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1.
J Craniofac Surg ; 33(4): 1250-1254, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-36041089

RESUMO

BACKGROUND: C342Y (Cys342Tyr) point mutation of FGFR2 (fibroblast growth factor receptor 2) is closely associated with the pathogenesis of Crouzon syndrome. The dura mater plays an important role in mediating the closure of cranial sutures. However, the underlying mechanisms of these pathological processes have been rarely investigated. in this study, the authors analyzed the effects of dura cells with FGFR2 mutations on the biological function of osteoblasts. METHODS: Dura cells and cranial osteoblasts from C57BL/6 mice were extracted and cultured. C342Y-FGFR2 mutant constructs were established via lentivirus and applied to infect dura cells. A co-cultured trans-well system with dura cells and osteoblasts was established. Three experimental groups were set up: oste group, Oste + Dura-vector group, and Oste + Dura-C342Y group. The expression levels of key factors in MEK (Mitogen-activated protein kinase kinase, MAPKK)/extracellular signal-regulated kinase (ERK) and Hippo pathway were detected by western blot and RT-qPCR (Real Time Quantitative PCR). Finally, a rescue experiment was carried out with small interference RUA. RESULTS: The proliferation level of osteoblasts in Oste + Dura- C342Y group was significantly up-regulated. Our studies indicated that the activation of MEK/ERK pathway in Oste + Dura-C342Y group could inhibit the Hippo pathway, lead to down-regulation of large tumor suppressor 1 and promote the activation and nuclear localization of yes-associated protein, and the results of rescue experiments showed a reverse expression trend, further confirming the effects of C342Y-FGFR2 mutation in dura cells on osteoblasts and its potential mechanism. CONCLUSIONS: This study suggested that the C342Y-FGFR2 mutation in dura cells could promote osteoblastic proliferation, and shown the crosstalk between MEK/ERK and Hippo pathways. As the regulatory machinery center, yes-associated protein might play a bridging role in these pathways, and might influence the pathogenesis of craniosynostosis by activating downstream transcriptional factors.


Assuntos
Osteoblastos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Proteínas de Sinalização YAP , Animais , Diferenciação Celular , Técnicas de Cocultura , Dura-Máter/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Via de Sinalização Hippo , Camundongos , Camundongos Endogâmicos C57BL , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Osteoblastos/citologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Proteínas de Sinalização YAP/genética
2.
Stem Cell Res Ther ; 13(1): 278, 2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35765036

RESUMO

BACKGROUND: Oncostatin M receptor (OSMR), as one of the receptors for oncostatin M (OSM), has previously been shown to mediate the stimulatory role of OSM in osteoclastogenesis and bone resorption. However, it remains to be clarified whether and how OSMR affects the differentiation of osteoblasts. METHODS: The expression level of OSMR during osteoblast and adipocyte differentiation was examined. The role of OSMR in the differentiation was investigated using in vitro gain-of-function and loss-of-function experiments. The mechanisms by which OSMR regulates bone cell differentiation were explored. Finally, in vivo function of OSMR in cell fate determination and bone homeostasis was studied after transplantation of OSMR-silenced bone marrow stromal cells (BMSCs) to the marrow of ovariectomized mice. RESULTS: OSMR was regulated during osteogenic and adipogenic differentiation of marrow stromal progenitor cells and increased in the metaphysis of ovariectomized mice. OSMR suppressed osteogenic differentiation and stimulated adipogenic differentiation of progenitor cells. Mechanistic investigations showed that OSMR inhibited extracellular signal-regulated kinase (ERK) and autophagy signaling. The downregulation of autophagy, which was mediated by ERK inhibition, suppressed osteogenic differentiation of progenitor cells. Additionally, inactivation of ERK/autophagy signaling attenuated the stimulation of osteogenic differentiation induced by Osmr siRNA. Furthermore, transplantation of BMSCs in which OSMR was silenced to the marrow of mice promoted osteoblast differentiation, attenuated fat accumulation and osteoclast differentiation, and thereby relieved the osteopenic phenotype in the ovariectomized mice. CONCLUSIONS: Our study has for the first time established the direct role of OSMR in regulating osteogenic differentiation of marrow stromal progenitor cells through ERK-mediated autophagy signaling. OSMR thus contributes to bone homeostasis through dual regulation of osteoblasts and osteoclasts. It also suggests that OSMR may be a potential target for the treatment of metabolic disorders such as osteoporosis.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular , Sistema de Sinalização das MAP Quinases , Subunidade beta de Receptor de Oncostatina M , Osteoblastos , Osteogênese , Animais , Autofagia/fisiologia , Diferenciação Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Subunidade beta de Receptor de Oncostatina M/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo
3.
J Musculoskelet Neuronal Interact ; 22(2): 242-250, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35642703

RESUMO

OBJECTIVES: a) To explore the expression of Foxf1 and NF-κB in bone tissue of ovariectomized rats with osteoporosis and b) to investigate the role and mechanism of NF-κB pathway regulated by Foxf1 gene in the differentiation and formation of rat osteoclasts and osteoblasts with cell experiments. METHODS: Ovariectomized rat model of osteoporosis was established with 3-month-old female SD rats. The rats were divided into sham group (n=10) and osteoporosis group (n=10). Real time fluorescent quantitative PCR and Western blot were used to detect the expression levels of Foxf1 and NF-κB genes and proteins in the femur tissues of rats and analyze their correlation. RESULTS: Both Foxf1 and NF- κB were highly expressed in the femur tissues. Upon the overexpression of Foxf1 gene in osteoblasts and osteoclasts in vitro, the gene and protein expression of NF-κB were also upregulated, significantly reducing the gene and protein expression levels of osteogenic factors, including ATF4, OCN, ALP and Runx2. CONCLUSIONS: Foxf1 gene could inhibit osteoblast formation and promote osteoclast differentiation by NF-κB pathway, which may increase the risk of osteoporosis in rats.


Assuntos
Fatores de Transcrição Forkhead , NF-kappa B , Osteoporose , Animais , Feminino , Fatores de Transcrição Forkhead/genética , NF-kappa B/genética , Osteoblastos/citologia , Osteoclastos/citologia , Osteoporose/genética , Osteoporose/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Regulação para Cima
4.
Int J Mol Sci ; 23(10)2022 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-35628403

RESUMO

Simulated microgravity (SMG) inhibits osteoblast differentiation (OBD) and induces bone loss via the inhibition of the Wnt/ß-catenin pathway. However, the mechanism by which SMG alters the Wnt/ß-catenin pathway is unknown. We previously demonstrated that SMG altered the focal adhesion kinase (FAK)-regulated mTORC1, AMPK and ERK1/2 pathways, leading to the inhibition of tumor cell proliferation/metastasis and promoting cell apoptosis. To examine whether FAK similarly mediates SMG-dependent changes to Wnt/ß-catenin in osteoblasts, we characterized mouse MC3T3-E1 cells cultured under clinostat-modeled SMG (µg) conditions. Compared to cells cultured under ground (1 g) conditions, SMG reduces focal adhesions, alters cytoskeleton structures, and down-regulates FAK, Wnt/ß-catenin and Wnt/ß-catenin-regulated molecules. Consequently, protein-2 (BMP2), type-1 collagen (COL1), alkaline-phosphatase activity and matrix mineralization are all inhibited. In the mouse hindlimb unloading (HU) model, SMG-affected tibial trabecular bone loss is significantly reduced, according to histological and micro-computed tomography analyses. Interestingly, the FAK activator, cytotoxic necrotizing factor-1 (CNF1), significantly suppresses all of the SMG-induced alterations in MC3T3-E1 cells and the HU model. Therefore, our data demonstrate the critical role of FAK in the SMG-induced inhibition of OBD and bone loss via the Wnt/ß-catenin pathway, offering FAK signaling as a new therapeutic target not only for astronauts at risk of OBD inhibition and bone loss, but also osteoporotic patients.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal , Osteoblastos , Ausência de Peso , Via de Sinalização Wnt , beta Catenina , Células 3T3 , Animais , Ativação Enzimática , Quinase 1 de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Microtomografia por Raio-X , beta Catenina/metabolismo
5.
Genes Immun ; 23(3-4): 141-148, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35581496

RESUMO

Enhancer of zeste homolog 2 (EZH2) has been noted to contribute to the pathogenesis of autoimmune diseases. This study sought to investigate the mechanism of EZH2 in osteoclast (OCL) and osteoblast (OBL) differentiation (OCLD/OBLD) and bone destruction in RA. The animal model of collagen-induced arthritis (CIA) was established, followed by arthritis index (AI) scoring and histological staining, and measurements of inflammatory cytokines levels. The number of OCLs was detected via Tartrate-resistant acid phosphatase (TRAP) staining, and levels of OBL markers were determined by Western blot analysis. Trimethylated histone H3 at lysine 27 (H3K27me3) expression and its enrichment in the Ndrg2 promoter were detected. Collaborative experiments were performed with GSK-J1 or sh-Ndrg2 in CIA mice with EZH2 knockdown. EZH2 was upregulated while Ndrg2 was downregulated in knee joint tissues of CIA mice. Silencing EZH2 reduced AI scores, pathological injury of the knee joint, levels of inflammatory cytokines, and TRAP-positive cells, and increased protein levels of RUNX2 and BMP2. EZH2 promoted H3K27me3 level in the Ndrg2 promoter to inhibit Ndrg2 transcription. H3K27me3 upregulation or Ndrg2 downregulation reversed the role of silencing EZH2 in bone destruction. Overall, EZH2 repressed OBLD and promoted OCLD to aggravate bone destruction in CIA mice through H3K27me3/Ndrg2.


Assuntos
Artrite Experimental , Proteína Potenciadora do Homólogo 2 de Zeste , Osteoblastos , Osteoclastos , Animais , Artrite Experimental/complicações , Artrite Experimental/genética , Osso e Ossos/patologia , Diferenciação Celular , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Histonas/metabolismo , Camundongos , Osteoblastos/citologia , Osteoclastos/citologia
6.
Am J Physiol Endocrinol Metab ; 322(6): E528-E539, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35466691

RESUMO

T-cell-like factor (TCF)7l2, a key effector of canonical Wnt signaling, is highly expressed in bone but nothing is known about its role in regulating osteoblast function. To test this, we generated mice with conditional disruption of Tcf7l2 gene in osteoblast lineages using Tcf7l2 floxed and Col1α2-Cre mice. Skeletal parameters were evaluated using heterozygous conditional knockdown (HCKD) mice since homozygous conditional knockout died during pregnancy or immediately after birth. At 5 wk of age, trabecular bone mass of long bones was reduced by 35% as measured by microcomputed tomography (µCT). Histology data showed a 42% reduction in femur trabecular bone mass caused by reduced bone formation. Knockdown of Tcf7l2 expression in osteoblasts decreased proliferation and differentiation by 20%-40%. Expression levels of genes (Hif1α, Vegf, and ß-catenin) targeted by TCF7L2 were decreased by 50% in Tcf7l2-deficient osteoblasts and bones of HCKD mice. We found that the Hif1α gene promoter contained multiple putative TCF7L2 motifs and stabilization of HIF1α protein levels rescued expression of TCF7L2 target genes and alkaline phosphatase (ALP) activity in Tcf7l2-deficient osteoblasts. Furthermore, Tcf7l2 overexpression increased proliferation in the presence of canonical Wnt3a that was not affected by ß-catenin inhibitor providing evidence for a noncanonical signaling in mediating TCF7L2 effects. Tcf7l2 expression was increased in response to mechanical strain (MS) in vitro and in vivo, and disruption of Tcf7l2 expression in osteoblasts reduced MS-induced ALP activity by 35%. We conclude that Tcf7l2, a mechanoresponsive gene, is an important regulator of osteoblast function acting, in part, via hypoxia signaling.NEW & NOTEWORTHY TCF7L2 is expressed by bone but it was not known whether TCF7L2 expression influenced bone development. By using a mouse model with conditional disruption of Tcf7l2 in osteoblast lineage cells, we have demonstrated for the first time, that TCF7L2 plays an important role in regulating osteoblasts via a noncanonical pathway.


Assuntos
Osteoblastos , Proteína 2 Semelhante ao Fator 7 de Transcrição , beta Catenina , Animais , Diferenciação Celular/fisiologia , Hipóxia Celular , Linhagem Celular , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/biossíntese , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Via de Sinalização Wnt , Microtomografia por Raio-X , beta Catenina/metabolismo
7.
Med Mol Morphol ; 55(3): 174-186, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35461467

RESUMO

Cleidocranial dysplasia (CCD) is a hereditary disorder associated with skeletal dysplasia and dental abnormalities. CCD arises from heterozygous loss of function mutations in the Runt-related transcription factor 2 (RUNX2) gene. Osteoporosis is often observed in CCD patients and conventional vitamin D supplementation is recommended. However, sufficient evidences have not been presented yet. This study investigated the role of RUNX2 in osteoblastic differentiation and sought to identify potential target genes for the treatment of osteoporosis associated with CCD, using induced pluripotent stem cell (iPSC) technology. We successfully established Runx2-/-, Runx2+/- and wild-type miPSCs from litter-matched mice and found poor Vdr expression in Runx2-/-cells. Significant down-regulation of osteoblastic differentiation in Runx2-/- miPSCs was observed. Gene expression array revealed unexpected results such as remarkable increase of Rankl expression and decrease of Vdr in Runx2-/- cells. Insufficient response to vitamin D in Runx2-/- cells was also observed. Our results suggest that RUNX2 functions as a regulator of Rankl and Vdr and thereby controls bone density. These findings also suggest that conventional vitamin D supplementation may not be as effective as previously expected, in the treatment of osteoporosis associated with CCD, and that inhibiting RANKL function might be worth considering as an alternative treatment strategy.


Assuntos
Displasia Cleidocraniana , Subunidade alfa 1 de Fator de Ligação ao Core , Células-Tronco Pluripotentes Induzidas , Osteoporose , Vitamina D , Animais , Diferenciação Celular , Displasia Cleidocraniana/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Osteoporose/genética , Vitamina D/farmacologia
8.
Cell Signal ; 95: 110335, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35461899

RESUMO

Osteoblast apoptosis is a prominent factor for disrupting skeletal homeostasis in multiple inflammatory bone diseases. METTL3, a key methyltransferase that catalyzes the N6-methyladenosine (m6A) modification of mRNA, has recently been shown to exert a critical role in osteogenic differentiation. However, the function of METTL3 in osteoblast apoptosis under inflammatory conditions remains elusive. In the present study, we observed that the total m6A level and METTL3 expression were upregulated in differentiated osteoblasts and downregulated after LPS stimulation. METTL3 knockdown induced a higher apoptotic rate in LPS-treated osteoblasts. The expression of the antiapoptotic protein BCL-2 decreased, and the apoptotic proteins cleaved Caspase-3, cleaved PARP-1 and cleaved Caspase-12 increased following METTL3 knockdown. Meanwhile, METTL3 silencing inhibited osteoblast proliferation and decreased osteogenic marker expression, ALP activity and mineralized nodules. RNA-seq analysis revealed that differentially expressed genes were significantly enriched in unfolded protein response pathways in METTL3-deficient cells. METTL3 depletion upregulated the expression of the ER stress-related markers, including p-PERK, p-eIF2α, p-IRE1α, GRP78, ATF4, CHOP and ATF6. Inhibition of ER stress by 4-PBA remarkably rescued METTL3 knockdown-induced apoptosis and promoted osteoblast proliferation and differentiation. Mechanistically, METTL3 depletion enhanced the expression and mRNA stability of Grp78, and similar results were observed after YTHDF2 knockdown. RIP-qPCR revealed that YTHDF2 directly interacted with Grp78 mRNA and that the interaction relied on METTL3. Taken together, our study demonstrated that METTL3 knockdown enhanced Grp78 expression through YTHDF2-mediated RNA degradation, which elicited ER stress, thereby promoting osteoblast apoptosis and inhibiting cell proliferation and differentiation under LPS-induced inflammatory condition.


Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Metiltransferases , Osteoblastos , Osteogênese , /genética , Humanos , Inflamação/metabolismo , Lipopolissacarídeos , Metiltransferases/genética , Metiltransferases/metabolismo , Osteoblastos/citologia , Osteogênese/fisiologia , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética
9.
J Mol Endocrinol ; 68(4): 195-207, 2022 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-35255002

RESUMO

During bone formation, mesenchymal progenitor cells mature into bone-forming osteoblasts after undergoing several stages of differentiation. Impaired bone formation is a predominant finding in glucocorticoid (GC)-induced osteoporosis (GIO). Osteoblasts at different stages of maturation can be affected by excessive endogenous or therapeutic GCs. Sex-determining region Y-box 2 (SOX2) is normally expressed in immature osteoblasts, but its overexpression can suppress osteoblast differentiation. This study aimed to evaluate whether GC affects SOX2 expression in osteoblasts, and whether SOX2 contributes to GC-induced inhibition of osteoblast differentiation. Treatment with GCs such as dexamethasone (Dex) or hydrocortisone enhanced SOX2 expression. Silencing SOX2 improved inhibition of GC-induced osteoblast differentiation, whereas SOX2 overexpression decreased mineralized nodule formation and RUNX2 and Osterix expression in MC3T3-E1 cells. On the contrary, when C3H10T1/2 uncommitted mesenchymal stem cells were subjected to SOX2 overexpression, RUNX2 expression increased. As a mechanism of Dex-induced SOX2 upregulation in preosteoblasts, we found that the STAT3 pathway or GC receptor (GR) is involved, using a GR antagonist, STAT3 regulators, and chromatin immunoprecipitation assays. Moreover, mice treated with Dex for 4 weeks showed a notable increase in SOX2 expression in the bones and an increased ratio of procollagen type 1 N-terminal propeptide to osteocalcin in the plasma than in control mice. This study demonstrated that GC enhances SOX2 expression in vitro in osteoblast and in vivo in the mice bone, which affects bone-forming activity differently depending on the differentiation stage of osteoblast-lineage cells. Our results provide new insights into prevention and treatment against impaired bone formation in GIO.


Assuntos
Glucocorticoides , Células-Tronco Mesenquimais , Osteoblastos , Fatores de Transcrição SOXB1 , Animais , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Células-Tronco Mesenquimais/citologia , Camundongos , Osteoblastos/citologia , Osteogênese/genética , Fatores de Transcrição SOXB1/genética
10.
FASEB J ; 36(4): e22231, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35230719

RESUMO

The dysfunction of osteogenesis is a key character in the pathogenesis of osteoporosis, but the network of signaling mechanisms in controlling the differentiation of osteoblast remain unclear. Thrap3 has been proved participating in various biological process, especially in the differentiation of stem cells. Here, we demonstrate that Thrap3 could promote osteogenesis through the inhibition of the degradation of Runx2, which is a key molecular structure in early osteoblast differentiation. Furthermore, we found that the osteogenesis enhancing capacity of Thrap3 was caused by physically binding with Sox9, inhibiting the transcriptional activity of Sox9, and then decreasing the decomposition-promoted effect of Sox9 on Runx2. Our data shows that Thrap3 promotes osteoblast differentiation through the Thrap3-Sox9-Runx2 axis. What we found may help for further clarifying the molecular mechanism of osteogenic differentiation and give a new potential therapeutic target for osteoporosis.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Proteínas de Ligação a DNA/fisiologia , Osteogênese/fisiologia , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/antagonistas & inibidores , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Osteoblastos/citologia , Fatores de Transcrição SOX9/fisiologia
11.
Bioengineered ; 13(2): 4201-4211, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35176943

RESUMO

Osteoporosis is a metabolic bone disease that significantly affects the quality of life and can even lead to death. In this study, we aimed to investigate the role of RAD51 recombinase (RAD51) in osteoblast and osteoclast differentiation. We analyzed differentially expressed genes using microarray analysis. The osteogenic differentiation capability was analyzed by alkaline phosphatase (ALP) staining and alizarin red staining assays. Osteogenesis and osteoclast related genes expression was detected using quantitative real-time PCR (qPCR) and Western blotting. The phosphorylation of Ataxia-telangiectasia mutated (ATM) and ATR serine/threonine kinase (ATR) was tested using Western blotting. The effect of RAD51 on osteoporosis was also explored in vivo. The results showed that RAD51 was downregulated in osteoporosis, but upregulated in differentiated osteoblasts. Overexpression of RAD51 enhanced the differentiation of osteoblasts and suppressed the formation of osteoclasts. Furthermore, p-ATM and p-ATR levels were upregulated in osteoblasts and downregulated in osteoclasts. RAD51 expression was reduced by the ATM/ATR pathway inhibitor AZ20. AZ20 treatment inhibited osteoblastogenesis and promoted osteoclastogenesis, whereas RAD51 reversed the effects induced by AZ20. Moreover, RAD51 improved bone microarchitecture in vivo. Taken together, ATM/ATR signaling-mediated RAD51 promoted osteogenic differentiation and suppressed osteoclastogenesis. These findings reveal a critical role for RAD51 in osteoporosis.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Osteoclastos/citologia , Osteogênese , Osteoporose/metabolismo , Rad51 Recombinase/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Humanos , Masculino , Camundongos , Células NIH 3T3 , Osteoblastos/citologia , Osteoblastos/enzimologia , Osteoclastos/enzimologia , Osteoporose/genética , Osteoporose/fisiopatologia , Rad51 Recombinase/genética , Ratos , Ratos Sprague-Dawley
12.
Development ; 149(3)2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35129199

RESUMO

Skeletal elements frequently associate with vasculature and somatosensory nerves, which regulate bone development and homeostasis. However, the deep, internal location of bones in many vertebrates has limited in vivo exploration of the neurovascular-bone relationship. Here, we use the zebrafish caudal fin, an optically accessible organ formed of repeating bony ray skeletal units, to determine the cellular relationship between nerves, bones and endothelium. In adult zebrafish, we establish the presence of somatosensory axons running through the inside of the bony fin rays, juxtaposed with osteoblasts on the inner hemiray surface. During development we show that the caudal fin progresses through sequential stages of endothelial plexus formation, bony ray addition, ray innervation and endothelial remodeling. Surprisingly, the initial stages of fin morphogenesis proceed normally in animals lacking either fin endothelium or somatosensory nerves. Instead, we find that sp7+ osteoblasts are required for endothelial remodeling and somatosensory axon innervation in the developing fin. Overall, this study demonstrates that the proximal neurovascular-bone relationship in the adult caudal fin is established during fin organogenesis and suggests that ray-associated osteoblasts pattern axons and endothelium.


Assuntos
Nadadeiras de Animais/fisiologia , Axônios/metabolismo , Endotélio/metabolismo , Organogênese/fisiologia , Peixe-Zebra/crescimento & desenvolvimento , Nadadeiras de Animais/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Endotélio/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator de Transcrição Sp7/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
13.
Cell Mol Life Sci ; 79(3): 158, 2022 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-35220463

RESUMO

Calvarial bone is one of the most complex sequences of developmental events in embryology, featuring a uniquely transient, pluripotent stem cell-like population known as the cranial neural crest (CNC). The skull is formed through intramembranous ossification with distinct tissue lineages (e.g. neural crest derived frontal bone and mesoderm derived parietal bone). Due to CNC's vast cell fate potential, in response to a series of inductive secreted cues including BMP/TGF-ß, Wnt, FGF, Notch, Hedgehog, Hippo and PDGF signaling, CNC enables generations of a diverse spectrum of differentiated cell types in vivo such as osteoblasts and chondrocytes at the craniofacial level. In recent years, since the studies from a genetic mouse model and single-cell sequencing, new discoveries are uncovered upon CNC patterning, differentiation, and the contribution to the development of cranial bones. In this review, we summarized the differences upon the potential gene regulatory network to regulate CNC derived osteogenic potential in mouse and human, and highlighted specific functions of genetic molecules from multiple signaling pathways and the crosstalk, transcription factors and epigenetic factors in orchestrating CNC commitment and differentiation into osteogenic mesenchyme and bone formation. Disorders in gene regulatory network in CNC patterning indicate highly close relevance to clinical birth defects and diseases, providing valuable transgenic mouse models for subsequent discoveries in delineating the underlying molecular mechanisms. We also emphasized the potential regenerative alternative through scientific discoveries from CNC patterning and genetic molecules in interfering with or alleviating clinical disorders or diseases, which will be beneficial for the molecular targets to be integrated for novel therapeutic strategies in the clinic.


Assuntos
Diferenciação Celular , Redes Reguladoras de Genes/genética , Osteogênese , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Crista Neural/citologia , Crista Neural/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
14.
J Bone Miner Metab ; 40(3): 434-447, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35195777

RESUMO

INTRODUCTION: The detailed mechanism of the process during bone healing of drill-hole injury has been elucidated, but a crucial factor in regulating drill-hole healing has not been identified. The transcription factor p53 suppresses osteoblast differentiation through inhibition of osterix expression. In present study, we demonstrate the effects of p53 deficiency on the capacity of MSCs and osteoblasts during drill-hole healing. MATERIALS AND METHODS: Mesenchymal stromal cells (MSCs) and osteoblasts were collected from bone marrow and calvaria of p53 knockout (KO) mice, respectively. The activities of cell mobility, cell proliferation, osteoblast differentiation, and wound healing of MSCs and/or osteoblasts were determined by in vitro experiments. In addition, bone healing of drill-hole injury in KO mice was examined by micro-CT and immunohistological analysis using anti-osterix, Runx2, and sclerostin antibodies. RESULTS: KO MSCs stimulated cell mobility, cell proliferation, and osteoblast differentiation. Likewise, KO osteoblasts enhanced cell proliferation and wound healing. KO MSCs and osteoblasts showed high potency in the inflammation and callus formation phases compared to those from wild-type (WT) mice. In addition, increased expression of osterix and Runx2 was observed in KO MSCs and osteoblasts that migrated in the drill-hole. Conversely, sclerostin expression was inhibited in KO mice. Eventually, KO mice exhibited high repairability of drill-hole injury, suggesting a novel role of p53 in MSCs and osteoblasts in improving bone healing. CONCLUSION: p53 Deficiency promotes bone healing of drill-hole injury by enhancing the bone-regenerative ability of MSCs and osteoblasts.


Assuntos
Regeneração Óssea , Subunidade alfa 1 de Fator de Ligação ao Core , Células-Tronco Mesenquimais , Osteoblastos , Proteína Supressora de Tumor p53 , Animais , Regeneração Óssea/fisiologia , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
15.
Calcif Tissue Int ; 110(6): 746-758, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35137272

RESUMO

Ebfs are a family of transcription factors regulating the differentiation of multiple cell types of mesenchymal origin, including osteoblasts. Global deletion of Ebf1 results in increased bone formation and bone mass, while global loss of Ebf2 leads to enhanced bone resorption and decreased bone mass. Targeted deletion of Ebf1 in early committed osteoblasts leads to increased bone formation, whereas deletion in mature osteoblasts has no effect. To study the effects of Ebf2 specifically on long bone development, we created a limb bud mesenchyme targeted Ebf2 knockout mouse model by using paired related homeobox gene 1 (Prrx1) Cre. To investigate the possible interplay between Ebf1 and Ebf2, we deleted both Ebf1 and Ebf2 in the cells expressing Prrx1. Mice with Prrx1-targeted deletion of Ebf2 had a very mild bone phenotype. However, deletion of both Ebf1 and Ebf2 in mesenchymal lineage cells lead to significant, age progressive increase in bone volume. The phenotype was to some extent gender dependent, leading to an increase in both trabecular and cortical bone in females, while in males a mild cortical bone phenotype and a growth plate defect was observed. The phenotype was observed at both 6 and 12 weeks of age, but it was more pronounced in older female mice. Our data suggest that Ebfs modulate bone homeostasis and they are likely able to compensate for the lack of each other. The roles of Ebfs in bone formation appear to be complex and affected by multiple factors, such as age and gender.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Osso e Ossos , Proteínas de Homeodomínio , Células-Tronco Mesenquimais , Transativadores , Fatores Etários , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Fenótipo , Fatores Sexuais , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
16.
Int J Mol Sci ; 23(3)2022 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-35163761

RESUMO

The main objective was to produce 3D printable hydrogels based on GelMA and hydroxyapatite doped with cerium ions with potential application in bone regeneration. The first part of the study regards the substitution of Ca2+ ions from hydroxyapatite structure with cerium ions (Ca10-xCex(PO4)6(OH)2, xCe = 0.1, 0.3, 0.5). The second part followed the selection of the optimal concentration of HAp doped, which will ensure GelMA-based scaffolds with good biocompatibility, viability and cell proliferation. The third part aimed to select the optimal concentrations of GelMA for the 3D printing process (20%, 30% and 35%). In vitro biological assessment presented the highest level of cell viability and proliferation potency of GelMA-HC5 composites, along with a low cytotoxic potential, highlighting the beneficial effects of cerium on cell growth, also supported by Live/Dead results. According to the 3D printing experiments, the 30% GelMA enriched with HC5 was able to generate 3D scaffolds with high structural integrity and homogeneity, showing the highest suitability for the 3D printing process. The osteogenic differentiation experiments confirmed the ability of 30% GelMA-3% HC5 scaffold to support and efficiently maintain the osteogenesis process. Based on the results, 30% GelMA-3% HC5 3D printed scaffolds could be considered as biomaterials with suitable characteristics for application in bone tissue engineering.


Assuntos
Materiais Biocompatíveis/farmacologia , Cério/química , Gelatina/química , Hidrogéis/farmacologia , Hidroxiapatitas/química , Metacrilatos/química , Osteoblastos/citologia , Animais , Materiais Biocompatíveis/química , Regeneração Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Hidrogéis/química , Camundongos , Osteogênese , Pós , Impressão Tridimensional , Engenharia Tecidual , Tecidos Suporte/química
17.
Chem Biol Interact ; 355: 109831, 2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-35120918

RESUMO

Bone disorders have become a global concern illustrated with decreased bone mineral density and disruption in microarchitecture of natural bone tissue organization. Natural compounds that promote bone health by augmenting osteoblast functions and suppressing osteoclast functions has gained much attention and offer greater therapeutic value compared to conventional therapies. Amongst several plant-based molecules, flavonoids act as a major combatant in promoting bone health through their multi-faceted biological activities such as antioxidant, anti-inflammatory, and osteogenic properties. They protect bone loss by regulating the signalling cascades involved in osteoblast and osteoclast functions. Flavonoids augment osteoblastogenesis and inhibits osteoclastogenesis through their modulation of various signalling pathways. This review discusses the role of various flavonoids and their molecular mechanisms involved in maintaining bone health by regulating osteoblast and osteoclast functions.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Flavonas/química , Flavonoides/farmacologia , Osteogênese/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Flavonas/farmacologia , Flavonoides/química , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo
18.
Int J Mol Sci ; 23(3)2022 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-35163828

RESUMO

Hyperactivation of the KEAP1-NRF2 axis is a common molecular trait in carcinomas from different origin. The transcriptional program induced by NRF2 involves antioxidant and metabolic genes that render cancer cells more capable of dealing with oxidative stress. The TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) is an important regulator of glycolysis and the pentose phosphate pathway that was described as a p53 response gene, yet TIGAR expression is detected in p53-null tumors. In this study we investigated the role of NRF2 in the regulation of TIGAR in human carcinoma cell lines. Exposure of carcinoma cells to electrophilic molecules or overexpression of NRF2 significantly increased expression of TIGAR, in parallel to the known NRF2 target genes NQO1 and G6PD. The same was observed in TP53KO cells, indicating that NRF2-mediated regulation of TIGAR is p53-independent. Accordingly, downregulation of NRF2 decreased the expression of TIGAR in carcinoma cell lines from different origin. As NRF2 is essential in the bone, we used mouse primary osteoblasts to corroborate our findings. The antioxidant response elements for NRF2 binding to the promoter of human and mouse TIGAR were described. This study provides the first evidence that NRF2 controls the expression of TIGAR at the transcriptional level.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias/genética , Osteoblastos/citologia , Monoéster Fosfórico Hidrolases/genética , Proteína Supressora de Tumor p53/genética , Células A549 , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glucosefosfato Desidrogenase/genética , Células HCT116 , Células HeLa , Humanos , Camundongos , NAD(P)H Desidrogenase (Quinona)/genética , Neoplasias/metabolismo , Osteoblastos/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas
19.
Nat Commun ; 13(1): 700, 2022 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-35121733

RESUMO

SP7/Osterix is a transcription factor critical for osteoblast maturation and bone formation. Homozygous loss-of-function mutations in SP7 cause osteogenesis imperfecta type XII, but neomorphic (gain-of-new-function) mutations of SP7 have not been reported in humans. Here we describe a de novo dominant neomorphic missense variant (c.926 C > G:p.S309W) in SP7 in a patient with craniosynostosis, cranial hyperostosis, and long bone fragility. Histomorphometry shows increased osteoblasts but decreased bone mineralization. Mice with the corresponding variant also show a complex skeletal phenotype distinct from that of Sp7-null mice. The mutation alters the binding specificity of SP7 from AT-rich motifs to a GC-consensus sequence (typical of other SP family members) and produces an aberrant gene expression profile, including increased expression of Col1a1 and endogenous Sp7, but decreased expression of genes involved in matrix mineralization. Our study identifies a pathogenic mechanism in which a mutation in a transcription factor shifts DNA binding specificity and provides important in vivo evidence that the affinity of SP7 for AT-rich motifs, unique among SP proteins, is critical for normal osteoblast differentiation.


Assuntos
Doenças Ósseas/genética , Osso e Ossos/metabolismo , Regulação da Expressão Gênica , Mutação , Fator de Transcrição Sp7/genética , Animais , Doenças Ósseas/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Criança , Células HEK293 , Humanos , Hibridização In Situ , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/metabolismo , Fator de Transcrição Sp7/metabolismo , Microtomografia por Raio-X
20.
Molecules ; 27(3)2022 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-35163879

RESUMO

Osteoporosis is a skeletal disease that is both systemic and silent characterized by an unbalanced activity of bone remodeling leading to bone loss. Rising evidences demonstrate that thyroid stimulating hormone (TSH) has an important role in the regulation on the metabolism of bone. However, TSH regulation on human osteoblast essential transcriptional factors has not been identified. Current study examined the role of TSH on human osteoblastic Runx2 expression and their functional genes by in vitro and in slico analysis. Human osteoblast like (HOS and SaoS-2) cells were cultured with DMEM and treated with hTSH at the concentration of 0.01 ng/mL and 10 ng/mL. After treatment, osteoblastic Runx2 and IGF-1R beta expression were studied using RT-PCR and western blot analysis. TSH treatment induced osteoblastic essential transcriptional factor, Runx2 in HOS and SaOS2 cells on 48 h duration and elevated the expression of IGF-IR ß gene and Protein in SaoS-2 cells. TSH also promotes Runx2 responsive genes such as ALP, Collagen and osteocalcin in SaOS2 cells on day 2 to day 14 of 10 ng/mL of treatment and favors' matrix mineralization matrix in these cells. In addition, TSH facilitated human osteoblastic cells to mineralize their matrix confirmed by day 21 of alizarin red calcium staining. In silico study was performed to check CREB and ELK1 interaction with Runx2. Results of in silico analysis showed that TSH mediated signalling molecules such as CREB and ELK1 showed interaction with Runx2 which involve in osteobalstic gene expression and differentiation. Present findings confirm that TSH promotes Runx2 expression, osteoblastic responsive genes and bone matrix formation.


Assuntos
Calcificação Fisiológica , Diferenciação Celular , Simulação por Computador , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoblastos/fisiologia , Osteogênese , Tireotropina/farmacologia , Matriz Óssea/citologia , Matriz Óssea/fisiologia , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Humanos , Técnicas In Vitro , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos
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