Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for activation of p38alpha and p38delta MAPK isoforms by TGF-beta 1 in murine mesangial cells.

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix (ECM) synthesis that leads to renal fibrosis. Intracellular signaling mechanisms involved in this process remain incompletely understood. Mitogen-activated protein kinase (MAPK) is a major stress signal-transducing pathway, and we have previously reported activation of p38 MAPK by TGF-beta1 in rat mesangial cells and its role in the stimulation of pro-alpha1(I) collagen. In this study, we further investigated the mechanism of p38 MAPK activation by TGF-beta1 and the role of MKK3, an upstream MAPK kinase of p38 MAPK, by examining the effect of targeted disruption of the Mkk3 gene. We first isolated glomerular mesangial cells from MKK3-null (Mkk3-/-) and wild-type (Mkk3+/+) control mice. Treatment with TGF-beta1 induced rapid phosphorylation of MKK3 as well as p38 MAPK within 15 min in cultured wild-type (Mkk3+/+) mouse mesangial cells. In contrast, TGF-beta1 failed to induce phosphorylation of either MKK3 or p38 MAPK in MKK3-deficient (Mkk3-/-) mouse mesangial cells, indicating that MKK3 is required for TGF-beta1-induced p38 MAPK activation. TGF-beta1 selectively activated the p38 MAPK isoforms p38alpha and p38delta in wild-type (Mkk3+/+) mesangial cells, but not in MKK3-deficient (Mkk3-/-) mesangial cells. Thus, activation of p38alpha and p38delta is dependent on the activation of upstream MKK3 by TGF-beta1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-beta1-stimulated up-regulation of pro-alpha1(I) collagen expression but not TGF-beta1 induction of fibronectin and PAI-1. These data demonstrate that the MKK3 is a critical component of the TGF-beta1 signaling pathway, and its activation is required for subsequent p38alpha and p38delta MAPK activation and collagen stimulation by TGF-beta1.

Transforming growth factor-␤1 (TGF-␤1) is a potent inducer of extracellular matrix (ECM) synthesis that leads to renal fibrosis. Intracellular signaling mechanisms involved in this process remain incompletely understood. Mitogen-activated protein kinase (MAPK) is a major stress signal-transducing pathway, and we have previously reported activation of p38 MAPK by TGF-␤1 in rat mesangial cells and its role in the stimulation of pro-␣1(I) collagen. In this study, we further investigated the mechanism of p38 MAPK activation by TGF-␤1 and the role of MKK3, an upstream MAPK kinase of p38 MAPK, by examining the effect of targeted disruption of the Mkk3 gene. We first isolated glomerular mesangial cells from MKK3null (Mkk3؊/؊) and wild-type (Mkk3؉/؉) control mice. Treatment with TGF-␤1 induced rapid phosphorylation of MKK3 as well as p38 MAPK within 15 min in cultured wild-type (Mkk3؉/؉) mouse mesangial cells. In contrast, TGF-␤1 failed to induce phosphorylation of either MKK3 or p38 MAPK in MKK3-deficient (Mkk3؊/؊) mouse mesangial cells, indicating that MKK3 is required for TGF-␤1-induced p38 MAPK activation. TGF-␤1 selectively activated the p38 MAPK isoforms p38␣ and p38␦ in wild-type (Mkk3؉/؉) mesangial cells, but not in MKK3-deficient (Mkk3؊/؊) mesangial cells. Thus, activation of p38␣ and p38␦ is dependent on the activation of upstream MKK3 by TGF-␤1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-␤1-stimulated up-regulation of pro-␣1(I) collagen expression but not TGF-␤1 induction of fibronectin and PAI-1. These data demonstrate that the MKK3 is a critical component of the TGF-␤1 signaling pathway, and its activation is required for subsequent p38␣ and p38␦ MAPK activation and collagen stimulation by TGF-␤1.
Mitogen-activated protein kinase (MAPK) 1 is a major signaling system that transduces a variety of extracellular signals through a cascade of intracellular protein phosphorylation and plays an important role in the regulation of cell growth, differentiation, apoptosis, and cellular responses to environmental stress (1,2). In mammals, three major subgroups of MAPK superfamily members have been identified: the extracellular signal-regulated kinases 1 and 2 (ERK1/2), also known as p44/42 MAPK, the c-Jun N-terminal kinase (JNK), and the p38 MAPK. ERK1/2 is prototypically activated through a Ras-dependent pathway by mitogenic stimuli and growth factors, such as epidermal growth factor and platelet-derived growth factor, to regulate cell proliferation and differentiation (3,4). Whereas, JNK and p38 MAPK are activated predominantly by environmental stresses such as osmotic changes, UV radiation, and heat shock, and also by a variety of cytokines including tumor necrosis factor-␣ (TNF-␣) and interleukin-1 (IL-1), and participate in regulation of cytokine production, T cell proliferation and differentiation, and apoptosis (5-7).
Recent investigations including ours have revealed that p38 MAPK can be activated by the multifunctional cytokine transforming growth factor-␤1 (TGF-␤1), which regulates numerous cellular functions including proliferation, differentiation, and apoptosis (8 -11). TGF-␤1 is also a potent inducer of extracellular matrix (ECM) synthesis and participates in pathological processes as a central mediator of tissue response to injury and progressive fibrosis in a variety of tissues, including the kidney (12). Moreover, activation of p38 MAPK has been demonstrated in several disease models, including inflammation, septic shock, ischemia, ischemia-reperfusion, and vascular injury (13,14). In these studies, p38 activation in key cell types correlated with disease initiation and progression, and inhibitors of p38 MAPK were shown to attenuate both p38 activation and disease severity. Recently, evidence for the involvement of activated p38 MAPK in renal response to injury has been accumulating as well. For instance, activation of p38 MAPK has been demonstrated in ischemic and ischemic-reperfused rat kidneys (15). Increased p38 MAPK activation was also detected in glomeruli isolated from rats with experimental proliferative glomerulonephritis and diabetic nephropathy (16,17).
Four major isoforms of p38 MAPK have been identified through molecular cloning studies: p38␣, p38␤, p38␥, and p38␦ (18,19). Each of the p38 isoforms contains the Thr-Gly-Tyr canonical site, and activation is associated with dual phosphorylation of the threonine and tyrosine residues. The p38␤, p38 ␥, and p38␦ share homology (73, 63, and 62% amino acid identity, respectively) with p38␣ (20). The p38␣ and p38␤ isoforms are ubiquitously expressed, whereas p38␦ has restricted expression predominantly to the kidney and lung, while p38␥ is primarily expressed in skeletal muscle (19,20). Thus, it has been suggested that these isoforms represent related, but dis-tinct MAPK subgroups, and the signal transduction pathways leading to p38 MAPK activation are diverse, overlapping, and depend on cell types and stressor. Activation of the p38 MAPK involves phosphorylation by upstream MAPK kinases (MAP-KKs) in the protein kinase cascade and include MKK3, MKK6, and possibly MKK4. Among the different MAPKK subtypes, MKK3 and MKK6 are thought to play the predominant role in activating p38 MAPK (21). Hence, the mechanism accounting for signaling specificity by the p38 MAPK pathway may involve the selectivity of MKK3 and/or MKK6 to activate distinct p38 MAPK isoforms.
In the present study, we explored the mechanism of activation of the p38 MAPK pathway by TGF-␤1 and the role of MKK3 by examining the effect of targeted disruption of the Mkk3 gene. Utilizing glomerular mesangial cells isolated from MKK3-null (Mkk3Ϫ/Ϫ) and wild-type (Mkk3ϩ/ϩ) control mice, we show that TGF-␤1 rapidly and strongly phosphorylated MKK3 and p38 MAPK and that activation of MKK3 is required for TGF-␤1-induced p38 MAPK activation. TGF-␤1 selectively activated the p38 MAPK isoforms, p38␣ and p38␦, and both were dependent on the activation of MKK3 by TGF-␤1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-␤1-stimulated up-regulation of pro-␣1(I) collagen expression. Taken together, our data demonstrate that the MKK3 functions as a critical component of TGF-␤1 signaling pathway and that its activation is required for subsequent p38␣ and p38␦ MAPK activation and collagen stimulation by TGF-␤1.
Murine Mesangial Cell Culture-Glomerular mesangial cells were isolated and characterized as previously described (10), from glomeruli of MKK3-null (Mkk3Ϫ/Ϫ) mice and wild-type (Mkk3ϩ/ϩ) control mice (22) and from C57BL/6 mice, using differential sieving technique with the following modifications. Following collagenase digestion, the cells were plated in RPMI 1640 medium (Mediatech) supplemented with 20% fetal bovine serum (Bio-Whittaker), insulin (10 g/ml), 5 units/ml penicillin, and 5 g/ml streptomycin, and incubated in a humidified atmosphere of 5% CO 2 and 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 Inc.), and negative staining for cytokeratin (Roche Molecular Biochemicals) and von Willebrand's factor (Dianova) as well as negative fluorescent acetylated LDL uptake (Biomedical Technologies Inc.) (10). Cells between 7 and 16 passages were used for the experiments. The targeted disruption of the Mkk3 gene by homologous recombination and the generation of Mkk3(Ϫ/Ϫ) mice were as previously described (22).
Western Blot Analysis-The cells (4 ϫ 10 4 ) were plated in 100-mm dishes and incubated in RPMI 1640 medium containing 20% fetal bovine serum, 10 g/ml insulin. Cells grown to subconfluence were rendered quiescent in medium containing 0.5% fetal bovine serum for 24 h, followed by treatment with TGF-␤1 (2 ng/ml) for varying time periods. Total cellular extracts were obtained by lysis of cells in buffer containing 1 M Tris, pH 6.8, 10% SDS, 10% glycerol, 1 M dithiothreitol, and bromphenol blue. 20-g protein samples from each group were boiled for 5 min and were loaded onto 10% SDS-PAGE gels, then electroblotted onto nitrocellulose membranes (Bio-Rad). The membranes were incubated overnight with phospho-p38 MAPK, phospho-MKK3/6, phospho-ATF2, phospho-p42/44 MAPK, and phospho-JNK rabbit polyclonal antibodies (1:1000), followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies for 1 h. Sig-nal development was carried out using LumiGLO (Cell Signaling Technologies) and exposure to x-ray film. All of the assays were repeated three times, and representative blots are shown. As a control, all of the blots were subjected to immunoblotting for corresponding nonphospho-p38 MAPK, -MKK3, -ATF2, -p44/42 MAPK, and -JNK rabbit polyclonal antibodies.
Immunoprecipitation⁄Western Blot-The cells (4 ϫ 10 4 ) were plated in 100-mm dishes and incubated in RPMI 1640 medium containing 20% fetal bovine serum, 10 g/ml insulin. Cells grown to subconfluence were rendered quiescent in medium containing 0.5% fetal bovine serum for 24 h, followed by treatment with TGF-␤1 (2 ng/ml) for 30 min. Cells were washed with ice-cold phosphate-buffered saline, followed by lysis in radioimmune precipitation assay buffer (1ϫ phosphate-buffered saline, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml aproptinin, 1 g/ml leupeptin, 1 g/ml pepstatin, 1 mM Na 3 VO 4 , 1 mM NaF). The cell lysates were passed through 21-gauge needles several times, then centrifuged for 15 min at 14,000 ϫ g at 4°C. The protein concentration was determined by BCA protein assay reagent kit (Pierce). 200-g protein samples for each group were incubated with anti-p38␣ (2 g/ml), anti-p38␤ (2 g/ml), and anti-p38␦ (4 g/ml) antibodies for 1 h on a rocker at 4°C. 20 l of protein A/G-agarose were added to each sample and were continued incubating on a rocker overnight at 4°C. The immunoprecipitate complexes were washed four times and mixed with 1ϫ electrophoresis buffer, then boiled for 2-3 min. The protein samples were loaded onto 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin and then incubated with mouse anti-phosphotyrosine antibodies (1 g/ml) overnight at 4°C. After incubation, the membranes were washed 4 -5 times (30 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-mouse secondary antibodies (1:3000) for 1 h at room temperature. Signal development was carried out using LumiGLO and exposure to x-ray film. Three independent experiments were performed with essentially the same results, and representative blots are shown in Fig. 3.
Northern Blot Analysis-The cells (4 ϫ 10 4 ) were plated in 100-mm dishes, and incubated in RPMI 1640 medium containing 20% fetal bovine serum, 10 g/ml insulin. After cells reached subconfluence, they were incubated in medium containing 0.5% fetal bovine serum in the absence or in the presence of TGF-␤1 (2 ng/ml) for 24 h. Total RNA in each group was isolated by cell lysis with TRIzol (Invitrogen) according to the manufacturer's instructions and was size-fractionated (15 g/ lane) on a 1% agarose, 2% formaldehyde gel in 20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA (pH 7.2). mRNA was transferred and UV cross-linked to nylon membranes (Gene Screen Plus; Dupont). The blots were prehybridized for 2 h in Church Gilbert's hybridization buffer (Quality Biological, Inc.) and hybridized overnight in the same solution containing the appropriate 32 P-labeled probe at 65°C. The blots were then washed two times in solution containing 0.5% bovine serum albumin, 5% SDS, 40 mM phosphate buffer (pH 7.0), and 1mM EDTA (pH 8.0) for 30 min each at 65°C, followed by 15-min washes with solution containing 1% SDS, 40 mM phosphate buffer (pH 7.0), and 1 mM EDTA (pH 8.0) at 65°C. The blots were exposed to Kodak X-AR film. Similar results were obtained from three independent experiments and representative blots are shown in Fig. 6. To control for relative equivalence of RNA loading, the same blots were hybridized with 32 P-labeled oligonucleotide probes corresponding to the 18 S rRNA as previously described (10).

Rapid Activation of MKK3 and p38 MAPK in Mouse Mesang-
ial Cells by TGF-␤1-We first determined whether the p38 MAPK pathway is activated by TGF-␤1 in cultured mouse mesangial cells. The levels of p38 MAPK protein expression in mouse mesangial cells treated with exogenous TGF-␤1 (2 ng/ ml) was determined by Western blot analyses using phospho-p38 MAPK and p38 MAPK antibodies. As shown in Fig. 1A, exposure to TGF-␤1 stimulated the phosphorylation of p38 MAPK within 15 min. We next determined whether the upstream MAPK kinase MKK3 was also rapidly activated by TGF-␤1 using Western blot analyses with phospho-MKK3/6 and MKK3 antibodies. Fig. 1B demonstrates that treatment with TGF-␤1 also resulted in a rapid increase in phosphorylation of MKK3 in mouse mesangial cells.

MKK3 Is Required for Activation of p38 MAPK by TGF-␤1 in
Mouse Mesangial Cells-To investigate the functional role of MKK3 in the activation of the p38 MAPK pathway, we examined the effect of targeted disruption of the Mkk3 gene. We isolated mesangial cells from wild-type (Mkk3ϩ/ϩ) and MKK3deficient (Mkk3Ϫ/Ϫ) mice, and first confirmed the specific absence of MKK3 activation in the Mkk3Ϫ/Ϫ mesangial cells. As shown in Fig. 2A, increases in phosphorylation of MKK3 were observed within 15 min of treatment with exogenous TGF-␤1 (2 ng/ml) in wild-type (Mkk3ϩ/ϩ) mouse mesangial cells. In contrast, no phosphorylated MKK3 proteins were detected in the MKK3-deficient (Mkk3Ϫ/Ϫ) mouse mesangial cells.
We next determined whether TGF-␤1 nonetheless activated p38 MAPK in the absence of activation of MKK3. Fig. 2B demonstrates that while treatment with exogenous TGF-␤1 (2 ng/ml) resulted in a rapid increase in phosphorylation of p38 MAPK in wild-type (Mkk3ϩ/ϩ) mouse mesangial cells, no increases in phosphorylation of p38 MAPK were observed within the same time period in the MKK3-deficient (Mkk3Ϫ/Ϫ) mouse mesangial cells. A higher concentration of TGF-␤1 (10 ng/ml) also resulted in rapid phosphorylation of MKK3 and p38 MAPK in the wild-type (Mkk3ϩ/ϩ) cells, but still failed to induce phosphorylation of either MKK3 or p38 MAPK in the Mkk3Ϫ/Ϫ cells (data not shown). Our data indicate that MKK3 is required for activation of p38 MAPK by TGF-␤1 in mouse mesangial cells. Immunoblotting with p38 MAPK antibodies was performed for loading controls, and Fig. 2C shows no changes in the expression of total (phosphorylation state-independent) p38 MAPK proteins.
Selective Activation of p38␣ and p38␦ Isoforms by TGF-␤1 Is Blocked in MKK3-deficient Mouse Mesangial Cells-We next sought to investigate the specific p38 MAPK isoform(s) activated by TGF-␤1 in mouse mesangial cells. The three major isoforms of p38 MAPK, p38␣, p38␤, and p38␦, were isolated by immunoprecipitation with the respective antibodies, and phosphorylation of the p38 isoforms by TGF-␤1 was detected by immunoblotting with anti-phosphotyrosine antibodies. In wildtype (Mkk3ϩ/ϩ) mouse mesangial cells, treatment with exogenous TGF-␤1 resulted in increases of phosphorylated forms of p38␣ and p38␦ MAPK isoforms (Fig. 3, A and C). However, in the MKK3-deficient (Mkk3Ϫ/Ϫ) mouse mesangial cells, the TGF-␤1-induced phosphorylation of p38␣ and p38␦ MAPK isoforms was abrogated, indicating that MKK3 mediates the activation of both p38␣ and p38␦. Interestingly, no appreciable increases in phosphorylation of p38␤ MAPK isoform were ob-served in either cell type (Fig. 3B). Immunoblotting with p38 MAPK antibodies for the detection of total (phosphorylation state-independent) p38 MAPK proteins was performed for loading controls (Fig. 3C). Our results demonstrate that TGF-␤1 selectively activates p38␣ and p38␦ isoforms in mesangial cells. Moreover, the results in MKK3-deficient (Mkk3Ϫ/Ϫ) mouse mesangial cells indicate that the predominant upstream MAPK kinase mediating the activation of p38 isoforms by TGF-␤1 is MKK3.

Activation of the Downstream Target-activating Transcription Factor 2 (ATF2) in Mouse Mesangial Cells by TGF-␤1 Is
Dependent of MKK3-To explore the downstream targets of activated MKK3-p38 MAPK by TGF-␤1, here we examined the activation of ATF2, a downstream nuclear target of p38 MAPK, and the effect of targeted disruption of the Mkk3 gene. In wild-type (Mkk3ϩ/ϩ) mouse mesangial cells, treatment with exogenous TGF-␤1 induced the phosphorylation of ATF2 (Fig.  4). However, in the MKK3-deficient (Mkk3Ϫ/Ϫ) mouse mesangial cells, the TGF-␤1-induced ATF2 phosphorylation was inhibited, suggesting that the activation of the downstream target ATF-2 by TGF-␤1 in mesangial cells is dependent on MKK3.
Activation of ERK1⁄2, but Not JNK, by TGF-␤1 in Mouse Mesangial Cells-Previous reports, including studies from our

TGF-␤1 Activates p38 via MKK3-dependent Pathway
laboratory have demonstrated that other MAPK family members ERK1/2 and JNK are activated by TGF-␤1 in various cell types (4,10,23,24). We sought to determine whether the targeted disruption of the Mkk3 gene caused any changes in the expression and activation of ERK1/2 and JNK. We observed that in both wild-type (Mkk3ϩ/ϩ) and MKK3-deficient (Mkk3Ϫ/Ϫ) mouse mesangial cells, phosphorylation of ERK1/2 was strongly induced by TGF-␤1 within 5 min of treatment (Fig. 5A). In contrast, no appreciable increases in phosphorylation of JNK were observed within the same time period in either of the cell types (Fig. 5B). Taken together, these data demonstrate that Mkk3 gene disruption caused a specific MKK3 deficiency in the absence of changes in the expression and activation of the other MAPKs ERK1/2 and JNK.

Inhibition of TGF-␤1-induced pro-␣1(I) Collagen Expression by MKK3 Deficiency in Mouse Mesangial Cells-Mesangial
cells are capable of synthesizing ECM proteins, and TGF-␤1 induces the synthesis of ECM proteins including collagen, fibronectin, laminin, and proteoglycans in various types of cells (10,25,26). In the present study, we investigated the role of the MKK3-p38 MAPK pathway in the induction of pro-␣1(I) collagen, fibronectin, and PAI-1 expression by TGF-␤1 in mouse mesangial cells. As shown in Fig. 6A, treatment with exoge-nous TGF-␤1 (2 ng/ml) increased pro-␣1(I) collagen mRNA expression in the control wild-type mouse mesangial cells. Similarly, exogenous TGF-␤1 also increased fibronectin (Fig. 6B) and PAI-1 (Fig. 6C) mRNA expression in the wild-type mesangial cells. However, in the MKK3-deficient mesangial cells, the same increase in pro-␣1(I) collagen expression was not observed, while both fibronectin and PAI-1 expression increased with TGF-␤1 treatment. A higher concentration of TGF-␤1 (10 ng/ml) also failed to induce pro-␣1(I) collagen expression in the

TGF-␤1 Activates p38 via MKK3-dependent Pathway
Mkk3Ϫ/Ϫ cells (data not shown). Together, our data suggest a critical role for the MKK3-p38 MAPK pathway in mediating collagen stimulation by TGF-␤1. DISCUSSION TGF-␤1, a 25-kDa homodimeric polypeptide, is a multifunctional cytokine that regulates a wide variety of cellular functions, including inhibition and stimulation of cell growth, as well as induction of cellular differentiation, and ECM protein synthesis. Recent evidence has suggested a fundamental role for TGF-␤1 as a critical mediator of tissue response to injury and pathological processes such as inflammation, wound repair, tumorigenesis, and tissue fibrosis (27)(28)(29)(30). However, the molecular mechanisms responsible for signaling the multiple TGF-␤1 actions remain incompletely understood. In the present study, we examined the role of a major stress-activated intracellular signaling system, the p38 MAPK pathway, in transducing TGF-␤1 actions. We first established that TGF-␤1 is capable of activating the p38 MAPK in glomerular mesangial cells, a major target cell type is a variety of renal glomerular injury. We show that treatment with TGF-␤1 rapidly (within 15 min) induced the phosphorylation of p38 MAPK in cultured murine mesangial cells (Fig. 1A). We also show a concurrent increase in the phosphorylation of the immediate upstream MAPK kinase MKK3 within the same time period of TGF-␤1 treatment (Fig. 1B). The rapid kinetics of activation of both MKK3 and p38 MAPK provide support for direct activation by TGF-␤1.
To explore the mechanism of activation of the p38 MAPK pathway by TGF-␤1 and the functional role of MKK3 in TGF-␤1 signal transduction, we employed a genetic approach of targeted gene disruption of the MKK3. We isolated mesangial cells from wild-type (Mkk3ϩ/ϩ) and MKK3-null (Mkk3Ϫ/Ϫ) mice, and first confirmed the specific absence of activated MKK3 protein in the Mkk3Ϫ/Ϫ mesangial cells, in contrast to the wild-type cells ( Fig. 2A). Moreover, in the absence of activation of MKK3, TGF-␤1 was unable to induce phosphorylation of p38 MAPK (Fig. 2B) indicating that activation of MKK3 by TGF-␤1 is a requirement for activation of p38 MAPK. The basal levels of phosphorylated p38 detected in Mkk3Ϫ/Ϫ mesangial cells reflect activities of MAPK kinases other than MKK3, such as MKK6 previously demonstrated to be expressed normally in Mkk3Ϫ/Ϫ mice (22). Activated p38 MAPKs phosphorylate specific downstream targets, including many transcription factors, translation factors, and other kinases to elicit cellular responses (1,2). Here, we investigated the effects of Mkk3 gene disruption on TGF-␤1 activation of a downstream nuclear target of p38 MAPK, ATF2. The observed inhibition of TGF-␤1induced activation of transcription factor ATF2 in MKK3-deficient (Mkk3Ϫ/Ϫ) mesangial cells suggests that the activation of the downstream target ATF2 by TGF-␤1 in mesangial cells is dependent on MKK3 (Fig. 4).
Although several MAPK kinases, MKK3, MKK6, and MKK4, may be capable of activating p38 MAPK, differences in the relative contribution of these protein kinases in activating p38 MAPK exist among various stimuli and cell types. For instance, MKK3 is the major activator of p38 in PC-12 cells exposed to osmotic stress, while MKK6 is the dominant activator of p38 in epithelial (KB) cells exposed to osmotic stress, TNF-␣, and IL-1, and in monocytes stimulated by bacterial lipopolysaccharide (31)(32)(33). Whereas MKK4 and MKK7 are thought to activate predominantly the JNK, although few instances of in vitro p38 activation via MKK4 have been reported (34 -36). The relative contribution of the different MKKs in TGF-␤1 activation of the p38 MAPK pathway has not been previously known, and our data demonstrate clearly that the MKK3 specifically activates p38 MAPK in response to stimulation by TGF-␤1 in glomerular mesangial cells. Thus, the differential signal transduction pathways initiated by various stimuli that lead to the activation of p38 MAPK by one or several specific MKKs may account for stimulus-specific and cell-specific responses.
Indeed, it is becoming increasingly apparent that the complex signaling mechanisms that mediate multiple TGF-␤1 actions are cell-and context-specific (27). Depending on the cell type and stimulus, activated p38 MAPK, like TGF-␤1, can exert opposing effects, for instance, to either promote or suppress apoptosis (37,38). These opposing effects of p38 MAPK may be explained by the fact that distinct p38 isoforms can have opposite effects in terms of regulating apoptosis. For example, p38␣ induces apoptosis of cardiac myocytes, HeLa epithelial cells and Jurkat T cells while p38␤ suppresses apoptosis in the same cell types (37,38). The expression of multiple p38 MAPK isoforms in mammalian cells, and the different profiles of p38 isoforms expressed in different cell types suggest that these MAPK isoforms differ in their physiological function. The p38 MAPK isoforms may, in addition, be coupled to different upstream signal transduction pathways. This would enable activation of specific p38 MAPK isoforms in response to different stimuli. Alternatively, these p38 MAPK isoforms may differ in their downstream substrate specificity. Such differences could allow coupling of different p38 MAPK isoforms to different signal transduction targets. Thus, the differential activation of the p38 MAPK isoforms can facilitate cell type-and stimulus-specific cellular responses, and may provide a mechanism mediating, in part, the multiple actions of TGF-␤1. In the present studies, we observed that TGF-␤1 selectively activated the p38 MAPK isoforms, p38␣ and p38␦ (but not p38␥) in wild-type mesangial cells (Fig. 3). In contrast, in MKK3-deficient (Mkk3Ϫ/Ϫ) mesangial cells TGF-␤1 was unable to activate either of the p38 isoforms, indicating that activation of the isoforms p38␣ and p38␦ was dependent on the activation of direct upstream MAPK kinase MKK3 by TGF-␤1. Although it had been reported in transformed HeLa and COS-7 cells, using constitutively activated or recombinant MKK3 and MKK6, that MKK3 activates p38␣ and p38␥, while MKK6 activates p38␣, p38␤, and p38␥ (39), little had been previously known if any in other cell types, particularly primary culture-derived cells such as mesangial cells, and in response to stimulation by TGF-␤1.
Recent studies, including those from our laboratory have demonstrated that other MAPK family members ERK1/2 and JNK are activated by TGF-␤1 in various cell types (4,10,23,24). We have previously reported the activation of ERK1/ERK2 by TGF-␤1 in cultured macrophages, and demonstrated a critical role of the ERK pathway in signaling the anti-apoptotic effects of TGF-␤1 in these cells (4). We have also reported that TGF-␤1 is capable of rapidly activating the ERK1/ERK2 and the p38 MAPK, but not JNK, in glomerular mesangial cells (10). Therefore, it was important that we confirmed that our targeted Mkk3 gene disruption caused a specific MKK3 deficiency in the absence of changes in the expression and activation of the other MAPKs ERK1/2 and JNK (Fig. 5).
Investigations of the p38 MAPK signaling pathway have widely utilized pharmacological inhibitors such as the pyridinylimidazole compound SB-203580 to inhibit the function of the p38 MAPK. Using this approach, we had observed that SB-203580 inhibited TGF-␤1-induced pro-␣1(I) collagen expression and suggested that this TGF-␤1 response was via the p38 MAPK-dependent pathway in cultured rat mesangial cells (10). However, recent findings indicate that SB-203580 can directly inhibit thromboxane synthase, and cyclooxygenases-1 and -2, and thus some of the effects of SB-203580 may not be specifically due to p38 kinase inhibition, but rather due to cyclooxygenase inhibition and alteration in arachidonic acid metabolism (40). In the present study, we utilized an alternative genetic approach of targeted Mkk3 gene disruption. Remarkably, MKK3 deficiency resulted in a selective disruption of TGF-␤1-stimulated up-regulation of pro-␣1(I) collagen expression (Fig. 6A), without changes in TGF-␤1-induced fibronectin, and PAI-1 expression in murine mesangial cells. (Fig. 6, B and C). These data affirm a critical and selective role for the MKK3-p38 MAPK pathway in mediating collagen stimulation by TGF-␤1. Moreover, SB-203580 inhibits p38␣ and p38␤ with in vitro IC 50 values of 0.3-0.6 M, but has no inhibitory action on p38␦ and p38␥ (41). Thus, our present findings of TGF-␤1 activation of p38␣ and p38␦, but not p38␤, together with our previous observations that TGF-␤1-induced pro␣1(I) collagen expression was also inhibited by SB-203580, support a MKK3-p38␣ MAPK-dependent pathway for collagen stimulation by TGF-␤1.
To date, little is known regarding the functional role of p38␦. Of particular interest is the relatively restricted expression of p38␦ predominantly to the kidney and lung and our present findings of rapid activation of p38␦ by TGF-␤1 in renal mesangial cells. Interestingly, recent studies report renal up-regulation (26-fold higher than controls) of the p38␦ isoform activity in the renal cortex of rats with anti-GBM glomerulonephritis (19), a model in which TGF-␤1 has been previously demonstrated to be induced.
In summary, our data provide strong evidence that TGF-␤1 rapidly and strongly activates MKK3 and that this activation of MKK3, but not MKK6, is required for the subsequent activation of p38 MAPK by TGF-␤1 in glomerular mesangial cells. The p38 MAPK isoforms, p38␣ and p38␦, but not p38␤, were selectively activated by TGF-␤1 and was dependent on MKK3 activation by TGF-␤1. Furthermore, MKK3 deficiency selectively inhibited TGF-␤1-stimulated up-regulation of pro-␣1(I) collagen expression. These data provide clear evidence that the MKK3 functions as a critical component in TGF-␤1 signaling and that its activation is a requirement for subsequent p38␣ and p38␦ MAPK activation and collagen stimulation by TGF-␤1.