ABSTRACT
Periodontitis is an inflammatory disease that affects tooth-supporting tissues. Chronic inflammation can progress to periodontitis, which results in loss of alveolar bone. Asarylaldehyde is a potential substance for bone metabolism present in natural compounds. Here, we propose the application of asarylaldehyde in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) to prevent bone loss. We investigated the effect of asarylaldehyde on hPDLSCs together with bone differentiation media in vitro. The osteogenic differentiation effect was observed after treatment of hPDLSCs with several concentrations of asarylaldehyde. After 21 days, osteogenic cells were identified by mineralization. We also observed that asarylaldehyde increased the mRNA expression of osteoblast-specific markers in hPDLSCs. Interestingly, asarylaldehyde regulated the levels of alkaline phosphatase (ALP) transcriptional activity through the p38/extracellular-signal-regulated kinase (ERK) signaling pathway. Notably, asarylaldehyde induced hPDLSCs to promote osteogenic differentiation. These results suggest that asarylaldehyde plays a key role in the osteogenic differentiation of hPDLSCs. Asarylaldehyde may be a good candidate for the application of natural compounds in future in periodontal regeneration.
Subject(s)
Aldehydes/pharmacology , Osteogenesis/drug effects , Periodontal Ligament/drug effects , Stem Cells/drug effects , Aldehydes/administration & dosage , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Gene Expression/drug effects , Humans , MAP Kinase Signaling System/drug effects , Osteogenesis/genetics , Osteogenesis/physiology , Periodontal Ligament/cytology , Periodontal Ligament/metabolism , Periodontitis/drug therapy , Periodontitis/pathology , Periodontitis/physiopathology , Phytotherapy , Regeneration/drug effects , Regeneration/physiology , Stem Cells/cytology , Stem Cells/metabolismABSTRACT
Berberine is a natural isoquinoline alkaloid present in various herbs and is effective against metabolic syndrome in the pre-diabetic stage and high insulin resistance. The present study aimed to determine the effectiveness of WJCPR11, a berberine derivative that is commonly used for diabetes treatment, in ameliorating insulin resistance and diabetes treatment. WJCPR11 promoted adipocyte differentiation to a higher extent than other berberine derivatives and showed no noticeable toxicity in its effective concentration range. It increased the mRNA expression levels and protein abundance of adipogenic markers, including peroxisome proliferator-activated receptor γ (PPARγ), glucose transporter type 4 (GluT4), and fatty acid synthase (FAS), and markedly enhanced the level of adiponectin, a distinct marker of insulin sensitivity. Meanwhile, the mRNA levels of inflammatory markers such as plasminogen activator inhibitor-1 (PAI-1), monocyte chemoattractant protein-1 (MCP-1), and interleukin 6 (IL-6) were reduced after WJCPR11 treatment. Furthermore, the tumor necrosis factor-α (TNF-α)-induced inhibition of adipocyte differentiation and downregulation of glucose uptake were markedly reversed by WJCPR11 treatment. Collectively, the findings of this study indicate that WJCPR11 has great potential for diabetes treatment.
Subject(s)
Adipocytes/cytology , Berberine/analogs & derivatives , Glucose/metabolism , Prediabetic State/drug therapy , Tumor Necrosis Factor-alpha/metabolism , Adipocytes/drug effects , Adipocytes/metabolism , Animals , Berberine/pharmacology , Cell Differentiation/physiology , Cells, Cultured , Insulin Resistance , Mice , Prediabetic State/metabolism , Prediabetic State/pathologyABSTRACT
Peroxisome proliferator-activated receptor gamma (PPARγ) is a master transcription factor in adipocyte differentiation, while distal-less homeobox 5 (Dlx5) is essential for initiating osteoblast differentiation by driving Runt-related transcription factor 2 expression. Considering that adipocytes and osteoblasts share common progenitors, there is a reciprocal correlation between bone and fat formation. However, the mechanism by which Dlx5 controls PPARγ remains unclear. We elucidated that Dlx5 physically binds to PPARγ during immunoprecipitation; in particular, the ligand-binding and DNA-binding domains of PPARγ were involved in the interaction. Transcriptional activity of PPARγ was significantly decreased by Dlx5 overexpression, whereas the opposite results were detected with Dlx5 knockdown. Rosiglitazone, a PPARγ agonist, further enhanced the PPARγ-induced transcriptional activity; however, Dlx5 overexpression effectively repressed the rosiglitazone-mediated increase in activity. Finally, DNA-binding affinity assay revealed that Dlx5 interrupts the interaction of PPARγ with the PPARγ response element promoter. In conclusion, our findings indicate that Dlx5 impedes PPARγ-induced activity, and it may be useful for managing diabetes drug-mediated obesity.
Subject(s)
Homeodomain Proteins/metabolism , PPAR gamma/metabolism , 3T3-L1 Cells , Adipocytes/physiology , Animals , Cell Differentiation/drug effects , Cell Differentiation/genetics , Mice , PPAR gamma/agonists , Promoter Regions, Genetic , Protein Domains , Rosiglitazone/pharmacology , Transcriptional ActivationABSTRACT
Berberine is an essential phytochemical for the treatment of various diseases; however, when used to treat osteoporosis, it has minor effect as compared with that of the currently available drugs. This study aimed to find a new compound that would have a better anti-osteoporotic effect than that of berberine. Based on structure and activity relationship study, we identified compound 2c, a berberine derivative, to be the most potent compound to affect osteoblast differentiation. Compound 2c was more effective than berberine and exhibited no toxicity within its effective concentration. Compound 2c increased, in a dose-dependent manner, ALP activity during osteoblast differentiation and enhanced the mRNA expression of osteogenic factors including ALP, Runx2, and Osterix. Furthermore, compound 2c increased the transcriptional activity induced by BMP4 on the ALP and BSP promoter. Taken together, compound 2c shows promise as a therapeutic agent for osteoporosis by promoting osteoblast differentiation.
Subject(s)
Berberine/analogs & derivatives , Berberine/pharmacology , Osteogenesis/drug effects , Animals , Cell Differentiation/drug effects , Cell Line , Gene Expression Regulation/drug effects , Mice , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoblasts/metabolismABSTRACT
2-Hydroxymelatonin is a metabolite produced when melatonin 2-hydroxylase catalyzes melatonin. Recent studies have reported the important roles of melatonin in bone metabolism. However, the roles of 2-hydroxymelatonin in bone metabolism remains poorly understood. The purpose of this study is to present evidence of the effect of 2-hydroxymelatonin on osteogenic differentiation in C2C12 cells. In this study, we demonstrated the synergistic stimulating effect of 2-hydroxymelatonin and bone morphogenetic protein (BMP)-4 on osteogenic differentiation in vitro, using alkaline phosphatase (ALP) staining, Alizarin red S (ARS) staining, qPCR, and luciferase reporter assay. The combination of 2-hydroxymelatonin and BMP-4 revealed a synergistic effect on osteogenic differentiation in vitro. This finding provides evidence that optimal concentrations of both 2-hydroxymelatonin and BMP-4 are beneficial for anabolic effects on bone in vitro.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Melatonin/analogs & derivatives , Osteogenesis/drug effects , Alkaline Phosphatase/genetics , Animals , Cell Differentiation/drug effects , Cell Line , Gene Expression Regulation, Developmental/drug effects , Melatonin/pharmacology , Mice , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoblasts/metabolismABSTRACT
Previous studies have suggested that platycodin D is implicated in bone biology and ameliorates osteoporosis development. Platycodin D repressed the osteoclast activity and enhanced bone mineral density in the mouse model. However, the effects of platycodin D on osteoblast differentiation have not been elucidated yet. In C3H10T1/2 cells, platycodin D upregulated osteogenic markers including alkaline phosphatase (ALP), bone sialoprotein, and collagen type 1 alpha 1, and transcription factors, such as Runx2 and osterix, subsequently enhancing the bone mineralization. In a molecular mechanism study, platycodin D induced ß-catenin nuclear accumulation by upregulating GSK3ß phosphorylation. Furthermore, platycodin D upregulated the ALP activity and enhanced the mineralization process in osteoblast cells via the sirtuin 1/ß-catenin pathways. Taken together, these results suggested that platycodin D could be an effective therapeutic compound against osteoporosis because of its regulatory effects during the osteoblast differentiation.
Subject(s)
Biomarkers/metabolism , Cell Differentiation/drug effects , Mesenchymal Stem Cells/drug effects , Osteoblasts/drug effects , Osteogenesis/drug effects , Saponins/pharmacology , Triterpenes/pharmacology , Alkaline Phosphatase/metabolism , Animals , Calcification, Physiologic/drug effects , Cell Line , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Integrin-Binding Sialoprotein/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mice , Molecular Structure , Osteoblasts/cytology , Saponins/chemistry , Transcription Factors/metabolism , Triterpenes/chemistry , Wnt Signaling Pathway/drug effects , beta Catenin/metabolismABSTRACT
Pseudoshikonin I (PSI), a novel biomaterial isolated from Lithospermi radix, has been recognized as an herbal medicine for the treatment of infectious and inflammatory diseases. Bone remodeling maintains a balance through bone resorption (osteoclastogenesis) and bone formation (osteoblastogenesis). Bone formation is generally attributed to osteoblasts. However, the effects of PSI on the bone are not well known. In this study, we found that the ethanol extracts of PSI induced osteoblast differentiation by increasing the expression of bone morphogenic protein 4 (BMP 4). PSI positively regulates the transcriptional expression and osteogenic activity of osteoblast-specific transcription factors such as Runx2 and Osterix. To identify the signaling pathways that mediate PSI-induced osteoblastogenesis, we examined the effects of serine-threonine kinase inhibitors that are known regulators of Osterix and Runx2. PSI-induced upregulation of Osterix and Runx2 was suppressed by treatment with AKT and PKA inhibitors. These results suggest that PSI enhances osteoblast differentiation by stimulating Osterix and Runx2 via the AKT and PKA signaling pathways. Thus, the activation of Runx2 and Osterix is modulated by PSI, thereby demonstrating its potential as a treatment target for bone disease.
Subject(s)
Core Binding Factor Alpha 1 Subunit/genetics , Ethanol/pharmacology , Lithospermum/chemistry , Osteoblasts/cytology , Sp7 Transcription Factor/genetics , Animals , Bone Morphogenetic Protein 4/metabolism , Bone Remodeling , Cell Differentiation/drug effects , Cell Line , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Mice , Naphthoquinones/chemistry , Osteoblasts/drug effects , Osteoblasts/metabolism , Plant Extracts/pharmacology , Sp7 Transcription Factor/metabolism , Transcription, Genetic/drug effectsABSTRACT
Berberine has been implicated to be involved in maintaining bone health due to its anti-oxidative and osteogenic properties. However, low potency and low bioavailability limit the clinical development of the drug. To overcome these obstacles, we previously synthesized a compound, Q8, which is a structural homolog of berberine. The present study examined the pharmacological functions of Q8 to evaluate its potential use in bone regeneration with respect to osteoblast differentiation. Here, we report that Q8 enhanced BMP4-induced alkaline phosphatase (ALP) activity and transcription from the ALP promoter. In addition, Q8 suppressed the expression and activity of PPARγ (a known negative regulator of osteogenesis due to its stimulatory effects on adipogenesis and its role as an adipogenic transcription factor), which in turn increases ß-catenin expression in the nucleus, and ultimately promotes osteoblast differentiation. Meanwhile, Q8 reversed the inhibitory effects of the PPARγ agonist, rosiglitazone, on osteoblast differentiation. This study demonstrated that Q8 promotes osteoblast differentiation via inhibition of PPARγ and the enhancement of osteoblast function by Q8 may contribute to the prevention for osteoporosis.
Subject(s)
Berberine/pharmacology , Osteogenesis/drug effects , Animals , Berberine/analogs & derivatives , Cell Differentiation , Cell Line , Glycogen Synthase Kinase 3 beta/metabolism , Mice , Myoblasts/metabolism , Osteoblasts/metabolism , Osteoporosis , PPAR gamma/metabolism , Phosphorylation , Rosiglitazone/pharmacologyABSTRACT
Platycodon grandiflorum root-derived saponins (Changkil saponins, CKS) are reported to have many pharmacological activities. In our latest research, CKS was proven to have a significant osteogenic effect. However, the detail molecular mechanism of CKS on osteoclastic differentiation has not been fully investigated. Administration of CKS considerably reduced OVX-induced bone loss, and ameliorated the reduction in plasma levels of alkaline phosphatase, calcium, and phosphorus observed in OVX mice. CKS also repressed the deterioration of bone trabecular microarchitecture. Interestingly, platycodin D, the most abundant and major pharmacological constituent of triterpenoid CKS, inhibited receptor activator of NF-κB ligand (RANKL)-induced activation of NF-κB, and ERK and p38 MAPK, ultimately repressing osteoclast differentiation. OVX-induced bone turnover was attenuated by CKS, possibly via repression of osteoclast differentiation by platycodin D, the active component of CKS. Platycodin D can be regarded as an antiosteoporotic candidate for treatment of osteoporosis diseases. J. Cell. Biochem. 118: 860-868, 2017. © 2016 Wiley Periodicals, Inc.
Subject(s)
MAP Kinase Signaling System/drug effects , NFATC Transcription Factors/metabolism , Osteogenesis/drug effects , Osteogenesis/physiology , Saponins/pharmacology , Triterpenes/pharmacology , Animals , Cell Differentiation/drug effects , Disease Models, Animal , Female , Humans , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred ICR , NF-kappa B/metabolism , Osteoclasts/cytology , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteoporosis, Postmenopausal/drug therapy , Osteoporosis, Postmenopausal/metabolism , Osteoporosis, Postmenopausal/pathology , Ovariectomy , Proto-Oncogene Proteins c-akt/metabolism , RANK Ligand/metabolism , RAW 264.7 Cells , Signal Transduction/drug effects , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The Q8 compound is a unique derivative of berberine. The present study investigated the functional role of Q8 to evaluate its potential for use in bone regeneration, especially in osteoblast differentiation. The safe concentration of Q8 increased BMP4-induced alkaline phosphatase (ALP) activity, and induced RNA expression of ALP, bone sialoprotein (BSP), and osteocalcin (OC). The activities of ALP-, BSP- and OC-luciferase reporters were also increased by Q8. During osteoblast differentiation, Q8 stabilized the Runx2 and Osterix protein abundance by blocking the ubiquitin-proteasome pathway, which in turn promoted Runx2 and Osterix induced transcriptional activity and subsequently increased the osteoblast differentiation. Meanwhile, depletion of Runx2 and Osterix markedly abolished the bone anabolic effect of Q8 on osteoblast differentiation. To evaluate the signal transduction pathway involved in the Q8-mediated regulation of Runx2 and Osterix, we examined the reporter assay using various kinase inhibitors. Treatment with a protein kinase A (PKA) inhibitor, H89 inhibited the Q8-mediated regulation of Runx2 and Osterix. Based on these findings, this study demonstrates that Q8 promotes the osteoblast differentiation by stabilization of Runx2/Osterix through the increased activation of PKA signaling. The enhancement of osteoblast function by Q8 may contribute to the prevention for osteoporosis.
Subject(s)
Berberine/analogs & derivatives , Berberine/pharmacology , Osteoblasts/cytology , Osteoblasts/drug effects , Osteogenesis/drug effects , Animals , Cell Differentiation/drug effects , Core Binding Factor Alpha 1 Subunit/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , HEK293 Cells , Humans , Mice , Osteoblasts/metabolism , Phosphorylation/drug effects , Protein Stability/drug effects , Sp7 Transcription Factor , Transcription Factors/metabolismABSTRACT
Osterix is an essential transcription factor for osteogenesis and is expressed in osteoblasts. Although Osterix has been shown to be induced by bone morphogenetic protein 4, the molecular mechanism underlying Osterix function during osteoblast differentiation remains unclear. Connexin43 (Cx43) is the most abundant gap junction protein in bone cells and plays a critical role in osteoblast differentiation. However, little is known about the functional interactions between Osterix and the Cx43 promoter. In the present study, we investigated the relationship between Osterix and Cx43 in HEK293 and C2C12 cells. Cx43 expression was significantly repressed by the addition of shRNA against Osterix, whereas overexpression of Osterix resulted in enhanced Cx43 expression. Furthermore, Osterix directly occupied the promoter region of Cx43 and subsequently increased Cx43 promoter activity in a dose-dependent manner. In addition, phosphorylation of the Ser76 and Ser80 residues in Osterix were found to be critical for its activity on the Cx43 promoter. Our results suggest that Osterix plays an important role in increasing bone morphogenetic protein 4-induced Cx43 activity.
Subject(s)
Bone Morphogenetic Protein 4/genetics , Connexin 43/genetics , Osteoblasts/metabolism , Transcription Factors/genetics , Animals , Bone Morphogenetic Protein 4/metabolism , Cell Line , Connexin 43/metabolism , Gene Expression Regulation , Genes, Reporter , HEK293 Cells , Humans , Luciferases/genetics , Luciferases/metabolism , Mice , Models, Biological , Myoblasts/cytology , Myoblasts/metabolism , Osteoblasts/cytology , Plasmids/chemistry , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Sp7 Transcription Factor , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , TransfectionABSTRACT
SIRT2 is a member of the sirtuin family of NAD(+)-dependent protein deacetylases. It is involved in metabolic homeostasis and has been linked to the progression of age-related diseases. Casitas B-lineage lymphoma (Cbl) proteins regulate signal transduction through many pathways and, consequently, regulate cell function and development. Cbl proteins are ubiquitin ligases that ubiquitinate and target many signaling molecules for degradation. The function of SIRT2 is modulated by post-translational modifications. However, the precise molecular signaling mechanism of SIRT2 through interactions with Cbl proteins has not yet been established. In this study, we investigated the potential regulation of SIRT2 function by the Cbl mammalian family members Cbl-b and c-Cbl. We found that Cbl-b and c-Cbl increased the protein level and stability of SIRT2 and that Cbl-b and c-Cbl interact with SIRT2. They were also found to regulate the deacetylase activity of SIRT2. Further investigation revealed that Cbl-mediated SIRT2 regulation occurred via ubiquitination of SIRT2.
Subject(s)
Proto-Oncogene Proteins c-cbl/physiology , Sirtuin 2/physiology , Cell Line , Humans , Protein Processing, Post-Translational , Tumor Suppressor Protein p53/metabolism , UbiquitinationABSTRACT
Dlx5 transcription factor plays important roles in osteoblast differentiation and its transcription is regulated by many osteogenic signals including BMP-2. Recent studies suggest that the function of Dlx5 is also regulated post-translationally by protein kinases such as p38 and CaMKII. Protein kinase A (PKA) is involved in several steps of osteoblast differentiation and its activity has been shown necessary, yet not sufficient, for BMP-induced osteoblast differentiation. PKA is a ubiquitous cellular kinase that phosphorylates serine and threonine residues(s) of target proteins. In this study, we investigated the potential regulation of Dlx5 function by PKA in osteoblast differentiation. We found that PKA phosphorylates Dlx5 and that PKA activation increases the protein stability, osteogenic activity and transcriptional activity of Dlx5. We also found that BMP-2 increases the protein level of Dlx5 in a PKA activity-dependent manner. These results suggest that PKA activity enhances the osteogenic function of Dlx5, at least in part, through protein stabilization and that BMP-2 regulates the osteogenic function of Dlx5, at least in part, through PKA.
Subject(s)
Cell Differentiation , Cyclic AMP-Dependent Protein Kinases/metabolism , Homeodomain Proteins/metabolism , Osteoblasts/cytology , Osteogenesis , Animals , Bone Morphogenetic Protein 2/metabolism , Cell Differentiation/genetics , Cell Line , Homeodomain Proteins/genetics , Humans , Mice , Osteoblasts/metabolism , Phosphorylation , Protein Processing, Post-Translational , Protein Stability , Transcription, GeneticABSTRACT
BACKGROUND/PURPOSE: Periodontal ligament stem cells (PDLSCs)-based regeneration therapy has received attention for its potential alternative applications in hard tissue and tooth. However, the environmental diversity of oral cavity that regulates PDLSCs differentiation has made it difficult to develop. Therefore, we investigated how high calcium concentrations in the oral environment influence osteogenic differentiation of human PDLSCs (hPDLSCs). MATERIALS AND METHODS: hPDLSCs collected from human molars were isolated and cultured with CaCl2. First, multi lineage differentiation potentials to osteogenic, chondrogenic, and adipogenic cells were investigated. Then, the effects of CaCl2 on both alkaline phosphatase (ALP) activity and bone mineralization were analyzed and the expression of mRNA and protein for osteogenic marker was explored. Further, luciferase assay was performed to evaluate CaCl2 could regulate the transcriptional activity on osteogenic differentiation in hPDLSCs. RESULTS: CaCl2 treatment at normal to high concentrations showed similar suppression of ALP activity, while mineralized nodule formation was decreased by CaCl2 treatment dose-dependently without affecting proliferation or cytotoxicity in hPDLSCs. We also observed that CaCl2 treatment repressed the mRNA expression and protein abundance of osteogenic genes and transcriptional factors. Notably, repression of the Runx2 level was significant, and CaCl2 treatment inhibited Runx2-mediated transcriptional activity on the osteoblast-specific element (OSE) and ALP promoters. CONCLUSION: High concentrations of calcium negatively regulate osteogenic differentiation of hPDLSCs, by repressing osteogenic gene expressions and transcriptional activity. Therefore, these conditions may be applicable to determine the physiologically appropriate concentration of calcium.
ABSTRACT
Protein acetyltransferases and deacetylases affect the activities of each other. This is well documented by the acetylation and inhibition of HDAC1 by p300, a transcriptional co-activator with protein acetyltransferase activity. However, the relationship between HDAC6 and p300 is poorly understood. HDAC6 is a class II histone deacetylase and differs from other members of HDAC family in that it contains two HDAC domains and an ubiquitin-binding motif. HDAC6 is a microtubule-associated deacetylase. It predominantly deacetylates non-histone proteins, including alpha-tubulin, and regulates cell motility. Here, we report that p300 interacts with and acetylates HDAC6 resulting down-regulation of HDAC6 deacetylase activity. Furthermore, we provide evidences that acetylation of HDAC6 by p300 inhibits tubulin deacetylation and suppression of Sp1 transcriptional activity by HDAC6. Our results demonstrate that p300 can inactivate HDAC6 by acetylation, and that p300 may regulate the activity of Sp1 indirectly through HDAC6 in addition to its direct modification of Sp1.
Subject(s)
Histone Deacetylases/metabolism , p300-CBP Transcription Factors/metabolism , Acetylation , Cell Line , Gene Expression Regulation , Genes, Reporter , Histone Deacetylase 6 , Histone Deacetylases/genetics , Humans , Luciferases/chemistry , Protein Interaction Mapping , Sp1 Transcription Factor/metabolism , rho GTP-Binding Proteins/geneticsABSTRACT
Histone deacetylases (HDACs) are subdivided into three classes--HDAC I, HDAC II, and Sir2. Sirt proteins are mammalian members of the Sir2 family of NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacetylases. The balance between acetylation and deacetylation of histone and non-histone proteins, regulated by protein acetyltransferases and deacetylases, affects the expression of genes involved in a variety of cellular processes. In addition, HDAC1 is acetylated and regulated by p300, a transcriptional co-activator with protein acetyltransferase activity, suggesting that protein acetyltransferases and deacetylases they control the activities of each other. Although the regulation of HDAC1 by p300 is well characterized, the relationship between Sir2 homologs and p300 is not understood. Here, we report that p300 interacts with Sirt2, a member of the Sir2 family, and triggers the acetylation and subsequent down-regulation of the deacetylation activity of Sirt2, and that the acetylation of Sirt2 by p300 relieves the inhibitory effect of Sirt2 on the transcriptional activity of p53. These observations demonstrate that p300 can inactivate Sirt2 by acetylation and that p300 may regulate the activity of p53 indirectly through Sirt2 in addition to its direct modification of p53.
Subject(s)
Histone Deacetylase Inhibitors , Histone Deacetylases/metabolism , Sirtuins/antagonists & inhibitors , Sirtuins/metabolism , p300-CBP Transcription Factors/metabolism , Acetylation , Cell Line , Down-Regulation , Humans , Protein Interaction Mapping , Protein Structure, Tertiary , Sirtuin 2 , Sirtuins/genetics , Transcription, Genetic , Tumor Suppressor Protein p53/metabolism , p300-CBP Transcription Factors/geneticsABSTRACT
Although the biological role of melatonin in osteogenic differentiation has been suggested, the mechanism of osteoblast differentiation remains unclear. Thus, the present study investigated the underlying molecular mechanisms based on osteoblast-specific transcription factors. We found that melatonin enhanced BMP-4-induced osteogenic differentiation and increased the expression of osteogenic markers, especially Osterix, which is an essential transcription factor for the differentiation of preosteoblasts into mature osteoblasts in the late stage of osteoblast differentiation. Melatonin treatment increased the expression of Osterix during osteoblast differentiation and stabilized its expression by the inhibition of ubiquitin-proteasome-mediated degradation of Osterix, leading to up-regulated Osterix transcriptional activity on the osteogenic promoter and promoting alkaline phosphatase activity and bone mineralization. Furthermore, treatment with protein kinase A (PKA) inhibitor H89 and protein kinase C (PKC) inhibitor Go6976 blocked the melatonin-induced transcriptional activity and phosphorylation of Osterix, indicating that melatonin regulates Osterix expression via the PKA and PKC signaling pathways. Overall, these findings suggest that melatonin directly regulates the late stage of osteoblast differentiation by enhancing Osterix expression; this provides further evidence of melatonin as a potent agent for treating osteoporosis.
Subject(s)
Melatonin/pharmacology , Osteoblasts/drug effects , Protein Stability/drug effects , Animals , Bone Morphogenetic Protein 4 , Carbazoles/pharmacology , Cell Differentiation/drug effects , Cell Line , Enzyme Inhibitors/pharmacology , Isoquinolines/pharmacology , Mice , Osteogenesis/drug effects , Sp7 Transcription Factor/genetics , Sp7 Transcription Factor/metabolism , Sulfonamides/pharmacology , Transcription FactorsABSTRACT
Chalcones, the biosynthetic precursors of flavonoids and isoflavonoids abundant in edible plants, possess a number of pharmacological properties, and there is growing evidence that chalcone derivatives inhibit TNF-α mediated insulin resistance. The aim of the present study was to define the effects of 4-methoxychalcone (4-MC) on adipocyte differentiation and to determine the underlying molecular mechanism. We investigated the effects of 4-MC on adipocyte differentiation and lipid accumulation, and expression of adipogenic genes in 3T3-L1 cells. Additionally, treatment with 4-MC significantly increased the PPARγ-induced transcriptional activity and 4-MC also enhanced the DNA binding affinity of PPARγ to the proliferator-activated receptor response elements (PPRE) at target promoters. Next, we tested the effect of 4-MC on the inhibition induced by TNF-α on adipocyte differentiation. Treatment with 4-MC enhanced the lipid accumulation and strongly up-regulated the expression of adipogenic markers, including PPARγ, aP2, FAS, and adiponectin during adipocyte differentiation. Finally, 4-MC attenuated the inhibitory effect of TNF-α on adipocyte differentiation and adiponectin expression and subsequently regulated the expression and secretion of various adipokines that are involved in insulin sensitivity. This study clearly demonstrates that 4-MC enhanced adipocyte differentiation, in part, by its potent effects on PPARγ activation and by its reverse effect on TNF-α.
Subject(s)
Adipocytes/drug effects , Adipogenesis/drug effects , Chalcones/pharmacology , PPAR gamma/metabolism , Tumor Necrosis Factor-alpha/metabolism , Adipocytes/cytology , Adipocytes/metabolism , Animals , Cell Differentiation , Mice , PPAR gamma/genetics , Response Elements/drug effects , Transcriptional Activation/drug effects , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Osterix is a novel bone-related transcription factor involved in osteoblast differentiation, and bone maturation. Because a reciprocal relationship exists between adipocyte and osteoblast differentiation of bone marrow derived mesenchymal stem cells, we hypothesized that Osterix might have a role in adipogenesis. Ablation of Osterix enhanced adipogenesis in 3T3-L1 cells, whereas overexpression suppressed this process and inhibited the expression of adipogenic markers including CCAAT/enhancer-binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). Further studies indicated that Osterix significantly decreased PPARγ-induced transcriptional activity. Using co-immunoprecipitation and GST-pull down analysis, we found that Osterix directly interacts with PPARγ. The ligand-binding domain (LBD) of PPARγ was responsible for this interaction, which was followed by repression of PPARγ-induced transcriptional activity, even in the presence of rosiglitazone. Taken together, we identified the Osterix has an important regulatory role on PPARγ activity, which contributed to the mechanism of adipogenesis.
Subject(s)
Adipocytes/physiology , Bone Marrow Cells/physiology , Mesenchymal Stem Cells/physiology , Osteoblasts/physiology , PPAR gamma/metabolism , Sp7 Transcription Factor/metabolism , Adipogenesis , Animals , Cell Differentiation , Cell Line , Mice , PPAR gamma/genetics , Protein Binding , RNA, Small Interfering/genetics , Sp7 Transcription Factor/genetics , Transcription, GeneticABSTRACT
Estrogen receptor α (ER-α), which is involved in bone metabolism and breast cancer, has been shown to have transcriptional targets. Dlx3 is essential for the skeletal development and plays an important role in osteoblast differentiation. Various osteogenic stimulators and transcription factors can induce the protein expression of Dlx3. However, the regulatory function of ER-α in the Dlx3 mediated osteogenic process remains unknown. Therefore, we investigated the regulation of Dlx3 and found that ER-α is a positive regulator of Dlx3 transcription in BMP2-induced osteoblast differentiation. We also found that ER-α interacts with Dlx3 and increases its transcriptional activity and DNA binding affinity. Furthermore, we demonstrated that the regulation of Dlx3 activity by ER-α is independent of the ligand (estradiol) binding domain. These results indicate that Dlx3 is a novel target of ER-α, and that ER-α regulates the osteoblast differentiation through modulation of Dlx3 expression and/or interaction with Dlx3.