RESUMEN

BACKGROUND: mir-RNAs play a role in regulating bone homeostasis. In this study we assessed the functional role of mir-RNA 150 in bone homeostasis. We also assess the effects of miR-150 deficiency on osteoblast and osteoclast differentiation and function using in vivo and in vitro approaches. METHODS: Wild type (WT) (C57BL/6J) and miR-150 KO mice were compared for a variety of parameters. Micro-CT imaging was conducted to quantify trabecular bone mass inferior to the distal growth plate of the femur. Von Kossa staining was performed for osteoblast culture mineralization. RT-qPCR, biochemical analysis and bone histomorphometry were utilized for quantification of relevant genes and serum protein measurements. Differentiation and function of osteoblasts and osteoclasts was performed using primarily cultures and assessed the cell autonomous response of mir-RNA-150 on cell differentiation and function. RESULTS: Mir-150 exhibited expression in a variety of tissues and increases progressively with age. Through micro-CT imaging, we found that KO mice presented reduced bone mass at 4, 8, and 16 weeks of age compared to WT mice. Furthermore, histomorphometric analysis revealed increased trabecular separation, decreased bone thickness, and decreased osteoblast number in KO compared to WT mice. Mir-150 deficiency also correlated with higher bone resorption, accompanied with significant increases in CTX-1 serum levels, and a decrease in cell apoptotic rate ex vivo. Additionally, miR-150 KO mice showed increased osteoblast differentiation and decreased osteoclastogenesis ex vivo. Luciferase assay showed increased Osteoactivin/GPNMB expression in miR-150 KO osteoblasts compared to WT cells. CONCLUSION: Our data suggests that miR-150 influences osteoblast and osteoclast functionality and differentiation; specifically, miR-150 serves as a negative regulator for osteoblasts and a positive regulator for osteoclasts by regulating, at least in part, Osteoactivin/GPNMB expression.

Asunto(s)

Resorción Ósea , MicroARNs , Animales , Resorción Ósea/genética , Diferenciación Celular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , Osteoblastos , Osteoclastos , Osteocitos , Osteogénesis/genética

RESUMEN

Osteoactivin is a heavily glycosylated protein shown to have a role in bone remodeling. Previous studies from our lab have shown that mutation in Osteoactivin enhances osteoclast differentiation but inhibits their function. To date, a classical receptor and a signaling pathway for Osteoactivin-mediated osteoclast inhibition has not yet been characterized. In this study, we examined the role of Osteoactivin treatment on osteoclastogenesis using bone marrow-derived osteoclast progenitor cells and identify a signaling pathway relating to Osteoactivin function. We reveal that recombinant Osteoactivin treatment inhibited osteoclast differentiation in a dose-dependent manner shown by qPCR, TRAP staining, activity and count. Using several approaches, we show that Osteoactivin binds CD44 in osteoclasts. Furthermore, recombinant Osteoactivin treatment inhibited ERK phosphorylation in a CD44-dependent manner. Finally, we examined the role of Osteoactivin on receptor activator of nuclear factor-κ B ligand (RANKL)-induced osteolysis in vivo. Our data indicate that recombinant Osteoactivin inhibits RANKL-induced osteolysis in vivo and this effect is CD44-dependent. Overall, our data indicate that Osteoactivin is a negative regulator of osteoclastogenesis in vitro and in vivo and that this process is regulated through CD44 and ERK activation.

Asunto(s)

Proteínas del Ojo/metabolismo , Receptores de Hialuranos/metabolismo , Sistema de Señalización de MAP Quinasas , Glicoproteínas de Membrana/metabolismo , Osteoclastos/citología , Transducción de Señal , Animales , Diferenciación Celular , Células Cultivadas , Masculino , Ratones Endogámicos C57BL , Osteoclastos/metabolismo , Ligando RANK/metabolismo , Proteínas Recombinantes/metabolismo

RESUMEN

UNLABELLED: Accumulating evidence over the last 40years suggests that silicate from dietary as well as silicate-containing biomaterials is beneficial to bone formation. However, the exact biological role(s) of silicate on bone cells are still unclear and controversial. Here, we report that orthosilicic acid (Si(OH)4) stimulated human mesenchymal stem cells (hMSCs) osteoblastic differentiation in vitro. To elucidate the possible molecular mechanisms, differential microRNA microarray analysis was used to show that Si(OH)4 significantly up-regulated microRNA-146a (miR-146a) expression during hMSC osteogenic differentiation. Si(OH)4 induced miR-146a expression profiling was further validated by quantitative RT-PCR (qRT-PCR), which indicated miR-146a was up-regulated during the late stages of hMSC osteogenic differentiation. Inhibition of miR-146a function by anti-miR-146a suppressed osteogenic differentiation of MC3T3 pre-osteoblasts, whereas Si(OH)4 treatment promoted osteoblast-specific genes transcription, alkaline phosphatase (ALP) production, and mineralization. Furthermore, luciferase reporter assay, Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence showed that Si(OH)4 decreased TNFα-induced activation of NF-κB, a signal transduction pathway that inhibits osteoblastic bone formation, through the known miR-146a negative feedback loop. Our studies established a mechanism for Si(OH)4 to promote osteogenesis by antagonizing NF-κB activation via miR-146a, which might be interesting to guide the design of osteo-inductive biomaterials for treatments of bone defects in humans. STATEMENT OF SIGNIFICANCE: Accumulating evidence over 40years suggests that silicate is beneficial to bone formation. However, the biological role(s) of silicate on bone cells are still unclear and controversial. Here, we report that Si(OH)4, the simplest form of silicate, can stimulate human mesenchymal stem cells osteoblastic differentiation. We identified that miR-146a is the expression signature in bone cells treated with Si(OH)4. Further analysis of miR-146a in bone cells reveals that Si(OH)4 upregulates miR-146a to antagonize the activation of NF-κB. Si(OH)4 was also shown to deactivate the same NF-κB pathway to suppress osteoclast formation. Our findings are important to the development of third-generation cell-and gene affecting biomaterials, and suggest silicate and miR-146a can be used as pharmaceuticals for bone fracture prevention and therapy.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , MicroARNs/biosíntesis , FN-kappa B/metabolismo , Osteoblastos/metabolismo , Ácido Silícico/farmacología , Animales , Humanos , Ratones , Osteogénesis/efectos de los fármacos , Células RAW 264.7

RESUMEN

The potential application of GPNMB/OA as a therapeutic target for lung cancer will require a greater understanding of the impact of GPNMB/OA ectodomain (ECD) protein shedding into tumor tissues. Thus, in this work we characterized GPNMB/OA expression and extent of shedding of its ECD protein while evaluating the impact on lung cancer progression using three non-small cell lung cancer (NSCLC) cell lines: A549, SK-MES-1 and calu-6. We observed a direct correlation (R2 = 0.89) between GPNMB/OA expression on NSCLC cells and the extent of GPNMB/OA ECD protein shedding. Meanwhile, siRNA-mediated knockdown of GPNMB/OA in cancer cells significantly reduced GPNMB/OA ECD protein shedding, migration, invasion and adhesion to extracellular matrix materials. Also, exogenous treatment of cancer cells (expressing low GPNMB/OA) with recombinant GPNMB/OA protein (rOA) significantly facilitated cell invasion and migration, but the effects of rOA was negated by inclusion of a selective RGD peptide. Further studies in athymic (nu/nu) mice-bearing calu-6 showed that intratumoral supplementation with rOA effectively facilitated in vivo tumor growth as characterized by a high number of proliferating cells (Ki67 staining) coupled with a low number of apoptotic cells. Taken together, our results accentuate the relevance of GPNMB/OA ECD protein shedding to progression of lung cancer. Thus, strategies that suppress GPNMB/OA expression on lung cancer cells as well as negate shedding of GPNMB/OA ECD protein are worthy of consideration in lung cancer therapeutics.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Movimiento Celular , Proliferación Celular , Neoplasias Pulmonares/patología , Glicoproteínas de Membrana/metabolismo , Animales , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Adhesión Celular , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Invasividad Neoplásica , Dominios Proteicos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Initial identification of osteoactivin (OA)/glycoprotein non-melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased threefold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post-fracture, and primary rat osteoblasts show increased OA expression during differentiation in vitro. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV-promoter (OA-Tg). Western blot analysis showed increased OA/gpnmb in OA-Tg osteoblasts, compared to wild-type (WT). In OA-Tg mouse femurs versus WT littermates, micro-CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA-Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over-expressed in osteoclasts in OA-Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX-1 and RANK-L, and decreased osteoclast numbers in OA-Tg, compared to WT mice, indicating decreased bone remodeling in OA-Tg mice. The proliferation rate of OA-Tg osteoblasts in vitro was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA-Tg osteoprogenitors. Quantitative RT-PCR analysis showed increased TGF-ß1 and TGF-ß receptors I and II expression in OA-Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo.

Asunto(s)

Remodelación Ósea/fisiología , Huesos/metabolismo , Diferenciación Celular/fisiología , Proteínas del Ojo/metabolismo , Glicoproteínas de Membrana/metabolismo , Osteoblastos/citología , Osteoclastos/citología , Animales , Densidad Ósea/fisiología , Remodelación Ósea/genética , Resorción Ósea/metabolismo , Diferenciación Celular/genética , Proteínas del Ojo/genética , Glicoproteínas de Membrana/genética , Ratones Transgénicos , Osteogénesis/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo

RESUMEN

We previously reported on the importance of osteoactivin (OA/Gpnmb) in osteogenesis. In this study, we examined the role of OA in osteoclastogenesis, using mice with a nonsense mutation in the Gpnmb gene (D2J) and wild-type controls (D2J/Gpnmb(+)). In these D2J mice, micro-computed tomography and histomorphometric analyses revealed increased cortical thickness, whereas total porosity and eroded surface were significantly reduced in D2J mice compared with wild-type controls, and these results were corroborated by lower serum levels of CTX-1. Contrary to these observations and counterintuitively, temporal gene expression analyses supported up-regulated osteoclastogenesis in D2J mice and increased osteoclast differentiation rates ex vivo, marked by increased number and size. The finding that MAPK was activated in early differentiating and mature D2J osteoclasts and that survival of D2J osteoclasts was enhanced and mediated by activation of the AKT-GSK3ß pathway supports this observation. Furthermore, this was abrogated by the addition of recombinant OA to cultures, which restored osteoclastogenesis to wild-type levels. Moreover, mix and match co-cultures demonstrated an induction of osteoclastogenesis in D2J osteoblasts co-cultured with osteoclasts of D2J or wild-type. Last, in functional osteo-assays, we show that bone resorption activity of D2J osteoclasts is dramatically reduced, and these osteoclasts present an abnormal ruffled border over the bone surface. Collectively, these data support a model whereby OA/Gpnmb acts as a negative regulator of osteoclast differentiation and survival but not function by inhibiting the ERK/AKT signaling pathways.

Asunto(s)

Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Proteínas del Ojo/genética , Glicoproteínas de Membrana/genética , Mutación , Osteoclastos/citología , Ligando RANK/fisiología , Animales , Remodelación Ósea , Ratones , Ratones Endogámicos DBA , Ligando RANK/metabolismo , Transducción de Señal , Microtomografía por Rayos X

RESUMEN

Osteoactivin (OA), also known as glycoprotein nmb (gpnmb) plays an important role in the regulation of osteoblast differentiation and function. OA induced osteoblast differentiation and function in vitro by stimulating alkaline phosphatase (ALP) activity, osteocalcin production, nodule formation, and matrix mineralization. Recent studies reported a role for OA in cell adhesion and integrin binding. In this study, we demonstrate that recombinant osteoactivin (rOA) as a matricellular protein stimulated adhesion, spreading and differentiation of MC3T3-E1 osteoblast-like cells through binding to αv ß1 integrin and heparan sulfated proteoglycans (HSPGs). MC3T3-E1 cell adhesion to rOA was blocked by neutralizing anti-OA or anti-αv and ß1 integrin antibodies. rOA stimulated-osteoblast adhesion was also inhibited by soluble heparin and sodium chlorate. Interestingly, rOA stimulated-osteoblast adhesion promoted an increase in FAK and ERK activation, resulting in the formation of focal adhesions, cell spreading and enhanced actin cytoskeleton organization. In addition, differentiation of primary osteoblasts was augmented on rOA coated-wells marked by increased alkaline phosphatase staining and activity. Taken together, these data implicate OA as a matricellular protein that stimulates osteoblast adhesion through binding to αv ß1 integrin and cell surface HSPGs, resulting in increased cell spreading, actin reorganization, and osteoblast differentiation with emphasis on the positive role of OA in osteogenesis.

Asunto(s)

Proteínas del Ojo/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Glicoproteínas de Membrana/metabolismo , Osteoblastos/fisiología , Receptores de Vitronectina/metabolismo , Células 3T3 , Citoesqueleto de Actina/fisiología , Fosfatasa Alcalina/biosíntesis , Animales , Anticuerpos/inmunología , Adhesión Celular , Diferenciación Celular , Línea Celular , Proliferación Celular , Cloratos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/biosíntesis , Proteínas del Ojo/genética , Proteínas del Ojo/inmunología , Quinasa 1 de Adhesión Focal/biosíntesis , Adhesiones Focales , Heparina/farmacología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Osteogénesis/fisiología , Unión Proteica , Ratas , Receptores de Vitronectina/inmunología , Proteínas Recombinantes

RESUMEN

We have previously identified osteoactivin (OA), encoded by Gpnmb, as an osteogenic factor that stimulates osteoblast differentiation in vitro. To elucidate the importance of OA in osteogenesis, we characterized the skeletal phenotype of a mouse model, DBA/2J (D2J) with a loss-of-function mutation in Gpnmb. Microtomography of D2J mice showed decreased trabecular mass, compared to that in wild-type mice [DBA/2J-Gpnmb(+)/SjJ (D2J/Gpnmb(+))]. Serum analysis showed decreases in OA and the bone-formation markers alkaline phosphatase and osteocalcin in D2J mice. Although D2J mice showed decreased osteoid and mineralization surfaces, their osteoblasts were increased in number, compared to D2J/Gpnmb(+) mice. We then examined the ability of D2J osteoblasts to differentiate in culture, where their differentiation and function were decreased, as evidenced by low alkaline phosphatase activity and matrix mineralization. Quantitative RT-PCR analyses confirmed the decreased expression of differentiation markers in D2J osteoblasts. In vitro, D2J osteoblasts proliferated and survived significantly less, compared to D2J/Gpnmb(+) osteoblasts. Next, we investigated whether mutant OA protein induces endoplasmic reticulum stress in D2J osteoblasts. Neither endoplasmic reticulum stress markers nor endoplasmic reticulum ultrastructure were altered in D2J osteoblasts. Finally, we assessed underlying mechanisms that might alter proliferation of D2J osteoblasts. Interestingly, TGF-ß receptors and Smad-2/3 phosphorylation were up-regulated in D2J osteoblasts, suggesting that OA contributes to TGF-ß signaling. These data confirm the anabolic role of OA in postnatal bone formation.

Asunto(s)

Proteínas del Ojo/genética , Glicoproteínas de Membrana/genética , Osteoblastos/fisiología , Osteocalcina/genética , Osteogénesis/genética , Transducción de Señal , Fosfatasa Alcalina/metabolismo , Animales , Animales Recién Nacidos , Apoptosis , Huesos/metabolismo , Huesos/patología , Diferenciación Celular/genética , Masculino , Ratones , Ratones Endogámicos DBA , Mutación , Osteoblastos/citología , Fenotipo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

Osteoactivin (OA) is a novel osteogenic factor important for osteoblast differentiation and function. Previous studies showed that OA stimulates matrix mineralization and transcription of osteoblast specific genes required for differentiation. OA plays a role in wound healing and its expression was shown to increase in post fracture calluses. OA expression was reported in muscle as OA is upregulated in cases of denervation and unloading stress. The regulatory mechanisms of OA in muscle and bone have not yet been determined. In this study, we examined whether OA plays a role in transdifferentiation of C2C12 myoblast into osteoblasts. Infected C2C12 with a retroviral vector overexpressing OA under the CMV promoter were able to transdifferentiate from myoblasts into osteoblasts. Immunofluorescence analysis showed that skeletal muscle marker MF-20 was severely downregulated in cells overexpressing OA and contained significantly less myotubes compared to uninfected control. C2C12 myoblasts overexpressing OA showed an increase in expression of bone specific markers such as alkaline phosphatase and alizarin red staining, and also showed an increase in Runx2 protein expression. We also detected increased levels of phosphorylated focal adhesion kinase (FAK) in C2C12 myoblasts overexpressing OA compared to control. Taken together, our results suggest that OA is able to induce transdifferentiation of myoblasts into osteoblasts through increasing levels of phosphorylated FAK.

Asunto(s)

Transdiferenciación Celular/genética , Proteínas del Ojo/genética , Glicoproteínas de Membrana/genética , Mioblastos/metabolismo , Osteoblastos/metabolismo , Fosfatasa Alcalina/biosíntesis , Animales , Línea Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Proteínas del Ojo/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/biosíntesis , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Glicoproteínas de Membrana/metabolismo , Ratones , Mioblastos/citología , Osteoblastos/citología