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Cyclosporin A Specifically Affects Nuclear PLCß₁ in Immunodepressed Heart Transplant Patients with Gingival Overgrowth

¹ Department of SAU&FAL, University of Bologna,
² Department of Dental Science, University of Bologna,
³ ITOI-CNR, Unit of Bologna, c/o IOR, Bologna, Italy; and
⁴ UCO of Dental Sciences, DMUN, University of Trieste, Via Stuparich, 1, 34129, Trieste, Italy;

^* corresponding author, lbreschi{at}units.it

	ABSTRACT

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One of the most commonly observed adverse effects of cyclosporin A (CsA) is the development of gingival overgrowth (GO). Fibroblasts are involved in GO, but the question why only a percentage of patients undergoing CsA treatment shows this side-effect remains unanswered. In a previous study, CsA has been demonstrated to induce over-expression of phospholipase C (PLC) ß₁ in fibroblasts of patients with clinical GO, in cells from both enlarged and clinically healthy gingival sites. In this work, we assessed the expression of PLCß isoforms to investigate whether the exaggerated fibroblast response to CsA related to increased PLCß₁ expression could also be detected in CsA-treated patients without clinical signs of GO. Our results support the hypothesis of a multi-factorial origin of gingival overgrowth, including specific changes within the gingival tissues orchestrating fibroblastic hyper-responsiveness as a consequence of a long-term in vivo exposure to cyclosporin A.

KEY WORDS: gingival overgrowth • cyclosporin A • phospholipase C • immunoblotting • immuno-TEM

	INTRODUCTION

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Cyclosporin A (CsA) is considered the immunosuppressive agent of choice in heart transplant patients (Seymour et al., 1997). One of the most commonly observed adverse effects of CsA is the development of gingival overgrowth (GO), which is characterized by a dense collagen stroma and epithelial hyperplasia (Somacarrera et al., 1994; Montebugnoli et al., 1996).

The precise mechanism of CsA-induced GO is uncertain. Analysis of data from the literature suggests that the incidence and severity of GO are dependent upon the interaction of several factors, including plaque control, level of gingival inflammation, extent of periodontal destruction, dosage and duration of CsA therapy, plasma and tissue concentrations of drug and its metabolites, age of patient, and possibly the underlying medical condition (Seymour et al., 1996; Montebugnoli and Bernardi, 1999; Montebugnoli et al., 2000, 2002).

There is wide evidence that fibroblasts are the cells mainly involved in GO, but the question why only a percentage of patients undergoing CsA treatment shows this side-effect remains unanswered (Bolzani et al., 2000; Hyland et al., 2003). In a previous study, CsA has been demonstrated to have a great in vitro influence on fibroblast metabolism of patients with clinical GO, both in cells from enlarged gingival sites and in cells from clinically healthy gingival sites (Breschi et al., 2000). From these results, it was not possible to explain whether the exaggerated reactivity to in vitro CsA treatment was a consequence of a genetically transmitted susceptibility to CsA, or whether it was a secondary effect of the long-term in vivo exposure to CsA.

However, analysis of the data underlined the lack of any close relationship between enhanced fibroblast activity and clinical signs of GO, supporting the hypothesis that factors other than CsA are involved in the pathogenesis of the CsA-induced GO.

It is known that the expression of nuclear phospholipase C(PLC)ß₁ is related to the cell-cycle progression, which is a key element in the control of a specific checkpoint of the G1 phase of the cell cycle (Martelli et al., 2004). In particular, we tested whether the signal transduction system based on PLC may be involved in GO development induced by CsA, by evaluating PLCß major isoform expression in primary cultures of fibroblasts from patients subjected to CsA treatment and showing partial (pGO) or full (fGO) GO, and patients without clinical evidence of GO (noGO).

The aim of the present study was to investigate whether the exaggerated fibroblast response to CsA, related to an increased PLCß₁ expression, could also be detected in CsA-treated patients without clinical signs of GO, as a means of disclosing a genetic transmission of the abnormal susceptibility.

	MATERIALS & METHODS

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Reagents
Reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise indicated.

Patients
Gingival biopsies were obtained from nine heart transplant patients undergoing CsA therapy for at least one year, either with or without clinical evidence of GO, classified according to the Hyperplastic Index (HI) (Montebugnoli et al.,1996). The study was approved by the local ethics committee; subjects’ rights were protected, and informed consent was given. Additional information on patient selection can be found on the Web Appendix. Biopsies (2 gingival fragments, measuring 2 x 2 x 2 mm, from each patient) were obtained from: (1) three patients (male; average age, 46 yrs) showing no clinical signs of GO (noGO) with an average HI of 16; (2) three patients (male; average age, 45 yrs) with some areas revealing no clinical signs of GO and others showing GO with an average HI of 35, thus classified as having partial GO (pGO); and (3) three patients (male; average age, 41 yrs) showing a full-mouth GO (fGO) with an average HI of 65. In patients with pGO, 2 gingival biopsies were performed: 1 from clinically healthy (pGO1) and 1 from enlarged tissue (pGO2).

Additional similar biopsies, as controls, were performed in three aged-matched patients (male; average age, 39 yrs) with clinically healthy gingiva (HP) and never having received CsA therapy.

Fibroblasts and Culture Conditions
Tissue specimens were cut, placed on Petri dishes with medium (199 HEPES Modification, containing 20% fetal calf serum [FCS], 100 UI/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL amphotericin B, ITS Liquid Media Supplement), and stored in an incubator with 5% CO₂ at 37°C.

Each cell culture at passage 5 was starved in the absence of FCS for 48 hrs to reach the G0 phase of the cell cycle. The expression of PLCß₁, PLCß₂, PLCß₃, and PLCß₄ was studied in different conditions: (1) after 1 hour’s administration of 20% FCS; (2) after 1 hour’s administration of CsA at 232 ng/mL (dosage corresponding to the mean blood concentration of CsA detected in the patients); and (3) without either FCS or CsA (serum-deprived cells) as the control group. Levels of PLCß₁, PLCß₂, PLCß₃, and PLCß₄ of each group were then analyzed by means of immunoblotting and immunocytochemistry.

Western Blotting Procedure
Cells were washed in phosphate-buffered saline (PBS) (pH 7.4) containing a complete protease inhibitor cocktail supplemented with 1.0 mM Na₃VO₄ and 20 nM okadaic acid. Cells underwent lysis at ~ 10⁷/mL in boiling electrophoresis sample buffer containing the protease inhibitor cocktail. Lysates were then syringed and boiled for 5 min to solubilize proteins. A 30-µg quantity of separated protein on SDS-PAGE was transferred to nitrocellulose sheets with the use of a semi-dry blotting apparatus. Expressions of PLCß₁, PLCß₂, PLCß₃, and PLCß₄ were assessed by Western blotting as described previously (Faenza et al., 2004). Additionally, enzyme expression was assessed in fibroblast nuclei from all tested cell samples. Nuclei isolation and purification were undertaken as described previously (Breschi et al., 2000; Faenza et al., 2004).

Electron Microscopy Immunocytochemistry
Glass-adherent fibroblasts were fixed with 1% glutaraldehyde in 0.1 M phosphate buffer for 30 min at 4°C, dehydrated up to 70% ethanol, and embedded in LR White Resin (Structure Probe, Inc./SPI Supplies, West Chester, PA, USA) at 0°C (Zini et al., 1996). Ultra-thin sections were pre-incubated with 5% normal goat serum in 0.05 M Tris-HCl, pH 7.6, 0.14 M NaCl, and 0.1% BSA (TBS I), incubated overnight at 4°C with the anti-PLCß₁ monoclonal antibody (UBI, Lake Placid, NY, USA), diluted 1:40 in TBS I, and then with a goat anti-mouse IgG conjugated with 10 nm colloidal gold particles (Amersham Life Science, Little Chalfont, UK), diluted 1:10 in 0.02 M Tris-HCl, pH 8.2, 0.14 M NaCl, and 0.1% BSA (TBS II) for 1 hr, and then amplified with a Silver Enhancer Kit (Amersham Life Science) (Zini et al., 1996). Controls consisted of samples incubated without the primary antibody. Before transmission electron microscopy (TEM) observation with a Zeiss EM 109, the thin sections were stained with aqueous uranyl acetate and lead citrate.

Quantitative Evaluation
At least 15 micrographs at the same magnification were obtained for each sample (at least 3 for each case). The labeling density (mean of gold particle numbers/µm² ± SD) was determined on 10 randomly chosen photographs for each case. The Mann-Whitney U test was used for comparing the labeling in CsA-treated and untreated fibroblasts from the same patient. The Kruskall-Wallis non-parametric test, followed by Dunn’s test for post hoc multiple comparisons, was used to compare label density in fibroblasts from different patients. These tests were calculated by the Monte Carlo method for small samples. P values less than 0.05 were considered significant. The post hoc statement of power was evaluated when differences were not significant.

	RESULTS

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Blotting analysis revealed no significant variations for PLCß₁, PLCß₂, PLCß₃, and PLCß₄, in FCS-treated cells (data not shown). Similarly, the administration of CsA to FCS-deprived cells showed no difference in PLCß₂, PLCß₃, and PLCß₄ expression (data not shown), while PLCß₁ revealed significant increases in fGO and pGO patients (in both pGO1 and pGO2 areas; Fig. 1, line A). Cell fractionation showed that the increase in PLCß₁ expression was due to a specific accumulation of the enzyme in the nuclear compartment (Fig. 1, line B). Indeed, densitometric analysis, correcting for the amount of protein loaded, indicated that, in the cytosolic compartment, there was no enzyme increase (data not shown).

View larger version (32K): [in this window] [in a new window]   Figure 1. Expression of the PLCß1 isoform during differentiation of primary human gingival fibroblasts. (A) Cells underwent lysis as described in MATERIALS & METHODS, and were subjected to SDS-PAGE, blotted, and immunoprobed with anti-PLCß1 antibody. A 20-µg quantity of protein was loaded for each lane. The results shown are representative of at least 3 independent determinations. (B) PLCß1 expression in purified nuclei from primary cultures of different fibroblasts populations. A 20-µg quantity of protein was loaded for each lane. (C) ß tubulin levels were used for normalization. The immunochemical analysis by Western blot on total homogenate and nuclear fraction does not give information on the precise localization of the enzyme, while, at the TEM level, the intranuclear and the intracytoplasmic distributions of the PLCß1 can be determined with high accuracy, and quantitative variations of the label distribution can be further investigated.

Immunolabeling at the TEM level revealed that labeling anti-PLCß₁ was present mainly at the nuclear level, while only a few gold particles were scattered in the cytoplasm. Within the nucleus, gold particles were localized in the interchromatin domains, while heterochromatin, the nucleolus, and the nuclear envelope were almost completely unlabeled (Figs. 2–4).

View larger version (62K): [in this window] [in a new window]   Figure 2. Fibroblasts from patients undergoing CsA-immunosuppressive therapy without any clinical signs of gingival overgrowth (noGO). (a,b) Immunocytochemistry of PLCß1 at the TEM level of FCS-deprived (2a; UN = untreated cells; HC = heterochromatin; IC = interchromatin; N = nucleolus; bar = 0.5 µm) and CsA-treated cells (2b; +CsA; bar = 0.5 µm). The analysis was performed on thin sections of cell monolayers, partially dehydrated, and embedded in LR White resin at low temperature, a procedure that ensures a reliable antigen detection. Due to the absence of osmium fixation and to the characteristics of the hydrophilic resin, the membranes are not electrondense. (c) Quantitative evaluation of the nuclear labeling density was performed with the Mann-Whitney U test (UN; n = 10) (+CsA; n = 10). Values are the means of examined samples ± SD. No significant difference was observed (power = 0.74).

View larger version (60K): [in this window] [in a new window]   Figure 3. Fibroblasts from healthy patients (HP) who never received any treatment with CsA. TEM micrographs of FCS-deprived fibroblasts (3a; UN = untreated cells; HC = heterochromatin; IC = interchromatin; N = nucleolus; bar = 0.5 µm) and CsA-treated ones (3b; +CsA; bar = 0.5 µm), both revealing scarce labeling related to interchromatin domains. (c) Quantitative evaluation of the nuclear labeling density was performed with the Mann-Whitney U test (UN; n = 10) (+CsA; n = 10). Values are the means of examined cases ± SD. No significant difference was observed (power = 0.71).

View larger version (118K): [in this window] [in a new window]   Figure 4. Fibroblasts from patients undergoing CsA immunosuppresive therapy and showing pGO or fGO. TEM micrographs of fibroblasts from not-clinically-enlarged gingival sites (pGO1; a,b; UN = untreated cells, +CsA = after CsA administration; HC = heterochromatin; IC = interchromatin; bar = 0.5 µm), from hypertrophic areas (pGO2; d,e; N = nucleolus; bar = 0.5 µm) of the same patient and from gingival biopsies of a patient exhibiting a full-mouth GO (fGO; g,h; bar = 0.5 µm). Untreated (UN; a,d,g) fibroblasts reveal scarce labeling, while CsA-treated cells (+CsA; b,e,h) show an intense labeling of nuclear domains, particularly localized in the interchromatin areas. (c,f,i) Quantitative evaluation by the Mann-Whitney U test of the nuclear label of untreated fibroblasts (UN; n = 10) and after CsA administration to the culture medium (+CsA; n = 10); values are the means of examined samples ± SD. A significant increase of PLCß1 nuclear labeling (P < 0.05; Mann-Whitney U test) was observed in treated fibroblasts with respect to untreated cells in all three cases examined.

Significant differences (P < 0.05; Kruskall-Wallis followed by Dunn’s test) were observed in CsA-treated samples from pGO patients (pGO1, pGO2 areas) and fGO patients with respect to HP and noGO, respectively. No significant differences (Kruskall-Wallis test) were observed in untreated samples (power = 0.77).

	DISCUSSION

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PLCß₁ is an enzyme located mostly in the nucleus and is a key intermediate involved in inositide signaling and elicited by growth factors or hormones. Nuclear PLCß₁ signaling responds to different stimuli acting independently from that at the plasma membrane, influencing functions such as pre-mRNA splicing and chromatin arrangement (Martelli et al., 2004). Moreover, a direct link between PLCß₁ and DNA synthesis has been demonstrated, and thus expression and activation of this enzyme represent a key step in mitotic cell response (Martelli et al., 2004).

In a previous study, we used PLCß₁ expression to investigate the effects of CsA on gingival fibroblasts from heart transplant patients with GO (Breschi et al., 2000). It has also been reported that in vivo and in vitro exposure to CsA, as well as to phenytoin, produces long-term effects on gingival fibroblast metabolism, which remain evident through multiple passages in vitro (Zebrowski et al., 1994; Henderson et al., 1997; Newell and Irwin, 1997; Soory and Kasasa, 1997).

Therefore, CsA could mimic endogenous signals for wound repair or remodeling, but, in the absence of any specific process, its effect must be threshold; however, when specific processes take place within localized and specific areas, the effects of CsA could act synergistically with endogenous signals, stimulating an excessive repair or overgrowth (Iacopino et al., 1997; Wondimu and Modéer, 1997). In support of this hypothesis, there are data documenting that inflammatory processes stimulate relevant modifications in the activity of the gingival fibroblasts via a series of endogenous signals (Mizel et al., 1981; Kjeldsen et al., 1993), and that elevated concentrations of these endogenous signals are detectable at sites of GO (Nares et al., 1996; Plemons et al., 1996).

We performed the present study to address the controversy as to whether fibroblast hyperactivity is genetically transmitted. The aim was to investigate if an in vitro exaggerated fibroblast response to CsA can also be detected in CsA-treated patients without clinical signs of GO (noGO). The study was designed to find a direct link between PLCß₁ expression and the exaggerated fibroblast response to CsA. To determine if the signaling transduction system based on PLC may be involved in the GO development induced by CsA, we evaluated PLCß₁ expression in primary cultures of fibroblasts from CsA-treated patients showing pGO or fGO and patients without clinical evidence of GO. The results obtained showed that PLCß₁ over-expression was found in CsA-treated patients with clinical signs of GO, not only in the areas showing GO but also in those without signs of GO. In contrast, the results obtained from fibroblasts of noGO patients showed that, in response to CsA in vitro, there was an unchanged expression of PLCß₁, which seems to contradict the hypothesis that the exaggerated fibroblast activity in CsA-treated patients is a prerogative of all CsA-treated subjects. On the contrary, they suggest that cells from enlarged gingival sites could be highly reactive because of an individual predisposition to CsA, which is present only in a given group of individuals and which identifies subjects at risk for developing GO.

The normal fibroblast responsiveness to CsA in noGO patients suggests that fibroblast metabolism may also be unaffected, despite long-term in vivo exposure to CsA. It has been demonstrated that gingival fibroblasts exhibit functional heterogeneity in response to various stimuli, and our results are in agreement with those of others, which emphasizes that subjects developing GO are characterized by gingival fibroblasts with abnormal susceptibility to CsA, while subjects not predisposed to GO have gingival fibroblasts unresponsive to CsA (Hassell et al., 1988).

In conclusion, the results of the present study may be in agreement with current opinions supporting the hypothesis of a multifactorial origin of the CsA-induced GO, which indicates either the presence of notably genetic factors controlling the interaction between CsA and fibroblasts, or of specific factors, such as inflammatory changes within the gingival tissues orchestrating the fibroblastic hyper-responsiveness as a possible consequence of a long-term in vivo exposure to CsA (Stashenko et al., 1991; Wikesjö et al., 1992; Cebeci et al., 1996; O’Valle et al., 1994). GO induced by CsA appears to be related to a signaling molecule such as nuclear PLCß₁, which is directly involved in the expression of cyclin D3 and, in turn, in the control of G1 phase progression (Faenza et al., 2000). This issue is strengthened by the fact that deletion of the PLCß₁ gene is somehow involved in human acute myeloid leukemia (LoVasco et al., 2004).

Further studies are currently in progress to investigate fibroblast metabolism in subjects before and after heart transplantation, to give us more insight into the relationship between CsA and GO.

	ACKNOWLEDGMENTS

 
The authors thank Mr. A. Valmori for technical assistance. This work was supported by grants from Italian MIUR Cofin 2002–2004 and the CARISBO Foundation, Bologna, Italy.

	FOOTNOTES

 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received April 29, 2004; Last revision January 26, 2005; Accepted April 8, 2005

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