Diphenylhydantoin plays a role in gene expression related to cytoskeleton and protein adhesion in human normal palate fibroblasts.

AIMS: Morphogenetic processes during palate development are related to extracellular matrix composition. The cell-extracellular matrix relation plays a role in cell activity and in gene expression. We studied the effect of diphenylhydantoin, a teratogen known to induce cleft palate in human newborns, on extracellular matrix production. We investigated whether diphenylhydantoin treatment caused any differences in glycosaminoglycans, collagen synthesis and gene expression in human normal palate fibroblasts. METHODS: Human palate fibroblasts were maintained for 24 hours in serum-free 199 medium containing 5 microg/mL (3)H-glucosamine or (3)H proline hydrochloride. Collagen and glycosaminoglycan classes were then measured using biochemical methods, gene expression with microarray analysis and cytoskeleton components with immunofluorescent antibodies and computer analysis. RESULTS: In normal fibroblasts diphenylhydantoin reduced collagen and glycosaminoglycan synthesis with a marked effect on sulphated glycosaminoglycans. There were also substantial decreases in tubulin, vimentin and alpha-actin staining and an increase of vinculin compared to controls. Diphenylhydantoin acted on several genes related to the synthesis of cytoskeleton and adhesion membrane proteins. It inhibited caderin, caveolin, RTK and alpha-actin, and increased nectin, cytoplasmatic FRG vinculin, ITGA, ITGB extracellular matrix ligand and EDG2 gene expression. DNA binding gene expression, which plays a role in cell growth and senescence, was activated. CONCLUSIONS: Since cell activity is dependent on the cell morphology and extracellular matrix composition, these findings indicate that in human normal palate fibroblasts diphenylhydantoin can modify cytoskeletal components and extracellular matrix-cell adhesion, with consequent effects on gene expression. These changes might be related to anomalous palate development.

Gene expression, cytoskeletal changes and extracellular matrix synthesis in human osteoblasts treated with cyclosporin A.

Cyclosporin A (CyA) is an immunosuppressive agent used to prevent allograft rejection, but unfortunately it causes adverse effects such as bone diseases, osteoporosis and osteomalacia. These pathologies involve an imbalance between synthesis, degradation and mineralization of extracellular matrix. CyA can modify extracellular matrix components such as glycosaminoglycans (GAG) and collagen fibers. In addition, normal cell activity is dependent on cell morphology and substrate cell attachment. We treated normal human osteoblasts with CyA and analyzed: (i) gene expression by a microarray method; (ii) extracellular GAG and collagen after (3)H-glucosamine and Western blot analysis; and (iii) cytoskeletal changes, using actin and tubulin fluorescent antibodies. CyA increased intra- and extracellular GAG and extracellular GAG classes such as hyaluronic acid, chondroitin sulphate, and dermatan sulphate; there was no noteworthy effect on heparan sulphate and the ratio of non-sulphated to sulphated GAG. In osteoblast cultures the drug reduced cytoskeletal actin, while tubulin did not change. In vivo the osteoblasts showed morphological changes with different extracellular matrix synthesis. Microarray analysis indicated the inhibition of gene pathways related to Wnt signaling molecules, and the cytoskeletal and focal adhesion cascade. In in vitro human osteoblasts CyA modified gene expression related to cytoskeletal pattern organization and cell morphology. Since in bone pathologies osteoblasts show different morphology related to cell size, these data suggest that in vivo osteoblast different functions could be dependent on alteration of osteoblast differentiation.

Toremifene decreases type I, type II and increases type III receptors in desmoid and fibroma and inhibits TGFbeta1 binding in desmoid fibroblasts.

Tissue infiltration is different in desmoid and fibroma tumours. Both produce high levels of transforming growth factor beta1 (TGFbeta1), which is related to extracellular matrix (ECM) accumulation which in turn regulates cell function and cell migration. Interactions between collagen, proteoglycans and cell surface fibronectin are involved in the assembly and functions of the ECM. As toremifene inhibits collagen and TGFbeta1 synthesis, we tested it in normal, desmoid and fibroma fibroblasts. We will report the changes in glycosaminoglycan (GAG) and collagen synthesis, TGFbeta1 activity, fibronectin mRNA expression and TGFbeta1 receptors after toremifene treatment in normal, fibroma and desmoid fibroblasts. We evaluated GAG and collagen synthesis with 3H-glucosamine and 3H-proline incorporation, TGFbeta1 activity with the ELISA method, TGFbeta1 receptor affinity with 125I-TGFbeta1 binding and total RNA with Northern blot analysis. GAG and collagen synthesis, TGFbeta1 activity and fibronectin levels were higher in fibroma and desmoid than normal fibroblasts. The increase was greater in desmoid than fibroma tumour cells. Toremifene treatment reduced GAG and collagen synthesis, TGFbeta1 activity and fibronectin levels in all cell cultures. The percentage reduction in GAG was similar in all cultures; the reduction in collagen synthesis and TGFbeta1 activity was the highest in desmoid fibroblasts. TGFbeta1 receptors were higher in fibroma and desmoid cells than controls. Toremifene reduced TGFbeta1 receptors only in desmoid fibroblasts, with no effect on the changes in type I, II, and III receptors. Our data show that toremifene modifies the ECM components that regulate cytokine activity and cell migration. The reduction in receptor number only in desmoid cells suggests that toremifene may reduce TGFbeta1's affinity for its receptors. Synthesis of a substance regulating protein kinase activity, which is directly involved in the link between TGFbeta1 and its receptors, cannot be excluded.

Downregulated gene expression in human palate fibroblasts after cyclosporin A treatment.

BACKGROUND: 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. METHODS: 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. RESULTS: 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

Glycosaminoglycan, collagen, and glycosidase changes in human osteoblasts treated with interleukin 1, and osteodystrophy.

Normal bone homeostasis involves a balance between osteoblast and osteoclast action, regulated by hormones and cytokine stimuli. Hemodialysis patients appear to have increased production of interleukin-1 (IL-1), interleukin-6 (IL-6) and glycosaminoglycans (GAG) in serum. IL-1 plays a role in the synthesis, degradation and degree of sulphatation of ECM components such as glycosaminoglycans. Also, continuous changes in the ECM involve enzymes such as beta-N-acetyl-d-glucosaminidase (beta-NAG) and beta-d-glucuronidase (beta-GLU) which act on different GAG classes and collagen fibers. We examined the effects of IL-1alpha on ECM synthesis and the related enzymes in human uremic osteoblast cultures. We also measured the levels of IL-1beta, and IL-6 and alkaline phosphatase activity. In biopsies of uremic bone there was less ECM deposition than resorption associated with changes in osteoblast morphology. In vitro osteoblast proliferation was higher (P< or =0.01), and extracellular GAG lower (P< or =0.01) than in controls. The enzyme beta-NAG was high (P< or =0.05) but there were no noteworthy changes in beta-GLU. ELISA of the medium indicated spontaneous production of IL-1beta and IL-6, which significantly increased after IL-1alpha treatment compared to controls. IL-1alpha reduced alkaline phosphatase activity (P< or =0.01) in uremic osteoblast cultures. IL-1 acts on osteoblasts with decreases in GAG synthesis and alkaline phosphatase activity, while beta-NAG increases. This lead to a reduction in the organic component in ECM and its mineralization, and to changes in the regulation of cytokine activity by GAG. The enzymatic breakdown might be facilitated by metabolic acidosis and failed osteoblast differentiation; these factors could be correlated with different degrees of osteodystrophy.

Desmoid and fibroma tumors differently respond to TGFbeta(1) stimulus and ECM macromolecule accumulation.

Desmoid and fibroma tumours are characterized by cell proliferation, glycosaminoglycan and collagen fibre accumulation, high levels of transforming growth factor beta(1) (TGFbeta(1)) and different patterns of tissue infiltration. TGFbeta(1) is related to extracellular matrix (ECM) composition which, in turn, regulates cell functions and cell migration. In this study we report changes in cell proliferation, glycosaminoglycan (GAG) and collagen synthesis, TGFbeta(1) mRNA expression and fibronectin levels in normal, desmoid and fibroma fibroblast cultures before and after TGFbeta(1) stimulation. Our data showed cell proliferation, GAG and collagen synthesis, transforming growth factor beta(1) mRNA expression and fibronectin levels were significantly higher in desmoid than in fibroma cultures. TGFbeta(1) treatment had no effect on cell proliferation, but increased TGFbeta(1) mRNA expression, GAG, fibronectin and collagen synthesis in desmoid and fibroma fibroblasts. Its effects were more marked in desmoid cells. Fibronectin favours cell migration, while changes in GAG composition alter cell behaviour and ECM organization. In conclusion our data suggest that the different patterns of infiltration in desmoid and fibroma tumours are due to changes in ECM components and cell-ECM interactions which can be ascribed to altered TGFbeta(1) mRNA expression and TGFbeta(1) activity.

Depletion of polyamines and increase of transforming growth factor-beta1, c-myc, collagen-type I, matrix metalloproteinase-1, and metalloproteinase-2 mRNA in primary human gingival fibroblasts.

BACKGROUND: The polyamines spermidine, spermine, and putrescine are known to be deeply linked with growth processes, gene expression, and extracellular matrix synthesis. Their cellular content depends primarily on the activity of the enzyme ornithine decarboxylase. High levels of ornithine decarboxylase and polyamines have been found in proliferative, inflammatory, and neoplastic pathologies of the oral cavity and in gingival fluid. Difluoromethylornithine (DFMO) selectively inhibits ornithine decarboxylase, thus depleting polyamine content and preventing cell proliferation and synthesis activity. The aim of this study was to investigate whether DFMO treatment could modify the genes involved in cell proliferation and extracellular matrix turnover. METHODS: Fibroblasts derived from non-inflamed gingiva were maintained in Dulbecco's modified Eagle's medium (DMEM) plus alpha-difluoromethylornithine for 4 days. At 0, 24, 48, 72, and 96 hours cell number was assessed, polyamine levels were quantified with high performance liquid chromatography (HPLC) method, and transforming growth factor-beta1 (TGF-beta1), c-myc, matrix metalloproteinases (MMP)-1 and 2, collagen type I (COL-I) and tissue inhibitor of matrix metalloproteinases (TIMP)-1 were evaluated by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Fibroblasts treated with DFMO significantly decreased cell proliferation, ornithine decarboxylase activity, and putrescine levels at all treatment times, spermidine after 72 and 96 hours, and spermine after 96 hours of culture. Total polyamines decreased (P < or =0.01) at 96 hours after DFMO treatment, while c-myc, TGF-beta1, MMP-1 and 2, COL-I mRNA significantly increased. Conversely, TIMP-1 did not show any significant change. The polyamines trend was not correlated to c-myc, TGF-beta1, MMP-1 and -2, and TIMP-1 mRNA levels. Transforming growth factor-beta1 and c-myc mRNA expression were related and correlated to MMP-1 and 2, COL-I and TIMP-1 mRNA trend after DFMO treatment. CONCLUSIONS: Our data show that as the polyamine content decreases, TGF-beta1, c-myc, MMP-1 and -2, and COL-I mRNA levels increase, therefore a negative regulatory role of the polyamines on the mRNA expression could be suggested.

Changes in polyamines, c-myc and c-fos gene expression in osteoblast-like cells exposed to pulsed electromagnetic fields.

Pulsed electromagnetic field (PEMF) stimulation promotes the healing of fractures in humans, though its effect is little known. The processes of tissue repair include protein synthesis and cell differentiation. The polyamines (PA) are compounds playing a relevant role in both protein synthesis processes and cell differentiation through c-myc and c-fos gene activation. Since several studies have demonstrated that PEMF acts on embryonic bone cells, human osteoblast-like cells and osteosarcoma TE-85 cell line, in this study we analyzed the effect on cell PAs, proliferation, and c-myc and c-fos gene expression of MG-63 human osteoblast-like cell cultures exposed to a clinically useful PEMF. The cells were grown in medium with 0.5 or 10% fetal calf serum (FCS). c-myc and c-fos gene expressions were determined by RT-PCR. Putrescine (PUT), spermidine (SPD), or spermine (SPM) levels were evaluated by HPLC. [(3)H]-thymidine was added to cultures for DNA analysis. The PEMF increased [(3)H]-thymidine incorporation (P < or = .01), while PUT decreased after treatment (P < or = .01); SPM and SPD were not significantly affected. c-myc was activated after 1 h and downregulated thereafter, while c-fos mRNA levels increased after 0.5 h and then decreased. PUT, SPD, SPM trends, and [(3)H]-thymidine incorporation were significantly related to PEMF treatment. These results indicate that exposure to PEMF exerts biological effects on the intracellular PUT of MG-63 cells and DNA synthesis, influencing the genes encoding c-myc and c-fos gene expression. These observations provide evidence that in vitro PEMF affects the mechanisms involved in cell proliferation and differentiation.

Ultrastructural changes in Langerhans cells in gingival overgrowth in cyclosporin A-treated renal transplant patients.

AIM: Our research was focused on the ultrastructural features of gingival epithelium in kidney transplant patients and, in particular, on Langerhans cells, with the aim of verifying whether ultrastructural modifications might explain gingival overgrowth. METHODS: Using electron microscopy and immunohistochemical S100 staining at optical microscopy, we examined gingival samples obtained from 18 kidney transplant patients who presented with gingival overgrowth following cyclosporin A treatment. RESULTS: The hyperkeratosis shown by the epithelium, and especially the absence of Birbeck granules in the Langerhans cells observed in serial sections, lead us to correlate these data to an immunodeficiency which affects the epithelium in the complex mechanism determining overgrowth. CONCLUSIONS: In our previous studies we attributed the responsibility of overgrowth to the connective tissue alone. However, in the light of the present results, we cannot exclude a contribution of the epithelium to gingival overgrowth.

Effects of interleukin-1beta on chondroblast viability and extracellular matrix changes in bovine articular cartilage explants.

Osteoarthritis is a degenerative disease of joint cartilage, characterized by the progressive and permanent degeneration of cartilage due to an imbalance in normal extracellular matrix turnover. Interleukin-1 beta is a proinflammatory agent, which is present in an elevated amount in osteoarthritic cartilage, and is thought to play a decisive role in osteoarthritis. Interleukin-1 beta acts as an important mediator of extracellular matrix changes where its activity is regulated by glycosaminoglycan composition. The aim of this study was to investigate the extracellular matrix changes in bovine cartilage explants following interleukin-1 beta treatment by morphological, histochemical and biochemical methods. Interleukin-1 beta stimulated the release of matrix sulfated proteoglycans in the culture medium, and significantly inhibited sulfated proteoglycan synthesis. These events were associated to a strong stimulation of nitric oxide production. Interleukin-1 beta-treated cartilage showed evident collagen fibers around the chondrocytes, together with diminished glycosaminoglycan sulfate content in the extracellular matrix of the explants. Moreover, the ultrastructure and viability of cells did not change in treated cartilage. Our data show that interleukin-1 beta modifies the ECM turnover without toxic effect on chondrocytes.

The effect of polyamines and dialysate fluid on extracellular matrix synthesis in VERO cell cultures.

BACKGROUND: Polyamines are involved in normal and pathological cell proliferation and differentiation. Like acid radicals, they play an important role in remodelling the extracellular matrix and are considered "uremic toxins". Proteins and glycosaminoglycans are essential components of the extracellular matrix, and contribute to normal mature organ functions. The aim of this study was to analyse the effects of free polyamines, dialysate fluid components and dialysis fluid on protein and extracellular glycosaminoglycan synthesis in VERO cell cultures. METHODS: The dialysate fluid components were separated with a Sephadex G15 column and the cultures were analysed after incorporation of 3H-leucine and 3H-glucosamine. Cultures were run at pH 7.0 and pH 7.4. The glycosaminoglycan classes were separated with a DEAE column, and polyamines were determined by high-performance liquid chromatography. Proteins and single glycosaminoglycan classes were quantified by a scintillator. DNA gel electrophoresis was done to detect chromatin fragmentation. RESULTS: Dialysate contained putrescine, spermidine and spermine, chromatography showing four peaks; only peaks I and II indicated polyamines at respectively Da 5000 and 1500. Polyamines are therefore linked to different carriers. There was an increase of protein and glycosaminoglycan synthesis with dialysis fluid and polyamines, but inhibition with peak II or dialysate. DNA gel electrophoresis showed no chromatin fragmentation. Findings at pH 7.0 and 7.4 were similar. CONCLUSIONS: It would appear that in uremic patients polyamines are conjugated to protein carriers of different molecular weights with different biological actions. As polyamines and dialysis fluid affect changes in extracellular matrix, they could be related to physiological organ functions. However, these in vitro data must be considered with the appropriate limitations when we try to extrapolate them to the in vivo situation.

Cyclosporin A and transforming growth factor beta modify the pattern of extracellular glycosaminoglycans without causing cytoskeletal changes in human gingival fibroblasts.

Cyclosporin A is a powerful immunosuppressive drug that has had a major impact on transplant therapy. It apparently links to different enzymatic pathways, and affects multiple enzymatic systems. Transforming growth factor beta induces the deposition of glycosaminoglycans, proteoglycans, and collagen fibers in the extracellular matrix. The aim of this study of normal and hypertrophic human gingival fibroblast cultures was to evaluate the cytoskeletal and extracellular changes in glycosaminoglycan secretion due to the presence of cyclosporin A and transforming growth factor beta. The results showed that there is an increase in total and individual classes of extracellular glycosaminoglycans in the presence of cyclosporin A and transforming growth factor beta, but the action of the latter was significantly greater. Immunohistochemical analysis of the cytoskeleton did not reveal any morphological differences between treated and control cells. Our data suggest that the biochemical changes in the extracellular matrix are caused more by cytokine, and that cyclosporin A does not induce any morphological changes in fibroblast cultures derived from hypertrophic and normal gingiva.