results in enhanced TGF- 1 -induced type 1 collagen expression in mouse mesangial cells. Mesangial cells transfected with wild-type Flag-p38 MAPK (p38 ) and dominant-negative p38 (p38 DN), as indicated, were cultured in medium supplemented with 15% FBS for 24 h and then rendered quiescent in medium containing 0.5% FBS for another 16 h, before treatment with exogenous TGF- 1 (2 ng/ml) for 24 h. Empty vector pcDNA3.1-transfected cells were used as controls. A : cell lysates were subjected to Western blotting with anti-mouse type 1 collagen antibodies and anti- -actin antibodies as loading controls. B : expression levels of type I collagen were quantitated and normalized to the expression levels in control untreated cells. Results represent means ± SE of 3 separate experiments. * P ) m8 p5 E6 {  \  e1 M) A- E4 \
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Fig. 6. Ectopic expression of TAB1 enhances TGF- 1 -induced type 1 collagen expression in mouse mesangial cells. Mesangial cells transfected with Flag-TAB1 or empty vector pcDNA3.1 were cultured in medium supplemented with 15% FBS for 24 h and then rendered quiescent in medium containing 0.5% FBS for 16 h, before treatment with exogenous TGF- 1 (2 ng/ml) for 24 h. A : cell lysates were prepared for Western blotting of type 1 collagen. The membranes were reblotted for -actin protein as a loading control. B : expression levels of type I collagen protein were quantitated and normalized to the expression levels in control untreated cells. Results represent means ± SE of 3 separate experiments. * P
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Constitutive expression of TAK1DN abrogates TGF- 1 -induced type I collagen expression. To determine the functional role of TAK1 in TGF- 1 -induced type I collagen expression in mouse mesangial cells, we examined the effects of increased TAK1 activity by constitutive expression of TAK1 or blockade of TAK1 by TAK1DN. In stably transfected mesangial cells TAK1WT#3, constitutive expression of wild-type TAK1 resulted in slightly enhanced type I collagen expression in the absence of TGF- 1 stimulation, while constitutive expression of TAK1DN in TAK1DN#7 cells strongly suppressed TGF- 1 -induced type I collagen expression compared with control cells (pcDNA) ( Fig. 7 ). These data indicate that the TAK1-MMK3-p38 MAPK signaling cascade mediates TGF- 1 -induced type I collagen expression in mesangial cells.
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Fig. 7. Inhibition of TGF- 1 -induced type 1 collagen expression by TAK1DN in mouse mesangial cells. Mesangial cells stably transfected with TAK1DN (TAK1DN#7), wild-type TAK1 (TAK1WT#3), and empty vector pcDNA3.1 were grown to subconfluence and rendered quiescent in the medium containing 0.5% FBS for 16 h followed by TGF- 1 (2 ng/ml) treatment for 48 h. A : cell lysates were subjected to Western blotting with anti-mouse type 1 collagen antibodies and anti- -actin antibodies as loading controls. B : expression levels of type I collagen protein were quantitated and normalized to the expression levels in control untreated cells. Results represent means ± SE of 3 separate experiments. * P
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DISCUSSION2 l, m% B# ~2 L! h' w

While it is well known that TGF- 1 stimulates the expression of genes encoding ECM proteins including type I collagen, the intracellular signaling mechanisms by which TGF- 1 stimulates this process remain incompletely understood. We have previously shown that TGF- type I receptor (T RI)-MKK3-p38 MAPK signaling axis mediates TGF- 1 -induced type I collagen expression in rat and mouse mesangial cells ( 6, 37 ). In this report, we demonstrate that TAK1 plays a crucial role as a predominant upstream activator of MKK3 in the TGF- 1 signaling pathway.% h# I. T/ K( |: ?; G  w7 W  Z

Although TAK1 was initially identified as a kinase activated by TGF- 1 stimulation in Mv1Lu and MC3T3 cells ( 29, 38 ), the same activation was not found in HeLa cells showing TAK1 activation by IL-1 stimulation ( 25 ), suggesting that TGF- -induced TAK1 activation occurs in a cell-specific manner. In this regard, we first examined whether TGF- 1 activates TAK1 in mesangial cells. Our data show that TAK1 was rapidly activated by TGF- 1 stimulation in mouse mesangial cells, and blockade of the TAK1 signaling pathway by constitutive expression of TAK1DN abrogated TGF- 1 -induced MKK3 activation, indicating that TAK1 is a major upstream mediator of TGF- 1 -induced MKK3 activation.  f. q! u7 [" J: Y

Interestingly, unlike MKK3, p38 MAPK activation by TGF- 1 stimulation was not completely abrogated by TAK1DN. It is possible that very low levels of phosphorylated MKK3 undetectable by Western blot analysis may be responsible for some of p38 MAPK activation by TGF- 1 stimulation in TAK1DN#7 cells. It is also possible that constitutive reduction of TAK1 activity triggers activation of other alternative signaling pathways to activate p38 MAPK. For instance, TAB1 can interact and activate p38 MAPK without participation of MKK3 or MKK6 ( 5, 7 ). We show that in mouse mesangial cells, coexpression of TAB1 and p38 MAPK revealed their interaction, resulting in phosphorylation of p38 MAPK in the absence of TGF- 1 stimulation. This interaction was very specific since coexpression of p38 MAPK and TAB1 did not show the same interaction (data not shown). In addition, it has been reported that the TAB1-directed activation of p38 MAPK has been observed in ischemic heart of Mkk3 -null mice ( 32 ). Therefore, it is suggested that the TAB1-directed activation of p38 MAPK might operate as an alternative path for p38 MAPK activation when either the TAK1 or MKK3/6 pathway is blocked.

In addition to phosphorylation, constitutive expression of TAK1DN significantly downregulated the steady-state protein levels of MKK3 and p38 MAPK. To our knowledge, this is the first demonstration that TAK1 can regulate MKK3 and p38 MAPK activity by controlling their steady-state protein levels as well as their phosphorylation levels. In general, MAPK signaling pathways including p38 MAPK are mainly and acutely regulated by cycles of phosphorylation and dephosphorylation of related kinases rather than by their expression levels. Moreover, there has been a paucity of reports showing the alterations of cellular concentration of MAPKs and their upstream activators, suggesting that these proteins may be expressed constitutively. In a recent report, however, an exceptional mechanism has been reported whereby p38 MAPK can negatively regulate the stability of MKK6 mRNA through AU-rich elements in the 3'-untranslated region (UTR) and thus control the steady-state concentration of one of its upstream activators ( 2 ). However, no AU-rich sequence has been found in both 5'- and 3'-UTR of MKK3. Moreover, Northern blot analysis showed that the constitutive expression of TAK1DN did not alter the steady-state mRNA levels of MKK3 and p38 MAPK, suggesting that the reduction of cellular concentration of MKK3 and p38 MAPK protein in mesangial cells stably expressing TAK1DN might be related to either synthesis or degradation of MKK3 and p38 MAPK protein.$ s- ~& y6 M7 }0 ?: u4 R; c! V" H

Constitutive expression of TAK1DN in mouse mesangial cells also resulted in the blockade of TGF- 1 -induced type I collagen expression. Ono et al. ( 18 ) reported similar results from SV40-induced transformed mouse mesangial cells (SV40 MES13) by stable expression of truncated TAK1. Evidence to support in vivo the profibrotic function of TAK1 is provided by a recent report of enhanced phosphorylation of p38 MAPK and development of interstitial fibrosis in the hearts of 9-day-old Tak1 transgenic mice ( 39 ). As previously mentioned, a MKK3-p38 MAPK signaling cascade is indispensable for the increased expression of type I collagen by TGF- 1 stimulation. Therefore, we propose that TAK1 plays a crucial role in TGF- 1 -induced type I collagen expression through the activation of a MKK3-p38 MAPK signaling cascade in mesangial cells.4 u* R* Q, ?; A1 o" n1 t( |0 Q

Previously, we have demonstrated that p38 MAPK activation is essential for TGF- 1 -induced type I collagen expression by using SB 203580, a p38 MAPK-specific chemical inhibitor and mesangial cells from Mkk3 knockout mouse. In the present study, we also confirmed the necessity of p38 MAPK activation via transient expression of dominant-negative mutant of p38 MAPK (p38 DN), resulting in blockade of TGF- 1 effect on type I collagen expression. Thus our present data provide consistent evidence that the activation of a TAK1-MKK3-p38 MAPK signaling cascade is responsible for TGF- 1 -induced type I collagen expression in mesangial cells. Interestingly, however, activation of p38 MAPK by the coexpression of TAB1 enhanced type I collagen expression only when the cells were stimulated with TGF- 1, suggesting that p38 MAPK activation itself might be necessary but not sufficient for the induction of type I collagen expression. Besides the TAK1-MKK3-p38 MAPK signaling pathway, other studies have demonstrated that the Smad signaling pathway is also involved in TGF- 1 -induced type I collagen expression ( 20, 22, 23, 27 ). Moreover, various collagen genes including COL1A1, COL3A1, COL5A2, and COL6A1 were identified as targets of Smad activation by using a combined cDNA microarray/promoter transactivation approach ( 35 ). However, our data showed that overexpression of p38 DN prevents the TGF- 1 -induced type I collagen expression, indicating that like p38 MAPK, TGF- -induced Smad activation is necessary but not sufficient for the induction of type I collagen expression. Therefore, we suggest that the TAK1-MKK3-p38 MAPK and Smad signaling pathways might cooperate in TGF- 1 -induced type I collagen expression. Indeed, recent reports support the importance of cross talk between the Smad and p38 MAPK pathways for the activation of gene expression by TGF- ( 1, 9 ). In addition, p38 MAPK activation possibly enhances Smad-dependent transcriptional activity via an increase in sumoylation of Smad4 ( 15 ). On the other hand, we have also shown that TGF- 1 -induced MKK3-p38 MAPK enhanced the expression vascular endothelial cell growth factor (VEGF) 164 isoform, which, in turn, increased type I collagen expression ( 36 ). Therefore, it is likely that an interplay of multiple signaling pathways activated by TGF- 1, including the TAK1/TAB1-MKK3-p38 MAPK signaling cascade, contributes to TGF- 1 -induced type I collagen expression.- j+ G8 n9 `) K2 i( R) d+ a
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GRANTS

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases R01-DK-57661 Grant to M. E. Choi; a Grant-in-Aid 0665379U from the American Heart Association to S. I. Kim; and the M. James Scherbenske Grant from the American Society of Nephrology to M. E. Choi.. N: A  R8 e6 h. C1 j( E: H# Q
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