Expression of SirT1 in human chondrocytes reduces apoptosis.
To determine whether SirT1 has a prosurvival effect on human chondrocytes, it was transiently overexpressed in these cells by Nucleofector technology, which resulted in 90–95% transfection efficiency using the solutions and electroporation program recommended by the manufacturer. As shown in Figure 1, SirT1 was efficiently overexpressed by day 2 posttransfection, as assessed by both Western blot (Figure 1A) and SirT1 activity assay (Figure 1B). The ectopically expressed SirT1 was also found to be targeted to the nucleus (data not shown). As shown in Figure 1C, passage 1 cells exhibited slightly reduced levels of α1(II) collagen and aggrecan gene expression, compared with levels in freshly isolated cells. The transfected cells were then monitored for apoptosis, using flow cytometry. Figure 1D shows that the level of apoptosis dropped 5.5-fold when SirT1 was expressed. Adding the SirT1 inhibitor nicotinamide to the culture at the time of transfection abolished the protective effect of SirT1 against apoptosis (Figure 1D). When an enzymatically inactive SirT1 mutant (SirT1H355Y) was expressed in chondrocytes, it had no effect on chondrocyte survival (data not shown).
Figure 1. SirT1 is a positive regulator of survival of osteoarthritic (OA) human chondrocytes. A and B, Extracts from passage 1 OA human chondrocytes transfected with either a SirT1 plasmid or pcDNA control (empty vector) were analyzed using immunoblotting (A) or SirT activity assays (B). C, Genes from freshly isolated chondrocytes and from passage 0 (P0), passage 1 (P1), and passage 4 (P4) chondrocytes were analyzed using real-time polymerase chain reaction. D and E, Transfected chondrocytes were left untreated or were treated with either nicotinamide (NAM; 1 mM) (D) or tumor necrosis factor α (TNFα; 10 ng/ml)/actinomycin D (ActD; 0.2 μg/ml) (E), and the percentage of apoptotic cells was assessed. F and G, Transfected chondrocytes were cultured under either low serum conditions (0.5% fetal bovine serum [FBS]) or high serum conditions (10% FBS) (F) or were plated at either low confluence (20%) or full confluence (100%) (G), and apoptosis was assessed at 24 hours. H, Passage 1 chondrocytes were transiently transfected with either a control small interfering RNA (siRNA) or SirT1 siRNA. Levels of SirT1 protein were determined by immunoblotting and quantitated by scanning densitometry. I, Transfected chondrocytes were treated with TNFα/ActD or were left untreated, and the percentage of apoptotic cells was assessed. Values in B–I are the mean and SD. ∗ = P < 0.05.
Download figure to PowerPoint
Apoptosis was induced in chondrocytes by adding TNFα and actinomycin D, which elevated the level of cell death more than 3-fold (Figure 1E). Overexpression of SirT1 in these cells significantly reduced this TNFα/actinomycin D–mediated apoptosis (Figure 1E). Apoptosis was also induced in chondrocytes cultured under low serum conditions (0.5% FBS) (Figure 1F) or at low confluence (20%) (Figure 1G). Under both physiologic apoptotic conditions, expression of SirT1 led to decreased apoptosis. A SirT1-specific siRNA was transfected into chondrocytes, which lowered the levels of endogenous SirT1 in nontransfected human chondrocytes by 3-fold (Figure 1H). This reduction in SirT1 resulted in a corresponding increase in the level of apoptosis in either the presence or the absence of TNFα/actinomycin D (Figure 1I). Taken together, these data indicate that SirT1 is an antiapoptotic protein in human chondrocytes and that this function requires its enzymatic activity.
Expression of SirT1 in chondrocytes leads to activation of the IGFR receptor/Akt pathway.
To investigate the mechanism by which SirT1 protects chondrocytes against apoptosis, the IGFR pathway was investigated (Figure 2A), since IGF-1 is a well-known survival factor for chondrocytes (25, 26) and since SirT1 has been shown to affect components of this pathway (13, 27, 28). Chondrocytes were transfected with the SirT1 expression plasmid, and IGFR levels were assessed. As shown in Figure 2B, total IGFR levels were unchanged by SirT1; however, levels of the activated/tyrosine phosphorylated form of the receptor, pIGFR (Tyr1135/1136), were significantly elevated in the presence of elevated SirT1 (Figure 2B). Activation of IGFR leads to phosphorylation of phosphatidylinositol 3-kinase (PI 3-kinase), which in turn leads to phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK-1) (29), as shown in Figure 2A. The levels of the phosphorylated forms of both PI 3-kinase and PDK-1 were significantly increased by SirT1, while there was no change in the levels of PI 3-kinase, PDK-1, or GAPDH (Figure 2C). Additionally, SirT1 did not change the phosphorylation status of pTEN (Figure 2C), a negative regulator of this pathway. Phosphorylated PDK-1 is known to phosphorylate the prosurvival kinase Akt on Thr308, resulting in Akt activation (29). As shown in Figure 2D, pAkt(Thr308) levels were significantly increased by SirT1.
Figure 2. Activation of insulin-like growth factor receptor (IGFR) and Akt in human chondrocytes expressing SirT1. A, Akt is phosphorylated by IGF-activated protein kinases and by mTOR. Phosphorylated Akt(Thr308) is a target of phosphorylated phosphatidylinositol 3-kinase (PI 3K), while pAkt(Ser473) is a target of phosphorylated mTOR. B–E, Passage 1 human chondrocytes were transiently transfected with either a SirT1 expression plasmid or pcDNA control (empty vector), and levels of phosphorylated and nonphosphorylated IGFR (B), phosphorylated and nonphosphorylated phosphatidylinositol 3-kinase and phosphoinositide-dependent protein kinase 1 (PDK-1) and phosphorylated pTEN (C), phosphorylated and nonphosphorylated Akt (D), and phosphorylated and nonphosphorylated mTOR (E) were assessed. Extracts were generated at 24 hours posttransfection and immunoblotted with the indicated antibodies. F, Chondrocytes were transfected and treated with nicotinamide (NAM; 1 mM), and extracts were generated and immunoblotted with the indicated antibodies. G, Chondrocytes were transfected, and extracts were generated and immunoblotted with the indicated antibodies. EGFR = epidermal growth factor receptor; PDGFR = platelet-derived growth factor receptor.
Download figure to PowerPoint
In order to achieve optimal activation of Akt, phosphorylation on Ser473 is also required (Figure 2A). As shown in Figure 2D, pAkt(Ser473) levels were significantly elevated by SirT1. The protein kinase responsible for phosphorylation of Ser473 is mTOR, which in turn needs to be phosphorylated in order to be activated. Assessment of the phosphorylation status of mTOR on residues Ser2448 and Ser2481 showed increased phosphorylation in the presence of SirT1 (Figure 2E), indicating mTOR was activated.
To confirm that SirT1 mediates the activation of Akt by phosphorylation on Thr308 and Ser473, the SirT1 inhibitor nicotinamide was added to the cultures following SirT1 transfection. In the presence of nicotinamide, phosphorylation of Akt on Ser473 and Thr308 was not elevated by SirT1 (Figure 2F). In additional control experiments, the IGFR antagonist AG1024 or the PI 3-kinase inhibitor LY294002 was added to cells following SirT1 transfection. These inhibitors significantly blocked the phosphorylation of Akt on Thr308 in the presence of elevated SirT1 (results not shown), indicating that activation of Akt by SirT1 occurs via IGFR and PI 3-kinase. Taken together, these data show that elevated expression of SirT1 in human chondrocytes leads to the activation of IGFR, which initiates a phosphorylation cascade culminating in phosphorylation of Akt on the two amino acid residues needed for activation.
We examined, as controls, the status of other tyrosine kinases potentially affected by SirT1. Figure 2G shows a modest increase in epidermal growth factor receptor tyrosine phosphorylation in the presence of SirT1; however, no increases were observed in tyrosine phosphorylation of platelet-derived growth factor receptor α (PDGFRα), PDGFRβ, or Src.
Activation of Akt by SirT1 leads to phosphorylation of the MDM2 protein and inhibition of the proapoptotic protein p53.
Activated Akt has multiple cellular targets that participate in cell survival, including MDM2, a protein that functions in part by binding and blocking the proapoptotic protein p53. It is known that activated Akt phosphorylates human MDM2 on Ser186 (Figure 3A), leading to increased affinity of MDM2 for p53 (30–32). As shown in Figure 3B, chondrocytes overexpressing SirT1 exhibited a significantly increased level of pMDM2(Ser186) compared with the level exhibited by control transfected cells, while the total levels of MDM2 in chondrocytes and in control cells did not vary. These data are consistent with fact that SirT1 activates Akt, which in turns leads to phosphorylation of MDM2.
Figure 3. Phosphorylation of MDM2 and inactivation of p53 in chondrocytes expressing SirT1. A, MDM2 is phosphorylated by Akt. B and C, Passage 1 human chondrocytes were transiently transfected with either a SirT1 expression plasmid or pcDNA control (empty vector). Extracts were generated at 24 hours posttransfection and immunoblotted (IB) with the indicated antibodies (B), and then, extracts were immunoprecipitated (IP) with an MDM2-specific antibody and immunoblotted and probed with the indicated antibodies (C). D, Chondrocytes were transfected with a control small interfering RNA (siRNA) or an MDM2 siRNA. Levels of MDM2 protein were determined by immunoblotting and quantitated by scanning densitometry. Chondrocytes were also transfected with a pcDNA control, the SirT1 plasmid, a control siRNA, and an MDM2 siRNA. The percentage of apoptotic cells was assessed by flow cytometry. E, Chondrocytes were transfected with either a pcDNA control or the SirT1 plasmid. Extracts were generated and immunoblotted with the indicated antibodies. F, Chondrocytes transfected with a pcDNA control (contr), a SirT1 expression plasmid, a p53-responsive promoter/luciferase construct, or a control promoter/luciferase construct were assayed for luciferase activity. Values in D and F are the mean and SD. ∗ = P < 0.05.
Download figure to PowerPoint
It has been demonstrated that phosphorylated MDM2 binds p53 more efficiently than does nonphosphorylated MDM2 (30). To determine if this was the case in our chondrocyte extracts, MDM2 was immunoprecipitated from extracts of the control and SirT1 transfected cells, which were then immunoblotted for MDM2, pMDM2, and p53 (Figure 3C). In cells expressing SirT1, there was a significant increase in the amount of pMDM2 in immunoprecipitated MDM2, while the total amount of MDM2 did not differ between the two conditions. Consistent with this increase in phosphorylated MDM2, there was a large increase in the amount of p53 in the immunoprecipitated extracts expressing SirT1, which contained elevated amounts of pMDM2. These data indicate that p53 associates more efficiently with phosphorylated MDM2, as reported previously (30).
If MDM2 mediates the survival effect of SirT1 in chondrocytes, then down-regulation of MDM2 by siRNA transfection should abolish the antiapoptotic effect of SirT1. Chondrocytes were transfected with either control siRNA or MDM2 siRNA, and levels of MDM2 protein were assessed. As shown in Figure 3D, the MDM2 siRNA reduced the level of MDM2 protein by ∼4-fold. When cells were cotransfected with the MDM2 siRNA and the SirT1 expression plasmid (Figure 3D), SirT1 was not nearly as effective at reducing the level of apoptosis in the presence of the MDM2 siRNA as it was in the presence of the control siRNA; the number of apoptotic cells increased 2-fold in the presence of the MDM2 siRNA. Thus, reducing MDM2 levels reduces the ability of SirT1 to block apoptosis.
While p53 can be blocked by its association with pMDM2, p53 also can be inactivated by SirT1-mediated deacetylation, as reported previously (12, 14). To determine if p53 was affected by SirT1 in human chondrocytes, the acetylation status of p53 was assessed. As shown in Figure 3E, SirT1 did not affect total levels of p53, but dramatically reduced the level of acetylated p53. Since p53 activity can be blocked by both pMDM2 binding and deacetylation, it was expected that p53 target gene expression would be reduced by SirT1. One such gene is p21, and as shown in Figure 3E, p21 protein levels were down-regulated by SirT1. Additionally, cells were transfected with a p53-responsive promoter/luciferase in the presence or absence of SirT1. As shown in Figure 3F, this p53-responsive promoter was significantly repressed in cells expressing SirT1, while a control promoter was not affected. Taken together, these data indicate that activation of Akt leads to phosphorylation of MDM2 and inactivation of the proapoptotic protein p53.
It is recognized that active Akt serves an antiapoptotic function by phosphorylating a number of proteins in addition to MDM2, including the FoxO family of proteins (33, 34) and Bad (35, 36). In our experiments, we observed no increased phosphorylation of Bad, FoxO1, FoxO3a, or FoxO4 following overexpression of SirT1 in chondrocytes (results not shown), indicating that, at least in chondrocytes, Akt activation by SirT1 may not lead to efficient phosphorylation of these proteins.
SirT1 represses expression of PTP1B, a proapoptotic protein that targets IGFR.
Our data indicate that SirT1 is able to activate the IGFR pathway. To determine if IGFR was activated by autocrine production of IGF-1 and IGF-2, the levels of these growth factors were assessed in chondrocytes expressing SirT1. The IGFs (IGF-1 in particular) are known to activate Akt (25), are a well-known component of cartilage, and can be secreted by chondrocytes (37). SirT1 had no effect on IGF-1 at either the RNA or the protein level (data not shown). SirT1 was able to induce IGF-2 in chondrocytes at both the RNA and protein levels. However pure IGF-2 had no effect on reducing the level of chondrocyte apoptosis (data not shown), while IGF-1 had an antiapoptotic effect that was consistent with the findings of previous studies (25, 26). These data suggest that activation of IGFR by SirT1 may be due to factors in addition to IGF-1.
One factor that has been demonstrated to block IGFR activity is PTP1B, which dephosphorylates IGFR, thereby inactivating it (38, 39). Importantly, SirT1 can repress expression of PTP1B, thereby enhancing insulin signaling (40). It was therefore thought that SirT1 activates the IGFR pathway in chondrocytes by repressing PTP1B. As shown in Figures 4A and B, SirT1 had a significant effect on repression of PTP1B at both the RNA and protein levels. When additional PTPs were assessed (Figure 4B), PTPα levels were not changed in cells expressing elevated SirT1, indicating that SirT1 does not affect it. Using immunoblotting, we could not detect PTPγ, PTPκ, or LAR in these chondrocyte extracts (results not shown).
Figure 4. Protein tyrosine phosphatase 1B (PTP1B) is a potent proapoptotic protein in human osteoarthritic chondrocytes, is repressed by SirT1, and reduces the level of activated insulin-like growth factor receptor (IGFR). A and B, RNA and protein extracts from passage 1 chondrocytes transfected with a SirT1 expression plasmid or pcDNA control were quantified using reverse transcriptase–polymerase chain reaction (A) or immunoblotting with the indicated antibodies (B). The relative protein levels from the immunoblotting analysis are also shown. C and D, Protein extracts from chondrocytes transfected with a PTP1B or PTP1B mutant (PTP1Bmut) expression plasmid or a pcDNA control were used in immunoblots with the indicated antibodies (C), and the percentage of apoptotic cells was assessed (D). E and F, Chondrocytes transfected with a control small interfering RNA (siRNA) or a PTP1B siRNA were immunoblotted (E), and the percentage of apoptotic cells was assessed (F). G, Chondrocytes transfected with the PTP1B expression plasmid and the PTP1B siRNA were assayed for viable adherent cell numbers. H–J, Chondrocytes were transfected with a control siRNA or a PTP1B siRNA. Cells were left untreated or were treated with tumor necrosis factor α (TNFα; 10 ng/ml)/actinomycin D (ActD; 0.2 μg/ml), and the percentage of apoptotic cells was assessed (H). Cells were cultured under either low serum conditions (0.5% fetal bovine serum [FBS]) or high serum conditions (10% FBS) (I) or were plated at either low confluence (20%) or full confluence (100%) (J), and apoptosis was assessed. K, Chondrocytes were transfected with the PTPα, PTPγ, or PTP1B expression plasmids. Extracts were immunoblotted, and the percentage of apoptotic cells was assessed. Values are the mean and SD. In A, B, D, F, and G, ∗ = P < 0.05. In H–K, ∗ = P < 0.025.
Download figure to PowerPoint
Since repression of PTP1B by SirT1 was associated with a decrease in apoptosis, it would be expected that increased expression of PTP1B would lead to increased apoptosis. PTP1B was therefore overexpressed in chondrocytes (Figure 4C). When the percentage of apoptotic cells was assayed (Figure 4D), it was clear that PTP1B was a potent proapoptotic protein, leading to a significant increase in chondrocyte cell death. This proapoptotic effect of PTP1B was dependent upon its enzyme activity, since expression of an enzymatically inactive mutant PTP1B in chondrocytes (Figure 4C) had no effect on apoptosis (Figure 4D).
When IGFR was assessed in cells expressing PTP1B, there was a clear down-regulation in levels of pIGFR(Tyr1135/1136), indicating that the phosphatase was effective in dephosphorylating the receptor. In addition, pAkt and pMDM2 levels were significantly decreased in cells expressing PTP1B (Figure 4C), indicating that tyrosine dephosphorylation markedly impaired the activation of Akt and MDM2. Serving as a control, the inactive PTP1B mutant had no effect on levels of pIGFR, pAkt, or pMDM2 (Figure 4C).
To further assess the role of PTP1B in the IGFR pathway, cells were transfected with a PTP1B siRNA. As shown in Figure 4E, PTP1B levels dropped significantly in cells expressing PTP1B siRNA. Correspondingly, phosphorylation of IGFR and Akt increased significantly in cells transfected with the PTP1B siRNA (Figure 4E). The percentage of apoptotic cells and the total number of viable cells following PTP1B siRNA transfection indicated that cell death dramatically declined, while the number of viable cells increased, when PTP1B levels were reduced (Figures 4F and G). Additionally, survival was assessed after PTP1B siRNA transfection and following induction by treatment with TNFα/actinomycin D (Figure 4H) or culture under low serum conditions (0.5%) (Figure 4I) and at low confluence (20%) (Figure 4J). The data showed a reduction in apoptosis under all conditions following PTP1B siRNA transfection and indicate that PTP1B is a potent negative regulator of chondrocyte survival.
We also determined whether other control PTPs were able to induce apoptosis. As shown in Figure 4K, PTPα and PTPγ were efficiently overexpressed in chondrocytes; however, only PTPα was able to induce apoptosis. These data indicate that only some PTPs induce chondrocyte apoptosis and that, of those tested, only PTP1B is regulated by SirT1.
SirT1 and PTP1B show inverse expression patterns in OA and normal cartilage samples.
Recent data indicate that SirT1 protein levels are down-regulated in chondrocytes from OA knee cartilage compared with levels in chondrocytes from normal knee cartilage, as assessed using immunoblotting (9). Immunohistochemical analysis of cartilage sections confirmed this finding. The staining intensity and number of cells staining for SirT1 were reduced in OA cartilage sections compared with the intensity and number of stained cells observed in similar sections from normal cartilage, and there was a significant decrease in the percentage of SirT1-positive cells in OA cartilage (Figure 5A).
Figure 5. SirT1 levels are reduced in osteoarthritic (OA) cartilage, in which protein tyrosine phosphatase 1B (PTP1B) levels are elevated. A–C, Normal and OA human cartilage samples were analyzed by immunohistochemistry to determine the presence of SirT1 (A), PTP1B (B), or matrix metalloproteinase 13 (MMP-13) (C). Images of the intermediate layer of cartilage are shown. Stained cells are reflective of >10-fold intensity above background, as determined using scanning densitometry. An average of 10 fields from 3 sections of 6 separate OA and normal cartilage samples were assessed. Each field was read by 2 individuals under blinded conditions. Immunoblots in B are from freshly isolated chondrocyte extracts from 2 normal and 2 OA cartilage samples probed with the indicated antibodies; the percentages of apoptotic cells in the cartilage samples were assessed by TUNEL assay. Values are the mean and SD. ∗ = P < 0.05. (Original magnification in A and C × 16.)
Download figure to PowerPoint
Since SirT1 represses PTP1B, a decrease in SirT1 levels in OA cartilage should lead to an up-regulation of PTP1B, which our findings confirmed (Figure 5B). The staining intensity and number of stained PTP1B-positive cells was significantly elevated in OA cartilage compared with normal cartilage. PTP1B levels were confirmed by Western blot, which revealed elevated PTP1B levels in freshly isolated chondrocyte extracts from OA patients. As shown in Figure 5B, there was a significant elevation in the percentage of SirT1-postive cells in OA cartilage. When the percentage of apoptotic cells in these sections was assessed, a relationship between PTP1B levels and apoptosis was evident (Figure 5B). MMP-13, serving as a control, was significantly expressed within the ECM in OA samples (Figure 5C) but very little MMP-13 was detected in the normal samples, findings that are consistent with the expression pattern of MMP-13 in OA cartilage, as reported previously (41, 42). Taken together, these data indicate that there is an inverse relationship in the expression patterns of SirT1 and PTP1B in OA and normal articular cartilage; SirT1 levels are high and PTP1B levels are low in normal cartilage, while SirT1 levels are low and PTP1B levels are high in OA cartilage.