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2.
Front Endocrinol (Lausanne) ; 15: 1342938, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39092287

RESUMO

Glucocorticoids (GC) and parathyroid hormone (PTH) are widely used therapeutic endocrine hormones where their effects on bone and joint arise from actions on multiple skeletal cell types. In osteocytes, GC and PTH exert opposing effects on perilacunar canalicular remodeling (PLR). Suppressed PLR can impair bone quality and joint homeostasis, including in GC-induced osteonecrosis. However, combined effects of GC and PTH on PLR are unknown. Given the untapped potential to target osteocytes to improve skeletal health, this study sought to test the feasibility of therapeutically mitigating PLR suppression. Focusing on subchondral bone and joint homeostasis, we hypothesize that PTH(1-34), a PLR agonist, could rescue GC-suppressed PLR. The skeletal effects of GC and PTH(1-34), alone or combined, were examined in male and female mice by micro-computed tomography, mechanical testing, histology, and gene expression analysis. For each outcome, females were more responsive to GC and PTH(1-34) than males. GC and PTH(1-34) exerted regional differences, with GC increasing trabecular bone volume but reducing cortical bone thickness, stiffness, and ultimate force. Despite PTH(1-34)'s anabolic effects on trabecular bone, it did not rescue GC's catabolic effects on cortical bone. Likewise, cartilage integrity and subchondral bone apoptosis, tartrate-resistant acid phosphatase (TRAP) activity, and osteocyte lacunocanalicular networks showed no evidence that PTH(1-34) could offset GC-dependent effects. Rather, GC and PTH(1-34) each increased cortical bone gene expression implicated in bone resorption by osteoclasts and osteocytes, including Acp5, Mmp13, Atp6v0d2, Ctsk, differences maintained when GC and PTH(1-34) were combined. Since PTH(1-34) is insufficient to rescue GC's effects on young female mouse bone, future studies are needed to determine if osteocyte PLR suppression, due to GC, aging, or other factors, can be offset by a PLR agonist.


Assuntos
Densidade Óssea , Remodelação Óssea , Glucocorticoides , Osteócitos , Hormônio Paratireóideo , Animais , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Hormônio Paratireóideo/farmacologia , Feminino , Masculino , Camundongos , Glucocorticoides/farmacologia , Remodelação Óssea/efeitos dos fármacos , Densidade Óssea/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Microtomografia por Raio-X
3.
Front Endocrinol (Lausanne) ; 15: 1359052, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39157681

RESUMO

Introduction: Changes to bone physiology play a central role in the development of osteoarthritis with the mechanosensing osteocyte releasing factors that drive disease progression. This study developed a humanised in vitro model to detect osteocyte responses to either interleukin-6, a driver of degeneration and bone remodelling in animal and human joint injury, or mechanical loading, to mimic osteoarthritis stimuli in joints. Methods: Human MSC cells (Y201) were differentiated in 3-dimensional type I collagen gels in osteogenic media and osteocyte phenotype assessed by RTqPCR and immunostaining. Gels were subjected to a single pathophysiological load or stimulated with interleukin-6 with unloaded or unstimulated cells as controls. RNA was extracted 1-hour post-load and assessed by RNAseq. Markers of pain, bone remodelling, and inflammation were quantified by RT-qPCR and ELISA. Results: Y201 cells embedded within 3D collagen gels assumed dendritic morphology and expressed mature osteocytes markers. Mechanical loading of the osteocyte model regulated 7564 genes (Padj p<0.05, 3026 down, 4538 up). 93% of the osteocyte transcriptome signature was expressed in the model with 38% of these genes mechanically regulated. Mechanically loaded osteocytes regulated 26% of gene ontology pathways linked to OA pain, 40% reflecting bone remodelling and 27% representing inflammation. Load regulated genes associated with osteopetrosis, osteoporosis and osteoarthritis. 42% of effector genes in a genome-wide association study meta-analysis were mechanically regulated by osteocytes with 10 genes representing potential druggable targets. Interleukin-6 stimulation of osteocytes at concentrations reported in human synovial fluids from patients with OA or following knee injury, regulated similar readouts to mechanical loading including markers of pain, bone remodelling, and inflammation. Discussion: We have developed a reproducible model of human osteocyte like cells that express >90% of the genes in the osteocyte transcriptome signature. Mechanical loading and inflammatory stimulation regulated genes and proteins implicated in osteoarthritis symptoms of pain as well as inflammation and degeneration underlying disease progression. Nearly half of the genes classified as 'effectors' in GWAS were mechanically regulated in this model. This model will be useful in identifying new mechanisms underlying bone and joint pathologies and testing drugs targeting those mechanisms.


Assuntos
Inflamação , Células-Tronco Mesenquimais , Osteoartrite , Osteócitos , Humanos , Osteócitos/metabolismo , Osteócitos/patologia , Osteoartrite/patologia , Osteoartrite/metabolismo , Inflamação/patologia , Inflamação/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Interleucina-6/metabolismo , Remodelação Óssea , Células Cultivadas , Diferenciação Celular
4.
J Bone Miner Res ; 39(9): 1253-1267, 2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-39126373

RESUMO

Osteogenesis imperfecta (OI) is a group of severe genetic bone disorders characterized by congenital low bone mass, deformity, and frequent fractures. Type XV OI is a moderate to severe form of skeletal dysplasia caused by WNT1 variants. In this cohort study from southern China, we summarized the clinical phenotypes of patients with WNT1 variants and found that the proportion of type XV patients was around 10.3% (25 out of 243) with a diverse spectrum of phenotypes. Functional assays indicated that variants of WNT1 significantly impaired its secretion and effective activity, leading to moderate to severe clinical manifestations, porous bone structure, and enhanced osteoclastic activities. Analysis of proteomic data from human skeleton indicated that the expression of SOST (sclerostin) was dramatically reduced in type XV patients compared to patients with COL1A1 quantitative variants. Single-cell transcriptome data generated from human tibia samples of patients diagnosed with type XV OI and leg-length discrepancy, respectively, revealed aberrant differentiation trajectories of skeletal progenitors and impaired maturation of osteocytes with loss of WNT1, resulting in excessive CXCL12+ progenitors, fewer mature osteocytes, and the existence of abnormal cell populations with adipogenic characteristics. The integration of multi-omics data from human skeleton delineates how WNT1 regulates the differentiation and maturation of skeletal progenitors, which will provide a new direction for the treatment strategy of type XV OI and relative low bone mass diseases such as early onset osteoporosis.


Osteogenesis imperfecta is a rare disease characterized by low bone mass, frequent fractures, and long bone deformity. Type XV osteogenesis imperfect is an autosomal recessive disorder caused by WNT1 variants, while heterozygous variants of WNT1 result in early onset osteoporosis. In this cohort study, we summarized the clinical features of 25 patients diagnosed with type XV osteogenesis imperfect. The WNT1 variants were confirmed by genetic test. Molecular assays were conducted to reveal the impact of variants on WNT1 protein activity and bone structure. We then compared the protein levels in bone tissues isolated from the type XV patients and patients with mild deformity using proteomic method, and found that the expression of SOST, mainly produced by mature osteoblasts and osteocytes, was dramatically reduced in type XV patients. We further compared the global mRNA expression levels in the skeletal cells using single-cell RNA sequencing. Analyses of these data indicated that more immature progenitors were identified and maturation of osteocytes was impaired with WNT1 loss-of-function. Our study helps to understand the underlying pathogenesis of type XV osteogenesis imperfecta.


Assuntos
Diferenciação Celular , Osteogênese Imperfeita , Proteína Wnt1 , Humanos , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Proteína Wnt1/metabolismo , Proteína Wnt1/genética , Masculino , Feminino , Criança , Mutação com Perda de Função , Pré-Escolar , Animais , Proteômica , Adolescente , Osteócitos/metabolismo , Osteócitos/patologia , Fenótipo , Camundongos , Multiômica
5.
JCI Insight ; 9(16)2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-39171528

RESUMO

Obesity can increase the risk of bone fragility, even when bone mass is intact. This fragility stems from poor bone quality, potentially caused by deficiencies in bone matrix material properties. However, cellular and molecular mechanisms leading to obesity-related bone fragility are not fully understood. Using male mouse models of obesity, we discovered TGF-ß signaling plays a critical role in mediating the effects of obesity on bone. High-carbohydrate and high-fat diets increase TGF-ß signaling in osteocytes, which impairs their mitochondrial function, increases cellular senescence, and compromises perilacunar/canalicular remodeling and bone quality. By specifically inhibiting TGF-ß signaling in mouse osteocytes, some of the negative effects of high-fat and high-carbohydrate diets on bones, including the lacunocanalicular network, perilacunar/canalicular remodeling, senescence, and mechanical properties such as yield stress, were mitigated. DMP1-Cre-mediated deletion of TGF-ß receptor II also blunted adverse effects of high-fat and high-carbohydrate diets on energy balance and metabolism. These findings suggest osteocytes are key in controlling bone quality in response to high-fat and high-carbohydrate diets. Calibrating osteocyte function could mitigate bone fragility associated with metabolic diseases while reestablishing energy balance.


Assuntos
Dieta Hiperlipídica , Obesidade , Osteócitos , Fator de Crescimento Transformador beta , Animais , Osteócitos/metabolismo , Dieta Hiperlipídica/efeitos adversos , Camundongos , Fator de Crescimento Transformador beta/metabolismo , Masculino , Obesidade/metabolismo , Transdução de Sinais , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Remodelação Óssea , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Osso e Ossos/metabolismo , Densidade Óssea/efeitos dos fármacos , Carboidratos da Dieta/efeitos adversos , Carboidratos da Dieta/administração & dosagem
6.
Acta Biomater ; 186: 141-155, 2024 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-39142531

RESUMO

Macrophages and osteocytes are important regulators of inflammation, osteogenesis and osteoclastogenesis. However, their interactions under adverse conditions, such as biomaterial-associated infection (BAI) are not fully understood. We aimed to elucidate how factors released from macrophages modulate osteocyte responses in an in vitro indirect 3D co-culture model. Human monocyte-derived macrophages were cultured on etched titanium disks and activated with either IL-4 cytokine (anti-inflammatory M2 phenotype) or Staphylococcus aureus secreted virulence factors to simulate BAI (pro-inflammatory M1 phenotype). Primary osteocytes in collagen gels were then stimulated with conditioned media (CM) from these macrophages. The osteocyte response was analyzed by gene expression, protein secretion, and immunostaining. M1 phenotype macrophages were confirmed by IL-1ß and TNF-α secretion, and M2 macrophages by ARG-1 and MRC-1.Osteocytes receiving M1 CM revealed bone inhibitory effects, denoted by reduced secretion of bone formation osteocalcin (BGLAP) and increased secretion of the bone inhibitory sclerostin (SOST). These osteocytes also downregulated the pro-mineralization gene PHEX and upregulated the anti-mineralization gene MEPE. Additionally, exhibited pro-osteoclastic potential by upregulating pro-osteoclastic gene RANKL expression. Nonetheless, M1-stimulated osteocytes expressed a higher level of the potent pro-osteogenic factor BMP-2 in parallel with the downregulation of the bone inhibitor genes DKK1 and SOST, suggesting a compensatory feedback mechanisms. Conversely, M2-stimulated osteocytes mainly upregulated anti-osteoclastic gene OPG expression, suggesting an anti-catabolic effect. Altogether, our findings demonstrate a strong communication between M1 macrophages and osteocytes under M1 (BAI)-simulated conditions, suggesting that the BAI adverse effects on osteoblastic and osteoclastic processes in vitro are partly mediated via this communication. STATEMENT OF SIGNIFICANCE: Biomaterial-associated infections are major challenges and the underlying mechanisms in the cellular interactions are missing, especially among the major cells from the inflammatory side (macrophages as the key cell in bacterial clearance) and the regenerative side (osteocyte as main regulator of bone). We evaluated the effect of macrophage polarization driven by the stimulation with bacterial virulence factors on the osteocyte function using an indirect co-culture model, hence mimicking the scenario of a biomaterial-associated infection. The results suggest that at least part of the adverse effects of biomaterial associated infection on osteoblastic and osteoclastic processes in vitro are mediated via macrophage-to-osteocyte communication.


Assuntos
Comunicação Celular , Macrófagos , Osteócitos , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos/microbiologia , Osteócitos/metabolismo , Osteócitos/efeitos dos fármacos , Técnicas de Cocultura , Infecções Relacionadas à Prótese/patologia , Infecções Relacionadas à Prótese/metabolismo , Infecções Relacionadas à Prótese/microbiologia , Meios de Cultivo Condicionados/farmacologia , Modelos Biológicos , Osteogênese/efeitos dos fármacos
7.
J Cell Biol ; 223(11)2024 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-39212624

RESUMO

Osteocytes' response to dynamic loading plays a crucial role in regulating the bone mass but quickly becomes saturated such that downstream induction of bone formation plateaus. The underlying mechanisms that downregulate osteocytes' sensitivity and overall response to loading remain unknown. In other cell types, purinergic signaling through the P2Y2 receptor has the potential to downregulate the sensitivity to loading by modifying cell stiffness through actin polymerization and cytoskeleton organization. Herein, we examined the role of P2Y2 activation in regulating osteocytes' mechanotransduction using a P2Y2 knockout cell line alongside conditional knockout mice. Our findings demonstrate that the absence of P2Y2 expression in MLO-Y4 cells prevents actin polymerization while increasing the sensitivity to fluid flow-induced shear stress. Deleting osteocytes' P2Y2 expression in conditional-knockout mice enabled bone formation to increase when increasing the duration of exercise. Overall, P2Y2 activation under loading produces a negative feedback loop, limiting osteocytes' response to continuous loading by shifting the sensitivity to mechanical strain through actin stress fiber formation.


Assuntos
Mecanotransdução Celular , Camundongos Knockout , Osteócitos , Receptores Purinérgicos P2Y2 , Estresse Mecânico , Animais , Receptores Purinérgicos P2Y2/metabolismo , Receptores Purinérgicos P2Y2/genética , Osteócitos/metabolismo , Camundongos , Linhagem Celular , Osteogênese , Transdução de Sinais , Actinas/metabolismo , Camundongos Endogâmicos C57BL
8.
Exp Cell Res ; 442(1): 114211, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-39147261

RESUMO

Blood vessel growth and osteogenesis in the skeletal system are coupled; however, fundamental aspects of vascular function in osteoblast-to-osteocyte transition remain unclear. Our study demonstrates that vascular smooth muscle cells (VSMCs), but not endothelial cells, are sufficient to drive bone marrow mesenchymal stromal cell-derived osteoblast-to-osteocyte transition via ß-catenin signaling and exosome-mediated communication. We found that VSMC-derived exosomes are loaded with transcripts encoding proteins associated with the osteocyte phenotype and members of the WNT/ß-catenin signaling pathway. In contrast, endothelial cell-derived exosomes facilitated mature osteoblast differentiation by reprogramming the TGFB1 gene family and osteogenic transcription factors osterix (SP7) and RUNX2. Notably, VSMCs express significant levels of tetraspanins (CD9, CD63, and CD81) and drive the intracellular trafficking of exosomes with a lower membrane zeta potential than those from other cells. Additionally, the high ATP content within these exosomes supports mineralization mechanisms, as ATP is a substrate for alkaline phosphatase. Osteocyte function was further validated by RNA sequencing, revealing activity in genes related to intermittent mineralization and sonic hedgehog signaling, alongside a significant increase in TNFSF11 levels. Our findings unveil a novel role of VSMCs in promoting osteoblast-to-osteocyte transition, thus offering new insights into bone biology and homeostasis, as well as in bone-related diseases. Clinically, these insights could pave the way for innovative therapeutic strategies targeting VSMC-derived exosome pathways to treat bone-related disorders such as osteoporosis. By manipulating these signaling pathways, it may be possible to enhance bone regeneration and improve skeletal health in patients with compromised bone structure and function.


Assuntos
Exossomos , Músculo Liso Vascular , Osteoblastos , Osteócitos , Osteogênese , beta Catenina , Osteoblastos/metabolismo , Osteoblastos/citologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologia , Exossomos/metabolismo , Animais , beta Catenina/metabolismo , beta Catenina/genética , Osteócitos/metabolismo , Osteócitos/citologia , Camundongos , Osteogênese/genética , Osteogênese/fisiologia , Miócitos de Músculo Liso/metabolismo , Diferenciação Celular , Humanos , Via de Sinalização Wnt , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Células Cultivadas , Transdução de Sinais , Camundongos Endogâmicos C57BL
9.
PLoS Biol ; 22(8): e3002753, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39163396

RESUMO

Cell-to-cell mitochondrial transfer has recently been shown to play a role in maintaining physiological functions of cell. We previously illustrated that mitochondrial transfer within osteocyte dendritic network regulates bone tissue homeostasis. However, the mechanism of triggering this process has not been explored. Here, we showed that stressed osteocytes in mice release adenosine diphosphate (ADP), resulting in triggering mitochondrial transfer from healthy osteocytes to restore the oxygen consumption rate (OCR) and to alleviate reactive oxygen species accumulation. Furthermore, we identified that P2Y2 and P2Y6 transduced the ADP signal to regulate osteocyte mitochondrial transfer. We showed that mitochondrial metabolism is impaired in aged osteocytes, and there were more extracellular nucleotides release into the matrix in aged cortical bone due to compromised membrane integrity. Conditioned medium from aged osteocytes triggered mitochondrial transfer between osteocytes to enhance the energy metabolism. Together, using osteocyte as an example, this study showed new insights into how extracellular ADP triggers healthy cells to rescue energy metabolism crisis in stressed cells via mitochondrial transfer in tissue homeostasis.


Assuntos
Difosfato de Adenosina , Homeostase , Mitocôndrias , Osteócitos , Animais , Osteócitos/metabolismo , Mitocôndrias/metabolismo , Camundongos , Difosfato de Adenosina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Consumo de Oxigênio , Metabolismo Energético , Camundongos Endogâmicos C57BL , Estresse Fisiológico
10.
Biofabrication ; 16(4)2024 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-39116896

RESUMO

Osteoporosis is the most common bone disorder, which is a highly dangerous condition that can promote bone metastases. As the current treatment for osteoporosis involves long-term medication therapy and a cure for bone metastasis is not known, ongoing efforts are required for drug development for osteoporosis. Animal experiments, traditionally used for drug development, raise ethical concerns and are expensive and time-consuming. Organ-on-a-chip technology is being developed as a tool to supplement such animal models. In this study, we developed a bone-on-a-chip by co-culturing osteoblasts, osteocytes, and osteoclasts in an extracellular matrix environment that can represent normal bone, osteopenia, and osteoporotic conditions. We then simulated bone metastases using breast cancer cells in three different bone conditions and observed that bone metastases were most active in osteoporotic conditions. Furthermore, it was revealed that the promotion of bone metastasis in osteoporotic conditions is due to increased vascular permeability. The bone-on-a-chip developed in this study can serve as a platform to complement animal models for drug development for osteoporosis and bone metastasis.


Assuntos
Neoplasias Ósseas , Dispositivos Lab-On-A-Chip , Osteoporose , Osteoporose/patologia , Osteoporose/tratamento farmacológico , Neoplasias Ósseas/secundário , Neoplasias Ósseas/patologia , Animais , Humanos , Osteoblastos/metabolismo , Técnicas de Cocultura , Camundongos , Osteoclastos/patologia , Osteoclastos/metabolismo , Osteócitos/patologia , Osteócitos/metabolismo , Osso e Ossos/patologia , Linhagem Celular Tumoral , Feminino
11.
ACS Appl Mater Interfaces ; 16(31): 40411-40427, 2024 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-39044386

RESUMO

The treatment of critical-sized bone defects caused by tumor removal, skeletal injuries, or infections continues to pose a major clinical challenge. A popular potential alternative solution to autologous bone grafts is a tissue-engineered approach that utilizes the combination of mesenchymal stromal/stem cells (MSCs) with synthetic biomaterial scaffolds. This approach aims to support new bone formation by mimicking many of the biochemical and biophysical cues present within native bone. Regrettably, osteocyte cells, crucial for bone maturation and homeostasis, are rarely produced within MSC-seeded scaffolds, thereby restricting the development of fully mature cortical bone from these synthetic implants. In this work, we have constructed a multimodal scaffold by combining electrospun poly(lactic-co-glycolic acid) (PLGA) fibrous scaffolds with poly(ethylene glycol) (PEG)-based hydrogels that mimic the functional unit of cortical bone, osteon (osteon-mimetic) scaffolds. These scaffolds were decorated with a novel bone morphogenic protein-6 (BMP6) peptide (BMP6p) after our findings revealed that the BMP6p drives higher levels of Smad signaling than the full-length protein counterpart, soluble or when bound to the PEG hydrogel backbone. We show that our osteon-mimetic scaffolds, in presenting concentric layers of BMP6p-PEG hydrogel overlaid on MSC-seeded PLGA nanofibers, promoted the rapid formation of osteocyte-like cells with a phenotypic dendritic morphology, producing early osteocyte markers, including E11/gp38 (E11). Maturation of these osteocyte-like cells was further confirmed by the observation of significant dentin matrix protein 1 (DMP1) throughout our bilayered scaffolds after 3 weeks, even when cultured in a medium without dexamethasone (DEX) or any other osteogenic supplements. These results demonstrate that these osteon-mimetic scaffolds, in presenting biochemical and topographical cues reminiscent of the forming osteon, can drive the formation of osteocyte-like cells in vitro from hBMSCs without the need for any osteogenic factor media supplementation.


Assuntos
Materiais Biomiméticos , Células-Tronco Mesenquimais , Nanofibras , Osteócitos , Osteogênese , Alicerces Teciduais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Alicerces Teciduais/química , Nanofibras/química , Humanos , Osteogênese/efeitos dos fármacos , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Osteócitos/citologia , Osteócitos/metabolismo , Osteócitos/efeitos dos fármacos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Proteína Morfogenética Óssea 6/química , Proteína Morfogenética Óssea 6/farmacologia , Proteína Morfogenética Óssea 6/metabolismo , Polietilenoglicóis/química , Diferenciação Celular/efeitos dos fármacos , Engenharia Tecidual/métodos , Hidrogéis/química , Hidrogéis/farmacologia
12.
J Bone Miner Res ; 39(8): 1174-1187, 2024 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-38959852

RESUMO

Intracellular phosphoinositide 3-kinase (PI3K) signaling is activated by multiple bone-active receptors. Genetic mutations activating PI3K signaling are associated with clinical syndromes of tissue overgrowth in multiple organs, often including the skeleton. While one formation is increased by removing the PI3K inhibitor (phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)), the effect of direct PI3K activation in the osteoblast lineage has not been reported. We introduced a known gain-of-function mutation in Pik3ca, the gene encoding the p110α catalytic subunit of PI3K, in osteocytes and late osteoblasts using the dentin matrix protein-1 Cre (Dmp1Cre) mouse and assessed the skeletal phenotype. Femur shape was grossly normal, but cortical thickness was significantly greater in both male and female Dmp1Cre.Pik3caH1047R mice, leading to almost doubled bone strength at 12 wk of age. Both sexes had smaller marrow areas from 6 wk of age. Female mice also exhibited greater cross-sectional area, which continued to increase until 24 wk of age, resulting in a further increase in bone strength. Although both male and female mice had increased endocortical mineralizing surface, only female mice had increased periosteal mineralizing surface. The bone formed in the Dmp1Cre.Pik3caH1047R mice showed no increase in intracortical remodeling nor any defect in cortical bone consolidation. In contrast, on both endocortical and periosteal surfaces, there was more lamellar bone formation, including highly organized osteocyte networks extending along the entire surface at a greater thickness than in control mice. In conclusion, direct activation of PI3Kα in cells targeted by Dmp1Cre leads to high cortical bone mass and strength with abundant lamellar cortical bone in female and male mice with no increase in intracortical remodeling. This differs from the effect of PTEN deletion in the same cells, suggesting that activating PI3Kα in osteoblasts and osteocytes may be a more suitable target to promote formation of lamellar bone.


Patients with genetic activation of enzymes called phosphoinositide-3 kinase (PI3K) have tissue overgrowth syndromes, where parts of the body become enlarged, sometimes including the skeleton. There are 2 types of mutations that cause this: one that directly activates the PI3K enzyme, and one that removes the normal brake on PI3K signaling (called PTEN). We tested the effect of directly activating a PI3K enzyme specifically in osteoblasts (the cells that form bone) and osteocytes (osteoblasts that make a network inside the bone tissue itself). We found that mice with these mutations had very strong bones with an outer shell that was thicker than usual. In both male and female mice, it became thicker on the inside of the shell, but in female mice it also became thicker on the outside, making the bones even stronger over time. The new bone was well-organized, which likely helped make the increase in bone strength so profound. This is very different to previous studies of mice with the other type of mutation in their bone-forming cells; they had a shell with many large holes (pores). This indicates that directly stimulating PI3K enzyme is more beneficial for bone than removing the PTEN brake.


Assuntos
Osso Cortical , Osteoblastos , Osteócitos , Animais , Osteócitos/metabolismo , Feminino , Masculino , Osteoblastos/metabolismo , Camundongos , Osso Cortical/metabolismo , Caracteres Sexuais , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/genética , Ativação Enzimática , Fosfatidilinositol 3-Quinases/metabolismo , Fêmur
13.
Mol Metab ; 88: 102000, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39074536

RESUMO

OBJECTIVE: The skeleton is one of the largest organs in the body, wherein metabolism is integrated with systemic energy metabolism. However, the bioenergetic programming of osteocytes, the most abundant bone cells coordinating bone metabolism, is not well defined. Here, using a mouse model with partial penetration of an osteocyte-specific PPARG deletion, we demonstrate that PPARG controls osteocyte bioenergetics and their contribution to systemic energy metabolism independently of circulating sclerostin levels, which were previously correlated with metabolic status of extramedullary fat depots. METHODS: In vivo and in vitro models of osteocyte-specific PPARG deletion, i.e. Dmp1CrePparγflfl male and female mice (γOTKO) and MLO-Y4 osteocyte-like cells with either siRNA-silenced or CRISPR/Cas9-edited Pparγ. As applicable, the models were analyzed for levels of energy metabolism, glucose metabolism, and metabolic profile of extramedullary adipose tissue, as well as the osteocyte transcriptome, mitochondrial function, bioenergetics, insulin signaling, and oxidative stress. RESULTS: Circulating sclerostin levels of γOTKO male and female mice were not different from control mice. Male γOTKO mice exhibited a high energy phenotype characterized by increased respiration, heat production, locomotion and food intake. This high energy phenotype in males did not correlate with "beiging" of peripheral adipose depots. However, both sexes showed a trend for reduced fat mass and apparent insulin resistance without changes in glucose tolerance, which correlated with decreased osteocytic responsiveness to insulin measured by AKT activation. The transcriptome of osteocytes isolated from γOTKO males suggested profound changes in cellular metabolism, fuel transport, mitochondria dysfunction, insulin signaling and increased oxidative stress. In MLO-Y4 osteocytes, PPARG deficiency correlated with highly active mitochondria, increased ATP production, and accumulation of reactive oxygen species (ROS). CONCLUSIONS: PPARG in male osteocytes acts as a molecular break on mitochondrial function, and protection against oxidative stress and ROS accumulation. It also regulates osteocyte insulin signaling and fuel usage to produce energy. These data provide insight into the connection between osteocyte bioenergetics and their sex-specific contribution to the balance of systemic energy metabolism. These findings support the concept that the skeleton controls systemic energy expenditure via osteocyte metabolism.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Metabolismo Energético , Osteócitos , PPAR gama , Animais , Osteócitos/metabolismo , Camundongos , Masculino , Feminino , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , PPAR gama/metabolismo , PPAR gama/genética , Estresse Oxidativo , Camundongos Knockout , Mitocôndrias/metabolismo , Camundongos Endogâmicos C57BL
14.
Chem Biol Interact ; 399: 111135, 2024 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-38971422

RESUMO

Iron overload is a risk factor for osteoporosis due to its oxidative toxicity. Previous studies have demonstrated that an excessive amount of iron increases osteocyte apoptosis and receptor activator of nuclear factor κ-B ligand (RANKL) production, which stimulates osteoclast differentiation in vitro. However, the effects of exogenous iron supplementation-induced iron overload on osteocytes in vivo and its role in iron overload-induced bone loss are unknown. This work aimed to develop an iron overloaded murine model of C57BL/6 mice by intraperitoneal administration of iron dextran for two months. The iron levels in various organs, bone, and serum, as well as the microstructure and strength of bone, apoptosis of osteocytes, oxidative stress in bone tissue, and bone formation and resorption, were assessed. The results showed that 2 months of exogenous iron supplementation significantly increased iron levels in the liver, spleen, kidney, bone tissue, and serum. Iron overload negatively affected bone microstructure and strength. Osteocyte apoptosis and empty lacunae rate were elevated by exogenous iron. Iron overload upregulated RANKL expression but had no significant impact on osteoprotegerin (OPG) and sclerostin levels. Static and dynamic histologic analyses and serum biochemical assay showed that iron overload increased bone resorption without significantly affecting bone formation. Exogenous iron promoted oxidative stress in osteocytes in vivo and in vitro. Additional supplementation of iron chelator (deferoxamine) or N-acetyl-l-cysteine (NAC) partially alleviated bone loss, osteocyte apoptosis, osteoclast formation, and oxidative stress due to iron overload. These findings, in line with prior in vitro studies, suggest that exogenous iron supplementation induces osteoclastogenesis and osteoporosis by promoting osteocyte apoptosis and RANKL production via oxidative stress.


Assuntos
Apoptose , Reabsorção Óssea , Ferro , Camundongos Endogâmicos C57BL , Osteócitos , Estresse Oxidativo , Ligante RANK , Animais , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Ligante RANK/metabolismo , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Camundongos , Ferro/metabolismo , Modelos Animais de Doenças , Masculino , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Sobrecarga de Ferro/induzido quimicamente , Osteoprotegerina/metabolismo , Acetilcisteína/farmacologia , Proteínas Adaptadoras de Transdução de Sinal
15.
J Struct Biol ; 216(3): 108111, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39059753

RESUMO

Osteocytes are the major actors in bone mechanobiology. Within bone matrix, they are trapped close together in a submicrometric interconnected network: the lacunocanalicular network (LCN). The interstitial fluid circulating within the LCN transmits the mechanical information to the osteocytes that convert it into a biochemical signal. Understanding the interstitial fluid dynamics is necessary to better understand the bone mechanobiology. Due to the submicrometric dimensions of the LCN, making it difficult to experimentally investigate fluid dynamics, numerical models appear as a relevant tool for such investigation. To develop such models, there is a need for geometrical and morphological data on the human LCN. This study aims at providing morphological data on the human LCN from measurement of 27 human femoral diaphysis bone samples using synchrotron radiation nano-computed tomography with an isotropic voxel size of 100 nm. Except from the canalicular diameter, the canalicular morphological parameters presented a high variability within one sample. Some differences in terms of both lacunar and canalicular morphology were observed between the male and female populations. But it has to be highlighted that all the canaliculi cannot be detected with a voxel size of 100 nm. Hence, in the current study, only a specific population of large canaliculi that could be characterize. Still, to the authors knowledge, this is the first time such a data set was introduced to the community. Further processing will be achieved in order to provide new insight on the LCN permeability.


Assuntos
Diáfises , Fêmur , Síncrotrons , Humanos , Fêmur/diagnóstico por imagem , Diáfises/diagnóstico por imagem , Feminino , Masculino , Osteócitos/metabolismo , Imageamento Tridimensional/métodos , Tomografia Computadorizada por Raios X/métodos , Idoso , Pessoa de Meia-Idade
16.
Endocrinology ; 165(8)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-39024412

RESUMO

Osteocytes are embedded in lacunae and connected by canaliculi (lacuno-canalicular network, LCN). Bones from mice with X-linked hypophosphatemia (Hyp), which have impaired production of 1,25 dihydroxyvitamin D (1,25D) and hypophosphatemia, have abnormal LCN structure that is improved by treatment with 1,25D or an anti-FGF23 targeting antibody, supporting roles for 1,25D and phosphate in regulating LCN remodeling. Bones from mice lacking the vitamin D receptor (VDR) in osteocytes (Vdrf/f;Dmp1Cre+) and mice lacking the sodium phosphate transporter 2a (Npt2aKO), which have low serum phosphate with high serum 1,25D, have impaired LCN organization, demonstrating that osteocyte-specific actions of 1,25D and hypophosphatemia regulate LCN remodeling. In osteoclasts, nuclear factor of activated T cells cytoplasmic 1 (NFATc1) is critical for stimulating bone resorption. Since osteocytes also resorb matrix, we hypothesize that NFATc1 plays a role in 1,25D and phosphate-mediated LCN remodeling. Consistent with this, 1,25D and phosphate suppress Nfatc1 mRNA expression in IDG-SW3 osteocytes, and knockdown of Nfatc1 expression in IDG-SW3 cells blocks 1,25D- and phosphate-mediated suppression of matrix resorption gene expression and 1,25D- and phosphate-mediated suppression of RANKL-induced acidification of the osteocyte microenvironment. To determine the role of NFATc1 in 1,25D- and phosphate-mediated LCN remodeling in vivo, histomorphometric analyses of tibiae from mice lacking osteocyte-specific Nfatc1 in Vdrf/f;Dmp1Cre+ and Npt2aKO mice were performed, demonstrating that bones from these mice have decreased lacunar size and expression of matrix resorption genes, and improved canalicular structure compared to Vdrf/f;Dmp1Cre+ and Npt2aKO control. This study demonstrates that NFATc1 is necessary for 1,25D- and phosphate-mediated regulation of LCN remodeling.


Assuntos
Remodelação Óssea , Fator de Crescimento de Fibroblastos 23 , Fatores de Transcrição NFATC , Osteócitos , Fosfatos , Vitamina D , Animais , Masculino , Camundongos , Remodelação Óssea/efeitos dos fármacos , Raquitismo Hipofosfatêmico Familiar/metabolismo , Raquitismo Hipofosfatêmico Familiar/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/metabolismo , Fatores de Transcrição NFATC/genética , Osteócitos/metabolismo , Osteócitos/efeitos dos fármacos , Fosfatos/metabolismo , Receptores de Calcitriol/metabolismo , Receptores de Calcitriol/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismo , Vitamina D/farmacologia , Vitamina D/análogos & derivados , Feminino
17.
Biochem Biophys Res Commun ; 727: 150315, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-38950493

RESUMO

In response to mechanical loading of bone, osteocytes produce nitric oxide (NO•) and decrease sclerostin protein expression, leading to an increase in bone mass. However, it is unclear whether NO• production and sclerostin protein loss are mechanistically linked, and, if so, the nature of their hierarchical relationship within an established mechano-transduction pathway. Prior work showed that following fluid-shear stress (FSS), osteocytes produce NOX2-derived reactive oxygen species, inducing calcium (Ca2+) influx. Increased intracellular Ca2+ results in calcium-calmodulin dependent protein kinase II (CaMKII) activation, which regulates the lysosomal degradation of sclerostin protein. Here, we extend our discoveries, identifying NO• as a regulator of sclerostin degradation downstream of mechano-activated CaMKII. Pharmacological inhibition of nitric oxide synthase (NOS) activity in Ocy454 osteocyte-like cells prevented FSS-induced sclerostin protein loss. Conversely, short-term treatment with a NO• donor in Ocy454 cells or isolated murine long bones was sufficient to induce the rapid decrease in sclerostin protein abundance, independent of changes in Sost gene expression. Ocy454 cells express all three NOS genes, and transfection with siRNAs targeting eNOS/Nos3 was sufficient to prevent FSS-induced loss of sclerostin protein, while siRNAs targeting iNOS/Nos2 mildly blunted the loss of sclerostin but did not reach statistical significance. Similarly, siRNAs targeting both eNOS/Nos3 and iNOS/Nos2 prevented FSS-induced NO• production. Together, these data show iNOS/Nos2 and eNOS/Nos3 are the primary producers of FSS-dependent NO•, and that NO• is necessary and sufficient for sclerostin protein control. Further, selective inhibition of elements within this sclerostin-controlling mechano-transduction pathway indicated that NO• production occurs downstream of CaMKII activation. Targeting Camk2d and Camk2g with siRNA in Ocy454 cells prevented NO• production following FSS, indicating that CaMKII is needed for NO• production. However, NO• donation (1min) resulted in a significant increase in CaMKII activation, suggesting that NO• may have the ability to tune CaMKII response. Together, these data support that CaMKII is necessary for, and may be modulated by NO•, and that the interaction of these two signals is involved in the control of sclerostin protein abundance, consistent with a role in bone anabolic responses.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Óxido Nítrico , Osteócitos , Óxido Nítrico/metabolismo , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Osteócitos/metabolismo , Camundongos , Estresse Mecânico , Camundongos Endogâmicos C57BL , Mecanotransdução Celular , Linhagem Celular , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo
18.
Curr Osteoporos Rep ; 22(4): 396-415, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38980532

RESUMO

PURPOSE OF REVIEW: Quantification of the morphology of osteocyte lacunae has become a powerful tool to investigate bone metabolism, pathologies and aging. This review will provide a brief overview of 2D and 3D imaging methods for the determination of lacunar shape, orientation, density, and volume. Deviations between 2D-based and 3D-based lacunar volume estimations are often not sufficiently addressed and may give rise to contradictory findings. Thus, the systematic error arising from 2D-based estimations of lacunar volume will be discussed, and an alternative calculation proposed. Further, standardized morphological parameters and best practices for sampling and segmentation are suggested. RECENT FINDINGS: We quantified the errors in reported estimation methods of lacunar volume based on 2D cross-sections, which increase with variations in lacunar orientation and histological cutting plane. The estimations of lacunar volume based on common practice in 2D imaging methods resulted in an underestimation of lacunar volume of up to 85% compared to actual lacunar volume in an artificial dataset. For a representative estimation of lacunar size and morphology based on 2D images, at least 400 lacunae should be assessed per sample.


Assuntos
Imageamento Tridimensional , Osteócitos , Humanos , Osteócitos/citologia , Imageamento Tridimensional/métodos , Densidade Óssea , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Processamento de Imagem Assistida por Computador/métodos
19.
Biochem Biophys Res Commun ; 730: 150391, 2024 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-39002199

RESUMO

Glucocorticoid-induced osteoporosis serves as a primary cause for secondary osteoporosis and fragility fractures, representing the most prevalent adverse reaction associated with prolonged glucocorticoid use. In this study, to elucidate the impact and underlying mechanisms of fluid shear stress (FSS)-mediated Piezo1 on dexamethasone (Dex)-induced apoptosis, we respectively applied Dex treatment for 6 h, FSS at 9 dyne/cm2 for 30 min, Yoda1 treatment for 2 h, and Piezo1 siRNA transfection to intervene in MLO-Y4 osteocytes. Western blot analysis was used to assess the expression of Cleaved Caspase-3, Bax, Bcl-2, and proteins associated with the PI3K/Akt pathway. Additionally, qRT-PCR was utilized to quantify the mRNA expression levels of these molecules. Hoechst 33258 staining and flow cytometry were utilized to evaluate the apoptosis levels. The results indicate that FSS at 9 dyne/cm2 for 30 min significantly upregulates Piezo1 in osteocytes. Following Dex-induced apoptosis, the phosphorylation levels of PI3K and Akt are markedly suppressed. FSS-mediated Piezo1 exerts a protective effect against Dex-induced apoptosis by activating the PI3K/Akt pathway. Additionally, downregulating the expression of Piezo1 in osteocytes using siRNA exacerbates Dex-induced apoptosis. To further demonstrate the role of the PI3K/Akt signaling pathway, after intervention with the PI3K pathway inhibitor, the activation of the PI3K/Akt pathway by FSS-mediated Piezo1 in osteocytes was significantly inhibited, reversing the anti-apoptotic effect. This study indicates that under FSS, Piezo1 in MLO-Y4 osteocytes is significantly upregulated, providing protection against Dex-induced apoptosis through the activation of the PI3K/Akt pathway.


Assuntos
Apoptose , Dexametasona , Canais Iônicos , Osteócitos , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Estresse Mecânico , Osteócitos/metabolismo , Osteócitos/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Camundongos , Canais Iônicos/metabolismo , Canais Iônicos/genética , Transdução de Sinais/efeitos dos fármacos , Dexametasona/farmacologia , Linhagem Celular
20.
Connect Tissue Res ; 65(4): 313-329, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38982804

RESUMO

AIM: As osteoblasts deposit a mineralized collagen network, a subpopulation of these cells differentiates into osteocytes. Biochemical and mechanical stimuli, particularly fluid shear stress (FSS), are thought to regulate this, but their relative influence remains unclear. Here, we assess both biochemical and mechanical stimuli on long-term bone formation and osteocytogenesis using the osteoblast-osteocyte cell line IDG-SW3. METHODS: Due to the relative novelty and uncommon culture conditions of IDG-SW3 versus other osteoblast-lineage cell lines, effects of temperature and media formulation on matrix deposition and osteocytogenesis were initially characterized. Subsequently, the relative influence of biochemical (ß-glycerophosphate (ßGP) and ascorbic acid 2-phosphate (AA2P)) and mechanical stimulation on osteocytogenesis was compared, with intermittent application of low magnitude FSS generated by see-saw rocker. RESULTS: ßGP and AA2P supplementation were required for mineralization and osteocytogenesis, with 33°C cultures retaining a more osteoblastic phenotype and 37°C cultures undergoing significantly higher osteocytogenesis. ßGP concentration positively correlated with calcium deposition, whilst AA2P stimulated alkaline phosphatase (ALP) activity and collagen deposition. We demonstrate that increasing ßGP concentration also significantly enhances osteocytogenesis as quantified by the expression of green fluorescent protein linked to Dmp1. Intermittent FSS (~0.06 Pa) rocker had no effect on osteocytogenesis and matrix deposition. CONCLUSIONS: This work demonstrates the suitability and ease with which IDG-SW3 can be utilized in osteocytogenesis studies. IDG-SW3 mineralization was only mediated through biochemical stimuli with no detectable effect of low magnitude FSS. Osteocytogenesis of IDG-SW3 primarily occurred in mineralized areas, further demonstrating the role mineralization of the bone extracellular matrix has in osteocyte differentiation.


Assuntos
Glicerofosfatos , Osteoblastos , Osteócitos , Estresse Mecânico , Glicerofosfatos/farmacologia , Glicerofosfatos/metabolismo , Osteoblastos/metabolismo , Osteoblastos/citologia , Animais , Osteócitos/metabolismo , Osteócitos/citologia , Linhagem Celular , Camundongos , Osteogênese/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Ácido Ascórbico/farmacologia , Ácido Ascórbico/metabolismo , Ácido Ascórbico/análogos & derivados
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