Inhibition of systemic sclerosis dermal fibroblast type I collagen production and gene expression by simvastatin

Abstract

Objective

To examine whether statins are capable of modulating collagen gene expression in cultured systemic sclerosis dermal fibroblasts.

Methods

Cultured dermal fibroblasts from 3 patients with diffuse systemic sclerosis of recent onset were treated with 5 μM and 10 μM of simvastatin for 3 or 4 days. Morphologic features, cytotoxicity, and type I collagen production and messenger RNA (mRNA) levels in the fibroblasts were examined. The effects of mevalonate, geranylgeranyl pyrophosphate (GGPP), and farnesyl pyrophosphate (FPP), which are lipids downstream from the hydroxymethylglutaryl–coenzyme A block, were also examined. Transient transfections with COL1A1 promoter-reporter constructs and electrophoretic gel mobility shift assays were utilized to examine COL1A1 transcription and Sp1 and CCAAT-box binding factor (CBF) binding.

Results

Simvastatin did not cause morphologic changes or cytotoxicity in the fibroblasts, even after 4 days of treatment. Type I collagen production and mRNA levels showed a potent and dose-related inhibition following 3 and 4 days of treatment. The inhibition of collagen gene expression by simvastatin was completely reversed by mevalonate and GGPP, but not by FPP. The statin effects occurred at the transcriptional level and involved the proximal COL1A1 promoter region encompassing −174 bp. A significant reduction in Sp1 and CBF binding activity was also found in simvastatin-treated cells.

Conclusion

Simvastatin is a powerful inhibitor of type I collagen gene expression in normal and systemic sclerosis fibroblasts. The pleiotropic protective effects of statins on various endothelial and immune cell functions in conjunction with their potent inhibitory effects on type I collagen gene expression suggest that statins may be effective therapeutic agents in systemic sclerosis.

Systemic sclerosis is characterized by the excessive deposition of collagen and other connective tissue components in skin and multiple internal organs, prominent and often severe alterations in the microvasculature, and humoral and cellular immune system abnormalities (for review, see refs. 1–3). Although the exact mechanisms involved in the pathogenesis of systemic sclerosis are not known, progressive cutaneous, visceral, and vascular fibrosis is responsible for most of the clinical manifestations and severity of the disease as well as the resultant mortality (4, 5). Numerous studies have shown that the pathologic increase in tissue collagen deposition is largely the result of increased expression of collagen genes (for review, see refs. 2 and3).

Fibroblasts from affected systemic sclerosis skin cultured in vitro produce excessive amounts of various collagens (6, 7) and display increased transcription of the corresponding genes (7–10). However, the mechanisms responsible for the increased transcriptional activity of collagen genes in systemic sclerosis fibroblasts are not fully understood. Transforming growth factor β (TGFβ) plays a crucial role in the development of tissue fibrosis (11), and a plausible hypothesis has suggested that this growth factor may be intimately involved in the pathogenesis of systemic sclerosis (12). The intracellular pathways involved are mediated through complex interactions between specific cell surface receptors and several intracellular proteins of the Smad family (13, 14). However, recent studies indicate that non-Smad pathways may also be involved in some of the effects of TGFβ (15).

Posttranslational protein isoprenylation is of fundamental importance in the regulation of cell survival, proliferation, and differentiation, as well as in cytoskeletal organization. Isoprenylation reactions also participate in the regulation of collagen genes and in some of the effects of TGFβ. Investigators in our group previously demonstrated that specific inhibition of geranylgeranyl transferase I with the compound GGTI298 caused a profound and selective inhibition of expression of type I and type III collagen genes in normal and systemic sclerosis dermal fibroblasts (16). Since statins also affect the synthesis of lipid intermediates involved in isoprenylation reactions (17, 18), we postulated that statins may also directly inhibit the expression of type I collagen genes, and examined this hypothesis in normal and systemic sclerosis dermal fibroblasts in vitro. The results obtained indicate that statins are powerful inhibitors of type I collagen gene expression in normal and systemic sclerosis fibroblasts, and this is likely attributable to their effects on geranylgeranyl prenylation of key intracellular mediators. Previous results demonstrating the pleiotropic protective effects of these drugs on various endothelial and immune cell functions (19–21) coupled with the potent inhibitory effects of statins on type I collagen gene expression suggest that these drugs may be effective therapeutic agents in systemic sclerosis and other diseases that are characterized by tissue fibrosis and vascular dysfunction.

PATIENTS AND METHODS

Fibroblast cultures.

Dermal fibroblast cell lines were established from patients with systemic sclerosis who fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) criteria for the classification of the disease (22). To avoid variability introduced by heterogeneity of the clinical characteristics and differences in the stage of evolution of systemic sclerosis, only cells from untreated patients with diffuse systemic sclerosis of recent onset and rapid progression were studied, as described previously (7, 16). The cell lines were obtained from full-thickness skin biopsy tissue surgically excised for diagnostic purposes from the leading edge of clinically apparent systemic sclerosis lesions. Normal fibroblast cell lines were obtained from age- and sex-matched individuals undergoing surgical procedures. All studies were approved by the institutional review board of Thomas Jefferson University.

For all studies, only early-passage fibroblasts (<12) were used, to avoid changes in their original phenotype during subculture. Cells were maintained in Dulbecco's modified Eagle's medium, containing 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA), 1% vitamins, 2 mM glutamine, and antibiotics, and were incubated at 37°C in a 5% CO₂ humidified atmosphere. The cultures were supplemented with 40 μg/ml L-ascorbic acid phosphate magnesium salt n-hydrate (Wako, Osaka, Japan) for 24 hours prior to the addition of the test compounds to optimize their level of collagen production, as described previously (23). At the conclusion of all experiments, the cells were counted and their viability was assessed by trypan blue exclusion and, in certain experiments, the MTT TOX-1 assay kit (Sigma, St. Louis, MO) was used, as described previously (24).

Treatment of cultured fibroblasts.

Simvastatin was purchased from Calbiochem (La Jolla, CA) in its active form. All other chemicals were of reagent grade. Three strains of systemic sclerosis fibroblasts and 3 strains of normal fibroblasts were studied. Normal and systemic sclerosis cells were plated in 6-well plates and cultured until confluent. Following preincubation with 40 μg/ml L-ascorbic acid phosphate magnesium salt n-hydrate, the cells were incubated with 5 μM and 10 μM of simvastatin in 10% FBS medium for 48 hours. The medium was then changed with fresh medium containing the same components and the cultures incubated for an additional 24 or 48 hours. The type I collagen present in the culture media was quantified by Western blotting using a specific anti-human type I collagen polyclonal antibody (Rockland, Gilbertsville, PA). Geranylgeranyl pyrophosphate (GGPP), farnesyl pyrophosphate (FPP), and mevalonic acid were from Sigma and were used at concentrations that have been shown in other studies to be sufficient for complete reversal of the inhibition of activity of the corresponding enzymes.

Northern hybridization.

Fibroblasts were grown to confluency and, following incubation either under control conditions or with simvastatin, total RNA was isolated with the RNeasy Mini Kit (Qiagen, Valencia, CA). Aliquots of total RNA (5–10 μg/well) were electrophoresed on 1.0% formaldehyde agarose gels. The RNA was then transferred to Hybond N+ membranes (Amersham Biosciences, Piscataway, NJ) and the filters hybridized to ³²P-radiolabeled human complementary DNA (cDNA) for COLlAl, as described previously (16, 24). Equivalent amounts of RNA were loaded and RNA loading and transfer were evaluated by probing with GAPDH cDNA. Equivalent loading and transfer were also verified by quantitative image analysis of ethidium bromide staining of ribosomal RNA in the same blots. The filters were analyzed using storage phosphor technology (Image-Quant version 5.1 software; Amersham Biosciences).

Transient transfections of normal and systemic sclerosis fibroblasts with COL1A1 promoter–chloramphenicol acetyltransferase (CAT) constructs.

Transient transfections of normal and systemic sclerosis fibroblasts were performed with the lipid-based FuGene 6 Kit (Roche, Indianapolis, IN) as described previously (25). Normal and systemic sclerosis fibroblasts were transfected at 80% confluence in 60-mm dishes with 2.5 μg of each plasmid. COL1A1 transcription was assessed by using deletion constructs of the COL1A1 promoter fused to the CAT reporter gene. All of the constructs examined ended at nucleotide +42 to ensure transcription in a proper reading frame, and their 5′ ends were at −675 bp, −174 bp, and −84 bp. The detailed procedures for preparation of these constructs have been described previously (26). Four hours later, fresh medium with or without 5 μM simvastatin was added, and the cells were harvested 72 hours after transfection; CAT activity was then determined in cell extracts. The efficiency of transfection was normalized by cotransfecting 0.2 μg of vector containing Escherichia coli β-galactosidase cDNA (pCMV β-galactosidase; Clontech, Palo Alto, CA) followed by assays of β-galactosidase enzymatic activity.

Electrophoretic gel mobility shift assay (EMSA).

Confluent cells in two 175-mm flasks were treated with 5 μM of simvastatin for 4 days in the same manner as that described above, and nuclear extracts were prepared using a kit from Sigma. EMSA was performed as described previously (24, 25). A −125-bp DNA fragment from the COL1A1 promoter, encompassing nucleotides −174 to −50, was used as a probe for the EMSA experiments. The fragment was obtained by polymerase chain reaction amplification with primers at −174 bp (5′-GGTGGACTCCCTTCCCTCCTC-3′) and at −50 bp (5′-AGGACCCCTGCCCCTCGGAGA-3′) of the human COLlA1 promoter. Radioactive probes were prepared by phosphorylation of the 5′ ends with polynucleotide kinase (Promega, Madison, WI) and α³²-ATP (Amersham Biosciences). Ten micrograms of nuclear extracts, 4 μg of poly(dI-dC), and 5 × 10⁴ counts per minute of radiolabeled probe were incubated for 20 minutes in a buffer containing 40 mM KCl, 15 mM HEPES, pH 7.9, 1 mM EDTA, 0.5 mM dithiothreitol, 1 mM MgCl₂, and 5% glycerol.

To test the specificity of binding, competition experiments using 100-fold molar excess of unlabeled Sp1 and CCAAT-box binding factor (CBF) consensus oligonucleotides and their mutated forms were performed. These oligonucleotides were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). EMSA was performed in 5% nondenaturing polyacrylamide gels as described previously (24, 25). Sp1 and CBF binding activities were quantified by densitometry (Image-Quant, version 5.1; Molecular Dynamics, Sunnyvale, CA).

Statistical analysis.

Results are expressed as the mean ± SEM. Student's t-test was used for comparing 2 groups of data, utilizing GraphPad software (San Diego, CA). P values less than 0.05 were considered significant.

RESULTS

Effects of simvastatin on the viability and morphologic features of normal and systemic sclerosis fibroblasts.

To examine the morphologic and/or cytotoxic effects of simvastatin, confluent cultures of normal and systemic sclerosis fibroblasts were treated with various simvastatin concentrations (1.25 μM, 2.5 μM, 5 μM, and 10 μM) for 24, 48, 72, and 96 hours. Morphologic changes were evaluated by dark field microscopy, while cytotoxicity was measured by an MTT reduction assay utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The highest concentration of simvastatin examined in these experiments (10 μM) did not cause morphologic changes; furthermore, there was no detectable cytotoxicity even after 4 days of treatment with the highest concentration (results not shown). Therefore, all subsequent experiments were performed with 5 μM and/or 10 μM of the drug for 3 or 4 days.

Effect of simvastatin on type I collagen production.

The effects of simvastatin on the expression of type I collagen were examined in 3 different systemic sclerosis cell lines and in 3 normal cell lines treated for 3 or 4 days with either 5 μM or 10 μM simvastatin (Figure 1). Simvastatin reduced type I collagen production in a dose-dependent manner in both cell types. In normal fibroblasts, simvastatin at a concentration of 5 μM reduced collagen production by 74% and 75% at days 3 and 4, respectively, and at a concentration of 10 μM, it reduced collagen production by 82% and 93% at days 3 and 4, respectively (Figures 1A and B). In systemic sclerosis fibroblasts, simvastatin treatment for 3 days at a concentration of 5 μM reduced collagen production by 77%, and at a concentration of 10 μM, by 84%. After 4 days, 5 μM simvastatin caused a 70% reduction and 10 μM caused an 86% reduction in type I collagen in SSc fibroblasts (Figures 1C and D). Therefore, the sensitivity to simvastatin was very similar between normal and SSc cells.

Figure 1. Effect of various concentrations of simvastatin (Simv) on normal and systemic sclerosis (SSc) dermal fibroblast type I collagen (COL1) production. A and B, Western blot and densitometric analysis of culture media from normal fibroblasts, after 3 days (A) and 4 days (B) of treatment with simvastatin. C and D, Western blot and densitometric analysis of culture media from SSc fibroblasts after 3 days (C) and 4 days (D) of treatment with simvastatin. Results are representative of 3 independent experiments, each performed in duplicate. Bars show the mean ± SEM percentage relative to the values in untreated (control) fibroblasts. ∗∗∗ = P < 0.0001 versus untreated control.

Down-regulation of COL1A1 steady-state mRNA levels by simvastatin.

To investigate whether the effects of simvastatin on type I collagen production were reflected at the COL1A1 mRNA level, confluent cultures of fibroblasts were treated with 5 μM or 10 μM of the drug for 3 and 4 days. The results are shown in Figure 2. In normal fibroblasts, following 3 days of simvastatin treatment at concentrations of 5 μM and 10 μM, COL1A1 transcripts were reduced by 75% and 87%, respectively (Figure 2A). Following 4 days of treatment with 5 μM or 10 μM of the drug, COL1A1 mRNA levels were reduced by 80% and more than 92%, respectively (Figure 2B). In all experiments, GAPDH mRNA levels were not affected. These data were strongly correlated with the protein measurements. In systemic sclerosis fibroblasts, 5 μM and 10 μM of simvastatin reduced COL1A1 mRNA levels after 3 and 4 days to a similar extent as that in normal fibroblasts (Figures 2C and D).

Figure 2. Northern hybridizations of total RNA from normal and SSc fibroblasts with cDNA for COL1A1, after treatment with simvastatin. A and B, Northern hybridization and densitometric analysis of normal fibroblasts after 3 days (A) and 4 days (B) of treatment. C and D, Northern hybridization and densitometric analysis of SSc fibroblasts after 3 days (C) and 4 days (D) of treatment. Densitometry values were corrected for differences in loading and transfer by normalization with the values obtained for GAPDH hybridizations. Northern blot results are representative of 3 independent experiments, each performed in duplicate. Bars show the mean ± SEM percentage relative to untreated (control) fibroblasts. ∗∗∗ = P < 0.0001 versus untreated control. See Figure 1 for definitions.

Effect of mevalonic acid on type I collagen expression and COL1A1 mRNA levels in simvastatin-treated systemic sclerosis fibroblasts.

Inhibition of hydroxymethylglutaryl–coenzyme A (HMG-CoA) reductase by simvastatin is known to reduce intracellular levels of mevalonate. To investigate the effect of exogenous addition of mevalonate to fibroblasts treated with simvastatin, systemic sclerosis fibroblasts were treated with 5 μM simvastatin with or without the addition of 500 μM mevalonic acid. Collagen production was measured at 2, 3, and 4 days of incubation. The results (Figures 3A and B) show that coincubation of systemic sclerosis fibroblasts with mevalonate completely abrogated the inhibitory effects of simvastatin on collagen production, thus indicating that these effects were directly caused by HMG-CoA reductase inhibition of the synthesis of mevalonate-derived lipid molecules, and could be fully reversed by addition of the metabolite immediately below the enzymatic block. This effect was also observed when COL1A1 mRNA levels were measured at 2, 3, and 4 days in 5 μM simvastatin–treated systemic sclerosis fibroblasts to which 500 μM mevalonic acid was added (Figures 3C and D). Similar effects were observed in normal fibroblasts (results not shown).

Figure 3. Effect of mevalonic acid on type I collagen production and steady-state COL1A1 mRNA levels in simvastatin-treated SSc fibroblasts. Western blot analysis (A) and densitometric analysis of the Western blots (B) were carried out on media of SSc fibroblasts after treatment with 5 μM simvastatin with or without 500 μM mevalonic acid for 2, 3, and 4 days. Northern hybridization (C) and densitometric analysis of the Northern blots (D) were carried out for COL1A1 mRNA in SSc fibroblasts after treatment with 5 μM simvastatin with or without 500 μM mevalonic acid. Values were corrected by normalization with the values obtained for GAPDH hybridizations. Densitometry results are representative of 2 independent experiments, each performed in duplicate. Bars show the mean ± SEM pixel density. ∗∗ = P < 0.001; ∗∗∗ = P < 0.0001, versus cultures without mevalonic acid. NS = not significant (see Figure 1 for other definitions).

Effects of FPP and GGPP on type I collagen production and COL1A1 mRNA levels in simvastatin-treated systemic sclerosis fibroblasts.

The role of the HMG-CoA downstream lipids FPP and GGPP on type I collagen production and mRNA levels was also evaluated in systemic sclerosis fibroblasts. For this purpose, the cells were treated with 5 μM simvastatin for 3 and 4 days with or without the addition of either 10 μM FPP or 10 μM GGPP. The results with regard to type I collagen production are shown in Figures 4A and B. As was observed with mevalonic acid, the addition of GGPP completely reversed the inhibitory effect of simvastatin on type I collagen production at both 3 and 4 days of treatment. In contrast, the addition of FPP failed to reverse the inhibitory effects of the statin on collagen production. Similar results were obtained when COL1A1 mRNA levels were measured at 3 and 4 days in 5 μM simvastatin–treated systemic sclerosis fibroblast cultures to which 10 μM of GGPP or 10 μM of FPP were added (Figures 4C and D). Moreover, testing of normal fibroblasts in the same manner produced similar results (not shown).

Figure 4. Effect of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) on type I collagen production and COL1A1 mRNA steady-state levels in SSc fibroblasts treated with simvastatin. Western blot (A) and densitometric analysis of the Western blots (from 3 separate experiments) (B) were carried out on media of 5 μM simvastatin–treated SSc fibroblasts cultured with either 10 μM FPP or 10 μM GGPP for either 3 or 4 days. Northern hybridization (C) and densitometric analysis of the Northern blots (from 3 separate experiments) (D) for COL1A1 mRNA, after normalization to GAPDH, were carried out on 5 μM simvastatin–treated SSc fibroblasts with or without FPP (10 μM) or GGPP (10 μM) after 3 and 4 days of treatment. Results from Western and Northern blotting are representative of 3 separate experiments. Bars show the mean ± SEM pixel density. ∗∗∗ = P < 0.0001 versus cultures without GGPP or FPP. NS = not significant (see Figure 1 for other definitions).

Effect of simvastatin on COL1A1 promoter activity in transient transfections.

Transient transfection experiments were performed to determine whether the effects of simvastatin on COL1A1 involved the transcriptional down-regulation of COL1A1. Three COL1A1 promoter constructs (−675 bp, −174 bp, and −84 bp) were transfected into 2 systemic sclerosis cell lines and 1 normal fibroblast cell line, and the cells were then treated with 5 μM simvastatin for 3 days. The results in systemic sclerosis cell cultures demonstrated that the promoter activities of the −675-bp and −174-bp COL1Al constructs were down-regulated by the statin, whereas the −84-bp construct was essentially not affected (Figures 5A and B). The results were similar between normal and systemic sclerosis fibroblasts. The most profound effect was observed with the −174-bp construct. An average of 3 separate experiments with systemic sclerosis cells revealed that simvastatin caused an ∼45% reduction in its promoter activity compared with that in untreated cells, as calculated by densitometric analysis of the combined results from the 3 cell lines.

Figure 5. Simvastatin treatment of SSc fibroblasts transiently transfected with COL1A1 promoter constructs. A, SSc fibroblasts were transfected with 2.5 μg of 1 of 3 different COL1A1 promoter constructs plus 0.2 μg of pCMV β-galactosidase DNA, and then either treated with 5 μM simvastatin or left untreated for 3 days. Results were normalized for transfection efficiency, and acetylated chloramphenicol activity was analyzed (as described in Patients and Methods). Results are representative of 3 independent experiments. Note that owing to the much higher transcriptional activity of the −174-bp construct, the image shown is from an underexposed gel (2 hours) as compared with the images obtained with the −675-bp and −84-bp constructs, which were exposed for 24 hours. B, Densitometric analysis of the transfection described in A, with results representative of 3 separate experiments. Bars show the mean ± SEM percentage of activity (shaded bars) relative to the values in each corresponding untreated control (open bars). ∗∗ = P < 0.001 versus untreated control. NS = not significant (see Figure 1 for other definitions).

Comparison of CBF and Sp1 binding activity in nuclear extracts from control and simvastatin-treated fibroblasts.

In previous studies, we have shown that Sp1 and CBF bind to a −125-bp fragment of the COL1A1 promoter, and we identified the formation of 4 DNA–protein complexes in nuclear extracts with this COL1A1 fragment. These complexes were identified as Cl, C2, C4 (corresponding to Sp1/Sp3 binding sites), and C3 (corresponding to a CBF binding site) (25–27). To compare the binding activity of CBF and Sp1 in nuclear extracts from control or 5 μM simvastatin–treated cells, we incubated nuclear extracts of treated or untreated systemic sclerosis fibroblasts with the −125-bp COL1A1 probe and performed EMSA with consensus oligonucleotides for Sp1 (Figures 6A and B) or CBF (Figures 6C and D) as competitors. To confirm the specificity of the DNA–protein complexes, mutated oligonucleotides for Sp1 and CBF were used (results not shown).

Figure 6. Electrophoretic gel mobility shift assay (EMSA) of nuclear extracts (NE) from systemic sclerosis skin fibroblasts interacting with a −125-bp DNA fragment from the COL1A1 promoter following treatment with 5 μM simvastatin (Simv) for 4 days. A, EMSA using competition with a 100-fold molar excess of wild-type (WT) Sp1 oligonucleotides. B, Densitometric analysis of results shown in lanes 5 and 6 and lanes 7 and 8 of A. Bars show the mean ± SEM pixel density from 3 separate experiments. C, EMSA using competition with a 100-fold molar excess of WT CCAAT-box binding factor (CBF) oligonucleotides. D, Densitometric analysis of results shown in lanes 5 and 6 and lanes 7 and 8 of C. Bars show the mean ± SEM pixel density from 3 separate experiments, each performed in duplicate. ∗∗ = P < 0.001; ∗∗∗ = P < 0.0001, versus untreated control.

Competition experiments with Sp1 consensus oligonucleotides enabled us to abolish Sp1 binding to the −125-bp COL1A1 fragment, and thus allowed us to evaluate the binding of only the CBF transcription factor (Figures 6A and B). It can be seen that CBF binding activity was reduced by ∼65% in nuclear extracts from simvastatin-treated fibroblasts. In the experiments in which CBF consensus competition was performed, the binding of CBF to the −125-bp COL1A1 DNA probe was abolished, allowing us to compare the effects of simvastatin on Sp1 binding activity (Figures 6C and D). The results obtained showed that simvastatin caused a level of inhibition of Sp1 binding to the −125-bp COL1A1 probe similar to the level of inhibition induced against CBF binding.

DISCUSSION

It has recently become apparent that posttranslational protein isoprenylation is of fundamental importance in the regulation of expression of numerous genes involved in cell survival, proliferation, and differentiation, as well as in cytoskeletal organization. These processes are regulated by the Rho and RAS superfamilies of proteins, which are small membrane-bound GTPases that require the attachment of a prenyl group (either farnesyl or geranylgeranyl) for their activity. It has also been demonstrated that isoprenylation may participate in the regulation of expression of interstitial collagen genes, as well as in some of the downstream effects of TGFβ. Indeed, specific inhibition of geranylgeranylation caused a significant reduction of collagen gene expression in normal fibroblasts as well as in fibroblasts from patients with systemic sclerosis (16). Other studies have shown that inhibition of HMG-CoA reductase with statins not only causes a potent inhibition of cholesterol biosynthesis, but also inhibits synthesis of lipid molecules required for isoprenylation (17, 18).

We therefore examined whether statins are capable of modulating type I collagen gene expression in cultured normal and systemic sclerosis dermal fibroblasts. The results demonstrated that treatment of systemic sclerosis cells with simvastatin (5–10 μM) caused a potent inhibition of COLlAl expression in these cells. The inhibition was not due to a generalized toxic effect, since it was completely abrogated when statin-treated cells were supplemented with the HMG-CoA downstream products mevalonic acid and GGPP. Of note was the observation that FPP, the other important downstream product of HMG-CoA, failed to reverse the inhibitory effects of the statin on COLlAl expression, indicating that geranylgeranylation, but not farnesyl prenylation, was involved. We further demonstrated that the statin acts ultimately at the transcriptional level, and that its effect on COL1A1 expression is mediated through a −174-bp gene segment in the proximal COLlAl promoter. Thus, it appears that the effects of this statin are mediated by COL1A1 promoter elements previously recognized as participants in the up-regulation of collagen gene expression in systemic sclerosis (9, 10). This possibility was explored further by the assessment of the binding activity of Sp1 and CBF, which are 2 transcriptional factors that play a crucial role in the regulation of COLlAl transcription, both under normal conditions and under the influence of TGFβ (25, 27–33).

It is well established that reduction and normalization of elevated cholesterol levels by statins promote primary and secondary prevention of ischemic, atherosclerotic-related events in patients with hypercholesterolemia (34–36). This effect was initially believed to be solely related to the cholesterol-lowering effects of the statins. However, there is increasing evidence that these agents may exert profound endothelial and vascular protective effects beyond their lipid-lowering action (19–21). These later clinical and experimental observations have led to a wealth of research studies examining the pleiotropic effects of statins, including modification of vascular disease and certain immune responses (for review, see ref. 21). Indeed, statins have been shown to down-regulate the expression of TGFβ1 by renal mesangial cells in models of renal nephropathy in which fibrosis is mediated by TGFβ1 and connective tissue growth factor (CTGF), and to reduce CTGF mRNA levels in normal human and rodent mesangial cell lines, in human renal fibroblast cell lines, and in human lung fibroblasts from patients with idiopathic pulmonary fibrosis (37–39).

To understand these pleiotropic effects, it is necessary to understand the metabolic pathways on which these agents exert their cholesterol-lowering effect. Statins inhibit the HMG-CoA reductase, which is the enzyme that catalyzes the formation of mevalonate from HMG-CoA, the earliest step in the production of cholesterol. Mevalonate is also the precursor of isoprenoid groups that are required to modify posttranslationally the Rho and Ras superfamilies of proteins by attaching a hydrophilic prenyl group, either farnesyl or geranylgeranyl. The Rho and Ras proteins are small membrane-bound GTPases, which are active in the GTP-bound state and inactive in the GDP-bound state. In their active state, these proteins regulate important cellular functions including cell survival, proliferation, and differentiation, as well as cytoskeletal organization, by relaying extracellular ligand–stimulated signals to cytoplasmic signaling cascades (40, 41). It is the inhibition of prenylation of the Rho- and Ras-related proteins by statins that is postulated to be responsible for some of the pleiotropic effects of this class of agents (42, 43).

We are very aware that the concentrations of simvastatin required in vitro in the systemic sclerosis fibroblast culture system to observe the effects on collagen gene expression are between 50 and 100 times greater than those achieved in plasma following oral administration of the drug at doses currently utilized for cholesterol-lowering effects. However, the simvastatin concentrations used in our study are very similar to those used in numerous other studies examining the pleiotropic effects of the drug in vitro. For example, the concentration of simvastatin considered effective for modulating cardiomyocyte proliferation was in the range of 10–20 μM (44) and the concentration required to influence apoptosis of malignant lung epithelial cell lines ranged from 8.4 μM to 45.4 μM depending on the cell line (45). Similarly, numerous other studies with mesenchymal cells utilized concentrations in the range of 1–10 μM for modulation of various cellular functions, including the expression of growth factors and the production of various extracellular matrix molecules (42).

It is likely that the exquisite sensitivity of the cholesterol biosynthetic pathway to the inhibitory effects of statins in vivo is related to the facility of the compounds for cell penetration, since, for example, liver cell membranes are extremely permeable to statins, whereas membranes from other cell types are more resistant. Another factor that may be responsible for the requirement of higher statin concentrations in the in vitro studies relates to the need to empty or substantially reduce the intracellular stores of cholesterol-derived lipid precursors to achieve the observed effects. It is likely that in vivo depletion of these lipid precursor pools can be accomplished following extended periods of treatment at lower concentrations of the drug; however, for in vitro studies, it is necessary that depletion of these storage pools be accomplished in a much shorter period of time (in hours), therefore requiring much higher concentrations of the drug.

It is worthwhile to mention that some in vivo human studies that used much higher statin doses than those used to lower cholesterol levels reached statin serum concentrations in the μM range. For example, a recent study to examine the effects of very high lovastatin doses on cerebral gliomas in human subjects showed that serum concentrations as high as 3.92 μM could be achieved (46). Thus, it may be feasible to reach, in vivo, the statin concentrations required to accomplish the effects that we have described herein by increasing the administered dose and by using statins that may have an improved or preferential ability to penetrate mesenchymal cell membranes.

The potent inhibitory effects of simvastatin on collagen demonstrated in the present study provide strong support to the testable hypothesis that statins may be of benefit in the treatment of systemic sclerosis, a disease in which the prominent endothelial cell dysfunction and vascular abnormalities have been suggested to play a crucial role in its pathogenesis and evolution. We therefore propose that statins, which are readily available and have been demonstrated to be remarkably safe, are a promising alternative for the treatment of systemic sclerosis, a disease for which few proven disease-modifying agents are available.

Acknowledgements

The expert assistance of Kate Salmon in the preparation of the manuscript is gratefully acknowledged.