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

Mona Dvir-Ginzberg,Viktoria Gagarina,Eun-Jin Lee,David J Hall

doi:10.1074/jbc.m803196200

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(alpha1) promoter in a Sox9-dependent fashion. Consistent with this association, SirT1 was targeted to the collagen 2(alpha1) enhancer and promoter, which in turn recruited the coactivators GCN5, PGC1alpha, 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.

Highlights

Transcriptional control over cartilage-specific gene expression plays a critical role in maintenance of the chondrocyte phenotype [1]
Given that there are more than 18 histone deacetylases (HDACs) genes distributed in at least four different classes, it is likely that multiple HDACs will affect the growth, differentiation, and survival of chondrocytes via transcriptional regulation of cartilage-specific genes
Because nothing is known of the role SirT1, nicotinamide phosphoribosyltransferase (NAMPT), and NAD play in chondrocyte biology, we have explored their functions in chondrocytes derived from knee joints of osteoarthritic patients

Summary

EXPERIMENTAL PROCEDURES

Cell Culture, and Transfections—Nicotinamide and resveratrol were purchased from Sigma. For an additional control we tested each set of experiments for expression of nonlineage specific genes (collagen 1 (␣1), fibronectin, NF␬B (p65); supplemental Figs. The SirT activity and luciferase assays were carried out in triplicate using 2 distinct cell lines generated from different transfections or cell sources (n ϭ 6). 1 and 2 are illustrated in the supplemental data for the equivalent experiments (n ϭ 6, triplicates of two cell lines/treatment). When the same analyses were performed for SirT1-M-expressing cells, it was clear that cartilage gene expression was significantly repressed as compared with the control cells (LSD, p Ͻ 0.05; Fig. 1F). QPCR was carried out on collagen 2a(␣1), collagen 2b(␣1), and aggrecan in the stably transfected cell lines To confirm that SirT1 had a positive effect on cartilage gene expression, transient expression experi-

RESULTS

DISCUSSION