Regulation of Cartilage-specific Gene Expression in Human Chondrocytes by SirT1 and Nicotinamide Phosphoribosyltransferase

Abstract

SirT1 is an NAD-dependent histone deacetylase that regulates gene expression, differentiation, development, and organism life span. Here we investigate the function of SirT1 in human chondrocytes derived from osteoarthritic patients. Elevation of SirT1 protein levels or activity in these chondrocytes led to a dramatic increase in cartilage-specific gene expression, whereas a reduction in SirT1 levels or activity significantly lowered cartilage gene expression. SirT1 associated with the cartilage-specific transcription factor Sox9, enhancing transcription from the collagen 2(␣1) promoter in a Sox9-dependent fashion. Consistent with this association, SirT1 was targeted to the collagen 2(␣1) enhancer and promoter, which in turn recruited the coactivators GCN5, PGC1␣, and p300. This led to elevated marks of active chromatin within the promoter; that is, acetylated histone K9/K14 and histone H4K5 as well as trimethylated histone H3K4. Finally, alterations in the NAD salvage pathway enzyme nicotinamide phosphoribosyltransferase led to changes in NAD levels, SirT activity, and cartilage-specific gene expression in human chondrocytes. SirT1, nicotinamide phosphoribosyltransferase, and NAD may, therefore, provide a positive function in human cartilage by elevating expression of genes encoding cartilage extracellular matrix.Transcriptional control over cartilage-specific gene expression plays a critical role in maintenance of the chondrocyte phenotype (1). Much effort has, therefore, gone into the characterization of chondrocyte-specific transcription factors such as Sox9, -5, and -6 (28). However, it is likely that other factors such as chromatin-modifying enzymes play important roles in controlling cartilage-specific gene expression. Chromatinmodifying enzymes, which include the histone acetyltransferases (HATs) 2 and the histone deacetylases (HDACs) can act as potent transcriptional coactivators and corepressors, respectively, for a variety of genes (2). HATs modify the core histones through acetylation of lysine residues, thereby relaxing chromatin for transcription initiation and elongation (3). HDACs remove the acetyl groups, leading to chromatin condensation and transcriptional repression (4). Additionally, acetylation and deacetylation of transcription factors provide another level of regulation over gene expression (2). In some contexts HDACs are more sensitive to environmental or developmental cues than the HATs and can provide a regulatory role in transcription. In this regard, HDACs have been demonstrated to control both cell proliferation and differentiation through the deacetylation of transcription factors, cytoplasmic proteins, and histones (5, 6). Although HDACs are critically involved in diverse biological processes, their function in chondrocyte biology and cartilage diseases have only recently been explored. Recent work indicates that inhibition of HDACs reduces the expression of matrix metalloproteinase in chondrocytes and fibroblasts (7) and, therefore, inhibits arthritis progression in animal models...

Acetylation reduces SOX 9 nuclear entry and ACAN gene transactivation in human chondrocytes

“…(2008). Cells were plated in 14‐cm 2 tissue culture dishes at a concentration of 1.5 × 10 6 cells per dish and were grown to confluence (passage 0 or 1) in DMEM (Sigma‐Aldrich, St Louis, MI, USA) containing 10% FCS and 1% penicillin–streptomycin (Beit‐Haemek Kibbutz, Israel).…”

“…Previous studies uncovered a key role for the nicotinamide adenine dinucleotide (NAD)‐dependent class III protein deacetylase SIRTUIN 1 (SIRT1) in cartilage homeostasis and gene expression (Dvir‐Ginzberg et al ., 2008; Oppenheimer et al ., 2014) While SIRT1 was shown to induce transcriptional repression in many cell types (Carafa et al ., 2012), the unexpected, contrasting discovery was made that it strikingly potentiated SOX9 gene targets (i.e. ACAN and COL2A1 ) in human chondrocytes (Dvir‐Ginzberg et al ., 2008; Dvir‐Ginzberg & Steinmeyer, 2013; Oppenheimer et al ., 2014).…”

“…ACAN and COL2A1 ) in human chondrocytes (Dvir‐Ginzberg et al ., 2008; Dvir‐Ginzberg & Steinmeyer, 2013; Oppenheimer et al ., 2014). In vitro studies showed that SIRT1 bound to and deacetylated SOX9, but did not affect SOX9 binding to the COL2A1 enhancer, as observed by chromatin immunoprecipitation assays of chondrocytes overexpressing wild‐type SIRT1 and a dominant negative H355Y SIRT1 mutant (Dvir‐Ginzberg et al ., 2008). Further studies suggested that most of SIRT1 action on COL2A1 expression occurred through recruitment of the histone methyl‐transferase SET7/9 and other histone acetyl transferases (i.e.…”

“…Further studies suggested that most of SIRT1 action on COL2A1 expression occurred through recruitment of the histone methyl‐transferase SET7/9 and other histone acetyl transferases (i.e. P300 and GCN5) to the promoter and enhancer sites of the gene (Dvir‐Ginzberg et al ., 2008; Oppenheimer et al ., 2014). On the other hand, the mechanisms whereby SIRT1 may regulate ACAN expression are yet unknown.…”

“…This study also revealed binding of SIRT1 to SOX9 in mesenchymal stem cells undergoing chondrogenic differentiation as well as in primary chondrocytes (Buhrmann et al ., 2014), a result consistent with our previous observations (Dvir‐Ginzberg et al ., 2008) . In vivo studies showed that loss of SIRT1 enzymatic function resulted in reduced levels of collagen type II and aggrecan in articular cartilage (Gabay et al ., 2013).…”