Aim—Connective tissue growth factor (ccn; ctgf) gene expression is upregulated in fibrotic renal glomeruli. Therefore, the regulation and pharmacological modulation of ccn2 (ctgf) mRNA expression was investigated in a human renal mesangial cell line.
Methods—A human renal mesangial cell line was cultured in vitro under standard conditions. After stimulation, RNA was extracted and ccn2 (ctgf) mRNA expression assessed by northern blot analysis.
Results—The expression of ccn2 (ctgf) mRNA was transiently upregulated by fetal calf serum. Very rapid onset but short lasting ccn2 (ctgf) mRNA expression was observed after stimulation with lysophosphatidic acid, a bioactive lipid, which activates G protein coupled receptors. Induction of ccn2 (ctgf) mRNA expression by transforming growth factor β (TGF-β) was more prolonged and lasted for more than one day. The small GTPases of the Rho family were essential for basal as well as induced ccn2 (ctgf) expression: preincubation of the cells with toxin B from Clostridium difficile abrogated ccn2 (ctgf) mRNA expression. HMG CoA reductase inhibitors, which are therapeutically used as lipid lowering drugs, interfere with the isoprenylation and thus activation of Rho proteins. Simvastatin, an HMG CoA reductase inhibitor, inhibited ccn2 (ctgf) mRNA expression in a concentration dependent manner (IC50: 1–2 μM).
Conclusion—Statins were identified as potent inhibitors of ccn2 (ctgf) mRNA expression in mesangial cells, and therefore might be of potential use to modulate the excessive ccn2 (ctgf) expression in mesangial cells related to glomerular fibrosis.
- connective tissue growth factor
- Rho proteins
- mesangial cells
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Glomerulosclerosis is associated with end stage renal disease of different pathological origins. It is characterised by the accumulation of extracellular matrix in the glomerular mesangium as a result of an imbalance of matrix synthesis and degradation. Transforming growth factor β (TGF-β) is one of the major cytokines involved in the pathogenesis of fibrotic disorders.1 Raised concentrations of TGF-β are seen in human and experimental glomerulosclerosis and antibodies against TGF-β attenuate the development of matrix accumulation. TGF-β may directly induce the expression of extracellular matrix proteins.2 However, many of the effects of TGF-β on matrix synthesis seem to be mediated by downstream mediators such as connective tissue growth factor (CCN2; CTGF).3
CCN2 (CTGF) is a member of the CCN (CCN1 (CYR61), CCN2 (CTGF), and CCN3 (NOV)) family of proteins, which are secreted matrix associated proteins involved in diverse biological activities such as the regulation of cell adhesion, migration, mitogenesis, differentiation, and survival.4 Raised concentrations of CCN2 (CTGF) are found in fibrotic lesions,5–8 and are suggested to be related to the development and progression of fibrotic diseases. In the kidney, ccn2 (ctgf) mRNA values were increased in most biopsies obtained from patients with various types of renal disease characterised by glomerulosclerosis and tubulointerstitial fibrosis.9 The expression of ccn2 (ctgf) in diabetic glomerulosclerosis has recently been confirmed in the db/db mouse model.10 In line with these results, in vitro expression of ccn2 (ctgf) is upregulated in mesangial cells cultured in high glucose medium.11 CCN2 (CTGF) is encoded by an immediate early response gene and its expression is regulated at the transcriptional and post-transcriptional level. It is induced by TGF-β, one of the major factors involved in the development of glomerular lesions of different pathogenesis.1, 12
Because of its role in glomerulosclerosis, CCN2 (CTGF) might be a target for therapeutic intervention. Therefore, model systems to study ccn2 (ctgf) expression are needed. In a recent study, we have shown in rat mesangial cells that the small GTPase RhoA is crucially involved in the regulation of ccn2 (ctgf) expression.13 It was the aim of our present study to characterise the regulation of ccn2 (ctgf) in a human mesangial cell line as a model system for pharmacological intervention to modulate excessive and potentially harmful ccn2 (ctgf) expression.
Material and methods
Recombinant human TGF-β was obtained from TEBU, Frankfurt, Germany. PD98059 was from Calbiochem, Bad Soden, Germany. Lysophosphatidic acid (LPA) was obtained from Sigma, Deisenhofen, Germany. Cell culture reagents were from Biochrom, Berlin, Germany, Fetal calf serum (FCS) was from Gibco, Eggenstein, Germany. Toxin B from Clostridium difficile was kindly provided by Drs F Hofmann and K Aktories, Freiburg, Germany.
The generation and characterisation of the human mesangial cell line has been described previously.14 The cells were grown in DMEM supplemented with 2 mM l-glutamine, 4.5 g/litre glucose, 100 U/ml penicillin and 100 μg/ml streptomycin containing 10% FCS. Mesangial cells (0.5–1.0 × 106 cells/10 ml) were plated in 100 mm Petri dishes in medium containing 10% FCS. At subconfluency (after three to four days) cells were serum starved in DMEM containing 0.5% FCS for two to three days.
NORTHERN BLOT ANALYSIS
Northern blot analysis was performed as described previously.15 After stimulation of the cells for the indicated times, total RNA was extracted according to the protocol of Chomczynski and Sacchi,16 with minor alterations. Usually, the RNA yield was about 30–40 μg/10 cm Petri dish. Separation of total RNA (10 μg/lane) was achieved by the use of 1.2% agarose gels containing 1.9% formaldehyde with 1× MOPS as gel running buffer. Separated RNA was transferred to nylon membranes by capillary blotting and fixed by baking at 80°C for two hours.
Hybridisation was performed with cDNA probes labelled with [32P]dCTP using the NonaPrimer kit from Appligene, Heidelberg, Germany. A full length cDNA of human ccn2 (ctgf) was kindly provided by N Wahab, London, UK.17 DNA–RNA hybrids were detected by autoradiography using Kodak X-Omat AR film. Quantitative analysis of the autoradiographs was performed by densitometric scanning (Froebel, Wasserburg, Germany). As a control for equal loading of the gels, the blotted 18S rRNA was stained with methylene blue (0.04% in 500 mM sodium acetate, pH 5.2) and directly quantitated by densitometry. All values were corrected for differences in RNA loading by calculating the ratio of the specific bands to 18S rRNA expression.
Mesangial cells were cultured in the presence of 10% FCS and then serum starved in 0.5% FCS. The high amounts of ccn2 (ctgf) mRNA detected in growing cells declined rapidly upon growth arrest (fig 1A). Low amounts of ccn2 (ctgf) were detectable for several days, even when the cells were cultured in the absence of serum (data not shown). ccn2 (ctgf) mRNA was reinducible when the cells were incubated with 10% FCS (fig 1B). The induction was transient with the maximum response seen after two hours. One of the factors that might be responsible for the increase in ccn2 (ctgf) mRNA expression is LPA, which is present in serum at micromolar concentrations.18 Incubation of mesangial cells with LPA rapidly induced ccn2 (ctgf) expression, with maximal steady state values after one to two hours, which declined thereafter (fig 1C). TGF-β, the classic stimulator of ccn2 (ctgf) expression in almost all cells tested thus far, induced ccn2 (ctgf) mRNA expression with different kinetics: little induction was seen after one hour, maximal expression was detectable for several hours (two to eight), and mRNA values were still raised after 20 hours (fig 1D). ccn2 (ctgf) mRNA was rather unstable. When the cells were treated with TGF-β for two hours and then further incubated in the presence of an inhibitor of transcription (DRB; 5,6 dichloro-1-β-D-ribofuranosylbenzimidazole), half of the mRNA disappeared within one to two hours (data not shown).
Recently, we have shown that the small GTPase RhoA is crucially involved in the regulation of ccn2 (ctgf) mRNA expression in primary rat mesangial cells.13 Inhibition of the proteins of the Rho family by toxin B from Clostridium difficile completely prevented the induction of ccn2 (ctgf) by LPA or TGF-β in the human mesangial cells (fig 2). It also affected the basal expression of ccn2 (ctgf), indicating that continuous cell activity is necessary to maintain low basal expression of ccn2 (ctgf) mRNA.
The modification of Rho proteins by geranylgeranylation is essential for their membrane location and thus activity. Inhibitors of HMG CoA reductase, termed statins, interfere with the synthesis of geranylgeranylpyrophosphate and thus the modification of Rho proteins. Mesangial cells were incubated with simvastatin overnight to reduce isoprenylation of newly synthesised Rho proteins. Incubation with simvastatin led to a decrease in basal ccn2 (ctgf) expression (fig 3 A,B). Furthermore, the induction of ccn2 (ctgf) by LPA or TGF-β was inhibited (fig 3 A,B). Inhibition was concentration dependent. Half maximal inhibition was similar with both stimuli and obtained with 1–2 μM simvastatin.
Mesangial cells have been shown to upregulate CCN2 (CTGF) synthesis in vivo in diseases associated with glomerulosclerosis.9 Therefore, we used a human mesangial cell line to characterise ccn2 (ctgf) mRNA expression. The regulation of ccn2 (ctgf) mRNA in human mesangial cells was comparable with that seen previously in rat mesangial cells13: ccn2 (ctgf) was induced by TGF-β, the most potent stimulus of ccn2 (ctgf) expression described thus far. Induction was long lasting, with increased values still detectable 24 to 48 hours after the addition of TGF-β. The half life of TGF-β stimulated ccn2 (ctgf) mRNA was one to two hours, comparable with that determined in cortisol stimulated osteoblasts19 and dexamethasone stimulated rat mesangial cells (M Eberlain et al, 2001, unpublished observation). Thus, although the long lasting increase in ccn2 (ctgf) mRNA values suggested an increase in mRNA stability, ongoing transcriptional activation seems to be a prerequisite for this phenomenon. A much more rapid and transient induction is seen with LPA as the stimulus. This bioactive lipid activates G protein coupled receptors of the edge family20 and thus activates signalling pathways distinct from TGF-β. Nevertheless, both signalling pathways were completely blocked by toxin B, an inhibitor of the small GTPases RhoA, Rac1, and cdc42. Rho proteins as essential regulators of ccn2 (ctgf) expression have previously been described in rat mesangial cells,13 and also in renal fibroblasts.20a This indicates that the involvement of Rho proteins in the regulation of ccn2 (ctgf) is not restricted to a special cell type or stimulus, but seems to be a general feature of ccn2 (ctgf) regulation.
HMG Co-A reductase is the key enzyme of cholesterol synthesis and inhibitors of this enzyme (statins) are widely used in the treatment of hypercholesterolaemia. Apart from their lipid lowering effects, these compounds also interfere with the isoprenylation of small GTPases of the Rho and Ras families.21 Statins have been shown to interfere with cellular proliferation,22, 23 with the integrity of the actin cytoskeleton,24 and with the induction of matrix proteins.25 In human mesangial cells, statins also interfere with the induction of certain inflammatory mediators such as the low density lipoprotein induced expression of interleukin 6,26 or the induction of monocyte chemoattractant protein 1.27, 28 We have now shown that CCN2 (CTGF), which by itself is an inducer of matrix proteins, is another target of statins. The concentration needed for half maximal inhibition was independent of the stimulus used, indicating a common target of statin action. Given the strong inhibition of CCN2 (CTGF) expression by toxin B it seems most likely that interference with the modification of Rho proteins is the basis of the observed suppression of CCN2 (CTGF) induction. This was confirmed in renal fibroblasts, where the inhibition of statins was overcome by geranylgeranylpyrophosphate, the substrate for the modification of RhoA (M Eberlain, 2001, unpublished observation).
CCN2 (CTGF) as a target of statin treatment may contribute to the beneficial effects of statins that have been reported in humans with different types of glomerulonephritis or renal impairment.29 Because CCN2 (CTGF) has been implicated in many fibrotic diseases, it may be of interest to evaluate statins as potential antifibrotic drugs in different pathological settings.
This work was supported by the Deutsche Forschungsgemeinschaft, SFB 423, TP B3.
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