Normal Human Gingival Fibroblasts Research Articles

Differential regulation of extracellular matrix proteoglycan (PG) gene expression. Transforming growth factor-beta 1 up-regulates biglycan (PGI), and versican (large fibroblast PG) but down-regulates decorin (PGII) mRNA levels in human fibroblasts in culture

Proteoglycans (PGs) comprise a group of extracellular matrix macromolecules which play an important role in matrix biology. In this study, normal human skin and gingival fibroblast cultures were incubated with transforming growth factor-beta 1 (TGF-beta 1), and the expression of three PGs, viz. biglycan (PGI), decorin (PGII), and versican (a large fibroblast proteoglycan) was examined. The results indicate that TGF-beta 1 (5 ng/ml) markedly increased the expression of biglycan (up to 24-fold) and versican (up to 6-fold) mRNAs and the enhancement of biglycan expression was coordinate with elevated type I procollagen gene expression in the same cultures. In contrast, the expression of decorin mRNA was markedly (up to approximately 70%) inhibited by TGF-beta 1. The response to TGF-beta 1 was similar in both skin and gingival fibroblasts, although the gingival cells were clearly more responsive to stimulation by TGF-beta 1 with respect to biglycan gene expression. Analysis of 35S-labeled proteoglycans in the culture media of skin and gingival fibroblasts also revealed stimulation of biglycan and versican production, and reduction in decorin production. Quantitation of both [35S]sulfate and [3H]leucine-labeled decorin in cell culture media by immunoprecipitation revealed a 50% reduction in decorin production in cell cultures treated with TGF-beta 1. This TGF-beta 1-elicited reduction was accompanied by an apparent increase in the size of the decorin molecules, although the size of the core protein was not altered, as judged by Western immunoblotting following chondroitinase ABC digestion. Analysis of the proteoglycans in the matrix and membrane fractions also revealed increased amounts of versican in cultures treated with TGF-beta 1. These results indicate differential regulation of PG gene expression in fibroblasts by TGF-beta 1, and these observations emphasize the role of PGs in the extracellular matrix biology and pathology.

Induction of interleukin-1 and -6 in human gingival fibroblast cultures stimulated with Bacteroides lipopolysaccharides

Normal human gingival fibroblasts stimulated in vitro by lipopolysaccharides (LPS) from oral Bacteroides species produced cell-free and cell-associated thymocyte-activating factors (TAF). Neutralization assays using antisera to human interleukin-1 alpha (HuIL-1 alpha), HuIL-1 beta, and HuIL-6 revealed that cell-free TAF was attributable mainly to IL-1 beta and that IL-6 augmented the TAF activity of IL-1 beta in the culture supernatant. Another factor(s), however, may also be involved in cell-free TAF. By contrast, the active entity of cell-associated TAF was ascribed to IL-1 alpha alone. Furthermore, IL-6 was detected mainly in the supernatant of fibroblast cultures stimulated with Bacteroides LPS. Fibroblasts pretreated with natural human beta or gamma interferon, but not those pretreated with alpha interferon, synthesized higher levels of cell-associated IL-1 alpha in response to stimulation by Bacteroides LPS; however, no interferons exhibited direct IL-1-inducing activity or synergistic IL-1-inducing activity with LPS. Endogenously induced beta interferon was suggested to be necessary for fibroblasts to produce cell-associated IL-1 alpha in response to Bacteroides LPS.

Effect of phenytoin and nifedipine on collagen gene expression in human gingival fibroblasts

Phenytoin (PHT), a widely used anticonvulsant, and nifedipine (NF), an anti-anginal drug, cause clinically similar gingival overgrowths in some patients. The aim of this work was to investigate their effects on collagen and protein synthesis and cellular proliferation in normal human gingival fibroblasts in vitro. Gingival fibroblasts were cultured from biopsies taken from three healthy individuals during operations on maxillary canines and incubated with various concentrations of NF (100 and 200 ng/ml) and PHT (5 and 10 micrograms/ml) for up to 7 days. The results showed that NF and PHT have a specific effect in reducing total protein and collagen synthesis but do not influence cell proliferation in healthy gingival fibroblasts in vitro. In addition the level of mRNA for type I collagen was decreased after incubation of the cells with the drugs for 1 or 2 days. The decrease in the level of type I collagen mRNA seemed to be specific since the level of type IV collagenase mRNA used as a reference RNA did not decrease.

Regulation of epidermal growth factor receptor metabolism in gingival fibroblasts by phenytoin in vitro

Normal human gingival fibroblasts derived from five children between 8 and 12 yr of age were cultured under serum-free conditions in the presence of epidermal growth factor (EGF) either alone or in combination with 5,5-diphenylhydantoin (phenytoin; PHT). DNA-synthesis, binding of EGF to its cell-surface receptor and internalisation of EGF-receptor-ligand complexes were studied. In normal gingival fibroblasts treated solely with EGF for 48 h, DNA synthesis increased significantly, as in cells treated solely with PHT. When EGF binding data was calculated according to Scatchard, it was found that the number of EGF receptors in fibroblasts increased significantly after PHT treatment. The number of EGF-receptors in untreated gingival fibroblasts varied from 147,000 to 170,000 receptors per cell whereas in PHT-treated fibroblasts the range was from 181,000 to 280,000. The study indicates that PHT regulates EGF-receptor metabolism in human gingival fibroblasts by increasing the number of cell-surface EGF-receptors which may contribute to the alteration of gingival connective tissue observed in patients undergoing PHT medication.

Comparison of the inhibitory effect of polyunsaturated fatty acids on prostaglandin synthesis. II. Fibroblasts

In a previous publication (1) we reported that PUFAs of the n-6 and n-3 series caused significant inhibition of synthesis of both PGE 2 (28.4–92.8%) and PGF 2α (24.4–84.0%) in the oral squamous carcinoma cell line SCC-25. In this report we describe the inhibitory effect of the same acids on PG synthesis in normal human gingival fibroblasts under the same experimental conditions. It was found that a combination of EPA + DCHA (6:4), DCHA and ALA caused significant reduction in synthesis of PGE 2 (10.1–87.8%) and PGF 2α (14.0–54.6%) at the four dose levels studied. The rank order of potency of acids in reduction of PG synthesis was: EPA + DCHA>DCHA>EPA>ALA>LA>DGLA>GLA. The data suggest that although PUFAs are effective inhibitors of PG synthesis by gingival fibroblasts and SCC-25, the fibroblast is less susceptible to the inhibitory effect of fatty acids.

Biochemical characterization of phenytoin-induced hyperplastic human gingival fibroblasts. Non-collagenous proteins biosynthesis

Phenytoin (PHT), administered as an anticonvulsant, has a side effect gingiva overgrowth in approximately 50% of patients. The present study was attempted to explore the biochemical mechanism on non-collagenous protein biosynthesis as affected by PHT. Responder cells (RES A3, RES C2) of a patient with gingival overgrowth were obtained by the method of Kawase et al. Normal human gingival fibroblasts (Gin-1), purchased from ATCC, were also used. All cells were inoculated at 1 x 10(4) cells/cm2 (12 multi-well plate or 60 mm tissue culture dish), and then cultured for 4, 8 and 12 days with or without PHT (5 micrograms/ml). Prior to harvesting at the indicated times, cells were incubated with 14C-amino acids (1.25 microCi/ml) for 24 hours. The 14C-labeled proteins were isolated from the cell layers including extracellular matrix, following Kurkinen et al. with a minor change. Each 14C-labeled fraction was dissolved in 3 ml of Aquasol-2 and the radioactivity by a liquid scintillation counter. The DNA content of cell layers affected by PHT was increased on Gin-1, RES A3 and RES C2 at the post-confluence, resulting also in an increase in cell number. Two morphologically different phenotypes of responder cells were observed, differing in nuclear and cell sizes. At 12 days culture, RES A3, were stimulated by PHT, showed increased synthesis of both total extractable proteins (EP) and binding proteins (BP) labeled with 14C-amino acids. Therefore, at least two distinct phenotypic responder cells are present in the PHT-induced overgrowth gingiva, alter the synthesis of non-collagenous proteins.

Interleukin 1 stimulation of plasminogen activator production in cultured gingival fibroblasts.

Addition of human interleukin‐1 (IL‐1) to normal human gingival fibroblasts in culture stimulated the production of plasminogen activator (PA). This stimulation could be detected as early as 3 hours after addition of IL‐1, was dosedependent and inhibited by antibody directed against IL‐1. Additional observations suggest that IL‐1 increases PA production. These include: (1) an increase in both intra‐ and extracellular PA activity, (2) the dependence of increased PA activity on RNA and protein synthesis and (3) an increase in immunologically detectable PA molecules by immunoperoxidase staining. Since IL‐1 has been reported to be present in human gingival fluid and PA activity has been associated with connective tissue matrix alterations, the results presented here may provide some insight as to how immune events are linked to soft tissue destruction associated with periodontitis.

The effects of phenytoin (5, 5‐diphenylhydantoin) on human gingival fibroblasts in culture

Macromolecular biosynthesis in monolayer cultures of normal and responder human gingival fibroblasts derived from tissue explants was evaluated in the presence or absence of phenytoin (5μg/ml) from the 5th to the 8th day after plating and a labeling period with 14C‐proline (0.2 μCi/ml) for the last 48 hours. Collagenase digestion and hydroxyproline determinations indicated an increase in collagen product. At least part of the increase may have been due to a drug‐modulated decrease in collagen degradation. Phenytoin (PHT) also stimulated synthesis of glycosaminoglycan as indicated by an approximately 2‐fold increase in sulfate‐labeled product. Ultrastructural examination revealed a pronounced change in the protein synthetic apparatus of PHT‐treated cells. Tritiated phenytoin bound to fibroblasts and appeared to undergo internalization as indicated by trypsin treatment.

The effects of platelet-derived transforming growth factor beta on normal human diploid gingival fibroblasts.

Studies of the effects of transforming growth factor (TGF) beta on normal human diploid gingival fibroblasts (HGF) have been carried out to determine possible physiological effects of this growth factor. Responses distinctly different from those characterized using established cell lines were observed. Whether alone, or in combination with EGF (2.5 ng/ml), human platelet-derived TGF-beta (0.1 ng/ml or 1.0 ng/ml) did not induce anchorage-independent growth of HGFs in soft agar assays. However, TGF-beta with EGF acted synergistically in promoting a 1.8-fold increase in anchorage-dependent proliferation of quiescent HGFs. At the same concentrations TGF-beta alone stimulated the incorporation of [35S]methionine into both cellular (cell-layer) and matrix (medium) proteins by as much as 3-fold and 1.7-fold respectively. Densitometric analysis of fluorographs of radiolabeled media proteins separated by SDS-PAGE revealed that the TGF-beta-stimulated protein synthesis was selective. However, synthesis of collagen, the major protein synthesized and secreted by HGFs, was stimulated by TGF-beta to the same extent as the average secreted protein. Protein synthesis and cell proliferation were significantly greater in subconfluent cells compared to confluent and multilayered cells. These effects are likely to reflect physiological activity of platelet-derived TGF-beta which may act to promote the wound healing response.

APhA House of Delegates Roster

The objective of the study was to investigate the antimicrobial effects of deglycyrrhizinated licorice root extracts (DG-LRE) against Streptococcus mutans UA159 in both the planktonic and biofilm phases by determining the minimum inhibitory concentration and minimum bactericidal concentration, and by performing time-kill kinetic, growth, adhesion, and biofilm assays. The cell toxicity of DG-LRE on normal human gingival fibroblast (NHGF) cells was tested using a methyl thiazolyl tetrazolium assay. This study showed that DG-LRE had strong antimicrobial activity against S. mutans in the planktonic phase with little cytotoxic effect on NHGF cells. In addition, DG-LRE significantly inhibited biofilm formation by S. mutans UA159 at concentrations over 4 μg/ml for glucose or 16 μg/ml for sucrose, respectively, regardless of the presence of saliva-coating. To the best of our knowledge, this is the first report to provide evidence that DG-LRE demonstrates antimicrobial activity against S. mutans. These results suggest that DG-LRE can be used in developing oral hygiene products, such as gargling solution and dentifrice to prevent human dental caries.

Letters

The objective of the study was to investigate the antimicrobial effects of deglycyrrhizinated licorice root extracts (DG-LRE) against Streptococcus mutans UA159 in both the planktonic and biofilm phases by determining the minimum inhibitory concentration and minimum bactericidal concentration, and by performing time-kill kinetic, growth, adhesion, and biofilm assays. The cell toxicity of DG-LRE on normal human gingival fibroblast (NHGF) cells was tested using a methyl thiazolyl tetrazolium assay. This study showed that DG-LRE had strong antimicrobial activity against S. mutans in the planktonic phase with little cytotoxic effect on NHGF cells. In addition, DG-LRE significantly inhibited biofilm formation by S. mutans UA159 at concentrations over 4 μg/ml for glucose or 16 μg/ml for sucrose, respectively, regardless of the presence of saliva-coating. To the best of our knowledge, this is the first report to provide evidence that DG-LRE demonstrates antimicrobial activity against S. mutans. These results suggest that DG-LRE can be used in developing oral hygiene products, such as gargling solution and dentifrice to prevent human dental caries.