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Christopher L. Murphy - One of the best experts on this subject based on the ideXlab platform.
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mir 1247 functions by targeting cartilage Transcription Factor Sox9
Journal of Biological Chemistry, 2013Co-Authors: Aida Martinezsanchez, Christopher L. MurphyAbstract:microRNAs are a large and essential class of gene regulators that play key roles in development, homeostasis, and disease. They are necessary for normal skeletal development, and their expression is altered in arthritis. However, the specific role of individual microRNAs is only beginning to be unraveled. Using microRNA expression profiling in healthy human articular cartilage cells (chondrocytes), we identified miR-1247 expression as highly correlated with that of the differentiated cell phenotype. Transcribed from the DLK1-DIO3 locus, the function of miR-1247 is completely unknown. In mice its expression level was relatively high in cartilage tissue, and correlated with cartilage-associated microRNA miR-675 across a range of 15 different mouse tissues. To further probe miR-1247 function, overexpression and inhibition studies were performed in isolated human chondrocytes. Modulation of miR-1247 was found to exert profound phenotypic effects altering expression levels of cartilage master regulator Transcription Factor Sox9. Sox9 is essential for cartilage development and subsequent function throughout life, and mutations in this gene result in severe dwarfism. Putative miR-1247 binding sites were further investigated using luciferase reporter assays, which indicated binding of miR-1247 to a highly conserved region in the coding sequence of Sox9 but not in its 3′-UTR. Interestingly, depletion of Sox9 in human chondrocytes resulted in increased levels of the mature, processed microRNA, suggesting a negative feedback loop between miR-1247 and its target Sox9. Background: The function of miR-1247 was heretofore unknown. Results: We show that miR-1247 directly targets Sox9, a Transcription Factor essential for cartilage formation and function. Conclusion: miR-1247 may be an important regulator of cartilage function. Significance: miR-1247 is a potential new target for joint repair.
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miR-1247 Functions by Targeting Cartilage Transcription Factor Sox9
Journal of Biological Chemistry, 2013Co-Authors: Aida Martinez-sanchez, Christopher L. MurphyAbstract:Abstract microRNAs are a large and essential class of gene regulators that play key roles in development, homeostasis, and disease. They are necessary for normal skeletal development, and their expression is altered in arthritis. However, the specific role of individual microRNAs is only beginning to be unraveled. Using microRNA expression profiling in healthy human articular cartilage cells (chondrocytes), we identified miR-1247 expression as highly correlated with that of the differentiated cell phenotype. Transcribed from the DLK1-DIO3 locus, the function of miR-1247 is completely unknown. In mice its expression level was relatively high in cartilage tissue, and correlated with cartilage-associated microRNA miR-675 across a range of 15 different mouse tissues. To further probe miR-1247 function, overexpression and inhibition studies were performed in isolated human chondrocytes. Modulation of miR-1247 was found to exert profound phenotypic effects altering expression levels of cartilage master regulator Transcription Factor Sox9. Sox9 is essential for cartilage development and subsequent function throughout life, and mutations in this gene result in severe dwarfism. Putative miR-1247 binding sites were further investigated using luciferase reporter assays, which indicated binding of miR-1247 to a highly conserved region in the coding sequence of Sox9 but not in its 3′-UTR. Interestingly, depletion of Sox9 in human chondrocytes resulted in increased levels of the mature, processed microRNA, suggesting a negative feedback loop between miR-1247 and its target Sox9.
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regulation of human chondrocyte function through direct inhibition of cartilage master regulator Sox9 by microrna 145 mirna 145
Journal of Biological Chemistry, 2012Co-Authors: Aida Martinezsanchez, Katarzyna Dudek, Christopher L. MurphyAbstract:Articular cartilage enables weight bearing and near friction-free movement in the joints. Critical to its function is the production of a specialized, mechanocompetent extracellular matrix controlled by master regulator Transcription Factor Sox9. Mutations in Sox9 cause campomelic dysplasia, a haploinsufficiency disorder resulting in severe skeletal defects and dwarfism. Although much is understood about how Sox9 regulates cartilage matrix synthesis and hence joint function, how this master regulator is itself regulated remains largely unknown. Here we identify a specific microRNA, miR-145, as a direct regulator of Sox9 in normal healthy human articular chondrocytes. We show that miR-145 directly represses Sox9 expression in human cells through a unique binding site in its 3′-UTR not conserved in mice. Modulation of miR-145 induced profound changes in the human chondrocyte phenotype. Specifically, increased miR-145 levels cause greatly reduced expression of critical cartilage extracellular matrix genes (COL2A1 and aggrecan) and tissue-specific microRNAs (miR-675 and miR-140) and increased levels of the hypertrophic markers RUNX2 and MMP13, characteristic of changes occurring in osteoarthritis. We propose miR-145 as an important regulator of human chondrocyte function and a new target for cartilage repair.
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inhibition of hypoxia inducible Factor targeting prolyl hydroxylase domain containing protein 2 phd2 enhances matrix synthesis by human chondrocytes
Journal of Biological Chemistry, 2010Co-Authors: Brendan L Thoms, Christopher L. MurphyAbstract:Human articular cartilage is an avascular tissue, and therefore it functions in a hypoxic environment. Cartilage cells, the chondrocytes, have adapted to this and actually use hypoxia to drive tissue-specific functions. We have previously shown that human chondrocytes enhance cartilage matrix synthesis in response to hypoxia specifically through hypoxia-inducible Factor 2α (HIF-2α)-mediated up-regulation of master regulator Transcription Factor Sox9, which in turn drives expression of the main cartilage-specific extracellular matrix genes. HIF-α isoforms are themselves regulated by specific prolyl hydroxylase domain-containing proteins, which target them for proteosomal degradation. In fact, prolyl hydroxylase domains are the direct oxygen sensors because they require molecular oxygen as a co-substrate. Here, we have identified PHD2 as the dominant isoenzyme regulating HIF-2α stability in human chondrocytes. Moreover, specific inhibition of PHD2 using RNA interference-mediated depletion caused an up-regulation of Sox9 and enhanced extracellular matrix protein production. Depletion of PHD2 resulted in greater HIF-2α levels and therefore enhanced Sox9-induced cartilage matrix production compared with the levels normally found in hypoxia (1% oxygen) implying that PHD2 inhibition offers a novel means to enhance cartilage repair in vivo. The need for HIF-specific hydroxylase inhibition was highlighted because treatment with the 2-oxoglutarate analogue dimethyloxalylglycine (which also inhibits the collagen prolyl 4-hydroxylases) prevented secretion of type II collagen, a critical cartilage matrix component.
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chondrogenic differentiation of murine embryonic stem cells effects of culture conditions and dexamethasone
Journal of Cellular Biochemistry, 2004Co-Authors: Hiroshi Tanaka, Christopher L. Murphy, Chiho Murphy, Mitsuhiro Kimura, Shinya Kawai, Julia M PolakAbstract:Pluripotent embryonic stem (ES) cells have the capability to differentiate to various cell types and may represent an alternative cell source for the treatment of cartilage defects. Here, we show that differentiation of ES cells toward the chondrogenic lineage can be enhanced by altering the culture conditions. Chondrogenesis was observed in intact embryoid body (EB) cultures, as detected by an increase in mRNA levels for aggrecan and Sox9 genes. Collagen IIB mRNA, the mature chondrocyte-specific splice variant, was absent at day 5, but appeared at later time points. Dexamethasone treatment of alginate-encapsulated EB cultures did not have a strong chondrogenic effect. Nor was chondrogenesis enhanced by alginate encapsulation compared to simple plating of EBs. However, disruption of day 5 EBs and culture as a micromass or pelleted mass, significantly enhanced the expression of the cartilage marker gene collagen type II and the Transcription Factor Sox9 compared to all other treatments. Histological and immunohistochemical analysis of pellet cultures revealed cartilage-like tissue characterized by metachromatically stained extracellular matrix and type II collagen immunoreactivity, indicative of chondrogenesis. These findings have potentially important implications for cartilage tissue engineering, since they may enable the increase in differentiated cell numbers needed for the in vitro development of functional cartilaginous tissue suitable for implantation. © 2004 Wiley-Liss, Inc.
Mateusz C Ambrozkiewicz - One of the best experts on this subject based on the ideXlab platform.
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polarity acquisition in cortical neurons is driven by synergistic action of Sox9 regulated wwp1 and wwp2 e3 ubiquitin ligases and intronic mir 140
Neuron, 2018Co-Authors: Kei Hori, Mateusz C Ambrozkiewicz, Manuela Schwark, Mika Kishimotosuga, Ekaterina Borisova, Andrea Salazarlazaro, Alexandra Rusanova, Bekir AltasAbstract:Summary The establishment of axon-dendrite polarity is fundamental for radial migration of neurons during cortex development of mammals. We demonstrate that the E3 ubiquitin ligases WW-Containing Proteins 1 and 2 (Wwp1 and Wwp2) are indispensable for proper polarization of developing neurons. We show that knockout of Wwp1 and Wwp2 results in defects in axon-dendrite polarity in pyramidal neurons, and their aberrant laminar cortical distribution. Knockout of miR-140, encoded in Wwp2 intron, engenders phenotypic changes analogous to those upon Wwp1 and Wwp2 deletion. Intriguingly, Transcription of the Wwp1 and Wwp2/miR-140 loci in neurons is induced by the Transcription Factor Sox9. Finally, we provide evidence that miR-140 supervises the establishment of axon-dendrite polarity through repression of Fyn kinase mRNA. Our data delineate a novel regulatory pathway that involves Sox9–[Wwp1/Wwp2/miR-140]-Fyn required for axon specification, acquisition of pyramidal morphology, and proper laminar distribution of cortical neurons.
Timothy E. Hardingham - One of the best experts on this subject based on the ideXlab platform.
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Directed differentiation of human embryonic stem cells toward chondrocytes
Nature Biotechnology, 2010Co-Authors: Rachel A. Oldershaw, Timothy E. Hardingham, Emma T. Lowe, Melissa A. Baxter, Nicola Bates, Lisa M Grady, Francesca Soncin, Daniel R. Brison, Susan J. KimberAbstract:We report a chemically defined, efficient, scalable and reproducible protocol for differentiation of human embryonic stem cells (hESCs) toward chondrocytes. HESCs are directed through intermediate developmental stages using substrates of known matrix proteins and chemically defined media supplemented with exogenous growth Factors. Gene expression analysis suggests that the hESCs progress through primitive streak or mesendoderm to mesoderm, before differentiating into a chondrocytic culture comprising cell aggregates. At this final stage, 74% (HUES1 cells) and up to 95-97% (HUES7 and HUES8 cells) express the chondrogenic Transcription Factor Sox9. The cell aggregates also express cell surface CD44 and aggrecan and deposit a sulfated glycosaminoglycan and cartilage-specific collagen II matrix, but show very low or no expression of genes and proteins associated with nontarget cell types. Our protocol should facilitate studies of chondrocyte differentiation and of cell replacement therapies for cartilage repair.
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Hyperosmolarity regulates Sox9 mRNA postTranscriptionally in human articular chondrocytes.
American Journal of Physiology-Cell Physiology, 2009Co-Authors: Simon R. Tew, Timothy E. Hardingham, Mandy J. Peffers, Tristan R. Mckay, Emma T. Lowe, Wasim S. Khan, Peter D. CleggAbstract:The Transcription Factor Sox9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cyclohex...
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regulation of Sox9 mrna in human articular chondrocytes involving p38 mapk activation and mrna stabilization
Journal of Biological Chemistry, 2006Co-Authors: Timothy E. HardinghamAbstract:Abstract Human articular chondrocytes rapidly lose their phenotype in monolayer culture. Recently we have shown that overexpression of the Transcription Factor Sox9 greatly enhanced re-expression of the phenotype in three-dimensional aggregate cultures. Here we show that endogenous Sox9 mRNA can be rapidly up-regulated in subcultured human articular chondrocytes if grown in alginate, in monolayer with cytochalasin D, or with specific inhibition of the RhoA effector kinases ROCK1 and -2, which all prevent actin stress fiber formation. Disruption of actin stress fibers using any of these redifferentiation stimuli also supported the superinduction of Sox9 by cycloheximide. The superinduction was blocked by inhibitors of the p38 MAPK signaling pathway and involved the stabilization of Sox9 mRNA. Furthermore stimulation of chondrocyte p38 MAPK activity with interleukin-1β resulted in increased levels of Sox9 mRNA, and this was again dependent on the absence of actin stress fibers in the cells. In this study of chondrocyte redifferentiation we have provided further evidence of the early involvement of Sox9 and have discovered a novel post-Transcriptional regulatory mechanism activated by p38 MAPK, which stabilized Sox9 mRNA.
Yuko Mori-akiyama - One of the best experts on this subject based on the ideXlab platform.
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Sox9 Is Required for the Differentiation of Paneth Cells in the Intestinal Epithelium
Gastroenterology, 2007Co-Authors: Yuko Mori-akiyama, Jiexin Zhang, Stanley R. Hamilton, Maaike Van Den Born, Johan H. Van Es, Henry P. Adams, Hans CleversAbstract:Background & Aims: The Transcription Factor Sox9 has been shown previously to have an essential role in the differentiation of a small number of discrete cell lineages. In the intestine, Sox9 is expressed in the epithelial cells of the crypts and is a target of Wnt signaling. Methods: To examine the function of Sox9 in the intestine, we inactivated the Sox9 gene in intestinal epithelial cells by generating mice that harbored a conditional Sox9 gene and a Villin-Cre transgene. Results: In the absence of Sox9, Paneth cells were not formed, but the differentiation of other intestinal epithelial cell types was unaffected. The lack of Sox9 also lead to crypt enlargement, to a marked increase in cell proliferation throughout the crypts, and to replacement of the Paneth cells by proliferating epithelial cells. Conclusions: We conclude that Sox9 is required for the differentiation of Paneth cells. Our results elucidate an essential step in the differentiation of gut epithelium.
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The Transcription Factor Sox9 is required for the differentiation of Paneth cells in the intestinal epithelium
Cancer Research, 2006Co-Authors: Yuko Mori-akiyama, Stanley R. Hamilton, Hans CleversAbstract:3955 In the crypts of the intestinal epithelium, undifferentiated multipotent stem cells give rise to proliferating transient cells, which differentiate into four epithelial cell types: enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Whereas Paneth cells migrate towards the bottom of the crypts, the other cell types migrate up the crypt-villi axis and are shed into the lumen. Sox9 is a target gene of Wnt/β-catenin in epithelial cells of the intestinal crypts and duplicates the pattern of expression of β-catenin in these cells, i.e. in stem cells, proliferative transient cells and Paneth cells. In other tissues Sox9 is required for the differentiation of a discrete number of distinct cell types including chondrocytes in the skeleton, glial cells in the nervous system, Sertoli cells in the male gonad, cells in the endocardial cushions in the heart and others. We have previously shown that Sox9 also inhibits proliferation of chondrocytes and have proposed that this occurs through the inhibition of the Transcriptional activity of β-catenin by Sox9. To clarify the function of Sox9 in the intestine, we inactivated the Sox9 gene in the intestine using the Cre recombinase /LoxP recombination system. In Villin-Cre transgenic mice, Cre is expressed in all intestinal epithelial cells, which enables the deletion of Sox9 gene throughout the small intestine. Immunohistochemical staining using Sox9 antibodies showed almost complete absence of Sox9 expression in the intestinal epithelium of Sox9floxed/floxed;VillinCre mice. Histological analysis of mutant miceshowed an overall normal morphology of crypts and villi, but the cells in the bottom of the crypts did not show the typical granules of Paneth cells. In the knock-out mice those cells were negative for Lysozyme, an early marker for Paneth cells. Whereas in wild type mice Paneth cells were negative for the proliferation marker Ki67, in Sox9 mutants cells in the bottom of the crypts were positive for Ki67. Thus in the mutant there is a marked expansion of the domain of proliferationg cells in the crypts. We also noted that the length from pylorus to cecum of the Sox9floxed/floxed;VillinCre mice is longer than that of littermate controls. Our results indicate that Sox9 is required for the differentiation of Paneth cells from progenitor cells and suggest the hypothesis that Sox9 may participate in the control of intestinal epithelial cell proliferation.
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The Transcription Factor Sox9 is degraded by the ubiquitin-proteasome system and stabilized by a mutation in a ubiquitin-target site.
Matrix Biology, 2005Co-Authors: Haruhiko Akiyama, Laura C. Bridgewater, Tetsu Kamitani, Xiaohong Yang, Roshini Kandyil, Marc Fellous, Yuko Mori-akiyamaAbstract:Sox9 is a Transcription Factor that is critical for chondrogenesis, testis determination, and development of several other organs in vertebrates. Thus the levels of Sox9 protein and its activity may be tightly regulated. Here we show that inhibitors of the 26S proteasome increase both the levels of Sox9 protein and its Transcriptional activity measured with Col2a1 promoter/enhancer construct in RCS cells and C3H10T1/2 cells. Indeed, in intact cells ubiquitination assays indicate that Sox9 is multiply ubiquitinated. The K398A mutation, which was introduced in a potential ubiquitin-binding site, increases the stability of Sox9 protein and its Transcriptional activity of Col2a1, Col11a2, and AMH promoter/enhancer constructs without affecting the subcellular localization and the DNA binding efficiency of Sox9. Pulse-chase experiments show that the increased Sox9 levels resulting from treatment with the MG132 proteasome inhibitor or from the K398A mutation produce stabilization of the protein. Our in vitro studies indicate that the ubiquitin-proteasome proteolytic system degrades Sox9 and regulates its Transcriptional activity.
Aida Martinezsanchez - One of the best experts on this subject based on the ideXlab platform.
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mir 1247 functions by targeting cartilage Transcription Factor Sox9
Journal of Biological Chemistry, 2013Co-Authors: Aida Martinezsanchez, Christopher L. MurphyAbstract:microRNAs are a large and essential class of gene regulators that play key roles in development, homeostasis, and disease. They are necessary for normal skeletal development, and their expression is altered in arthritis. However, the specific role of individual microRNAs is only beginning to be unraveled. Using microRNA expression profiling in healthy human articular cartilage cells (chondrocytes), we identified miR-1247 expression as highly correlated with that of the differentiated cell phenotype. Transcribed from the DLK1-DIO3 locus, the function of miR-1247 is completely unknown. In mice its expression level was relatively high in cartilage tissue, and correlated with cartilage-associated microRNA miR-675 across a range of 15 different mouse tissues. To further probe miR-1247 function, overexpression and inhibition studies were performed in isolated human chondrocytes. Modulation of miR-1247 was found to exert profound phenotypic effects altering expression levels of cartilage master regulator Transcription Factor Sox9. Sox9 is essential for cartilage development and subsequent function throughout life, and mutations in this gene result in severe dwarfism. Putative miR-1247 binding sites were further investigated using luciferase reporter assays, which indicated binding of miR-1247 to a highly conserved region in the coding sequence of Sox9 but not in its 3′-UTR. Interestingly, depletion of Sox9 in human chondrocytes resulted in increased levels of the mature, processed microRNA, suggesting a negative feedback loop between miR-1247 and its target Sox9. Background: The function of miR-1247 was heretofore unknown. Results: We show that miR-1247 directly targets Sox9, a Transcription Factor essential for cartilage formation and function. Conclusion: miR-1247 may be an important regulator of cartilage function. Significance: miR-1247 is a potential new target for joint repair.
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regulation of human chondrocyte function through direct inhibition of cartilage master regulator Sox9 by microrna 145 mirna 145
Journal of Biological Chemistry, 2012Co-Authors: Aida Martinezsanchez, Katarzyna Dudek, Christopher L. MurphyAbstract:Articular cartilage enables weight bearing and near friction-free movement in the joints. Critical to its function is the production of a specialized, mechanocompetent extracellular matrix controlled by master regulator Transcription Factor Sox9. Mutations in Sox9 cause campomelic dysplasia, a haploinsufficiency disorder resulting in severe skeletal defects and dwarfism. Although much is understood about how Sox9 regulates cartilage matrix synthesis and hence joint function, how this master regulator is itself regulated remains largely unknown. Here we identify a specific microRNA, miR-145, as a direct regulator of Sox9 in normal healthy human articular chondrocytes. We show that miR-145 directly represses Sox9 expression in human cells through a unique binding site in its 3′-UTR not conserved in mice. Modulation of miR-145 induced profound changes in the human chondrocyte phenotype. Specifically, increased miR-145 levels cause greatly reduced expression of critical cartilage extracellular matrix genes (COL2A1 and aggrecan) and tissue-specific microRNAs (miR-675 and miR-140) and increased levels of the hypertrophic markers RUNX2 and MMP13, characteristic of changes occurring in osteoarthritis. We propose miR-145 as an important regulator of human chondrocyte function and a new target for cartilage repair.