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NDT Advance Access published online on August 22, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn462
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Mycophenolic acid inhibits the autocrine PDGF-B synthesis and PDGF-BB-induced mRNA expression of Egr-1 in rat mesangial cells

Danuta Sabuda-Widemann, Bernd Grabensee, Christina Schwandt and Cornelia Blume

Department for Nephrology, Heinrich-Heine Universität, Düsseldorf, Germany

Correspondence and offprint requests to: Cornelia Blume, Klinik für Nephrologie, Heinrich-Heine Universität, Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany. Tel: +49511-5849434; Fax: +4911-5849446; E-mail: cornelia.blume{at}cityweb.de



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Uncontrolled mesangial cell (MC) proliferation within the context of glomerular disease contributes to the development of glomerulosclerosis. Mesangial autocrine growth factor stimulation has been described as a pathogenic factor. We investigated the effects of mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil (MMF), on proliferation factors of cultured rat MCs. MPA was tested on the expression of platelet-derived growth factor-B (PDGF-B) and its receptor β (PDGFR-β), the immediate early gene (IEG) c-fos and the early growth response gene-1 (Egr-1), and AP-1 activation.

Methods. Growth-arrested rat MCs were stimulated with 10% fetal calf serum (FCS) or 10–25 ng/ml platelet-derived growth factor-BB (PDGF-BB) in the presence or absence of MPA (0.019–10 µM) with or without guanosine (100 µM). MC proliferation was quantified by 5-bromo-2'-deoxyuridine (BrdU) incorporation and direct cell counting. Cytotoxicity of MPA was evaluated using the MTT and LDH tests. Protein expression of PDGF-B and its receptor PDGFR-β was quantified by western blot analysis. The effect of MPA on gene expression of PDGF-B, Egr-1 and c-fos was determined by the reverse transcriptase–polymerase chain reaction (RT–PCR). AP-1 activation was analysed by an electrophoretic mobility shift assay (EMSA).

Results. Exposure of MCs to MPA caused a concentration-dependent inhibition of FCS-induced cell proliferation (cell number increase) with an IC50 of 0.44 ± 0.03 µM and DNA synthesis with an IC50 of 0.52 ± 0.02 µM without cell cytotoxicity in the therapeutic range. MPA decreased the PDGF-B protein expression and mRNA self-induction of PDGF-B but did not alter the protein expression of PDGFR-β. MPA strongly inhibited the PDGF-BB-induced mRNA expression of Egr-1 decreasing to 7.6 ± 2.5% after 30 min (P ≤ 0.001) and to 4.7 ± 3.1% after 1 h (P ≤ 0.05), both being compared to the maximal expression induced by PDGF-BB. PDGF-BB-induced c-fos expression under MPA was unchanged after 30 min and decreased to 57 ± 26% after 1 h (n.s.). MPA treatment did not affect PDGF-BB-induced AP-1 activity determined after 1 h and 2 h. The inhibitory MPA effect on PDGF-BB-induced PDGF-B expression was not significantly restored by guanosine (56 ± 18% versus 32 ± 17% after 2 h, n.s.), and MPA inhibition of PDGF-BB-induced Egr-1 expression was not reversed by exogenous guanosine.

Conclusions. Treatment of cultured MCs with MPA inhibits MC proliferation correlating with a downregulation of the PDGF-B gene and protein expression and a suppression of Egr-1 mRNA expression. Since exogenous guanosine was not able to reverse the inhibitory MPA effect on PDGF-B and Egr-1 expression, we conclude that the antiproliferative effect of MPA on MCs may not solely depend on dGTP depletion but on a specific interference with the autocrine PDGF-B synthesis and Egr-1 expression of MCs.

Keywords: AP-1; Egr-1; mesangial cell proliferation; MPA; PDGF



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Mycophenolate mofetil (MMF), the morpholinoethyl ester of mycophenolic acid (MPA), is an immunosuppressive agent, which was first used for the prevention of transplant rejection. The antiproliferative action of MPA on lymphocytes results from the reversible inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH), which catalyzes the first reaction in the de novo synthesis of GMP. Inhibition of this enzyme leads to a depletion of the intracellular GTP and dGTP pools and to the suppression of cell mitosis of lymphocytes. As has been shown earlier, MPA exerts an antiproliferative effect on non-lymphatic cells such as vascular muscle cells or mesangial cells (MCs) [1–3].

This antiproliferative effect of MPA on MCs may contribute to the therapeutic use of its prodrug MMF in glomerulonephritis (GN) in patients, especially the mesangioproliferative forms. Increased proliferation of glomerular MCs and enhanced matrix deposition are characteristic features of several types of experimental and human GN as well as in chronic rejection of renal transplants [4]. Many inflammatory substances have been identified that induce MC proliferation, for instance, various peptide growth factors, cytokines or vasoactive amines. Among these, platelet-derived growth factor (PDGF) seems to be one of the most potent mitogens for glomerular MCs and is expressed in both experimental and human GN [5,6]. The observation that many growth factors or cytokines induce PDGF production by MCs suggests that PDGF may act in an autocrine manner to stimulate MC proliferation [7]. As reported, antagonism of the PDGF-B chain with neutralizing antibodies or PDGF-B aptamers [8] led to the reduction of MC proliferation and matrix accumulation in a rat model of mesangioproliferative nephritis, the anti-Thy-1.1 model. It has been shown by Terada et al. that the PDGF-B chain and β-receptor mRNA expression was significantly higher in glomeruli of patients with mesangioproliferative GN (e.g. IgA nephropathy) as compared with those in glomeruli of other GN forms or in normal glomeruli [9].

PDGF-BB binds to its receptor eliciting a broad spectrum of intracellular events including (1) dimerization and autophosphorylation of the PDGF receptor [5,10]; (2) activation of intracellular signal transduction pathways such as phosphatidylinositol (PI) 3-kinase and phospholipase C (PLC) {gamma}(1), and Ras–Raf–mitogen-activated protein kinase (MAPK) and MEK–ERK-1/2 (extracellular signal-regulated kinase) and JNK as well as (3) induction of transcription factors such as c-fos or early growth response gene-1 (Egr-1) [11].

It has been suggested by Hauser et al. [2] that the mechanism of the inhibiting action of MPA on MCs is caused by GTP depletion due to an inhibition of the de novo purine synthesis similar as in lymphatic cells. In contrast, it has been described by Morris [12] that the antiproliferative effect of MMF is not only dependent on the inhibition of IMPDH but also on the capacity of cells to synthesize purine nucleotides by the salvage pathway rather than the de novo pathway of guanosine synthesis. It is therefore possible that MPA effectively inhibits proliferation of non-lymphatic cells by other mechanism than IMPDH inhibition. The aim of this study was to examine whether MPA exerts specific effects on proliferation factors such as PDGF-B with its β receptor as well as c-fos and Egr-1 and the binding activity of AP-1.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals
MPA (Roche AG, USA) and guanosine (Sigma Aldrich Chemie, Germany) were solubilized in dimethyl sulfoxide (DMSO; Sigma Aldrich Chemie) with a final concentration of 0.1%. The cell culture media DMEM and RPMI 1640, fetal calf serum (FCS), penicillin/streptomycin solution and trypsin/EDTA were obtained from Gibco Life Technologies (Germany). Insulin and bovine serum albumin (BSA) for cell culture were procured from Sigma Aldrich Chemie. PDGF-BB (Cell Concept, Germany) was dissolved in phosphate-buffered saline (PBS; Biochrom, Germany) supplemented with 0.1% BSA. Chemicals for the reverse transcriptase–polymerase chain reaction (RT–PCR) were sourced by the following companies: First-Strand Buffer and M-MLV Reverse Transcriptase from Invitrogen Life Technologies; DTT from Sigma Aldrich Chemie; Random-Primer (pd(N)6), DNase/RNase-free BSA, dNTPs and RNase-free DNase from Amersham Pharmacia Biotechnology; Recombinant RNasin Ribonuclease Inhibitor from Promega; and HotStar Taq DNA Polymerase Kit (including enzyme, PCR-Buffer and Q-solution) from Qiagen (Hilden, Germany). All specific primers were synthesized by Interactiva (Germany). The primary antibodies for immunodetection were procured by the following companies: anti-PDGF-B polyclonal rabbit IgG and anti-β-tubulin polyclonal rabbit IgG from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-PDGFR-β monoclonal mouse IgG from BD Biosciences. The secondary antibodies horseradish peroxidase-conjugated, goat anti-rabbit IgG and sheep anti-mouse IgG were obtained from Santa Cruz Biotechnology. The protease inhibitors were sourced by Roche Molecular Biochemicals. [{gamma}32P]-deoxynucleotides were obtained from Amersham Pharmacia Biotechnology and T4 polynucleotide kinase was procured by BioLabs (New England). All other chemicals were obtained either from Merck (Darmstadt, Germany), Sigma (München, Germany), Roth (Karlsruhe, Germany) or Fluka (Buchs, Switzerland).

Cell culture
Rat glomerular MCs were cultured as previously described [13]. Cells were grown in the RPMI 1640 medium containing Glutamax-I, 25 mM HEPES and phenol red supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml insulin and 10% heat-inactivated FCS. MCs were used for the experiments between passages 10 and 20. Before MPA treatment and specific growth stimulation, MCs were growth arrested by serum deprivation in DMEM without FCS containing Glutamax-I, 4.5 g/l glucose and phenol red supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml insulin and 0.1% BSA.

Determination of DNA synthesis
A total of 5 x 103 MCs/well were seeded onto 96-well plates and grown to semiconfluence in RPMI with 10% FCS for 24 h. Cells were washed once with PBS before growth arresting in DMEM without FCS for 72 h. Quiescent MCs were stimulated with 10% FCS and treated with different concentrations of MPA (0.019–10 µM) in the absence or presence of guanosine (25–125 µM) for 48 h. DNA synthesis was quantified by 5-bromo-2'-deoxyuridine (BrdU) incorporation into proliferating cells over 2 h (Roche Diagnostics, Mannheim, Germany).

Determination of cell number
A total of 2.5 x 105 MCs/well were seeded onto 6-well plates and growth arrested as previously described. Quiescent MCs were exposed to a fresh medium with different concentrations of MPA (0.019–10 µM) for 1 h. FCS (10%) was added and cells were incubated for 48 h. Thereafter, MCs were washed with PBS, trypsinized, centrifugated and resuspended in a fresh medium without FCS. Cell growth was quantified by counting of intact viable cells after trypan blue (Biochrom, Germany) staining using a haemocytometer.

Detection of cell viability (MTT test) and the cell lysis (LDH test)
A total of 1 x 104 MCs/well were seeded onto 96-well plates in RPMI with 10% FCS for 24 h. Cells were washed with PBS and growth arrested as previously described. Quiescent cells were treated with different concentrations of MPA (0.019–10 µM) in DMEM with 1% FCS for 48 h before cell viability was quantified using the MTT test (Roche Diagnostics). Cell lysis was tested using the LDH test following the standard procedures (Roche Diagnostics) after 24 h.

Protein extraction and western blot analysis
Quiescent MCs grown in 10-cm dishes were incubated with MPA (0.1 µM, 1 µM or 2.5 µM) for 1 h and stimulated with 10% FCS for 24 h. Cells were washed with ice-cold PBS and lysed in a lysis buffer containing 20 mM Tris–HCl (pH 7.5), 1 mM ethylenediaminetetra-acetic acid (EDTA), 1 mM ethylenglycol-bis(aminoethylenether)-tetra-acetic acid (EGTA), 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 25 µg/ml leupeptin and 2 mM dithiothreitol (DTT). MC lysates were incubated for 15 min on ice and homogenized and centrifuged at 13 000 g at 4°C for 4 min. The protein concentration of the supernatants was determined using the Bradford assay (Bio-Rad, Munich, Germany).

Equal amounts of total MC protein (15 µg) were suspended in a 5:1 ratio in a sixfold sample buffer [150 mM Tris–HCl, pH 6.8, 40% (v/v) glycerol, 12% sodium dodecyl sulfate (SDS), 15% mercaptoethanol, 0.12% bromphenol blue, 0.004% pyrolin] and denaturated. Protein samples were subjected to SDS–PAGE in a running electrode buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at 200 V for 1 h using the Mini-Protean II cell (Bio-Rad) and immunoblotted onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Bedford, MA, USA) in a transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) at 15–20 V for 1 h using a Trans-Blot SD semi-dry electrophoresis transfer cell (Bio-Rad).

Membranes were blocked in Tris-buffered saline (TBS) Tween 0.2% (pH 7.6, 20 mM Tris, 140 mM NaCl) (TBS-T) with 3% non-fat dry milk and 3% BSA at room temperature for 1 h. Then, primary antibodies (anti-PDGF-B polyclonal rabbit IgG diluted 1:250, anti-PDGFR-ß monoclonal mouse IgG diluted 1:50, anti-β-tubulin polyclonal rabbit IgG diluted 1:1000) were applied and incubated at room temperature for 1 h or overnight at 4°C. Membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG or sheep anti-mouse IgG secondary antibodies at room temperature for up to 1 h and washed with TBS-T. For visualization, the ECL System (Amarsham Pharmacia) was used according to the manufacturer's instructions. The intensity of the bands representing PDGF-B (26 kDa), PDGFR-β (180 kDa) and β-tubulin (54 kDa) protein was evaluated using an imaging densitometer and software ScanPack 2.0 (Biometra, Göttingen, Germany).

RNA isolation and cDNA synthesis
MCs were grown in 6-well plates in the RPMI medium with 10% FCS until subconfluency, and growth arrested in serum-free DMEM for 72 h. Stimulation with 10–25 ng/ml PDGF-BB followed after treatment with 2.5 µM MPA in the absence or presence of 100 µM guanosine for 1 h. The concentration of guanosine neutralizing effects of MPA was determined by a preliminary study (data not shown). At indicated times, total RNA was isolated, using the QIAshredder and RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacture's instructions. RNA concentration was determined by UV absorption at 260 nm. Integrity of RNA was assessed by gel electrophoresis. After DNase treatment, 1 µg of total RNA was reverse transcripted in a final volume of 20 µl containing a first-strand buffer (50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2), 0.5 mM dNTPs, 12 mM DTT, 0.105 mg/ml BSA, 20 U RNasin, 50 ng/µl primer pd (N)6 hexamer by 200 U M-MLV reverse transcriptase using a Biometra Thermal Cycler (10 min at 25°C, 50 min at 42°C, 5 min at 95°C).

PCR
2.5–5.0% of newly synthesized cDNA was used as a template for PCR performed in a final volume of 25 µl containing a PCR buffer (10 mM Tris–HCl, pH 8.7, 50 mM KCl, 1.5–2.5 mM MgCl2), 0.2 mM dNTPs, 0.3–0.6 µM of each 5' and 3' primer, 0.05–0.1 µM of each 5' and 3' primer of a housekeeping gene GAPDH as internal standard, and 1.25 U Hot Star Taq DNA-Polymerase. For the PDGF-B– or Egr-1–PCR, 5 µl of 5 x Q-Solution was added to suppress unspecific synthesis. The amplification was performed using a Biometra Thermal Cycler according to the conditions shown in Table 1. Primers sequences, sources and sizes of PCR products are listed in Table 2.


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  		Table 1 Conditions of PCR

 

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  		Table 2 Primer sequences and sizes of PCR products

 
A probe of each reaction mixture was electrophoresed on 1.8% agarose gels. PCR products were stained with ethidium bromide and visualized using the Gel Doc TM 1000 UV fluorescent gel documentation system (Biorad, Germany). Appropriate bands were densitometrially analysed using quantification software of Raytest (Tina 2.09; Straubenhardt, Germany). The optical density of single bands of PDGF-B, c-fos and Egr-1 was compared to that of GAPDH detected in the same sample.

The sequences of PCR products were evaluated using an ABI Prism-Sequencer and were identical with those reported in a gene sequence bank. The sequence for c-fos was specific as reported for rat c-fos (Genbank X06769.1 [GenBank] ), the sequence for Egr-1 was specific as reported for rat Egr-1 (Genbank J04154.1 [GenBank] ), the sequence for PDGF-B was specific as reported for mouse PDGF-B (Genbank XM_122916.1) and the sequence for GAPDH was specific as reported for rat GAPDH (Genbank NM_017008 [GenBank] .1).

Nuclear protein extraction and electrophoretic mobility shift assay (EMSA)
At indicated times (1 h, 2 h) after MPA pretreatment (2.5 µM) for 1 h and PDGF-BB stimulation (25 ng/ml), rat MCs were incubated in ice-cold PBS with 0.1 mM EDTA for 5 min, harvested by scraping and centrifuged. Cell pellets resuspended in an ice-cold buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 10 µg/ml Aprotinin, 1 µg/ml pepstatin, 0.5 µg/ml Leupeptin, 0.5 mM DTT, 0.5 mM PMSF) were incubated on ice for 15 min before 10% IGEPAL-CA 630 was added. The resulting pellets after centrifugation at 13 000 rpm were resuspended in an ice-cold buffer B (20 mM Hepes pH 7.9, 25% glycerol, 0.4 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 µg/ml Aprotinin, 1 µg/ml pepstatin, 0.5 µg/ml Leupeptin, 0.5 mM DTT, 0.5 mM PMSF) before nuclear proteins were extracted by vigorous shaking for 20 min. Protein lysates were centrifuged at 1300 r.p.m. for 5 min and supernatants containing nuclear proteins were frozen using liquid nitrogen and stored at –80°C. Protein concentration was determined according to the Bradford method.

Nuclear extracts were assayed for AP-1 binding activity using [ {gamma}32P]-ATP end-labelled oligonucleotides with a binding site for AP-1 c-Jun homodimer and Jun/fos heterodimer (5-CGC TTG ATG ACT CAG CCG GAA-3'). The complementary DNA strands were end-labelled by T4 polynucleotide kinase. Binding reactions were performed on ice for 30 min with 5 µg of nuclear protein in 20 µl of binding buffer [20 mM Hepes, pH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 4% Ficol, 0.25 mg/ml BSA, 2 mg poly(dI-dC)] and [ {gamma}32P]-ATP end-labelled oligonucleotides (50 000 cpm/reaction). Protein–DNA complexes were separated from unbound DNA by electrophoresis on 6% native polyacrylamide gels using a running buffer 0.5 x TBE. Gels were vacuum dried, wrapped in plastic, and signals were detected by a phosphoimager (Fuji 1000, Tokyo, Japan).

Statistical analysis
All data are presented as means ± standard deviation (SD). Statistical differences were assessed using one-way factorial ANOVA followed by post hoc tests (multiple comparisons tests). P ≤ 0.05 was considered statistically significant.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Effect of MPA on MC proliferation and antagonistic effect of guanosine
The effect of the immunosuppressant on FCS-stimulated cell proliferation was evaluated using cell counting (Figure 1) and analysis of DNA synthesis (data not shown). After 48 h, FCS significantly increased the cell number by 5.5-fold versus unstimulated control MCs (data not shown). The addition of MPA to FCS-stimulated MCs promoted a significant dose-dependent reduction of MC proliferation with an IC50 of 0.44 ± 0.03 µM (means ± SD, n = 3). MPA inhibition of cell proliferation in FCS-stimulated MCs was significant at a concentration of 0.313 µM (P ≤ 0.001). Maximal MPA inhibition of >90% of proliferating MCs as compared to FCS-stimulated MCs was achieved at 1.25 µM (P ≤ 0.001) coinciding with the MPA effect on DNA synthesis. In parallel, BrdU incorporation into DNA of MCs was enhanced by fourfold in FCS-stimulated MCs (10%) as compared to unstimulated cells (results not shown). Treatment of MCs with different concentrations of MPA (0.019–10 µM) caused a concentration-dependent decrease of FCS-stimulated DNA synthesis, a dosage range representing the therapeutic range of this immunosuppressant in humans. The inhibiting concentration of 50% (IC50) of the FCS-induced BrdU incorporation by MPA was quantified as 0.52 ± 0.02 µM (means ± SD, n = 6). Maximal inhibition of DNA synthesis was present at 1.25 µM (P ≤ 0.001). Additionally, cell viability and cell lysis in MPA-treated MCs were tested. We were able to affirm previous observations [2], that the growth-inhibitory effect of MPA was not associated with cytotoxicity. No significant cell lysis (LDH test) could be detected up to a concentration of 10 µM MPA. Furthermore, cell viability was unaltered according to the MTT test (data not shown).


Figure 1
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  		Fig. 1 MPA inhibition of MC proliferation, dose–response curve. Subconfluent quiescent rat MCs were stimulated with FCS (10%) and exposed to different concentrations of MPA (0.019–10 µM) for 48 h. Cell number was determined. Numbers were corrected for the cell count at time point 0. Values are means ± SD of three independent experiments. ***P ≤ 0.001 versus FCS- and vehicle-treated MCs (FCS + DMSO).

 
The addition of guanosine antagonized the antiproliferative effect of 2.5 µM MPA on BrdU incorporation of MCs stimulated with 10% FCS in a significant way, but guanosine itself had an additional proliferative effect on FCS-stimulated MCs up to a concentration of 100 µM guanosine. At 100 µM guanosine, DNA synthesis of FCS-stimulated MCs was equal with or without the presence of 2.5 µM MPA. We therefore used 100 µM guanosine for all further experiments (Figure 2).


Figure 2
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  		Fig. 2 Effect of guanosine on DNA synthesis in FCS-stimulated rat MCs without or with MPA treatment. Subconfluent quiescent rat MCs were stimulated with FCS (10%) in the absence or presence of different concentrations of guanosine (25–125 µM) without or with MPA (2.5 µM) for 48 h. DNA synthesis was determined by BrdU incorporation over 2 h. Data are means ± SD of a typical experiment with six replicate samples. °°°P ≤ 0.001 versus control without stimulus, ***P ≤ 0.001 versus FCS with 0.1% DMSO (FCS + vehicle), ++P ≤ 0.01, +++P ≤ 0.001 versus FCS with MPA.

 
Effect of MPA on autocrine PDGF-B protein synthesis and PDGFR-ß
To study the influence of MPA on mesangial PDGF-B protein synthesis, western blot analyses were performed. As shown in Figure 3, PDGF-B protein expression was significantly increased upon incubation with FCS (10%) by 4.2-fold (Experiment B) and 4.6-fold (Experiment A) as compared to unstimulated MCs (P ≤ 0.001, n = 3). Incubation of FCS-stimulated MCs with 0.1 µM MPA led to a significant decrease of PDGF-B protein expression to a value of 2.1 of the unstimulated MC control (P ≤ 0.05, n = 3). A concentration of 2.5 µM MPA further decreased PDGF-B protein expression to the 1.4-fold of the unstimulated MC control. Altogether, MPA inhibition of PDGF-B protein expression was strongly significant as compared with FCS-stimulated, vehicle-treated MCs (P ≤ 0.01, n = 3).


Figure 3
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  		Fig. 3 Effect of MPA on PDGF-B protein expression in FCS-stimulated rat MCs. Quiescent MCs were stimulated with FCS (10%) in the absence or presence of MPA (experiment A: 2.5 µM MPA, experiment B: 0.1 and 1 µM MPA). After 24-h incubation, MCs were harvested and protein was extracted. Fifteen micrograms protein per lane was size fractionated by SDS–PAGE and immunoblotted using PDGF-B- and β-tubulin-specific antibodies. Blots were analysed by enhanced chemiluminescence system (ECL) and the intensities of the bands were evaluated using densitometry. The intensity of the bands representing PDGF-B was compared with the intensity of the bands representing β-tubulin used as an internal reference protein. Upper figures A and B: immunoblots for PDGF-B (upper blots) and for β-tubulin (lower blots). Lanes as indicated, controls: 0.5% FCS, vehicle: 0.1% DMSO. Lower figures (diagrams): densitometric quantification of PDGF-B/β-tubulin protein expression in western blot experiments (A and B, respectively). The unstimulated control was defined as 1. Data are means ± SD of three experiments. °°°P ≤ 0.001 versus control; *P ≤ 0.05; **P ≤ 0.01 versus FCS + vehicle.

 
Protein expression of the PDGF receptor β (PDGFR-β) remained unaltered by MPA (data not shown).

Effect of MPA on autocrine PDGF-B mRNA synthesis
PDGF-BB (25 ng/ml) stimulation of vehicle-treated MCs led to a maximal increase of PDGF-B mRNA steady-state levels at 2 and 4 h as compared with unstimulated, vehicle-treated MCs (P ≤ 0.05, n = 6, Figure 4). This finding confirmed the existence of an autocrine mesangial PDGF-B mRNA production after PDGF-BB stimulation. MC incubation with 2.5 µM MPA led to an inhibition of PDGF-BB-induced PDGF-B mRNA expression at all indicated times up to 12 h. MPA inhibition was significant at 2 and 4 h of incubation in PDGF-BB-stimulated MCs as compared with the PDGF-BB-stimulated, vehicle-treated MC control (P ≤ 0.01, n = 6, Figure 4).


Figure 4
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  		Fig. 4 Effect of MPA on PDGF-B mRNA expression in PDGF-BB-stimulated cultured rat MCs. Quiescent rat MCs were stimulated with PDGF-BB (25 ng/ml) with or without pre-treatment (1 h) with MPA (2.5 µM). At indicated time points (30 min, 1, 2, 4, 8 and 12 h), total RNA was extracted and reverse transcription was performed starting with 1 µg of total RNA. The resulting cDNA was used for PCR amplification with primers specific for PDGF-B and for the housekeeping gene GAPDH as internal control. Products were separated on 1.8% agarose gels and stained with ethidium bromide (upper figure). The figure shows a representative SDS–PAGE with amplification products for PDGF-B (542 bp) and GAPDH (343 bp). Lanes as indicated, controls: 0.5% FCS, vehicle: 0.1% DMSO. Lower figure: relative ratio of PDGF-B and GAPDH mRNA expression after densitometric quantification. Results are mean values of two independent experiments with three replicate RT–PCRs ± SD. All data are normalized to the control at 1 h. °P ≤ 0.05 versus control and **P ≤ 0.01 versus PDGF-BB with vehicle.

 
Exogenous guanosine did not significantly reverse the inhibitory effect of MPA on PDGF-BB (10 ng/ml)-induced PDGF-B mRNA expression (n.s., n = 6, Figure 5). In detail, MPA treatment significantly reduced the PDGF mRNA expression to 32 ± 17% of the maximum (100%) in PDGF-BB-stimulated, vehicle-treated MCs (P ≤ 0.001, n = 6). In comparison, the addition of guanosine (100 µM) to MPA-treated (2.5 µM), PDGF-BB-stimulated MCs did not significantly restore PDGF-B mRNA reaching levels of 56 ± 18% of the maximal expression. The PDGF-B mRNA expression of unstimulated, vehicle-treated MCs amounted to 18 ± 9%.


Figure 5
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  		Fig. 5 PDGF-BB-induced mRNA expression of PDGF-B in MPA-treated rat MCs in the absence or presence of guanosine. Growth-arrested rat MCs were treated with MPA (2.5 µM) in the presence or absence of guanosine (Guo) (100 µM) for 1 h and stimulated with PDGF-BB (10 ng/ml). After 2-h incubation, total RNA was extracted and reverse transcription was performed as described in Figure 4. Upper figure: representative SDS–PAGE with the amplification products for PDGF-B (542 bp) and GAPDH (343 bp). Lanes as indicated, control: 0.5% FCS, vehicle: 0.1% DMSO. Lower figure: relative ratio of PDGF-B and GAPDH mRNA expression after densitometric quantification. Results are mean values of three independent experiments with duplicate RT–PCRs ± SD, normalized compared with the maximal mRNA expression of PDGF-BB-stimulated cells as 100%. °°°P ≤ 0.001 versus control, and *P ≤ 0.05, ***P ≤ 0.001 versus PDGF-BB with vehicle.

 
Effect of MPA on PDGF-BB-induced immediate early gene (IEG) expression in MCs
PDGF-BB (10 ng/ml) stimulation of MCs led to a transient increase of mRNA steady-state levels of the IEGs Egr-1 and c-fos (Figures 6 and 7). After stimulation of MCs with PDGF-BB for 30 min, the expression of Egr-1 and c-fos mRNA got maximal and reached a significantly increased level of 24-fold for Egr-1 (P ≤ 0.001) and 8-fold for c-fos (P ≤ 0.01) as compared to unstimulated MCs. One hour after PDGF-BB addition to MCs, mRNA steady-state levels of Egr-1 and c-fos were still significantly increased as compared to the unstimulated control (P ≤ 0.05 and P ≤ 0.01, respectively), whereas 4 h after PDGF-BB stimulation, Egr-1 and c-fos mRNA were not detectable any more (data not shown).


Figure 6
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  		Fig. 6 Effect of MPA on PDGF-BB-induced mRNA expression of Egr-1 in cultured rat MCs in the absence or presence of guanosine. MCs were prepared as described in Figure 5. At indicated time points, total RNA was isolated and reverse transcription was performed as described in Figure 4. cDNA was used for PCR amplification with primers specific for Egr-1 and the housekeeping gene GAPDH as internal control. PCR products were separated on 1.8% agarose gels and stained with ethidium bromide. Upper figure: representative SDS–PAGE with the amplification products for Egr-1 (441 bp) and GAPDH (343 bp). Lanes as indicated, controls: 0.5% FCS, vehicle: 0.1% DMSO. Lower figure: relative ratio of Egr-1 and GAPDH mRNA expression after densitometric quantification. Results are mean values of three independent experiments with duplicate RT–PCRs ± SD normalized to the control at 30 min. °P ≤ 0.05, °°°P ≤ 0.001 versus control and *P ≤ 0.05, ***P ≤ 0.001 versus PDGF-BB plus vehicle.

 

Figure 7
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  		Fig. 7 Effect of MPA on PDGF-BB-induced mRNA expression of c-fos in cultured rat MCs in the absence or presence of guanosine. MCs were prepared as previously described (Figure 5), and RNA isolated at indicated time points was reverse transcribed as described in Figure 4. cDNA was used for PCR amplification with primers specific for c-fos and the housekeeping gene GAPDH as internal control. Products were separated on 1.8% agarose gels and stained with ethidium bromide. Upper figure: representative SDS–PAGE with the amplification products for c-fos (432 bp) and GAPDH (343 bp). Lanes as indicated, controls: 0.5% FCS, vehicle: 0.1% DMSO. Lower figure: relative ratio of c-fos and GAPDH mRNA expression after densitometric quantification normalized to the control at time point 0. Results are mean values of three independent experiments with duplicate RT–PCRs ± SD. °°P ≤ 0.05 versus control.

 
MPA treatment of MCs caused a significant decrease in Egr-1 mRNA expression to 7.6 ± 2.5% after 30 min (P ≤ 0.001) and to 4.7 ± 3.1% after 1 h (P ≤ 0.05) as compared to the maximal expression induced by PDGF-BB (100%). The inhibitory MPA effect on PDGF-BB-induced Egr-1 expression was not reversed by exogenous guanosine (Figure 6). Guanosine alone exerted an inhibitory effect on Egr-1 mRNA expression in PDGF-BB activated MCs; the expression quantified 25 ± 16% of the maximum after 30 min (P ≤ 0.001) and 22 ± 17% after 1 h (n.s.).

In MPA-treated, PDGF-BB-stimulated MCs, the c-fos mRNA expression was unchanged after 30 min and decreased to 57 ± 26% after 1 h (n.s.; Figure 7). Guanosine alone had no effect on c-fos expression in PDGF-BB-stimulated MCs until 30 min, but led to a decrease in the c-fos steady-state level after 1 h (57% ± 15% of PDGF-BB-activated PDGF-B expression, n.s.).

Effect of MPA on PDGF-BB-stimulated AP-1 activation
In order to examine whether MPA affects AP-1 activation, EMSA was performed. For this purpose, radiolabelled oligonucleotides with a consensus AP-1 binding site were used. As demonstrated in Figure 8, the stimulation of MCs with PDGF-BB (25 ng/ml) strongly induced the formation of AP-1-connecting complexes. At the times of examination (1 h and 2 h), the PDGF-BB-induced AP-1 activation of MCs was not affected by MPA (2.5 µM).


Figure 8
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  		Fig. 8 Effect of MPA on the activation of AP-1 by PDGF-BB. Quiescent rat MCs were stimulated with PDGF-BB (25 ng/ml) after pre-incubation (1 h) with MPA (2.5 µM) or vehicle. At indicated times, nuclear proteins were extracted and analysed in an electrophoretic mobility shift assay using [ {gamma}32P]-ATP end-labelled oligonucleotides. This experiment was performed twice with concordant results.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
MC proliferation can be advantageous for the physiological reconstitution of the kidney, but leads in an uncontrolled form to renal fibrosis. Rat MCs served as a model for this process occurring within several forms of GN as IgA nephritis or lupus nephritis.

Our results confirmed that up to 10 µM of the immunosuppressant MPA block MC proliferation in a non-toxic way, equivalent to the therapeutic frame for humans. In our study, the inhibiting concentration of 50% of the DNA synthesis in MCs stimulated with FCS (10%) was determined at 0.52 ± 0.02 µM for MPA, and 1.25 µM MPA reduced FCS-stimulated MC growth by over 90% (Figure 1) as reported earlier for rat or human MCs [1,2]. Furthermore, in a model of nephritis rats, MPA led to significant amelioration of glomerular histology, synthesis of alpha-smooth muscle actin, extracellular matrix deposition and glomerular hypertrophy as well as a significantly reduced proteinuria [1]. Findings of human MCs showed that MPA inhibits the matrix formation after FCS or TGF-beta stimulation e.g. of collagen I or fibronectin in an observation period of up to 14 days [3]. Hauser et al. suggested an IMPDH-associated effect since the MPA-induced cell growth reduction could be counteracted by the addition of the nucleoside guanosine [2]. In other studies on MC, the addition of extracellular nucleotides led to an activation of MAPKs [18]. Because non-lymphatic cells not only solely depend upon the de novo purine synthesis but also possess a salvage pathway, it is most probable that the IMPDH-dependent purine depletion is only part of the inhibitory effect of MPA on MC. Furthermore, Hauser et al. found no apoptosis induced by MPA up to a concentration of 10 µM [2]. Therefore, the underlying mechanisms behind the early antiproliferative MPA effect on renal cells still remained unclear. We suggest that MPA could interfere in early mitotic processes based on transcription factors such as Egr-1 or c-fos or on mitogens such as PDGF. Here we studied whether MPA influences the autocrine PDGF-BB synthesis in MCs induced by certain growth factors such as PDGF-BB, EGF, TNF alpha or bFGF.

In our study, 48 h after PDGF-BB stimulation, BrdU incorporation into the DNA of proliferating MC was enhanced by 3.5-fold (data not shown), and this effect was paralleled by an upregulation of the autocrine synthesis of the PDGF-BB protein and PDGF-B mRNA in MCs (Figures 3–5). Mesangial PDGF-BB protein synthesis was significantly and concentration dependently reduced by MPA (Figure 3), and MPA strongly inhibited the PDGF-B transcript all over 12 h after PDGF-B mRNA induction (Figure 4). Treatment with MPA (2.5 µM) decreased PDGF-B mRNA of MCs stimulated with PDGF-BB (25 ng/ml) to the level of the unstimulated MC control (Figure 4), concordant with the proliferation assays (Figure 1). The addition of guanosine to MPA-treated, PDGF-BB-stimulated MCs did not significantly restore the suppressing effect of MPA on PDGF-B mRNA transcription (Figure 5). We conclude that the antiproliferative effect of MPA on PDGF-BB-evoked MC proliferation might also be due to a suppression of PDGF-B mRNA transcription and PDGF-BB protein expression.

The antiproliferative effect of certain drugs includes the blocking of PDGF receptors. For instance, the effect of the anti-platelet drug trapidil on MCs is caused by a competitive binding to the specific receptors of PDGF-BB and by a modified transcription of PDGFR-β [19]. Furthermore, blocking of the autophosphorylation of the PDGF receptors with the tyrosine–kinase inhibitor genistein led to an inhibited DNA synthesis [20]. In the presented work, we did not find a significantly altered protein expression of PDGFR-β exerted by MPA after a 24-h incubation of FCS (10%)-stimulated MCs with MPA (2.5 µM). Therefore, the MPA-induced growth inhibition of stimulated MCs is probably not due to a modification of PDGFR-β.

PDGF-BB binding to its receptors leads to receptor dimerization and autophosphorylation hereby activating certain transcription factors such as Egr-1 or AP-1 [5,10,11]. We investigated whether MPA influences the expression of the IEGs in PDGF-BB-stimulated MCs and whether these changes can be antagonized by exogenous guanosine. Enhanced Egr-1 induction occurs in cultured MCs and, for instance, within the proliferative anti-Thy-1.1-nephritis in rats in vivo [21]. A significantly increased expression of c-fos was shown in patients with lupus nephritis, IgA nephritis or focal segmental sclerosis [22].

We found a significant Egr-1 expression in PDGF-BB-stimulated rat MCs (Figure 6). Treatment of these proliferating MCs with MPA (2.5 µM) led to a suppression of Egr-1 that was not antagonized by the addition of exogenous guanosine up to 1 h. These results suggest that MPA not only suppresses MC proliferation by IMPDH inactivation but MPA inhibition might also include Egr-1 suppression.

Because Egr-1 acts as a transcriptional regulator for many genes, there could be several pathways involved in the MPA effect. Downregulation of Egr-1 may affect PDGF-B mRNA expression thereby breaking the autocrine PDGF-BB synthesis cascade of MCs. Furthermore, Egr-1 is involved in the transcriptional regulation of the thymidine kinase gene thereby influencing DNA synthesis [23]. Egr-1 upregulated the expression of cyclin D1 via interaction with its promoter as shown in Chinese hamster ovary cells [24]. Within rat anti-Thy-1-nephritis, MPA led to a reduced mRNA expression of cyclins B, D1, D2, D3 as well as an increased mRNA expression of the cyclin-dependent kinase (CDK) inhibitor p27kip1 [25]. As a consequence, the CDK complexes are inhibited and the retinoblastoma protein (pRb) regulating G1/S transition remains hypophosphorylated and acts as growth restrictive by binding the transcription factor E2F [26]. Altogether, these Egr-1-dependent processes interfere with the proliferation of cells.

Our results showed a significant induction of c-fos mRNA in PDGF-BB-stimulated MCs (Figure 7). MPA did not inhibit PDGF-BB-induced c-fos transcription (Figure 7). Furthermore, MPA did not change the PDGF-BB-induced AP-1 activation 1 and 2 h after stimulation of MCs using an EMSA (Figure 8).

Altogether, we suggest that the reduced Egr-1 expression is a consequence of the MPA-inhibited PDGF-BB-mediated pathways whereas c-fos and AP-1 activation is not influenced by MPA.

Following PDGF-BB activation of its receptors, several transcription factors are stimulated [5,10]. PDGF-BB-activated signal pathways involve activated G proteins and small G proteins as ras, rac or Rho [27], but MPA had no effect on the ATP-stimulated Ca2+ release mediated by purine receptors and on trimeric GTP protein binding in human MCs [2]. In vascular smooth muscle cells, MPA inhibited the PDGF-BB-induced cell proliferation at least partially via cellular ROS levels and ERK 1/2 and p38 MAPK signals [28], and these pathways were not significantly antagonized by exogenous guanosine. Therefore, we suppose that MPA inhibits MC growth partially by blocking the ERK1/2 pathway.

Taken together, our results show that MPA concentrations within the therapeutic range for humans exert a non-toxic and early-occurring antiproliferative effect in stimulated rat MCs. In contrast to earlier findings, our results suggest that this antiproliferative MPA effect might not only be due to IMPDH-dependent purine depletion, but might additionally be based upon an inhibition of the autocrine PDGF-B synthesis of MCs as well as the PDGF-BB-dependent upregulation of the transcription factor Egr-1 and of PDGF-B mRNA steady-state levels. Further studies have to show to what extent the newly found influence of MPA on IEGs and PDGF in MCs are independent from IMPDH inhibition.

The molecular findings on the immediate antiproliferative efficacy of MPA on renal cells emphasize the therapeutic potency of this immunosuppressant in GN.



   Acknowledgments
 
We thank Joseph Pfeilschifter, director of the pharmacentrum Frankfurt, for the kind gift of cultured rat mesangial cells from his lab for these experiments. We also thank his co-workers, Karl Friedrich Beck and his colleagues, for helpful discussions as well as for their kind assistance in performing the electrophoretic mobility shift assay.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 26. 9.07
Accepted in revised form: 22. 7.08


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