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1.
J Oral Sci ; 66(1): 15-19, 2024 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-38008425

RESUMO

PURPOSE: After tooth extraction, preservation of the alveolar ridge by socket grafting attenuates bone resorption. Runt-related transcription factor 2 (RUNX2) and SP7/Osterix (OSX) are transcription factors playing an important role in osteoblast differentiation. The purpose of this study was to evaluate the effects of carbonate apatite (CO3Ap) on osteoblast-related gene and protein expression after socket grafting. METHODS: Alveolar bone and new bone after CO3Ap grafting were collected at the time of implant placement. Levels of mRNA for RUNX2, SP7/OSX, bone morphogenetic protein 2 (BMP2), BMP7 and platelet derived growth factor B were determined by real-time PCR. Immunostaining was performed using antibodies against RUNX2, SP7/OSX, vimentin and cytokeratin. To evaluate bone resorption rates, cone-beam CT (CBCT) imaging was performed after socket grafting and before implant placement. RESULTS: CBCT imaging showed that the average degree of bone resorption at the CO3Ap graft site was 7.15 ± 3.79%. At the graft sites, levels of SP7/OSX and BMP2 mRNA were significantly increased. Replacement of CO3Ap with osteoid was evident histologically, and in the osteoid osteoblast-like cells were stained for SP7/OSX and vimentin. CONCLUSION: These results show that gene expression of both SP7/OSX and BMP2 can be induced by CO3Ap, suggesting that increased expression of SP7/OSX and vimentin may be involved in the BMP pathway.


Assuntos
Apatitas , Proteína Morfogenética Óssea 2 , Reabsorção Óssea , Humanos , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Vimentina/genética , Vimentina/metabolismo , Vimentina/farmacologia , Diferenciação Celular , Osteoblastos/metabolismo , Processo Alveolar/cirurgia , RNA Mensageiro/metabolismo , Reabsorção Óssea/metabolismo , Expressão Gênica , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Fator de Transcrição Sp7/farmacologia
2.
Int J Mol Sci ; 24(20)2023 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-37894935

RESUMO

Deubiquitinases (DUBs) are essential for bone remodeling by regulating the differentiation of osteoblast and osteoclast. USP17 encodes for a deubiquitinating enzyme, specifically known as ubiquitin-specific protease 17, which plays a critical role in regulating protein stability and cellular signaling pathways. However, the role of USP17 during osteoblast differentiation has not been elusive. In this study, we initially investigated whether USP17 could regulate the differentiation of osteoblasts. Moreover, USP17 overexpression experiments were conducted to assess the impact on osteoblast differentiation induced by bone morphogenetic protein 4 (BMP4). The positive effect was confirmed through alkaline phosphatase (ALP) expression and activity studies since ALP is a representative marker of osteoblast differentiation. To confirm this effect, Usp17 knockdown was performed, and its impact on BMP4-induced osteoblast differentiation was examined. As expected, knockdown of Usp17 led to the suppression of both ALP expression and activity. Mechanistically, it was observed that USP17 interacted with Osterix (Osx), which is a key transcription factor involved in osteoblast differentiation. Furthermore, overexpression of USP17 led to an increase in Osx protein levels. Thus, to investigate whether this effect was due to the intrinsic function of USP17 in deubiquitination, protein stabilization experiments and ubiquitination analysis were conducted. An increase in Osx protein levels was attributed to an enhancement in protein stabilization via USP17-mediated deubiquitination. In conclusion, USP17 participates in the deubiquitination of Osx, contributing to its protein stabilization, and ultimately promoting the differentiation of osteoblasts.


Assuntos
Osteoblastos , Osteogênese , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Osteogênese/genética , Osteoblastos/metabolismo , Diferenciação Celular/genética , Estabilidade Proteica , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo
3.
Cell Tissue Res ; 393(2): 265-279, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37247031

RESUMO

Osteoblast differentiation is regulated by various transcription factors, signaling molecules, and posttranslational modifiers. The histone acetyltransferase Mof (Kat8) is involved in distinct physiological processes. However, the exact role of Mof in osteoblast differentiation and growth remains unknown. Herein, we demonstrated that Mof expression with histone H4K16 acetylation increased during osteoblast differentiation. Inhibition of Mof by siRNA knockdown or small molecule inhibitor, MG149 which is a potent histone acetyltransferase inhibitor, reduced the expression level and transactivation potential of osteogenic key markers, Runx2 and Osterix, thus inhibiting osteoblast differentiation. Besides, Mof overexpression also enhanced the protein levels of Runx2 and Osterix. Mof could directly bind the promoter region of Runx2/Osterix to potentiate their mRNA levels, possibly through Mof-mediated H4K16ac to facilitate the activation of transcriptional programs. Importantly, Mof physically interacts with Runx2/Osterix for the stimulation of osteoblast differentiation. Yet, Mof knockdown showed indistinguishable effect on cell proliferation or apoptosis in MSCs and preosteoblast cells. Taken together, our results uncover Mof functioning as a novel regulator of osteoblast differentiation via the promotional effects on Runx2/Osterix and rationalize Mof as a potential therapeutic target, like possible application of inhibitor MG149 for the treatment of osteosarcoma or developing specific Mof activator to ameliorate osteoporosis.


Assuntos
Osteogênese , Fatores de Transcrição , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Histona Acetiltransferases/metabolismo , Osteoblastos , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Camundongos
4.
Int J Mol Sci ; 24(5)2023 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-36901736

RESUMO

Epigenetic modifications are critical for cell differentiation and growth. As a regulator of H3K9 methylation, Setdb1 is implicated in osteoblast proliferation and differentiation. The activity and nucleus localization of Setdb1 are regulated by its binding partner, Atf7ip. However, whether Atf7ip is involved in the regulation of osteoblast differentiation remains largely unclear. In the present study, we found that Atf7ip expression was upregulated during the osteogenesis of primary bone marrow stromal cells and MC3T3-E1 cells, and was induced in PTH-treated cells. The overexpression of Atf7ip impaired osteoblast differentiation in MC3T3-E1 cells regardless of PTH treatment, as measured by the expression of osteoblast differentiation markers, Alp-positive cells, Alp activity, and calcium deposition. Conversely, the depletion of Atf7ip in MC3T3-E1 cells promoted osteoblast differentiation. Compared with the control mice, animals with Atf7ip deletion in the osteoblasts (Oc-Cre;Atf7ipf/f) showed more bone formation and a significant increase in the bone trabeculae microarchitecture, as reflected by µ-CT and bone histomorphometry. Mechanistically, Atf7ip contributed to the nucleus localization of Setdb1 in MC3T3-E1, but did not affect Setdb1 expression. Atf7ip negatively regulated Sp7 expression, and through specific siRNA, Sp7 knockdown attenuated the enhancing role of Atf7ip deletion in osteoblast differentiation. Through these data, we identified Atf7ip as a novel negative regulator of osteogenesis, possibly via its epigenetic regulation of Sp7 expression, and demonstrated that Atf7ip inhibition is a potential therapeutic measure for enhancing bone formation.


Assuntos
Epigênese Genética , Osteogênese , Animais , Camundongos , Osteogênese/genética , Fator de Transcrição Sp7/genética , Diferenciação Celular/genética , Osteoblastos/metabolismo , Proteínas Repressoras/genética
5.
Curr Osteoporos Rep ; 21(2): 241-252, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36881265

RESUMO

PURPOSE OF REVIEW: The purpose of this review is to summarize the different roles of the transcription factor SP7 in regulating bone formation and remodeling, discuss current studies in investigating the causal relationship between SP7 mutations and human skeletal disease, and highlight potential therapeutic treatments that targeting SP7 and the gene networks that it controls. RECENT FINDINGS: Cell-type and stage-specific functions of SP7 have been identified during bone formation and remodeling. Normal bone development regulated by SP7 is strongly associated with human bone health. Dysfunction of SP7 results in common or rare skeletal diseases, including osteoporosis and osteogenesis imperfecta with different inheritance patterns. SP7-associated signaling pathways, SP7-dependent target genes, and epigenetic regulations of SP7 serve as new therapeutic targets in the treatment of skeletal disorders. This review addresses the importance of SP7-regulated bone development in studying bone health and skeletal disease. Recent advances in whole genome and exome sequencing, GWAS, multi-omics, and CRISPR-mediated activation and inhibition have provided the approaches to investigate the gene-regulatory networks controlled by SP7 in bone and the therapeutic targets to treat skeletal disease.


Assuntos
Osteogênese Imperfeita , Osteogênese , Humanos , Osteogênese/genética , Osteogênese Imperfeita/genética , Osso e Ossos , Mutação , Transdução de Sinais/genética , Fator de Transcrição Sp7/genética
6.
Biochim Biophys Acta Mol Basis Dis ; 1869(3): 166636, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36584722

RESUMO

The mandible is an important component of the craniofacial bones, whose development is regulated by complex molecular networks and involves the well-coordinated development of the bone, cartilage, and teeth. Previously, we demonstrated that Krüppel-like factor 4 (KLF4) promoted dentinogenesis and osteogenesis, but it was enigmatic whether Klf4 participated in the development of the mandible. In this study, the Sp7-Cre; Klf4f/+ mice exhibited underdeveloped mandibles and insufficient elongation of the mandibular incisor when compared with Klf4f/+ and Sp7-Cre mice. Moreover, morphological and molecular analysis showed that the alveolar bone mass was significantly decreased in KLF4 deficient mice, accompanied by reduced expression of osteoblast-related genes. Meanwhile, the KLF4 deficient mice had decreased expression of receptor activator of nuclear factor kappa-Β ligand (RANKL) and no significant change of osteoprotegerin (OPG) in the alveolar bone near the mandibular incisor. Simultaneously, the osteoclastogenesis in the alveolar bone of KLF4 deficient mice was attenuated, which was demonstrated by a diminished number of tartrate-resistant acid phosphatase positive (TRAP+), matrix metallopeptidase 9 positive (MMP9+), and cathepsin K positive (CTSK+) multinucleated osteoclasts, respectively. Collectively, our study suggested that Klf4 participated in mandibular development, and Klf4 in Sp7+ lineage affected osteogenesis directly and osteoclastogenesis indirectly.


Assuntos
Glicoproteínas , Incisivo , Camundongos , Animais , Glicoproteínas/metabolismo , Incisivo/metabolismo , Fosfatase Ácida , Haploinsuficiência , Mandíbula/metabolismo , Fator de Transcrição Sp7
8.
Calcif Tissue Int ; 111(5): 519-534, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35731246

RESUMO

Indian hedgehog (Ihh) is an indispensable paracrine factor for proper tissue patterning, skeletogenesis, and cellular proliferation. Recent genetic studies have revealed critical roles of chondrocyte-derived Ihh in regulating chondrocyte proliferation, hypertrophy and cartilage ossification. However, the functions of Sp7-expressing cell-derived Ihh in osteoblast differentiation and bone formation remain unclear. Sp7 is an essential transcription factor for osteoblast differentiation. In the current study, we generated Sp7-iCre; Ihhfl/fl mice, in which the Ihh gene was specifically deleted in Sp7-expressing cells to investigate the roles of Ihh. Ihh ablation in Sp7-expressing cells resulted in a dwarfism phenotype with severe skeletal dysplasia and lethality at birth, but with normal joint segmentation. Sp7-iCre; Ihhfl/fl mice had fewer osteoblasts, almost no cortical and trabecular bones, smaller skulls, and wider cranial sutures. Additionally, the levels of osteogenesis- and angiogenesis-related genes, and of major bone matrix protein genes were significantly reduced. These results demonstrated that Ihh regulates bone formation in Sp7-expressing cells. Ihh deficiency in primary osteoblasts cultured in vitro inhibited their proliferation, differentiation, and mineralization ability, and reduced the expression of osteogenesis-related genes. Moreover, the deletion of Ihh also attenuated the Bmp2/Smad/Runx2 pathway in E18.5 tibial and primary osteoblasts. The activity of primary osteoblasts in mutant mice was rescued after treatment with rhBMP2. In summary, our data revealed that Ihh in Sp7-expressing cells plays an indispensable role in osteoblast differentiation, mineralization, and embryonic osteogenesis, further implicated that its pro-osteogenic role may be mediated through the canonical Bmp2/Smad/Runx2 pathway.


Assuntos
Nanismo , Osteogênese , Animais , Diferenciação Celular , Proliferação de Células , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Nanismo/genética , Nanismo/metabolismo , Proteínas Hedgehog/metabolismo , Camundongos , Osteoblastos/metabolismo , Osteogênese/fisiologia , Fenótipo , Fator de Transcrição Sp7/metabolismo , Fatores de Transcrição/genética
9.
Mol Ther ; 30(10): 3226-3240, 2022 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-35642253

RESUMO

Circular RNAs (circRNAs) play an important role in biological activities, especially in regulating osteogenic differentiation of stem cells. However, no studies have reported the role of circRNAs in early osseointegration. Here we identified a new circRNA, circRNA422, from rat bone marrow mesenchymal stem cells (BMSCs) cultured on sandblasted, large-grit, acid-etched titanium surfaces. The results showed that circRNA422 significantly enhanced osteogenic differentiation of BMSCs with increased expression levels of alkaline phosphatase, the SP7 transcription factor (SP7/osterix), and lipoprotein receptor-related protein 5 (LRP5). Silencing of circRNA422 had opposite effects. There were two SP7 binding sites on the LRP5 promoter, indicating a direct regulatory relationship between SP7 and LRP5. circRNA422 could regulate early osseointegration in in vivo experiments. These findings revealed an important function of circRNA422 during early osseointegration. Therefore, circRNA422 may be a potential therapeutic target for enhancing implant osseointegration.


Assuntos
Células-Tronco Mesenquimais , Osteogênese , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/farmacologia , Animais , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osseointegração/genética , Osteogênese/genética , RNA Circular/genética , Ratos , Fator de Transcrição Sp7/metabolismo , Titânio/química , Titânio/metabolismo , Titânio/farmacologia
10.
Signal Transduct Target Ther ; 7(1): 155, 2022 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-35538062

RESUMO

Maxillofacial bone defects are commonly seen in clinical practice. A clearer understanding of the regulatory network directing maxillofacial bone formation will promote the development of novel therapeutic approaches for bone regeneration. The fibroblast growth factor (FGF) signalling pathway is critical for the development of maxillofacial bone. Klotho, a type I transmembrane protein, is an important components of FGF receptor complexes. Recent studies have reported the presence of Klotho expression in bone. However, the role of Klotho in cranioskeletal development and repair remains unknown. Here, we use a genetic strategy to report that deletion of Klotho in Osx-positive mesenchymal progenitors leads to a significant reduction in osteogenesis under physiological and pathological conditions. Klotho-deficient mensenchymal progenitors also suppress osteoclastogenesis in vitro and in vivo. Under conditions of inflammation and trauma-induced bone loss, we find that Klotho exerts an inhibitory function on inflammation-induced TNFR signaling by attenuating Rankl expression. More importantly, we show for the first time that Klotho is present in human alveolar bone, with a distinct expression pattern under both normal and pathological conditions. In summary, our results identify the mechanism whereby Klotho expressed in Osx+-mensenchymal progenitors controls osteoblast differentiation and osteoclastogenesis during mandibular alveolar bone formation and repair. Klotho-mediated signaling is an important component of alveolar bone remodeling and regeneration. It may also be a target for future therapeutics.


Assuntos
Desenvolvimento Ósseo , Osso e Ossos , Proteínas Klotho , Células-Tronco Mesenquimais , Osteogênese , Desenvolvimento Ósseo/fisiologia , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Proteínas Klotho/metabolismo , Maxila/crescimento & desenvolvimento , Maxila/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Fator de Transcrição Sp7
11.
Int J Mol Sci ; 23(10)2022 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-35628456

RESUMO

Osteoblast differentiation is a tightly regulated process in which key transcription factors (TFs) and their target genes constitute gene regulatory networks (GRNs) under the control of osteogenic signaling pathways. Among these TFs, Sp7 works as an osteoblast determinant critical for osteoblast differentiation. Following the identification of Sp7 and a large number of its functional studies, recent genome-scale analyses have made a major contribution to the identification of a "non-canonical" mode of Sp7 action as well as "canonical" ones. The analyses have not only confirmed known Sp7 targets but have also uncovered its additional targets and upstream factors. In addition, biochemical analyses have demonstrated that Sp7 actions are regulated by chemical modifications and protein-protein interaction with other transcriptional regulators. Sp7 is also involved in chondrocyte differentiation and osteocyte biology as well as postnatal bone metabolism. The critical role of SP7 in the skeleton is supported by its relevance to human skeletal diseases. This review aims to overview the Sp7 actions in skeletal development and maintenance, particularly focusing on recent advances in our understanding of how Sp7 functions in the skeleton under physiological and pathological conditions.


Assuntos
Doenças Ósseas , Sistema Musculoesquelético , Osteoblastos , Fator de Transcrição Sp7 , Doenças Ósseas/genética , Humanos , Sistema Musculoesquelético/metabolismo , Osteoblastos/metabolismo , Osteogênese/genética , Esqueleto/metabolismo , Fator de Transcrição Sp7/genética
12.
Int J Mol Sci ; 23(8)2022 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-35457117

RESUMO

Yin Yang 2 (YY2) is a paralog of YY1, a well-known multifunctional transcription factor containing a C-terminal zinc finger domain. Although the role of YY1 in various biological processes, such as the cell cycle, cell differentiation and tissue development, is well established, the function of YY2 has not been fully determined. In this study, we investigated the functional role of YY2 during osteoblast differentiation. YY2 overexpression and knockdown increased and decreased osteoblast differentiation, respectively, in BMP4-induced C2C12 cells. Mechanistically, YY2 overexpression increased the mRNA and protein levels of Osterix (Osx), whereas YY2 knockdown had the opposite effect. To investigate whether YY2 regulates Osx transcription, the effect of YY2 overexpression and knockdown on Osx promoter activity was evaluated. YY2 overexpression significantly increased Osx promoter activity in a dose-dependent manner, whereas YY2 knockdown had the opposite effect. Furthermore, vectors containing deletion and point mutations were constructed to specify the regulation site. Both the Y1 and Y2 sites were responsible for YY2-mediated Osx promoter activation. These results indicate that YY2 is a positive regulator of osteoblast differentiation that functions by upregulating the promoter activity of Osx, a representative osteogenic transcription factor in C2C12 cells.


Assuntos
Osteogênese , Yin-Yang , Diferenciação Celular/genética , Osteoblastos/metabolismo , Osteogênese/genética , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
Bone ; 160: 116400, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35367406

RESUMO

Mutations in SP7 (encoding osterix) have been identified as a rare cause of recessive osteogenesis imperfecta ('OI type XII') and in one case of dominant juvenile Paget's disease. We present the first description of young adult siblings with OI due to a unique heterozygous mutation in SP7. The phenotype was characterized by fragility fractures (primarily of the long bone diaphyses), poor healing, scoliosis, and dental malocclusion. Both siblings had very low cortical volumetric bone mineral density on peripheral quantitative computed tomography of the radius (z-scores -6.6 and - 6.7 at the diaphysis), porous cortices, and thin cortices at the radial metaphysis. Histomorphometry demonstrated thin cortices and low bone turnover with reduced osteoblast function. Both siblings were heterozygous for a missense variant affecting a highly conserved zinc finger domain of osterix (c.1019A > C; p.Glu340Ala) on DNA sequencing. Co-transfection of plasmids carrying the SP7 mutation with DLX5 and a luciferase reporter demonstrated that this variant impacted gene function (reduced transcription co-activation compared to wild-type SP7). The low cortical density and cortical porosity seen in our patients are consistent with previous reports of individuals with SP7 mutations. However, the low bone turnover in our patients contrasts with the high turnover state seen in previously reported patients with SP7 mutations. This report indicates that dominant variants in SP7 can give rise to OI. The predominant feature, low cortical density, is common in patients with other SP7 mutations, however other features appear to depend on the specific variant.


Assuntos
Osteíte Deformante , Osteogênese Imperfeita , Densidade Óssea/genética , Remodelação Óssea , Osso e Ossos , Heterozigoto , Humanos , Mutação , Osteogênese Imperfeita/diagnóstico por imagem , Osteogênese Imperfeita/genética , Fator de Transcrição Sp7/genética
14.
Int J Mol Sci ; 23(6)2022 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-35328592

RESUMO

The relationship of lacunocanalicular network structure and mechanoresponse has not been well studied. The lacunocanalicular structures differed in the compression and tension sides, in the regions, and in genders in wild-type femoral cortical bone. The overexpression of Sp7 in osteoblasts resulted in thin and porous cortical bone with increased osteoclasts and apoptotic osteocytes, and the number of canaliculi was half of that in the wild-type mice, leading to a markedly impaired lacunocanalicular network. To investigate the response to unloading, we performed tail suspension. Unloading reduced trabecular and cortical bone in the Sp7 transgenic mice due to reduced bone formation. Sost-positive osteocytes increased by unloading on the compression side, but not on the tension side of cortical bone in the wild-type femurs. However, these differential responses were lost in the Sp7 transgenic femurs. Serum Sost increased in the Sp7 transgenic mice, but not in the wild-type mice. Unloading reduced the Col1a1 and Bglap/Bglap2 expression in the Sp7 transgenic mice but not the wild-type mice. Thus, Sp7 transgenic mice with the impaired lacunocanalicular network induced Sost expression by unloading but lost the differential regulation in the compression and tension sides, and the mice failed to restore bone formation during unloading, implicating the relationship of lacunocanalicular network structure and the regulation of bone formation in mechanoresponse.


Assuntos
Reabsorção Óssea , Osteócitos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Densidade Óssea , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismo , Osteócitos/metabolismo , Fator de Transcrição Sp7/metabolismo
15.
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
16.
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
17.
FASEB J ; 36(2): e22115, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35032415

RESUMO

Bone loss is a hallmark of inflammatory bone diseases caused by aberrantly activated osteoclasts (OCLs). Studies have shown that OCLs exhibit various phenotypes and functions due to variations in the source(s) of precursor cells, cytokine expressions, and microenvironment-dependent factors. During these conditions, inflammatory osteoclasts (iOCLs) lose their immune-suppressive effect relative to OCLs under physiological conditions. This induces TNF α-producing CD4+ T cells in an antigen-dependent manner and finally leads to cascade amplification of iOCLs. OCL-derived exosomes have been reported to regulate OCL formation and inhibit the osteoblast activity. However, the specific function and mechanism of iOCL-derived exosomes on osteoblast have not been studied yet. In the present study, we compare the osteoblast promoting activities of iOCL-derived exosomes and OCL-derived exosomes. We found that iOCLs exosomes specifically target osteoblasts through ephrinA2/EphA2. Mechanistically, the lncRNA LIOCE is enriched in iOCL exosomes and promotes the osteoblast activity after being incorporated into osteoblasts. Furthermore, our results revealed that exosomal lncRNA LIOCE stabilizes osteogenic transcription factor Osterix by interacting and reducing the ubiquitination level of Osterix. This study demonstrated that the bone loss is alleviated in the inflammatory osteolysis mice model after injection of iOCL exosomes encapsulating lncRNA LIOCE. The role of exosomes encapsulating lncRNA LIOCE in promoting bone formation was well established in the rat bone repair model. Our results indicate that iOCL-derived exosomal lncRNA LIOCE promotes bone formation by upregulating Osx expression, and thus, the exosomes encapsulating lncRNA LIOCE may be an effective strategy to increase bone formation in osteoporosis and other bone metabolic disorders.


Assuntos
Exossomos/genética , Inflamação/genética , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Osteogênese/genética , RNA Longo não Codificante/genética , Fator de Transcrição Sp7/genética , Células 3T3 , Animais , Diferenciação Celular/genética , Linhagem Celular , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteólise/genética , Osteoporose/genética , Ratos , Fatores de Transcrição/genética , Ubiquitinação/genética , Regulação para Cima/genética
18.
Biochem Biophys Res Commun ; 587: 9-15, 2022 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-34861472

RESUMO

OBJECTIVE: The role of circadian clock in cementogenesis is unclear. This study examines the role of REV-ERBs, one of circadian clock proteins, in proliferation, migration and mineralization of cementoblasts to fill the gap in knowledge. METHODS: Expression pattern of REV-ERBα in cementoblasts was investigated in vivo and in vitro. CCK-8 assay, scratch wound healing assay, alkaline phosphatase (ALP) and alizarin red S (ARS) staining were performed to evaluate the effects of REV-ERBs activation by SR9009 on proliferation, migration and mineralization of OCCM-30, an immortalized cementoblast cell line. Furthermore, mineralization related markers including osterix (OSX), ALP, bone sialoprotein (BSP) and osteocalcin (OCN) were evaluated. RESULTS: Strong expression of REV-ERBα was found in cellular cementum around tooth apex. Rev-erbα mRNA oscillated periodically in OCCM-30 and declined after mineralization induction. REV-ERBs activation by SR9009 inhibited proliferation but promoted migration of OCCM-30 in vitro. Results of ALP and ARS staining suggested that REV-ERBs activation negatively regulated mineralization of OCCM-30. Mechanically, REV-ERBs activation attenuated the expression of OSX and its downstream targets including ALP, BSP and OCN. CONCLUSIONS: REV-ERBs are involved in cementogenesis and negatively regulate mineralization of cementoblasts via inhibiting OSX expression. Our study provides a potential target regarding periodontal and cementum regeneration.


Assuntos
Relógios Biológicos/genética , Calcificação Fisiológica/genética , Cemento Dentário/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Cementogênese/efeitos dos fármacos , Cementogênese/genética , Cemento Dentário/citologia , Cemento Dentário/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Humanos , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Pirrolidinas/farmacologia , Transdução de Sinais , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Tiofenos/farmacologia
19.
J Craniofac Surg ; 33(3): 956-961, 2022 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-34456284

RESUMO

OBJECTIVES: To investigate whether and how the long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) sponges microRNA-96 (miR-96) to achieve the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). METHODS: Protein levels were detected by Western blot. Mineralized bone matrix formation was studied by alizarin red staining. Metastasis-associated lung adenocarcinoma transcript 1, miR-96, and osteogenesis-related Messenger RNA expression was assessed by Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). The interactions between miR-96 and osterix (Osx), MALAT1, and miR-96 were determined by luciferase reporter assay. RESULTS: The expression of MALAT1 was upregulated whereas that of miR-96 was downregulated in osteogenic hBMSCs. In addition, the expression of MALAT1 significantly decreased whereas that of miR-96 increased in the hBMSCs of osteoporosis (OP) patients. qRT-PCR and alizarin red staining assays showed that MALAT1 silencing or miR-96 overexpression inhibits hBMSC osteogenic differentiation and vice versa. overexpression of miR-96 reversed the promotive effect of MALAT1 on the osteogenic differentiation of hBMSCs. Dual luciferase report assay verified that miR-96 is a regulatory target of MALAT1 and that Osx is a gene target of miR-96. CONCLUSIONS: Taken together, the results demonstrate that MALAT1 promotes the osteogenic differentiation of hBMSCs by regulating the miR-96/Osx axis. Our study provides novel mechanistic insights into the critical role of lncRNA MALAT1 as a microRNA sponge in OP patients and sheds new light on lncRNA-directed diagnostics and therapeutics in OP.


Assuntos
Células-Tronco Mesenquimais , MicroRNAs , Osteoblastos , Osteoporose , RNA Longo não Codificante , Fator de Transcrição Sp7 , Medula Óssea , Diferenciação Celular/genética , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Osteoblastos/citologia , Osteogênese/genética , RNA Longo não Codificante/genética , Fator de Transcrição Sp7/genética
20.
Anal Cell Pathol (Amst) ; 2021: 7890674, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34868829

RESUMO

BACKGROUND: Long noncoding RNAs (lncRNAs) are dysregulated in periodontitis development and involved in osteogenesis. The current study was aimed at investigating the function of lncRNA ANRIL in periodontal ligament cells (PDLCs) and potential molecular mechanisms. METHODS: Firstly, the level of ANRIL was tested by qPCR. Then, PDLCs were treated with a mineralizing solution to induce osteogenic differentiation. ALP activity was measured, and protein levels of BMP2, Osterix, and OCN were measured by Western blot. A target of ANRIL was verified using dual-luciferase reporter assay. miR-7 level was measured by qPCR, and the signals of the NF-κB pathway were tested by Western blot. RESULTS: ANRIL expression was downregulated in PDL tissues. Next, ALP activity and protein levels of BMP2, Osterix, and OCN were increased to show that PDLCs were differentiated. ANRIL level was increased in differential PDLCs, in which knockdown inhibited osteogenic differentiation. Then, miR-7 was found as a target of ANRIL. The miR-7 level was upregulated in PDL tissues and reduced in differential PDLCs. Inhibition of miR-7 suppressed ALP activity and BMP2, Osterix, and OCN expression. Moreover, inhibition of miR-7 reversed the effects on the osteogenic differentiation induced by knockdown of ANRIL. Besides, the levels of p-P65 and p-IκBα were elevated by ANRIL downregulation and were rescued by suppressing miR-7. CONCLUSIONS: Knockdown of ANRIL inhibited osteogenic differentiation via sponging miR-7 through the NF-κB pathway, suggesting that ANRIL might be a therapeutic target for periodontitis.


Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica , MicroRNAs/genética , NF-kappa B/metabolismo , Osteogênese/genética , Ligamento Periodontal/metabolismo , RNA Longo não Codificante/genética , Western Blotting , Proteína Morfogenética Óssea 2/metabolismo , Células Cultivadas , Regulação para Baixo , Células HEK293 , Humanos , MicroRNAs/metabolismo , Ligamento Periodontal/citologia , Interferência de RNA , RNA Longo não Codificante/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fator de Transcrição Sp7/metabolismo
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