Objective SirT1 has been previously implicated in the regulation of human cartilage homeostasis and chondrocyte survival. Exposing human osteoarthritic chondrocytes to TNFα generates a stable and enzymatically inactive 75kDa form of SirT1 (75SirT1) via Cathepsin B-mediated cleavage. Because 75SirT1 is resistant to further degradation, we assumed it has a distinct role in osteoarthritis (OA) pathology, which we sought out to identify in this study. Methods OA and normal human chondrocytes were analyzed for the presence of Cathepsin B and 75SirT1. Confocal imaging of SirT1 monitored its subcellular trafficking following TNFα stimulation. Co-immunofluorescent staining was carried out for Cathepsin B, mitochondrial Cox IV and Lysosome-associated membrane protein I (LAMP-I) together with SirT1. Human chondrocyte were tested for apoptosis via FACS analysis and immunoblotting for caspase 3 and 8. Human chondrocyte mitochondrial extracts were obtained and analyzed for 75SirT1/Cytochrome C association. Results Confocal imaging and immunoblot analyses following TNFα challenge of human chondrocytes, demonstrated that 75SirT1 was exported to the cytoplasm and colocalized with the mitochondrial membrane. Consistently, immunoprecipitation and immunoblot analyses revealed that 75SirT1 is enriched in mitochondrial extracts and associates with Cytochrome C, following TNFα stimulation. Preventing nuclear export of 75SirT1 or reducing levels of FLSirT1 and 75SirT1 augmented chondrocyte apoptosis in the presence of TNFα Cathepsin B and 75SirT1 were elevated in OA vs. normal chondrocytes. Additional analyses shows that human chondrocytes exposed to OA-derived synovial fluid generate the 75SirT1 fragment. Conclusion These data suggest that 75SirT1 promotes chondrocyte survival following exposure to proinflammatory cytokines.

The amelogenin protein is considered as the major molecular marker of developing ectodermal enamel. Recent data suggest other roles for amelogenin beyond structural regulation of enamel mineral crystal growth. Here we describe our novel discovery of amelogenin expression in long bone cells, in cartilage cells, in cells of the epiphyseal growth plate, and in bone marrow stromal cells. Anat Rec, 290:455-460, 2007. 2007 Wiley-Liss, Inc.

Type II collagen is a key cartilaginous extracellular protein required for normal endochondral development and cartilage homeostasis. COL2A1 gene expression is positively regulated by the NAD-dependent protein deacetylase Sirtuin 1 (SirT1), through its ability to bind chromatin regions of the COL2A1 promoter and enhancer. Although SirT1/Sox9 binding on the enhancer site of COL2A1 was previously demonstrated, little is known about its functional role on the gene promoter site. Here, we examined the mechanism by which promoter-associated SirT1 governs COL2A1 expression. Human chondrocytes were encapsulated in three-dimensional (3D) alginate beads where they exhibited upregulated COL2A1 mRNA expression and increased levels of SirT1 occupancy on the promoter and enhancer regions, when compared to monolayer controls. Chromatin immunoprecipitation (ChIP) analyses of 3D cultures showed augmented levels of the DNA-binding transcription factor SP1, and the histone methyltransferase Set7/9, on the COL2A1 promoter site. ChIP reChIP assays revealed that SirT1 and Set7/9 form a protein complex on the COL2A1 promoter region of 3D-cultured chondrocytes, which also demonstrated elevated trimethylated lysine 4 on histone 3 (3MeH3K4), a hallmark of Set7/9 methyltransferase activity. Advanced passaging of chondrocytes yielded a decrease in 3MeH3K4 and Set7/9 levels on the COL2A1 promoter and reduced COL2A1 expression, suggesting that the SirT1/Set7/9 complex is preferentially formed on the COL2A1 promoter and required for gene activation. Interestingly, despite SirT1 occupancy, its deacetylation targets (ie, H3K9/14 and H4K16) were found acetylated on the COL2A1 promoter of 3D-cultured chondrocytes. A possible explanation for this phenotype is the enrichment of the histone acetyltransferases P300 and GCN5 on the COL2A1 promoter of3 D-cultured chondrocytes. Our study indicates that Set7/9 prevents the histone deacetylase activity of SirT1, potentiating euchromatin formation on the promoter site of COL2A1 and resulting in morphology-dependent COL2A1 gene transactivation.

Tuftelin has been suggested to play an important role during the development and mineralization of enamel, but its precise function is still unclear. This article reviews major milestones in the discovery, structural characterization, expression, localization, and conservation of tuftelin in different vertebrate species. It focuses on the structure of the human tuftelin gene, which has recently been deciphered [12]. It describes the exon-intron organization, sizes and structure, the promoter structure, and the newly discovered alternatively spliced human tooth-bud tuftelin mRNA transcripts. It also examines information on the structural motifs in the human-derived tuftelin protein and how they relate to tuftelin from other species. It reviews our recent results on the transcription of tuftelin mRNA and protein expression in several nonmineralizing soft tissues, using reverse-transcription polymerase chain reaction (RT-PCR) followed by DNA cloning and sequencing, indirect immunohistochemistry, immunohistochemistry combined with confocal microscopy, and in situ hybridization. These results and earlier Northern blot results show that tuftelin, in addition to being expressed in the developing and mineralizing tooth, is also expressed in several nonmineralizing soft tissues, suggesting that tuftelin has a universal function and/or a multifunctional role.

ObjectivePrevious work has established that the deacetylase sirtuin-1 (SIRT1) is cleaved by cathepsin B in chondrocytes subjected to proinflammatory stress, yielding a stable but inactive N-terminal (NT) polypeptide (75SIRT1) and a C-terminal (CT) fragment. The present work examined if chondrocyte-derived NT-SIRT1 is detected in serum and may serve as an investigative and exploratory biomarker of osteoarthritis (OA).MethodsWe developed a novel ELISA assay to measure the ratio of NT to CT of SIRT1 in the serum of human individuals and mice subjected to post-traumatic OA (PTOA) or age-dependent OA (ADOA). We additionally monitored NT/CT SIRT1 in mice subject to ADOA/PTOA followed by senolytic clearance. Human chondrosenescent and non-senescent chondrocytes were exposed to cytokines and analysed for apoptosis and NT/CT SIRT1 ratio in conditioned medium.ResultsWild-type mice with PTOA or ADOA of moderate severity exhibited increased serum NT/CT SIRT1 ratio. In contrast, this ratio remained low in cartilage-specific Sirt1 knockout mice despite similar or increased PTOA and ADOA severity. Local clearance of senescent chondrocytes from old mice with post-traumatic injury resulted in a lower NT/CT ratio and reduced OA severity. While primary chondrocytes exhibited NT/CT ratio increased in conditioned media after prolonged cytokine stimulation, this increase was not evident in cytokine-stimulated chondrosenescent cells. Finally, serum NT/CT ratio was elevated in humans with early-stage OA.ConclusionsIncreased levels of serum NT/CT SIRT1 ratio correlated with moderate OA in both mice and humans, stemming at least in part from non-senescent chondrocyte apoptosis, possibly a result of prolonged inflammatory insult.