Stimulation of pro-alpha 1(I) collagen by TGF-beta 1 in mesangial cells: role of the p38 MAPK pathway

Beek Yoke Chin1, Amir Mohsenin2, Su Xia Li3, Augustine M. K. Choi4, and Mary E. Choi2

1 Toxicological Sciences, Environmental Health Sciences, and 3 Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205; and Sections of 4 Pulmonary and Critical Care Medicine and 2 Nephrology, Department of Internal Medicine, Yale University School of Medicine and the Veterans Affairs Connecticut Healthcare Systems, New Haven, Connecticut 06520


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor-beta 1 (TGF-beta 1) is a potent inducer of extracellular matrix protein synthesis and a key mediator of renal fibrosis. However, the intracellular signaling mechanisms by which TGF-beta 1 stimulates this process remain incompletely understood. In this report, we examined the role of a major stress-activated intracellular signaling cascade, belonging to the mitogen-activated protein kinase (MAPK) superfamily, in mediating TGF-beta 1 responses in rat glomerular mesangial cells, using dominant-negative inhibition of TGF-beta 1 signaling receptors. We first stably transfected rat glomerular mesangial cells with a kinase-deleted mutant TGF-beta type II receptor (Tbeta R-IIM) designed to inhibit TGF-beta 1 signaling in a dominant-negative fashion. Next, expression of Tbeta R-IIM mRNA was confirmed by Northern analysis. Cell surface expression and ligand binding of Tbeta R-IIM protein were demonstrated by affinity cross-linking with 125I-labeled-TGF-beta 1. TGF-beta 1 rapidly induced p38 MAPK phosphorylation in wild-type and empty vector (pcDNA3)-transfected control mesangial cells. Interestingly, transfection with dominant-negative Tbeta R-IIM failed to block TGF-beta 1-induced p38 MAPK phosphorylation. Moreover, dominant-negative Tbeta R-IIM failed to block TGF-beta 1-stimulated pro-alpha 1(I) collagen mRNA expression and cellular protein synthesis, whereas TGF-beta 1-induced extracellular signal-regulated kinase (ERK) 1/ERK2 activation and antiproliferative responses were blocked by Tbeta R-IIM. In the presence of a specific inhibitor of p38 MAPK, SB-203580, TGF-beta 1 was unable to stimulate pro-alpha 1(I) collagen mRNA expression in the control and Tbeta R-IIM-transfected mesangial cells. Finally, we confirmed that both p38 MAPK activation and pro-alpha 1(I) collagen stimulation were TGF-beta 1 effects that were abrogated by dominant-negative inhibition of TGF-beta type I receptor. Thus we show first demonstration of p38 MAPK activation by TGF-beta 1 in mesangial cells, and, given the rapid kinetics, this TGF-beta 1 effect is likely a direct one. Furthermore, our findings suggest that the p38 MAPK pathway functions as a component in the signaling of pro-alpha 1(I) collagen induction by TGF-beta 1 in mesangial cells.

transforming growth factor-beta receptor; signal transduction; mitogen-activated protein kinase; matrix


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TRANSFORMING GROWTH FACTOR-beta 1 (TGF-beta 1) is a member of a superfamily of multifunctional cytokines that participates in a wide array of biological activities such as development and wound repair, as well as pathological processes (2). TGF-beta 1 is a potent inducer of extracellular matrix (ECM) protein synthesis and accumulation and has been implicated as the key mediator of fibrogenesis in a variety of tissues (3). In the kidney, the critical role of TGF-beta 1 has been well recognized in several renal diseases characterized by progressive glomerular ECM accumulation leading to the development of glomerulosclerosis. In kidneys of both patients and experimental animals with glomerulonephritis, increased TGF-beta 1 expression has been observed (4, 32). Moreover, suppression of excess ECM deposition and glomerulosclerosis by neutralizing TGF-beta 1 antibody has been shown in experimental glomerulonephritis (4). Enhanced glomerular expression of TGF-beta 1 has also been shown in both human and experimental diabetic nephropathy (45). Furthermore, in vivo transfection of the TGF-beta 1 gene into the rat kidney has been shown to induce glomerulosclerosis (24). Similarly, the development of glomerulosclerosis has been described in mice expressing the transgene for TGF-beta 1 under the control of a murine albumin promoter (Alb/TGF-beta 1) and that have elevated circulating levels of TGF-beta 1 (25).

TGF-beta 1 induces the synthesis of a number of ECM proteins, including types I and III collagen, fibronectin, laminin, and proteoglycans (23, 36). Type I collagen is a major structural component of the ECM, which is synthesized by fibroblasts and vascular smooth muscle cells and in the kidney by glomerular mesangial cells, considered to be centrally involved in progressive glomerular ECM accumulation associated with chronic sclerotic processes (12, 18). We and others have previously reported increased collagen synthesis by cultured glomerular mesangial cells in response to TGF-beta 1 (9, 18). Although mesangial cells in culture express types I, III, IV, and V collagen, ~95% of the total collagen synthesized is type I, which has been identified in both the secreted and cell-associated fractions, and it has been suggested that the process of cell culture induces an inflammatory or sclerosing phenotype in the glomerular mesangial cell (19, 39). Induction of type I collagen in the glomerular mesangium has been well demonstrated in experimental models of glomerulosclerosis. For instance, in the Alb/TGF-beta 1 transgenic mice that develop glomerulosclerosis, markedly increased immunostaining of type I collagen, and type III collagen to a lesser degree, is seen in the glomerulus within capillary loops and the mesangium, whereas near normal expression of type IV collagen is observed (30).

Despite the ever-growing body of data demonstrating TGF-beta 1 effects on ECM induction and development of glomerulosclerosis, the intracellular signaling mechanisms by which TGF-beta 1 stimulates this process remain poorly understood. Molecular cloning has revealed that biological actions of TGF-beta 1 are mediated by two transmembrane Ser/Thr kinases, types I and II, which are coexpressed by most cells, including glomerular mesangial cells (9, 28). The initiation of signaling involves the binding of TGF-beta 1 to TGF-beta type II receptor (Tbeta R-II), a constitutively active Ser/Thr kinase, resulting in the recruitment and phosphorylation of TGF-beta type I receptor (Tbeta R-I) to produce a heteromeric signaling complex, which in turn activates downstream signaling pathways (40, 44). Recently, an emerging body of evidence implicates the intracellular mitogen-activated protein kinase (MAPK) as an important TGF-beta 1 signaling pathway (1, 6, 20).

The MAPK is a major signaling system used by eukaryotic cells to transduce extracellular signals to intracellular responses (38, 41). The activation of the MAPKs requires a well-coordinated cascade of three protein kinase reactions that transduce signals by sequential phosphorylation and activation of the next kinase in their respective pathways (38). The MAPK cascades display evolutionary conservation and are implicated to play essential roles in the signal transduction of many biological events such as the regulation of cell growth, differentiation, and apoptosis and cellular responses to environmental stresses. The best characterized of the MAPKs is the ERK cascade, which consists of the extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) and which is protypically activated by mitogenic stimuli (31). The other two MAPK family members, the stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) and the p38 MAPK, are activated predominantly by cellular stresses or inflammatory signals (13, 26).

TGF-beta 1 has been demonstrated in various cell types to be capable of activating each of the three major MAPK superfamily members. We have recently demonstrated the requirement of the ERK pathway in signaling anti-apoptotic effects of TGF-beta 1 to promote cell survival of macrophages (6). This investigation was thus undertaken to determine whether TGF-beta 1 activates specific MAPK signaling pathway(s) in cultured rat mesangial cells and whether this MAPK activation mediates TGF-beta 1-induced stimulation of type I collagen, using the strategy of dominant-negative inhibition of TGF-beta 1 signaling receptors. We have successfully used this strategy previously to inhibit Tbeta R-II-mediated signaling in glomerular endothelial cells and in macrophages (6, 8). In the present study, we show that TGF-beta 1 rapidly induced the phosphorylation of p38 MAPK in cultured rat glomerular mesangial cells. However, transfection with dominant-negative mutant of Tbeta R-II (Tbeta R-IIM) failed to block the TGF-beta 1-induced p38 MAPK phosphorylation and the TGF-beta 1-stimulated pro-alpha 1(I) chain of type I procollagen mRNA and cellular protein synthesis. Moreover, a specific chemical inhibitor of p38 MAPK, SB-203580, blocked TGF-beta 1-induced pro-alpha 1(I) collagen mRNA. Our data, taken together, indicate that TGF-beta 1 directly and rapidly activates the p38 MAPK and that the p38 MAPK functions as a component in TGF-beta 1 signaling of pro-alpha 1(I) collagen induction in mesangial cells.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents. Recombinant human TGF-beta 1 was obtained from R & D Systems (Minneapolis, MN). The phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, and phospho-activating transcription factor-2 (ATF-2) (Thr71) rabbit polyclonal antibodies, purified ATF-2 fusion protein, phospho-p44/42 MAPK (Thr202/Tyr204), phospho-SAPK/JNK (Thr183/Tyr185), and SAPK/JNK rabbit polyclonal antibodies were purchased from New England Biolabs (Beverly, MA). The specific inhibitor of p38 MAPK, SB-203580, was obtained from Calbiochem (San Diego, CA), and the MAPK kinase (MEK) 1 inhibitor PD-098059 was from New England Biolabs.

Constructs. A truncated TGF-beta type II receptor construct (Tbeta R-IIM), lacking the cytoplasmic Ser/Thr kinase domain but containing the full transmembrane-spanning and extracellular domains, was generated by PCR using a rat Tbeta R-II cDNA as the template, as previously described (8), with the following modifications. Primer sequences were sense primer 5'-GTTTGAATTCGACGGGGGCTGCCATG-3' and antisense primer 5'-TTCTACTGTTACCGTGTCCATCACCACCATCATCACTAGCGGCCGCGGGCC-3' and contained the sequences for the restriction enzymes EcoR I and Not I, respectively (underlined), for directional cloning and a stop codon in the antisense primer. The PCR amplified product was cloned into pcDNA3 (Invitrogen). Confirmation that the clone contained correct directionality and in-frame sequences of the PCR product was obtained by complete sequencing using the dideoxy chain termination technique with Sequenase 2.0 (Amersham).

A truncated Tbeta R-I construct (Tbeta R-IM) that lacks the cytoplasmic GS and kinase domains but contains the transmembrane and extracellular domains was generated by PCR using rat Tbeta R-I cDNA (7, 10) as a template; a similar strategy as described for the kinase-deleted Tbeta R-II construct (Tbeta R-IIM) was used and cloned in pcDNA3. Primer sequences used to amplify the Tbeta R-IM construct were sense primer 5'-ACGGGGTACCCCATGGAGGCGGC GTCGGCTGCTTT-3' and antisense primer 5'-GCGCTCTAGAGCGCCTATGGCACGCGGTG GTGAATGACA-3' and contained the sequences for the restriction enzymes Kpn I and Xba I, respectively (underlined), for directional cloning and a stop codon in the antisense primer.

Cell culture. Glomerular mesangial cells were isolated from the renal cortex of ~150-g Sprague-Dawley rats as previously described (9), using the standard sieving technique with the following modifications. After collagenase digestion, the cells were plated in RPMI 1640 medium (GIBCO-BRL), supplemented with 20% FBS (HyClone), 5 U/ml penicillin, and 5 µg/ml streptomycin, and incubated in a humidified atmosphere of 5% CO2 and 95% air at 37°C. After 96 h, the serum in the medium was changed to 15% FBS and cells propagated. All newly isolated cells were characterized by immunostaining for anti-vimentin (Dako) and anti-myosin antibodies (Zymed) and negative staining for cytokeratin (Boehringer-Mannheim) and von Willebrand's factor (Dianova) as well as negative fluorescent acetylated low-density lipoprotein uptake (Biomedical Technologies). Cell labeling was performed according to the manufacturer's protocol. Once the cells were established in culture, they were maintained in RPMI 1640 with 15% FBS, 5 U/ml penicillin, and 5 µg/ml streptomycin. Cells between passages 5 and 15 were used for the experiments. In experiments involving exogenous TGF-beta 1 treatment, cells grown to 90% confluence were placed in RPMI medium containing 0.5% FBS in the presence or absence of human TGF-beta 1 (R & D). In experiments using the p38 and MEK1 inhibitors, cells were preincubated for 1 h in the absence or presence of 10 µM SB-203580 or PD-098059, respectively, before treatment with or without exogenous TGF-beta 1.

Stable transfection of rat mesangial cells. To generate clones that stably expressed Tbeta R-IIM or Tbeta R-IM, cells were transfected using Lipofectin (GIBCO-BRL) as follows. Cells grown to ~50% confluency on six-well plates were incubated with 1-5 µg DNA (Tbeta R-IIM or Tbeta R-IM constructs ligated in pcDNA3) in RPMI and 5-10 µl Lipofectin suspension at 37°C in a 5% CO2 atmosphere. Control cells were incubated with pcDNA3 (not containing Tbeta R-IIM) and Lipofectin. After a 24-h incubation, RPMI medium containing 20% FBS was added to each well to make a final concentration of 10% FBS and was incubated for another 24 h. Next, the DNA- and Lipofectin-containing medium was changed to 10% FBS in RPMI (no antibiotics) and incubated for 24 h. To select for stable transfectants, cells were treated with 400 µg/ml geneticin (GIBCO-BRL) in RPMI medium containing 15% FBS, and the medium was changed every 2-3 days. G418-resistant clones emerging ~14 days after lipofection were subcloned using ring cylinders, expanded, and maintained in RPMI medium containing 15% FBS, 200 µg/ml geneticin, 5 U/ml penicillin, and 5 µg/ml streptomycin. Four independent stably transfected clones containing Tbeta R-IIM, two clones containing Tbeta R-IM, and three clones containing empty vector were expanded and used in all subsequent experiments, and data from representative clones are presented. Confirmation of respective mRNA expression of Tbeta R-IIM and Tbeta R-IM was obtained by RT-PCR and Northern analyses.

Northern blot analysis. Total RNA was isolated by cell lysis with TRIzol (GIBCO-BRL) according to the manufacturer's instructions and was size-fractionated (10 µg/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 ultravioletly linked to a nylon membrane (Gene Screen Plus; Dupont). The blots were prehybridized for 2 h at 65°C in 1% BSA (Sigma), 7% SDS, 0.5 M phosphate buffer (pH 7.0), and 1 mM EDTA (pH 8.0) and hybridized overnight in the same solution containing the appropriate 32P-labeled probe at 65°C. The blots were then washed two times in solution containing 0.5% BSA, 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 four 15-min washes with 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 5 film. The Tbeta R-II and Tbeta R-I cDNA probes were previously described 2.8-kb rat Tbeta R-II full-length cDNA and Xba I fragment of rat Tbeta R-I cDNA (9, 10). The human pro-alpha 1(I) collagen, fibronectin 1, and plasminogen activator inhibitor (PAI)-1 cDNA probes were obtained from ATCC. To control for relative equivalence of RNA loading, the blots were hybridized with 32P-labeled beta -actin cDNA (Clontech) probe or an oligonucleotide probe corresponding to the 18S rRNA as previously described (10).

Covalent labeling of TGF-beta receptors. Cells grown on 100-mm plates were affinity labeled with 400 pM 125I-labeled TGF-beta 1 (Amersham) in the presence or absence of 100 nM unlabeled TGF-beta 1 (R & D) and covalently cross-linked using disuccinimidyl suberate (Pierce), as previously described (8, 9). The cells were subsequently lysed with 100 µl of 1% Triton X-100, 10 mM Tris (pH 7.4), 1 mM EDTA, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ ml aprotinin, and 10 µg/ml pepstatin, and sample loading buffer (sucrose, 0.01% bromphenol blue, 2% beta -mercaptoethanol, and 5 mM EDTA) was added (1:1 vol/vol) and boiled for 5 min followed by 12% SDS-PAGE. A sample of rainbow-colored protein molecular weight markers (Amersham) was loaded in an adjacent lane. The gel was stained with Coomassie brilliant blue (Bio-Rad) to visualize equivalence in protein loading and destained before autoradiography.

[3H]thymidine and [3H]leucine incorporation. Cells (104) were plated in 24-well dishes, incubated in medium containing 15% FBS, and grown to subconfluence. Medium was then changed to serum-free RPMI for 48 h, followed by incubation in 0.5% FBS in the presence or absence of TGF-beta 1 (2 ng/ml). After 45 h, the medium was removed, and cells were exposed for 3 h to 1 µCi/ml [3H]thymidine in RPMI 1640 medium containing 2% bovine platelet-poor plasma-derived serum at 37°C. The cells were washed three times with RPMI and then extracted three times with ice-cold 6% TCA followed by solubilization in 1 N NaOH and were counted in a Packard liquid scintillation counter. For [3H]leucine incorporation, [3H]leucine (1 µCi/ml) was added to the cells 40 h after initiation of TGF-beta 1 treatment. At the end of an additional 8-h incubation, the supernatant was removed, and the cell proteins were precipitated on the plate with ice-cold 6% TCA and washed three times with fresh TCA. The cells were then washed one time with water-saturated ether, dried, solubilized in 1 N NaOH, and subjected to scintillation counting. For all experiments, cell numbers were determined in replicate plates. Data shown are mean values of triplicate determinations ± SE, corrected for cell number, and are expressed as percentage of baseline incorporation in cells not treated with TGF-beta 1. Each of the experiments was repeated three times (n = 3) and gave essentially the same results.

Western blot analysis. Total cellular extracts were obtained by lysis of cells in buffer containing 1% Nonidet P-40, 20 mM Tris (pH 8.0), 150 mM NaCl, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml aprotinin. Protein concentrations of the cell lysates were determined by Coomassie blue dye-binding assay (Bio-Rad). An equal volume of 2× SDS loading buffer [0.125 mM Tris · HCl (pH 7.4), 4% SDS, and 20% glycerol] was added, and the samples were boiled for 5 min. Protein samples (100 µg) were resolved on a 12% SDS-PAGE and then electroblotted onto nitrocellulose membranes (Bio-Rad). The membranes were incubated with phospho-p38 MAPK, phospho-p44/42 MAPK, or phospho-SAPK/JNK rabbit polyclonal antibodies (1:1,000) for 1.5 h followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit antibody for 1.5 h. Signal development was carried out using LumiGLO (New England Biolabs) and was exposed to X-ray film. All of the assays were repeated three times. As a control, all of the blots were subjected to immunoblotting for corresponding nonphospho-p38 MAPK, p44/42 MAPK, or SAPK/JNK rabbit polyclonal antibodies.

p38 MAPK activity assays. Kinase assays were performed as described by Marais et al. (27) with minor modifications. Briefly, cells were lysed in buffer containing 20 mM Tris · HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta -glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM PMSF and sonicated. Protein concentrations were determined as described for Western analysis. Total protein (200 µg) samples were incubated with phospho-p38 MAPK rabbit polyclonal antibody (1:50) overnight on a rocker at 4°C. Protein A-Sepharose beads (Pharmacia Biotech) were then added to immunoprecipitate (IP) the activated p38 MAPK complex. The IP pellets were incubated with 1 µg ATF-2 fusion protein in the presence of 100 µM ATP and a kinase buffer containing 25 mM Tris · HCl (pH 7.5), 5 mM beta -glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, and 10 mM MgCl2. The reaction was terminated with SDS loading buffer [62.5 mM Tris · HCl (pH 6.8), 2% wt/vol SDS, 10% glycerol, 50 mM DTT, and 0.1% wt/vol bromphenol blue]. The samples were analyzed on a 12% SDS-PAGE and electroblotted as described for Western blot. p38 MAPK activity was assayed by detection of phosphorylated ATF-2 using a phospho-ATF-2 rabbit polyclonal antibody (1:1,000). After overnight incubation with the primary antibody at 4°C, the membrane was incubated for 1 h with an HRP-conjugated anti-rabbit secondary antibody (1:2,000) at room temperature with gentle rocking. The proteins were subsequently detected using LumiGLO (New England Biolabs) and exposed to X-ray film. All of the assays were repeated three times.

Statistical analysis. Statistical significance of the experimental data for the [3H]thymidine and [3H]leucine incorporation assays was determined by the Student's t-test for paired data. P values <0.05 were considered significant. Data are presented as means ± SE of triplicate determinations.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Expression of dominant-negative Tbeta R-IIM in rat mesangial cells. First, to demonstrate that the stably transfected rat mesangial cells expressed Tbeta R-IIM mRNA, Northern blot analysis of total RNA isolated from wild-type and transfected mesangial cells was performed using the Tbeta R-II cDNA probe. As shown in Fig. 1A, a 5.5-kb band was detected in all cells, corresponding to endogenously expressed Tbeta R-II mRNA. However, an additional band of ~1.8 kb in size was detected only in the cells transfected with Tbeta R-IIM, and not in wild-type cells or in mock-transfected cells with empty vector pcDNA3. Next, cell surface expression of Tbeta R-IIM protein and ligand binding were determined by affinity cross-linking with 125I-labeled TGF-beta 1. As shown in Fig. 1B, an intensely labeled band of ~46 kDa in molecular mass was detected in the cells transfected with Tbeta R-IIM, in addition to the ~89- and 69-kDa bands corresponding to endogenously expressed Tbeta R-II and Tbeta R-I, respectively.


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Fig. 1.   Expression of dominant-negative transforming growth factor-beta (TGF-beta ) type II receptor (Tbeta R-IIM) in rat mesangial cells. A: Northern analysis of total RNA (10 µg/lane) from wild-type (WT) rat mesangial cells (lane 1), cells transfected with empty vector pcDNA3 (lane 2), or Tbeta R-IIM (lane 3) hybridized with a 32P-labeled rat Tbeta R-II cDNA probe. A 5.5-kb mRNA was detected, corresponding to wild-type Tbeta R-II in all lanes. In lane 3, an additional 1.8-kb mRNA was observed, corresponding to Tbeta R-IIM. Similar densities for the beta -actin signals indicate approximate equivalence of RNA loading. B: affinity cross-linking of 125I-labeled TGF-beta 1 to cell surface receptors. Lanes 1 and 2, control mesangial cells transfected with empty vector pcDNA3 cross-linked with 125I-labeled TGF-beta 1 in the presence or absence of unlabeled TGF-beta 1, respectively. Lanes 3 and 4, transfected mesangial cells carrying Tbeta R-IIM in the presence or absence of unlabeled TGF-beta 1, respectively. Specifically labeled bands are observed at ~98 and 70 kDa corresponding to wild-type Tbeta R-II and Tbeta R-I, respectively. In addition, a specifically labeled band of ~46 kDa is observed only in the transfected cells expressing Tbeta R-IIM.

Regulated expression of TGF-beta receptor mRNA by TGF-beta 1. We next determined whether the expression of TGF-beta receptors in rat mesangial cells was regulated by TGF-beta 1. Northern blot analysis of total RNA isolated from control mesangial cells transfected with empty vector pcDNA3 demonstrated a dose-dependent decrease in Tbeta R-II mRNA abundance (Fig. 2A), but not Tbeta R-I mRNA (Fig. 2B), in response to treatment with exogenous TGF-beta 1. In mesangial cells transfected with dominant-negative Tbeta R-IIM, this dose-dependent decrease in Tbeta R-II mRNA expression was blocked. The lower molecular mass mRNA corresponding to Tbeta R-IIM was again detected only in the cells transfected with Tbeta R-IIM. Thus our data demonstrate differential regulation of the expression of the two signaling receptors by TGF-beta 1.


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Fig. 2.   Regulation of Tbeta R-II and TGF-beta type I receptor (Tbeta R-I) mRNA expression by TGF-beta 1 in rat mesangial cells. A: Northern analysis of Tbeta R-II mRNA expression in transfected mesangial cells carrying empty vector pcDNA3 or Tbeta R-IIM in the absence (lanes 1 and 7) or in the presence of increasing doses of exogenous TGF-beta 1, as indicated, for 24 h. Additional 1.8-kb mRNA corresponding to Tbeta R-IIM is noted in the Tbeta R-IIM transfected cells. B: same blot as in A probed with Tbeta R-I cDNA. C: similar densities for the 18S rRNA signals indicate approximate equivalence of RNA loading.

Stimulation of pro-alpha 1(I) collagen mRNA expression by TGF-beta 1. TGF-beta 1 induces ECM protein synthesis in mesangial cells. As shown in Fig. 3A, treatment with exogenous TGF-beta 1 increased pro-alpha 1(I) collagen mRNA expression in a dose-dependent fashion in control mesangial cells transfected with empty vector pcDNA3. Similarly, exogenous TGF-beta 1 also increased fibronectin (Fig. 3B) and PAI-1 (Fig. 3C) mRNA expression in a dose-dependent fashion in the empty vector-transfected cells. Remarkably, in mesangial cells transfected with dominant-negative Tbeta R-IIM, a dose-dependent increase in pro-alpha 1(I) collagen mRNA expression was also observed upon treatment with exogenous TGF-beta 1. However, TGF-beta 1 induced expression of fibronectin and PAI-1 mRNA was inhibited in cells transfected with Tbeta R-IIM.


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Fig. 3.   Dose-dependent induction pro-alpha 1(I) collagen mRNA expression by TGF-beta 1 in cells transfected with empty vector pcDNA3 or with dominant-negative Tbeta R-IIM. Northern analysis of mRNA from mesangial cells in the presence of increasing doses of exogenous TGF-beta 1 with pro-alpha 1(I) collagen (A), fibronectin (FN; B), and plasminogen activator inhibitor (PAI)-1 (C) cDNA probes. Total RNA from mesangial cells transfected with empty vector pcDNA3 or Tbeta R-IIM, in the absence (lanes 1 and 7) or in the presence of increasing doses of exogenous TGF-beta 1, as indicated, for 24 h. Normalization for RNA loading was by 18S rRNA signals (D).

Effect of TGF-beta 1 on mesangial cell [3H]thymidine and [3H]leucine incorporation. Treatment of wild-type rat mesangial cells in culture with exogenous TGF-beta 1 (2 ng/ml for 48 h) significantly inhibited [3H]thymidine incorporation to 18 ± 10% of baseline (Fig. 4A). Inhibition of [3H]thymidine incorporation was similarly observed in empty vector transfected cells treated with exogenous TGF-beta 1. Conversely, treatment with exogenous TGF-beta 1 increased [3H]leucine incorporation to 140 ± 9% of baseline in wild-type mesangial cells (Fig. 4B) and in empty vector transfected cells. In transfected cells carrying Tbeta R-IIM, the inhibitory effect on [3H]thymidine incorporation by exogenous TGF-beta 1 was blocked (Fig. 4A) but the stimulatory effect on [3H]leucine incorporation was not blocked (Fig. 4B).


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Fig. 4.   Effects of TGF-beta 1 on mesangial cell proliferation and protein synthesis. Wild-type rat mesangial cells (filled bars) and mesangial cells transfected with dominant-negative mutant receptor construct Tbeta R-IIM (hatched bars) were incubated in the absence or presence of exogenous TGF-beta 1 (2 ng/ml for 48 h). Incorporation of [3H]thymidine (A) and [3H]leucine (B) was determined. Data represent mean values of triplicate determinations and are expressed as %baseline ± SE. *P < 0.005, Tbeta R-IIM transfected cells compared with wild-type cells, Student's t-test, n = 3 experiments.

Activation of p38 MAPK in rat mesangial cells by TGF-beta 1. Previous reports, including studies from our laboratory, have suggested that TGF-beta 1 exerts its biological effects via the MAPK signaling pathway in certain cell types (1, 6, 20). We first determined the levels of p38 MAPK protein expression in rat mesangial cells treated with exogenous TGF-beta 1 (2 ng/ml) by Western analyses, using phospho-p38 MAPK and p38 MAPK antibodies. The phospho-p38 MAPK antibodies detect specifically the phosphorylated forms of p38 MAPK, whereas the p38 MAPK antibodies detect total (phosphorylation state-independent) p38 MAPK proteins. As shown in Fig. 5, increases in phosphorylation of p38 MAPK were observed within 30 min of stimulation with exogenous TGF-beta 1 in the wild-type rat mesangial cells. Moreover, the time-dependent increases in phosphorylated p38 MAPK were also observed in mesangial cells transfected with dominant-negative Tbeta R-IIM but not in cells transfected with dominant-negative Tbeta R-IM.


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Fig. 5.   Activation of p38 MAPK by TGF-beta 1 in rat mesangial cells. Lysates from wild-type rat mesangial cells (WT) or cells transfected with dominant-negative Tbeta R-IIM or with Tbeta R-IM incubated in the absence (Ctl) or in the presence of exogenous TGF-beta 1 (2 ng/ml) for the indicated times were subjected to Western analysis using phospho-p38 mitogen-activated protein kinase (MAPK; top in each set) or p38 MAPK (bottom in each set) antibodies as described under EXPERIMENTAL PROCEDURES. Bands are detected corresponding to phosphorylated forms of p38 MAPK (top in each set) and total p38 MAPK (bottom in each set).

We next examined whether the induction of p38 MAPK phosphorylation by TGF-beta 1 was associated with an increase in p38 MAPK activity using an immunocomplex kinase assay. Lysates from wild-type rat mesangial cells incubated in the absence or presence of exogenous TGF-beta 1 (2 ng/ml) were first subjected to immunoprecipitation using phospho-p38 MAPK antibodies. The resultant active p38 MAPK IP was then allowed to phosphorylate ATF-2 fusion protein, and kinase activity was assayed by the detection of phosphorylated ATF-2 by Western blot analysis. As was the case with the TGF-beta 1-induced increase in phosphorylated p38 MAPK (Fig. 5), an increase of the phosphorylated form of ATF-2 was observed within 30 min of stimulation with exogenous TGF-beta 1 (2 ng/ml) in wild-type rat mesangial cells (Fig. 6).


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Fig. 6.   Immunocomplex kinase assay. Lysates from wild-type rat mesangial cells incubated in the absence (Ctl) or in the presence of exogenous TGF-beta 1 (2 ng/ml) for the indicated times were analyzed for p38 MAPK activity as described under EXPERIMENTAL PROCEDURES. p38 MAPK activity was assayed by immunoprecipitation with phospho-p38 MAPK antibody followed by detection of phosphorylation of ATF-2 fusion protein by Western blotting with phospho-ATF-2 (Thr71) antibody.

Induction of ERK1/ERK2 phosphorylation in rat mesangial cells by TGF-beta 1. To determine whether TGF-beta 1 activated the ERK1/ERK2 pathway in cultured rat mesangial cells, we performed Western analyses using phospho-p44/42 MAPK polyclonal antibodies. The phospho-p44/42 MAPK antibodies detect specifically the phosphorylated forms of ERK1/ERK2 proteins. As shown in Fig. 7, increases in phosphorylation of ERK1 and ERK2 proteins were observed in wild-type mesangial cells as early as 30 min after stimulation with exogenous TGF-beta 1 (2 ng/ml). However, this rapid induction of ERK1/ERK2 phosphorylation was blocked in the Tbeta R-IIM-transfected mesangial cells.


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Fig. 7.   Induction of extracellular signal-regulated kinase (ERK) 1/ERK2 phosphorylation by TGF-beta 1 in rat mesangial cells. Cell lysates from wild-type and Tbeta R-IIM-transfected rat mesangial cells treated with exogenous TGF-beta 1 (2 ng/ml) for the indicated times were subjected to Western analysis using phospho-p44/42 MAPK (top) or p44/42 MAPK (bottom) antibodies as described under EXPERIMENTAL PROCEDURES. In each lane, two bands are detected to ERK1/ERK2. Control cell extracts from the p44/42 MAPK assay kit (New England Biolabs) served as a positive control (+Ctl).

Effect of TGF-beta 1 on SAPK/JNK phosphorylation in rat mesangial cells. We also determined whether TGF-beta 1 activated the SAPK/JNK pathway by Western analyses using phospho-SAPK/JNK rabbit polyclonal antibodies in cultured rat mesangial cells. In contrast to the p38 MAPK and the ERK1/ERK2, there were no appreciable increases in the phosphorylation of SAPK/JNK within the same time periods of TGF-beta 1 treatment (Fig. 8). As a control, the same blots were immunoblotted with SAPK/JNK rabbit polyclonal antibodies to detect levels of total (phosphorylation state-independent) SAPK/JNK proteins.


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Fig. 8.   TGF-beta 1 does not activate SAPK/JNK in rat mesangial cells. Cell lysates from wild-type rat mesangial cells, treated with exogenous TGF-beta 1 (2 ng/ml) for the indicated times, were subjected to Western analysis using phospho-SAPK/JNK (top) or SAPK/JNK (bottom) antibodies as described under EXPERIMENTAL PROCEDURES. Control cell extracts from the SAPK/JNK assay kit (New England Biolabs) served as a positive control.

Inhibition of TGF-beta 1-induced pro-alpha 1(I) collagen expression by SB-203580, a specific inhibitor of p38 MAPK. Given that TGF-beta 1 induced increases in both pro-alpha 1(I) collagen mRNA expression and p38 MAPK phosphorylation and transfection with a dominant-negative Tbeta R-IIM failed to inhibit both of these TGF-beta 1-effects, we posed the question whether the p38 MAPK pathway was involved in mediating TGF-beta 1-induced pro-alpha 1(I) collagen mRNA expression. In the presence of SB-203580, a specific inhibitor of p38 MAPK, exogenous TGF-beta 1 was unable to induce pro-alpha 1(I) collagen mRNA expression in either wild-type (WT) or Tbeta R-IIM-transfected mesangial cells (Fig. 9), whereas, PD-098059, a specific inhibitor of MEK1 that prevents the activation of ERK1/ERK2 pathway, failed to inhibit TGF-beta 1-induced pro-alpha 1(I) collagen mRNA expression. At 10 µM concentrations of PD-098059 in mesangial cells, we have observed inhibition of ERK activation by TGF-beta 1. Moreover, in cells transfected with a dominant-negative Tbeta R-IM, TGF-beta 1-induced pro-alpha 1(I) collagen mRNA expression was inhibited as was TGF-beta 1-induced p38 MAPK phosphorylation.


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Fig. 9.   Effect of p38 MAPK inhibitor SB-203580 on TGF-beta 1-induced pro-alpha 1(I) collagen mRNA expression in rat mesangial cells. Total RNA from wild-type, Tbeta R-IIM-transfected, and Tbeta R-IM-transfected rat mesangial cells pretreated with (+) or without (-) SB-203580 (10 µM) or PD-098059 (10 µM) and incubated in the presence (+) or absence (-) of exogenous TGF-beta 1 (2 ng/ ml) for 24 h were subjected to Northern blot hybridization with 32P-labeled pro-alpha 1(I) collagen cDNA probe. SB-203580 is a highly specific inhibitor of p38 MAPK and does not inhibit the SAPK/JNK and ERK1/ERK2. PD-098059 is a specific inhibitor of mitogen/extracellular signal-regulated kinase 1 and prevents downstream activation of ERK1/ERK2.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The critical role of TGF-beta 1 in the pathogenesis of glomerulosclerosis has been well recognized, and although it is well known that TGF-beta 1 stimulates the transcription and synthesis of ECM proteins, including type I collagen, the intracellular signaling mechanisms by which TGF-beta 1 stimulates this process remain poorly understood. This study examined the role of the MAPK signaling cascades in mediating TGF-beta 1 responses in rat glomerular mesangial cells using dominant-negative inhibition of TGF-beta signaling receptors. Given that heteromeric complex formation and phosphorylation of Tbeta R-I by Tbeta R-II are thought to be essential for propagation of TGF-beta 1 signals, a mutant kinase-deleted Tbeta R-IIM is predicted to inhibit Tbeta R-II-dependent signals by virtue of competition for Tbeta R-I binding (5, 8, 43). We have previously used this strategy of dominant-negative inhibition of Tbeta R-II-mediated signaling in glomerular endothelial cells and in macrophages (6, 8). Thus we stably transfected cultured rat glomerular mesangial cells with dominant-negative mutant Tbeta R-IIM. The mRNA expression of Tbeta R-IIM in the transfected mesangial cells was demonstrated by Northern blot analysis (Fig. 1A). We next confirmed that the Tbeta R-IIM protein was expressed at the cell surface and that it strongly bound the TGF-beta 1 ligand by affinity labeling and cross-linking with 125I-labeled-TGF-beta 1 (Fig. 1B). We then examined the effects on TGF-beta 1 responses by dominant-negative inhibition of the Tbeta R-II-dependent signaling pathway in mesangial cells.

We first observed differential regulation of the expression of the two signaling receptors by TGF-beta 1 in mesangial cells. We had previously shown that the Tbeta R-II mRNA abundance in mesangial cells was reduced within 24 h by exogenous TGF-beta 1, suggesting that TGF-beta 1 negatively regulates expression of its own receptor (9). The present study demonstrated that exogenous TGF-beta 1 downregulated Tbeta R-II mRNA in a dose-dependent fashion, an effect blocked by transfection of Tbeta R-IIM but not Tbeta R-I mRNA (Fig. 2). Thus modulation of relative levels of expression of the two receptors may play an important role in determining how cells interpret extracellular signals.

We next observed dose-dependent induction of PAI-1 mRNA and fibronectin mRNA by exogenous TGF-beta 1 in the wild-type and empty vector pcDNA3-transfected control mesangial cells. As predicted, both of these TGF-beta 1 effects were inhibited in transfected cells carrying dominant-negative Tbeta R-IIM. However, pro-alpha 1(I) collagen mRNA increased in response to exogenous TGF-beta 1 in both control and Tbeta R-IIM-transfected cells (Fig. 3). Moreover, transfection of the dominant-negative Tbeta R-IIM in mesangial cells blocked the growth inhibitory effects of TGF-beta 1, as determined by [3H]thymidine incorporation, but failed to block the induction of protein synthesis in mesangial cells, as determined by [3H]leucine incorporation (Fig. 4). Remarkably, this apparent differential inhibition of TGF-beta 1 responses by Tbeta R-IIM was also noted when we examined the effects of TGF-beta 1 on activation of the major MAPK pathways.

As shown in Fig. 5, treatment of mesangial cells with exogenous TGF-beta 1 resulted in a rapid induction of p38 MAPK phosphorylation that was not inhibited by transfection of the dominant-negative Tbeta R-IIM. In contrast, exogenous TGF-beta 1 also rapidly induced phosphorylation of ERK1/ERK2 in mesangial cells, but this induction was blocked by the dominant-negative Tbeta R-IIM (Fig. 7). Thus our findings indicate that overexpression of the dominant-negative Tbeta R-IIM selectively inhibits some TGF-beta 1 effects, such as growth inhibition and ERK1/ERK2 activation, but not its induction of pro-alpha 1(I) collagen mRNA or p38 MAPK activation. Two earlier studies had suggested seemingly separate signaling pathways for TGF-beta 1-mediated growth inhibition and ECM induction. Chen et al. (5) overexpressed a dominant-negative Tbeta R-II in Mv1.Lu cells and reported abolition of TGF-beta 1-mediated inhibition of both cell proliferation and pRB phosphorylation but not TGF-beta 1-mediated induction of ECM. Saitoh et al. (37) observed that phosphorylation of Ser172 and Thr176 residues of Tbeta R-I was essential for the TGF-beta 1-mediated growth inhibitory effect but not for TGF-beta 1 induction of ECM in Mv1.Lu cells. Indeed, the apparent disparity observed in all of these studies may be due to different threshold levels of receptor activity (or receptor protein density) and the signal transduction requirement for the various TGF-beta 1 actions. Alternatively, it is also possible that the differential inhibition by Tbeta R-IIM is because certain TGF-beta 1 responses involve Tbeta R-I-mediated signals that do not require Tbeta R-II kinase-dependent phosphorylation. For instance, the Ser172 and Thr176 residues of Tbeta R-I, which were shown to be essential for growth inhibition by TGF-beta 1, are not phosphorylated (37). A molecular mechanism remains to be elucidated in this case.

In contrast to the ERK pathway, p38 MAPK was not activated primarily by mitogens but by cellular stresses and inflammatory cytokines in various cell types. The p38 MAPK was first isolated as a 38-kDa protein that was rapidly tyrosine phosphorylated by bacterial lipopolysaccharide stimulation (15). p38 MAPK activation has also been observed in response to other environmental stress such as heat shock and hyperosmolarity and in inflammatory responses such as tumor necrosis factor-alpha -stimulated neutrophils and human umbilical vein endothelial cells (11, 34, 47). In addition, a number of growth factors and cytokines have now also been shown to be capable of inducing the activation of p38 MAPK, including granulocyte macrophage colony-stimulating factor, fibroblast growth factor, and insulin-like growth factor, as well as TGF-beta 1 (33). Despite growing evidence that TGF-beta 1 induces activation of p38 MAPK in a number of cell types such as fibroblasts, neutrophils, and smooth muscle cells, paucity of data exists for glomerular mesangial cells (17, 21, 35). In the present studies, given the rapid kinetics observed for TGF-beta 1-induced activation of p38 MAPK (within 15 min of TGF-beta 1 treatment) in mesangial cells, the effect of TGF-beta 1 on the p38 MAPK pathway is likely a direct one, perhaps through TGF-beta -activated kinase 1 (TAK1; see Refs. 16, 29, 46). Because, in our studies, the transfection of dominant-negative Tbeta R-IIM failed to inhibit TGF-beta 1-induced p38 MAPK phosphorylation, we sought to confirm that the p38 MAPK activation in mesangial cells was indeed TGF-beta 1 induced. Here, we used dominant-negative inhibition of Tbeta R-I by transfection of mesangial cells with a truncated Tbeta R-IM construct lacking the GS and the kinase domains. The activation of p38 MAPK by TGF-beta 1 was completely abrogated by dominant-negative Tbeta R-IM, indicating that signaling by Tbeta R-I is required for this TGF-beta 1 response.

Given that TAK1 can also lead to the activation of JNK and in a number of cell types, including Hep G2, Chinese hamster ovary, and Madin-Darby canine kidney cell lines, TGF-beta 1 has been shown to activate SAPK/JNK. We also determined if TGF-beta 1 induced SAPK/JNK activation (16, 42). However, no apparent activation of JNK was observed within the same time periods of TGF-beta 1 treatment in mesangial cells (Fig. 8). The failure of rapid activation of JNK by TGF-beta 1 was also observed in the studies by Hanafusa et al. (16) in C2C12 cells.

Based on our data that dominant-negative Tbeta R-IIM selectively inhibits some but not all TGF-beta 1 effects in mesangial cells and the associated findings that the Tbeta R-IIM failed to block TGF-beta 1-mediated induction of pro-alpha 1(I) collagen mRNA and protein synthesis, as well as activation of p38 MAPK, we posed the question whether the p38 MAPK pathway was involved in mediating TGF-beta 1-induced pro-alpha 1(I) collagen. We hypothesized that blockade of the p38 MAPK cascade would result in failure of TGF-beta 1 to induce pro-alpha 1(I) collagen mRNA. Thus we used a specific chemical inhibitor of the p38 MAPK cascade known as SB-203580. As shown in Fig. 9, in the presence of SB-203580, exogenous TGF-beta 1 was unable to induce pro-alpha 1(I) collagen mRNA expression in mesangial cells. In contrast, blockade of the ERK cascade by PD-098059, a specific inhibitor of MEK1 that prevents downstream activation of ERK1/ERK2, did not prevent TGF-beta 1 induction of pro-alpha 1(I) collagen mRNA. Thus our data demonstrate the critical involvement of the p38 MAPK in the TGF-beta 1 signaling pathway in glomerular mesangial cells. Moreover, our findings suggest that the p38 MAPK functions as a component in the signaling of pro-alpha 1(I) collagen induction by TGF-beta 1. Furthermore, as with our findings of inhibition of TGF-beta 1-induced p38 MAPK activation by dominant-negative Tbeta R-IM, we also observed inhibition of TGF-beta 1-induced pro-alpha 1(I) collagen mRNA expression in cells expressing the mutant Tbeta R-IM and indicate that signaling by Tbeta R-I is required also for this TGF-beta 1 response.

Reports in the literature suggesting a possible role of the p38 MAPK cascade in the pathogenesis of glomerulosclerosis such as in diabetic complications are only beginning to come forth. For instance, Igarashi et al. (22) recently reported activation of p38 MAPK in rat aortic smooth muscle cells by hyperglycemia and in aorta from streptozotocin-induced diabetic rats, implicating involvement of the p38 MAPK pathway in the development of diabetic vascular complications. Interestingly, their studies showed that even a moderately elevated glucose concentration (16.5 mM) significantly increased p38 MAPK but not ERK1/ERK2 activities. Increased levels of phosphorylated p38 MAPK were also detected in glomeruli isolated from rats with streptozotocin-induced diabetes compared with nondiabetic control rats and insulin-treated diabetic rats (14). In addition, activation of p38 MAPK was detected in isolated glomeruli exposed to high glucose concentrations of 25 mmol/l compared with 5.6 mmol/l (14). To our knowledge, this is the first demonstration of rapid activation of the p38 MAPK by TGF-beta 1 in glomerular mesangial cells, and the data suggest the MAPKs as important TGF-beta 1 signaling pathways involved in the tissue injury response. Our findings suggest that TGF-beta 1 induces pro-alpha 1(I) collagen expression via the p38 MAPK-dependent pathway. Furthermore, this TGF-beta 1-induced collagen expression requires Tbeta R-I-mediated signaling but is not dependent on Tbeta R-II kinase.


    ACKNOWLEDGEMENTS

This work was supported in part by National Institutes of Health (NIH) Grant R01 DK-57661-01, Grant-in-Aid no. 0051319T from the American Heart Association (AHA), and a Veterans Affairs Career Development Award to M. E. Choi. A. M. K. Choi was supported by NIH Grants R01 HL-55330, R01 HL-60234, and R01 AI-42365, and an AHA Established Investigator Award. B. Y. Chin was supported by a Laboratory Scholarship from Toxicological Sciences at the Johns Hopkins University School of Hygiene and Public Health.


    FOOTNOTES

Address for reprint requests and other correspondence: M. E. Choi, Univ. of Pittsburgh, Renal-Electrolyte Division, 3550 Terrace St., A919 Scaife Hall, Pittsburgh, PA 15261 (E-mail: choim{at}pitt.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 21 August 2000; accepted in final form 2 November 2000.


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