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Cell Biology International (2005) 29, 422–428 (Printed in Great Britain)

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Pioglitazone attenuates TGF-β1-induction of fibronectin synthesis and its splicing variant in human mesangial cells via activation of peroxisome proliferator-activated receptor (PPAR)γ

Atsuko Maeda, Satoshi Horikoshi, Tomohito Gohda, Toshinao Tsuge, Kunimi Maeda and Yasuhiko Tomino^*

Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan

Abstract

The peroxisome proliferator-activated receptor (PPAR)γ is expressed not only in adipose tissue but also in macrophages/monocytes and plays important roles in acute/chronic inflammation. Transforming growth factor (TGF)-β is a common pathogenic indicator of sclerosis because it induces the accumulation of extracellular matrix (ECM) in the glomerular mesangium of the kidney. Among components of the ECM, fibronectin (FN) is an acute reactant in inflammation, and isoforms of it produced by splicing of gene variants appear during abnormal conditions such as wound healing. In this study, we examined the effects of pioglitazone, a PPARγ agonist, on TGF-β1-induced FN synthesis in cultured mesangial cells using RT-PCR and Western blot analysis. We also analyzed its splicing variant, extra domain (ED) A, containing FN (EDA⁺FN). TGF-β1 enhanced the production of both FN and EDA⁺ FN and down-regulated PPARγ expression. Pioglitazone reversed both these effects of TGF-β1. These findings suggest that PPARγ activation by pioglitazone may affect the TGF-β1-induced FN accumulation observed in the glomerular mesangium in cases of glomerulosclerosis, although further in vivo experiments are needed to evaluate this inference.

Keywords: PPARγ, Pioglitazone, Fibronectin, Fibronectin extra domain (ED) A, Mesangial cells, TGF-β.

^*Corresponding author. Tel./fax: +81 3 5802 1604.

1 Introduction

In many studies, extracellular matrix (ECM) accumulation has been shown to be a central feature of various progressive glomerulonephritides, and transforming growth factor (TGF)-β is well known as an important common pathogenic mediator. In anti-thymocyte serum injected rats, mesangial matrix expansion paralleled both elevated proteoglycan synthesis and TGF-β expression over time. Exogenous TGF-β mimicked these effects, and stimulation was blocked by TGF-β antiserum (Border et al., 1991; Okuda et al., 1990). Fibronectin (FN) is an ECM glycoprotein, levels of which are increased during cell adhesion, migration, differentiation and proliferation. Three distinct splice sites have been identified and termed extra domain (ED) A, EDB and IIICS in humans, or EIIIA, EIIIB and V in rats. EDA and EDB are encoded by a single exon within the type III homology domain. In general, alternative splicing of FN is developmentally regulated. The EDA⁺ FN, EDB⁺ FN and IIICS⁺ FN isoforms are usually seen in fetal cells and tumor cells and during wound healing but not in normal adult cells (Borsi et al., 1987). Previous studies have shown that aging and growth factors such as TGF-β up-regulate the alternative splicing of FN pre-mRNA in the EDA, EDB and type III connecting sequence exons in vivo and in vitro (Magnuson et al., 1991).

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors belonging to the nuclear receptor gene superfamily. The PPARs have three isoforms (α, δ and γ), which differ in tissue distribution and ligand specificity. In recent years, PPARγ has been shown to play a major regulatory role not only in lipid and glucose metabolism but also in cellular proliferation and inflammation (Jiang et al., 1998; Ricote et al., 1998). Several studies have demonstrated that PPARγ inhibits such inflammatory mediators as interleukin (IL)-1β, IL-6, IL-12, tumor necrosis factor (TNF)-α, nitric oxide (NO) and cyclooxygenase-2 (COX-2). All these have been shown to play pivotal roles in the pathogenesis of many types of glomerulonephritis (Alleva et al., 2002; Inoue et al., 2000; Jiang et al., 1998; Tanaka et al., 1999). Some studies have suggested that the anti-inflammatory effects of PPARγ might be useful for treatment in vivo (Kawamoto et al., 2000; Reilly et al., 2000a). However, the role of PPARγ activation in ECM accumulation in mesangial cells and its participation in human glomerulosclerosis generally have not been fully evaluated.

In the present study, we observed the effects of various doses of TGF-β1 on FN and EDA⁺ FN synthesis, since different optimal doses of TGF-β have been reported in cell culture experiments. Moreover, to explore the effects of pioglitazone on abnormal ECM production, we examined PPARγ mRNA and protein expression in cultured human mesangial cells. We also explored the effect of pioglitazone as an agonist of PPARγ in TGF-β1-induced FN and EDA⁺ FN synthesis.

2 Materials and methods

2.1 Cell culture

Primary human mesangial cells purchased from Clontics (Walkersville, MD, USA) were maintained in 50% Dulbecco's modified Eagle's medium/50% Ham's F12 containing 2 mM glutamine, 100 U/ml penicillin, 10 μg/ml streptomycin, 5 μg/ml insulin and 10 μg/ml transferrin, with or without 20% heat-inactivated fetal calf serum (Gibco, Grand Island, NY, USA). The cells were grown in culture dishes and studied between passages 6 and 10.

TGF-β1 was purchased from R & D System (Minneapolis, MN, USA). The cells were pretreated in serum-free medium (SFM) for 8 h and then treated with 0, 0.5, 1.0 or 5.0 ng/ml of TGF-β1 in SFM for 16 h. Cells were harvested and RNA and protein extractions were prepared for further experiments.

Pioglitazone was a gift from Takeda Chemical Industries (Osaka, Japan). The cells were pre-incubated in SFM for 8 h and treated with 1.0 ng/ml TGF-β1 alone in SFM or in the presence of either 10⁻⁵, 5×10⁻⁶ or 10⁻⁶ mol/l pioglitazone for 16 h (Reilly et al., 2000, 2001). Quiescent cells without pioglitazone in SFM served as untreated controls. The cytotoxicity of pioglitazone in 0.5% DMSO was tested after treatment with 5×10⁻⁶ mol/l pioglitazone in 0.5% of DMSO, 0.5% of DMSO alone or 5.0 ng/ml of TGF-β1 in PBS for 16 h using a Cell Counting Kit (Dojindo Co., Kumamoto, Japan). Since the kit measures the absorbance of tetrazolium-formazan dye after treatment of the cells with dehydrogenase, the results reflect the activity of non-specific cellular enzymes in the living cells.

2.2 RNA preparation and semi-quantitative reverse-transcription/polymerase chain reaction (RT-PCR) analysis

Total RNA was extracted from the cultured cells by a single-step guanidinium thiocyanate-phenol chloroform method using Trizol (Invitrogen, Carlsbad, CA, USA). Two micrograms of total RNA were converted to cDNA using oligo (dT) primers (Invitrogen, Carlsbad, CA, USA) and reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The single-strand cDNA was amplified in a Gene Amp PCR system (model 9600, Perkin-Elmer, Norwalk, CT) as follows: initial determination at 94 °C, for 5 min, amplification (1 min at 94 °C, 1 min at 54 °C, 1 min at 72 °C) and final extension at 72 °C for 5 min. The numbers of amplification cycles were 19, 24 and 30 for FN, EDA and PPARγ, respectively. The amplifications were linear within these ranges of cycles.

The sequences of the PCR primers (from 5′ to 3′) were as follows: GCA GAG GCA TAA GGT TCG GG (hFN sense), CAG GAG CAA ATG GCA CCG AG (hFN antisense), GGA GAG AGT CAG CCT CTG GTT CAG (EDA sense) (Ting et al., 2000), TGT CCA CTG GGC GCT CAG GCT TGT G (EDA antisense) (Ting et al., 2000), TCT CTC CGT AAT GGA AGA CC (PPARγ sense), CCC CTA CAG AGT ATT ACG (PPARγ antisense), CCA CCC ATG GCA AAT TCC ATG GCA (human GAPDH sense), and TCT AGA CGG CAG GTC AGG TCC ACC (GAPDH antisense). The amplification products were separated by electrophoresis on 2.0% agarose gels and visualized by ethidium bromide staining. Gels were scanned using Master Scan (Scanalytics, Billerica, MA, USA), and the signal intensity of bands was measured and normalized to that of the GAPDH band.

2.3 Western blot analysis

Samples for Western blot analysis were prepared as previously described (Marx et al., 1998). In brief, harvested cells were lysed in 10 mM Hepes (pH 7.9), 1.5 mM MgCl2, 10 mM KCl and 0.5% Nonidet P-40. Nuclei were pelleted at 13,000×g for 5 min and lysed in 20 mM Hepes (pH 7.9), 1.5 mM MgCl2, 420 mM NaCl, and 0.2 mM ethylenediaminetetraacetate (EDTA). The supernatants from the first centrifugation were used as the cellular extracts for FN and EDA⁺ FN protein analysis. After the second centrifugation (13,000×g for 5 min), the supernatant was diluted with an equal volume of 20 mM Hepes (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 20% glycerol and used as the nuclear extract for PPARγ protein analysis.

Protein concentrations in both cellular and nuclear extracts were determined using the bicinchroninic acid method (Pierce, Rockford, IL, USA). Twenty micrograms of protein were electrophoresed on SDS-polyacrylamide gel. The proteins separated by 7.5% polyacrylamide were electroblotted on to a polyvinylidene difluoride (PVDF) membrane (Millipore, Yonezawa, Japan). The membranes were incubated in blocking solution (Block Ace, Dainippon Pharm., Osaka, Japan) at 4 °C overnight and then incubated with mouse monoclonal anti-human FN antibody (Chemicon International, Temecula, CA, USA), rabbit anti-human EDA antibody (Abcan, Cambridge, UK) or rabbit polyclonal anti-human PPARγ (H-100) antiserum (Santa Cruz Biotechnology, CA, USA) at room temperature for 1 h. After washing with 0.1% Tween 20 containing PBS, the membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (Cappel, Aurora, OH, USA) for EDA and PPARγ, or anti-mouse IgG antisera (Cappel, Aurora, OH, USA) for FN, at room temperature for 1 h. The signal was detected by an enhanced chemiluminescence system (ECL-plus, Amersham Pharmacia Biotech, Buckinghamshire, UK) and visualized by a luminescent image analyzer (model LAS-1000 plus, Fuji Film, Tokyo, Japan). The signal intensities of the bands were determined.

2.4 Analysis of data

All experiments were repeated at least three times, and the results are given as mean±standard deviation (SD). Analysis of variance (ANOVA) and Bonferroni/Dunn analysis with Stat View 4.0 on a Macintosh were used for statistical determinations; a level of P<0.05 was considered statistically significant.

3 Results

During all experiments, pioglitazone did not change the number, viability (data not shown) and GAPDH mRNA expression of the cells. The expression of both FN and EDA⁺ FN mRNA increased in a dose-dependent manner in response to treatment with 0.5, 1.0 and 5.0 ng/ml of TGF-β1 for 16 h (Fig. 1), and Western blotting analysis showed that FN protein expression increased similarly (Fig. 2). However, the expression of PPARγ mRNA was dose-dependently suppressed by the same concentrations of TGF-β1 (Fig. 3). Since the effects of 1.0 and 5.0 ng/ml TGF-β1 were indistinguishable, 1.0 ng/ml was used in subsequent experiments.

Fig. 1

Effects of different dosages of TGF-β1 on FN (A) and EDA⁺ FN mRNA (B) expression in human mesangial cells. After 16 h incubation with 0, 0.5, 1.0, or 5.0 ng/ml TGF-β1, the effects were evaluated by RT-PCR analysis. N, negative control using water. The intensity of the bands was measured by an image analyzer and the relative amounts of FN and EDA± FN mRNA were normalized with GAPDH mRNA. The relative amount in the unstimulated control was taken as 1. Values present the mean±SD from three separate dishes. (A) *P<0.005 and **P<0.05 vs TGF-β1 unstimulated control. (B) *P<0.005 vs TGF-β1 unstimulated control.

Fig. 2

Effects of different dosages of TGF-β1 on FN protein expression in human mesangial cell cultures. After 16 h incubation with 0, 0.5, 1.0, or 5.0 ng/ml TGF-β1, the effects were evaluated by Western blot analysis. The intensity of bands of FN protein was measured by an image analyzer. The relative amount of the unstimulated control was taken as 1. Values present the mean±SD from three separate dishes. (A) *P<0.005 and **P<0.05 vs TGF-β1 unstimulated control. (B) *P<0.005 vs TGF-β1 unstimulated control.

Fig. 3

Effects of different dosages of TGF-β1 on PPARγ mRNA expression in human mesangial cells. After 16 h incubation with 0, 0.5, 1.0, or 5.0 ng/ml TGF-β1, the effects were evaluated by RT-PCR analysis. N, negative control using water. The intensity of the bands of PPARγ mRNA was measured by an image analyzer and the relative amounts in unstimulated control was taken as 1. Values present the means±SD from four separate dishes. *P<0.005 vs TGF-β1 unstimulated control.

Pioglitazone (10⁻⁵, 5×10⁻⁶ and 10⁻⁶ mol/l) dose-dependently attenuated the augmentation of FN and EDA⁺ FN mRNA expression induced by 1.0 ng/ml of TGF-β1 (Fig. 4A,B; P<0.05), and the enhancement of FN and EDA⁺ FN protein expression was correspondingly suppressed (Fig. 5A,B; P<0.005, P<0.05, respectively). The suppression of FN mRNA was statistically significant only after treatment with 10⁻⁵ mol/l of pioglitazone (P<0.05).

Fig. 4

Effects of TGF-β1 and pioglitazone on FN (A) and EDA⁺ FN (B) mRNA expression in human mesangial cells. The relative amounts of FN and EDA⁺ FN mRNA were measured with an image analyzer after normalization with GAPDH. The relative amount in the untreated control was taken as 1. Values represent the means±SD from three separate dishes. *P<0.05 vs TGF-β1 stimulation without pioglitazone.

Fig. 5

Effects of TGF-β1 and pioglitazone on FN (A) and EDA⁺ FN (B) protein expression in human mesangial cells. The relative amounts of FN and EDA⁺ FN protein were measured by an image analyzer and the relative amount in the untreated control was taken as 1. Values represent the means±SD from four separate dishes. (A) *P<0.005 vs TGF-β1 stimulation without pioglitazone. (B) *P<0.05 vs TGF-β1 stimulation without pioglitazone.

The number of mesangial cells did not change during these experiments, indicating that these concentrations of pioglitazone were not cytotoxic (data not shown). Moreover, pioglitazone (5×10⁻⁵ and 10⁻⁶ mol/l) significantly reversed the suppression of PPARγ mRNA (Fig. 6; P<0.005) and protein (Fig. 7; P<0.005, P<0.05, respectively) expression by 1.0 ng/ml TGF-β1. When the cells were treated with 10⁻⁵ mol/l pioglitazone in the absence of TGF-β1, no significant change was observed in either FN or PPARγ expression.

Fig. 6

Effects of TGF-β1 and pioglitazone on PPARγ mRNA expression in human mesangial cells. The relative amount of PPARγ mRNA was measured by an image analyzer after normalization with GAPDH. The relative amount in the untreated control was taken as 1. Values represent the means±SD from three separate dishes. *P<0.05 vs TGF-β1 stimulation without pioglitazone. N, negative control using water.

Fig. 7

Effects of TGF-β1 and pioglitazone on PPARγ protein expression in human mesangial cells. The relative amounts of FN and EDA⁺ FN protein were measured with an image analyzer and the relative amount in the untreated control was taken as 1. Values represent the means±SD from four separate dishes. *P<0.005 and **P<0.05 vs TGF-β1 stimulation without pioglitazone.

4 Discussion

The stimulatory effect of TGF-β1 on ECM in kidney cell lines is in agreement with previous reports (Mackay et al., 1989; Poncelet and Schnaper, 1998). The expression of α1 (I), α1 (III) and α1 (IV) collagen mRNA was increased dose-dependently when primary cultured human tubular epithelial cells were stimulated with TGF-β at concentrations between 0.5 and 5.0 ng/ml (Poncelet and Schnaper, 1998). However, less than 1.0 ng/ml TGF-β was required to increase the expression of types I and IV collagen at the protein level in the same cell line. Poncelet and Schnaper (1998) suggested that the concentrations of TGF-β required to achieve the same stimulatory effect on ECM expression differed with cell lineage, culture conditions and/or the transcription/translation rates in vitro. Therefore, in this study, we initially examined the concentrations of TGF-β1 required to stimulate FN mRNA and protein expressions in human mesangial cells. Dose-dependent up-regulation was observed at 0.5, 1.0 and 5.0 ng/ml. In addition to the stimulatory effect on FN, TGF-β changes the alternative splicing forms of FN pre-mRNAs in vivo and in vitro. Among these splicing variants, the EDA but not the EDB segment is active in promoting cell spreading, adhesion and migration through interaction with integrin α5β1 (Manabe et al., 1997). EDA containing FN also elicits proteoglycan release in cartilage and induces MMP-1 in chondrocytes and MMP-1, -3 and -9 in synovial cells (Saito et al., 1999). These results suggest that TGF-β regulates FN production by controlling splicing. Therefore, we examined the effect of TGF-β on the expression of the EDA isoform and whole FN simultaneously in human mesangial cells. We found that EDA⁺ FN mRNA expression was dose-dependently up-regulated by TGF-β1 in the same way as whole FN, suggesting active modulation of cell adhesion, migration and spreading, although to confirm this we need to examine these activities by measuring the expression of MMPs. In vivo studies demonstrated EDA⁺ FN only in the mesangium in the normal adult human nephron (Laitinen et al., 1991), and temporal over-expression associated with mesangial cell proliferation and infiltration of lymphocytes was found in experimental mesangioproliferative nephritis (Alonso et al., 1999). These results suggest that EDA⁺ FN may play an important role in glomerular inflammation.

Recently, PPARγ was reported to be a negative regulator of macrophage activation (Ricote et al., 1998), and it had anti-inflammatory effects on such cytokines as TNF-α, IL-1α, IL-1β and IL-6 (Jiang et al., 1998). Therefore, we examined the effect of pioglitazone, a PPARγ agonist, on TGF-β-induced FN and EDA⁺ FN production. We found that pioglitazone attenuated this induction at both the mRNA and protein levels. Interestingly, treatment with pioglitazone in the absence of TGF-β had no effect on FN, EDA⁺ FN or PPARγ expression. This unique profile of pioglitazone, replacing over-expression of FN through PPARγ activation when TGF-β is in excess, may indicate its value as an anti-inflammatory drug. Fu et al. (2003) demonstrated that early stimulation of PPARγ expression by TGF-β was mediated via the ERK/Erg-1 signaling pathway, whereas TGF-β-mediated late inhibition of PPARγ expression occurred via AP-1 and Smad3 in human aortic smooth muscle cells. However, further analysis of the signaling pathway leading to PPARγ expression by TGF-β in human mesangial cells is needed, because the pleiotropic effect of pioglitazone seems to vary from cell types and culture conditions (Fu et al., 2003).

Several recent reports have demonstrated that 15d-PGJ2, a natural ligand of PPARγ, has anti-inflammatory effects independent of PPARγ, involving direct inhibition, or inhibition via mitogen-activated protein kinase and the NF-κB pathway (Harris et al., 2002; Rossi et al., 2000; Simonin et al., 2002). Troglitazone does not affect NF-κB activation but modulates TNF-α production (Simonin et al., 2002) and attenuates the inhibitory action of TNF-α on PPARγ expression. These results suggest that thiazolidinedions, unlike 15d-PGJ2, modulate the effects of inflammatory cytokines through activation of PPARγ.

In summary, we report that activation of PPARγ by pioglitazone attenuates TGF-β1-induced EDA⁺ FN, an active alternative splicing product. It is suggested that pioglitazone may be useful as a therapeutic reagent for glomerulonephritides, although more precise in vivo studies will be needed to substantiate this view.

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Received 10 August 2004/16 December 2004; accepted 25 January 2005