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Cyclosporin A Affects Signaling Events Differentially in Human Gingival Fibroblasts
A. Bostrom,
H. Bharath,
A. Saulewicz and
A.S. Narayanan*
Department of Pathology, Box 357470, University of Washington School of Medicine, Seattle, WA 98195-7470, USA;
Correspondence: * corresponding author, sampath{at}u.washington.edu
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ABSTRACT
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Gingival overgrowth is a common side-effect of the administration of cyclosporin A (CSA), phenytoin, and calcium blockers. To identify the signaling mechanisms possibly involved in the overgrowth, we examined how CSA affects the activities of MAP kinases and transcription factors in human gingival fibroblasts (HGF). The HGF were treated with CSA and TNF- or PDGF. DNA-binding activity of NFAT, NF B, and AP-1 transcription factors was determined by gel shift assay, and JNK, p38, and ERK1 and ERK2 activation was assessed by Western blot analysis of immunoprecipitates. The CSA inhibited NFAT, NFB, and p38 and JNK activities; however, ERK1 and ERK2 were not affected significantly. AP-1 activity increased ~ 4.5-fold. Our results indicate that CSA affects signaling molecules in HGF differently from other cell types, and that a CSA-induced increase in AP-1 activity may affect the expression of fibrogenic molecules in gingiva and promote gingival overgrowth.
Key Words: gingival overgrowth • cyclosporin A • MAP kinases • AP-1 • fibroblasts
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INTRODUCTION
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Gingival overgrowth is a common side-effect of the chronic use of the immunosuppressive drug cyclosporin A, the anti-epileptic drug diphenylhydantoin (phenytoin), and calcium blockers. The most significant biochemical feature of gingival overgrowth and hereditary gingival fibromatosis, a genetically heterogeneous condition, is increased fibroblast activity and overproduction and deposition of collagens, proteoglycans, and other extracellular matrix components (Narayanan and Bartold, 1996; Seymour et al., 1996). The deposition of excess matrix components is due to a shift in the balance between synthesis and degradation of matrix components toward synthesis and accumulation. This is believed to be largely due to TGF-β, which activates the synthesis of collagen and other extracellular matrix components and suppresses the production of matrix metalloproteinases (MMPs) (Atamas, 2002; Werner and Grose, 2003). Diminished production of IFN- , a lymphokine which regulates collagen production, by T-cells may also contribute to collagen accumulation (Atamas, 2002; Werner and Grose, 2003). In addition, selection of fibroblast subpopulations characterized by high steady-state collagen synthesis levels has also been suggested as a mechanism contributing to gingival overgrowth and other fibroses (Narayanan and Bartold, 1996; Phan, 2003).
Cyclosporin A is used to prevent graft rejection in organ transplant patients and to treat inflammatory diseases such as bullous pemphigoid, psoriasis, and rheumatoid arthritis. Its use is associated with gingival overgrowth, interstitial fibrosis of the kidneys, and acceleration of atherogenesis (Seymour et al., 1996; Johnson et al., 1999); however, the molecular mechanisms involved are not clearly understood. Cyclosporin A binds to cytoplasmic cyclophilin and inhibits the Ca++-calmodulin-dependent serine-threonine phosphatase, calcineurin (Ho et al., 1996; Jørgensen et al., 2003). In activated T-cells, calcineurin inhibition prevents dephosphorylation of the transcription factor, nuclear factor of activated T-cells-c (NFATc), and its translocation to the nucleus, and this results in inhibition of transcription of IL-2, IFN-, and other immunoregulatory genes. In cells and tissues treated with cyclosporin A, collagen synthesis is higher, but collagenase expression is decreased (Schincaglia et al., 1992; Sugano et al., 1998; Bolzani et al., 2000). Cyclosporin-A-treated tissues and cells also contain higher TGF-β levels (Shin et al., 1998; Cotrim et al., 2002). The expression of both TGF-β and collagenase genes is regulated by AP-1 sites (Roberts et al., 1991; Vincenti and Brinckerhoff, 2002), and TGF-β represses collagenase expression via AP-1 complexes (Mauviel et al., 1996). AP-1 activity is regulated by JNK, p38, and ERK1/ERK2 MAP kinases (Kyriakis and Avruch, 2001), and these kinases also mediate collagenase gene expression (Reunanen et al., 1998; Reuben et al., 2002). These observations indicate that cyclosporin-A-induced changes in AP-1 and MAP-kinase activities may play a central role in the evolution of gingival overgrowth. Because fibroblasts are the cell type responsible for synthesizing matrix components in fibrosis, we have examined how cyclosporin A affects these signaling activities in gingival fibroblasts.
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MATERIALS & METHODS
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Cell Culture
Gingival fibroblasts were obtained from healthy human donors after informed consent was obtained, in accordance with protocols approved by the University of Washington Human Subjects IRB. Cells between 8 and 15 passages were grown and maintained in Dulbecco-Vogt medium containing 10% fetal bovine serum (Yonemura et al., 1993). Confluent cultures in 10-cm Petri plates were pre-treated with 10 µg/mL cyclosporin A in serum-free medium for 24 hrs, activated with fresh medium containing cyclosporin A and 10 ng/mL TNF- (PeproTech, Rocky Hills, NJ, USA) or PDGF (R & D Systems, Minneapolis, MN, USA), and harvested after indicated times. Controls contained no cyclosporin A, but had the same concentration of alcohol vehicle.
Assay of Kinase Activities
Cells were scraped in ice-cold phosphate-buffered saline, collected by centrifugation at 500 g, and underwent lysis in 0.5 mL of 50 mmol/L Tris HCL, pH 7.4, containing 150 mmol/L NaCl, 1% Triton-X100, 50 mmol/L β-glycerophosphate, 10% glycerol, 2 mmol/L EDTA, 1 mmol/L orthovanadate, 1 mmol/L each of benzamidine, phenylmethylsulfonyl fluoride, and N-ethylmaleimide, and 1 µg/mL each of pepstatin and leupeptin. JNK, p38, and ERK1/2 were immunoprecipitated from 50-µL cell lysates by the addition of 10 µL of antibody to unphosphorylated proteins and 25 µL of sepharose-conjugated protein A+G. After overnight incubation at 4°C, immunoprecipitates were separated by NaDodSO4-polyacrylamide gel electrophoresis (12.5% gels) (Saito and Narayanan, 1999). Separated proteins were electroblotted onto nitrocellulose membranes, blocked with 5% non-fat dry milk, and incubated with monoclonal antibodies to phosphorylated and unphosphorylated p38, JNK, and ERK1/2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; Cell Signaling Technology, Beverly, MA, USA). After incubation with HRP-conjugated secondary antibodies, cross-reacting proteins were visualized by Enhanced Chemiluminescence reagent. The blots were scanned, and band intensities were calculated by an NIH Image program (version 1.61). Values were normalized for protein loading.
Gel Shift Assay
Oligonucleotides were labeled with [32P]dATP with the use of the Klenow fragment of DNA polymerase (Promega Life Science, Madison, WI, USA). Cells underwent lysis in 10 mmol/L HEPES, pH 7.9, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl fluoride, and 1.0 µg/mL each of pepstatin and leupeptin, and nuclei were separated by centrifugation at 4500 rpm for 15 min in a microcentrifuge. The nuclei underwent lysis in 20 mmol/L HEPES, pH 7.9, 25% glycerol, 1.5 mmol/L MgCl2, 1.2 mol/L KCl, 0.5 mmol/L DTT, 0.2 mmol/L EDTA, 0.5 mmol/L phenylmethylsulfonyl fluoride, and 1.0 µg/mL each of pepstatin and leupeptin, and nuclear extracts were separated by centrifugation at 14,000 rpm for 30 min. Five µg of nuclear extracts were incubated at 37°C for 15 min with 1.23 µg of poly(dI/dC), 20 mmol/L Tris, pH 7.5, 100 mmol/L NaCl, 1.6 mmol/L DTT, 0.05% NP40, and 2.46 µg bovine serum albumin in a final volume of 22 µL. NaCl concentration was then adjusted to 200 mmol/L, 5 – 10 x 105 cpm of double-stranded [32P]dATP-oligonucleotide (NFAT from Santa Cruz Biotechnology, Santa Cruz, CA, USA; AP-1 and NFB from Promega Life Science, Madison, WI, USA) was added, and the reaction mixture was incubated for 30 min at room temperature. It was then separated in non-denaturing polyacrylamide gels (6%) at 100 V for 3 to 5 hrs with the use of pH 8.0 Tris-borate-EDTA buffer. After electrophoresis, the gels were dried, and protein bands were detected by autoradiography. Band intensities were quantified after densitometry, as described above.
Transfection and Luciferase-reporter Assay
Human gingival fibroblasts at 40–80% confluent cell density in 24-well plates were transfected with pAP1-luciferase cDNA (obtained from Dr. Friedemann Schaub, Department of Pathology, University of Washington) and co-transfected with β-actin Renilla luciferase at a 1:33 ratio. Transfections were done with 0.8 µg DNA/well for 3 hrs in the presence of Lipofectamine reagent, according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). After 24–48 hrs, the transfected cells were treated as indicated, subjected to lysis in passive lysis buffer (Promega), and read in a Luminometer. Values were normalized for Renilla luciferase.
Statistical Analysis
Experiments were repeated at least three times. Quantitative comparisons were performed by Student’s t test, and a p value < 0.05 was considered statistically significant.
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RESULTS
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In preliminary experiments, we found that basal steady-state DNA-binding activity of transcription factors and MAP kinase activities were too low for meaningful comparisons (data not shown); therefore, we determined the cyclosporin A effect in cells activated by either TNF- or PDGF.
We first determined how cyclosporin A affects the DNA-binding activity of NFAT and NFB transcription factors in human gingival fibroblasts. We exposed the cells to 10 ng/mL TNF- for 4 hrs, prepared nuclear extracts, and determined DNA-binding activity by gel shift assay. The results showed that, as expected, NFAT and NFB activities were less in cells exposed to cyclosporin A (Fig. 1 ). The DNA-binding activities of NFAT and NFB in cultures treated with cyclosporin A were 40 ± 26% (p < 0.05, n = 3; Fig. 1A) and 21 ± 11% (p < 0.005, n = 3; Fig. 1B) as much as those of controls without cyclosporin A, respectively. Similar results were obtained for cells treated with PDGF (data not shown).
One reason for the decrease in NFAT activity may be activation of MAP kinases, especially p38, which counteract calcineurin dephosphorylation (Kyriakis and Avruch, 2001; Crabtree and Olson, 2002). Therefore, we determined if cyclosporin A increases p38 and JNK activation. Cells were treated with TNF- and cyclosporin A, p38 and JNK kinases were immunoprecipitated, and the extent of their phosphorylation was assessed by Western blot analysis with antibodies to phosphorylated proteins. The activation of p38 was less in cells treated with cyclosporin A; after 20 min, these cells had 40 ± 24% as much phosphorylated-p38 as cells without cyclosporin A (p < 0.05, n = 3) (Fig. 2, lanes "a", "b"). The activation remained suppressed after 4 hrs (lanes "c", "d").
The band intensities of phosphorylated-p56 and -p46 components of JNK in cells treated with cyclosporin A and TNF- were 49 ± 9% (p < 0.005, n = 3) and 42 ± 6% (p < 0.005, n = 3) as much as control cultures, respectively (lanes "a" and "b", Fig. 3). These bands appeared to remain reduced after 4 hrs (lane "c", upper panel); however, correction for protein loading showed that they did not differ significantly from controls (lower panel).
Both JNK and p38 enhance the transcriptional activity of AP-1 transcription factor by direct phosphorylation. AP-1 is a major regulator of transcription of collagenase, which is decreased in tissues and cells exposed to cyclosporin A (Sugano et al., 1998; Bolzani et al., 2000; Vincenti and Brinckerhoff, 2002). Therefore, we determined if cyclosporin A inhibition of p38 and JNK leads to a decrease in AP-1 activity. Nuclear extracts were prepared from cyclosporin-A-treated and control cells, and DNA-binding activity was assessed by gel shift assay. The results showed that nuclear extracts from cyclosporin-A-treated cells manifested higher levels of AP-1 binding activity (Fig. 4A). The activity was higher in cells treated with TNF- as well as PDGF, which is a major mitogen for, and induces, c-jun expression in gingival fibroblasts (Yonemura et al., 1993) (Fig. 4A). The shifted band intensity in cyclosporin-A-treated cells was 435 ± 52% as much as controls minus cyclosporin A (p < 0.005, n = 3). The increase in DNA-binding activity paralleled transaction activity, because cells transfected with the AP-1-luciferase reporter construct contained 3.0 times as many luciferase units as controls (Fig. 4B).
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Figure 4. AP-1 activity of human gingival fibroblasts treated with and without 10 µg/mL cyclosporin A and 10 ng/mL TNF- or PDGF. (A) Gel shift assay; 5 µg of nuclear proteins were incubated with labeled [32P]ATP-labeled oligonucleotide probe, separated, and visualized by autoradiography, as described in the "METHODS". For competition, unlabeled nucleotide was added at 100-fold molar excess. Arrows indicate shifted complexes. (B) Luciferase reporter activity normalized for β-actin Renilla luciferase control. Mean ± SD of triplicates is shown (p < 0.025).
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We examined if the cyclosporin-A-induced increase in AP-1 binding activity is associated with increased activities of ERK-1 and ERK-2, because these kinases increase AP-1 activity by up-regulating c-fos gene transcription (Shaulian and Karin, 2001). Western blot analysis of immunoprecipitates showed that ERK-1 and ERK-2 activation levels in cyclosporin-A-exposed cultures were marginally higher (126 ± 11% for ERK1 and 119 ± 31% for ERK2; p > 0.05, n = 3), but the increase was not statistically significant (data not shown).
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DISCUSSION
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In activated T-cells, the predominant outcome of cyclosporin A action is the inhibition of calcineurin, which prevents the dephosphorylation of NFATc and its translocation to the nucleus, and thus abolishes its transcriptional activity (Ho et al., 1996; Jørgensen et al., 2003). The cyclosporin A also decreases NFB activity in the T-cells. Analysis of our data shows that cyclosporin A has a similar effect in human gingival fibroblasts, and it decreases the DNA-binding activity of both NFAT and NFB. However, the decrease in NFB activity in gingival fibroblasts is in contrast to bronchial epithelial cells and kidneys, in which the activity increases after cyclosporin A treatment (Aoki and Kao, 1997; Asai et al., 2003). The decrease in DNA-binding activity could result either from less binding activity or a decrease in protein levels, or both.
The MAP kinases, especially p38, can oppose NFATc translocation to the nucleus by phosphorylating serines within the SP-repeats at the N-terminus (Crabtree and Olson, 2002), thereby decreasing its activity in the nucleus. They can also have the opposite effect and increase its transcriptional activity by phosphorylating at the transactivation domain (Crabtree and Olson, 2002). In the gingival fibroblasts, inhibition of p38 and JNK activation by cyclosporin A could decrease NFAT activity through supplementing calcineurin inhibition, and by inhibition of NFAT phosphorylation. The reduction in NFB activity may be due to inhibition of the transcriptional induction of the p50 subunit and c-Rel protein components, inhibition of calcineurin-Ca++ response, or decreased IB degradation (Crabtree, 2001).
Cyclosporin A decreased p38 and JNK activities and did not affect ERK1/2. These effects are expected to decrease AP-1 activity; however, we observed a four-fold increase. At least 2 factors can account for the increase: Cyclosporin A inhibition of calcineurin could prevent dephosphorylation and inactivation of Elk1, the kinase that induces transcriptional activity and c-fos expression, thereby increasing AP-1 activity. Alternatively, NFB and NFAT transcription factors physically and functionally interact with AP-1 (Crabtree, 2001; Macián et al., 2001); therefore, their inhibition by cyclosporin A could increase the net available AP-1 activity. In contrast to our results, AP-1 activity was reported to be less in human gingival fibroblasts exposed to cyclosporin A (Sugano et al., 1998); this may be because Sugano et al. determined AP-1 activity 10 min after activation with LPS, whereas we treated the cells with PDGF and TNF- for 6 hrs. Cyclosporin A also increases AP-1 activity in smooth-muscle cells (Murakami et al., 2003).
Cyclosporin-A-induced increase in AP-1 activity can explain the higher levels of TGF-β in cells and tissues exposed to cyclosporin A, because AP-1 is a major regulator of TGF-β gene expression (Roberts et al., 1991; Shin et al., 1998; Cotrim et al., 2002). However, this is not consistent with the reduction in activity and mRNA levels of collagenase, which is regulated by AP-1 (Sugano et al., 1998; Bolzani et al., 2000; Vincenti and Brinckerhoff, 2002). These observations indicate that changes in levels of TGF-β, tissue inhibitors of metalloproteinases and other MMP inhibitors, and other factors may contribute to decreased collagenase activity in the presence of cyclosporin A.
In gingival fibroblasts, JNK and p38 MAP kinases and NFAT and NFB transcription factors are inhibited by cyclosporin A, and AP-1 is activated; these effects presumably contribute to the evolution of fibrosis. In contrast, cyclosporin A inhibition of NFAT3 protects the heart from hypertrophic response (Molkentin et al., 1998). In smooth-muscle cells, whether cyclosporin A inhibits JNK phosphorylation is dependent upon the inducer (Saito et al., 2002). These observations indicate that the effect of cyclosporin A on signaling events varies in different cell types, and that the outcome depends upon cellular context and responding cell type. They also indicate that evolution of gingival overgrowth and fibrosis involves interactions among the outcomes of the effect of cyclosporin A on fibroblasts, other resident cells, and inflammatory cells.
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ACKNOWLEDGMENTS
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This work was supported by NIH grants DE 13069 and DE-08229. We thank Hanah Kim for technical assistance.
Received for publication October 13, 2004.
Revision received March 2, 2005.
Accepted for publication March 6, 2005.
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Journal of Dental Research, Vol. 84, No. 6,
532-536 (2005)
DOI: 10.1177/154405910508400609
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ABSTRACT
INTRODUCTION
