Perlecan-enriched intercellular space of junctional epithelium provides primary infrastructure for leukocyte migration through squamous epithelial cells.

The aim of this study was to demonstrate the presence of intraepithelial stroma represented by extracellular matrix (ECM) deposits in the junctional epithelium to clarify its function as a scaffold for leukocyte migration through epithelial cells. Twenty-three biopsy specimens from the gingiva including the junctional epithelium were examined to determine comparative protein and gene level expression profiles for keratin and ECM molecules between the junctional epithelium and the gingival epithelium using immunohistochemistry and in situ hybridization. Intraepithelial leukocyte types and frequencies were also determined and compared between the junctional and gingival epithelia. In the junctional epithelium, which was positive for keratin 19, perlecan was strongly deposited in intercellular space of the whole epithelial layer, while it was faintly positive around the parabasal layer of the gingival epithelium. Perlecan mRNA signals were enhanced to a greater degree in both epithelial and inflammatory cells within the junctional epithelium. In the junctional epithelium, greater numbers of neutrophils and macrophages were found as compared with the gingival epithelium. Our results showed that perlecan is the primary ECM molecule comprising intraepithelial stroma of the junctional epithelium, in which leukocytes may migrate on ECM scaffolds in intercellular space toward the surface of the gingival sulci or pockets.

Differential gene and protein expression of tissue inhibitors of metalloproteinases (TIMP)-3 and TIMP-4 in gingival tissues from drug induced gingival overgrowth.

OBJECTIVES: The purpose of this study was to analyse mRNA expression and protein localization of tissue inhibitors of metalloproteinases (TIMP)-3 and TIMP-4 in gingival tissues removed from drug (calcium-channel blocker) induced gingival overgrowth and periodontitis patients. DESIGN: Employing RT-PCR, we evaluated TIMP-3 and TIMP-4 mRNA levels of 20 human gingival tissue samples taken from patients suffering gingival overgrowth (GO) and periodontitis (P). Then, using immunohistochemistry we investigated the TIMP-3 and TIMP-4 protein localization of five sample tissues from each group. RESULTS: TIMP-4 mRNA levels in GO-gingiva tended to be lower than in P-gingiva but the results differed little (p = 0.22). Varying degrees of inflammation in the protein localization of TIMP-3 and TIMP-4 were found. TIMP-4 immunoreactivity (IR) was weak in the endothelial cells, fibroblasts, epithelial basal and parabasal cells while the degree of inflammation differed as well. TIMP-3 and TIMP-4 IR in inflammatory cells, including lymphocytes, plasma cells, and macrophages, were faint and intense respectively. For P-gingiva, both TIMP-3 and TIMP-4 IR expression was weak in the endothelial cells, fibroblasts, basal and parabasal epithelial layers. Expression of TIMP-3 was faint in the inflammatory cells, whereas TIMP-4 IR was strong. CONCLUSION: Our findings suggest that TIMP-3 and TIMP-4 expression differs in GO and P-gingival tissues, both of which are potentially involved in pathogenesis.

Altered gene expression levels of matrix metalloproteinases and their inhibitors in periodontitis-affected gingival tissue.

BACKGROUND: The balance between the degradation and synthesis of extracellular matrix determines periodontal attachment levels and alveolar bone matrix concentration in periodontal diseases. Matrix metalloproteinases (MMPs) are known to degrade periodontal ligamental attachment and bone matrix proteins. The purpose of this study was to examine the effect of different expression levels of MMPs and their inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), in periodontitis. METHODS: Sixteen inflamed gingival tissue samples from subjects with generalized chronic periodontitis and 14 control tissue samples from systemically and periodontally healthy subjects were evaluated. The total RNA was extracted, and the transcript levels for MMP-1, -3, -9, and -13 and TIMP-1, -2, -3, and -4 relative to beta-actin were determined by quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Gene transcript levels for MMP-1 and TIMP-4 were significantly higher in periodontitis-affected gingival tissues (P <0.05). MMP-3, -9, and -13 and TIMP-1 mRNAs also were elevated in periodontitis; however, the difference was not statistically significant. TIMP-2 and -3 mRNA levels were similar in healthy and diseased gingivae. The ratios of MMP-1/TIMP-2 (P <0.01), MMP-3/TIMP-2 (P <0.05), MMP-9/TIMP-2 (P <0.05), and MMP-1/TIMP-3 (P <0.01) from periodontitis lesions were significantly higher than those in the control tissues. CONCLUSIONS: Upregulated MMP expression and an increased MMP/TIMP ratio indicate that a potential imbalance between degradation and synthesis of extracellular matrix persists in periodontitis-affected gingival tissues. This process may be responsible for increased tissue breakdown in periodontitis.

The 2G allele of promoter region of matrix metalloproteinase-1 as an essential pre-condition for the early onset of oral squamous cell carcinoma.

BACKGROUND: Matrix metalloproteinase (MMP) is known to be involved in the initial and progressive stages of cancer development, and in the aggressive phenotypes of cancer. This study examines the association of single nucleotide polymorphisms in promoter regions of MMP-1 and MMP-3 with susceptibility to oral squamous cell carcinoma (OSCC). METHODS: We compared 170 Japanese OSCC cases and 164 healthy controls for genotypes of MMP-1 and MMP-3. RESULTS: The frequency of the MMP-1 2G allele was higher and that of the 1G homozygote was lower in the OSCC cases (p = 0.034). A multivariate logistic regression analysis revealed that subjects who were 45 years old or older had a significantly increased (2.47-fold) risk of OSCC (95%CI 1.47-4.14, p = 0.0006), and those carrying the MMP-1 2G allele had a 2.30-fold risk (95%CI 1.15-4.58, p = 0.018), indicating independent involvement of these factors in OSCC. One of the key discoveries of this research is the apparent reduction of the MMP-1 1G/1G and 1G/2G genotype distributions among the early onset OSCC cases under the ages of 45 years. It should be noted that the tongue was the primary site in 86.2% of these early onset cases. This could suggest the specific carcinogenic mechanisms, i.e. specific carcinogenic stimulations and/or genetic factors in the tongue. CONCLUSION: Since the 2G allele is a majority of the MMP-1 genotype in the general population, it seems to act as a genetic pre-condition in OSCC development. However this report suggests a crucial impact of the MMP-1 2G allele in the early onset OSCC.

Alterations of gene expression in human neutrophils induced by smoking cessation.

OBJECTIVE: The purpose of the present study was to investigate the effect of smoking cessation on the peripheral neutrophil mRNA expression levels for inflammatory cytokines, chemokine, growth factor and matrix metalloproteinase (MMP). MATERIAL AND METHODS: Sixteen male smokers (aged 22-39 [25.3+/-4.0] years), with no clinical signs of periodontal and systemic diseases, were recruited. The experiment was performed before (baseline) and at 1, 4 and 8 weeks after smoking cessation. The status of smoking and smoking cessation was verified by exhaled carbon monoxide (CO) concentration and serum cotinine concentration. Neutrophils were isolated from each subjects' peripheral blood, then the cell was stimulated with N-formyl-methionyl-leucyl-phenylalanine (FMLP). The mRNA expression levels for interleukin (IL)-1 beta, IL-8, tumor necrosis factor (TNF)-alpha, vascular endothelial growth factor (VEGF) and MMP-8 were analyzed by semiquantitative reverse transcription-polymerase chain reactions. The same experiment was performed on 11 non-smoking controls (four female and seven male), aged 23-27 (24.4+/-1.2) years. RESULTS: Eleven of 16 smokers successfully completed smoking cessation for 8 weeks. At 1 day after smoking cessation, there was a statistically significantly lower CO concentration than at baseline (p<0.01). Also, cotinine concentration markedly decreased at the second measurement, which was taken at 1 week. All of the analyzed mRNA expression levels of neutrophils from smokers were statistically significantly lower than that in non-smokers (p<0.01: IL-1 beta, IL-8, VEGF; p<0.05: TNF-alpha, MMP-8). The MMP-8 mRNA levels were statistically significantly increased at 8 weeks after smoking cessation compared with the baseline (p<0.05). Although the other mRNA expression levels were also elevated gradually from the baseline, they did not reach the statistically significant levels at 8 weeks after smoking cessation. CONCLUSION: The results showed that the neutrophil transcript levels in smokers were generally lower than those in non-smokers, which could be related to an impairment of neutrophils by smoking effects. The significant increase of MMP-8 mRNA levels were associated with the effects of smoking cessation, while recovery of the other mRNA levels seemed to require a bit longer period beyond 8 weeks after smoking cessation.

Matrix metalloproteinase-1 and -3 gene promoter polymorphisms in Japanese patients with periodontitis.

BACKGROUND/AIMS: Matrix metalloproteinase (MMP)-1 and MMP-3 have important roles in the connective tissue remodelling and destruction processes in periodontitis. MMP-1 1G/2G (-1607) and MMP-3 5A/6A (-1171) polymorphisms have been identified and appear to influence the transcription of the genes. The aim of this study was to investigate whether these gene promoter polymorphisms were associated with the susceptibility to periodontitis. MATERIAL AND METHODS: Genomic DNA was obtained from 37 generalised aggressive, 205 slight-to-severe generalised chronic-periodontitis patients and 142 healthy subjects. All subjects were non-smoking Japanese. We genotyped by using TaqMan PCR assay. The statistics were analysed by chi2-test. RESULTS: We found no significant differences in genotype distributions, allele frequencies, carriage rates and haplotype frequencies in the MMP-1 and the MMP-3 gene promoter polymorphisms among all groups. The distributions of MMP-1 and MMP-3 genotypes in our study were different from those of previously reported in Caucasians or Brazilians, but consistent with previously reported in Japanese. CONCLUSION: Our data did not support the hypothesis that MMP-1 and/or MMP-3 gene promoter polymorphisms influenced the susceptibility to periodontitis in Japanese patients, indicating MMP-1 and MMP-3 expressions were regulated by complex processes such as cytokine network in periodontal disease rather than gene polymorphisms.