Sirtuin 1 Is a Negative Regulator of Parathyroid Hormone Stimulation of Matrix Metalloproteinase 13 Expression in Osteoblastic Cells

Background: Parathyroid hormone (PTH) stimulates MMP13 in osteoblasts. Results: Sirtuin 1 (SIRT1) activation suppresses PTH stimulation of Mmp13 expression, and this process is mediated by the AP-1 complex. Conclusion: SIRT1 is a novel suppressor of PTH stimulation of MMP13 expression. Significance: Activation of SIRT1 and suppression of MMP13 may prolong the anabolic bone-forming period of PTH treatment in osteoporotic patients. Parathyroid hormone (PTH) is the only current anabolic treatment for osteoporosis in the United States. PTH stimulates expression of matrix metalloproteinase 13 (MMP13) in bone. Sirtuin 1 (SIRT1), an NAD-dependent deacetylase, participates in a variety of human diseases. Here we identify a role for SIRT1 in the action of PTH in osteoblasts. We observed increased Mmp13 mRNA expression and protein levels in bone from Sirt1 knock-out mice compared with wild type mice. PTH-induced Mmp13 expression was significantly blocked by the SIRT1 activator, resveratrol, in osteoblastic UMR 106-01 cells. In contrast, the SIRT1 inhibitor, EX527, significantly enhanced PTH-induced Mmp13 expression. Two h of PTH treatment augmented SIRT1 association with c-Jun, a component of the transcription factor complex, activator protein 1 (AP-1), and promoted SIRT1 association with the AP-1 site of the Mmp13 promoter. This binding was further increased by resveratrol, implicating SIRT1 as a feedback inhibitor regulating Mmp13 transcription. The AP-1 site of the Mmp13 promoter is required for PTH stimulation of Mmp13 transcriptional activity. When the AP-1 site was mutated, EX527 was unable to increase PTH-stimulated Mmp13 promoter activity, indicating a role for the AP-1 site in SIRT1 inhibition. We further showed that SIRT1 deacetylates c-Jun and that the cAMP pathway participates in this deacetylation process. These data indicate that SIRT1 is a negative regulator of MMP13 expression, SIRT1 activation inhibits PTH stimulation of Mmp13 expression, and this regulation is mediated by SIRT1 association with c-Jun at the AP-1 site of the Mmp13 promoter.


Parathyroid hormone (PTH) is the only current anabolic treatment for osteoporosis in the United States. PTH stimulates expression of matrix metalloproteinase 13 (MMP13) in bone. Sirtuin 1 (SIRT1), an NAD-dependent deacetylase, participates in a variety of human diseases.
Here we identify a role for SIRT1 in the action of PTH in osteoblasts. We observed increased Mmp13 mRNA expression and protein levels in bone from Sirt1 knock-out mice compared with wild type mice. PTH-induced Mmp13 expression was significantly blocked by the SIRT1 activator, resveratrol, in osteoblastic UMR 106-01 cells. In contrast, the SIRT1 inhibitor, EX527, significantly enhanced PTH-induced Mmp13 expression. Two h of PTH treatment augmented SIRT1 association with c-Jun, a component of the transcription factor complex, activator protein 1 (AP-1), and promoted SIRT1 association with the AP-1 site of the Mmp13 promoter. This binding was further increased by resveratrol, implicating SIRT1 as a feedback inhibitor regulating Mmp13 transcription. The AP-1 site of the Mmp13 promoter is required for PTH stimulation of Mmp13 transcriptional activity. When the AP-1 site was mutated, EX527 was unable to increase PTH-stimulated Mmp13 promoter activity, indicating a role for the AP-1 site in SIRT1 inhibition. We further showed that SIRT1 deacetylates c-Jun and that the cAMP pathway participates in this deacetylation process. These data indicate that SIRT1 is a negative regulator of MMP13 expression, SIRT1 activation inhibits PTH stimulation of Mmp13 expression, and this regulation is mediated by SIRT1 association with c-Jun at the AP-1 site of the Mmp13 promoter.
Parathyroid hormone (PTH) 2 is a polypeptide hormone that is produced by the parathyroid gland. It is an essential regulator of calcium homeostasis and a major regulator of bone remodeling (1) in both normal and pathological conditions. Daily intermittent injections of PTH promote bone formation in osteoporotic patients (2). On the other hand, excessive PTH, as seen in patients with hyperparathyroidism, causes bone loss (3). PTH stimulates bone formation by binding to its receptors on osteoblasts (4). Additionally, PTH increases transcription and secretion of the collagenase, MMP13, in osteoblasts (5,6).
MMP13 is a key enzyme that degrades type I collagen in bone matrix. It is critical for endochondral ossification and bone formation because Mmp13 knock-out mice have delayed endochondral bone formation, vascularization of their primary ossification centers, and ossification (7). However, excess MMP13 is associated with pathological conditions. MMP13 is highly expressed in osteoarthritis (8), rheumatoid arthritis (9), and tumor metastasis (10).
PTH stimulates MMP13 expression in bone tissue in vivo (5) as well as in osteoblastic cells in vitro (6,11). Upon PTH treatment of UMR 106-01 cells, an initial increase of Mmp13 mRNA was observed at 2 h, and maximal induction was seen at 4 h, which declined to ϳ30% of maximum by 8 h (6).
We and others found that the AP-1 transcription complex is essential to the process of PTH stimulation of Mmp13 gene expression. In response to PTH, Mmp13 gene transcription was significantly attenuated by mutation of AP-1 binding sites (12). We have previously shown that PTH stimulation of MMP13 expression is regulated by histone acetylases p300, p300/CBPassociated factor (PCAF), and histone deacetylase 4 (HDAC4) (13)(14)(15).
HDACs are a class of enzymes that remove acetyl groups from an ⑀-N-acetyl lysine amino acid on a histone, thus regu-* This work was supported, in whole or in part, by National Institutes of Health lating gene expression. SIRT1 is one of the class III HDACs, being an NAD ϩ -dependent protein deacetylase. Mammals have seven sirtuins (SIRT1-7) (16). Of the seven sirtuins, SIRT1 has been the most investigated. SIRT1 targets not only histones but also a variety of other proteins.
Sirtuins have been identified as a family of antiaging genes and have been shown to increase lifespan in lower organisms. SIRT1 activity declines with age (17), suggesting the association of SIRT1 with age-related diseases. One of the age-related diseases is bone loss. Therefore, it is likely that SIRT1 functions in bone.
In the present study, we determined the function of SIRT1 in the action of PTH stimulation of MMP13 transcription in osteoblasts. We propose that SIRT1 negatively regulates this process. Indeed, we found that SIRT1 activation blocked, whereas SIRT1 inhibition or deletion enhanced, PTH stimulation of Mmp13 mRNA. This regulation is in part mediated by SIRT1 association with Jun at the AP-1 site of the Mmp13 promoter.

EXPERIMENTAL PROCEDURES
Animals-All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of New York University and Ottawa Health Research Center Institute. One-month-old wild type C57BL/6 mice were utilized to isolate primary osteoblasts from cortical bone chips, and nine mice were utilized in these experiments.
Sirt1-deficient mice on C57BL/6 genetic background were kindly provided by Dr. Michael W. McBurney at Ottawa Health Research Center Institute. RNA extractions were from frozen femurs of 2.5-7-month-old Sirt1 ϩ/ϩ and Sirt1 Ϫ/Ϫ male and female mice (25). Four Sirt1 ϩ/ϩ and 10 Sirt1 Ϫ/Ϫ mice were utilized for RNA analysis. Two mice per group were utilized for histology and protein extraction, respectively. Histology sections and protein extractions were from femora and tibiae of 3-month-old female Sirt1 ϩ/ϩ and Sirt1 Ϫ/Ϫ mice.
Cell Culture-The rat osteoblastic UMR 106-01 cells were cultured in Eagle's minimum essential medium with 5% fetal bovine serum (FBS), 25 mM Hepes, pH 7.4, 1% nonessential amino acids, 100 units/ml penicillin, and 100 g/ml streptomycin. Primary osteoblasts were isolated from bone chips of femora/tibiae of 1-month-old male wild type mice and were cultured in ␣-MEM plus 10% FBS, 100 units/ml penicillin, and 100 g/ml streptomycin in 100-mm dishes. At confluence, the cells were plated at a density of 2 ϫ 10 5 cells/well of 6-well plates. After reaching 90% confluence, cells were changed to medium with 0% FBS and treated with 10 M EX527 or vehicle overnight. The next day, cells were treated with 10 Ϫ8 M PTH or vehicle for 2 h before total protein was extracted or 4 h before RNA extraction. Resveratrol was added to the cells alone or together with PTH post-serum starvation. HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 100 g/ml streptomycin and 10% fetal bovine serum.
Luciferase Assay-For luciferase assays, UMR 106-01 cells were plated in 12-well plates overnight before transfecting with either wild type Mmp13 promoter construct (100 ng/well in 12-well plates) or Mmp13 promoter construct with mutation of the AP-1 site (12). Two days after transfection, cells were serum-deprived overnight. Cells were then either pretreated with resveratrol for 2 h or with EX527 overnight before PTH (10 Ϫ8 M for resveratrol experiments, 10 Ϫ9 M for EX527 experiments, rat PTH(1-34); Sigma) treatment for 6 h. Our previous studies have shown that 10 Ϫ8 M PTH elicits the maximal stimulation of Mmp13 gene expression (6). Because we expected an enhancement of Mmp13 gene expression after co-treatment with PTH and SIRT1 inhibitor, we decided to use 10 Ϫ9 M PTH in the EX527 experiment. The cell lysates were harvested and analyzed immediately for luciferase activity with the luciferase assay reagent (Promega) and an OptiCompII luminometer (MGM Instruments, Inc., Hamden, CT). Luciferase activity was normalized to the amount of protein, which was determined by the Bio-Rad protein assay.
Chromatin Immunoprecipitation Assays-UMR 106-01 cells were treated with medium (control) or PTH (10 Ϫ8 M) and/or resveratrol (4.5 M) for 4 h. The cells were fixed for 10 min at room temperature with medium containing 0.8% formaldehyde solution. The cells were then washed with ice-cold PBS containing protease inhibitors and 1 mM formaldehyde and resuspended in SDS lysis buffer for 10 min on ice. The samples were then sonicated to reduce the DNA length to 0.5-1 kbp. The cellular debris was removed by centrifugation, and the supernatant was diluted 10-fold in buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, and protease inhibitors). For PCR analysis, aliquots (1:100) of total chromatin were saved before immunoprecipitation. Prior to chromatin precipitation, the samples were precleared with 80 l of a 25% (v/v) suspension of DNA-coated protein A/G-agarose for 30 min at 4°C. The supernatant was used for immunoprecipitation with the appropriate antibody (anti-SIRT1 (Upstate Biotechnology, Inc.), anti-c-Jun (Santa Cruz Biotechnology), or anti-c-Fos (Santa Cruz Biotechnology)) or with IgG (Santa Cruz Biotechnology) as a negative control for ChIP analysis overnight at 4°C. For ChIP-reChIP assays, the immunoprecipitated complexes obtained by ChIP with anti-c-Jun antibody (Cell Signaling) were eluted in ChIP elution buffer and then diluted 5-fold and subjected to the ChIP procedure with anti-SIRT1 (Abcam) or IgG as negative control. Then the DNA was subjected to PCR with primers Ϫ84/Ϫ2 that amplified the AP-1 site of the rat Mmp13 promoter, 5Ј-ACTCACTAGGAAGTGAACAC-3Ј (forward) and 5Ј-TCCCAGGGCAAGCATTCTCTA-3Ј (reverse) (13). Input DNA (1:100) is a positive control for the assay. Data are shown as -fold change compared with the IgG control samples.
Protein Immunoprecipitation and Western Blot Analyses-For protein immunoprecipitation, UMR 106-01 cells were seeded in 100-mm dishes. After reaching 90% confluence, cells were serum-deprived overnight. The cells were then treated with 10 Ϫ8 M PTH for 4 h. At the end of the culture, whole cell lysates were harvested with RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 0.1% SDS, and protease inhibitors) and were incubated in RIPA buffer for 15 min at 4°C before centrifuging for 15 min at 14,000 rpm. Protein concentrations were measured in the supernatants by the Bio-Rad protein assay. The lysates were precleared with IgG for 30 min at 4°C, and then equal amounts of protein lysates were incubated with anti-c-Jun antibody (0.5 g of antibody/1 mg of total protein; BD Biosciences) overnight at 4°C. The lysates were then incubated with protein A/G-agarose beads (Santa Cruz Biotechnology) for 2 h before centrifugation at 2,500 rpm at 4°C to pull down the immunoprecipitation complexes. The beads were washed with RIPA buffer three times and were then boiled in SDS loading buffer for 5 min. Samples were fractionated by SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad). Membranes were blocked for 1 h with 5% nonfat dried milk and then incubated overnight at 4°C with anti-SIRT1 (1:2,000; Abcam) or anti-c-Jun (1:2,000; BD Biosciences). Membranes were then incubated with appropriate secondary antibodies (Santa Cruz Biotechnology) for 45 min at room temperature. Blots were developed with Super Signal reagents (Thermo Scientific).
For c-Jun acetylation experiments, HEK293 cells were plated overnight and then transfected with c-Jun with Sirt1 or vector FLAG plasmids. p300 plasmids were co-transfected to increase c-Jun acetylation (29). To detect c-Jun acetylation in UMR osteoblastic cells, cells were transfected with c-Jun together with p300 on the second day of cell plating. Two hundred ng of each plasmid was transfected in 10-mm culture dishes. All transfections were facilitated with GeneJammer Transfection Reagent (Agilent Technologies) following the manufacturer's instructions. Sirt1 and vector FLAG plasmids were kindly provided by Dr. Paolo Sassone-Corsi (University of California). p300 plasmids were kindly provided by Dr. Deyu Fang (Northwestern University) and c-Jun was from Dr. Michael J. Birrer (Massachusetts General Hospital). Seventy-two h post-transfection, cells were changed to serum-free medium, which additionally contained the HDAC inhibitor trichostatin A (Sigma) at 400 nM and the SIRT1 inhibitor nicotinamide (Sigma) at 20 mM. The next day, cells were treated with 8-bromo-cAMP (0.2 mM; Sigma) for 2 h for HEK cells or 10 Ϫ8 M PTH or vehicle for 2 h for UMR cells, and whole cell lysates were harvested with RIPA buffer. RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 0.1% SDS, and protease inhibitors) was freshly made and had 1 mM DTT (Sigma), 400 nM trichostatin A, and 10 mM nicotinamide. Supernatants were precleared with IgG at 4°C for 15 min and then incubated with anti-c-Jun antibody (0.5 g of antibody/1 mg of total protein; BD Biosciences) for 1 h, followed by incubation with protein A/G-agarose beads for an additional 2 h. The beads were washed three times with lysis buffer, dissolved with 4ϫ Laemmli's buffer, and boiled for 5 min. After SDS-PAGE fractionation and transfer to PVDF membranes, they were blocked for 1 h with 5% nonfat dried milk and then incubated overnight at 4°C with acetylated lysine antibody (1:1,000; Cell Signaling). Blots were developed with SuperSignal West Femto chemiluminescent substrate (Thermo Scientific). Membranes were then blotted with anti-c-Jun (1:2000; BD Biosciences). To detect MMP13 protein in bone, Western blots were performed with protein extracted from the whole bones of femur and tibia (including marrow) from 3-month-old female mice (n ϭ 2). Membranes were first blotted with the MMP13 antibody (Abcam), and then blots were reprobed with ␤-actin antibody (Cell Signaling). Western blot bands were analyzed by ImageJ (National Institutes of Health, Bethesda, MD).
Immunohistochemistry-For histological analysis, femora and tibiae were harvested from 3-month-old female Sirt1 ϩ/ϩ and Sirt1 Ϫ/Ϫ mice. Samples were fixed in 4% paraformaldehyde overnight and decalcified in 10% EDTA, pH 7.5, for 4 weeks at 4°C. Samples were soaked in 10% sucrose for 2 h, 20% sucrose for 6 h, and then 30% sucrose overnight; embedded in optimal cutting temperature compound; and frozen. The sections (10 m) were cut longitudinally to the femur and tibia. To evaluate the localization and protein levels of MMP13, we performed immunohistochemical staining by the ABC staining system (Santa Cruz Biotechnology). The sections were washed with PBS and then immersed in methanol containing 1% hydrogen peroxide to block endogenous peroxidase activity. The sections were then blocked with 2% blocking serum and then with anti-MMP13 rabbit antibody (Abcam) at 4°C overnight. Sections were then washed three times and incubated with biotin-labeled goat anti-rabbit IgG at room temperature for 30 min. Control reactions were performed by omitting primary antibody. Sections were mounted with Cytoseal 60 (Thermo Scientific).
Statistical Analysis-Results were expressed as the means Ϯ S.E. Differences were analyzed using a Mann and Whitney U test for analysis between two groups or one-way analysis of variance using the Tukey HSD (honestly significant difference) test for three or more groups, and differences were considered significant at p Ͻ 0.05.

Increased Mmp13 mRNA and Protein Level in Sirt1
Knockout Mice-To test our hypothesis that SIRT1 is a negative regulator of MMP13, we first examined the mRNA expression of Mmp13 in bones of wild type and Sirt1 Ϫ/Ϫ mice. Total RNA was extracted from the metaphyseal region of the femurs of Sirt1 ϩ/ϩ or Sirt1 Ϫ/Ϫ mice. We observed that Mmp13 mRNA expression was significantly increased in Sirt1 Ϫ/Ϫ mice (Fig.  1A), indicating that SIRT1 is a negative regulator of MMP13 expression in bone. We did not observe a significant difference in Mmp13 mRNA expression by gender, or from age 2.5 to 7 months. We next determined MMP13 protein levels in whole bones from 3-month-old female mice. We found MMP13 protein increased in bones from Sirt1 Ϫ/Ϫ mice compared with wild type mice (Fig. 1B). To determine the localization of MMP13, we performed immunohistochemistry. MMP13 was detected mainly in the growth plate region (Fig. 1C). We observed increased MMP13 staining in both the tibia and femoral growth plate region in Sirt1 Ϫ/Ϫ mice compared with wild type mice.
In Sirt1 Ϫ/Ϫ mice, we also observed significantly increased femoral mRNA expression of osteocalcin and a 7.8-fold increase of bone sialoprotein compared with wild type (Fig. 1, D  and E). In separate studies with Sirt1 osteoblast-specific knockout mice, we have also found that SIRT1 regulates extracellular matrix proteins, including type I collagen, and bone sialoprotein (data not shown). 3 SIRT1 Activator Blocked and SIRT1 Inhibitor Enhanced Mmp13 Expression Induced by PTH-We next determined the effects of pharmacological modulation of SIRT1 on PTH stimulation of Mmp13 expression in osteoblastic UMR 106-01 cells. Consistent with our previous findings, PTH highly stimulated Mmp13 mRNA expression (Fig. 2). The SIRT1 activator, resveratrol, significantly blocked Mmp13 expression stimulated by PTH in a dose-dependent manner ( Fig. 2A). In support of these data, SIRT1 overexpression decreased PTH stimulation of Mmp13 mRNA (Fig. 2B). On the other hand, the SIRT1 inhibitor, EX527, enhanced Mmp13 mRNA expression induced by PTH (Fig. 2C).
We further examined the effect of EX527 on BMSCs from Sirt1 osteoblast-specific knock-out (Sirt1 ObϪ/Ϫ ) mice. EX527 enhanced Mmp13 mRNA expression in control cells, whereas we did not observe enhancement with EX527 treatment in Sirt1 ObϪ/Ϫ cells (Fig. 2D). These data suggest that EX527 is specific for SIRT1. 3 Y. Fei, T. Nakatani, and N. C. Partridge, manuscript in preparation. Of note, protein levels of SIRT1 did not change with PTH stimulation (Fig. 2E). These data suggest that SIRT1 is a suppressor of PTH stimulation of Mmp13 transcription.
SIRT1 Associates with the AP-1 Site of the Mmp13 Promoter-To dissect the molecular mechanisms by which SIRT1 regulates Mmp13 expression, we performed ChIP assays of the rat Mmp13 promoter. We postulated that the AP-1 site is critical for SIRT1 to regulate Mmp13 expression. Notably, PTH treatment for 4 h significantly increased SIRT1 binding to the AP-1 site of the Mmp13 promoter (Fig. 3A). This is after maximal transcription of Mmp13 has been induced at 2 h (6). The SIRT1 activator, resveratrol, further enhanced SIRT1 binding to the AP-1 site of the Mmp13 promoter (Fig. 3A), which may be why it is a such a potent inhibitor of Mmp13 transcription. Additionally, we observed that PTH treatment enhanced the AP-1 components, c-Fos and c-Jun, binding to the AP-1 site of the Mmp13 promoter (Fig. 3B).

SIRT1 Associates with c-Jun in Osteoblasts and PTH Treatment Enhances the Association between These
Proteins-Based on our ChIP assay data indicating that PTH promoted SIRT1 association with the AP-1 site of the Mmp13 promoter, we then performed immunoprecipitation assays to determine the pro-tein association of SIRT1 with c-Jun in osteoblastic UMR 106-01 cells. It should be noted that SIRT1 does not associate with c-Fos (data not shown). We found that SIRT1 interacts with c-Jun in these cells and that PTH treatment significantly stimulates SIRT1 binding to c-Jun (Fig. 4, A and B). It should be noted that SIRT1 protein level does not change following PTH treatment (Fig. 2D). We confirmed the finding of PTH stimulation of SIRT1 interaction with c-Jun in primary osteoblasts, which were isolated from mouse femoral bone chips (Fig. 4C). In addition, we observed that the SIRT1 inhibitor, EX527, attenuated the interaction between SIRT1 and c-Jun (Fig. 4C) in primary osteoblasts.

SIRT1 Deacetylates c-Jun and 8-Bromo-cAMP Stimulates Jun
Deacetylation-Because we observed that SIRT1 associated with c-Jun and SIRT1 is a protein deacetylase, we next determined the acetylation of c-Jun with anti-acetyllysine antibody. We found that co-transfection of Sirt1 with c-Jun significantly reduced c-Jun acetylation (Fig. 4, D and E) in HEK293 cells. Additionally, 8bromo-cAMP treatment decreased c-Jun acetylation (Fig. 4, D and  E) as well, suggesting that the cAMP pathway is involved in this process. Further, we observed that PTH treatment down-regulated c-Jun acetylation in UMR cells (Fig. 4, F and G).

PTH Treatment Increased SIRT1 and c-Jun Association with the AP-1 Site of the Mmp13 Promoter-We further performed
ChIP-reChIP analysis of SIRT1 and c-Jun, and we found that PTH treatment increased the association of SIRT1 and c-Jun to the AP-1 site of the Mmp13 promoter (Ϫ84/Ϫ2) (Fig. 4H).
The AP-1 Site of the Mmp13 Promoter Is Essential for SIRT1 Modulation of PTH Stimulation of Mmp13 Promoter Activity-We next determined the effect of pharmacological modulation of SIRT1 on Mmp13 promoter activity stimulated by PTH. The hormone at 10 Ϫ8 M maximally stimulates Mmp13 promoter activity in osteoblastic UMR 106-01 cells. Two h of pretreatment with resveratrol (45 M) significantly blocked the PTH stimulation of Mmp13 promoter activity (Fig. 5A). To determine the importance of the AP-1 site in the role of SIRT1 action, we transfected UMR 106-01 cells with an Mmp13 promoter luciferase construct with the AP-1 site mutated. As we have reported previously (12), this Mmp13 promoter construct is not able to bind the AP-1 factors c-Jun and c-Fos. Transcriptional activity of the Mmp13 promoter with the AP-1 site mutation was reduced. PTH treatment was still able to stimulate its transcriptional activity significantly (p Ͻ 0.01); however, pretreatment with the SIRT1 activator, resveratrol (45 M), did not inhibit Mmp13 promoter activity (Fig. 5A). Conversely, we examined the effect of the SIRT1 inhibitor, EX527, on Mmp13 transcriptional activity stimulated by PTH (10 Ϫ9 M). EX527 at both 0.1 and 1 M significantly enhanced Mmp13 transcriptional activity stimulated by PTH, but this enhancement was not observed with the AP-1 mutant promoter (Fig. 5B). These data suggest that activation of SIRT1 down-regulates and inhibition of SIRT1 enhances PTH-stimulated Mmp13 promoter activity in osteoblastic cells. These data also indicate that the AP-1 site of the Mmp13 promoter is essential for SIRT1 modulation of Mmp13 transcriptional activity stimulated by PTH.

DISCUSSION
Our current study demonstrates that SIRT1 is a critical factor suppressing Mmp13 expression stimulated by PTH (Fig. 6).
In the initial response, PTH stimulation recruits the AP-1 transcription factor complex to the AP-1 site of the Mmp13 promoter and enhances transcription of Mmp13. We speculate that there is negative feedback at later stages of PTH treatment; SIRT1 is recruited to the AP-1 site, interacts with c-Jun, and suppresses Mmp13 expression. Our data present a novel mechanism, by which PTH stimulation of MMP13 expression in osteoblastic cells is modulated by SIRT1. According to this model, activation of SIRT1 and suppression of MMP13 may prolong the anabolic bone-forming period of PTH treatment in osteoporotic patients. Here, we report for the first time that SIRT1 negatively regulates MMP13 expression stimulated by PTH, and this process requires the AP-1 site in osteoblastic cells.
In the present study, we have shown that activation of SIRT1 by resveratrol significantly down-regulates Mmp13 gene expression, stimulated by PTH, in osteoblastic cells. EX527, a potent SIRT1 inhibitor, is widely utilized in physiological studies. Inhibition of SIRT1 by EX527 significantly enhanced Mmp13 gene expression stimulated by PTH. Further, we observed that Sirt1 knock-out mice display a significant increase in Mmp13 gene expression and protein abundance in bone. In separate studies, we have specifically deleted Sirt1 in osteoblasts in vivo. We observed that Mmp13 mRNA expression increased in bones of osteoblast-specific knock-out mice compared with controls. 3 Therefore, these data suggest that SIRT1 alone has the capacity to regulate Mmp13 expression.
Protein levels of MMP13 do not always match the mRNA expression. We have established that MMP13 is a highly regulated secreted extracellular protein. We have previously shown that osteoblastic cells respond to PTH treatment. We observed an initial increase of Mmp13 mRNA at 2 h, a maximal induction at 4 h, and ϳ30% reduction of the maximum by 8 h (6).
At the protein level, we showed that UMR cells respond to PTH by secreting MMP13. This reaches a maximal concentration extracellularly 12-24 h after PTH stimulation but then declines to undetectable levels by 96 h (30). Neither spontaneous nor cell-mediated extracellular degradation could account for this disappearance because the enzyme maintained stability in both fresh and conditioned media (30).
We have identified that a specific receptor in osteoblastic cells functions to eliminate extracellular MMP13 (30). Binding of MMP13 to this receptor and low density lipoprotein receptor-related protein 1 is coupled to the internalization and eventual degradation of the enzyme.
We also showed decreased binding, internalization, and degradation of MMP13 after 24 h of PTH treatment; these activities returned to control values after 48 h and were increased substantially after 96 h of treatment of PTH. We further demonstrated that degradation of MMP13 required receptor-mediated endocytosis and sequential processing by endosomes and lysosomes (31). The subsequent internalization and degradation of MMP13 requires the low density lipoprotein receptorrelated protein, LRP1 (32). After confluence, the cells were plated in 6-well plates at 2 ϫ 10 5 cells/well. Upon reaching 90% confluence, the cells were serum-deprived and treated with 10 M EX527 or vehicle overnight. The next day, the cells were treated with PTH 10 Ϫ8 M for 2 h before total protein extraction. D, SIRT1 deacetylates c-Jun, and 8-bromo-cAMP treatment reduces c-Jun acetylation. HEK293 cells were co-transfected with c-Jun and Sirt1 or vector FLAG plasmids. Seventytwo h after transfection, the cells were treated with 8-bromo-cAMP for 2 h, and whole cell lysates were harvested for analysis. E, quantification of c-Jun acetylation from three independent experiments. The value of the FLAG control group was set as 1.  Therefore, PTH regulates the expression and synthesis of Mmp13 mRNA and MMP13 protein as well as the abundance and functioning of the MMP13 receptor and the intracellular degradation of its ligand. The coordinate changes in the secretion of MMP13 and expression of the receptor determine the net abundance of the enzyme in the extracellular space (30,31). Thus, protein levels of MMP13 do not always match mRNA expression. The data in this report indicate that SIRT1 is a repressor of Mmp13 gene expression in bone and osteoblastic cells.
In addition to MMP13, SIRT1 also targets other extracellular proteins, including osteocalcin and bone sialoprotein, in global SIRT1 knock-out and in SIRT1 osteoblast-specific knock-out mice. Our current study focuses on the function of SIRT1 in the action of PTH stimulation of Mmp13 transcription in the osteoblast.
Aging is tightly associated with osteoporosis in both women and men. Recent research into the science of aging has identified one family of antiaging genes, the sirtuins. The sirtuins are a highly conserved family of NAD ϩ -dependent deacetylases that have been shown to increase lifespan in lower organisms. Mammals have seven sirtuins (SIRT1-7) (16). Of the seven sirtuins, SIRT1, SIRT2, SIRT6, and SIRT7 are located in the nucleus (33). In addition to SIRT1, to our knowledge, SIRT6 is the only other sirtuin that has been reported to function in bone or cartilage (34,35). We did not detect a significant difference in Sirt6 mRNA in osteoblasts of Sirt1 ObϪ/Ϫ and control Sirt1 flox/flox mice (data not shown), suggesting that there was no compensation by SIRT6. SIRT1 is involved in a variety of human diseases (33,36,37), including bone diseases. SIRT1 has many functions in bone; global Sirt1 deletion (19,38,39) and osteoblast-specific Sirt1 deletion (18,20,38) lead to reduced bone formation. Potentially, SIRT1 regulates osteoblast differentiation and bone formation through regulation of the tran-scription factor Runx2 (40,41). Others have reported that SIRT1 associates with Runx2 and reduces its acetylation status (40). SIRT1 activation with resveratrol promotes osteoblast differentiation in vitro (40 -43) and prevents bone loss in vivo (44). These observations are the compelling systematic biological rationale for our focus on SIRT1 in bone.
We understand that there are controversial reports on resveratrol activating SIRT1 (45). However, there is a growing body of studies that have shown that the effects of resveratrol depend on SIRT1. Effects of resveratrol are lost after knocking down or inhibiting SIRT1 in cell culture studies (46 -48). The benefits of resveratrol require SIRT1 in vivo. The ability of resveratrol to improve glucose homeostasis is impaired when hypothalamic SIRT1 is inhibited (49). The ability of resveratrol to activate AMP-activated protein kinase and increase mitochondrial function is lost in SIRT1 knock-out mice; further, overexpression of SIRT1 mimics the effects of resveratrol on mitochondria and AMP-activated protein kinase activation (27).
Moreover, recent studies provide direct evidence of SIRT1 activation by resveratrol and other sirtuin-activating compounds (42,50). Interaction of SIRT1 with certain substrates allows activation of SIRT1 by resveratrol and other sirtuin-activating compounds. Hubbard et al. (50) identified a critical amino acid, Glu-230, in SIRT1 required for these effects. Mouse myoblasts reconstituted with SIRT1 mutated at this amino acid lost their responsiveness to resveratrol and other SIRT1 activators (50). These studies provide strong evidence that resveratrol is a SIRT1 activator, and the effects of resveratrol depend on SIRT1 both in vitro and in vivo. Therefore, there is strong evidence that resveratrol can directly activate SIRT1 (50), although it is possible that resveratrol may indirectly activate SIRT1 through the AMP-activated protein kinase (51).
Activation of the cAMP pathway is one key means by which PTH modulates gene transcription in osteoblasts (1). We observed that 8-bromo-cAMP stimulates c-Jun deacetylation by SIRT1. In addition, PTH has been shown to increase NAD ϩ production in vivo (52), which is required for SIRT1 activity. SIRT1 associates with c-Jun and decreases its acetylation, thereby reducing AP-1 transcriptional activity and decreasing Mmp13 gene transcription. SIRT1 has been reported to repress MMP13 expression in chondrocytes. Sirt1 overexpression significantly inhibited the up-regulation of MMP13 caused by IL-1␤ (53). Conversely, reduced SIRT1 protein levels are associated with increased protein levels of MMP13 in osteoarthritic cartilage (53)(54)(55)(56). MMP13 preferentially degrades type II collagen in cartilage but also degrades type I collagen in bone.
Previous studies in our group have reported that PTH potently induced MMP13 expression in osteoblastic cells (6). PTH regulates MMP13 gene transcription through a protein kinase A-dependent pathway (57). Under basal conditions, gene transcription of Mmp13 is repressed by the presence of HDAC4, which binds to Runx2 at the runt domain binding site (15). PTH treatment causes HDAC4 to dissociate from Runx2 on the Mmp13 promoter, and Runx2 then recruits the histone acetyltransferases, p300 and PCAF (13,14). Newly synthesized c-Fos/c-Jun subsequently bind to the AP-1 site and interact with CBP and with proteins bound to Runx2, allowing maximal transcription of Mmp13 (58).
Our previous studies showed that c-Fos and c-Jun are the most physiologically relevant AP-1 members in regulating Mmp13 mRNA expression in osteoblasts (11). Mmp13 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that the AP-1 and runt domain binding sites are responsible for Mmp13 gene transcription. The AP-1 and runt domain binding sites bind members of the AP-1 family and Runx2 transcription factors. We have identified Runx2 binding to the runt domain binding site. We have also investigated various members of the AP-1 family: Fra-1, Fra-2, FosB, c-Fos, c-Jun, JunB, and JunD. We identified JunD in addition to a Fos-related antigen binding to the AP-1 site. Our studies showed that JunD expression remains fairly constant during osteoblast differentiation. Overexpressing Fra-2 and JunD repressed Runx2-induced Mmp13 promoter activity. Overexpression of both c-Fos and c-Jun in osteoblasts or Runx2 increased Mmp13 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and Runx2 synergistically increased Mmp13 promoter activity (11). Based on these studies, we focused on c-Fos and c-Jun in the present project.
SIRT1 has been shown to associate with c-Jun and suppress the transcriptional activity of the AP-1 factor in other cell systems: maintaining T cell tolerance (29), inhibiting cyclooxygenase-2 expression in macrophages (59), and inhibiting MMP9 expression in mouse embryonic fibroblasts (60). Here we report that SIRT1 suppresses MMP13 expression stimulated by PTH in osteoblastic cells, and this process is mediated through the AP-1 site of the Mmp13 promoter.