RESUMO
O-glycosylation is a conserved posttranslational modification that impacts many aspects of organismal viability and function. Recent studies examining the glycosyltransferase Galnt11 demonstrated that it glycosylates the endocytic receptor megalin in the kidneys, enabling proper binding and reabsorption of ligands, including vitamin D-binding protein (DBP). Galnt11-deficient mice were unable to properly reabsorb DBP from the urine. Vitamin D plays an essential role in mineral homeostasis and its deficiency is associated with bone diseases such as rickets, osteomalacia, and osteoporosis. We therefore set out to examine the effects of the loss of Galnt11 on vitamin D homeostasis and bone composition. We found significantly decreased levels of serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, consistent with decreased reabsorption of DBP. This was accompanied by a significant reduction in blood calcium levels and a physiologic increase in parathyroid hormone (PTH) in Galnt11-deficient mice. Bones in Galnt11-deficient mice were smaller and displayed a decrease in cortical bone accompanied by an increase in trabecular bone and an increase in a marker of bone formation, consistent with PTH-mediated effects on bone. These results support a unified model for the role of Galnt11 in bone and mineral homeostasis, wherein loss of Galnt11 leads to decreased reabsorption of DBP by megalin, resulting in a cascade of disrupted mineral and bone homeostasis including decreased circulating vitamin D and calcium levels, a physiological increase in PTH, an overall loss of cortical bone, and an increase in trabecular bone. Our study elucidates how defects in O-glycosylation can influence vitamin D and mineral homeostasis and the integrity of the skeletal system.
Assuntos
Osso e Ossos , Homeostase , Polipeptídeo N-Acetilgalactosaminiltransferase , Vitamina D , Animais , Masculino , Camundongos , Osso e Ossos/anatomia & histologia , Osso e Ossos/química , Osso e Ossos/metabolismo , Cálcio/metabolismo , Glicosilação , Homeostase/genética , Hormônio Paratireóideo/metabolismo , Vitamina D/metabolismo , Vitamina D/análogos & derivados , Proteína de Ligação a Vitamina D/metabolismoRESUMO
Many studies have been conducted to elucidate the role of Type VI collagen in muscle and tendon, however, its role in oral tissues remains unclear. In this study, an α2(VI) deficient mouse (Col6α2-KO) model was used to examine the role of Type VI collagen in oral tissues. Tissue volume and mineral density were measured in oral tissues by µCT. Proteome analysis was performed using protein extracted from alveolar bone. In addition, alveolar bone was evaluated with a periodontitis induced model. µCT analysis showed the Col6α2-KO mice had less volume of alveolar bone, dentin and dental pulp, while the width of periodontal ligament (PDL) was greater than WT. The mineral density in alveolar bone and dentin were elevated in Col6α2-KO mice compared with WT. Our proteome analysis showed significant changes in proteins related to ECM organization and elevation of proteins associated with biomineralization in the Col6α2-KO mice. In induced periodontitis, Col6α2-KO mice had greater alveolar bone loss compared with WT. In conclusion, Type VI collagen has a role in controlling biomineralization in alveolar bone and that changes in the ECM of alveolar bone could be associated with greater bone loss due to periodontitis.
Assuntos
Perda do Osso Alveolar , Periodontite , Camundongos , Animais , Colágeno Tipo VI/genética , Proteoma , Camundongos Knockout , Perda do Osso Alveolar/metabolismoRESUMO
Biglycan (Bgn) and Fibromodulin (Fmod) are small leucine rich proteoglycans (SLRPs) which are abundant in the extra-cellular matrix (ECM) of mineralized tissues. We have previously generated a Bgn/Fmod double knock-out (DKO) mouse model and found it has a 3-fold increase in osteoclastogenesis compared with Wild type (WT) controls, resulting in a markedly low bone mass (LBM) phenotype. To try and rescue/repair the LBM phenotype of Bgn/Fmod DKO mice by suppressing osteoclast formation and activity, 3- and 26-week-old Bgn/Fmod DKO mice and age/gender matched WT controls were treated with OPG-Fc for 6 weeks after which bone parameters were evaluated using DEXA, micro-computed tomography (µCT) and serum biomarkers analyses. In the appendicular skeleton, OPG-Fc treatment improved some morphometric and geometric parameters in both the trabecular and cortical compartments in Bgn/Fmod DKO female and male mice, especially in the repair module. For many of the skeletal parameters analyzed, the Bgn/Fmod DKO mice were more responsive to the treatment than their WT controls. In addition, we found that OPG-Fc treatment was not able to prevent or ameliorate the formation of ectopic ossification, which are common lesions seen in aged joints and are one of the phenotypical hallmarks of our Bgn/Fmod DKO model. Analysis of skull bones, specifically the occipital bone, showed the treatment recovered some parameters of LBM phenotype in the craniofacial skeleton, more so in the younger rescue module. Using OPG-Fc as treatment alleviated, yet did not completely restore, the severe osteopenia and mineralized tissue structural abnormalities that Bgn/Fmod DKO mice suffer from.
Assuntos
Biglicano/deficiência , Osso e Ossos/efeitos dos fármacos , Fibromodulina/deficiência , Fragmentos Fc das Imunoglobulinas/farmacologia , Osteoprotegerina/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Esqueleto/efeitos dos fármacos , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Osso e Ossos/metabolismo , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Fenótipo , Esqueleto/metabolismoRESUMO
Temporomandibular joint (TMJ) osteoarthritis (OA) is a complex disease that affects both cartilage and subchondral bone. It is accompanied by loss of extracellular matrix (ECM) and may be controlled by bone morphogenetic protein-2 (BMP-2). We analyzed the effect of BMP-2 in both cartilage and subchondral bone in a TMJ-OA animal model that is deficient in biglycan (Bgn) and fibromodulin (Fmod) (Bgn-/-Fmod-/-). Whole mandibles were dissected from 3-week-old wild-type (WT) and Bgn-/-Fmod-/- mice and incubated with and without 250 µg/mL BMP-2 for 2 days using an explant culture system. Condyle growth was measured by microCT and the expression levels of cartilage and bone-related genes were analyzed using RT-PCR or by immunohistochemistry from condyles that contained an intact cartilage/subchondral bone interface. Osteoclast activity was estimated by tartrate-resistant acid phosphatase (TRAP) staining and by TRAP, Rankl, and Adamts4 mRNA expression levels. Our results showed that most parameters examined were slightly up-regulated in WT samples treated with BMP-2, and this up-regulation was significantly enhanced in the Bgn-/-Fmod-/- mice. The up-regulation of both catabolic and anabolic agents did not appear to positively affect the overall growth of Bgn-/-Fmod-/- condyles compared to WT controls. In summary, the up-regulation of both anabolic and catabolic genes in the WT and Bgn-/-Fmod-/- TMJs treated with BMP-2 suggests that BMP increases matrix turnover in the condyle, and, further, that Bgn and Fmod could have protective roles in regulating this process.
Assuntos
Biglicano/metabolismo , Proteína Morfogenética Óssea 2/genética , Matriz Extracelular/metabolismo , Osteoartrite/genética , Articulação Temporomandibular/patologia , Animais , Proteína Morfogenética Óssea 2/metabolismo , Modelos Animais de Doenças , Feminino , Fibromodulina , Humanos , Camundongos , Camundongos Knockout , Osteoartrite/metabolismoRESUMO
WISP1/CCN4 (hereafter referred to as WISP1), a member of the CCN family, is found in mineralized tissues and is produced by osteoblasts and their precursors. In this study, Wisp1-deficient (Wisp1(-/-)) mice were generated. Using dual-energy x-ray absorptiometry, we showed that by 3 months, the total bone mineral density of Wisp1(-/-) mice was significantly lower than that of WT mice. Further investigation by micro-computed tomography showed that female Wisp1(-/-) mice had decreased trabecular bone volume/total volume and that both male and female Wisp1(-/-) mice had decreased cortical bone thickness accompanied by diminished biomechanical strength. The molecular basis for decreased bone mass in Wisp1(-/-) mice arises from reduced bone formation likely caused by osteogenic progenitors that differentiate poorly compared with WT cells. Osteoclast precursors from Wisp1(-/-) mice developed more tartrate-resistant acid phosphatase-positive cells in vitro and in transplants, suggesting that WISP1 is also a negative regulator of osteoclast differentiation. When bone turnover (formation and resorption) was induced by ovariectomy, Wisp1(-/-) mice had lower bone mineral density compared WT mice, confirming the potential for multiple roles for WISP1 in controlling bone homeostasis. Wisp1(-/-) bone marrow stromal cells had reduced expression of ß-catenin and its target genes, potentially caused by WISP1 inhibition of SOST binding to LRP6. Taken together, our data suggest that the decreased bone mass found in Wisp1(-/-) mice could potentially be caused by an insufficiency in the osteodifferentiation capacity of bone marrow stromal cells arising from diminished Wnt signaling, ultimately leading to altered bone turnover and weaker biomechanically compromised bones.
Assuntos
Remodelação Óssea , Osso e Ossos/metabolismo , Proteínas de Sinalização Intercelular CCN/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Via de Sinalização Wnt , Alelos , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Feminino , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/metabolismo , Osteoclastos/metabolismo , RNA Mensageiro/metabolismo , Receptores de LDL/metabolismo , Recombinação Genética , Células Estromais/citologia , Proteínas Supressoras de Tumor/metabolismo , Microtomografia por Raio-XRESUMO
The repair of injured tendons remains a great challenge, largely owing to a lack of in-depth characterization of tendon cells and their precursors. We show that human and mouse tendons harbor a unique cell population, termed tendon stem/progenitor cells (TSPCs), that has universal stem cell characteristics such as clonogenicity, multipotency and self-renewal capacity. The isolated TSPCs could regenerate tendon-like tissues after extended expansion in vitro and transplantation in vivo. Moreover, we show that TSPCs reside within a unique niche predominantly comprised of an extracellular matrix, and we identify biglycan (Bgn) and fibromodulin (Fmod) as two critical components that organize this niche. Depletion of Bgn and Fmod affects the differentiation of TSPCs by modulating bone morphogenetic protein signaling and impairs tendon formation in vivo. Our results, while offering new insights into the biology of tendon cells, may assist in future strategies to treat tendon diseases.
Assuntos
Matriz Extracelular/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Tendões/citologia , Adipogenia , Animais , Biglicano , Diferenciação Celular , Separação Celular/métodos , Células Cultivadas , Criança , Condrogênese , Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibromodulina , Genes Reporter , Histocitoquímica , Humanos , Imuno-Histoquímica , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Osteogênese , Proteoglicanas/metabolismo , Transplante de Células-Tronco , Tendões/cirurgia , Transplante HomólogoRESUMO
Although extracellular control of canonical Wnt signaling is crucial for tissue homeostasis, the role of the extracellular microenvironment in modulating this signaling pathway is largely unknown. In the present study, we show that a member of the small leucine-rich proteoglycan family, biglycan, enhances canonical Wnt signaling by mediating Wnt function via its core protein. Immunoprecipitation analysis revealed that biglycan interacts with both the canonical Wnt ligand Wnt3a and the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6), possibly via the formation of a trimeric complex. Biglycan-deficient cells treated with exogenous Wnt3a had less Wnt3a retained in cell layers compared with WT cells. Furthermore, the Wnt-induced levels of LRP6 phosphorylation and expression of several Wnt target genes were blunted in biglycan-deficient cells. Both recombinant biglycan proteoglycan and biglycan core protein increased Wnt-induced ß-catenin/T cell-specific factor-mediated transcriptional activity, and this activity was completely inhibited by Dickkopf 1. Interestingly, recombinant biglycan was able to rescue impaired Wnt signaling caused by a previously described missense mutation in the extracellular domain of human LRP6 (R611C). Furthermore, biglycan's modulation of canonical Wnt signaling affected the functional activities of osteoprogenitor cells, including the RUNX2-mediated transcriptional activity and calcium deposition. Use of a transplant system and a fracture healing model revealed that expression of Wnt-induced secreted protein 1 was decreased in bone formed by biglycan-deficient cells, further suggesting reduced Wnt signaling in vivo. We propose that biglycan may serve as a reservoir for Wnt in the pericellular space and modulate Wnt availability for activation of the canonical Wnt pathway.
Assuntos
Biglicano/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Biglicano/deficiência , Biglicano/genética , Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Crânio/metabolismo , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo , beta Catenina/metabolismoRESUMO
Endochondral bone development and regeneration relies on activation and proliferation of periosteum derived-cells (PDCs). Biglycan (Bgn), a small proteoglycan found in extracellular matrix, is known to be expressed in bone and cartilage, however little is known about its influence during bone development. Here we link biglycan with osteoblast maturation starting during embryonic development that later affects bone integrity and strength. Biglycan gene deletion reduced the inflammatory response after fracture, leading to impaired periosteal expansion and callus formation. Using a novel 3D scaffold with PDCs, we found that biglycan could be important for the cartilage phase preceding bone formation. The absence of biglycan led to accelerated bone development with high levels of osteopontin, which appeared to be detrimental to the structural integrity of the bone. Collectively, our study identifies biglycan as an influencing factor in PDCs activation during bone development and bone regeneration after fracture.
RESUMO
For many years there has been a keen interest in developing regenerative treatment for temporomandibular joint-osteoarthritis (TMJ-OA). Currently, there is no consensus treatment due to the limited self-healing ability of articular cartilage and lack of understanding of the complex mechanisms regulating cartilage development in the TMJ. Endochondral ossification, the process of subchondral bone formation through chondrocyte differentiation, is critical for TMJ growth and development, and is tightly regulated by the composition of the extracellular matrix (ECM). Type VI collagen is a highly expressed ECM component in the TMJ cartilage, yet its specific functions are largely unknown. In this study, we investigated α2(VI)-deficient (Col6a2-knockout [KO]) mice, which are unable to secret or incorporate type VI collagen into their ECM. Compared with wild-type (WT) mice, the TMJ condyles of Col6a2-KO mice exhibit decreased bone volume/tissue volume (BV/TV) and a larger bone marrow space, suggesting the α2(VI)-deficient condyles have a failure in endochondral ossification. Differentiating chondrocytes are the main source of bone cells during endochondral ossification. Our study shows there is an increased number of chondrocytes in the proliferative zone and decreased Col10-expressing chondrocytes in Col6a2-KO cartilage, all pointing to abnormal chondrocyte differentiation and maturation. In addition, RNA sequencing (RNAseq) analysis identified distinct gene expression profiles related to cell cycle and ECM organization that were altered in the mutant condyles. These data also suggest that bone morphogenetic protein 2 (BMP2) activity was deregulated during chondrocyte differentiation. Immunohistochemical analysis indicated an upregulation of Col2 and Acan expression in Col6a2-KO cartilage. Moreover, the expression of pSmad1/5/8 and Runx2 was decreased in the Col6a2-KO cartilage compared with WT controls. Taken together, our data indicate that type VI collagen expressed in the TMJ cartilage is important for endochondral ossification, possibly by modulating the ECM and altering/disrupting signaling pathways important for TMJ chondrocyte differentiation. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
RESUMO
Methylmalonic acidemia (MMA) is a severe and potentially lethal autosomal recessive inborn error of metabolism most frequently caused by mutations in the methylmalonyl-CoA mutase (MMUT) gene. Proof-of-concept adeno-associated virus (AAV) gene therapy studies using mouse models of MMA have demonstrated promise for this therapeutic approach but translation to the clinic could be limited by preexisting capsid immunity and vector potency. Here we explore the efficacy of a novel clade E capsid, 44.9, as a serotype for systemic AAV gene therapy for MMA. An anti-AAV44.9 neutralizing antibody (NAb) survey in adult volunteers (n = 19) and a large cohort of MMA patients (n = 48) revealed a seroprevalence rate of â¼26% and 13%, respectively. The efficacy of AAV44.9 gene delivery was examined in two murine models of MMA, representing neonatal lethal and juvenile phenotypes of MMA. Systemic delivery of the AAV44.9-Mmut vector prevented lethality and lowered disease-related metabolites in MMA mice. Tissue biodistribution and transgene expression studies in treated MMA mice showed that AAV44.9 was efficient at transducing the liver and heart. In summary, we establish that AAV44.9 exhibits a low prevalence of preexisting NAb in humans, is highly efficacious in the treatment of clinically severe MMA mouse models and is therefore a promising vector for clinical translation.
RESUMO
Tendon is a vital musculoskeletal tissue that is prone to degeneration. Proper tendon maintenance requires complex interactions between extracellular matrix components that remain poorly understood. Collagen VI and biglycan are two matrix molecules that localize pericellularly within tendon and are critical regulators of tissue properties. While evidence suggests that collagen VI and biglycan interact within the tendon matrix, the relationship between the two molecules and its impact on tendon function remains unknown. We sought to elucidate potential coordinate roles of collagen VI and biglycan within tendon by defining tendon properties in knockout models of collagen VI, biglycan, or both molecules. We first demonstrated co-expression and co-localization of collagen VI and biglycan within the healing tendon, providing further evidence of cooperation between the two molecules during nascent tendon matrix formation. Deficiency in collagen VI and/or biglycan led to significant reductions in collagen fibril size and tendon mechanical properties. However, collagen VI-null tendons displayed larger reductions in fibril size and mechanics than seen in biglycan-null tendons. Interestingly, knockout of both molecules resulted in similar properties to collagen VI knockout alone. These results indicate distinct and non-additive roles for collagen VI and biglycan within tendon. This work provides better understanding of regulatory interactions between two critical tendon matrix molecules.
RESUMO
The temporomandibular joint is critical for jaw movements and allows for mastication, digestion of food, and speech. Temporomandibular joint osteoarthritis is a degenerative disease that is marked by permanent cartilage destruction and loss of extracellular matrix (ECM). To understand how the ECM regulates mandibular condylar chondrocyte (MCC) differentiation and function, we used a genetic mouse model of temporomandibular joint osteoarthritis that is deficient in two ECM proteins, biglycan and fibromodulin (Bgn(-/0)Fmod(-/-)). Given the unavailability of cell lines, we first isolated primary MCCs and found that they were phenotypically unique from hyaline articular chondrocytes isolated from the knee joint. Using Bgn(-/0) Fmod(-/-) MCCs, we discovered the early basis for temporomandibular joint osteoarthritis arises from abnormal and accelerated chondrogenesis. Transforming growth factor (TGF)-beta1 is a growth factor that is critical for chondrogenesis and binds to both biglycan and fibromodulin. Our studies revealed the sequestration of TGF-beta1 was decreased within the ECM of Bgn(-/0) Fmod(-/-) MCCs, leading to overactive TGF-beta1 signal transduction. Using an explant culture system, we found that overactive TGF-beta1 signals induced chondrogenesis and ECM turnover in this model. We demonstrated for the first time a comprehensive study revealing the importance of the ECM in maintaining the mandibular condylar cartilage integrity and identified biglycan and fibromodulin as novel key players in regulating chondrogenesis and ECM turnover during temoporomandibular joint osteoarthritis pathology.
Assuntos
Condrogênese/genética , Proteínas da Matriz Extracelular/fisiologia , Matriz Extracelular/metabolismo , Osteoartrite/genética , Proteoglicanas/fisiologia , Transtornos da Articulação Temporomandibular/genética , Articulação Temporomandibular/patologia , Animais , Biglicano , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Matriz Extracelular/genética , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibromodulina , Masculino , Côndilo Mandibular/metabolismo , Côndilo Mandibular/patologia , Camundongos , Camundongos Knockout , Osteoartrite/metabolismo , Osteoartrite/patologia , Proteoglicanas/genética , Proteoglicanas/metabolismo , Articulação Temporomandibular/metabolismo , Transtornos da Articulação Temporomandibular/metabolismo , Transtornos da Articulação Temporomandibular/patologiaRESUMO
Fibromodulin (Fmod) is a keratan sulfate small leucine-rich proteoglycan which is enriched in bones and teeth. In order to determine its functions on bone and tooth mineralization we characterized the phenotype of Fmod-deficient (Fmod-KO) mice using a new-generation microfocus computerized tomography system (micro-CT) and software allowing advanced visualization of 3-D data. Three-week-old and 10- week-old Fmod-KO mandibles and teeth were compared with those of age-matched wild-type (WT) mice. In both young and mature mice the Fmod-KO mandibles were hypomineralized, especially the posterior (proximal) part of the mandible as it appeared to be the main target of the molecule deficiency whereas less extensive alterations were found in the alveolar bone. In transverse sections, larger marrow spaces were observed in the Fmod-KO mice compared with age-matched young or mature WT mice. Quantitative evaluation of the pulp volume of the first molar and 3-D reconstructions suggested that dentinogenesis was diminished in 3-week-old Fmod-KO teeth. In contrast, increased dentin formation was found in 10-week-old Fmod-KO mice and it was accompanied by a reduced pulp volume. Thus, the differential effects of Fmod deficiency on bones and teeth appear to diverge in adult mice. This may result from the previously reported differences in the molecular weight of Fmod in the 2 tissues or from compensatory mechanisms due to the overexpression of DSP and DMP-1 in the dental pulp of Fmod-KO. It is also possible that a single molecule plays diverging roles in a tissue-specific or region-specific manner.
Assuntos
Proteínas da Matriz Extracelular/deficiência , Mandíbula/diagnóstico por imagem , Proteoglicanas/deficiência , Dente/diagnóstico por imagem , Microtomografia por Raio-X , Animais , Polpa Dentária/diagnóstico por imagem , Polpa Dentária/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibromodulina , Processamento de Imagem Assistida por Computador , Mandíbula/metabolismo , Camundongos , Camundongos Knockout , Dente Molar/diagnóstico por imagem , Proteoglicanas/metabolismo , Fatores de Tempo , Dente/metabolismoRESUMO
Type VI collagen is well known for its role in muscular disorders, however its function in bone is still not well understood. To examine its role in bone we analyzed femoral and vertebral bone mass by micro-computed tomography analysis, which showed lower bone volume/total volume and trabecular number in Col6α2-KO mice compared with WT. Dynamic histomorphometry showed no differences in trabecular bone formation between WT and Col6α2-KO mice based on the mineral appositional rate, bone formation rate, and mineralizing perimeter. Femoral sections were assessed for the abundance of Tartrate Resistant Acid Phosphatase-positive osteoclasts, which revealed that mutant mice had more osteoclasts compared with WT mice, indicating that the primary effect of Col6a2 deficiency is on osteoclastogenesis. When bone marrow stromal cells (BMSCs) from WT and Col6α2-KO mice were treated with rmTNFα protein, the Col6α2-KO cells expressed higher levels of TNFα mRNA compared with WT cells. This was accompanied by higher levels of p-p65, a down-stream target of TNFα, suggesting that BMSCs from Col6α2-KO mice are highly sensitive to TNFα signaling. Taken together, our data imply that Col6a2 deficiency causes trabecular bone loss by enhancing osteoclast differentiation through enhanced TNFα signaling.
Assuntos
Osso Esponjoso/crescimento & desenvolvimento , Osso Esponjoso/patologia , Colágeno Tipo VI/genética , Osteogênese/genética , Fator de Necrose Tumoral alfa/metabolismo , Animais , Densidade Óssea/genética , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Linhagem Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoclastos/citologia , Osteogênese/fisiologia , Células RAW 264.7 , Transdução de Sinais , Células Estromais/metabolismo , Fator de Transcrição RelA/metabolismo , Microtomografia por Raio-XRESUMO
Anti-angiogenic therapies have generated significant interest for their potential to combat tumor growth. However, tumor overproduction of pro-angiogenic ligands can overcome these therapies, hampering success of this approach. To circumvent this problem, we target the resynthesis of phosphoinositides consumed during intracellular transduction of pro-angiogenic signals in endothelial cells (EC), thus harnessing the tumor's own production of excess stimulatory ligands to deplete adjacent ECs of the capacity to respond to these signals. Using zebrafish and human endothelial cells in vitro, we show ECs deficient in CDP-diacylglycerol synthase 2 are uniquely sensitive to increased vascular endothelial growth factor (VEGF) stimulation due to a reduced capacity to re-synthesize phosphoinositides, including phosphatidylinositol-(4,5)-bisphosphate (PIP2), resulting in VEGF-exacerbated defects in angiogenesis and angiogenic signaling. Using murine tumor allograft models, we show that systemic or EC specific suppression of phosphoinositide recycling results in reduced tumor growth and tumor angiogenesis. Our results suggest inhibition of phosphoinositide recycling provides a useful anti-angiogenic approach.
Assuntos
Inibidores da Angiogênese/farmacologia , Endotélio Vascular/metabolismo , Fosfatidilinositóis/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismo , Aloenxertos/efeitos dos fármacos , Animais , Bovinos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Diacilglicerol Colinofosfotransferase/deficiência , Diacilglicerol Colinofosfotransferase/metabolismo , Endotélio Vascular/efeitos dos fármacos , Deleção de Genes , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos Knockout , Modelos Biológicos , Neovascularização Fisiológica/efeitos dos fármacos , Especificidade de Órgãos , Transdução de Sinais , Peixe-ZebraRESUMO
The extracellular matrix of newborn, 7- and 21-day-old fibromodulin-deficient (Fmod KO) mice was compared with age-matched wild-type (WT) mice. Western blotting of proteins from 21-day-old WT mice revealed that the molecular weight of Fmod is smaller in dental tissues (approx. 40 kDa) compared to alveolar bone extracts (approx. 52 kDa). Dentin matrix protein1 (DMP1) was slightly increased in Fmod KO versus WT tooth extracts. After chondroitinase ABC digestion, dentin sialophosphoprotein (DSPP) appeared as 2 strong bands (approx. 150 and 70 kDa) in incisors from 21-day-old Fmod KO mice, whereas the smaller-sized species of DSPP was nearly absent in WT molars and no difference was detected between WT and KO mice in molars. Dentin mineralization was altered in newborn and 7-day-old KO mice, but seemed normal in 21-day-old KO mice. DMP1 and DSPP may be involved in compensatory mechanisms. The enamel had a twisted appearance and looked porous at day 21 in KO incisor, and the outer aprismatic layer was missing in the molar. Alveolar bone formation was enhanced in Fmod KO mice at days 0 and 7, whereas no difference was detected at day 21. We conclude that Fmod may control dental tissue formation and early maturation, where it acts mostly as an inhibitor in alveolar bone accumulation, excerpting its effects only at early developing stages. These dual functions may be related to the different forms of Fmod found in bone versus teeth.
Assuntos
Proteínas da Matriz Extracelular/deficiência , Osteogênese/fisiologia , Proteoglicanas/deficiência , Dente/metabolismo , Animais , Western Blotting , Esmalte Dentário/citologia , Esmalte Dentário/metabolismo , Dentina/citologia , Dentina/ultraestrutura , Proteínas da Matriz Extracelular/metabolismo , Fibromodulina , Camundongos , Proteoglicanas/metabolismo , Dente/citologiaRESUMO
Understanding the mechanisms that control cutaneous wound healing is crucial to successfully manage repair of damaged skin. The goal of the current study was to uncover novel extracellular matrix (ECM) components that control the wound healing process. Full thickness skin defects were created in mice and used to show CCN4 up-regulation during wound-healing as early as 1â¯day after surgery, suggesting a role in inflammation and subsequent dermal migration and proliferation. To determine how CCN4 could regulate wound healing we used Ccn4-KO mice and showed they had delayed wound closure accompanied by reduced expression of Col1a1 and Fn mRNA. Boyden chamber assays using Ccn4-deficient dermal fibroblasts showed they have reduced migration and proliferation compared to WT counterparts. To confirm CCN4 has a role in proliferation and migration of dermal cells, siRNA knockdown and transduction of CCN4 adenoviral transduction were used and resulted in reduced or enhanced migration of human adult dermal fibroblast (hADF) cells respectively. The induced migration of the dermal fibroblasts by CCN4 appears to work via α5ß1 integrin receptors that further stimulates down-stream ERK/JNK signaling. The regulation of CCN4 by TNF-α prompted us look further at their potential relationship. Treatment of hADFs with CCN4 and TNF-α alone or together showed CCN4 counteracted the inhibition of TNF-α on COL1A1 and FN mRNA expression and the stimulation of TNF-α on MMP-1 and MMP3 mRNA expression. CCN4 appeared to counterbalance the effects of TNF-α by inhibiting downstream NF-κB/p-65 signaling. Taken together we show CCN4 stimulates dermal fibroblast cell migration, proliferation and inhibits TNF-α stimulation, all of which could regulate wound healing.
Assuntos
Proteínas de Sinalização Intercelular CCN/genética , Derme/citologia , Integrina alfa5beta1/metabolismo , Proteínas Proto-Oncogênicas/genética , Fator de Necrose Tumoral alfa/metabolismo , Cicatrização , Animais , Proteínas de Sinalização Intercelular CCN/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Derme/metabolismo , Modelos Animais de Doenças , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Humanos , Camundongos , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Biglycan (Bgn) and decorin (Dcn) are highly expressed in numerous tissues in the craniofacial complex. However, their expression and function in the cranial sutures are unknown. In order to study this, we first examined the expression of biglycan and decorin in the posterior frontal suture (PFS), which predictably fuses between 21 and 45 days post-natal and in the non-fusing sagittal (S) suture from wild-type (Wt) mice. Our data showed that Bgn and Dcn were expressed in both cranial sutures. We then characterized the cranial suture phenotype in Bgn deficient, Dcn deficient, Bgn/Dcn double deficient, and Wt mice. At embryonic day 18.5, alizarin red/alcian blue staining showed that the Bgn/Dcn double deficient mice had hypomineralization of the frontal and parietal craniofacial bones. Histological analysis of adult mice (45-60 days post-natal) showed that the Bgn or Dcn deficient mice had no cranial suture abnormalities and immunohistochemistry staining showed increased production of Dcn in the PFS from Bgn deficient mice. To test possible compensation of Dcn in the Bgn deficient sutures, we examined the Bgn/Dcn double deficient mice and found that they had impaired fusion of the PFS. Semi-quantitative RT-PCR analysis of RNA from 35 day-old mice revealed increased expression of Bmp-4 and Dlx-5 in the PFS compared to their non-fusing S suture in Wt tissues and decreased expression of Dlx-5 in both PF and S sutures in the Bgn/Dcn double deficient mice compared to the Wt mice. Failure of PFS fusion and hypomineralization of the calvaria in the Bgn/Dcn double deficient mice demonstrates that these extracellular matrix proteoglycans could have a role in controlling the formation and growth of the cranial vault.
Assuntos
Suturas Cranianas/anormalidades , Proteínas da Matriz Extracelular/deficiência , Proteoglicanas/deficiência , Azul Alciano , Animais , Antraquinonas , Biglicano , Suturas Cranianas/embriologia , Suturas Cranianas/crescimento & desenvolvimento , Suturas Cranianas/metabolismo , Decorina , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Gravidez , Proteoglicanas/genética , Proteoglicanas/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Coloração e RotulagemRESUMO
Biglycan (Bgn) and Fibromodulin (Fmod) are subtypes of the small leucine-rich family of proteoglycans (SLRP). In this study we examined the skeletal phenotype of BgnFmod double knockout (BgnFmod KO) mice and found they were smaller in size and have markedly reduced bone mass compared to WT. The low bone mass (LBM) phenotype is the result of both the osteoblasts and osteoclasts from BgnFmod KO mice having higher differentiation potential and being more active compared to WT mice. Using multiple approaches, we showed that both Bgn and Fmod directly bind TNFα as well as RANKL in a dose dependent manner and that despite expressing higher levels of both TNFα and RANKL, BgnFmod KO derived osteoblasts cannot retain these cytokines in the vicinity of the cells, which leads to elevated TNFα and RANKL signaling and enhanced osteoclastogenesis. Furthermore, adding either Bgn or Fmod to osteoclast precursor cultures significantly attenuated the cells ability to form TRAP positive, multinucleated giant cells. In summary, our data indicates that Bgn and Fmod expressed by the bone forming cells, are novel coupling ECM components that control bone mass through sequestration of TNFα and/or RANKL, thereby adjusting their bioavailability in order to regulate osteoclastogenesis.
Assuntos
Biglicano/genética , Fibromodulina/genética , Osteogênese/genética , Ligante RANK/genética , Proteoglicanos Pequenos Ricos em Leucina/genética , Fator de Necrose Tumoral alfa/genética , Animais , Densidade Óssea/genética , Osso e Ossos/metabolismo , Diferenciação Celular/genética , Humanos , Camundongos , Camundongos Knockout , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismoRESUMO
Bone mass is maintained by a fine balance between bone formation by osteoblasts and bone resorption by osteoclasts. Although osteoblasts and osteoclasts have different developmental origins, it is generally believed that the differentiation, function, and survival of osteoclasts are regulated by osteogenic cells. We have previously shown that the extracellular matrix protein, biglycan (Bgn), plays an important role in the differentiation of osteoblast precursors. In this paper, we showed that Bgn is involved in regulating osteoclast differentiation through its effect on osteoblasts and their precursors using both in vivo and in vitro experiments. The in vivo osteolysis experiment showed that LPS (lipopolisaccharide)-induced osteolysis occurred more rapidly and extensively in bgn deficient mice compared to wild type (WT) mice. To further understand the mechanism of action, we determined the effects of Bgn on 1alpha, 25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3))-induced osteoclast differentiation and bone resorption in an co-culture of calvariae-derived pre-osteoblasts and osteoclast precursors derived from spleen or bone marrow. Time course and dose response experiments showed that tartrate-resistant acid phosphatase-positive multinuclear cells appeared earlier and more extensively in the co-cultures containing calvarial cells from bgn deficient mice than WT mice, regardless of the genotype of osteoclast precursors. The osteoblast abnormality that stimulated osteoclast formation appeared to be independent of the differential production of soluble RANKL and OPG and, instead, due to a decrease in osteoblast maturation accompanied by increase in osteoblastic proliferation. In addition to the imbalance between differentiation and proliferation, there was a differential decrease in secretory leukocyte protease inhibitor (slpi) in bgn deficient osteoblasts treated with 1,25-(OH)(2)D(3). These findings point to a novel molecular factor made by osteoblasts that could potentially be involved in LPS-induced osteolysis.