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1.  MiRNA-140 acts as a liver tumor suppressor by controlling NF-κB activity via directly targeting Dnmt1 expression 
Hepatology (Baltimore, Md.)  2013;57(1):162-170.
MicroRNAs (miRNAs) are small RNAs that regulate the expression of specific target genes. While deregulated miRNA expression levels have been detected in many tumors, whether miRNA functional impairment is also involved in carcinogenesis remains unknown. We investigated whether deregulation of miRNA machinery components and subsequent functional impairment of miRNAs are involved in hepatocarcinogenesis. Among miRNA-containing ribonucleoprotein complex components, reduced expression of DDX20 was frequently observed in human hepatocellular carcinomas, in which enhanced NF-κB activity is believed to be closely linked to carcinogenesis. Because DDX20 normally suppresses NF-κB activity by preferentially regulating the function of the NF-κB-suppressing miRNA-140, we hypothesized that impairment of miRNA-140 function may be involved in hepatocarcinogenesis. Dnmt1 was identified as a direct target of miRNA-140, and increased Dnmt1 expression in DDX20-deficient cells hypermethylated the promoters of metallothionein genes, resulting in decreased metallothionein expression leading to enhanced NF-κB activity. MiRNA-140-knockout mice were prone to hepatocarcinogenesis and had a phenotype similar to that of DDX20 deficiency, suggesting that miRNA-140 plays a central role in DDX20 deficiency-related pathogenesis.
Conclusion
These results indicate that miRNA-140 acts as a liver tumor suppressor, and that impairment of miRNA-140 function due to a deficiency of DDX20, a miRNA machinery component, could lead to hepatocarcinogenesis.
doi:10.1002/hep.26011
PMCID: PMC3521841  PMID: 22898998
microRNA; HCC; DDX20; ribonucleoprotein; methallothionein
2.  Production of Sry knockout mouse using TALEN via oocyte injection 
Scientific Reports  2013;3:3136.
Recently developed transcription activator-like effector nuclease (TALEN) technology has enabled the creation of knockout mice, even for genes on the Y chromosome. In this study, we generated a knockout mouse for Sry, a sex-determining gene on the Y chromosome, using microinjection of TALEN RNA into pronuclear stage oocytes. As expected, the knockout mouse had female external and internal genitalia, a female level of blood testosterone and a female sexually dimorphic nucleus in the brain. The knockout mouse exhibited an estrous cycle and performed copulatory behavior as females, although it was infertile or had reduced fertility. A histological analysis showed that the ovary of the knockout mouse displayed a reduced number of oocytes and luteinized unruptured follicles, implying that a reduced number of ovulated oocytes is a possible reason for infertility and/or reduced fertility in the KO mouse.
doi:10.1038/srep03136
PMCID: PMC3817445  PMID: 24190364
3.  Targeted Gene Deletion of miRNAs in Mice by TALEN System 
PLoS ONE  2013;8(10):e76004.
Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.
doi:10.1371/journal.pone.0076004
PMCID: PMC3797721  PMID: 24146809
4.  The AERO System: A 3D-Like Approach for Recording Gene Expression Patterns in the Whole Mouse Embryo 
PLoS ONE  2013;8(10):e75754.
We have recently constructed a web-based database of gene expression in the mouse whole embryo, EMBRYS (http://embrys.jp/embrys/html/MainMenu.html). To allow examination of gene expression patterns to the fullest extent possible, this database provides both photo images and annotation data. However, since embryos develop via an intricate process of morphogenesis, it would be of great value to track embryonic gene expression from a three dimensional perspective. In fact, several methods have been developed to achieve this goal, but highly laborious procedures and specific operational skills are generally required. We utilized a novel microscopic technique that enables the easy capture of rotational, 3D-like images of the whole embryo. In this method, a rotary head equipped with two mirrors that are designed to obtain an image tilted at 45 degrees to the microscope stage captures serial images at 2-degree intervals. By a simple operation, 180 images are automatically collected. These 2D images obtained at multiple angles are then used to reconstruct 3D-like images, termed AERO images. By means of this system, over 800 AERO images of 191 gene expression patterns were captured. These images can be easily rotated on the computer screen using the EMBRYS database so that researchers can view an entire embryo by a virtual viewing on a computer screen in an unbiased or non-predetermined manner. The advantages afforded by this approach make it especially useful for generating data viewed in public databases.
doi:10.1371/journal.pone.0075754
PMCID: PMC3797748  PMID: 24146773
5.  Structural and Functional Maturation of Distal Femoral Cartilage and Bone during Postnatal Development and Growth in Humans and Mice 
SYNOPSIS
Introduction
The size and shape of joints markedly affect their biomechanical properties, but the macroscopic 3-dimensional (3-D) mechanism and extent of cartilage and joint maturation during normal growth are largely unknown.
Aims
The purposes of this study were to qualitatively illustrate the development of the cartilage-bone interface in the knee during postnatal growth in humans and C57BL/6 wild-type mice, and to quantitatively define the 3-D shape using statistical shape modeling as well as to assess growth strain rates in the mouse distal femur.
Methods
Clinical computed tomography (CT) scans of asymptomatic knees (0.3–0.6mm in-plane resolution, 0.63mm slice thickness) were obtained from six patients between 4 to 12 years old. Micro-CT scans of mouse knees (9μm isotropic resolution) were from twenty-one mice between 12 to 120 days postnatal. Human and mouse images were compared qualitatively with 2-D images and 3-D reconstructions. Mouse femora shape parameters were determined with statistical shape modeling, and strain rates and directions during growth were mapped.
Results
The attainment of cartilage-bone interface shape of the distal femur and proximal tibia were qualitatively similar in humans and mice, with marked differences in growth plate morphology. Mouse distal femur shape was described by 11 independent parameters that accounted for >90% of total shape variation during growth. Each shape parameter described changes in specific anatomical regions of the distal femur and varied with age. Shape parameters and strains in the medial and lateral condyles, as well as intercondylar notch, varied greatly between postnatal days 16 to 30. Directions of growth strain across ages corresponded well with the appearance of anatomical landmarks within the distal femur.
Conclusion
Accurate quantification of the cartilage-bone interface geometry is imperative for furthering the understanding of the macroscopic mechanisms of cartilage maturation and overall joint development.
doi:10.1016/j.ocl.2012.01.005
PMCID: PMC3321216  PMID: 22480467
distal femur; postnatal development; growth strain; joint shape; statistical shape modeling
6.  Macro view of microRNA’s function in Osteoarthritis 
Nature reviews. Rheumatology  2012;8(9):543-552.
Osteoarthritis (OA) is the most common musculoskeletal disorder. It is a complex and multifaceted disease, characterized by the degradation of articular cartilage and joint inflammation. Although few pathogenesis pathways have been characterized, current knowledge is incomplete and has not led to effective approaches for prevention or treatment. These limitations can be overcome by advances in the understanding of molecular mechanisms that are involved in the maintenance and destruction of articular cartilage. Understanding extracellular regulators and intracellular signaling mechanisms in joint cells that control cartilage homeostasis has the potential to lead to the identification of new therapeutic targets for OA. Recently, non-coding RNAs, miRNAs, was noted to act as novel regulatory molecules that regulated the expression of several target genes. The major role of miRNAs is to control development and tissue homeostasis through the ‘fine-tuning’ of the gene expression. Several miRNAs exhibit a tissue- or developmental stage-specific expression pattern and have been associated with diseases such as cancer and cardiovascular disorders. This review is based on our observations that miRNAs play an important role in cartilage homeostasis, and is to summarize the information on miRNA involved in OA pathogenesis, and its clinical approach.
doi:10.1038/nrrheum.2012.128
PMCID: PMC3572197  PMID: 22890245
Osteoarthritis; Cartilage; Chondrocytes; microRNA; miR-140
7.  Cellular and extracellular matrix changes in anterior cruciate ligaments during human knee aging and osteoarthritis 
Introduction
Anterior cruciate ligament (ACL) degeneration is observed in most osteoarthritis (OA)-affected knee joints. However, the specific spatial and temporal relations of these changes and their association with extracellular matrix (ECM) degeneration are not well understood. The objective of this study was to characterize the patterns and relations of aging-related and OA-associated changes in ACL cells and the ECM.
Methods
Human knee joints from 80 donors (age 23 through 94) were obtained at autopsy. ACL degeneration was assessed histologically by using a quantitative scoring system. Tissue sections were analyzed for cell density, cell organization, ECM components, ECM-degrading enzymes and markers of differentiation, proliferation, and stem cells.
Results
Total cell number in normal ACL decreased with aging but increased in degenerated ACL, because of the formation of perivascular cell aggregates and islands of chondrocyte-like cells. Matrix metalloproteinase (MMP)-1, -3, and -13 expression was reduced in aging ACL but increased in degenerated ACL, mainly in the chondrocyte-like cells. Collagen I was expressed throughout normal and degenerated ACL. Collagen II and X were detected only in the areas with chondroid metaplasia, which also expressed collagen III. Sox9, Runt-related transcription factor 2 (Runx2), and scleraxis expression was increased in the chondrocyte-like cells in degenerated ACL. Alpha-smooth muscle actin (α-SMA), a marker of myofibroblasts and the progenitor cell marker STRO-1, decreased with aging in normal ACL. In degenerated ACL, the new cell aggregates were positive for α-SMA and STRO-1.
Conclusions
ACL aging is characterized by reduced cell density and activation. In contrast, ACL degeneration is associated with cell recruitment or proliferation, including progenitor cells or myofibroblasts. Abnormally differentiated chondrocyte-like cell aggregates in degenerated ACL produce abnormal ECM and may predispose to mechanical failure.
doi:10.1186/ar4165
PMCID: PMC3672799  PMID: 23406989
8.  MicroRNA-125b regulates the expression of aggrecanase-1 (ADAMTS-4) in human osteoarthritic chondrocytes 
Introduction
Increased expression of aggrecanase-1 (ADAMTS-4) has emerged as an important factor in osteoarthritis (OA) and other joint diseases. This study aimed to determine whether the expression of ADAMTS-4 in human chondrocytes is regulated by miRNA.
Methods
MiRNA targets were identified using bioinformatics. Chondrocytes were isolated from knee cartilage and treated with interleukin-1 beta (IL-1β). Gene expression was quantified using TaqMan assays and protein production was determined by immunoblotting. Luciferase reporter assay was used to verify interaction between miRNA and target messenger RNA (mRNA).
Results
In silico analysis predicted putative target sequence of miR-125b on ADAMTS-4. MiR-125b was expressed in both normal and OA chondrocytes, with significantly lower expression in OA chondrocytes than in normal chondrocytes. Furthermore, IL-1β-induced upregulation of ADAMTS-4 was suppressed by overexpression of miR-125b in human OA chondrocytes. In the luciferase reporter assay, mutation of the putative miR-125b binding site in the ADAMTS-4 3'UTR abrogated the suppressive effect of miR125.
Conclusions
Our results indicate that miR-125b plays an important role in regulating the expression of ADAMTS-4 in human chondrocytes and this identifies miR-125b as a novel therapeutic target in OA.
doi:10.1186/ar4164
PMCID: PMC3672767  PMID: 23406982
9.  MAML1 Enhances the Transcriptional Activity of Runx2 and Plays a Role in Bone Development 
PLoS Genetics  2013;9(1):e1003132.
Mastermind-like 1 (MAML1) is a transcriptional co-activator in the Notch signaling pathway. Recently, however, several reports revealed novel and unique roles for MAML1 that are independent of the Notch signaling pathway. We found that MAML1 enhances the transcriptional activity of runt-related transcription factor 2 (Runx2), a transcription factor essential for osteoblastic differentiation and chondrocyte proliferation and maturation. MAML1 significantly enhanced the Runx2-mediated transcription of the p6OSE2-Luc reporter, in which luciferase expression was controlled by six copies of the osteoblast specific element 2 (OSE2) from the Runx2-regulated osteocalcin gene promoter. Interestingly, a deletion mutant of MAML1 lacking the N-terminal Notch-binding domain also enhanced Runx2-mediated transcription. Moreover, inhibition of Notch signaling did not affect the action of MAML1 on Runx2, suggesting that the activation of Runx2 by MAML1 may be caused in a Notch-independent manner. Overexpression of MAML1 transiently enhanced the Runx2-mediated expression of alkaline phosphatase, an early marker of osteoblast differentiation, in the murine pluripotent mesenchymal cell line C3H10T1/2. MAML1−/− embryos at embryonic day 16.5 (E16.5) had shorter bone lengths than wild-type embryos. The area of primary spongiosa of the femoral diaphysis was narrowed. At E14.5, extended zone of collagen type II alpha 1 (Col2a1) and Sox9 expression, markers of chondrocyte differentiation, and decreased zone of collagen type X alpha 1 (Col10a1) expression, a marker of hypertrophic chondrocyte, were observed. These observations suggest that chondrocyte maturation was impaired in MAML1−/− mice. MAML1 enhances the transcriptional activity of Runx2 and plays a role in bone development.
Author Summary
To identify new molecules involved in bone and cartilage development and/or homeostasis, we utilized approximately 10,000 arrayed and addressable cDNA clones, which allowed systematic, efficient, and unbiased screening of cDNAs encoding factors that could activate critical bone differentiation activity via activation of Runx2, master regulator of bone development. We analyzed MAML1−/− mice to investigate the role of MAML1 in bone development. MAML1−/− embryos at embryonic day 14.5 and 16.5 had shorter bone lengths than wild-type embryos. The area of primary spongiosa of the femoral diaphysis was narrowed, indicated that chondrocyte maturation was impaired. This revealed that MAML1 plays an important role in proper bone development and may provide us with a new basis for identifying potential therapeutic targets for bone diseases.
doi:10.1371/journal.pgen.1003132
PMCID: PMC3542067  PMID: 23326237
10.  A Systems Approach and Skeletal Myogenesis 
Skeletal myogenesis depends on the strict regulation of the expression of various gene subsets. Therefore, the understanding of genome wide gene regulation is imperative for elucidation of skeletal myogenesis. In recent years, systems approach has contributed to the understanding of various biological processes. Our group recently revealed the critical genome network of skeletal myogenesis by using a novel systems approach combined with whole-mount in situ hybridization (WISH) database, high-throughput screening, and microarray analysis. In this paper, we introduce our systems approach for understanding the myogenesis regulatory network and describe the advantages of systems approach.
doi:10.1155/2012/759407
PMCID: PMC3443578  PMID: 22991503
13.  DNA binding - dependent glucocorticoid receptor activity promotes adipogenesis via krüppel-like factor 15 gene expression 
Glucocorticoids, such as dexamethasone (Dex), have been used as in vitro inducers of adipogenesis. However, the roles of the glucocorticoid receptor (GR) in adipogenesis have not been well characterized yet. Here we show that inhibition of GR activity using the GR antagonist RU486 prevents human mesenchymal stem cell (hMSC) and mouse embryonic fibroblast (MEF) differentiation into adipocytes. Moreover, in MEFs isolated from GR knockout (GRnull) and GRdim mice deficient in GR DNA-binding activity, adipogenesis was blocked. We identified GRE sites in the first intron of KLF15 by bioinformatical promoter analysis and confirmed their functional relevance by demonstrating GR interaction by chromatin immunoprecipitation. Moreover transfection of MEFs with siRNA for KLF15 significantly attenuated the expressions of adipogenic-marker genes and the lipid accumulation. Our results provide a new mechanism for understanding glucocorticoids dependent adipogenesis and that GR promotes adipogenesis via KLF15 gene expression as a transcriptional direct target.
doi:10.1038/labinvest.2010.170
PMCID: PMC3025047  PMID: 20956975
adipogenesis; glucocorticoid receptor; human mesenchymal stem cell; mouse embryonic fibroblast; bioinformatics analysis; Krüppel-like factor 15
14.  A Systems Approach Reveals that the Myogenesis Genome Network Is Regulated by the Transcriptional Repressor RP58 
Developmental cell  2009;17(6):836-848.
SUMMARY
We created a whole-mount in situ hybridization (WISH) database, termed EMBRYS, containing expression data of 1520 transcription factors and cofactors expressed in E9.5, E10.5, and E11.5 mouse embryos—a highly dynamic stage of skeletal myogenesis. This approach implicated 43 genes in regulation of embryonic myogenesis, including a transcriptional repressor, the zinc-finger protein RP58 (also known as Zfp238). Knockout and knockdown approaches confirmed an essential role for RP58 in skeletal myogenesis. Cell-based high-throughput transfection screening revealed that RP58 is a direct MyoD target. Microarray analysis identified two inhibitors of skeletal myogenesis, Id2 and Id3, as targets for RP58-mediated repression. Consistently, MyoD-dependent activation of the myogenic program is impaired in RP58 null fibroblasts and downregulation of Id2 and Id3 rescues MyoD’s ability to promote myogenesis in these cells. Our combined, multi-system approach reveals a MyoD-activated regulatory loop relying on RP58-mediated repression of muscle regulatory factor (MRF) inhibitors.
doi:10.1016/j.devcel.2009.10.011
PMCID: PMC3110151  PMID: 20059953
15.  The myogenic transcriptional network 
Cellular and Molecular Life Sciences  2011;68(11):1843-1849.
Myogenesis has been a leading model for elucidating the molecular mechanisms that underlie tissue differentiation and development since the discovery of MyoD. During myogenesis, the fate of myogenic precursor cells is first determined by Pax3/Pax7. This is followed by regulation of the myogenic differentiation program by muscle regulatory factors (Myf5, MyoD, Myog, and Mrf4) to form muscle tissues. Recent studies have uncovered a detailed myogenic program that involves the RP58 (Zfp238)-dependent regulatory network, which is critical for repressing the expression of inhibitor of DNA binding (Id) proteins. These novel findings contribute to a comprehensive understanding of the muscle differentiation transcriptional program.
doi:10.1007/s00018-011-0629-2
PMCID: PMC3092062  PMID: 21318263
Myogenesis; Pax; MyoD; RP58; Ids
16.  Correlation between MMP-13 and HDAC7 expression in human knee osteoarthritis 
Recent studies demonstrate that histone deacetylase (HDAC) inhibitors therapeutically prevent cartilage degradation in osteoarthritis (OA). Matrix metalloproteinase-13 (MMP-13) plays an important role in the pathogenesis of this disease and in the present study we investigated the correlation between HDACs and MMP-13. We found that HDAC inhibitor trichostatin A (TSA) could suppress both IL-1 dependent and independent MMP-13 mRNA expression (real time PCR) in human knee chondrocytes. Comparing the expression of different HDACs in cartilage from OA patients and healthy donors, HDAC7 showed a significant elevation in cartilage from OA patients. These results were confirmed by immunohistochemistry. Knockdown of HDAC7 by siRNA in SW 1353 human chondrosarcoma cells strongly suppressed IL-1 dependent induction of MMP-13 gene expression.
In conclusion, elevated HDAC7 expression in human OA may contribute to cartilage degradation via promoting MMP-13 gene expression and inhibition of HDACs by TSA or the selective inhibition of HDAC7 could be used therapeutically to stop OA progression.
doi:10.1007/s10165-009-0224-7
PMCID: PMC2818344  PMID: 19784544
17.  Tissue Neogenesis and STRO-1 Expression in Immature and Mature Articular Cartilage 
This study determined the potential for neo tissue formation and the role of STRO-1+ cells in immature versus mature articular cartilage. Cartilage explants from immature and mature bovine knee joints were cultured for up to 12 weeks and stained with Safranin O, for type II collagen and STRO-1. Bovine chondrocyte pellet cultures and murine knee joints at the age of 2 weeks and 3 months and surgically injured cartilage were analyzed for changes in STRO-1 expression patterns. Results show that immature explants contained more STRO-1+ cells than mature explants. After 8 weeks in culture, immature explants showed STRO-1+ cell proliferation and newly formed tissue, which contained glycosaminoglycan and type II collagen. Mature cartilage explants showed only minimal cell expansion and neo tissue formation. Pellet cultures with chondrocytes from immature cartilage showed increased glycosaminoglycan synthesis and STRO-1+ staining as compared to pellets from mature chondrocytes. The frequency of STRO-1+ cells in murine knee joints significantly declined with joint maturation. Following surgical injury, immature explants had higher potential for tissue repair than mature explants. In conclusion, these findings suggest that the high percentage of STRO-1+ cells in immature cartilage changes with joint maturation. STRO-1+ cells have the potential to form new cartilage spontaneously and after tissue injury.
doi:10.1002/jor.20944
PMCID: PMC2905317  PMID: 19603515
Tissue neogenesis; cartilage; stem cells; STRO-1; aging
18.  MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates IL-1 responses 
Arthritis and rheumatism  2009;60(9):2723-2730.
OBJECTIVE
MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. The miRNAs exhibit tissue-specific expression patterns and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNAs expressed in articular chondrocytes, determine changes in osteoarthritic cartilage and address the function of miR-140.
METHODS
To identify miRNAs specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative PCR with human articular chondrocytes compared to human mesenchymal stem cells (MSC). The expression pattern of miR-140 was monitored during chondrogenic differentiation of hMSC in pellet cultures and in human articular cartilage from normal and osteoarthritic knee joints. We tested effects of IL-1β on miR-140 expression. Double-strand (ds) miR-140 was transfected into chondrocytes to analyze changes in the expression of genes associated with osteoarthritis.
RESULTS
Microarray analysis showed that miR-140 has the largest difference in expression between chondrocytes and MSC. During chondrogenesis cultures of MSC miR-140 expression increased in parallel with Sox9 and Col2a1. Normal human articular cartilage expressed miR-140 and this was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1β suppressed miR-140 expression. Transfection of chondrocytes with ds-miR-140 downregulated IL-1β-induced ADAMTS-5 expression and rescued the IL-1β –dependent repression of Aggrecan gene expression.
CONCLUSION
This study shows that miR-140 has a chondrocyte differentiation-related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1β may contribute to the abnormal gene expression pattern characteristic of OA.
doi:10.1002/art.24745
PMCID: PMC2806094  PMID: 19714579
microRNA; chondrocytes; mesenchymal stem cells; cartilage; osteoarthritis
19.  Sox9 directly promotes Bapx1 gene expression to repress Runx2 in chondrocytes 
Experimental cell research  2009;315(13):2231-2240.
The transcription factor, Sry-related High Mobility Group (HMG) box containing gene 9 (Sox9), plays a critical role in cartilage development by initiating chondrogenesis and preventing the subsequent maturation process called chondrocyte hypertrophy. This suppression mechanism by Sox9 on late-stage chondrogenesis partially results from the inhibition of Runt-related transcription factor 2 (Runx2), the main activator of hypertrophic chondrocyte differentiation. However, the precise mechanism by which Sox9 regulates late chondrogenesis is poorly understood.
In the present study, the transcriptional repressor vertebrate homolog of Drosophila bagpipe (Bapx1) was found to be a direct target of Sox9 for repression of Runx2 expression in chondrocytes. We identified a critical Sox9 responsive region in the Bapx1 promoter via a luciferase reporter assay. Analysis by chromatin immunoprecipitation and electrophoretic mobility shift assays indicated that Sox9 physically bound to this region of the Bapx1 promoter. Consistent with the notion that Bapx1 and Sox9 act as negative regulators of chondrocyte hypertrophy by regulating Runx2 expression, transient knockdown of Sox9 or Bapx1 expression by shRNA in chondrocytes increased Runx2 expression, as well as expression of the late chondrogenesis marker, Col10a1. Furthermore, while over-expression of Sox9 decreased Runx2 and Col10a1 expressions, simultaneous transient knockdown of Bapx1 diminished that Sox9 over-expressing effect.
Our findings reveal that the molecular pathway modulated by Bapx1 links two major regulators in chondrogenesis, Sox9 and Runx2, to coordinate skeletal formation.
doi:10.1016/j.yexcr.2009.03.008
PMCID: PMC2696577  PMID: 19306868
Sox9; Bapx1; Runx2; chondrocyte; chondrogenesis; shRNA
20.  Smad3 activates the Sox9-dependent transcription on chromatin 
Transforming growth factor (TGF)-β has an essential role for the Sry-type high-mobility-group box (Sox)-regulated chondrogenesis. Chondrogenic differentiation is also controlled by chromatin-mediated transcription. We have previously reported that TGF-β-regulated Smad3 induces chondrogenesis through the activation of Sox9-dependent transcription. However, the cross-talk between TGF-β signal and Sox9 on chromatin-mediated transcription has not been elucidated. In the present study, we investigated the activity of Smad3, Sox9, and coactivator p300 using an in vitro chromatin assembly model. Luciferase reporter assays revealed that Smad3 stimulated the Sox9-mediated transcription in a TGF-β-dependent manner. Recombinant Sox9 associated with phosphorylated Smad3/4 and recognized the enhancer region of type II collagen gene. In vitro transcription and S1 nuclease assays showed that Smad3 and p300 cooperatively activated the Sox9-dependent transcription on chromatin template. The combination treatment of phosphorylated Smad3, Sox9, and p300 were necessary for the activation of chromatin-mediated transcription. These findings suggest that TGF-β signal Smad3 plays a key role for chromatin remodeling to induce chondrogenesis via its association with Sox9.
doi:10.1016/j.biocel.2008.10.032
PMCID: PMC2674534  PMID: 19041414
Chondrogenesis; Chromatin; p300; Smad3; Sox9
21.  Real-time functional imaging for monitoring miR-133 during myogenic differentiation 
MicroRNAs (miRNAs) are a class of non-coding small RNAs that act as negative regulators of gene expression through sequence-specific interactions with the 3′ untranslated regions (UTRs) of target mRNA and play various biological roles. miR-133 was identified as a muscle-specific miRNA that enhanced the proliferation of myoblasts during myogenic differentiation, although its activity in myogenesis has not been fully characterized. Here, we developed a novel retroviral vector system for monitoring muscle-specific miRNA in living cells by using a green fluorescent protein (GFP) that is connected to the target sequence of miR-133 via the UTR and a red fluorescent protein for normalization. We demonstrated that the functional promotion of miR-133 during myogenesis is visualized by the reduction of GFP carrying the miR-133 target sequence, suggesting that miR-133 specifically down-regulates its targets during myogenesis in accordance with its expression. Our cell-based miRNA functional assay monitoring miR-133 activity should be a useful tool in elucidating the role of miRNAs in various biological events.
doi:10.1016/j.biocel.2009.04.018
PMCID: PMC2806093  PMID: 19398036
Myogenesis; miR-133; Retroviral vector; Fluorescent protein; Live-cell imaging
22.  Expression of microRNA-146 in osteoarthritis cartilage 
Arthritis and rheumatism  2009;60(4):1035-1041.
Objective
A role of microRNAs, which are ∼22- nucleotide non coding RNAs, has recently been recognized in human diseases. The objective of this study was to identify the expression pattern of microRNA-146 (miR-146) in cartilage from patients with osteoarthritis (OA).
Methods
The expression of miR-146 in cartilage from 15 patients with OA was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization. Induction of the expression of miR-146 by cultures of normal human articular chondrocytes following stimulation with interleukin-1β (IL-1β) was examined by quantitative RT-PCR.
Results
All cartilage samples were divided into three groups according to a modified Mankin scale; grade I: 0 - 5, grade II: 6 - 10, grade III: 11 - 14. In OA cartilage samples of grade I, the expression of miR-146a and Col2a1 was significantly higher than that of other groups (p<0.05). In OA cartilage of grades II and III, the expression of miR-146a and Col2a1 decreased while the expression of MMP13 was elevated in grade II. These data show that miR-146a is expressed intensely in cartilage with a low Mankin grade, and that miR-146a expression decreases in accordance with level of MMP13 expression. Section in situ hybridization of pri-miR-146a revealed that pri-miR-146a is expressed in chondrocytes in all layers, especially in the superficial layer where it is intensely expressed. The expression of miR-146 was markedly elevated by IL-1β stimulation in human chondrocytes in vitro.
Conclusion
This study shows that miR-146 is intensely expressed in low grade OA cartilage, and that its expression is induced by stimulation of IL-1β. MiR-146 might play a role in OA cartilage pathogenesis.
doi:10.1002/art.24404
PMCID: PMC2670476  PMID: 19333945
23.  Expression of MicroRNA-146 in Rheumatoid Arthritis Synovial Tissue 
Arthritis and rheumatism  2008;58(5):1284-1292.
Objective
Several microRNA, which are ~22-nucleotide noncoding RNAs, exhibit tissue-specific or developmental stage–specific expression patterns and are associated with human diseases. The objective of this study was to identify the expression pattern of microRNA-146 (miR-146) in synovial tissue from patients with rheumatoid arthritis (RA).
Methods
The expression of miR-146 in synovial tissue from 5 patients with RA, 5 patients with osteoarthritis (OA), and 1 normal subject was analyzed by quantitative reverse transcription–polymerase chain reaction (RT-PCR) and by in situ hybridization and immunohistochemistry of tissue sections. Induction of miR-146 following stimulation with tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) of cultures of human rheumatoid arthritis synovial fibroblasts (RASFs) was examined by quantitative PCR and RT-PCR.
Results
Mature miR-146a and primary miR-146a/b were highly expressed in RA synovial tissue, which also expressed TNFα, but the 2 microRNA were less highly expressed in OA and normal synovial tissue. In situ hybridization showed primary miR-146a expression in cells of the superficial and sublining layers in synovial tissue from RA patients. Cells positive for miR-146a were primarily CD68+ macrophages, but included several CD3+ T cell subsets and CD79a+ B cells. Expression of miR-146a/b was markedly up-regulated in RASFs after stimulation with TNFα and IL-1β.
Conclusion
This study shows that miR-146 is expressed in RA synovial tissue and that its expression is induced by stimulation with TNFα and IL-1β. Further studies are required to elucidate the function of miR-146 in these tissues.
doi:10.1002/art.23429
PMCID: PMC2749927  PMID: 18438844
24.  Arginine methyltransferase CARM1/PRMT4 regulates endochondral ossification 
Background
Chondrogenesis and subsequent endochondral ossification are processes tightly regulated by the transcription factor Sox9 (SRY-related high mobility group-Box gene 9), but molecular mechanisms underlying this activity remain unclear. Here we report that coactivator-associated arginine methyltransferase 1 (CARM1) regulates chondrocyte proliferation via arginine methylation of Sox9.
Results
CARM1-null mice display delayed endochondral ossification and decreased chondrocyte proliferation. Conversely, cartilage development of CARM1 transgenic mice was accelerated. CARM1 specifically methylates Sox9 at its HMG domain in vivo and in vitro. Arg-methylation of Sox9 by CARM1 disrupts interaction of Sox9 with beta-catenin, regulating Cyclin D1 expression and cell cycle progression of chondrocytes.
Conclusion
These results establish a role for CARM1 as an important regulator of chondrocyte proliferation during embryogenesis.
doi:10.1186/1471-213X-9-47
PMCID: PMC2754437  PMID: 19725955
25.  Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis 
Introduction
Recent findings suggest that articular cartilage contains mesenchymal progenitor cells. The aim of this study was to examine the distribution of stem cell markers (Notch-1, Stro-1 and VCAM-1) and of molecules that modulate progenitor differentiation (Notch-1 and Sox9) in normal adult human articular cartilage and in osteoarthritis (OA) cartilage.
Methods
Expression of the markers was analyzed by immunohistochemistry (IHC) and flow cytometry. Hoechst 33342 dye was used to identify and sort the cartilage side population (SP). Multilineage differentiation assays including chondrogenesis, osteogenesis and adipogenesis were performed on SP and non-SP (NSP) cells.
Results
A surprisingly high number (>45%) of cells were positive for Notch-1, Stro-1 and VCAM-1 throughout normal cartilage. Expression of these markers was higher in the superficial zone (SZ) of normal cartilage as compared to the middle zone (MZ) and deep zone (DZ). Non-fibrillated OA cartilage SZ showed reduced Notch-1 and Sox9 staining frequency, while Notch-1, Stro-1 and VCAM-1 positive cells were increased in the MZ. Most cells in OA clusters were positive for each molecule tested. The frequency of SP cells in cartilage was 0.14 ± 0.05% and no difference was found between normal and OA. SP cells displayed chondrogenic and osteogenic but not adipogenic differentiation potential.
Conclusions
These results show a surprisingly high number of cells that express putative progenitor cell markers in human cartilage. In contrast, the percentage of SP cells is much lower and within the range of expected stem cell frequency. Thus, markers such as Notch-1, Stro-1 or VCAM-1 may not be useful to identify progenitors in cartilage. Instead, their increased expression in OA cartilage implicates involvement in the abnormal cell activation and differentiation process characteristic of OA.
doi:10.1186/ar2719
PMCID: PMC2714136  PMID: 19500336

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