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1.
Nat Biomed Eng ; 5(8): 926-940, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34373601

RESUMO

Current protocols for the differentiation of human pluripotent stem cells (hPSCs) into chondrocytes do not allow for the expansion of intermediate progenitors so as to prospectively assess their chondrogenic potential. Here we report a protocol that leverages PRRX1-tdTomato reporter hPSCs for the selective induction of expandable and ontogenetically defined PRRX1+ limb-bud-like mesenchymal cells under defined xeno-free conditions, and the prospective assessment of the cells' chondrogenic potential via the cell-surface markers CD90, CD140B and CD82. The cells, which proliferated stably and exhibited the potential to undergo chondrogenic differentiation, formed hyaline cartilaginous-like tissue commensurate to their PRRX1-expression levels. Moreover, we show that limb-bud-like mesenchymal cells derived from patient-derived induced hPSCs can be used to identify therapeutic candidates for type II collagenopathy and we developed a method to generate uniformly sized hyaline cartilaginous-like particles by plating the cells on culture dishes coated with spots of a zwitterionic polymer. PRRX1+ limb-bud-like mesenchymal cells could facilitate the mass production of chondrocytes and cartilaginous tissues for applications in drug screening and tissue engineering.


Assuntos
Proteínas de Homeodomínio/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/transplante , Condrogênese , Doenças do Colágeno/terapia , Meios de Cultura/química , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Pluripotentes/metabolismo , Polímeros/química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Antígenos Thy-1/metabolismo , Engenharia Tecidual
2.
Biofactors ; 2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34418170

RESUMO

This study aimed to reveal the possible mechanisms by which O-linked-N-acetylglucosaminylation (O-GlcNAcylation) regulates osteoblast differentiation using a series of bioinformatics-oriented experiments. To examine the influence of O-GlcNAcylation levels on osteoblast differentiation, osteoblastic MC3T3-E1 cells were treated with O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) inhibitors. Correlations between the levels of O-GlcNAcylation and the expression of osteogenic markers as well as OGT were evaluated by qPCR and western blotting. The O-GlcNAcylated proteins assumed to correlate with Runx2 expression were retrieved from several public databases and used for further bioinformatics analysis. Following the findings of the bioinformatics analysis, intracellular calcium ([Ca2+ ]i ) was monitored in the cells treated with OGT and OGA inhibitors using a confocal laser-scanning microscope (CLS). The interaction effect between O-GlcNAcylation and [Ca2+ ]i on osteogenic marker expression was determined using stable OGT knockdown MC3T3-E1 cells. O-GlcNAcylation was positively associated with osteoblast differentiation. The time-course profile of global O-GlcNAcylated proteins showed a distinctive pattern with different molecular weights during osteoblast differentiation. The expression pattern of several O-GlcNAcylated proteins was significantly similar to that of Runx2 expression. Bioinformatic analysis of the retrieved Runx2-related-O-GlcNAcylated-proteins revealed the importance of [Ca2+ ]i . CLS showed that alteration of O-GlcNAcylation rapidly changed [Ca2+ ]i in MC3T3-E1 cells. O-GlcNAcylation and [Ca2+ ]i showed an interaction effect on the expression of osteogenic markers. OGT knockdown disrupted the [Ca2+ ]i -induced expression changes of osteogenic markers. O-GlcNAcylation interacts with [Ca2+ ]i and elicits osteoblast differentiation by regulating the expression of osteogenic markers.

3.
Int J Cancer ; 149(8): 1593-1604, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34152598

RESUMO

Lung adenocarcinoma (LUAD) is the most common types among lung cancers generally arising from terminal airway and understanding of multistep carcinogenesis is crucial to develop novel therapeutic strategy for LUAD. Here we used human induced pluripotent stem cells (hiPSCs) to establish iHER2-hiPSCs in which doxycycline induced the expression of the oncoprotein human epidermal growth factor receptor 2 (HER2)/ERBB2. Lung progenitors that differentiated from iHER2-hiPSCs, which expressed NKX2-1/TTF-1 known as a lung lineage maker, were cocultured with human fetal fibroblast and formed human lung organoids (HLOs) comprising alveolar type 2-like cells. HLOs that overexpressed HER2 transformed to tumor-like structures similar to atypical adenomatous hyperplasia, which is known for lung precancerous lesion and upregulated the activities of oncogenic signaling cascades such as RAS/RAF/MAPK and PI3K/AKT/mTOR. The degree of morphological irregularity and proliferation capacity were significantly higher in HLOs from iHER2-hiPSCs. Moreover, the transcriptome profile of the HLOs shifted from a normal lung tissue-like state to one characteristic of clinical LUAD with HER2 amplification. Our results suggest that hiPSC-derived HLOs may serve as a model to recapitulate the early tumorigenesis of LUAD and would provide new insights into the molecular basis of tumor initiation and progression.


Assuntos
Adenocarcinoma de Pulmão/patologia , Carcinogênese , Regulação Neoplásica da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/patologia , Neoplasias Pulmonares/patologia , Organoides/patologia , Receptor ErbB-2/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Organoides/metabolismo , Receptor ErbB-2/genética , Transcriptoma , Células Tumorais Cultivadas
4.
J Clin Invest ; 131(6)2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33555272

RESUMO

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with inferior outcome compared with that of B cell ALL. Here, we show that Runt-related transcription factor 2 (RUNX2) was upregulated in high-risk T-ALL with KMT2A rearrangements (KMT2A-R) or an immature immunophenotype. In KMT2A-R cells, we identified RUNX2 as a direct target of the KMT2A chimeras, where it reciprocally bound the KMT2A promoter, establishing a regulatory feed-forward mechanism. Notably, RUNX2 was required for survival of immature and KMT2A-R T-ALL cells in vitro and in vivo. We report direct transcriptional regulation of CXCR4 signaling by RUNX2, thereby promoting chemotaxis, adhesion, and homing to medullary and extramedullary sites. RUNX2 enabled these energy-demanding processes by increasing metabolic activity in T-ALL cells through positive regulation of both glycolysis and oxidative phosphorylation. Concurrently, RUNX2 upregulation increased mitochondrial dynamics and biogenesis in T-ALL cells. Finally, as a proof of concept, we demonstrate that immature and KMT2A-R T-ALL cells were vulnerable to pharmacological targeting of the interaction between RUNX2 and its cofactor CBFß. In conclusion, we show that RUNX2 acts as a dependency factor in high-risk subtypes of human T-ALL through concomitant regulation of tumor metabolism and leukemic cell migration.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Animais , Linhagem Celular Tumoral , Quimiotaxia de Leucócito , Criança , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/metabolismo , Progressão da Doença , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Hematopoese , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Biogênese de Organelas , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais
5.
Int J Mol Sci ; 21(19)2020 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-32987737

RESUMO

Medication-related osteonecrosis of the jaw (MRONJ) is a severe pathological condition associated mainly with the long-term administration of bone resorption inhibitors, which are known to induce suppression of osteoclast activity and bone remodeling. Bone Morphogenetic Protein (BMP)-2 is known to be a strong inducer of bone remodeling, by directly regulating osteoblast differentiation and osteoclast activity. This study aimed to evaluate the effects of BMP-2 adsorbed onto beta-tricalcium phosphate (ß-TCP), which is an osteoinductive bioceramic material and allows space retention, on the prevention and treatment of MRONJ in mice. Tooth extraction was performed after 3 weeks of zoledronate (ZA) and cyclophosphamide (CY) administration. For prevention studies, BMP-2/ß-TCP was transplanted immediately after tooth extraction, and the mice were administered ZA and CY for an additional 4 weeks. The results showed that while the tooth extraction socket was mainly filled with a sparse tissue in the control group, bone formation was observed at the apex of the tooth extraction socket and was filled with a dense connective tissue rich in cellular components in the BMP-2/ß-TCP transplanted group. For treatment studies, BMP-2/ß-TCP was transplanted 2 weeks after tooth extraction, and bone formation was followed up for the subsequent 4 weeks under ZA and CY suspension. The results showed that although the tooth extraction socket was mainly filled with soft tissue in the control group, transplantation of BMP-2/ß-TCP could significantly accelerate bone formation, as shown by immunohistochemical analysis for osteopontin, and reduce the bone necrosis in tooth extraction sockets. These data suggest that the combination of BMP-2/ß-TCP could become a suitable therapy for the management of MRONJ.


Assuntos
Materiais Biocompatíveis/uso terapêutico , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/tratamento farmacológico , Conservadores da Densidade Óssea/uso terapêutico , Proteína Morfogenética Óssea 2/uso terapêutico , Regeneração Óssea/efeitos dos fármacos , Fosfatos de Cálcio/uso terapêutico , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/uso terapêutico
6.
Sci Rep ; 10(1): 9921, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32555437

RESUMO

Core Binding Factors (CBFs) are a small group of heterodimeric transcription factor complexes composed of DNA binding proteins, RUNXs, and a non-DNA binding protein, CBFB. The LH surge increases the expression of Runx1 and Runx2 in ovulatory follicles, while Cbfb is constitutively expressed. To investigate the physiological significance of CBFs, we generated a conditional mutant mouse model in which granulosa cell expression of Runx2 and Cbfb was deleted by the Esr2Cre. Female Cbfbflox/flox;Esr2cre/+;Runx2flox/flox mice were infertile; follicles developed to the preovulatory follicle stage but failed to ovulate. RNA-seq analysis of mutant mouse ovaries collected at 11 h post-hCG unveiled numerous CBFs-downstream genes that are associated with inflammation, matrix remodeling, wnt signaling, and steroid metabolism. Mutant mice also failed to develop corpora lutea, as evident by the lack of luteal marker gene expression, marked reduction of vascularization, and excessive apoptotic staining in unruptured poorly luteinized follicles, consistent with dramatic reduction of progesterone by 24 h after hCG administration. The present study provides in vivo evidence that CBFs act as essential transcriptional regulators of both ovulation and luteinization by regulating the expression of key genes that are involved in inflammation, matrix remodeling, cell differentiation, vascularization, and steroid metabolisms in mice.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Subunidade beta de Fator de Ligação ao Core/fisiologia , Fertilidade , Células da Granulosa/metabolismo , Infertilidade Feminina/fisiopatologia , Luteinização , Ovulação , Animais , Feminino , Células da Granulosa/citologia , Camundongos , Camundongos Knockout , Reprodução
7.
Nat Commun ; 11(1): 2289, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385263

RESUMO

The osteoblast differentiation capacity of skeletal stem cells (SSCs) must be tightly regulated, as inadequate bone formation results in low bone mass and skeletal fragility, and over-exuberant osteogenesis results in heterotopic ossification (HO) of soft tissues. RUNX2 is essential for tuning this balance, but the mechanisms of posttranslational control of RUNX2 remain to be fully elucidated. Here, we identify that a CK2/HAUSP pathway is a key regulator of RUNX2 stability, as Casein kinase 2 (CK2) phosphorylates RUNX2, recruiting the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which stabilizes RUNX2 by diverting it away from ubiquitin-dependent proteasomal degradation. This pathway is important for both the commitment of SSCs to osteoprogenitors and their subsequent maturation. This CK2/HAUSP/RUNX2 pathway is also necessary for HO, as its inhibition blocked HO in multiple models. Collectively, active deubiquitination of RUNX2 is required for bone formation and this CK2/HAUSP deubiquitination pathway offers therapeutic opportunities for disorders of inappropriate mineralization.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Ossificação Heterotópica/metabolismo , Osteogênese , Adulto , Idoso , Animais , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Diferenciação Celular , Displasia Cleidocraniana/genética , Displasia Cleidocraniana/patologia , Feminino , Deleção de Genes , Haploinsuficiência/genética , Membro Posterior/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Ossificação Heterotópica/genética , Ossificação Heterotópica/patologia , Osteoblastos/metabolismo , Fosforilação , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo
8.
Biochem Biophys Res Commun ; 526(1): 213-217, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32204914

RESUMO

The Cre-loxP recombination system is widely used to generate genetically modified mice for biomedical research. Recently, a highly efficient photoactivatable Cre (PA-Cre) based on reassembly of split Cre fragments has been established. This technology enables efficient DNA recombination that is activated upon blue light illumination with spatiotemporal precision. In this study, we generated a tTA-dependent photoactivatable Cre-loxP recombinase knock-in mouse model (TRE-PA-Cre mice) using a CRISPR/Cas9 system. These mice were crossed with ROSA26-tdTomato mice (Cre reporter mouse) to visualize DNA recombination as marked by tdTomato expression. We demonstrated that external noninvasive LED blue light illumination allows efficient DNA recombination in the liver of TRE-PA-Cre:ROSA26-tdTomato mice transfected with tTA expression vectors using hydrodynamic tail vein injection. The TRE-PA-Cre mouse established here promises to be useful for optogenetic genome engineering in a noninvasive, spatiotemporal, and cell-type specific manner in vivo.


Assuntos
Técnicas de Introdução de Genes , Engenharia Genética , Genoma , Integrases/metabolismo , Optogenética , Animais , Sequência de Bases , DNA/genética , Feminino , Luz , Masculino , Camundongos Endogâmicos C57BL , Modelos Animais , Tetraciclina/farmacologia
9.
Addict Biol ; 25(1): e12723, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30734456

RESUMO

In drug addiction, environmental stimuli previously associated with cocaine use readily elicit cocaine-associated memories, which persist long after abstinence and trigger cocaine craving and consumption. Although previous studies suggest that the medial prefrontal cortex (mPFC) is involved in the expression of cocaine-addictive behaviors, it remains unclear whether excitatory and inhibitory neurons in the mPFC are causally related to the formation and retrieval of cocaine-associated memories. To address this issue, we used the designer receptors exclusively activated by designer drugs (DREADD) technology combined with a cocaine-induced conditioned place preference (CPP) paradigm. We suppressed mPFC neuronal activity in a cell-type- and timing-dependent manner. C57BL/6J wild-type mice received bilateral intra-mPFC infusion of an adeno-associated virus (AAV) expressing inhibitory DREADD (hM4Di) under the control of CaMKII promotor to selectively suppress mPFC pyramidal neurons. GAD67-Cre mice received bilateral intra-mPFC infusion of a Cre-dependent AAV expressing hM4Di to specifically silence GABAergic neurons. Chemogenetic suppression of mPFC pyramidal neurons significantly attenuated both the acquisition and expression of cocaine CPP, while suppression of mPFC GABAergic neurons affected neither the acquisition nor expression of cocaine CPP. Moreover, chemogenetic inhibition of mPFC glutamatergic neurons did not affect the acquisition and expression of lithium chloride-induced conditioned place aversion. These results suggest that the activation of glutamatergic, but not GABAergic, neurons in the mPFC mediates both the formation and retrieval of cocaine-associated memories.


Assuntos
Transtornos Relacionados ao Uso de Cocaína/fisiopatologia , Cocaína/farmacologia , Neurônios GABAérgicos/efeitos dos fármacos , Memória/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/fisiopatologia , Animais , Modelos Animais de Doenças , Inibidores da Captação de Dopamina/farmacologia , Eletrofisiologia , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos C57BL
10.
Commun Biol ; 2: 346, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31552299

RESUMO

The pathophysiological role of mammalian target of rapamycin complex 1 (mTORC1) in neurodegenerative diseases is established, but possible therapeutic targets responsible for its activation in neurons must be explored. Here we identified solute carrier family 38a member 1 (SNAT1, Slc38a1) as a positive regulator of mTORC1 in neurons. Slc38a1 flox/flox and Synapsin I-Cre mice were crossed to generate mutant mice in which Slc38a1 was selectively deleted in neurons. Measurement of 2,3,5-triphenyltetrazolium chloride (TTC) or the MAP2-negative area in a mouse model of middle cerebral artery occlusion (MCAO) revealed that Slc38a1 deficiency decreased infarct size. We found a transient increase in the phosphorylation of p70S6k1 (pp70S6k1) and a suppressive effect of rapamycin on infarct size in MCAO mice. Autophagy inhibitors completely mitigated the suppressive effect of SNAT1 deficiency on neuronal cell death under in vitro stroke culture conditions. These results demonstrate that SNAT1 promoted ischemic brain damage via mTOR-autophagy system.


Assuntos
Sistema A de Transporte de Aminoácidos/antagonistas & inibidores , Sistema A de Transporte de Aminoácidos/metabolismo , Autofagia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Sistema A de Transporte de Aminoácidos/genética , Animais , Infarto Cerebral/etiologia , Infarto Cerebral/metabolismo , Infarto Cerebral/patologia , Expressão Gênica , Loci Gênicos , Genoma , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuroproteção , Especificidade de Órgãos
11.
Biochem Biophys Res Commun ; 516(4): 1229-1233, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31300199

RESUMO

Global gene deletion studies have established that Runt-related transcription factor-2 (Runx2) is essential during skeletogenesis for osteoblastic differentiation in both intramembranous and endochondral ossification processes. However, the postnatal significance of Runx2 in vivo is poorly understood because a global Runx2 deletion causes perinatal lethality. In this study, we generated tamoxifen-induced Runx2 global deficient mice by crossing Runx2flox mice with ROSA26-CreERT2 mice (Rosa26-CreERT2; Runx2flox/flox). Four-week-old mice were intraperitoneally treated with tamoxifen for five consecutive days, sacrificed, and analyzed six weeks after tamoxifen administration. Deletion of Runx2 led to low bone mass, which is associated with decreased bone formation and bone resorption as well as excessive bone marrow adiposity. Collectively, postnatal Runx2 absolutely plays an important role in maintaining the homeostasis of bone tissues not only in bone mass, but also in the bone marrow environment.


Assuntos
Adipócitos/citologia , Densidade Óssea , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Deleção de Genes , Adiposidade , Envelhecimento , Animais , Células da Medula Óssea/citologia , Cruzamentos Genéticos , Modelos Animais de Doenças , Feminino , Genótipo , Masculino , Camundongos , Camundongos Transgênicos , Osteoporose , Fenótipo , Tamoxifeno/farmacologia , Tíbia , Microtomografia por Raio-X
12.
Yakugaku Zasshi ; 139(6): 867-871, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31155527

RESUMO

The mesenchymal stem cell (MSC) is a type of tissue stem cell. In clinical studies, cultured MSCs have shown important therapeutic effects on diseases via both the reduction of neurological defects and the regulation of immune responses. However, in vivo MSC localization, function, and properties are poorly understood; therefore, the molecular understanding of MSC hierarchy is less advanced compared to hematopoietic stem cell hierarchy. Runt-related transcription factor 2 (Runx2) is an essential transcriptional regulator of osteoblast differentiation from MSCs. Runx2 deficiency in Paired-related homeobox 1 (Prrx1)-derived cells (Runx2Prrx1-/- mice) results in defective intramembranous ossification. Double-positive cells for Prrx1-GFP, and stem cell antigen-1 (Sca1) (Prrx1+Sca1+ cells) in the calvaria, express Runx2 at lower levels, and are more homogeneous and primitive compared with Prrx1+Sca1- cells. Our results suggest that osteoblast differentiation in vivo may begin at the Prrx1+Sca1+ MSC stage, with sequential progression to Prrx1+Sca1- cells, followed by Osterix+Prrx1-Sca1- osteoblast precursors, which eventually form mature α1(I)-collagen+ osteoblasts. This research will enable us to better understand the in vivo molecular biology features of MSCs, leading to their therapeutic applications for tissue repair and regeneration.


Assuntos
Linhagem da Célula , Descoberta de Drogas , Células-Tronco Mesenquimais , Camundongos/genética , Medicina Regenerativa , Animais , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Proteínas de Homeodomínio/fisiologia , Células-Tronco Mesenquimais/fisiologia , Biologia Molecular , Osteoblastos , Osteogênese/genética
14.
Nihon Yakurigaku Zasshi ; 153(2): 67-72, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-30745516

RESUMO

Mesenchymal stem cell (MSC) is a type of tissue stem cell. In clinical studies, cultured MSCs have shown important therapeutic effects on diseases via the reduction of neurological defects and regulation of immune responses. However, in vivo MSC localization, function, and properties are poorly understood; therefore, the molecular understanding of MSCs hierarchy is less advanced compared to hematopoietic stem cell hierarchy. To address these issues, we developed a method that enables us to visualize MSCs, manipulate their function, and analyze their molecular biology in vivo. Paired-related homeobox 1 (Prrx1)-positive cells are transiently observed during limb skeletal development in mice. Prrx1-positive cells form heterogeneous populations comprising multiple mesenchymal progenitors with different lineages that are developing into osteoblasts, chondrocytes, adipocytes, fibroblasts, and tendon and ligament cells. Our results suggest that osteoblast differentiation in the calvaria begins at the Prrx1+Sca1+ MSC stage with sequential progression to Prrx1+Sca1- cells, then Osterix+Prrx1-Sca1- osteoblast precursors, which eventually form mature α1(I)-collagen+ osteoblasts. Using Runt-related transcription factor 2 (Runx2) conditional knockout mice, furthermore, we found that the essential period of Runx2 function in intramembranous ossification likely begins at the Prrx1+Sca1+ MSC stage and ends at the Osterix+Prrx1-Sca1- osteoblast precursor stage (before mature the α1(I)-collagen+ osteoblasts appear). This approach will enable us to understand the in vivo molecular biology features of MSCs, leading to their therapeutic applications for tissue repair and regeneration. This development can also contribute to the field of pluripotent stem cell by enabling the transplantation of lineage-restricted mesenchymal progenitors.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteogênese , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Knockout
15.
Biochem Biophys Res Commun ; 509(4): 1028-1033, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30660360

RESUMO

Runt-related transcription factor 2 (Runx2), also known as core binding factor 1 (Cbfa1), is a multifunctional transcription factor and an essential master gene controlling osteoblast differentiation. We previously demonstrated the in vivo functions of Runx2 in mesoderm-derived cells. However, no studies have been conducted on Runx2 function during the differentiation of neural crest (NC)-derived cells in vivo. Wingless-type MMTV integration site family member 1 (Wnt1) is expressed in the NC, and Wnt1-Cre efficiently targets craniofacial NC-derived cells. Runx2 deficiency in cells of the Wnt1 lineage (referred henceforth as Runx2wnt1-/- within mice) resulted in defective ossification in certain regions, primarily in the anterior half of the craniofacial bones, including the frontal bone, jugal bone, squamous temporal bone, mandible, maxilla, and nasal bone. The skeletal analysis also revealed that heterozygous Runx2wnt1+/- embryos had an impaired closure of the frontal bone at the metopic suture and lacked the secondary palate in spite of otherwise normal ossification. This result suggests that ossification at the central part of the frontal bone is more dependent on Runx2 expression in comparison to other areas. These results indicate that Runx2 is indispensable not only for mesoderm-derived cells but also for NC-derived cells to differentiate during intramembranous ossification after migration to their destination from the neural plate border. Moreover, this implies that there are different levels of dependency on Runx2 expression for successful ossification between NC-derived cells that have migrated to different locations.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Crista Neural/citologia , Osteogênese , Animais , Diferenciação Celular , Movimento Celular , Anormalidades Craniofaciais/etiologia , Embrião de Mamíferos , Camundongos , Crista Neural/embriologia , Proteína Wnt1/metabolismo
16.
J Cell Physiol ; 234(5): 6679-6687, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30341902

RESUMO

Runx2 plays an essential role in embryonic disc tissue development in mice. However, the role of runt-related transcription factor 2 (Runx2) in postnatal disc tissue growth and development has not been defined. In the present studies, we generated Runx2 conditional knockout (KO) mice (Runx2Agc1ER ), in which Runx2 was deleted in Aggrecan-expressing cells in disc tissue at postnatal 2-weeks of age. We then analyzed changes in disc tissue growth and development using histology and immunohistochemical methods in 3-month-old mice. We found that large vacuolated notochordal cells were accumulated in the nucleus pulposus (NP) in Runx2 KO mice. The growth plate cartilage tissue in the disc was thicker in Runx2 KO mice. We also found a significant upregulation of Indian hedgehog (Ihh) expression in the cells in NP cells and in annulus fibrosus cells of Runx2 KO mice. These results demonstrated that Runx2 may play an important role in postnatal disc tissue development through interacting with Ihh signaling.


Assuntos
Anel Fibroso/crescimento & desenvolvimento , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Degeneração do Disco Intervertebral/patologia , Disco Intervertebral/crescimento & desenvolvimento , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Condrócitos/metabolismo , Lâmina de Crescimento/metabolismo , Proteínas Hedgehog/metabolismo , Disco Intervertebral/patologia , Camundongos Transgênicos , Núcleo Pulposo/patologia
17.
Bioorg Med Chem ; 27(2): 383-393, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30563725

RESUMO

Several malignant tumors and fibrotic diseases are associated with PDGFRß overexpression and excessive signaling, making this receptor attractive for molecular targeting and imaging approaches. A series of benzo[d]imidazole-quinoline derivatives were designed and synthesized to develop radioiodinated compounds as PDGFRß-specific imaging probes. The structure activity relationship (SAR) evaluation of the designed compounds was performed. Among them, 2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]-8-(piperazin-1-yl)quinoline (5a) and 4-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}morpholine (5d) exhibited a relatively high PDGFRß-TK inhibitory potency, whereas iodinated 5a derivative 5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]-8-(piperazin-1-yl)quinoline (8) exhibited a superior inhibitory potency as PDGFRß inhibitor than iodinated 5d derivative 4-{5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}morpholine (11). Furthermore, [125I]8 and [125I]11 were synthesized and evaluated for PDGFRß radioligand ability, both in vitro and in vivo. Cellular uptake experiments showed that [125I]8 had a higher uptake in BxPC3-luc cells as PDGFRß-positive cells than [125I]11. Incubation of [125I]8 after pretreatment of PDGFRß ligands significantly reduced the uptake of [125I]8. In biodistribution experiments using tumor-bearing mice, [125I]8 accumulation in the tumor 1 h postinjection was higher than that of the benzo[d]imidazol-quinoline derivative [125I]IIQP, used in our previous research. These results indicate that [125I]8 could be a promising PDGFRß imaging agent. Although its clinical application requires further structural modifications, the results obtained in this research may be useful for the development of PDGFRß-specific radioligands.


Assuntos
Benzimidazóis/farmacologia , Quinolinas/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Benzimidazóis/síntese química , Benzimidazóis/química , Benzimidazóis/farmacocinética , Linhagem Celular Tumoral , Desenho de Fármacos , Feminino , Humanos , Radioisótopos do Iodo/química , Ligantes , Masculino , Camundongos Endogâmicos BALB C , Estrutura Molecular , Neoplasias/diagnóstico por imagem , Quinolinas/síntese química , Quinolinas/química , Quinolinas/farmacocinética , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Relação Estrutura-Atividade , Distribuição Tecidual
18.
J Bone Miner Res ; 34(2): 327-332, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30352125

RESUMO

Bone morphogenetic protein 2 (BMP-2) is widely known as a potent growth factor that promotes bone formation. However, an increasing number of studies have demonstrated side effects of BMP-2 therapy. A deeper understanding of the effect of BMP-2 on cells other than those involved directly in bone remodeling is of fundamental importance to promote a more effective delivery of BMP-2 to patients. In this study, we aimed to investigate the effect of BMP-2 in the marrow environment. First, BMP-2 adsorbed onto titanium implants was delivered at the tooth extraction socket (marrow-absent site) or in the mandible marrow of beagle dogs. BMP-2 could induce marked bone formation around the implant at the tooth extraction socket. Surprisingly, however, no bone formation was observed in the BMP-2-coated titanium implants inserted in the mandible marrow. In C57BL/6 mice, BMP-2 adsorbed in freeze-dried collagen pellets could induce bone formation in marrow-absent calvarial bone. However, similar to the canine model, BMP-2 could not induce bone formation in the femur marrow. Analysis of osteoblast differentiation using Col1a1(2.3)-GFP transgenic mice revealed a scarce number of osteoblasts in BMP-2-treated femurs, whereas in the control group, osteoblasts were abundant. Ablation of femur marrow recovered the BMP-2 ability to induce bone formation. In vitro experiments analyzing luciferase activity of C2C12 cells with the BMP-responsive element and alkaline phosphatase activity of MC3T3-E1 osteoblasts further revealed that bone marrow cells inhibit the BMP-2 effect on osteoblasts by direct cell-cell contact. Collectively, these results showed that the effect of BMP-2 in inducing bone formation is remarkably repressed by marrow cells via direct cell-cell contact with osteoblasts; this opens new perspectives on the clarification of the side-effects associated with BMP-2 application. © 2018 American Society for Bone and Mineral Research.


Assuntos
Células da Medula Óssea/metabolismo , Proteína Morfogenética Óssea 2 , Microambiente Celular/efeitos dos fármacos , Materiais Revestidos Biocompatíveis , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Titânio , Animais , Células da Medula Óssea/patologia , Proteína Morfogenética Óssea 2/química , Proteína Morfogenética Óssea 2/farmacologia , Microambiente Celular/genética , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Cães , Feminino , Fêmur/metabolismo , Fêmur/patologia , Humanos , Camundongos , Camundongos Transgênicos , Osteoblastos/patologia , Osteogênese/genética , Titânio/química , Titânio/farmacologia
19.
Neurochem Res ; 43(11): 2047-2054, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30203400

RESUMO

Glioblastoma (GBM) is the most aggressive and lethal form of brain tumor. However, therapeutic strategies against malignant gliomas have not been completely established. Runt-related transcription factor 2 (Runx2) is an essential gene for skeletal development but its regulatory role in the malignant progression of glioma remains unclear. Here we investigated expression levels of RUNX2 in glioma tissues and its regulatory effects on aberrant growth of glioma cells. RUNX2 mRNA levels were higher in GBM tissues than that of normal brains or low-grade gliomas. RUNX2 protein was detected in five out of seven human GBM cell lines and its level was positively correlated with proliferative capacity. Stable transduction of dominant-negative Runx2 in rat-derived C6 glioma cells not only inhibited the promoter activity containing Runx2 response element, but also decreased mRNA expression levels of Runx2 target genes, such as Mmp13 and Spp1, as well as the proliferative capacity. Furthermore, transient introduction of Runx2-targeted siRNAs into C6 glioma cells significantly decreased mRNA expression levels of Mmp13 and Spp1 and the proliferative capacity. Furthermore, Runx2 knockdown suppressed both Ccnd1 mRNA expression and activation of the Ccnd1 promoter by forskolin, a PKA-activating reagent, in C6 glioma cells. Our results demonstrate that cross-talk between cAMP/PKA signaling and RUNX2 promotes a malignant phenotype of glioma cells.


Assuntos
Neoplasias Encefálicas/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Glioma/metabolismo , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Glioma/genética , Humanos , Ratos
20.
Development ; 145(14)2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-29986870

RESUMO

Erk5 belongs to the mitogen-activated protein kinase (MAPK) family. Following its phosphorylation by Mek5, Erk5 modulates several signaling pathways in a number of cell types. In this study, we demonstrated that Erk5 inactivation in mesenchymal cells causes abnormalities in skeletal development by inducing Sox9, an important transcription factor of skeletogenesis. We further demonstrate that Erk5 directly phosphorylates and activates Smurf2 (a ubiquitin E3 ligase) at Thr249, which promotes the proteasomal degradation of Smad proteins and phosphorylates Smad1 at Ser206 in the linker region known to trigger its proteasomal degradation by Smurf1. Smads transcriptionally activated the expression of Sox9 in mesenchymal cells. Accordingly, removal of one Sox9 allele in mesenchymal cells from Erk5-deficient mice rescued some abnormalities of skeletogenesis. These findings highlight the importance of the Mek5-Erk5-Smurf-Smad-Sox9 axis in mammalian skeletogenesis.


Assuntos
Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Osteogênese , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Diferenciação Celular , Condrogênese , Humanos , Mesoderma/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteólise , Crânio/anormalidades , Ubiquitina/metabolismo , Ubiquitinação
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