Induction of dental pulp fibroblast matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 gene expression by interleukin-1alpha and tumor necrosis factor-alpha through a prostaglandin-dependent pathway

Matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) are involved in the degradation of extracellular matrix in many inflammatory diseases. Little is known regarding the expression of these mediators in dental pulp fibroblasts. The effects of proinflammatory cytokines (interleukin (IL)-1alpha and tumor necrosis factor-alpha (TNF-alpha)) and prostaglandin E2 (PGE2) on pulp fibroblast MMP-1 and TIMP-1 gene expression were investigated. Northern hybridization showed that IL-1alpha and TNF-alpha induced significant MMP-1 gene expression, with only little effect on TIMP-1 gene. Exogenous PGE2, however, upregulated TIMP-1 mRNA synthesis but not MMP-1. Concomitant addition of IL-1alpha and PGE2 or TNF-alpha and PGE2 suppressed MMP-1 mRNA production, compared with the groups treated with IL-1alpha or TNF-alpha alone. In contrast, PGE2 enhanced the upregulatory effects of TIMP-1 mRNA by IL-1alpha or TNF-alpha. Furthermore, cytokine stimulation of MMP-1 and TIMP-1 gene expressions can be enhanced or blocked by indomethacin, respectively, and reversed by exogenous PGE2. These results suggested that cytokine-stimulated MMP-1 and TIMP-1 gene expression in dental pulp fibroblasts was mediated, at least in part, by a prostaglandin-dependent pathway. The differential regulation of IL-1alpha or TNF-alpha-induced MMP-1 and TIMP-1 mRNA synthesis, as well as the direct upregulation of TIMP-1 gene expression by PGE2, also implied that prostaglandin may serve as a protective mechanism from excessive tissue breakdown during pulpitis.     

Effect of phenytoin on collagen accumulation by human gingival fibroblasts exposed to TNF-alpha in vitro

OBJECTIVE: Tumor necrosis factor (TNF)-alpha is associated with chronic gingival inflammation and reported to induce gingival overgrowth (GO), while phenytoin (PHT) is also known to be a causative agent of GO. We examined the synergistic effect of PHT and TNF-alpha on collagen metabolism in human gingival fibroblasts (HGFs). MATERIALS AND METHODS: HGFs were cultured with TNF-alpha and PHT. Quantitative real-time RT-PCR was employed to determine the mRNA levels for collagen, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and integrin subunits. Cellular collagen endocytosis was determined using a flow-cytometry. RESULTS: The proliferation of HGFs was not affected by TNF-alpha or PHT individually, whereas both synergistically increased collagen accumulation in HGFs. Further, collagen mRNA expression was not increased by TNF-alpha or PHT, although together they markedly prevented cellular collagen endocytosis, associated with the suppression of alpha2beta1-integrin mRNA expression. The mRNA expression of MMP-1 and-2 was suppressed by PHT, while TIMP-1 mRNA expression was enhanced by both TNF-alpha and PHT. CONCLUSION: Our results suggest that TNF-alpha and PHT together cause impaired collagen metabolism by suppression of enzymatic degradation with MMPs/TIMP-1 and integrin-mediated endocytosis. These synergistic effects may also be involved in TNF-alpha- and PHT-induced collagen accumulation, leading to GO.     

In vitro expression of matrix metalloproteinase-1, tissue inhibitor of metalloproteinase-1 and transforming growth factor-beta1 in human periodontal ligament fibroblasts

Extracellular matrix remodelling is mediated via matrix metalloproteinases (MMPs) and their regulatory factors such as tissue inhibitors of metalloproteinases (TIMPs) and transforming growth factor-beta (TGF-beta). The regulation of MMPs is thought to be associated with cytoskeletal changes. In this study, cytoskeletal changes in human periodontal ligament fibroblasts (PDFs) were induced using cytochalasin B (CB) which reorganizes actin microfilaments reversibly, and colchicine which disrupts microtubules irreversibly. The levels of MMP-1, TIMP-1 and TGF-beta secreted by the CB- or colchicine-treated PDFs were measured using an enzyme-linked immunosorbent assay. Differences between experimental and control groups were tested using analysis of variance (ANOVA) and Scheffé's ad hoc test. Although CB treatment did not significantly increase MMP-1 expression over the controls, colchicine treatment significantly increased the expression of MMP-1 (P < 0.01) in a time-dependent manner compared with the controls. Both CB and colchicine showed a time-dependent increase in TIMP-1 and TGF-beta1 expression and a dose-dependent increase in TIMP-1 and TGF-beta1 expression until threshold compared with the controls (P < 0.05, P < 0.01 and P < 0.001). In addition, CB treatment produced significantly increased TGF-beta1 expression over the controls (P < 0.05 and P < 0.001) from lower doses, with this effect occurring at earlier time points compared with colchicine treatment.     

MMPs, IL-1, and TNF are regulated by IL-17 in periodontitis

Periodontitis is characterized by periodontal tissue destruction. Since interleukin-17 (IL-17) has been reported to up-regulate IL-1beta and tumor necrosis factor-alpha (TNF-alpha), it was hypothesized that it is increased in periodontitis and up-regulates these cytokines and tissue-destructive matrix metalloproteinases (MMP) in local migrant and resident cells. Immunocytochemistry disclosed elevated IL-1beta, TNF-alpha, and IL-17 levels in periodontitis. These cytokines induced proMMP-1 and especially MMP-3 in gingival fibroblasts, whereas MMP-8 and MMP-9 were not induced. IL-17 was less potent as a direct MMP inducer than IL-1beta and TNF-alpha, but it induced IL-1beta and TNF-alpha production from macrophages, and IL-6 and IL-8 from gingival fibroblasts. In accordance with these findings, immunocytochemistry disclosed that MMP-1 and MMP-3 were increased in periodontitis. Gingival fibroblasts may play an important role in tissue destruction in periodontitis via cytokine-inducible MMP-1 and MMP-3 production, in which IL-17 plays a role as a key regulatory cytokine.     

Expression profile of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs in mature human odontoblasts and pulp tissue

Previous studies have demonstrated that (at least) matrix metalloproteinase (MMP)-2, -8, -9, -14 and -20 are expressed by human odontoblasts. Here, we analysed the expression of 19 MMPs and their specific tissue inhibitors (TIMP)-1, -2 and -3) -1, -2 and -3 in mature human odontoblasts and pulp tissue. Since MMP-20 is almost exclusively expressed by the dentin-pulp complex cells, we further analysed the effect of transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMPs)-2 on its expression. Matrix metalloproteinase-9 served as a positive control for growth factor responsiveness. It was found that MMP-1, -2, -9, -10, -11, -13, -14, -15, -16, -17, -19, -20 and -23, in addition to TIMP-1, -2 and -3 were expressed by both odontoblasts and pulp tissue. Neither MMP-3 nor MMP-12 were expressed in odontoblasts or pulp tissue, and MMP-7, -8, -24 and -25 were expressed only in the odontoblasts; MMP-2, -10, -11, -14 and -20 were expressed more abundantly by odontoblasts, whereas pulp tissue expressed more MMP-13 and MMP-17. Transforming growth factor-beta1 (1 ng ml(-1)) and BMP-2 (100 ng ml(-1)) did not markedly affect MMP-20 mRNA expression. In contrast, TGF-beta1 alone and with BMP-2 significantly upregulated MMP-9 mRNA by 2.4-fold and by 2.6-fold, respectively, in odontoblasts, while in pulp tissue no effects could be detected. The wide-scale expression of MMPs and TIMPs by mature human odontoblasts and pulp tissue suggests that they may participate in dentin matrix organization prior to mineralization, and that growth factors may further control dentin matrix modeling by differentially regulating individual MMPs.     

Cytokines, matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 in gingival crevicular fluid from patients with Papillon-Lefèvre syndrome

The aim of the present study was to compare concentrations of cytokines, matrix metalloproteinases (MMPs) and a metalloproteinase inhibitor (TIMP-1) in gingival crevicular fluids (GCF) from sites with gingival inflammation in 28 young patients with Papillon-Lefèvre syndrome (PLS), and in age- and gender-matched controls. Each group consisted of 17 females and 11 males with a mean age of 11.0 years (range 4-22 years). In both groups, anterior upper sites with a clinical diagnosis of gingival inflammation and with pockets < or = 3 mm were selected for sampling of GCF, which was carried out with filter disks inserted into the gingival crevice until saturated. The concentrations of cytokines (IL-1alpha, IL-1beta, TNF-alpha, and IL-8), matrix metalloproteinases (MMP-1, MMP-3, MMP-8, and MMP-9), and their tissue inhibitor (TIMP-1) were analysed using commercial ELISA kits. Significantly higher levels of IL-1beta (P < 0.001) and MMP-8 (P < 0.05) were disclosed among the PLS patients compared with their controls, while the opposite was found for IL-8 (P < 0.05) and MMP-1 (P < 0.001). The individual variations were considerable in both groups. When comparing the expression of cytokines, MMPs, and TIMP-1 in PLS patients with clinically active and non-active periodontitis, the non-active PLS patients showed significantly higher values of IL-1beta than the patients with active periodontal disease (ANOVA, P < 0.01). In conclusion, this study was unable to demonstrate a clear-cut pathognomonic expression of cytokines or MMPs in patients with PLS, but further studies on cytokine and MMP output are warranted.     

Cytokine expression pattern in compression and tension sides of the periodontal ligament during orthodontic tooth movement in humans

Orthodontic tooth movement is achieved by the remodeling of periodontal ligament (PDL) and alveolar bone in response to mechanical loading and is believed to be mediated by several host mediators, such as cytokines. By means of real-time polymerase chain reaction (PCR), we studied the pattern of expression of mRNA encoding several pro- and anti-inflammatory cytokines in relation to several extracellular matrix and bone remodeling markers, in tension (T) and compression (C) sides of the PDL of human teeth subjected to rapid maxillary expansion. The PDL of normal teeth was used as a control. The results showed that both T and C sides exhibited significantly higher expression of all targets when compared with controls, except for type I collagen (COL-I) and tissue inhibitor of metalloproteinase-1 (TIMP-1) on the C side. Comparing C and T sides, the C side exhibited higher expression of tumor necrosis factor-alpha (TNF-alpha), receptor activator of nuclear factor-kappaB ligand (RANKL), and matrix metalloproteinase-1 (MMP-1), whereas the T side presented higher expression of interleukin-10 (IL-10), TIMP-1, COL-I, osteoprotegerin (OPG), and osteocalcin (OCN). The expression of transforming growth factor-beta (TGF-beta) was similar in both C and T sides. Our data demonstrate a differential expression of pro- and anti-inflammatory cytokines in compressed and stretched PDL during orthodontic tooth movement.     

Expression of mRNAs encoding for alpha and beta integrin subunits, MMPs, and TIMPs in stretched human periodontal ligament and gingival fibroblasts

The biological mechanisms of tooth movement result from the cellular responses of connective tissues to exogenous mechanical forces. Among these responses, the degradation of the extracellular matrix takes place, but the identification of the molecular basis as well as the components implicated in this degradation are poorly understood. To contribute to this identification, we subjected human fibroblasts obtained from the periodontal ligament (PDLs) and from the gingiva (HGFs) to a continuous stretch to quantify the mRNAs encoding for various metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and alpha and beta integrin subunits. Both cell lines reacted by inducing the expression of the mRNAs encoding for MMP-1, MMP-2, TIMP-1, and TIMP-2, while other mRNAs did not vary (MT1-MMP, TIMP-3) or were not expressed (MMP-9). PDLs expressed selectively the mRNAs encoding for alpha4 and alphav, with no difference measurable under stretching, while the mRNAs encoding for alpha6 and beta1 were increased and the one encoding for alpha5 was decreased. HGFs increased the mRNAs encoding for alpha2, alpha6, beta1, and beta3 and decreased the one encoding for alpha3. Analysis of our data indicated that stretched HGFs and PDLs induced the same pattern of mRNAs encoding for MMPs and TIMPs but differed for those encoding various integrin subunits, known to act as protein receptors in mechanotransduction.     

Regulation of matrix metalloproteinase-2 production by cytokines and pharmacological agents in human pulp cell cultures.

Type IV matrix metalloproteinases (MMPs) are members of the family of MMPs and are thought to play an important role in degradation of extracellular components. Human pulp cells can secrete and produce these enzymes. Recent evidence shows that MMPs may play a role in pulpal inflammation. To date little is known regarding the regulation of MMPs in human pulp cell cultures. The purpose of this study was to determine the effects of cytokines (interleukin-1 and transforming growth factor-beta (TGF-beta), protein synthesis inhibitor cycloheximide (CD), and protein kinase C inhibitors (H7 and Go6976) on the secretion and production of MMPs by human pulp cell cultures using gelatin zymography. The main gelatinase secreted by human pulp cells migrated at 72 kDa and represented MMP-2. Minor gelatinolytic bands were also observed at 92 kDa regions that correspond to MMP-9. After an 8-day culture period TGF-beta, CD, H7, and Go6976 were found to depress MMP-2 production. The inhibition decreased in an order of CD > H7 > TGF-beta > Go6976. IL-1 was found to elevate MMP-2 production. Human pulp cells, however treated with either cytokines or pharmacological agents had no effect on the pattern of MMP-9 produced or secreted in either cell extracts or conditioned medium fractions. These observations suggest that the cytokines and pharmacological agents can regulate MMP-2 produced by human pulp cells. Inflammatory cytokines stimulate the production of elevated levels of MMP-2 and MMP-2 might play a role in pulpal inflammation. In addition agents that target protein synthesis or the protein kinase C pathway in human pulp cells inhibit MMP-2 production, and such inhibition may contribute to the pathogenesis of pulpal inflammation. Such inhibition might contribute to therapeutic efficacy.     

Nicotine increases the collagen-degrading ability of human gingival fibroblasts

BACKGROUND AND OBJECTIVE: The objective of this study was to determine the effects that nicotine and the combination of nicotine and Porphyromonas gingivalis supernatant have on human gingival fibroblast-mediated collagen degradation. MATERIAL AND METHODS: Human gingival fibroblasts were cultured with 25-500 microg/ml of nicotine in collagen-coated six-well plates. On days 1-5, the conditioned media was collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The cells were then removed and the collagen cleavage visualized by Coomassie blue staining. To examine the combined effect, 250 microg/ml of nicotine and 10% v/v culture supernatant of P. gingivalis ATCC 33277 were added to the human gingival fibroblasts. The mRNA levels of multiple MMPs and TIMPs were monitored. RESULTS: Nicotine increased the human gingival fibroblast-mediated collagen cleavage. The MMP-14 and MMP-2 produced by the nicotine-treated human gingival fibroblasts more readily underwent zymogen activation. Nicotine treatment resulted in TIMP-2 redistribution to the cell surface. The mRNAs of multiple MMPs and TIMPs were unaltered by nicotine. An additive collagen cleavage effect was observed when the human gingival fibroblasts were treated with both nicotine and P. gingivalis. CONCLUSION: Nicotine increased human gingival fibroblast-mediated collagen degradation, in part through the activation of membrane-associated MMPs. Nicotine and P. gingivalis had an additive effect on human gingival fibroblast-mediated collagen degradation.     

Effect of avocado and soybean unsaponifiables on gelatinase A (MMP-2), stromelysin 1 (MMP-3), and tissue inhibitors of matrix metalloproteinase (TIMP- 1 and TIMP-2) secretion by human fibroblasts in culture.

BACKGROUND: In inflamed periodontal tissues, gingival fibroblasts are able to express matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). They can also respond to growth factors and cytokines. In this study, the in vitro effects of avocado and soybean unsaponifiable residues (ASU), their fractions (avocado unsaponifiable [ASF] or soy unsaponifiable [SSF]) on MMP-2 and MMP-3, and the activity and secretion of their inhibitors TIMP-1 and TIMP-2 were investigated using cultured human gingival fibroblasts. METHODS: Gingival fibroblasts were cultured for 72 hours with ASU, ASF, and SSF at concentrations of 0. 1, 0.5, 2.5, 5, and 10 microgram/ml of culture medium, after pretreatment or no pretreatment for 1 hour with interleukin-1beta (IL-1beta). MMP-2 and MMP-3 were detected and quantified in the culture media after zymography and image analysis. TIMP-1, TIMP-2, MMP-2, and MMP-3 were also evidenced by dot blotting and quantified by image analysis. RESULTS: In the absence of IL-1beta, a slight decrease in the secretion of MMP-2 was observed with lower doses of ASU, ASF, and SSF. The decrease of MMP-3 secretion was clearly marked with all fractions especially at low concentrations (0.1 and 2.5 microgram/ml). A slight decrease in TIMP-2 secretion was seen for low doses of ASU, ASF, and SSF, while a small increase was seen at higher concentrations. Concerning TIMP-1, no significant variation was observed in culture medium for low concentrations, and a decrease was noted for 5 and 10 microgram/ml of ASU, ASF, and SSF. As anticipated, IL-1beta induced a marked release of MMP-2, MMP-3, and TIMP-1, but no variation for TIMP-2 was seen. ASU, ASF, and SSF reversed the IL-1beta effect on gingival fibroblasts for MMP-2 and MMP-3, particularly with doses varying from 0.1 to 2.5 microgram/ml and for TIMP-1, particularly with doses varying from 2.5 to 10 microgram/ml. CONCLUSIONS: These findings suggest a potential role for avocado and soy unsaponifiable extracts to prevent the deleterious effects of IL-1beta that occur during periodontal diseases.     

MMP-7 and TIMP-1, New Targets in Predicting Poor Wound Healing in Apical Periodontitis

Introduction

Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) are strongly associated with tissue destruction because of inflammation. In this study, we investigated the expression of MMPs and TIMPs messenger RNA and protein levels in apical periodontitis lesions.

Methods

Tissue samples from patients presenting clinical signs of chronic apical abscess (CAA) or asymptomatic apical periodontitis (AAP) were collected postoperatively and used for gene expression analysis of MMP-2, -3, -7, -9, -14, -16, and -25; TIMP-1; and TIMP-2 in real-time polymerase chain reaction. Immunohistochemistry was also performed to detect the expression of MMP-7 and TIMP-1 proteins. Lastly, U-937 cells were induced to terminal differentiation into macrophages, infected with purified Escherichia coli lipopolysaccharide, and assessed for the expression of MMP-7 and TIMP-1 using immunocytochemistry and confocal microscopy.

Results

Significantly higher messenger RNA levels were found for all genes in AAP and CAA samples when compared with healthy control samples (P < .001). AAP cases exhibited significantly higher TIMP-1 when compared with CAA cases, whereas CAA cases showed higher MMP-2, MMP-7, and MMP-9 messenger RNA levels (P < .05). We also detected positive the expression of MMP-7 and TIMP-1 proteins in the tissue samples. The expression of both MMP-7 and TIMP-1 were increased in lipopolysaccharide-stimulated cells compared with nonstimulated cells and appear to colocalize in the Golgi apparatus.

Conclusions

MMPs appear to have an influential role in CAA cases in which ongoing tissue destruction is observed. TIMPs are preferentially associated with AAP, perhaps as a subsequent defense mechanism against excessive destruction. Taken together, our findings implicate MMP and TIMP molecules in the dynamics of inflammatory periapical lesion development.     

Localization of MMP-2, MMP-9, TIMP-1, and TIMP-2 in human coronal dentine

Objectives

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) play important roles in dentine formation, caries progression and hybrid layer degradation. This study tested the hypothesis that the distribution and concentrations of MMP-2, MMP-9, TIMP-1 and TIMP-2 are different at different depths of human coronal dentine, including odontoblasts.

Methods

Protein localization was performed using immunohistochemistry. Co-localization of the MMPs and their inhibitors was conducted using immunofluorescence double labelling. Protein concentrations were measured by ELISA and gelatinolytic potential was assessed with gelatine zymography.

Results

MMP-2 was the main gelatinase in dentine and was concentrated in the odontoblasts, deep dentine and the dentinoenamel junction. TIMP-2 was co-localized with MMP-2 mainly in the odontoblasts but its concentration was low. Both MMP-9 and TIMP-1 showed a decreasing distribution from the deep to the superficial dentine layers; however, the concentration of TIMP-1 was much higher than that of MMP-9. The gelatinolytic potential of dentine protein extracts decreased gradually from deep to superficial dentine.

Conclusions

The concentrations and distribution patterns of MMP-2, MMP-9, TIMP-1 and TIMP-2, and the gelatinolytic potential of dentine matrix are variable along different dentine depths. Thus, differential collagen degradation potentials may be expected depending upon the depth in which dentine is exposed.