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Anne M Delany - One of the best experts on this subject based on the ideXlab platform.
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a single nucleotide polymorphism in Osteonectin 3 untranslated region regulates bone volume and is targeted by mir 433
2015Co-Authors: Neha S Dole, Catherine B Kessler, Douglas J Adams, Kristina Kapinas, Renata C Pereira, Anne M DelanyAbstract:: Osteonectin/SPARC is one of the most abundant noncollagenous extracellular matrix proteins in bone, regulating collagen fiber assembly and promoting osteoblast differentiation. Osteonectin-null and haploinsufficient mice have low-turnover osteopenia, indicating that Osteonectin contributes to normal bone formation. In male idiopathic osteoporosis patients, Osteonectin 3' untranslated region (UTR) single-nucleotide polymorphism (SNP) haplotypes that differed only at SNP1599 (rs1054204) were previously associated with bone mass. Haplotype A (containing SNP1599G) was more frequent in severely affected patients, whereas haplotype B (containing SNP1599C) was more frequent in less affected patients and healthy controls. We hypothesized that SNP1599 contributes to variability in bone mass by modulating Osteonectin levels. Osteonectin 3' UTR reporter constructs demonstrated that haplotype A has a repressive effect on gene expression compared with B. We found that SNP1599G contributed to an miR-433 binding site, and miR-433 inhibitor relieved repression of the haplotype A, but not B, 3' UTR reporter construct. We tested our hypothesis in vivo, using a knock-in approach to replace the mouse Osteonectin 3' UTR with human haplotype A or B 3' UTR. Compared with haplotype A mice, bone Osteonectin levels were higher in haplotype B mice. B mice displayed higher bone formation rate and gained more trabecular bone with age. When parathyroid hormone was administered intermittently, haplotype B mice gained more cortical bone area than A mice. Cultured marrow stromal cells from B mice deposited more mineralized matrix and had higher osteocalcin mRNA compared with A mice, demonstrating a cell-autonomous effect on differentiation. Altogether, SNP1599 differentially regulates Osteonectin expression and contributes to variability in bone mass, by a mechanism that may involve differential targeting by miR-433. This work validates the findings of the previous candidate gene study, and it assigns a physiological function to a common Osteonectin allele, providing support for its role in the complex trait of skeletal phenotype. © 2014 American Society for Bone and Mineral Research.
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bone matrix Osteonectin limits prostate cancer cell growth and survival
2012Co-Authors: Kristina Kapinas, Catherine B Kessler, Katie M Lowther, Karissa Tilbury, Jay R Lieberman, Jennifer S Tirnauer, Paul J Campagnola, Anne M DelanyAbstract:There is considerable interest in understanding prostate cancer metastasis to bone and the interaction of these cells with the bone microenvironment. Osteonectin/SPARC/BM-40 is a collagen binding matricellular protein that is enriched in bone. Its expression is increased in prostate cancer metastases, and it stimulates the migration of prostate carcinoma cells. However, the presence of Osteonectin in cancer cells and the stroma may limit prostate tumor development and progression. To determine how bone matrix Osteonectin affects the behavior of prostate cancer cells, we modeled prostate cancer cell-bone interactions using the human prostate cancer cell line PC-3, and mineralized matrices synthesized by wild type and Osteonectin-null osteoblasts in vitro. We developed this in vitro system because the structural complexity of collagen matrices in vivo is not mimicked by reconstituted collagen scaffolds or by more complex substrates, like basement membrane extracts. Second harmonic generation imaging demonstrated that the wild type matrices had thick collagen fibers organized into longitudinal bundles, whereas Osteonectin-null matrices had thinner fibers in random networks. Importantly, a mouse model of prostate cancer metastases to bone showed a collagen fiber phenotype similar to the wild type matrix synthesized in vitro. When PC-3 cells were grown on the wild type matrices, they displayed decreased cell proliferation, increased cell spreading, and decreased resistance to radiation-induced cell death, compared to cells grown on Osteonectin-null matrix. Our data support the idea that Osteonectin can suppress prostate cancer pathogenesis, expanding this concept to the microenvironment of skeletal metastases.
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mir 29 suppression of Osteonectin in osteoblasts regulation during differentiation and by canonical wnt signaling
2009Co-Authors: Kristina Kapinas, Catherine B Kessler, Anne M DelanyAbstract:The matricellular protein Osteonectin, secreted protein acidic and rich in cysteine (SPARC, BM-40), is the most abundant non-collagenous matrix protein in bone. Matricellular proteins play a fundamental role in the skeleton as regulators of bone remodeling. In the skeleton, Osteonectin is essential for the maintenance of bone mass and for balancing bone formation and resorption in response to parathyroid hormone (PTH). It promotes osteoblast differentiation and cell survival. Mechanisms regulating the expression of Osteonectin in the skeleton and in other tissues remain poorly understood. We found that the proximal region of the mouse Osteonectin 3' untranslated region (UTR) contains a well-conserved, dominant regulatory motif that interacts with microRNAs (miRs)-29a and -29c. Transfection of osteoblastic cells with miR-29a inhibitors increased Osteonectin protein levels, whereas transfection of miR-29a precursor RNA decreased Osteonectin. miR-29a and -29c were increased during osteoblastic differentiation in vitro. The up-regulation of these miRNAs correlated with decreased Osteonectin protein during the matrix maturation and mineralization phases of late differentiation. In contrast, Osteonectin transcript levels remained relatively constant during this process, implying repression of translation. Treatment of osteoblasts with LiCl induced miR-29a and -29c expression and decreased Osteonectin synthesis. When cells were treated with Dickkopf-1 (Dkk-1), miR-29a and -29c expression was repressed. These data suggest that canonical Wnt signaling, which is increased during osteoblastic differentiation, induces expression of miR-29. Osteonectin and miR-29 are co-expressed in extra-skeletal tissues, and the post-transcriptional mechanisms regulating Osteonectin in osteoblasts are likely to be active in other cell systems.
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accentuated osteoclastic response to parathyroid hormone undermines bone mass acquisition in Osteonectin null mice
2008Co-Authors: Luciene M Reis, Catherine B Kessler, Douglas J Adams, Joseph A Lorenzo, Vanda Jorgetti, Anne M DelanyAbstract:Abstract Matricellular proteins play a unique role in the skeleton as regulators of bone remodeling, and the matricellular protein Osteonectin (SPARC, BM-40) is the most abundant non-collagenous protein in bone. In the absence of Osteonectin, mice develop progressive low turnover osteopenia, particularly affecting trabecular bone. Polymorphisms in a regulatory region of the Osteonectin gene are associated with bone mass in a subset of idiopathic osteoporosis patients, and these polymorphisms likely regulate Osteonectin expression. Thus it is important to determine how Osteonectin gene dosage affects skeletal function. Moreover, intermittent administration of parathyroid hormone (PTH) (1–34) is the only anabolic therapy approved for the treatment of osteoporosis, and it is critical to understand how modulators of bone remodeling, such as Osteonectin, affect skeletal response to anabolic agents. In this study, 10 week old female wild type, Osteonectin-haploinsufficient, and Osteonectin-null mice (C57Bl/6 genetic background) were given 80 μg/kg body weight/day PTH(1–34) for 4 weeks. Osteonectin gene dosage had a profound effect on bone microarchitecture. The connectivity density of trabecular bone in Osteonectin-haploinsufficient mice was substantially decreased compared with that of wild type mice, suggesting compromised mechanical properties. Whereas mice of each genotype had a similar osteoblastic response to PTH treatment, the osteoclastic response was accentuated in Osteonectin-haploinsufficient and Osteonectin-null mice. Eroded surface and osteoclast number were significantly higher in PTH-treated Osteonectin-null mice, as was endosteal area. In vitro studies confirmed that PTH induced the formation of more osteoclast-like cells in marrow from Osteonectin-null mice compared with wild type. PTH treated Osteonectin-null bone marrow cells expressed more RANKL mRNA compared with wild type. However, the ratio of RANKL:OPG mRNA was somewhat lower in PTH treated Osteonectin-null cultures. Increased expression of RANKL in response to PTH could contribute to the accentuated osteoclastic response in Osteonectin−/− mice, but other mechanisms are also likely to be involved. The molecular mechanisms by which PTH elicits bone anabolic vs. bone catabolic effects remain poorly understood. Our results imply that Osteonectin levels may play a role in modulating the balance of bone formation and resorption in response to PTH.
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Osteonectin sparc polymorphisms in caucasian men with idiopathic osteoporosis
2008Co-Authors: Anne M Delany, Donald J Mcmahon, J S Powell, D A Greenberg, Etah S KurlandAbstract:Animal models suggest a role for Osteonectin/SPARC in determination of bone mass. We found haplotypes consisting of three single nucleotide polymorphisms (SNPs) in the 3′ untranslated region (UTR) of the Osteonectin gene are associated with bone density in Caucasian men with idiopathic osteoporosis. Osteonectin is a matricellular protein regulating matrix assembly, osteoblast differentiation, and survival. Animal studies indicate that Osteonectin is essential for normal bone mass. The 3′ UTR is a regulatory region controlling mRNA stability, trafficking, and translation, and we determined whether Osteonectin 3′ UTR haplotypes could be associated with bone mass and/or idiopathic osteoporosis. Single strand conformation polymorphism and allele-specific PCR analysis were used to assess alleles at Osteonectin cDNA bases 1046, 1599, and 1970, using genomic DNA from middle-aged Caucasian men with idiopathic, low turnover osteoporosis (n = 56) and matched controls (n = 59). Bone density was measured by DXA at spine, hip and radius. Allele and haplotype frequencies were analyzed by Chi square analysis and Fisher’s exact test. Five common Osteonectin 3′ UTR haplotypes were identified. The frequency of one haplotype (1046C-1599C-1970T) was higher in controls compared with patients, and this haplotype was also associated with higher bone densities at multiple sites in patients. In contrast, a second haplotype (1046C-1599G-1970T) was associated with lower bone densities in patients at multiple sites. Osteonectin regulates skeletal remodeling and bone mass in animals, and haplotypes in the 3′ UTR of this gene are associated with bone density in Caucasian men with idiopathic osteoporosis.
Amy D Bradshaw - One of the best experts on this subject based on the ideXlab platform.
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sparc Osteonectin in mineralized tissue
2016Co-Authors: Emilie Moore Rosset, Amy D BradshawAbstract:Abstract Secreted protein acidic and rich in cysteine (SPARC/Osteonectin/BM40) is one of the most abundant non-collagenous protein expressed in mineralized tissues. This review will focus on elucidating functional roles of SPARC in bone formation building upon results from non-mineralized cells and tissues, the phenotype of SPARC-null bones, and recent discoveries of human diseases with either dysregulated expression of SPARC or mutations in the gene encoding SPARC that give rise to bone pathologies. The capacity of SPARC to influence pathways involved in extracellular matrix assembly such as procollagen processing and collagen fibril formation as well as the capacity to influence osteoblast differentiation and osteoclast activity will be addressed. In addition, the potential for SPARC to regulate cross-linking of extracellular matrix proteins by members of the transglutaminase family of enzymes is explored. Elucidating defined biological functions of SPARC in terms of bone formation and turnover are critical. Further insight into specific cellular mechanisms involved in the formation and homeostasis of mineralized tissues will lead to a better understanding of disease progression.
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sparc Osteonectin in mineralized tissue
2016Co-Authors: Emilie Moore Rosset, Amy D BradshawAbstract:Abstract Secreted protein acidic and rich in cysteine (SPARC/Osteonectin/BM40) is one of the most abundant non-collagenous protein expressed in mineralized tissues. This review will focus on elucidating functional roles of SPARC in bone formation building upon results from non-mineralized cells and tissues, the phenotype of SPARC-null bones, and recent discoveries of human diseases with either dysregulated expression of SPARC or mutations in the gene encoding SPARC that give rise to bone pathologies. The capacity of SPARC to influence pathways involved in extracellular matrix assembly such as procollagen processing and collagen fibril formation as well as the capacity to influence osteoblast differentiation and osteoclast activity will be addressed. In addition, the potential for SPARC to regulate cross-linking of extracellular matrix proteins by members of the transglutaminase family of enzymes is explored. Elucidating defined biological functions of SPARC in terms of bone formation and turnover are critical. Further insight into specific cellular mechanisms involved in the formation and homeostasis of mineralized tissues will lead to a better understanding of disease progression.
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sparc Osteonectin functions to maintain homeostasis of the collagenous extracellular matrix in the periodontal ligament
2010Co-Authors: Jessica M Trombetta, Amy D BradshawAbstract:Expression of secreted protein acidic and rich in cysteine (SPARC)/Osteonectin, a collagen-binding matricellular protein, is frequently associated with tissues with high rates of collagen turnover, such as bone. In the oral cavity, expression of SPARC/Osteonectin has been localized to the periodontal ligament (PDL), a collagen-rich tissue with high rates of collagen turnover. The PDL is critical for tooth position within the alveolar bone and for absorbing forces generated by chewing. To characterize the function of SPARC/Osteonectin in PDL, SPARC/Osteonectin expression in murine PDL was evaluated by immunochemistry at 1, 4, 6, and >18 months. Highest levels of SPARC/Osteonectin were detected at 1 and >18 months, with decreased levels associated with adult (4-6 months) PDL. To determine whether the absence of SPARC/Osteonectin expression influenced cellular and fibrillar collagen content in PDL, PDL of SPARC-null mice was evaluated using histological stains and compared with that of wild-type (WT). Our results demonstrated decreased numbers of nuclei in PDL of SPARC-null mice at 1 month. In addition, decreased collagen volume fractions were found at 1 and >18 months and decreases in thick collagen fiber volume fraction were detected at 4, 6, and >18 months in SPARC-null PDL. The greatest differences in cell number and in collagen content between SPARC-null and WT PDL coincided with ages at which levels of SPARC/Osteonectin expression were highest in WT PDL, at 1 and >18 months. These results support the hypothesis that SPARC/Osteonectin is critical in the control of tissue collagen content and indicate that SPARC/Osteonectin is necessary for PDL homeostasis.
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Osteonectin null mutation compromises osteoblast formation maturation and survival
2003Co-Authors: Anne M Delany, Amy D Bradshaw, Ivo Kalajzic, Helene E Sage, Ernesto CanalisAbstract:Osteonectin, also known as SPARC (secreted protein acidic and rich in cysteine) or BM-40, is one of the most abundant noncollagenous proteins in bone. Analysis of Osteonectin-null mice revealed that Osteonectin is necessary for the maintenance of bone mass and normal remodeling, as Osteonectin-null mice have decreased osteoblast number and bone formation rate. Cultures of bone marrow stromal cells and osteoblasts from control and Osteonectin-null mice were used to determine the cellular basis for the mutant phenotype. We found that marrow stroma from Osteonectin-null mice contains fewer osteoblastic precursors than that of control mice, and the Osteonectin-null mutation did not affect the proliferation rate of stromal cells or osteoblasts. Whereas Osteonectin-null cells could adopt an osteoblastic phenotype, a smaller proportion of these cells expressed markers of a fully differentiated osteoblast. Mutant cells exhibited decreased formation of mineralized nodules, as well as diminished expression of osteocalcin mRNA and response to PTH. Furthermore, Osteonectin-null cells showed an increased tendency to form adipocytes, with enhanced expression of the adipocytic markers adipsin and CCAAT/enhancer binding protein delta. Osteonectin-null cells were also more susceptible to environmental stresses. These data indicate that Osteonectin is important for osteoblast formation, maturation, and survival.
Jiianghuei Jeng - One of the best experts on this subject based on the ideXlab platform.
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butyrate stimulates histone h3 acetylation 8 isoprostane production rankl expression and regulated osteoprotegerin expression secretion in mg 63 osteoblastic cells
2018Co-Authors: Meichi Chang, Yunnjy Chen, Yunchia Lian, Beien Chang, Chihchia Huang, Weiling Huang, Yuhwa Pan, Jiianghuei JengAbstract:Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium, etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), Osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2–4 mM) for 3 days markedly stimulated Osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1–4 mM) may stimulate ALP, Osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction.
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Butyrate Stimulates Histone H3 Acetylation, 8-Isoprostane Production, RANKL Expression, and Regulated Osteoprotegerin Expression/Secretion in MG-63 Osteoblastic Cells
2018Co-Authors: Meichi Chang, Yunnjy Chen, Yunchia Lian, Beien Chang, Chihchia Huang, Weiling Huang, Yuhwa Pan, Jiianghuei JengAbstract:Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium, etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), Osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2⁻4 mM) for 3 days markedly stimulated Osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1⁻4 mM) may stimulate ALP, Osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction
Chia Ling Hsieh - One of the best experts on this subject based on the ideXlab platform.
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Osteonectin Promoter-Mediated Suicide Gene Therapy of Prostate Cancer.
2018Co-Authors: Wan Chi Hsiao, Shian Ying Sung, Leland W. K. Chung, Chia Ling HsiehAbstract:Suicide gene therapy using the herpes simplex virus thymidine kinase (HSV-tk) gene, combined with the prodrug ganciclovir (GCV) medication, is a promising approach for the treatment of malignant tumors, including prostate cancer. The success of this therapeutic strategy requires tissue- or tumor-specific gene expression and efficient gene delivery. In this chapter, we describe the experimental protocols of key methodologies, including promoter construction, reporter assay, adenoviral vector construction and preparation, HSV-tk enzymatic assay and cytotoxicity assay to evaluate the specificity and efficacy of Osteonectin promoter-mediated HSV-tk/GCV suicide gene therapy of prostate cancer.
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co targeting prostate cancer epithelium and bone stroma by human Osteonectin promoter mediated suicide gene therapy effectively inhibits androgen independent prostate cancer growth
2016Co-Authors: Shian Ying Sung, Leland W. K. Chung, Junn Liang Chang, Kuan Chou Chen, Shauh Der Yeh, Yun Ru Liu, Chia Yen Hsueh, Chia Ling HsiehAbstract:Stromal-epithelial interaction has been shown to promote local tumor growth and distant metastasis. We sought to create a promising gene therapy approach that co-targets cancer and its supporting stromal cells for combating castration-resistant prostate tumors. Herein, we demonstrated that human Osteonectin is overexpressed in the prostate cancer epithelium and tumor stroma in comparison with their normal counterpart. We designed a novel human Osteonectin promoter (hON-522E) containing positive transcriptional regulatory elements identified in both the promoter and exon 1 region of the human Osteonectin gene. In vitro reporter assays revealed that the hON-522E promoter is highly active in androgen receptor negative and metastatic prostate cancer and bone stromal cells compared to androgen receptor-positive prostate cancer cells. Moreover, in vivo prostate-tumor-promoting activity of the hON-522E promoter was confirmed by intravenous administration of an adenoviral vector containing the hON-522E promoter-driven luciferase gene (Ad-522E-Luc) into mice bearing orthotopic human prostate tumor xenografts. In addition, an adenoviral vector with the hON-522E-promoter-driven herpes simplex virus thymidine kinase gene (Ad-522E-TK) was highly effective against the growth of androgen-independent human prostate cancer PC3M and bone stromal cell line in vitro and in pre-established PC3M tumors in vivo upon addition of the prodrug ganciclovir. Because of the heterogeneity of human prostate tumors, hON-522E promoter-mediated gene therapy has the potential for the treatment of hormone refractory and bone metastatic prostate cancers.
Yuhwa Pan - One of the best experts on this subject based on the ideXlab platform.
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butyrate stimulates histone h3 acetylation 8 isoprostane production rankl expression and regulated osteoprotegerin expression secretion in mg 63 osteoblastic cells
2018Co-Authors: Meichi Chang, Yunnjy Chen, Yunchia Lian, Beien Chang, Chihchia Huang, Weiling Huang, Yuhwa Pan, Jiianghuei JengAbstract:Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium, etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), Osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2–4 mM) for 3 days markedly stimulated Osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1–4 mM) may stimulate ALP, Osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction.
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Butyrate Stimulates Histone H3 Acetylation, 8-Isoprostane Production, RANKL Expression, and Regulated Osteoprotegerin Expression/Secretion in MG-63 Osteoblastic Cells
2018Co-Authors: Meichi Chang, Yunnjy Chen, Yunchia Lian, Beien Chang, Chihchia Huang, Weiling Huang, Yuhwa Pan, Jiianghuei JengAbstract:Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium, etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), Osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2⁻4 mM) for 3 days markedly stimulated Osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1⁻4 mM) may stimulate ALP, Osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction
