35043794
Epigenetic silencing of HDAC1 by miR-449a upregulates Runx2 and promotes osteoblast differentiation.
Human-induced pluripotent (iPS) cells can be induced to differentiate into osteoblasts, but the process is inefficient and time-consuming. Previous studies indicated a close association between the expression of Runx2 and osteoblast differentiation, and established that the transcriptional activation of the Runx2 gene was closely associated with histone acetylation. microRNA-449a (miR-449a) represses HDAC1 expression, thereby regulating histone acetylation. In the present study, whether the expression of miR-449a enhanced the generation of osteoblasts from human iPS cells was investigated. Introduction of miR-449a into human iPS cells resulted in the expression of osteoblast markers after only four days, compared to eight days for untransfected human iPS cells. Differentiation to osteoblasts was associated with a reduction in HDAC1 expression, and higher levels of histone acetylation, particularly at the binding sites on the Runx2 promoter in the human miR-449a-transfected iPS cells. Silencing of endogenous HDAC1 expression by exogenous miR-449a therefore maintains histone acetylation status, stimulates Runx2 gene expression and rapidly promotes osteoblast differentiation.
5236100
High efficiency of reprogramming CD34⁺ cells derived from human amniotic fluid into induced pluripotent stem cells with Oct4.
Although many techniques can be used to generate multitype-induced pluripotent stem (iPS) cells from multitype seed cells, improving the efficiency and shortening the period of cell reprogramming remain troublesome issues. In this study, to generate iPS cells, CD34⁺ cells, isolated from human amniotic fluid cells (HuAFCs) by flow cytometry, were infected with retroviruses carrying only one reprogramming factor (Oct4) and cultured on human amniotic epithelial cell (HuAEC) feeder layers. Approximately 4 to 5 days after viral infection, some embryonic stem cell (ESC)-like colonies appeared among the feeder cells. These colonies were positive for alkaline phosphatase and expressed high levels of ESC pluripotent markers (Nanog, Sox2, Oct4, CD133, and Rex1). Moreover, these iPS cells exhibited high levels of telomerase activity and had normal karyotypes. Additionally, these cells could differentiate into cell types from all 3 germ layers in vivo and in teratomas. In summary, we report a novel way of iPS generation that uses CD34⁺ HuAFCs as seed cells. Using this method, we can generate human iPS cells with greater efficiency and safety (the oncogenic factors, c-Myc and Klf4, were not used), and using the minimum number of reprogramming factors (only one factor, Oct4). Besides, HuAECs were used as feeder layers to culture human iPS cells, which could not only avoid contamination with heterogeneous proteins, but also maintain iPS cells in a self-renewing and undifferentiated state for a long time.
23125747
Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression.
Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal.
26560555
Bone tissue engineering of induced pluripotent stem cells cultured with macrochanneled polymer scaffold.
A reliable source of osteogenic cells is an essential factor for bone tissue engineering. In this study, human-induced pluripotent stem cells (hiPSCs) without an embryoid body step were cultured in macrochanneled poly(caprolactone) (PCL) scaffolds prepared using a robotic dispensing technique, after which osteogenesis was promoted by the addition of exogenous osteogenic factors. The osteogenesis of the hiPSCs was demonstrated based on the detection of osteogenic molecules, such as osteopontin, using flow cytometry analysis, quantitative polymerase chain reaction and western blotting. Thereafter, the cell-scaffold constructs were transplanted into the subcutaneous site of male athymic mice. At 4 weeks after implantation, histological assays (hematoxylin & eosin staining, Alizarin red staining, and osteocalcin immunostaining) were conducted to determine the bone induction of hiPSCs. The results indicated a production of pronounced levels of extracellular matrices and their mineral deposition within the cell-scaffold implant, suggesting possible in vivo bone induction by the hiPSCs-based tissue engineering approach. The results presented here provide useful information regarding the tissue engineering of bone utilizing hiPSCs in conjunction with cell-supporting scaffolds.
1565219
Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors
Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.
8531539
Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors
Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells, capable of germline transmission, from mouse somatic cells by transduction of four defined transcription factors. Here, we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. Furthermore, these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts.
15475140
Effects of TGF-β1 and VEGF-A transgenes on the osteogenic potential of bone marrow stromal cells in vitro and in vivo
An exogenous supply of growth factors and bioreplaceable scaffolds may help bone regeneration. The aim of this study was to examine the effects of TGF-β1 and VEGF-A transgenes on the osteogenic potential of bone marrow stromal cells. Rat bone marrow stromal cells were transfected with plasmids encoding mouse TGF-β1 and/or VEGF-A complementary DNAs and cultured for up to 28 days. Furthermore, collagen scaffolds carrying combinations of the plasmids-transfected cells were implanted subcutaneously in rats. The transgenes increased alkaline phosphatase activity, enhanced mineralized nodule formation, and elevated osteogenic gene expressions in vitro. In vivo, messenger RNA expression of osteogenic genes such as BMPs and Runx2 elevated higher by the transgenes. The data indicate that exogenous TGF-β1 and VEGF-A acted synergistically and could induce osteoblastic differentiation of bone marrow stromal cells in both cell culture and an animal model. The results may provide valuable information to optimize protocols for transgene-and-cell-based tissue engineering.
24630813
Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells
The development of regenerative therapies for cartilage injury has been greatly aided by recent advances in stem cell biology. Induced pluripotent stem cells (iPSCs) have the potential to provide an abundant cell source for tissue engineering, as well as generating patient-matched in vitro models to study genetic and environmental factors in cartilage repair and osteoarthritis. However, both cell therapy and modeling approaches require a purified and uniformly differentiated cell population to predictably recapitulate the physiological characteristics of cartilage. Here, iPSCs derived from adult mouse fibroblasts were chondrogenically differentiated and purified by type II collagen (Col2)-driven green fluorescent protein (GFP) expression. Col2 and aggrecan gene expression levels were significantly up-regulated in GFP+ cells compared with GFP− cells and decreased with monolayer expansion. An in vitro cartilage defect model was used to demonstrate integrative repair by GFP+ cells seeded in agarose, supporting their potential use in cartilage therapies. In chondrogenic pellet culture, cells synthesized cartilage-specific matrix as indicated by high levels of glycosaminoglycans and type II collagen and low levels of type I and type X collagen. The feasibility of cell expansion after initial differentiation was illustrated by homogenous matrix deposition in pellets from twice-passaged GFP+ cells. Finally, atomic force microscopy analysis showed increased microscale elastic moduli associated with collagen alignment at the periphery of pellets, mimicking zonal variation in native cartilage. This study demonstrates the potential use of iPSCs for cartilage defect repair and for creating tissue models of cartilage that can be matched to specific genetic backgrounds.
31401452
Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells.
Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after, but not before, 14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair.
3073434
Involvement of MicroRNAs in Regulation of Osteoblastic Differentiation in Mouse Induced Pluripotent Stem Cells
Backgoround MicroRNAs (miRNAs), which regulate biological processes by annealing to the 3′-untranslated region (3′-UTR) of mRNAs to reduce protein synthesis, have been the subject of recent attention as a key regulatory factor in cell differentiation. The effects of some miRNAs during osteoblastic differentiation have been investigated in mesenchymal stem cells, however they still remains to be determined in pluripotent stem cells. Methodology/Principal Findings Bone morphogenic proteins (BMPs) are potent activators of osteoblastic differentiation. In the present study, we profiled miRNAs during osteoblastic differentiation of mouse induced pluripotent stem (iPS) cells by BMP-4, in which expression of important osteoblastic markers such as Rux2, osterix, osteopontin, osteocalcin, PTHR1 and RANKL were significantly increased. A miRNA array analysis revealed that six miRNAs including miR-10a, miR-10b, miR-19b, miR-9-3p, miR-124a and miR-181a were significantly downregulated. Interestingly, miR-124a and miR-181a directly target the transcription factors Dlx5 and Msx2, both of which were increased by about 80-and 30-fold, respectively. In addition, transfection of miR-124a and miR-181a into mouse osteo-progenitor MC3T3-E1 cells significantly reduced expression of Dlx5, Runx2, osteocalcin and ALP, and Msx2 and osteocalcin, respectively. Finally, transfection of the anti-miRNAs of these six miRNAs, which are predicted to target Dlx5 and Msx2, into mouse iPS cells resulted in a significant increase in several osteoblastic differentiation markers such as Rux2, Msx2 and osteopontin. Conclusions/Significance In the present study, we demonstrate that six miRNAs including miR-10a, miR-10b, miR-19b, miR-9-3p, miR-124a and miR-181a miRNAs, especially miR-124a and miR-181a, are important regulatory factors in osteoblastic differentiation of mouse iPS cells.
13635771
A Specific Oligodeoxynucleotide Promotes the Differentiation of Osteoblasts via ERK and p38 MAPK Pathways
A specific oligodeoxynucleotide (ODN), ODN MT01, was found to have positive effects on the proliferation and activation of the osteoblast-like cell line MG 63. In this study, the detailed signaling pathways in which ODN MT01 promoted the differentiation of osteoblasts were systematically examined. ODN MT01 enhanced the expression of osteogenic marker genes, such as osteocalcin and type I collagen. Furthermore, ODN MT01 activated Runx2 phosphorylation via ERK1/2 mitogen-activated protein kinase (MAPK) and p38 MAPK. Consistently, ODN MT01 induced up-regulation of osteocalcin, alkaline phosphatase (ALP) and type I collagen, which was inhibited by pre-treatment with the ERK1/2 inhibitor U0126 and the p38 inhibitor SB203580. These results suggest that the ERK1/2 and p38 MAPK pathways, as well as Runx2 activation, are involved in ODN MT01-induced up-regulation of osteocalcin, type I collagen and the activity of ALP in MG 63 cells.
18162236
Nell-1, a key Functional Mediator of Runx2, Partially Rescues Calvarial Defects in Runx2+/− Mice
Mesenchymal stem cell commitment to an osteoprogenitor lineage requires the activity of Runx2, a molecule implicated in the etiopathology of multiple congenital craniofacial anomalies. Through promoter analyses, we have recently identified a new direct transcriptional target of Runx2, Nell‐1, a craniosynostosis (CS)–associated molecule with potent osteogenic properties. This study investigated the mechanistic and functional relationship between Nell‐1 and Runx2 in regulating osteoblast differentiation. The results showed that spatiotemporal distribution and expression levels of Nell‐1 correlated closely with those of endogenous Runx2 during craniofacial development. Phenotypically, cross‐mating Nell‐1 overexpression transgenic (CMV‐Nell‐1) mice with Runx2 haploinsufficient (Runx2+/−) mice partially rescued the calvarial defects in the cleidocranial dysplasia (CCD)–like phenotype of Runx2+/− mice, whereas Nell‐1 protein induced mineralization and bone formation in Runx2+/− but not Runx2−/− calvarial explants. Runx2‐mediated osteoblastic gene expression and/or mineralization was severely reduced by Nell‐1 siRNA oligos transfection into Runx2+/+ newborn mouse calvarial cells (NMCCs) or in N‐ethyl‐N‐nitrosourea (ENU)–induced Nell‐1−/− NMCCs. Meanwhile, Nell‐1 overexpression partially rescued osteoblastic gene expression but not mineralization in Runx2 null (Runx2−/−) NMCCs. Mechanistically, irrespective of Runx2 genotype, Nell‐1 signaling activates ERK1/2 and JNK1 mitogen‐activated protein kinase (MAPK) pathways in NMCCs and enhances Runx2 phosphorylation and activity when Runx2 is present. Collectively, these data demonstrate that Nell‐1 is a critical downstream Runx2 functional mediator insofar as Runx2‐regulated Nell‐1 promotes osteoblastic differentiation through, in part, activation of MAPK and enhanced phosphorylation of Runx2, and Runx2 activity is significantly reduced when Nell‐1 is blocked or absent. © 2011 American Society for Bone and Mineral Research.
23675045
Osterix Regulates Calcification and Degradation of Chondrogenic Matrices through Matrix Metalloproteinase 13 (MMP13) Expression in Association with Transcription Factor Runx2 during Endochondral Ossification*
Background: Molecular mechanisms controlling the late stages of endochondral ossification are unclear. Results: Osterix functions as a downstream and transcriptional partner of Runx2 and induces MMP13 during chondrocyte differentiation. Conclusion: Osterix is essential for late-stage endochondral ossification. Significance: Osterix affects the ossification of cartilage matrices and matrix vesicles and might be involved in the development of osteoarthritis and related disorders. Endochondral ossification is temporally and spatially regulated by several critical transcription factors, including Sox9, Runx2, and Runx3. Although the molecular mechanisms that control the late stages of endochondral ossification (e.g. calcification) are physiologically and pathologically important, these precise regulatory mechanisms remain unclear. Here, we demonstrate that Osterix is an essential transcription factor for endochondral ossification that functions downstream of Runx2. The global and conditional Osterix-deficient mice studied here exhibited a defect of cartilage-matrix ossification and matrix vesicle formation. Importantly, Osterix deficiencies caused the arrest of endochondral ossification at the hypertrophic stage. Microarray analysis revealed that matrix metallopeptidase 13 (MMP13) is an important target of Osterix. We also showed that there exists a physical interaction between Osterix and Runx2 and that these proteins function cooperatively to induce MMP13 during chondrocyte differentiation. Most interestingly, the introduction of MMP13 stimulated the calcification of matrices in Osterix-deficient mouse limb bud cells. Our results demonstrated that Osterix was essential to endochondral ossification and revealed that the physical and functional interaction between Osterix and Runx2 were necessary for the induction of MMP13 during endochondral ossification.
17574652
Runx2 Haploinsufficiency Ameliorates the Development of Ossification of the Posterior Longitudinal Ligament
Ossification of the Posterior Longitudinal Ligament (OPLL) is a disease that is characterized by the ectopic calcification of the ligament; however, the pathogenesis of OPLL remains to be investigated. We attempted to identify the in vivo role of Runx2, a master regulator of osteoblast differentiation and skeletal mineralization, in the pathogenesis of OPLL. The expression of Runx2 in the ligament was examined using in situ hybridization and immunohistochemistry and by monitoring the activity of a LacZ gene that was inserted into the Runx2 gene locus. To investigate the functional role of Runx2, we studied ENPP1ttw/ttw mice, a mouse model of OPLL, that were crossed with heterozygous Runx2 mice to decrease the expression of Runx2, and we performed histological and quantitative radiological analyses using 3D-micro CT. Runx2 was expressed in the ligament of wild-type mice. The induction of Runx2 expression preceded the development of ectopic calcification in the OPLL-like region of the ENPP1ttw/ttw mice. Runx2 haploinsufficiency ameliorated the development of ectopic calcification in the ENPP1ttw/ttw mice. Collectively, this study demonstrated that Runx2 is expressed in an OPLL-like region, and its elevation is a prerequisite for developing the complete OPLL-like phenotype in a mouse model of OPLL.
17664322
CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation
Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage.
10806755
The cancer‐related transcription factor Runx2 modulates cell proliferation in human osteosarcoma cell lines
Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre‐osteoblast proliferation by affecting cell cycle progression in the G1 phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G1/S phase transition, and Runx2 is again up‐regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle‐regulated with respect to the G1/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre‐established levels in a given cell type triggers one or more anti‐proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth. J. Cell. Physiol. 228: 714–723, 2013. © 2012 Wiley Periodicals, Inc.
22024889
Histone deacetylase inhibitor trichostatin A promotes the osteogenic differentiation of rat adipose-derived stem cells by altering the epigenetic modifications on Runx2 promoter in a BMP signaling-dependent manner.
Adult stem cells reside in many types of tissues and adult stem cell-based regenerative medicine holds great promise for repair of diseased tissues. Recently, adipose-derived stem cells (ADSCs) were found to be an appealing alternative to bone marrow stem cells (BMSCs) for tissue-engineered bone regeneration. Compared with BMSCs, ADSCs can be easily and abundantly available from adipose tissue. However, our previous study has discovered an important phenomenon that BMSCs have greater osteogenic potential than ADSCs in vitro. In this study, we aimed to explore its mechanism and improve the osteogenic potential of ADSCs for bone tissue regeneration. It has been reported that the epigenetic states could contribute to lineage-specific differentiation of adult stem cells. We observed that the epigenetic changes of BMSCs were much greater compared with ADSCs after a 3-day osteogenic induction. Runt-related transcription factor 2 (Runx2) is essential for osteoblast differentiation and bone formation. We found that BMSCs underwent more obvious epigenetic changes on the Runx2 promoter than ADSCs after osteogenic induction. These results suggest the epigenetic regulation involvement in Runx2 expression, and thus osteogenesis. We subsequently used a histone deacetylase inhibitor, trichostatin A (TSA), to promote the osteogenesis capacity of ADSCs. The results showed that TSA promoted rat ADSCs osteogenic differentiation by altering the epigenetic modifications on the Runx2 promoter in a bone morphogenetic protein signaling-dependent manner.
6687245
Histone deacetylases activate hepatocyte growth factor signaling by repressing microRNA-449 in hepatocellular carcinoma cells.
BACKGROUND & AIMS
Histone deacetylation regulates chromatin remodeling and transcriptional down-regulation of specific genomic regions; it is altered in many types of cancer cells. We searched for microRNAs (miRs) that are affected by histone deacetylation and investigated the effects in hepatocellular carcinoma (HCC) cells.


METHODS
HCC cell lines (HepG2, HLE, HLF, and Huh7) and immortalized liver cell lines (THLE-2 and THLE-3) were incubated with the histone deacetylase inhibitor trichostatin A. Differentially expressed messenger RNAs (mRNAs) and miRs were identified by expression profiling. Small interfering RNAs were used to reduce levels of histone deacetylases (HDAC)1-3, and HCC cell lines were transfected with miR-449. We evaluated growth of xenograft tumors from modified cells in nude mice. Cells were analyzed by immunoblot and luciferase reporter assays. We analyzed HCC samples from 23 patients.


RESULTS
HDAC1-3 were up-regulated in HCC samples from patients. In cell lines, inhibition of HDAC significantly increased levels of hsa-miR-449a. c-MET mRNA, which encodes the receptor tyrosine kinase for hepatocyte growth factor, is a target of miR-449. Incubation of HCC cells with trichostatin A or transfection with miR-449 reduced expression of c-MET and phosphorylation of extracellular signal-regulated kinases 1 and 2 (downstream effectors of c-MET), increased apoptosis, and reduced proliferation. Huh-7 cells transfected with miR-449 formed tumors more slowly in mice than cells expressing control miRs. HCC samples from patients had lower levels of miR-449 and higher levels of c-MET than human reference.


CONCLUSIONS
In HCC cells, up-regulation of HDAC1-3 reduces expression of miR-449. miR-449 binds c-MET mRNA to reduce its levels, promoting apoptosis and reducing proliferation of liver cells. Expression of miR-449 slows growth of HCC xenograft tumors in mice; this miR might function as a tumor suppressor.
206750123
Combining microRNA-449a/b with a HDAC inhibitor has a synergistic effect on growth arrest in lung cancer.
Histone deacetylases (HDACs) play a crucial role in tumorigenesis. Over-expression of HDACs has been reported in lung cancer. The mechanism of highly expressed HDAC1 in lung cancer has yet not been determined. In the present study, we showed that miR-449a/b regulates HDAC1 by directly binding with the 3' untranslated region of the HDAC1. The expression of miR-449a/b was down-regulated and the expression of HDAC1 was up-regulated in primary lung cancer. The down expression of miR-449a/b might be one mechanism for over-expression of HDAC1 in lung cancer. miR-449a/b inhibited cell growth and anchorage-independent growth. Furthermore, co-treatment with miR-449a and HDAC inhibitors had a significant growth reduction compared with HDAC inhibitor mono-treatment. These results suggest that miR-449a/b may have a tumor suppressor function and might be a potential therapeutic candidate in patients with primary lung cancer.
23931251
miR-449a targets HDAC-1 and induces growth arrest in prostate cancer
Histone deacetylases (HDACs) are frequently overexpressed in broad range of cancer types, where they alter cellular epigenetic programming to promote cell proliferation and survival. However, the mechanism by which HDACs become overexpressed in human cancers remains somewhat of a mystery. In this study, we investigated the expression and functional significance of miR-449a in prostate cancer cells. Using real-time PCR, we found that miR-449a is downregulated in prostate cancer tissues relative to patient-matched control tissue. Introduction of miR-449a into PC-3 prostate cancer cells resulted in cell-cycle arrest, apoptosis and a senescent-like phenotype. In silico analysis of 3′-UTR regions identified a number of genes involved in cell-cycle regulation as putative targets of miR-449a. Using a luciferase 3′-UTR reporter system, we established that HDAC-1 (histone deacetylase 1), a gene that is frequently overexpressed in many types of cancer, is a direct target of miR-449a. Further, our data indicate that miR-449a regulates cell growth and viability in part by repressing the expression of HDAC-1 in prostate cancer cells. Our findings provide new insight into the function of miRNA in regulating HDAC expression in normal versus cancerous tissue.
16764938
Low microRNA-199a expression in human amniotic epithelial cell feeder layers maintains human-induced pluripotent stem cell pluripotency via increased leukemia inhibitory factor expression.
Human-induced pluripotent stem (iPS) cells share the same key properties as embryonic stem cells, and may be generated from patient- or disease-specific sources, which makes them attractive for personalized medicine, drug screens, or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state is a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, as they express endogenous leukemia inhibitory factor (LIF) at high levels. Here, we examined the effect of exogenous microRNA-199a regulation on endogenous LIF expression in HuAECs, and in turn on human iPS cell pluripotency. We found that HuAECs feeder cells transfected with microRNA-199a mutant expressed LIF at high levels, allowing iPS to maintain a high level of alkaline phosphatase activity in long-term culture and form teratomas in severe combined immunodeficient mice. The expression of stem cell markers was increased in iPS cultured on HuAECs feeder cells transfected with the microRNA-199a mutant, compared with iPS cultured on HuAECs transfected with microRNA-199a or mouse embryo fibroblasts. Taken together, these results suggested that LIF expression might be regulated by microRNA-199a, and LIF was a crucial component in feeder cells, and also was required for maintenance of human iPS cells in an undifferentiated, proliferative state capable of self-renewal.
7502867
MicroRNA‐199a targets CD44 to suppress the tumorigenicity and multidrug resistance of ovarian cancer‐initiating cells
In ovarian cancer, CD44+/CD117+ stem cells, also known as cancer‐initiating cells (CICs), are highly proliferative, have a low degree of differentiation, and are resistant to chemotherapeutics. Therefore, the CD44+/CD117+ subpopulation is thought to be an important target for novel therapeutic strategies. In this study, we investigated the role of microRNA‐199a (miR‐199a) in ovarian cancer stem cells. Luciferase reporter gene assays confirmed that miR‐199a targets CD44 via an miR‐199a‐binding site in the 3′‐UTR. CD44+/CD117+ ovarian CICs were enriched from human primary ovarian tumor tissues and confirmed by flow cytometric sorting. miR‐199a was cloned and transfected into ovarian CICs. CD44 mRNA and protein expression was significantly decreased in miR‐199a‐transfected ovarian CICs as compared with miR‐199a mutant‐transfected and untransfected cells. Cell cycle analysis, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide proliferation assays, the colony formation assay and the transwell migration assay indicated that miR‐199a significantly affected cell cycle regulation and suppressed the proliferation and invasive capacity of ovarian CICs in vitro. miR‐199a significantly increased the chemosensitivity of ovarian CICs to cisplatin, pacitaxel, and adriamycin, and reduced mRNA expression of the multidrug resistance gene ABCG2 as compared with miR‐199a mutant‐transfected and untransfected cells. The expression of stemness markers was also significantly reduced in miR‐199a‐transfected CICs as compared with miR‐199a mutant‐transfected and untransfected ovarian cells. Furthermore, xenograft experiments confirmed that miR‐199a suppressed the growth of xenograft tumors formed by ovarian CICs in vivo. Thus, expression of endogenous mature miR‐199a may prevent tumorigenesis in human ovarian cancer by regulating expression of its target gene CD44.
6076960
microRNA-182 inhibits the proliferation and invasion of human lung adenocarcinoma cells through its effect on human cortical actin-associated protein.
Lung cancer is one of the main causes of cancer death worldwide. The cortactin gene, CTTN, may play a pivotal role in the proliferation and invasion of tumors. A microRNA (miR-182) was cloned and used to study the expression of CTTN and its regulatory effects on the proliferation and invasion of the lung cancer cell line, A549. Cortactin protein and CTTN mRNA expression decreased in A549 cells that were transfected with the miR-182 expression plasmid. A cell proliferation assay indicated that miR-182 expression affected cell cycle regulation and suppressed proliferation of lung cancer cells in vitro. In addition, xenograft experiments confirmed the suppression of tumor growth in vivo, which was due to the promotion of apoptosis. In conclusion, endogenous mature miR-182 expression may have an important role in the pathogenesis of lung cancer through its interference with the target gene CTTN by epigenetic modification.
30300993
Selective Signaling by Akt1 Controls Osteoblast Differentiation and Osteoblast-Mediated Osteoclast Development
ABSTRACT Maintaining optimal bone integrity, mass, and strength throughout adult life requires ongoing bone remodeling, which involves coordinated activity between actions of bone-resorbing osteoclasts and bone forming-osteoblasts. Osteoporosis is a disorder of remodeling in which bone resorption outstrips deposition, leading to diminished bone mass and an increased risk of fractures. Here we identify Akt1 as a unique signaling intermediate in osteoblasts that can control both osteoblast and osteoclast differentiation. Targeted knockdown of Akt1 in mouse primary bone marrow stromal cells or in a mesenchymal stem cell line or genetic knockout of Akt1 stimulated osteoblast differentiation secondary to increased expression of the osteogenic transcription factor Runx2. Despite enhanced osteoblast differentiation, coupled osteoclastogenesis in Akt1 deficiency was markedly inhibited, with reduced accumulation of specific osteoclast mRNAs and proteins and impaired fusion to form multinucleated osteoclasts, defects secondary to diminished production of receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (m-CSF), critical osteoblast-derived osteoclast differentiation factors. Delivery of recombinant lentiviruses encoding Akt1 but not Akt2 to Akt1-deficient osteoblast progenitors reversed the increased osteoblast differentiation and, by boosting accumulation of RANKL and m-CSF, restored normal osteoclastogenesis, as did the addition of recombinant RANKL to conditioned culture medium from Akt1-deficient osteoblasts. Our results support the idea that targeted inhibition of Akt1 could lead to therapeutically useful net bone acquisition, and they indicate that closely related Akt1 and Akt2 exert distinct effects on cellular differentiation pathways.
45945170
microRNA-155 regulates angiotensin II type 1 receptor expression in umbilical vein endothelial cells from severely pre-eclamptic pregnant women.
Angiotensin II is critical in pre-eclampsia pathogenesis. In addition, microRNA-155 regulates angiotensin II type 1 receptor expression. We have explored the function of microRNA-155 in pre-eclamptic pregnant women. Human umbilical vein endothelial cells were isolated and cultured from healthy puerperant women and pre-eclampsia patients. The cells were transfected with a mature microRNA-155 plasmid. The effect of microRNA-155 was assessed by Northern blotting, in situ hybridization, quantitative real-time PCR, immunofluorescent staining and Western blotting. In addition, activation of extracellular signal-regulated kinase 1/2 was assessed by co-immunoprecipitation and Western blotting. Severely pre-eclamptic pregnant women expressed less mature miR-155 compared to healthy pregnant women. In addition, angiotensin II type 1 receptor expression decreased substantially in healthy cells and miR-155-transfected cells compared to miR-155-mutant-transfected cells and cells from pre-eclamptic patients. Mature miR-155 reduced angiotensin II-induced extracellular signal-regulated kinase 1/2 activation. In conclusion, endogenous mature miR-155 expression may be an important contributor to the pathogenesis of severe pre-eclampsia.
14238430
Chromatin: the interface between extrinsic cues and the epigenetic regulation of muscle regeneration.
Muscle regeneration provides a paradigm by which to study how extrinsic signals coordinate gene expression in somatic stem cells (satellite cells) by directing the genome distribution of chromatin-modifying complexes. Understanding the signal-dependent control of the epigenetic events underlying the transition of muscle stem cells through sequential regeneration stages holds the promise to reveal new targets for selective interventions toward repairing diseased muscles. This review describes the latest findings on how regeneration cues are integrated at the chromatin level to build the transcription network that regulates progression of endogenous muscle progenitors throughout the myogenic program. In particular, we describe how specific epigenetic signatures can confer responsiveness to extrinsic cues on discrete regions of the muscle stem cell genome.
27651946
Signaling to the chromatin during skeletal myogenesis: novel targets for pharmacological modulation of gene expression.
Cellular differentiation entails an extensive reprogramming of the genome toward the expression of discrete subsets of genes, which establish the tissue-specific phenotype. This program is achieved by epigenetic marks of the chromatin at particular loci, and is regulated by environmental cues, such as soluble factors and cell-to-cell interactions. How the intracellular cascades convert the myriad of external stimuli into the nuclear information necessary to reprogram the genome toward specific responses is a question of biological and medical interest. The elucidation of the signaling converting cues from outside the cells into chromatin modifications at individual promoters holds the promise to unveil the targets for selective pharmacological interventions to modulate gene expression for therapeutic purposes. Enhancing muscle regeneration and preventing muscle breakdown are important goals in the therapy of muscular diseases, cancer-associated cachexia and aging-associated sarcopenia. We will summarize the recent progress of our knowledge of the regulation of gene expression by intracellular cascades elicited by external cues during skeletal myogenesis. And will illustrate the potential importance of targeting the chromatin signaling in regenerative medicine--e.g. to boost muscle regeneration.
24850064
The Polycomb Ezh2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation.
The Ezh2 protein endows the Polycomb PRC2 and PRC3 complexes with histone lysine methyltransferase (HKMT) activity that is associated with transcriptional repression. We report that Ezh2 expression was developmentally regulated in the myotome compartment of mouse somites and that its down-regulation coincided with activation of muscle gene expression and differentiation of satellite-cell-derived myoblasts. Increased Ezh2 expression inhibited muscle differentiation, and this property was conferred by its SET domain, required for the HKMT activity. In undifferentiated myoblasts, endogenous Ezh2 was associated with the transcriptional regulator YY1. Both Ezh2 and YY1 were detected, with the deacetylase HDAC1, at genomic regions of silent muscle-specific genes. Their presence correlated with methylation of K27 of histone H3. YY1 was required for Ezh2 binding because RNA interference of YY1 abrogated chromatin recruitment of Ezh2 and prevented H3-K27 methylation. Upon gene activation, Ezh2, HDAC1, and YY1 dissociated from muscle loci, H3-K27 became hypomethylated and MyoD and SRF were recruited to the chromatin. These findings suggest the existence of a two-step activation mechanism whereby removal of H3-K27 methylation, conferred by an active Ezh2-containing protein complex, followed by recruitment of positive transcriptional regulators at discrete genomic loci are required to promote muscle gene expression and cell differentiation.
22568928
The oncogenic role of Yin Yang 1.
Yin Yang 1 (YY1) is a transcription factor with diverse and complex biological functions. YY1 either activates or represses gene transcription, depending on the stimuli received by the cells and its association with other cellular factors. Since its discovery, a biological role for YY1 in tumor development and progression has been suggested because of its regulatory activities toward multiple cancer-related proteins and signaling pathways and its overexpression in most cancers. In this review, we primarily focus on YY1 studies in cancer research, including the regulation of YY1 as a transcription factor, its activities independent of its DNA binding ability, the functions of its associated proteins, and mechanisms regulating YY1 expression and activities. We also discuss the correlation of YY1 expression with clinical outcomes of cancer patients and its target potential in cancer therapy. Although there is not a complete consensus about the role of YY1 in cancers based on its activities of regulating oncogene and tumor suppressor expression, most of the currently available evidence supports a proliferative or oncogenic role of YY1 in tumorigenesis.
6366559
Essential Dosage-Dependent Functions of the Transcription Factor Yin Yang 1 in Late Embryonic Development and Cell Cycle Progression
ABSTRACT Constitutive ablation of the Yin Yang 1 (YY1) transcription factor in mice results in peri-implantation lethality. In this study, we used homologous recombination to generate knockout mice carrying yy1 alleles expressing various amounts of YY1. Phenotypic analysis of yy1 mutant embryos expressing ∼75%, ∼50%, and ∼25% of the normal complement of YY1 identified a dosage-dependent requirement for YY1 during late embryogenesis. Indeed, reduction of YY1 levels impairs embryonic growth and viability in a dose-dependent manner. Analysis of the corresponding mouse embryonic fibroblast cells also revealed a tight correlation between YY1 dosage and cell proliferation, with a complete ablation of YY1 inducing cytokinesis failure and cell cycle arrest. Consistently, RNA interference-mediated inhibition of YY1 in HeLa cells prevents cytokinesis, causes proliferative arrest, and increases cellular sensitivity to various apoptotic agents. Genome-wide expression profiling identified a plethora of YY1 target genes that have been implicated in cell growth, proliferation, cytokinesis, apoptosis, development, and differentiation, suggesting that YY1 coordinates multiple essential biological processes through a complex transcriptional network. These data not only shed new light on the molecular basis for YY1 developmental roles and cellular functions, but also provide insight into the general mechanisms controlling eukaryotic cell proliferation, apoptosis, and differentiation.