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Cell Biology International (2005) 29, 422428 (Printed in Great Britain)
Pioglitazone attenuates TGF-β
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
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-β
Keywords: PPARγ, Pioglitazone, Fibronectin, Fibronectin extra domain (ED) A, Mesangial cells, TGF-β.
*Corresponding author. Tel./fax: +81 3 5802 1604.
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-β
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.
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-β
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 MgCl
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.
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-β
Effects of different dosages of TGF-β
Effects of different dosages of TGF-β
Effects of different dosages of TGF-β
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-β
Effects of TGF-β
Effects of TGF-β
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-β
Effects of TGF-β
Effects of TGF-β
The stimulatory effect of TGF-β
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-PGJ
In summary, we report that activation of PPARγ by pioglitazone attenuates TGF-β
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Received 10 August 2004/16 December 2004; accepted 25 January 2005doi:10.1016/j.cellbi.2005.01.005