Mona Dvir-Ginzberg

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 enhances survival of human osteoarthritic chondrocytes by repressing protein tyrosine phosphatase 1B and activating the insulin-like growth factor receptor pathway.

OBJECTIVE The protein deacetylase SirT1 inhibits apoptosis in a variety of cell systems by distinct mechanisms, yet its role in chondrocyte death has not been explored. We undertook the present study to assess the role of SirT1 in the survival of osteoarthritic (OA) chondrocytes in humans. METHODS SirT1, protein tyrosine phosphatase 1B (PTP1B), and PTP1B mutant expression plasmids as well as SirT1 small interfering RNA (siRNA) and PTP1B siRNA were transfected into primary human chondrocytes. Levels of apoptosis were determined using flow cytometry, and activation of components of the insulin-like growth factor receptor (IGFR)/Akt pathway was assessed using immunoblotting. OA and normal knee cartilage samples were subjected to immunohistochemical analysis. RESULTS Expression of SirT1 in chondrocytes led to increased chondrocyte survival in either the presence or the absence of tumor necrosis factor alpha/actinomycin D, while a reduction of SirT1 by siRNA led to increased chondrocyte apoptosis. Expression of SirT1 in chondrocytes led to activation of IGFR and the downstream kinases phosphatidylinositol 3-kinase, phosphoinosite-dependent protein kinase 1, mTOR, and Akt, which in turn phosphorylated MDM2, inhibited p53, and blocked apoptosis. Activation of IGFR occurs at least in part via SirT1-mediated repression of PTP1B. Expression of PTP1B in chondrocytes increased apoptosis and reduced IGFR phosphorylation, while down-regulation of PTP1B by siRNA significantly decreased apoptosis. Examination of cartilage from normal donors and OA patients revealed that PTP1B levels are elevated in OA cartilage in which SirT1 levels are decreased. CONCLUSION For the first time, it has been demonstrated that SirT1 is a mediator of human chondrocyte survival via down-regulation of PTP1B, a potent proapoptotic protein that is elevated in OA cartilage.

Increased apoptotic chondrocytes in articular cartilage from adult heterozygous SirT1 mice

Objective A growing body of evidence indicates that the protein deacetylase, SirT1, affects chondrocyte biology and survival. This report aims to evaluate in vivo attributes of SirT1 in cartilage biology of 129/J murine strains. Methods Heterozygous haploinsufficient (SirT1+/−) and wild-type (WT; SirT1+/+) 129/J mice aged 1 or 9 months were systematically compared for musculoskeletal features, scored for osteoarthritis (OA) severity, and monitored for chondrocyte apoptosis in articular cartilage. Sections of femorotibial joints were stained for type II collagen and aggrecan. Protein extracts from articular chondrocytes were isolated and immunoblotted for SirT1 and active caspase 3. Results Phenotypic observations show that, at 1 month of age, SirT1+/− mice were smaller than WT and showed a significant decrease in full-length SirT1 (FLSirT1; 110 kDa) protein levels. Levels of FLSirT1 were further decreased in both strains at 9 months. Immunoblot assays for 9-month-old strains revealed the presence of the inactive cleaved SirT1 variant (75 SirT1; 75 kDa) in WT mice, which was undetected in age-matched SirT1+/− mice. Nine-month-old SirT1+/− mice also showed increased OA and increased levels of apoptosis compared with age-matched WT mice. Conclusion The data suggest that the presence of 75 SirT1 may prolong viability of articular chondrocytes in adult (9-month-old) mice.

Tumor necrosis factor α–mediated cleavage and inactivation of SirT1 in human osteoarthritic chondrocytes

OBJECTIVE The protein deacetylase SirT1 positively regulates cartilage-specific gene expression, while the proinflammatory cytokine tumor necrosis factor α (TNFα) negatively regulates these same genes. This study was undertaken to test the hypothesis that SirT1 is adversely affected by TNFα, resulting in altered gene expression. METHODS Cartilage-specific gene expression, SirT1 activity, and results of chromatin immunoprecipitation analysis at the α2(I) collagen enhancer site were determined in RNA, protein extracts, and nuclei of human osteoarthritic chondrocytes left untreated or treated with TNFα. Protein extracts from human chondrocytes transfected with epitope-tagged SirT1 that had been left untreated or had been treated with TNFα were analyzed by immunoblotting with SirT1 and epitope-specific antibodies. The 75-kd SirT1-reactive protein present in TNFα-treated extracts was identified by mass spectroscopy, and its amino-terminal cleavage site was identified via Edman sequencing. SirT1 activity was assayed following an in vitro cathepsin B cleavage reaction. Cathepsin B small interfering RNA (siRNA) was transfected into chondrocytes left untreated or treated with TNFα. RESULTS TNFα-treated chondrocytes had impaired SirT1 enzymatic activity and displayed 2 forms of the enzyme: a full-length 110-kd protein and a smaller 75-kd fragment. The 75-kd SirT1 fragment was found to lack the carboxy-terminus. Cathepsin B was identified as the TNFα-responsive protease that cleaves SirT1 at residue 533. Reducing cathepsin B levels via siRNA following TNFα exposure blocked the generation of the 75-kd SirT1 fragment. CONCLUSION These data indicate that TNFα, a cytokine that mediates joint inflammation in arthritis, induces cathepsin B-mediated cleavage of SirT1, resulting in reduced SirT1 activity. This reduced SirT1 activity correlates with the reduced cartilage-specific gene expression evident in these TNFα-treated cells.

CB2 Cannabinoid Receptor Targets Mitogenic Gi Protein–Cyclin D1 Axis in Osteoblasts

CB2 is a Gi protein–coupled receptor activated by endo‐ and phytocannabinoids, thus inhibiting stimulated adenylyl cyclase activity. CB2 is expressed in bone cells and Cb2 null mice show a marked age‐related bone loss. CB2‐specific agonists both attenuate and rescue ovariectomy‐induced bone loss. Activation of CB2 stimulates osteoblast proliferation and bone marrow derived colony‐forming units osteoblastic. Here we show that selective and nonselective CB2 agonists are mitogenic in MC3T3 E1 and newborn mouse calvarial osteoblastic cultures. The CB2 mitogenic signaling depends critically on the stimulation of Erk1/2 phosphorylation and de novo synthesis of MAP kinase–activated protein kinase 2 (Mapkapk2) mRNA and protein. Further downstream, CB2 activation enhances CREB transcriptional activity and cyclin D1 mRNA expression. The CB2‐induced stimulation of CREB and cyclin D1 is inhibitable by pertussis toxin, the MEK‐Erk1/2 inhibitors PD098059 and U0126, and Mapkapk2 siRNA. These data demonstrate that in osteoblasts CB2 targets a Gi protein–cyclin D1 mitogenic axis. Erk1/2 phosphorylation and Mapkapk2 protein synthesis are critical intermediates in this axis.

Induced differentiation and maturation of newborn liver cells into functional hepatic tissue in macroporous alginate scaffolds

The present work explores cell cultivation in macroporous alginate scaffolds as a means to reproduce hepatocyte terminal differentiation in vitro. Newborn rat liver cell isolates, consisting of proliferating hepatocytes and progenitors, were seeded at high cell density of 125 × 106/cm3 within the scaffold and then cultivated for 6 wk in chemically defined medium. Within 3 days, the alginate‐seeded cells expressed genes for mature liver enzymes, such as trypthophan oxygenase, secreted a high level of albumin, and performed phase I drug metabolism. The cells formed compacted spheroids, establishing homotypic and heterotypic cell‐ to‐cell interactions. By 6 wk, the spheroids developed into organoids, with an external mature hepatocyte layer covered by a laminin layer encasing inner vimen‐ tin‐positive cells within a laminin‐rich matrix also containing collagen. The hepatocytes presented a distinct apical surface between adjacent cells and a basolateral surface with microvilli facing extracellular matrix de‐posits. By contrast, viable adherent cells within collagen scaffolds presenting the identical porous structure did not express adult liver enzymes or secrete albumin after 6 wk. This study thus illustrates the benefits of cell cultivation in macroporous alginate scaffolds as an effective promoter for the maturation of newborn liver cells into functional hepatic tissue, capable of maintaining prolonged hepatocellular functions.—Dvir‐Ginzberg, M., Elkayam, T., Cohen, S. Induced differentiation and maturation of newborn liver cells into functional hepatic tissue in macroporous alginate scaffolds. FASEB J. 22, 1440–1449 (2008)

Sirtuin 1 enzymatic activity is required for cartilage homeostasis in vivo in a mouse model

OBJECTIVE We and others previously demonstrated that sirtuin 1 (SIRT-1) regulates apoptosis and cartilage-specific gene expression in human chondrocytes and mouse models. This study was undertaken to determine if SIRT-1 enzymatic activity plays a protective role in cartilage homeostasis in vivo, by investigating mice with SIRT-1 mutations to characterize their cartilage. METHODS Articular cartilage was harvested from the paws and knees of 5- and 6-month-old wild-type (WT) mice and mice homozygous for SIRT-1tm2.1Mcby (SIRT-1y/y), an allele carrying a point mutation that encodes a SIRT-1 protein with no enzymatic activity (y/y mice). Mice ages 2 days old and 6-7 days old were also examined. Mouse joint cartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures. RESULTS We found that articular cartilage tissue sections from y/y mice of up to 6 months of age contained reduced levels of type II collagen, aggrecan, and glycosaminoglycan compared to sections from WT mice. In contrast, protein levels of matrix metalloproteinase 8 (MMP-8), MMP-9, and MMP-13 were elevated in the cartilage of y/y mice. In addition, chondrocyte apoptosis was elevated in SIRT-1 mutant mice as compared to their WT littermates. Consistent with these observations, protein tyrosine phosphatase 1b was elevated in the y/y mice. CONCLUSION Our in vivo findings in this animal model demonstrate that mice with defective SIRT-1 also have defective cartilage, with elevated rates of cartilage degradation with age. Hence, normal cartilage homeostasis requires enzymatically active SIRT-1 protein.

Sirt1-deficient mice exhibit an altered cartilage phenotype.

OBJECTIVE We previously demonstrated that Sirt1 regulates apoptosis in cartilage in vitro. Here we attempt to examine in vivo cartilage homeostasis, using Sirt1 total body knockout (KO) mice. METHOD Articular cartilage was harvested from hind paws of 1-week and 3-week-old mice carrying wild type (WT) or null Sirt1 gene. Knees of Sirt1 haploinsufficient mice also were examined, at 6 months. Joint cartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures. RESULTS We found that articular cartilage tissue sections from Sirt1 KO mice up to 3 weeks of age exhibited low levels of type 2 collagen, aggrecan, and glycosaminoglycan content. In contrast, protein levels of MMP-13 were elevated in the Sirt1 KO mice, leading to a potential increase of cartilage breakdown, already shown in the heterozygous mice. Additional results showed elevated chondrocyte apoptosis in Sirt1 KO mice, as compared to WT controls. In addition to these observations, PTP1b (protein tyrosine phosphatase b) was elevated in the Sirt1 KO mice, in line with previous reports. CONCLUSION The findings from this animal model demonstrated that Sirt1 KO mice presented an altered cartilage phenotype, with an elevated apoptotic process and a potential degradative cartilage process.

75-kd sirtuin 1 blocks tumor necrosis factor α–mediated apoptosis in human osteoarthritic chondrocytes

OBJECTIVE Sirtuin 1 (SirT1) has been implicated in the regulation of human cartilage homeostasis and chondrocyte survival. Exposing human osteoarthritic (OA) chondrocytes to tumor necrosis factor α (TNFα) generates a stable and enzymatically inactive 75-kd form of SirT1 (75SirT1) via cathepsin B-mediated cleavage. Because 75SirT1 is resistant to further degradation, we hypothesized that it has a distinct role in OA, and the present study was undertaken to identify this role. METHODS The presence of cathepsin B and 75SirT in OA and normal human chondrocytes was analyzed. Confocal imaging of SirT1 was used to monitor its subcellular trafficking following TNFα stimulation. Coimmunofluorescence staining for cathepsin B, mitochondrial cytochrome oxidase subunit IV, and lysosome-associated membrane protein 1 together with SirT1 was performed. Human chondrocytes were tested for apoptosis by fluorescence-activated cell sorter analysis and immunoblotting for caspases 3 and 8. Human chondrocyte mitochondrial extracts were obtained and analyzed for 75SirT1-cytochrome c association. RESULTS Confocal imaging and immunoblot analyses following TNFα challenge of human chondrocytes demonstrated that 75SirT1 was exported to the cytoplasm and colocalized with the mitochondrial membrane. Consistent with this, immunoprecipitation and immunoblot analyses revealed that 75SirT1 is enriched in mitochondrial extracts and associates with cytochrome c following TNFα stimulation. Preventing nuclear export of 75SirT1 or reducing levels of full-length SirT1 and 75SirT1 augmented chondrocyte apoptosis in the presence of TNFα. Levels of cathepsin B and 75SirT1 were elevated in OA versus normal chondrocytes. Additional analyses showed that human chondrocytes exposed to OA-derived synovial fluid generated the 75SirT1 fragment. CONCLUSION These data suggest that 75SirT1 promotes chondrocyte survival following exposure to proinflammatory cytokines.

A chronic low dose of [Delta]9-tetrahydrocannabinol (THC) restores cognitive function in old mice

The balance between detrimental, pro-aging, often stochastic processes and counteracting homeostatic mechanisms largely determines the progression of aging. There is substantial evidence suggesting that the endocannabinoid system (ECS) is part of the latter system because it modulates the physiological processes underlying aging. The activity of the ECS declines during aging, as CB1 receptor expression and coupling to G proteins are reduced in the brain tissues of older animals and the levels of the major endocannabinoid 2-arachidonoylglycerol (2-AG) are lower. However, a direct link between endocannabinoid tone and aging symptoms has not been demonstrated. Here we show that a low dose of Δ9-tetrahydrocannabinol (THC) reversed the age-related decline in cognitive performance of mice aged 12 and 18 months. This behavioral effect was accompanied by enhanced expression of synaptic marker proteins and increased hippocampal spine density. THC treatment restored hippocampal gene transcription patterns such that the expression profiles of THC-treated mice aged 12 months closely resembled those of THC-free animals aged 2 months. The transcriptional effects of THC were critically dependent on glutamatergic CB1 receptors and histone acetylation, as their inhibition blocked the beneficial effects of THC. Thus, restoration of CB1 signaling in old individuals could be an effective strategy to treat age-related cognitive impairments.