Characterization of a DNA binding site that mediates the stimulatory effect of cyclosporin-A on type III collagen expression in renal cells
Roberta Oleggini1,
Luca Musante1,
Stefania Menoni1,
Gerardo Botti1,
Marco Di Duca1,
Michela Prudenziati2,
Alba Carrea1,
Roberto Ravazzolo2 and
Gian Marco Ghiggeri1,
1 Nephrology Section, G. Gaslini Childrens Hospital,
2 Laboratory of Molecular Genetics, G. Gaslini Childrens Hospital and Department of Oncology Biology and Genetics, University of Genova, Italy
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Abstract
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Background. Previous work from our laboratory demonstrated upregulation of type III collagen by cyclosporin A (CsA) in a cellular model of renal fibroblasts in vitro, suggesting that a mechanism of gene transcriptional activation might be responsible for collagen accumulation in renal fibrosis resulting from chronic CsA treatment.
Methods. We analysed in the same cellular model: (i) COL3A1 mRNA expression by RTPCR; (ii) COL3A1 promoter activity by transfection of renal fibroblasts with constructs containing promoter fragments of different length fused to a reporter gene; (iii) expression of transcription factors by western blot analysis; (iv) DNAprotein binding by gel retardation assays with nuclear extracts from CsA-treated and untreated cells; and (v) site-directed mutagenesis of COL3A1 promoter to verify the role of a short DNA segment as CsA responsive element.
Results. CsA induced a 35-fold increase in COL3A1 mRNA that was paralleled by a stimulation of the COL3A1 promoter. Degradation of COL3A1 mRNA was comparable in CsA-treated and -untreated cells. The target region was first limited to a 178 bp fragment from -117 to +61 (pFV1). By gel retardation, utilizing several oligonucleotides that covered the whole length of pFV1, we detected a factor able to bind the promoter DNA (oligo 31) in nuclear extracts after 3 h treatment with CsA. The binding was absent in untreated cells and it was not detected when a 10-base mutation was introduced in oligonucleotide 31. Finally, the same substitution mutation at the site of binding of this factor abolished the stimulatory effect of CsA on COL3A1 promoter. Some transcription factors, whose potential binding sites are included in the above promoter fragment, were induced by CsA treatment either soon (3 h) or late (2472 h) after treatment and were detected by western blot analysis.
Conclusions. CsA induces the synthesis of type III collagen by stimulating a pathway leading to activation of COL3A1 promoter and upregulation of COL3A1 mRNA. A short promoter fragment, proximal to the transcription start site, is the target of CsA stimulation.
Keywords: COL3A1; cyclosporin; renal fibrosis; type III collagen
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Introduction
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Renal scarring and loss of renal function are the two major drawbacks of long-term treatment with cyclosporin A (CsA) of both organ grafts and mild immunological diseases [14]. Accumulation of extracellular matrix, mainly type I and type III collagens, is the basic histopathological hallmark of chronic CsA nephropathy. This finding is common to other nephropathies that evolve towards renal failure, although it is not characteristic of a specific pathogenetic entity [5,6]. This has prompted further investigation on the molecular mechanism of CsA-mediated renal toxicity with the long-term aim of deriving some pathophysiological conclusions. Different possible pathways may be envisaged, such as stimulation of the reninangiotensin system, resulting in increased levels of angiotensin II [79], TGF-ß [1012], or PDGF [13], all of which are recognized fibrogenic substances [14,15]. These possibilities are supported by already available experimental observations, such as CsA induction of renin release by the renal cortex in vitro and in vivo and reduction of tubulointerstitial fibrosis by blockade of angiotensin II in rats treated with CsA. On the other hand, CsA induction of collagen synthesis, mainly type III collagen, in various renal cell systems in vitro [16] and in vivo [17] might suggest a more direct role of the drug in stimulating renal accumulation of fibrogenic collagens. Previous work was directed at demonstrating increased expression of type III collagen, a homotrimer of
1(III) chains, in fibrotic kidney tissues after CsA treatment; no study has yet been performed to investigate an effect at the transcriptional level. The present study was planned to investigate the molecular mechanism responsible for the effect of CsA on the transcription of COL3A1, the gene encoding for type III collagen. In particular, we describe the localization of a CsA-responsive element in a region of the COL3A1 promoter proximal to the transcription start site and the induction of a DNA binding factor that recognizes a specific sequence inside this promoter region.
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Subjects and methods
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Cell culture
Monkey renal interstitial fibroblasts (CV1) were obtained from the American Type Culture Collection (Rockville, Maryland, USA) and were grown under a humidified atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 1% non-essential amino acids, 1% glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. The effect of CsA was determined by addition of various drug concentrations (from 0.5 to 200 ng/ml) after removal of serum from cell culture, in order to avoid uncontrolled binding of the drug to serum components giving rise to variable results; experiments were also performed in the presence of serum.
Constructs
PCR amplification from human genomic DNA was used to amplify fragments of the COL3A1 5' flanking region. A 1436 bp fragment pRup4, from -1375 to +61 relative to the transcription start site, was obtained by PCR using the following pairs of primers: sense 5' GAT CAA GCT TGC AAG TTT GCC ACT GTC CAG T 3' and antisense 5' CTC AAG CTT AGC ACC ATC AAG TTG TTC CC 3', both carrying the HindIII restriction site at their 5 end. A 318 bp fragment pRup6 (-257 to +61) was obtained by PCR by utilizing the sense primer 5' GCG AAG CTT CTA TAC GTT CCT AAG T 3', also carrying the HindIII restriction site, and the same antisense primer as above. The amplified fragments were inserted into the pGL2-basic vector (Promega Inc., Madison, WI) upstream of the luciferase reporter gene, after digestion with HindIII, giving rise to the pRup4 and the pRup6 constructs. The pFV1 construct was obtained from pRup6 after digestion with Ssp1 and removal of the DNA sequence from -318 to -117 (see Figure 1a
).

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Fig. 1. (A)COL3A1 promoter constructs utilized in the study. The three constructs (pRup4, pRup6, and pFV1) were of different lengths and spanned the entire DNA sequences between -1375 and +61. The sequence of oligonucleotide 31 was contained in the shortest fragment (pFV1). In pFV131m the sequence relative to oligo 31 was mutated according to the sequence in bold. (B) COL3A1 promoter sequence between -117 and +61. Lines indicate the oligonucleotides used for DNAprotein binding assay; the arrow indicates the transcription start site.
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Mutagenesis
Plasmid pGL231 m was made by site-directed mutagenesis using a two-step procedure based on PCR [19] to introduce substitutions into the original pFV1 plasmid, using the following pair of mutated primers: 5' TAC TTA GCT CCT CTT GGG TGC GGC 3', sense; 5' GCC GCA CCC AAG AGG AGC TAA GTA 3', antisense, in which the substituted bases are in bold.
Transient transfections
Cells were maintained in DMEM medium supplemented with 10% fetal calf serum, 100 mU/ml penicillin and 100 µg/ml streptomycin and were maintained in a humidified atmosphere at a density of 0.75x106 cells in 10 cm diameter plastic dishes. Transfections were performed as described by Chen and Okayama [20], using 5 µg of plasmid DNA in 1 ml of calcium phosphate coprecipitate. Plasmid pRL-Renilla Luciferase (Promega 0.4 µg) was always cotransfected as an internal standard. Twenty four hours after DNA addition, cells were washed twice with phosphate-buffered saline (PBS), re-fed with serum-free fresh medium with or without CsA, and further incubated for the indicated time. Transfection efficiency was evaluated by determining renilla luciferase activity (Dual Luciferase Renilla Assay System, Promega). All transfections were repeated at least three times. Results are expressed as mean±SD.
mRNA
Total cellular RNA was isolated by the UltraspecTM RNA isolation system (Biotecx Laboratories Inc. Houston, Texas, USA). For cDNA synthesis, 1st Strand cDNA Synthesis kit for RTPCR-AMV (Boehringer Mannheim.GmbH, Mannheim, Germany) was used. The amount of COL3A1 mRNA transcripts was determined by RTPCR. As monkey G3PDH was not available, aldolase mRNA was chosen as the internal standard of a constitutively expressed housekeeping gene for comparison and normalization.
The following primers were used for the detection of mRNA transcripts: COL3A1 primers sense 5' CTG GAC CAA AAG GTG ATG CTG 3' and antisense 5' TGC CAG GGA ATC CTC GAT GTC 3'. Aldolase sense primer 5' GGC AAG GGC ATC CTG GCT GCA GA 3', and antisense 5' TAA CGG GCC AGA ACA TTG GCA TT 3'. Densitometric evaluation of COL3A1 and aldolase mRNAs was done with LKB Ultrascan XL laser densitometer. COL3A1 mRNA was normalized for aldolase mRNA; the levels of COL3A1 mRNA accumulation were expressed as fold increase at various times after CsA cell treatment vs control untreated cells. In order to exclude any difference in degradation kinetics of RNA between CsA-treated and untreated cells, COL3A1 mRNA stability was assayed by actinomycin D as described [21]. Accordingly, cells were incubated with 0.5 ng/ml CsA for 24 h and were then treated with 5 µ/ml actinomycin D mRNA was evaluated at several times, from 1 to 24 h, after actinomycin D treatment.
Preparation of cellular and nuclear extracts
Total extracts from both untreated cells and cells incubated with CsA (0.5 ng/ml for 172 h), were lysed in RIPA buffer (1xphosphate buffer saline, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mmol/l phenylmethylsulphonyl fluoride). After 30 min at 4°C, the mixture was centrifuged at 14,000 r.p.m. for 15 min and the supernatant was treated as whole cell extract for western blot. Nuclear extracts were prepared according to the method of Dignam et al. [22]. All steps were carried out at +4°C, in the presence of phenylmethylsulphonyl fluoride (0.5 mmol/l).
Western blot analysis
Cellular extracts were subjected to SDSPAGE (816% polyacrylamide) and western blot analysis according to a previously described procedure [23]. Chemiluminescent detection was carried out according to the manufacturer's specifications (ECL Super-signal, Ultra, Pierce, Rockford, IL), using peroxidase-conjugated antirabbit-Ig (1 : 10 000). Antibodies recognizing several transcriptional factors (STAT 13, NF-
B p 65-p 50, c-Rel, c-Fos, c-Jun, ATF1,AP2
, CEBPß) were used at 1 : 100010 000 dilution. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Gel retardation assay
For DNA binding assay, double-stranded oligonucleotides corresponding to various elements (oligonucleotides 11, 21, 31, 41) in the COL3A1-promoter were used as 32P-labelled probes or as unlabelled competitors (see Figure 1b
). Oligonucleotides were end-labelled with [
-32P]ATP (Amersham, Little Chalfont, UK) and T4 polynucleotide kinase (New England Laboratories, Beverly, USA) and were purified by Quick SpinTM Columns (Boehringer). Gel retardation was performed as previously described [24]: nuclear extracts (5 µg protein), from both control and CsA-treated cells in a total volume of 15 µl containing 20 mmol/l TrisHCl (pH 7.5), 100 mmol/l NaCl, 0.35 mmol/l dithiothreitol, 0.5 mmol/l phenylmethylsulphonyl fluoride, 10% glycerol and 1µg poly (dIdC), were incubated with 10 fmol 32P-labelled probe. Competition experiments were performed in the presence of a 100400-fold molar excess of unlabelled competitor oligonucleotide. The bound complexes were separated from the free probe by electrophoresis on 5% polyacrylamide gels. Complexes were visualized by autoradiography of the dried gels.
For supershift assays, specific antibodies were included in the pre-incubation mixture prior to the addition of the probe and were loaded onto polyacrylamide gels; a lane containing pre-immune serum was always included as control.
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Results
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COL3A1 mRNA expression
Previous results showing an induction of type III collagen synthesis by CsA in renal fibroblasts [16] suggested that gene transcription might be involved. To verify such a possibility, COL3A1 mRNA expression in CV1 cells was evaluated after CsA treatment for different times and using increasing drug concentrations. Figure 2 (a and c)
shows the time course of COL3A1 mRNA expression obtained by stimulating cells with 0.5 ng/ml CsA, that was the optimal condition for response and was corresponding to the best doseresponse effect on type III collagen protein [1616]. We observed an increasing mRNA accumulation up to 35-fold with respect to untreated cells, with a peak at 48 h. Further experiments were done with actinomycin D to evaluate the stability of mRNA and to exclude a different degradation kinetics of mRNA between CsA-treated and untreated cells. As shown in Figure 2b
, the stability of COL3A1 mRNA was comparable in both cases, with the exception of an initial increased slope of the curve in the former case.

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Fig. 2. (AC) Time-course of COL3A1 mRNA expression in CV1 renal fibroblastic cells treated with CsA. (A) Cells were treated for several times (up to 5 days) with constant 0.5 ng/ml CsA (upper panel). The house-keeping gene utilized to normalize COL3A1 was aldolase. The results are given as the ratio of COL3A1 mRNA expression between CsA-treated and untreated cells after normalization for aldolase. (B) After 24 h treatment with 0.5 ng/ml CsA, cells were treated with actinomycin D and then COL3A1 mRNA was evaluated. Results are given for CsA-treated and control cells. (C) Example of a gel showing COL3A1 and aldolase mRNA expression by cells treated with CsA at different concentration and for different times.
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Effect of CsA on COL3A1 transcriptional activity
To clarify whether the COL3A1 mRNA induction by CsA might be ascribed to a mechanism of activation of gene transcription, transient transfection experiments in the same CV1 renal fibroblastic cells were performed. We utilized three constructs containing the luciferase reporter gene fused to COL3A1 promoter fragments of different length spanning an interval from nucleotide -1375 to +61 relative to the transcription start point. These constructs were utilized to detect the minimal promoter fragment responsive to CsA. In Figure 3
it is shown that CsA produced the maximal effect on pFV1 that is the shortest construct. Figure 4
(lower panel) describes the doseresponse effect of increasing concentrations of CsA from 0.5 to 5.0 ng/ml for 24 h after cell transfection, showing a maximum of the luciferase activity at 0.5. Further increase of drug concentration up to 200 ng/ml or decrease below 0.1 ng/ml resulted in low levels of activation. The effect of incubation at different times is described in Figure 4
(upper panel), showing a maximum at 24 h after transfection and then a plateau; however, the difference in pFV1 activity was roughly the same at 24, 48 and 72 h.

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Fig. 3. COL3A1 promoter activity in CsA-treated CV1 cells. Results of transfection with several constructs of different length (pRup4, pRup6, pFVl, pFV1M) were calculated as ratio of COL3A1 promoter activity between CsA-treated and untreated cells. The dose of CsA was 0.5 ng/ml in all cases. pFV1M construct incorporates a 10-base substitution mutation in the original pFV1 according to the sequence of 31 m (see Figure 1 ). Data are given as mean±SD.
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Fig. 4. Time and dose-dependent effect of CsA on COL3A1 promoter constructs activity. The results are expressed as described in the legend to Figure 3.
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DNAprotein binding assay
We then tried to identify, inside the pFV1 promoter sequence, a DNA element able to bind a factor(s) present in nuclear extracts of CV1 cells after CsA treatment. Four oligonucleotides (oligo 11, 21, 41 and 31) that covered portions of the pFV1 sequence potentially significant for factor binding were synthesized and incubated with nuclear extracts, in order to detected DNAprotein interaction. A retarded complex was found by incubating the double-strand labelled oligonucleotide 31 (101 to -78) with nuclear extracts from CsA-treated cells (Figure 5
, lanes 1 and 2). No other complex with differential appearance between CsA-treated and untreated cells was found using the other oligonucleotides. The complex detected with the 31 oligonucleotide was due to a sequence-specific DNAprotein interaction, since it was competed by a 100400-fold excess of the same 31 unlabelled oligonucleotide (Figure 5
, lanes 35) while the mutated form (31 m) did not compete (Figure 5
, lanes 68). Other retarded complexes present in the picture appear to be due to non-specific binding. Moreover, mutation at the 31 oligo abolished the binding with nuclear extract from cells stimulated with CsA (Figure 6
).

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Fig. 5. Gel retardation assay of nuclear extracts from CsA-treated and untreated CV1 cells. Lanes 1 and 2 show the comparison between nuclear extracts prepared from untreated or CsA-treated cells, using the oligonucleotide 31 (10 fmol) as a labelled probe. Lanes 38 show the effect of the indicated unlabelled competitor oligonucleotides 31 and 31 m at 100-, 200-, or 400-fold excess with respect to the molar concentration of the labelled oligonucleotide 31.
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Fig. 6. The mutated 31m oligonucleotide is unable to bind the CsA-induced factor. In lanes 1 and 2 the wild type labelled 31 oligonucleotide was used, whereas the mutant 31 m oligonucleotide was used in lanes 3 and 4.
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Site-directed mutagenesis in COL3A1 promoter
To verify whether this DNA protein interaction was meaningful for promoter response to CsA, we introduced a 10 bp substitution mutation inside the region of oligo 31. Once introduced in the promoter fragment fused to the reporter gene to generate plasmid pFVI 31 m, this mutation abolished responsiveness of the original pFVI to CsA action (Figure 3
), thus indicating that the -101 to -78 promoter fragment contains a cis-acting DNA element responsive to CsA.
CsA induction of transcription factors
On the basis of the results suggesting a mechanism of transcriptional activation and of computer analysis of the promoter sequence contained in our pFV1 construct (Figure 1
), we found that a number of transcription factors could find potential recognition sites in this promoter fragment. In particular, sites for AP2, C/EBPß and two GC-boxes were found between -96 and -78, corresponding to the oligonucleotide 31 sequence, a site for the Stat family between -70 and -62, sites for NF-AT and AP1 between -57 and -43, a NF-
B site between -9 and +l. We performed western blot analysis of CV1 cell extracts at different times after treatment with CsA, using antibodies against the factors that might bind these recognition sites, and the expression pattern is shown in Figure 7
. It appears that the drug elicits two types of response, early (within 6 h) and late (peak between 24 and 72 h). Factors induced in the early phase were c-Jun, c-Fos and c-Rel, whereas Stat 1, Stat 3, AP2
were observed in increased amounts in the late phase.

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Fig. 7. Transcription factors induced by CsA treatment of CV1 renal fibroblasts. Cellular extracts from CV1 cells incubated with CsA (0.5 ng/ml) for different times from 0 to 72 h were analysed by western blot, using antibodies against the indicated transcription factors. Early, late, or no response are shown.
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Temptative characterization of the interacting factor
Based on the localization of a DNA-responsive element to CsA within the COL3A1 promoter and on the results of transcription factor induction, we attempted to characterize the nuclear factor that binds oligonucleotide 31. The experimental approach was to incubate nuclear extract activated by CsA with oligo 31 and with antibodies raised against c-Jun, c-Fos, AP2, C/EBPß, Stat 1, Stat 3, p65 and p50 NF-
B, and c-Rel transcription factors in the gel retardation assays. Our results were negative (not shown), thus excluding the direct participation of these in the complex formed by the oligonucleotide 31 and proteins contained in nuclear extract from CsA-treated CV1 cells.
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Discussion
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The utilization of CsA as an antirejection drug has considerably improved allograft survival rates. Unfortunately CsA has adverse side-effects on both renal function and structure, which limit a more widespread utilization of the drug in kidney allograft. The first effect of CsA is mediated by the perturbation of renal haemodynamics, which may acutely decrease renal function, is dose related, and reverts after dose reduction. Chronic CsA nephropathy is, on the other hand, a major challenge in long-term therapy with the drug; it is characterized by tubulointerstitial fibrosis in a striped pattern and is associated with hyaline degeneration of the afferent arteries. This picture is, in many aspects, indistinguishable from other pathological conditions such as chronic rejection [5]. In spite of some recent advances, the pathogenesis of CsA nephrotoxicity remains hypothetical. One accredited possibility involves a regulatory loop [26,27] in which CsA stimulates the expression of fibrogenic cytokines, such as AngII or TGF-ß [8,11] and, through intermediate steps that are still unknown, activates collagen expression. Both AngII and TGF-ß are, in fact, key fibrogenic substances implicated in fibrosis in a number of chronic diseases of the kidney and other organs. Shihab et al. [12] recently demonstrated that the block of AngII decreases TGF-ß expression and matrix proteins deposition in chronic CsA nephropathy, thus suggesting a relationship between the drug and this fibrogenic cytokine.
Another interesting hypothesis is that CsA, in parallel with changes in renal haemodynamics, could finely regulate renal cell homeostasis by modulating transcription factors through a mechanism that involves binding with cyclophilin and calcineurin [28,29]. This mechanism has been described to explain the causal relationships between CsA treatment and the expression of Il-6 by fibroblasts [30]; in this case, the CsA-calcineurin complex acts by activating NF-
B or by inhibiting I
B
degradation.
We have recently demonstrated [16] that CsA stimulates the synthesis of collagen III in an in vitro renal cell model (CV1 fibroblasts) and, in the present work, we have started to investigate the molecular mechanism implicated in such a regulation, by analysing the effect of CsA on COL3A1 transcription in the same cell model. The basic finding is that CsA stimulates COL3A1 mRNA expression and, in parallel, up-regulates the activity of a 180-bp DNA fragment of COL3A1 promoter immediately upstream of the transcription start site. A second point was to characterize the DNA binding site within the fragment (pFV1) that is responsive to CsA. We utilized gel retardation experiments with oligonucleotides, designed on the basis of potential binding sites in the COL3A1 promoter. Only for one of these oligonucleotides (-101 to -78) binding to a factor present in nuclear extracts from CsA-treated cells was observed while it was absent in untreated cells. By introducing a 10-bp substitution mutation in the oligonucleotide sequence, the formation of the DNAprotein complex was abolished, indicating a specific site for the binding. Finally, when the same mutation was introduced in the pFV1 construct of COL3A1, the activation induced by CsA in the wild type promoter was abolished, thus demonstrating a central role at this site, in the stimulation by CsA of COL3A1 promoter activity. The presence on COL3A1 promoter sequence of a specific cis-acting element responsive to CsA strongly suggests the existence of a mechanism of activation in which CsA stimulates the expression of a nuclear DNA-binding factor which binds the COL3A1 promoter.
With the objective of identifying and characterizing a CsA-modulated transcription factor(s) that acts on this DNA binding site, we adopted a two-step experimental approach. The first step was to evaluate the cellular expression of some transcription factors whose binding sites are similar to DNA sequences present in the promoter fragment regulated by CsA. This was done by western blot analysis of cell extracts prepared at different times after CsA treatment. The list of transcription factors included members of the STAT (1 and 3), NF-
B (p65, p50, c-Rel) and AP1 (c-Jun, c-Fos) superfamilies as well as the AP2
complex and C/EBPß. CsA treatment induced a rapid increase (within 6 h) of c-Jun, c-Fos, and c-Rel expression, followed by a delayed (2472 h) increase in Stat 1, Stat 3 and NF-
B p50. These are ubiquitous factors and their expression could be modulated by CsA through some complex pathways of intracellular signalling. At the same time, a few of the aforementioned factors (AP2, C/EBPß) recognize DNA sequences with similarities for the DNA binding site defined by oligo 31. To ascertain whether there was any relationship between the expression of these transcription factors and the specific retarded complex obtained with the -101 to -78 promoter sequence, we evaluated its eventual modification by the antinuclear factor antibodies. However, we were unable to characterize further the transcription factor that mediates the activation of COL3A1 since none of the antibodies gave positive results. This point remains a key goal for future studies.
In conclusion, we were able to demonstrate a stimulatory effect of CsA on a short segment of COL3A1 promoter in which a DNA binding site responsive to the drug was characterized. The mechanisms of activation by CsA of COL3A1 promoter may provide a contribution to clarify all factors and events underlying the pathogenesis of renal fibrosis induced by long-term treatment with the drug.
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Acknowledgments
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We wish to thank Dr F. Ramirez for providing the COL3A1 clone containing the promoter sequence. This work was supported by a grant of the Italian Ministry of Health to G. Gaslini Institute (Progetto Finalizzato H 1290059) and by a CNR target Project on Biotechnology to RR.
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Notes
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Correspondence and offprint requests to: Dr Gian Marco Ghiggeri, Nephrology Section, G. Gaslini Childrens Hospital, Largo G. Gaslini 5, 16148 Genova, Italy. 
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Received for publication: 14.10.98
Revision received 31.12.99.