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

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(α1) promoter in a Sox9-dependent fashion. Consistent with this association, SirT1 was targeted to the collagen 2(α1) enhancer and promoter, which in turn recruited the coactivators GCN5, PGC1α, 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. 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(α1) promoter in a Sox9-dependent fashion. Consistent with this association, SirT1 was targeted to the collagen 2(α1) enhancer and promoter, which in turn recruited the coactivators GCN5, PGC1α, 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 (1Lefebvre V. Smits P. Birth Defects Res. C Embryo Today. 2005; 75: 200-212Crossref PubMed Scopus (390) Google Scholar). Much effort has, therefore, gone into the characterization of chondrocyte-specific transcription factors such as Sox9, -5, and -6 (28Lefebvre V. Li P. de Crombrugghe B. EMBO J. 1998; 17: 5718-5733Crossref PubMed Scopus (682) Google Scholar). However, it is likely that other factors such as chromatin-modifying enzymes play important roles in controlling cartilage-specific gene expression. Chromatin-modifying enzymes, which include the histone acetyltransferases (HATs) 2The abbreviations used are: HAThistone acetyltransferaseHDAChistone deacetylaseNAMnicotinamideqPCRquantitative PCRNAMPTnicotinamide phosphoribosyltransferasesiRNAsmall interfering RNAChIPchromatin immunoprecipitationRTreverse transcriptionGAPDHglyceraldehyde-3-phosphate dehydrogenaseLSDleast significant differenceOAosteoarthriticIPimmunoprecipitate and the histone deacetylases (HDACs) can act as potent transcriptional coactivators and corepressors, respectively, for a variety of genes (2Roth S.Y. Denu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1660) Google Scholar). HATs modify the core histones through acetylation of lysine residues, thereby relaxing chromatin for transcription initiation and elongation (3Hasan S. Hottiger M.O. J. Mol. Med. 2002; 80: 463-474Crossref PubMed Scopus (67) Google Scholar). HDACs remove the acetyl groups, leading to chromatin condensation and transcriptional repression (4Ruijter A. Gennip A. Caron H.N. Kemp S. Kuilenburg A. Biochem. J. 2003; 370: 737-749Crossref PubMed Scopus (2543) Google Scholar). Additionally, acetylation and deacetylation of transcription factors provide another level of regulation over gene expression (2Roth S.Y. Denu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1660) Google Scholar). histone acetyltransferase histone deacetylase nicotinamide quantitative PCR nicotinamide phosphoribosyltransferase small interfering RNA chromatin immunoprecipitation reverse transcription glyceraldehyde-3-phosphate dehydrogenase least significant difference osteoarthritic immunoprecipitate 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 (5Johnstone R.W. Licht J.D. Cancer Cell. 2003; 4: 13-18Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 6McKinsey T.A. Zhang C.L. Olson E.N. Curr. Opin. Cell Biol. 2002; 14: 763-772Crossref PubMed Scopus (162) Google Scholar). 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 (7Young D.A. Lakey R.L. Pennington C.J. Jones D. Kevorkian L. Edwards D.R. Cawston T.E. Clark I.M. Arthritis Res. Ther. 2005; 7: 503-512Crossref PubMed Google Scholar) and, therefore, inhibits arthritis progression in animal models (8Chung Y.L. Lee M.Y. Wang A.J. Yao L.F. Mol. Ther. 2003; 8: 707-717Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 9Nishida K. Komiyama T. Miyazawa S. Shen Z.N. Furumatsu T. Doi H. Yoshida A. Yamana J. Yamamura M. Ninomiya Y. Inoue H. Asahara H. Arthritis Rheum. 2004; 50: 3365-3376Crossref PubMed Scopus (192) Google Scholar). Additionally, it has been demonstrated that HDAC4, a Class II enzyme, plays a critical role in the onset of chondrocyte hypertrophy during endochondral ossification (10Vega R.B. Matsuda K. Oh J. Barbosa A.C. Yang X. Meadows E. McAnally J. Pomajzi C. Shelton J.M. Richardson J.A. Karsenty G. Olson E.N. Cell. 2004; 119: 555-566Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). It would appear then that HDACs regulate genes involved in both inflammation and chondrocyte differentiation. Given that there are more than 18 HDAC 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. Of particular interest with regard to cartilage biology are the NAD-dependent Class III HDACs comprised of SirT1–7. Within this group SirT1 has been extensively studied and is known to play a crucial part in regulating cell differentiation, proliferation, survival, and organism longevity (11Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1333) Google Scholar, 12Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. Annu. Rev. Biochem. 2006; 75: 435-465Crossref PubMed Scopus (606) Google Scholar). Class III HDACs nicotinamide and from NAD in the of thereby a potent of SirT1 by to a within the enzyme (11Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1333) Google Scholar, 12Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. Annu. Rev. Biochem. 2006; 75: 435-465Crossref PubMed Scopus (606) Google Scholar). NAD can from via dehydrogenase as well as by the NAD salvage pathway plays a role in de of NAD K. A. S. 2002; PubMed Scopus Google Scholar). this pathway NAD through the of salvage pathway enzymes, nicotinamide phosphoribosyltransferase and nicotinamide A. 2003; PubMed Scopus Google Scholar, H. S. D.A. 2006; PubMed Scopus Google Scholar). is known to have a affect NAD A. A. Wang T. A. B. Y. Wolberger C. J. S. Cell Full Text Full Text PDF PubMed Scopus Google of expression is important to the salvage pathway enzymes have been known to life likely through their SirT1 activity H. S. D.A. 2006; PubMed Scopus Google Scholar, E. C. C. Barbosa J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). is known of the role SirT1, and NAD play in chondrocyte we have their in chondrocytes derived from of osteoarthritic patients. that SirT1 in the of cartilage-specific gene expression and is the Cell and and from was from the and for SirT1 by and used to the was from and used to the was to transcription initiation of the collagen 2(α1) promoter This a from M. the collagen 2(α1) promoter and enhancer to a L. H. M. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). chondrocytes with in the of and in with the chondrocytes from the of osteoarthritic by the was to A. Full Text Full Text PDF PubMed Scopus Google Scholar). in at a of and to or In cartilage gene expression was at which it chondrocytes to of human chondrocytes as by A. Full Text Full Text PDF PubMed Scopus Google Scholar). chondrocyte the in with and of Cell expression and of M. R.L. S. P. Y. E. R.L. V. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). SirT1 expression was from expression was a of de was and as M. R.L. S. P. Y. E. R.L. V. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). from and used in the NAD NAD levels a NAD to NAD levels to the protein SirT activity was a was used to Denu J.M. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) with was to the cell to the and cell then as with the of A. to an with and acetylated a was to some then for at and the by of a with and was a at a was a from to are as of of from to the immunoprecipitation the to the was a PCR PCR RNA used to of RNA was to a with to to and the of the PCR are in was in PCR of and as by A. Full Text Full Text PDF PubMed Scopus Google Scholar). are in an control we of for expression of genes and cell protein and to A. A. 2005; 73: PubMed Scopus Google Scholar). and used for and are in in with and in for and was for in then with a of a was of levels of to least significant difference was to the within a levels of the the of a was used to SirT1 and was used for SirT1 and and was for and in the with and is as was used to in SirT1 and and from cell or cell SirT activity and in cell from different or cell in and from of cell quantitative PCR for and are in the for the of cell changes in SirT1 levels or activity cartilage gene expression. human chondrocytes with a SirT1 expression at used in or SirT activity RNA from the in was used in with the human of gene expression with and chondrocytes with for at which RNA was and used in with the human chondrocytes with a SirT1 and and used in for SirT1 and or for of SirT activity RNA was from the the in which was used in with the human chondrocytes with for at which RNA was and used with the human in and repression of gene expression. in the the and the is by an are of four was in for Elevation of SirT1 the that cartilage-specific gene expression in chondrocytes is by SirT1, osteoarthritic human chondrocytes with a SirT1 SirT1 or an was used as a as well as SirT1 and and a for SirT1 activity, the levels of a known for SirT1 M. Guarente L. PubMed Scopus Google it that the with the and control cell and which as a in cell in indicates a significant increase of SirT activity in SirT1 with and that the SirT1 is active within these chondrocytes. SirT activity with that it in a as J. D. A. Guarente L. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, H. E.N. Guarente L. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the of SirT1 cartilage-specific gene expression. RNA was and used in for of cartilage genes collagen and collagen and cartilage matrix protein significantly expression than the control the for it was that cartilage gene expression was significantly as with the control In cell we expression of the genes collagen and and changes in RNA levels SirT1, and cell and Additionally, was collagen collagen and in the cell and collagen collagen and to by SirT1 and by that SirT1 a positive cartilage gene expression, expression and in chondrocytes. SirT1 expression was in chondrocytes. in and SirT1 protein levels and SirT activity elevated in the chondrocytes. the SirT1 is as by significantly gene expression for the genes in with the control for that collagen collagen and by SirT1 In a of the was to chondrocytes to SirT1 J. D. A. Guarente L. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). in this in the of cartilage gene expression. that collagen collagen and by these that by or of SirT1, cartilage gene expression is in human chondrocytes. an control we expression of the genes collagen and in to SirT1 and was gene expression C and of SirT1 the of the a SirT1 was into chondrocytes to SirT1 protein in SirT1 levels and activity by that SirT1 protein levels significantly lowered cartilage-specific gene expression that SirT1 collagen 2(α1) and a to the chondrocytes with which is a of SirT1 (11Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1333) Google Scholar, 12Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. Annu. Rev. Biochem. 2006; 75: 435-465Crossref PubMed Scopus (606) Google Scholar). a reduction in cartilage gene expression in that expression of collagen 2(α1) and in chondrocytes In these that SirT1 levels or activity a cartilage gene expression. of genes collagen and in SirT1 and gene expression and with the that SirT1 expression. SirT1 with and from the 2(α1) that SirT1 regulates cartilage gene expression. it is well that expression of cartilage genes is by the transcription factor (1Lefebvre V. Smits P. Birth Defects Res. C Embryo Today. 2005; 75: 200-212Crossref PubMed Scopus (390) Google it was that SirT1 with Sox9, thereby enhancing transcriptional activity, through was it was in and in human chondrocytes derived from It was then with a and then in the was with a and the other was with an in is by the it is an acetylated with Sox9, acetylation and transcription from the collagen 2(α1) and human chondrocytes with the or expression and used in with a in and the and with a or an with Sox9, SirT1, or both the and SirT1 expression and used in with a or SirT1 the with a Sox9, SirT1, or as control the levels of SirT1 and with in the with Sox9, SirT1, or an for by an for as well as an for SirT1 by an for the SirT1 and at for with NAD or with and then and with or are of four SirT1 deacetylation (11Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1333) Google Scholar) is with a for deacetylation within This is to that within histones and and an of the collagen in the and SirT1 and expression as with the collagen in and human chondrocytes of to the the and expression as with the collagen in human chondrocytes. the SirT1 and expression into human chondrocytes with the collagen in the of of as a promoter was into the as in for SirT1 and with the SirT1 expression and was from the and the for SirT1 that SirT1 with in that only was SirT1 was to with it in SirT1 was and the for in the that with these that and SirT1 are to in was from the and the for acetylated a was the SirT1 was with Sox9, the of the acetylated was the of these that the of acetylated was by SirT1 was expression and immunoprecipitation of both and SirT1 and demonstrated that and SirT1 protein levels by in that SirT1 Sox9, with or SirT1, and the in G. S. P. J. A. S. C. Guarente D. 4: Scopus Google the then and then with NAD or is only active with then and for or in the of in the of NAD there was a reduction in the of by the whereas the level of was that SirT1 is active in and is in the of to by and Guarente (11Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1333) Google Scholar) to a SirT1 deacetylation This the of and is the group are to lysine is and, it is a of the of the association, with the collagen 2(α1) to L. H. M. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google as this is a well known gene (1Lefebvre V. Smits P. Birth Defects Res. C Embryo Today. 2005; 75: 200-212Crossref PubMed Scopus (390) Google Scholar). with the SirT1 expression in the of the collagen of the collagen is in in and for and human respectively, SirT1 affect transcription from collagen 2(α1) promoter, whereas was to transcription in human chondrocytes. both SirT1 and to the promoter which was to significant in both cell a the promoter the gene was by Sox9, SirT1, or and an human chondrocytes with SirT1 expression in the or of the was to the collagen 2(α1) In a human chondrocytes with of SirT1, a reduction in the transcription of collagen 2(α1) in the of In as in in the or of SirT1, is to the of these that active SirT1 is for of the collagen 2(α1) SirT1, the SirT1 GCN5, and to the 2(α1) SirT1 and in and affect transcription from the collagen 2(α1) promoter, used to SirT1 is targeted to regulatory in the collagen 2(α1) the promoter as well as the within the of the collagen 2(α1) used in the the to in the human collagen 2(α1) which is for cartilage-specific transcription (1Lefebvre V. Smits P. Birth Defects Res. C Embryo Today. 2005; 75: 200-212Crossref PubMed Scopus (390) Google Scholar). are as S. P. PubMed Scopus Google Scholar). the and chondrocytes. a in the a PCR the enhancer was significantly elevated in the SirT1 and to the control that the SirT1 and are targeted to this in an to RNA an in the PCR for the promoter in the cell whereas the control that SirT1 was in the the and with a SirT1 SirT1 and in the the by which SirT1 collagen we such as histone enzymes in the in SirT1 and are the enhancer with the levels in the control with the of SirT1 with active SirT1 to the transcriptional coactivators and to the enhancer as SirT1 have elevated and the enhancer to the control and the level of the which to the that SirT1 and are known to S. T. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). However, that SirT1 to a increase in the enhancer with that active SirT1 is for of to this In these that active SirT1 can the of and to the of the promoter that SirT1, the is targeted to the promoter PGC1α, GCN5, and are the promoter only in the active that PGC1α, GCN5, and are recruited to the promoter by active histone by the promoter as in it was that and levels within the promoter in the with the control and would that the of PGC1α, GCN5, and enzymes to the promoter has a of histone have to the histone that to an increase in the enhancer an increase in in or in the SirT1 in that through of proteins, SirT1 acetylation and of critical histone in the of the histone appear to active SirT1 as the SirT1 affect histone of important of SirT1 function is the of NAD as a NAD is to from which at levels in human chondrocytes K. A. S. 2002; PubMed Scopus Google Scholar). to NAD is via the NAD salvage pathway A. 2003; PubMed Scopus Google Scholar, H. S. D.A. 2006; PubMed Scopus Google Scholar). NAD from by the salvage pathway enzymes and nicotinamide is to in the A. A. Wang T. A. B. Y. Wolberger C. J. S. Cell Full Text Full Text PDF PubMed Scopus Google we plays a role in the regulation of cartilage gene expression. therefore, used chondrocytes. the activity of leading to a reduction in NAD levels and an in in M. Cancer Res. 2003; Google Scholar). NAD was in it was that the lowered NAD levels by and SirT activity levels by cartilage-specific gene expression was as in the levels of collagen and collagen significantly in the these a was to in of the with an led to a reduction in protein levels by whereas the protein control was SirT activity and NAD levels in significantly as with the control and Finally, cartilage-specific gene expression was as in the levels of collagen and collagen significantly in we expression of the gene collagen and in the and and changes in RNA levels and In these that plays a positive role in regulating NAD levels, SirT activity, and cartilage-specific gene expression in human chondrocytes. SirT1 was as a of gene expression (11Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1333) Google Scholar) and has been demonstrated to affect transcription via deacetylation of both histones M. Guarente L. PubMed Scopus Google Scholar) and transcription factors, and J. D. A. 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SirT1 and of elevated In to the of SirT1 cartilage gene expression, the expression of these cartilage genes. this to act in a with to gene expression, to cell survival and J. D. A. Guarente L. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, H. E.N. Guarente L. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). It is that with the activity of the the SirT1 to the enzyme K. X. A. Biol. 2003; PubMed Scopus Google Scholar, T. D. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google the with the SirT1, in activity, thereby cartilage gene expression. SirT1 has it was that is through transcription which regulates expression of cartilage-specific genes (1Lefebvre V. Smits P. Birth Defects Res. 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PubMed Scopus Google Scholar) to regulate activity, as in deacetylation of and may, therefore, of the critical in transcription of collagen that the was to these to the promoter, that activity of SirT1 is for this that the of SirT1 with and the enhancer in collagen 2(α1) via a to the and the transcription factor to the and are for collagen 2(α1) transcription C. C. L. G. P. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, T. M. Y. Asahara H. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). is targeted to the collagen 2(α1) promoter is by the that significantly to the promoter in the of SirT1 SirT1 is to and a transcription factor to the collagen 2(α1) and would then likely to the histones and for transcription likely from these is that have a for and PGC1α, which to NAD is for activity of SirT1, we the of the salvage pathway enzyme E. C. C. Barbosa J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) cartilage-specific gene expression. by the or in a NAD levels, SirT activity, and chondrocyte-specific gene expression. This would that is critically involved in the maintenance of NAD and that it cartilage-specific gene expression through SirT1 in cartilage from and we an in SirT1 levels and a levels in This that of are to an in SirT1 and In the for the that SirT1 plays a positive role in the regulation of cartilage-specific gene expression in human chondrocytes and that at least for collagen 2(α1) promoter SirT1 transcription by of a of a role for the NAD salvage pathway enzyme in transcriptional for the human cartilage for the of the SirT1 and and M. for the of the for and for with for the with