Age-Related Gene and Protein Expression in Mouse Mandibular Condyle Analyzed by Cap Analysis of Gene Expression and Immunohistochemistry.

INTRODUCTION: Aging, an inevitable physiological process, leads to morphological and histological degenerative changes in the mandibular condylar cartilage (MCC); however, the molecular mechanism has not yet been elucidated, and little information is available on age-related factors. Therefore, this study was designed to identify age-related factors by investigating the age-related differentially expressed genes (DEGs) and localization of their translated protein expression in the mandibular condyle. METHODS: Mandibular condyles were collected from 10- and 50-week-old mice. Total RNA was extracted from the samples and then analyzed using cap analysis of gene expression (CAGE) to identify age-related DEGs. Gene ontology (GO) enrichment analysis was performed to determine which biological processes were most affected by aging in terms of gene expression using Metascape. The mandibular condyle samples were processed for histology to investigate morphological changes caused by aging and for immunohistochemistry to localize the protein expression encoded by age-related genes identified with CAGE. Semi-quantitative immunohistochemistry was performed to assess age-related extracellular matrix (ECM) protein levels in the MCC. The histological sections were also used for Alcian blue histochemistry to detect glycosaminoglycans (GAGs). RESULTS: GO enrichment analysis revealed that the genes related to "extracellular matrix organization," including Acan, Col1a1, Col1a2, Col2a1, Mmp3, Mmp9, and Mmp13, were most differentially expressed in the aged mandibular condyle. Among these seven genes, Mmp3 was upregulated, and the others were downregulated with aging. Histological examination showed the age-related morphological and histological changes in the MCC. Immunohistochemical investigation showed the localization of matrix metalloproteinases (MMPs)-3, -9, and -13 and their substrate proteins, aggrecan, type I collagen, and type II collagen, in the mandibular condyle at 10 and 50 weeks, indicating different localizations between the young and the aged. In the aged MCC, semi-quantitative immunohistochemistry showed a significant decrease in the aggrecan protein level, and Alcian blue histochemistry showed a decrease in GAGs. CONCLUSION: MMP-3, MMP-9, and MMP-13 contribute to the remodeling of the ECM of the MCC and subchondral bone during aging by degrading ECM proteins at specific times and sites under the regulation of their production and secretion.

Expression of matrix metalloproteinase-3 and -10 is up-regulated in the periodontal tissues of aged mice.

OBJECTIVE: The present study was designed to investigate the whole transcriptome of periodontal tissues of both young and aged mice to identify the characteristic up-regulation of protease genes with aging and to localize their translated protein products in the periodontal tissues. BACKGROUND: The metzincin protease superfamily is composed of matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases, and a disintegrin and metalloproteinases with thrombospondin motifs. Up-regulation of these extracellular matrix-degrading proteases has been implicated in senescence of tissues and organs, including the skin. However, few studies have investigated the expression profiles of these proteases and potential involvement in aging of periodontal tissues. METHODS: Periodontal tissues with the surrounding mandibular bones were collected from 50- and 10-week-old mice. Total RNA was extracted from the periodontal tissue and analyzed by cap analysis of gene expression (CAGE) to identify differentially expressed genes encoding the metzincin proteases. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate the CAGE results, and the phenotypic expression of proteases involved in aging was localized via immunohistochemical analysis. RESULTS: The CAGE results showed that the expression levels of MMP-3, -10, and -12 were up-regulated at 50 weeks. Subsequent qRT-PCR analysis showed that the gene expression levels of MMP-3 and -10 were significantly increased with age. MMP-10 immunoreactivity was localized exclusively in the cementum and alveolar bone adjacent to the periodontal ligament and was stronger and broader in aged mice than young mice. MMP-3 immunoreactivity was localized in the periodontal ligaments at both 10 and 50 weeks. CONCLUSION: In the present study, we demonstrated that the expression of MMP-3 and -10 increased with aging and identified their characteristic localizations in aged periodontal tissues.