Differential effect of Cyclosporin A and FK506 on SPARC mRNA expression by human gingival fibroblasts
ABSTRACT 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.
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.
SPARC mRNA levels tended to increase 72 h after CsA treatment, whilst they are undetectable in FK506-treated fibroblasts, compared to VH.
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.
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ABSTRACT: Abstract Objective: We aimed to characterize the fibroblast phenotype of patients by analyzing gene and protein expression of cleft lip and/or cleft palate fibroblasts in relation to collagen turnover and extracellular matrix remodeling. Patients: Human palatal fibroblasts were obtained from three healthy subjects without cleft lip and/or cleft palate and from three subjects with nonsyndromic cleft lip and/or cleft palate. Collagen turnover-related gene and protein expression were analyzed by real-time polymerase chain reaction, Western and dot blots, and sodium dodecyl sulfate zymography. Results: Cleft lip and/or cleft palate fibroblasts, compared with controls, displayed a down-regulation of collagens type I and III messenger RNA (p < .0001 and p < .001, respectively) but an opposite tendency to increase protein levels. Cleft lip and/or cleft palate cells had higher lysyl hydroxylase-2b messenger RNA levels expressed in relation to collagen type I messenger RNA, down-regulated matrix metalloproteinase-1, tissue inhibitor of matrix metalloproteinase-1, and Secreted Protein Acidic and Rich in Cysteine messenger RNA (p < .0001 and p < .01, respectively). Pro-matrix metalloproteinase-1 tended to decrease, and pro-matrix metalloproteinase-2 and -9 were down-regulated (p < .01, p < .05, respectively), as was Secreted Protein Acidic and Rich in Cysteine protein expression (p < .05). Conclusions: Our results suggest that the cleft lip and/or cleft palate fibroblast phenotype is characterized by a tendency toward interstitial collagen deposition due to posttranslational modifications, such as decreased collagen degradation by matrix metalloproteinases and increased collagen cross-links. These findings may contribute to the knowledge of the cleft lip and/or cleft palate fibroblast phenotype and may be useful to the surgeon when considering the potential wound contraction and subsequent undesired scarring in cleft lip and/or cleft palate ocurring after the surgical closure of a cleft palate.The Cleft Palate-Craniofacial Journal 07/2010; 47(4):393-9. DOI:10.1597/07-196.1 · 1.11 Impact Factor
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ABSTRACT: Cyclosporin A is a powerful immunosuppressive drug with considerable impact on transplants and is able to modify extracellular matrix (ECM) composition. It has recently been demonstrated that cyclosporin A stimulates the production of the cytokine family. Cytokines such as interleukin, transforming growth factor beta(1), and bone morphogenetic protein induce the deposition of glycosaminoglycans (GAGs), proteoglycans, and collagen fibers in the connective ECM. ECM composition is very important for normal tissue development and function. In this work, we examine the effects caused by cyclosporin A on cultures of normal human palate fibroblasts in order to evaluate interleukin, transforming growth factor beta II, and bone morphogenetic protein II membrane receptor induction and extracellular GAG changes such as hyaluronic acid, heparin sulfate, and chondroitin sulfate. Palate fibroblasts were maintained for 24 h in serum-free 199 medium containing 5 microg/mL (3)H glucosamine hydrochloride. After this time, TGF II and BMP II receptors were determined by microarray analysis and GAG classes by the biochemical method. The results show that TGFbeta(1) II and BMP II membrane receptors are significantly inhibited in cyclosporin A-treated cultures as compared to controls, whereas IL-1R2 membrane receptors are stimulated. The behavior of total intra- and extracellular GAGs is significantly increased in cyclosporin A-treated cultures, whereas the ratio between non-sulfated/sulfated GAGs decreases (p <or=0.01) vis-à-vis controls. Because they form a highly complicated macromolecular network in the ECM, which provides an indication of cell function and gene expression and modulates growth factor activities, GAG changes are related to modification of ECM functions. Our data show that cyclosporin A causes biochemical changes to ECM through alterations in cytokines and respective membrane receptor linkages.Archives of Medical Research 10/2007; 38(7):717-22. DOI:10.1016/j.arcmed.2007.03.007 · 2.41 Impact Factor
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ABSTRACT: Gingival enlargement frequently occurs in transplant patients receiving immunosuppressive drugs. It was hypothesized that gingival enlargement associated with cyclosporin use results from increases in the number of fibroblasts and the volume of extracellular matrix. SPARC (secreted protein, acidic, and rich in cysteine) regulates cell-matrix interactions, binding to structural matrix proteins, and is induced by cyclosporin A (CsA). The aim of the study was to determine whether there is an association between SPARC genotypes and gingival enlargement in kidney transplant patients given CsA. Sixty-two unrelated kidney transplant patients with gingival overgrowth and 124 control transplant patients without overgrowth were enrolled into the study. Gingival overgrowth was assessed at 6 months after transplantation. All patients were given CsA as a principal immunosuppressive agent during the post-transplant period. SPARC polymorphism was determined using polymerase chain reaction-restriction fragments length polymorphism assay. In kidney transplant patients with gingival overgrowth, the mean score of gingival overgrowth was 1.42+/-0.63, whereas in control subjects it was 0. The distribution of SPARC 998C>G alleles among all kidney transplant patients, as well as in the two study subgroups, did not differ significantly from Hardy-Weinberg equilibrium. The frequencies of the 998G allele (24.2% versus 18.5%) and of 998G allele carriers (40.3% versus 33.1%) among individuals with gingival overgrowth was higher compared to the control group, but the differences did not reach the statistical difference. The risk for gingival overgrowth was highest among patients carrying the 998GG genotype (OR 2.25), but it did not differ significantly from the risks associated with the other genotypes. No association between SPARC gene polymorphism and gingival overgrowth was revealed in kidney transplant patients who were administered CsA.Journal of Periodontology 11/2007; 78(11):2185-9. DOI:10.1902/jop.2007.070115 · 2.57 Impact Factor