- Email: [email protected]
Differential effect of Cyclosporin A and FK506 on SPARC mRNA expression by human gingival fibroblasts
Biomedicine & Pharmacotherapy 59 (2005) 249–252 http://france.elsevier.com/direct/BIOPHA/
Original article
Differential effect of Cyclosporin A and FK506 on SPARC mRNA expression by human gingival fibroblasts Nicoletta Gagliano a,*, Claudia Moscheni a, Carlo Torri a, Claudia Dellavia b, Giordano Stabellini a, Virgilio F. Ferrario b, Magda Gioia a a
Department of Human Morphology – LITA, University of Milan, Via Fratelli Cervi 93, 20090 Segrate, Milan, Italy b Department of Human Morphology, University of Milan, Milan, Italy Received 10 May 2004; accepted 14 June 2004 Available online 28 April 2005
Abstract Background. – Secreted protein acidic and rich in cysteine (SPARC) is a glycoprotein that mediates cell–matrix interactions. In adults, its expression is mostly limited to tissue undergoing remodeling. During the development of Cyclosporin A (CsA)-induced gingival overgrowth (GO) a remodeling of the connective compartment occurs. By contrast, clinical trials showed that FK506 is not related to GO. SPARC expression and its involvement in GO is unknown. Our aim was, therefore, to analyze the effect of CsA and FK506 on SPARC gene expression. Methods. – Cultured human gingival fibroblasts were incubated with CsA, FK506 or with their vehicle (VH) for 24, 48 and 72 h. SPARC gene expression was determined by RT-PCR. Results. – SPARC mRNA levels tended to increase 72 h after CsA treatment, whilst they are undetectable in FK506-treated fibroblasts, compared to VH. Conclusion. – This gene expression profile is consistent with the involvement of SPARC in the mechanisms leading to the development of CsA-induced GO. By contrast, the undetectable SPARC mRNA levels in FK506-treated fibroblasts suggest that FK506 may be associated with a role of ECM stabilization, that does not induce GO. © 2005 Elsevier SAS. All rights reserved. Keywords: SPARC; Cyclosporin A; FK506
1. Introduction Secreted protein acidic and rich in cysteine (SPARC), also termed osteonectin, BM-40 or 43K protein, is a multifunctional glycoprotein that belongs to the matricellular proteins, defined as a group of extracellular regulatory macromolecules that mediates cell–matrix interactions but does not contribute significantly to extracellular matrix (ECM) structure [1]. SPARC is involved in a variety of diverse biological processes. Due to its counteradhesive properties, SPARC regulates focal cell adhesion and cell–matrix interactions binding to structural matrix proteins, such as collagens and vitronectin [2,3]. In vitro experiments showed that SPARC also inhib* Corresponding author. Tel.: +39 02 503 30462; fax: +39 02 503 30452. E-mail address: [email protected] (N. Gagliano). 0753-3322/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.biopha.2004.06.005
its the cell cycle [4], modulates growth factors and regulates cell differentiation [5]. SPARC is expressed at high levels in bone tissue, but is widely distributed in many other tissues, such as connective tissues, including the soft and mineralized tissue of teeth and their supporting periodontal tissues [6,7]. In adults, the expression of SPARC is mostly limited to tissue undergoing morphogenesis or remodeling due to wound healing, disease or natural processes [2]. In response to injury or tissue remodeling, SPARC would modify ECM reassembly and ECM cell interactions, facilitating proliferation, recruitment and differentiation of connective tissue cells [3]. A disturbance in connective tissue homeostasis has been suggested as one of the most relevant events occurring in the development of drug-induced gingival overgrowth (GO). In a significant number of cases (25–81%) the administration of the immunosuppressive drug Cyclosporin A (CsA) induces
250
N. Gagliano et al. / Biomedicine & Pharmacotherapy 59 (2005) 249–252
GO, a severe pathology that interferes with normal oral functions. CsA-induced GO has been extensively studied in humans as well as in animal experimental models, and is characterized by fibroblast proliferation and increased accumulation of ECM components, in particular of interstitial collagen, in the gingival connective compartment [8,9]. Up to now the mechanisms involved have not yet been fully determined. However, results of experimental investigations, although often controversial, indicate interstitial collagen as the main target of CsA; and, in particular, an altered collagen degradation pathway seems to be responsible for this pathology [10–12]. FK506 is a potent immunosuppressive agent used to prevent graft rejection and to treat autoimmune diseases [13]. It has been used successfully to prevent renal, liver and cardiac allograft rejection. Interestingly FK506, unlike CsA, does not appear to produce GO [14] and clinical trials showed that in patients immunosuppressed with FK506, the pathology spontaneously resolved when CsA was discontinued or converted to FK506 [15]. Considered the biological functions of SPARC during ECM remodeling and the different effect of CsA and FK506 in GO development, we aimed at analyzing in primary cell cultures of human gingival fibroblasts the relationship between SPARC gene expression and gingival collagen turnover, for the first time, after CsA and FK506 treatment.
2. Materials and methods 2.1. Cell culture Human gingival fibroblasts were obtained from the premolar area of the upper dental arch from four healthy volunteers (two males and two females) aged 20–27 years. All individuals displayed clinically normal gingiva without evidence of inflammation, hyperplasia or history of drug use associated with GO. Samples were obtained according to procedures approved by the local ethics committee. Informed consent was obtained from each subject. Gingival biopsies were washed with sterile phosphate buffered saline (PBS), plated in T-25 flasks, incubated in DMEM supplemented with 10% heat-inactivated foetal bovine serum (FBS) 10 U/ml of penicillin, 10 mg/ml streptomycin and 25 µg/ml amphotericin B at 37 °C in humidified atmosphere containing 5% CO2. When fibroblasts outgrow from the explant, they were trypsinized (0.1% trypsin–0.02% EDTA) for secondary passages. Cells were fed with fresh medium twice a week. Triplicate cultures were carried out for each sample and treatment. 2.2. Treatment of gingival fibroblasts with CsA or FK506 When human gingival fibroblasts between the fourth and the fifth passage grew to confluence in T-75 flasks, the culture medium was replaced with serum-free DMEM containing CsA (800 ng/ml) [16] or FK506 (1 µM) dissolved in a
Table 1 Synthetic oligonucleotide primers utilized for RT-PCR Gene
Sequence
GAPDH
5′-ATTCCATGGCACCGTCAAGGCT 3′-TCAGGTCCACCACTGACACGTT 5′-ACCATGAGGGCCTGGATC 3′-GGAGTGGATTTAGATCACAAG
SPARC
Product size (bp) 571 936
vehicle (VH) (0.008% ethanol, 0.0016% Tween-20). The dose of CsA used in our study has been previously reported to influence fibroblast proliferation and ECM turnover [16], in particular the mechanisms involved in COL-I degradation pathway [12]. The cultures were then incubated at 37 °C for 24, 48 and 72 h. Cultured fibroblasts of each sample incubated in VH served as control. At established time intervals the cell culture supernatants were collected and the fibroblasts were washed in PBS, trypsinized and harvested by centrifugation (100 × g, 5 min). 2.3. RT-PCR Total RNA was extracted from fibroblasts by a modification of the guanidine isothiocynate method (Extract-All, Eurobio, France). After DNase I digestion, 1 µg of total RNA was reverse-transcribed in 20 µl final volume reaction mix (Promega, Italy). The primer sequences utilized for RT-PCR are listed in Table 1. Amplification reactions were conducted in a final volume of 25 µl containing 2.5 µl of cDNA, 200 µM of the four dNTPs, 100 pmol of each primer, 2.5 U of Taq DNA polymerase (Taq 2000, Stratagene). The RT-PCR products were resolved by electrophoresis in 1% agarose gels, stained with ethidium bromide and quantified in duplicate by densitometric analysis (Image Pro-Plus). 2.4. Statistical analysis Data obtained from triplicate experiments and expressed by mean ± standard error (S.E.M.), were analyzed by one way analysis of variance (ANOVA), and Student post hoc test. P values less than 0.05 were considered significant.
3. Results 3.1. Cell viability and proliferation The observation by phase contrast microscopy of VH, CsA and FK506-treated cultured fibroblasts did not reveal any noticeable differences in their morphology. The data concerning cell proliferation show that fibroblasts after 48 and 72 h of CsA treatment increased their cell proliferation, compared with VH for the same time interval (data in press in Biomedicine and Pharmacotherapy). Conversely, FK506 treatment decreased fibroblast proliferation for all considered time inter-
N. Gagliano et al. / Biomedicine & Pharmacotherapy 59 (2005) 249–252
251
Fig. 1. SPARC gene expression after CsA and FK506 administration. a) Representative RT-PCR for SPARC. Each lane represents one fibroblast strain. Each amplified cDNA was normalized on GAPDH gene expression. b) Bar graph showing SPARC mRNA levels after densitometric analysis of amplification products bands. Changes in mRNA are expressed as normalized optical densities relative to GAPDH mRNA. Data, expressed as normalized optical densities, are mean ± S.E.M. for four triplicate samples. *: See Section 4.
vals (data submitted for publication to Journal of Clinical Periodontology). 3.2. SPARC gene expression SPARC mRNA levels were not affected in CsA-treated fibroblasts compared to VH, even if they tended to be higher 72 h after CsA treatment (+12%, P = ns); conversely, they become undetectable after FK506 administration for all time intervals. Representative RT-PCR of SPARC mRNA levels are shown in Fig. 1a, and their quantitative steady-state levels after densitometric analysis are shown in Fig. 1b. 4. Discussion and conclusion Interstitial collagen is one of the major ECM components. Under physiological conditions collagen content is the result of a dynamic balance between synthesis and degradation by matrix metalloproteinases (MMPs), a family of proteolytic enzymes responsible for the remodeling and degradation of ECM [17,18]. If these regulatory mechanisms are deranged, an increase in collagen in the connective compartment may occur, leading to GO. SPARC is an important multifunctional glycoprotein that modulates cellular interactions with ECM by its binding to structural matrix proteins [2]. One of its actions regards the regulation of ECM and matrix MMP production [19,20]. In adults, the expression of SPARC is limited to tissues undergoing repair or remodeling. In particular, SPARC is abundantly expressed in fibroblasts [21].
Our results show that SPARC is similarly expressed in VH and CsA-treated gingival fibroblasts until 48 h after drug administration, and thereafter it tended to increase, even if without statistical differences, in CsA-treated fibroblasts compared to VH. This pattern of expression is consistent with previously reported data regarding the effect of CsA on gingival interstitial collagen: CsA induces ECM remodeling characterized by COL-I accumulation and fibroblasts proliferation [10–12]. Conversely, SPARC mRNA levels are undetectable in FK506-treated fibroblasts, compared to CsAtreated ones. Since the expression of SPARC is restricted to sites of ECM turnover and is virtually undetectable in normal cells within their established ECM, we can hypothesize that in some way FK506 induces a sort of gingival ECM homeostasis stabilization, probably subsequent to decreased tissue remodeling. This suggestion is consistent with the observation that VH gingival fibroblasts, however, express basal SPARC mRNA levels, since healthy gingival connective tissue is a remodeling tissue with high turnover [7]. Interestingly, we observed that, among our CsA-treated fibroblasts 72 h after drug administration, there was one sample (see * in Fig. 1a) with independent behavior and similar to that observed in FK506-treated fibroblasts concerning MMP-1 collagenolytic levels (unpublished data): this sample, unlike all the other CsA-treated fibroblasts, was characterized by high levels of interstitial collagensase (data in press in Biomedicine and Pharmacotherapy) and undetectable SPARC mRNA levels, as observed in all FK506-treated samples. This observation is in agreement with the hypothesis that considers different genetically-based responses of
252
N. Gagliano et al. / Biomedicine & Pharmacotherapy 59 (2005) 249–252
fibroblasts in COL turnover after CsA-administration as one of the major determinants in the pathogenesis of CsAinduced GO [12,22]. In this context, we can speculate that a relationship between the above described phenotype of the CsA-treated fibroblasts displaying a FK506-similar “phenotype” and the absence of GO development probably exists, even if additional determinations are necessary to confirm this finding. Considered as a whole, since FK506 does not induce GO, our results are consistent with lowered SPARC mRNA levels in FK506-treated fibroblasts, compared with VH. However, why SPARC mRNA levels in FK506-treated fibroblasts are undetectable under levels observed in VH still have to be elucidated.
[7]
Acknowledgements
[14]
We thank Mr. Jerry Carson for the English revision of the manuscript. This work was supported by a FIRST grant.
[15]
References [1] [2] [3]
[4]
[5]
[6]
Bornstein P, Sage HE. Matricellular proteins: extracellular modulators of cell function. Curr Opin Cell Biol 2002;14:608–16. Brekken RA, Sage EH. SPARC, a matricellular protein: at the crossroads of cell–matrix communication. Matrix Biol 2001;19:816–27. Bradshaw AD, Sage EH. SPARC, a matricellular protein that functions in cellular differentiation and tissue response to injury. J Clin Invest 2001;107:1049–54. Funk SE, Sage EH. Differential effects of SPARC and cationic SPARC peptides on DNA synthesis by endothelial cells and fibroblasts. J Cell Physiol 1993;154:53–63. Bassuk JA, Birkebak T, Rothmier JD, Clark JM, Bradshaw A, Muchowski PJ, et al. Disruption of the SPARC locus in mice alters the differentiation of lenticular epithelial cells and leads to cataract formation. Exp Eye Res 1999;68:321–31. Sodek J, Zhu B, Huynh MH, Brown TJ, Ringuette M. Novel functions of the matricellular proteins osteopontin and osteonectin/SPARC. Connect Tissue Res 2002;43:308–19.
[8] [9] [10]
[11]
[12]
[13]
[16]
[17] [18] [19]
[20]
[21]
[22]
Bartold PM, Walsh LJ, Narayanan AS. Molecular and cell biology of the gingiva. Periodontol 2000 2000;24:28–55. Hassell TM, Hefti AF. Drug-induced gingival overgrowth: old problem, new problem. Crit Rev Oral Biol Med 1991;2:103–37. Seymour RA, Jacobs DJ. Cyclosporin and the gingival tissues. J Clin Periodontol 1992;19:1–11. Bolzani G, Della Coletta R, Martelli JH, De Almeida OP, Graner E. Cyclosporin A inhibits production and activity of matrix metalloproteinases by gingival fibroblasts. J Periodontol Res 2000;35:51–8. Kataoka M, Shimizu Y, Kunikiyo K, Asahara Y, Yamashita K, Ninomiya M, et al. Cyclosporin A decreases the degradation of type I collagen in rat gingival overgrowth. J Cell Physiol 2000;182:351–8. Hyland PL, Traynor PS, Myrillas TT, Marley JJ, Linden GJ, Winter P, et al. The effects of cyclosporin on the collagenolytic activity of gingival fibroblasts. J Periodontol 2003;74:437–45. Faulds D, Goa KL, Benfield P. Cyclosporin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in immunoregulatory disorders. Drugs 1993;45:953–1040. Mckaig SJ, Kelly D, Shaw L. Investigation of the effect of FK506 (tacrolimus) and cyclosporin on gingival overgrowth following paediatric liver transplantation. Int J Paediatr Dent 2002;12:398–403. Busque S, Demers P, St-Louis G, Boily JG, Tousignant J, Lemieux F, et al. Conversion from Neoral (cyclosporine) to tacrolimus of kidney transplant recipients for gingival hyperplasia or hypertrichosis. Transplant Proc 1998;30:1247–8. Esposito C, Fornoni A, Cornacchia F, Bellotti N, Fasoli G, Foschi A, et al. Cyclosporine induces different responses in human epithelial, endothelial and fibroblast cell cultures. Kidney Int 2000;58:123–30. Woessner Jr. FJ. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991;5:2145–54. Birkedal-Hansen H. Role of matrix metalloproteinases in human periodontal diseases. J Periodontol 1993;64:474–84. Hasselaar P, Loskutoff DJ, Sawdey M, Sage EH. SPARC induces the expression of type 1 plasminogen activator inhibitor in cultured bovine aortic endothelial cells. J Biol Chem 1991;266:13178–84. Tremble PM, Lane TF, Sage EH, Werb Z. SPARC, a secreted protein associated with morphogenesis and tissue remodeling, induces expression of metalloproteinases in fibroblasts through a novel extracellular matrix-dependent pathway. J Cell Biol 1993;121:1433–44. Porter PL, Sage EH, Lane TF, Funk SE, Gown AM. Distribution of SPARC in normal and neoplastic human tissue. J Histochem Cytochem 1995;43:791–800. Tipton DA, Stricklin GP, Dabbous MK. J Cell Biochem 1991;46:152–65.
Original article
Differential effect of Cyclosporin A and FK506 on SPARC mRNA expression by human gingival fibroblasts Nicoletta Gagliano a,*, Claudia Moscheni a, Carlo Torri a, Claudia Dellavia b, Giordano Stabellini a, Virgilio F. Ferrario b, Magda Gioia a a
Department of Human Morphology – LITA, University of Milan, Via Fratelli Cervi 93, 20090 Segrate, Milan, Italy b Department of Human Morphology, University of Milan, Milan, Italy Received 10 May 2004; accepted 14 June 2004 Available online 28 April 2005
Abstract Background. – Secreted protein acidic and rich in cysteine (SPARC) is a glycoprotein that mediates cell–matrix interactions. In adults, its expression is mostly limited to tissue undergoing remodeling. During the development of Cyclosporin A (CsA)-induced gingival overgrowth (GO) a remodeling of the connective compartment occurs. By contrast, clinical trials showed that FK506 is not related to GO. SPARC expression and its involvement in GO is unknown. Our aim was, therefore, to analyze the effect of CsA and FK506 on SPARC gene expression. Methods. – Cultured human gingival fibroblasts were incubated with CsA, FK506 or with their vehicle (VH) for 24, 48 and 72 h. SPARC gene expression was determined by RT-PCR. Results. – SPARC mRNA levels tended to increase 72 h after CsA treatment, whilst they are undetectable in FK506-treated fibroblasts, compared to VH. Conclusion. – This gene expression profile is consistent with the involvement of SPARC in the mechanisms leading to the development of CsA-induced GO. By contrast, the undetectable SPARC mRNA levels in FK506-treated fibroblasts suggest that FK506 may be associated with a role of ECM stabilization, that does not induce GO. © 2005 Elsevier SAS. All rights reserved. Keywords: SPARC; Cyclosporin A; FK506
1. Introduction Secreted protein acidic and rich in cysteine (SPARC), also termed osteonectin, BM-40 or 43K protein, is a multifunctional glycoprotein that belongs to the matricellular proteins, defined as a group of extracellular regulatory macromolecules that mediates cell–matrix interactions but does not contribute significantly to extracellular matrix (ECM) structure [1]. SPARC is involved in a variety of diverse biological processes. Due to its counteradhesive properties, SPARC regulates focal cell adhesion and cell–matrix interactions binding to structural matrix proteins, such as collagens and vitronectin [2,3]. In vitro experiments showed that SPARC also inhib* Corresponding author. Tel.: +39 02 503 30462; fax: +39 02 503 30452. E-mail address: [email protected] (N. Gagliano). 0753-3322/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.biopha.2004.06.005
its the cell cycle [4], modulates growth factors and regulates cell differentiation [5]. SPARC is expressed at high levels in bone tissue, but is widely distributed in many other tissues, such as connective tissues, including the soft and mineralized tissue of teeth and their supporting periodontal tissues [6,7]. In adults, the expression of SPARC is mostly limited to tissue undergoing morphogenesis or remodeling due to wound healing, disease or natural processes [2]. In response to injury or tissue remodeling, SPARC would modify ECM reassembly and ECM cell interactions, facilitating proliferation, recruitment and differentiation of connective tissue cells [3]. A disturbance in connective tissue homeostasis has been suggested as one of the most relevant events occurring in the development of drug-induced gingival overgrowth (GO). In a significant number of cases (25–81%) the administration of the immunosuppressive drug Cyclosporin A (CsA) induces
250
N. Gagliano et al. / Biomedicine & Pharmacotherapy 59 (2005) 249–252
GO, a severe pathology that interferes with normal oral functions. CsA-induced GO has been extensively studied in humans as well as in animal experimental models, and is characterized by fibroblast proliferation and increased accumulation of ECM components, in particular of interstitial collagen, in the gingival connective compartment [8,9]. Up to now the mechanisms involved have not yet been fully determined. However, results of experimental investigations, although often controversial, indicate interstitial collagen as the main target of CsA; and, in particular, an altered collagen degradation pathway seems to be responsible for this pathology [10–12]. FK506 is a potent immunosuppressive agent used to prevent graft rejection and to treat autoimmune diseases [13]. It has been used successfully to prevent renal, liver and cardiac allograft rejection. Interestingly FK506, unlike CsA, does not appear to produce GO [14] and clinical trials showed that in patients immunosuppressed with FK506, the pathology spontaneously resolved when CsA was discontinued or converted to FK506 [15]. Considered the biological functions of SPARC during ECM remodeling and the different effect of CsA and FK506 in GO development, we aimed at analyzing in primary cell cultures of human gingival fibroblasts the relationship between SPARC gene expression and gingival collagen turnover, for the first time, after CsA and FK506 treatment.
2. Materials and methods 2.1. Cell culture Human gingival fibroblasts were obtained from the premolar area of the upper dental arch from four healthy volunteers (two males and two females) aged 20–27 years. All individuals displayed clinically normal gingiva without evidence of inflammation, hyperplasia or history of drug use associated with GO. Samples were obtained according to procedures approved by the local ethics committee. Informed consent was obtained from each subject. Gingival biopsies were washed with sterile phosphate buffered saline (PBS), plated in T-25 flasks, incubated in DMEM supplemented with 10% heat-inactivated foetal bovine serum (FBS) 10 U/ml of penicillin, 10 mg/ml streptomycin and 25 µg/ml amphotericin B at 37 °C in humidified atmosphere containing 5% CO2. When fibroblasts outgrow from the explant, they were trypsinized (0.1% trypsin–0.02% EDTA) for secondary passages. Cells were fed with fresh medium twice a week. Triplicate cultures were carried out for each sample and treatment. 2.2. Treatment of gingival fibroblasts with CsA or FK506 When human gingival fibroblasts between the fourth and the fifth passage grew to confluence in T-75 flasks, the culture medium was replaced with serum-free DMEM containing CsA (800 ng/ml) [16] or FK506 (1 µM) dissolved in a
Table 1 Synthetic oligonucleotide primers utilized for RT-PCR Gene
Sequence
GAPDH
5′-ATTCCATGGCACCGTCAAGGCT 3′-TCAGGTCCACCACTGACACGTT 5′-ACCATGAGGGCCTGGATC 3′-GGAGTGGATTTAGATCACAAG
SPARC
Product size (bp) 571 936
vehicle (VH) (0.008% ethanol, 0.0016% Tween-20). The dose of CsA used in our study has been previously reported to influence fibroblast proliferation and ECM turnover [16], in particular the mechanisms involved in COL-I degradation pathway [12]. The cultures were then incubated at 37 °C for 24, 48 and 72 h. Cultured fibroblasts of each sample incubated in VH served as control. At established time intervals the cell culture supernatants were collected and the fibroblasts were washed in PBS, trypsinized and harvested by centrifugation (100 × g, 5 min). 2.3. RT-PCR Total RNA was extracted from fibroblasts by a modification of the guanidine isothiocynate method (Extract-All, Eurobio, France). After DNase I digestion, 1 µg of total RNA was reverse-transcribed in 20 µl final volume reaction mix (Promega, Italy). The primer sequences utilized for RT-PCR are listed in Table 1. Amplification reactions were conducted in a final volume of 25 µl containing 2.5 µl of cDNA, 200 µM of the four dNTPs, 100 pmol of each primer, 2.5 U of Taq DNA polymerase (Taq 2000, Stratagene). The RT-PCR products were resolved by electrophoresis in 1% agarose gels, stained with ethidium bromide and quantified in duplicate by densitometric analysis (Image Pro-Plus). 2.4. Statistical analysis Data obtained from triplicate experiments and expressed by mean ± standard error (S.E.M.), were analyzed by one way analysis of variance (ANOVA), and Student post hoc test. P values less than 0.05 were considered significant.
3. Results 3.1. Cell viability and proliferation The observation by phase contrast microscopy of VH, CsA and FK506-treated cultured fibroblasts did not reveal any noticeable differences in their morphology. The data concerning cell proliferation show that fibroblasts after 48 and 72 h of CsA treatment increased their cell proliferation, compared with VH for the same time interval (data in press in Biomedicine and Pharmacotherapy). Conversely, FK506 treatment decreased fibroblast proliferation for all considered time inter-
N. Gagliano et al. / Biomedicine & Pharmacotherapy 59 (2005) 249–252
251
Fig. 1. SPARC gene expression after CsA and FK506 administration. a) Representative RT-PCR for SPARC. Each lane represents one fibroblast strain. Each amplified cDNA was normalized on GAPDH gene expression. b) Bar graph showing SPARC mRNA levels after densitometric analysis of amplification products bands. Changes in mRNA are expressed as normalized optical densities relative to GAPDH mRNA. Data, expressed as normalized optical densities, are mean ± S.E.M. for four triplicate samples. *: See Section 4.
vals (data submitted for publication to Journal of Clinical Periodontology). 3.2. SPARC gene expression SPARC mRNA levels were not affected in CsA-treated fibroblasts compared to VH, even if they tended to be higher 72 h after CsA treatment (+12%, P = ns); conversely, they become undetectable after FK506 administration for all time intervals. Representative RT-PCR of SPARC mRNA levels are shown in Fig. 1a, and their quantitative steady-state levels after densitometric analysis are shown in Fig. 1b. 4. Discussion and conclusion Interstitial collagen is one of the major ECM components. Under physiological conditions collagen content is the result of a dynamic balance between synthesis and degradation by matrix metalloproteinases (MMPs), a family of proteolytic enzymes responsible for the remodeling and degradation of ECM [17,18]. If these regulatory mechanisms are deranged, an increase in collagen in the connective compartment may occur, leading to GO. SPARC is an important multifunctional glycoprotein that modulates cellular interactions with ECM by its binding to structural matrix proteins [2]. One of its actions regards the regulation of ECM and matrix MMP production [19,20]. In adults, the expression of SPARC is limited to tissues undergoing repair or remodeling. In particular, SPARC is abundantly expressed in fibroblasts [21].
Our results show that SPARC is similarly expressed in VH and CsA-treated gingival fibroblasts until 48 h after drug administration, and thereafter it tended to increase, even if without statistical differences, in CsA-treated fibroblasts compared to VH. This pattern of expression is consistent with previously reported data regarding the effect of CsA on gingival interstitial collagen: CsA induces ECM remodeling characterized by COL-I accumulation and fibroblasts proliferation [10–12]. Conversely, SPARC mRNA levels are undetectable in FK506-treated fibroblasts, compared to CsAtreated ones. Since the expression of SPARC is restricted to sites of ECM turnover and is virtually undetectable in normal cells within their established ECM, we can hypothesize that in some way FK506 induces a sort of gingival ECM homeostasis stabilization, probably subsequent to decreased tissue remodeling. This suggestion is consistent with the observation that VH gingival fibroblasts, however, express basal SPARC mRNA levels, since healthy gingival connective tissue is a remodeling tissue with high turnover [7]. Interestingly, we observed that, among our CsA-treated fibroblasts 72 h after drug administration, there was one sample (see * in Fig. 1a) with independent behavior and similar to that observed in FK506-treated fibroblasts concerning MMP-1 collagenolytic levels (unpublished data): this sample, unlike all the other CsA-treated fibroblasts, was characterized by high levels of interstitial collagensase (data in press in Biomedicine and Pharmacotherapy) and undetectable SPARC mRNA levels, as observed in all FK506-treated samples. This observation is in agreement with the hypothesis that considers different genetically-based responses of
252
N. Gagliano et al. / Biomedicine & Pharmacotherapy 59 (2005) 249–252
fibroblasts in COL turnover after CsA-administration as one of the major determinants in the pathogenesis of CsAinduced GO [12,22]. In this context, we can speculate that a relationship between the above described phenotype of the CsA-treated fibroblasts displaying a FK506-similar “phenotype” and the absence of GO development probably exists, even if additional determinations are necessary to confirm this finding. Considered as a whole, since FK506 does not induce GO, our results are consistent with lowered SPARC mRNA levels in FK506-treated fibroblasts, compared with VH. However, why SPARC mRNA levels in FK506-treated fibroblasts are undetectable under levels observed in VH still have to be elucidated.
[7]
Acknowledgements
[14]
We thank Mr. Jerry Carson for the English revision of the manuscript. This work was supported by a FIRST grant.
[15]
References [1] [2] [3]
[4]
[5]
[6]
Bornstein P, Sage HE. Matricellular proteins: extracellular modulators of cell function. Curr Opin Cell Biol 2002;14:608–16. Brekken RA, Sage EH. SPARC, a matricellular protein: at the crossroads of cell–matrix communication. Matrix Biol 2001;19:816–27. Bradshaw AD, Sage EH. SPARC, a matricellular protein that functions in cellular differentiation and tissue response to injury. J Clin Invest 2001;107:1049–54. Funk SE, Sage EH. Differential effects of SPARC and cationic SPARC peptides on DNA synthesis by endothelial cells and fibroblasts. J Cell Physiol 1993;154:53–63. Bassuk JA, Birkebak T, Rothmier JD, Clark JM, Bradshaw A, Muchowski PJ, et al. Disruption of the SPARC locus in mice alters the differentiation of lenticular epithelial cells and leads to cataract formation. Exp Eye Res 1999;68:321–31. Sodek J, Zhu B, Huynh MH, Brown TJ, Ringuette M. Novel functions of the matricellular proteins osteopontin and osteonectin/SPARC. Connect Tissue Res 2002;43:308–19.
[8] [9] [10]
[11]
[12]
[13]
[16]
[17] [18] [19]
[20]
[21]
[22]
Bartold PM, Walsh LJ, Narayanan AS. Molecular and cell biology of the gingiva. Periodontol 2000 2000;24:28–55. Hassell TM, Hefti AF. Drug-induced gingival overgrowth: old problem, new problem. Crit Rev Oral Biol Med 1991;2:103–37. Seymour RA, Jacobs DJ. Cyclosporin and the gingival tissues. J Clin Periodontol 1992;19:1–11. Bolzani G, Della Coletta R, Martelli JH, De Almeida OP, Graner E. Cyclosporin A inhibits production and activity of matrix metalloproteinases by gingival fibroblasts. J Periodontol Res 2000;35:51–8. Kataoka M, Shimizu Y, Kunikiyo K, Asahara Y, Yamashita K, Ninomiya M, et al. Cyclosporin A decreases the degradation of type I collagen in rat gingival overgrowth. J Cell Physiol 2000;182:351–8. Hyland PL, Traynor PS, Myrillas TT, Marley JJ, Linden GJ, Winter P, et al. The effects of cyclosporin on the collagenolytic activity of gingival fibroblasts. J Periodontol 2003;74:437–45. Faulds D, Goa KL, Benfield P. Cyclosporin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in immunoregulatory disorders. Drugs 1993;45:953–1040. Mckaig SJ, Kelly D, Shaw L. Investigation of the effect of FK506 (tacrolimus) and cyclosporin on gingival overgrowth following paediatric liver transplantation. Int J Paediatr Dent 2002;12:398–403. Busque S, Demers P, St-Louis G, Boily JG, Tousignant J, Lemieux F, et al. Conversion from Neoral (cyclosporine) to tacrolimus of kidney transplant recipients for gingival hyperplasia or hypertrichosis. Transplant Proc 1998;30:1247–8. Esposito C, Fornoni A, Cornacchia F, Bellotti N, Fasoli G, Foschi A, et al. Cyclosporine induces different responses in human epithelial, endothelial and fibroblast cell cultures. Kidney Int 2000;58:123–30. Woessner Jr. FJ. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991;5:2145–54. Birkedal-Hansen H. Role of matrix metalloproteinases in human periodontal diseases. J Periodontol 1993;64:474–84. Hasselaar P, Loskutoff DJ, Sawdey M, Sage EH. SPARC induces the expression of type 1 plasminogen activator inhibitor in cultured bovine aortic endothelial cells. J Biol Chem 1991;266:13178–84. Tremble PM, Lane TF, Sage EH, Werb Z. SPARC, a secreted protein associated with morphogenesis and tissue remodeling, induces expression of metalloproteinases in fibroblasts through a novel extracellular matrix-dependent pathway. J Cell Biol 1993;121:1433–44. Porter PL, Sage EH, Lane TF, Funk SE, Gown AM. Distribution of SPARC in normal and neoplastic human tissue. J Histochem Cytochem 1995;43:791–800. Tipton DA, Stricklin GP, Dabbous MK. J Cell Biochem 1991;46:152–65.