RESUMEN
Disruption of the finely tuned osteoblast-osteoclast balance is the underlying basis of several inflammatory bone diseases, such as osteomyelitis, osteoporosis, and septic arthritis. Prolonged and unrestrained exposure to inflammatory environment results in reduction of bone mineral density by downregulating osteoblast differentiation. Earlier studies from our laboratory have identified that Anacardic acid (AA), a constituent of Cashew nut shell liquid that is used widely in traditional medicine, has potential inhibitory effect on gelatinases (MMP2 and MMP9) which are over-expressed in numerous inflammatory conditions (Omanakuttan et al. in Mol Pharmacol, 2012 and Nambiar et al. in Exp Cell Res, 2016). The study demonstrated for the first time that AA promotes osteoblast differentiation in lipopolysaccharide-treated osteosarcoma cells (MG63) by upregulating specific markers, like osteocalcin, receptor activator of NF-κB ligand, and alkaline phosphatase. Furthermore, expression of the negative regulators, such as nuclear factor-κB, matrix metalloproteinases (MMPs), namely MMP13, and MMP1, along with several inflammatory markers, such as Interleukin-1ß and Nod-like receptor protein 3 were downregulated by AA. Taken together, AA expounds as a novel template for development of potential pharmacological therapeutics for inflammatory bone diseases.
Asunto(s)
Ácidos Anacárdicos/farmacología , Enfermedades Óseas/tratamiento farmacológico , Inflamasomas/antagonistas & inhibidores , Osteoblastos/efectos de los fármacos , Osteocalcina/agonistas , Ligando RANK/agonistas , Enfermedades Óseas/metabolismo , Enfermedades Óseas/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Inflamasomas/metabolismo , FN-kappa B/antagonistas & inhibidores , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Ligando RANK/metabolismoRESUMEN
BACKGROUND: Poor bone quality, increased fracture risks, and impaired bone healing are orthopedic comorbidities of type 1 diabetes (T1DM). Standard osteogenic growth factor treatments are inadequate in fully rescuing retarded healing of traumatic T1DM long bone injuries where both periosteal and bone marrow niches are disrupted. We test the hypotheses that osteogenesis of bone marrow-derived stromal cells (BMSCs) and periosteum-derived cells (PDCs), two critical skeletal progenitors in long bone healing, are both impaired in T1DM and that they respond differentially to osteogenic bone morphogenetic proteins (BMPs) and/or insulin-like growth factor-1 (IGF-1) rescue. METHODS: BMSCs and PDCs were isolated from Biobreeding Diabetes Prone/Worcester rats acquiring T1DM and normal Wistar rats. Proliferation, osteogenesis, and adipogenesis of the diabetic progenitors were compared with normal controls. Responses of diabetic progenitors to osteogenesis rescue by rhBMP-2/7 heterodimer (45 or 300 ng/ml) and/or rhIGF-1 (15 or 100 ng/ml) in normal and high glucose cultures were examined by alizarin red staining and qPCR. RESULTS: Diabetic BMSCs and PDCs proliferated slower and underwent poorer osteogenesis than nondiabetic controls, and these impairments were exacerbated in high glucose cultures. Osteogenesis of diabetic PDCs was rescued by rhBMP-2/7 or rhBMP-2/7 + rhIGF-1 in both normal and high glucose cultures in a dose-dependent manner. Diabetic BMSCs, however, only responded to 300 ng/nl rhBMP-2/7 with/without 100 ng/ml rhIGF-1 in normal but not high glucose osteogenic culture. IGF-1 alone was insufficient in rescuing the osteogenesis of either diabetic progenitor. Supplementing rhBMP-2/7 in high glucose osteogenic culture significantly enhanced gene expressions of type 1 collagen (Col 1), osteocalcin (OCN), and glucose transporter 1 (GLUT1) while suppressing that of adipogenic marker peroxisome proliferator-activated receptor gamma (PPARγ) in diabetic PDCs. The same treatment in high glucose culture only resulted in a moderate increase in Col 1, but no significant changes in OCN or GLUT1 expressions in diabetic BMSCs. CONCLUSIONS: This study demonstrates more effective osteogenesis rescue of diabetic PDCs than BMSCs by rhBMP-2/7 with/without rhIGF-1 in a hyperglycemia environment, underscoring the necessity to tailor biochemical therapeutics to specific skeletal progenitor niches. Our data also suggest potential benefits of combining growth factor treatment with blood glucose management to optimize orthopedic therapeutic outcomes for T1DM patients.
Asunto(s)
Proteína Morfogenética Ósea 2/farmacología , Proteína Morfogenética Ósea 7/farmacología , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Factor I del Crecimiento Similar a la Insulina/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colágeno Tipo I/agonistas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Transportador de Glucosa de Tipo 1/agonistas , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Osteocalcina/agonistas , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis/genética , PPAR gamma/antagonistas & inhibidores , PPAR gamma/genética , PPAR gamma/metabolismo , Periostio/efectos de los fármacos , Periostio/metabolismo , Periostio/patología , Cultivo Primario de Células , Ratas , Ratas Endogámicas BB , Ratas Wistar , Proteínas Recombinantes/farmacologíaRESUMEN
AIMS. To study the proliferation of osteoblasts and genes expression under normal glucose, high glucose, and metformin (Met). METHODS. MG63 osteoblast-like cells were cultured in osteogenic medium supplemented with normal glucose (glucose 5.5 mmol/L) or high glucose (glucose 16.7 mmol/L) and metformin + high glucose (Met 300 µmol/L + glucose 16.7 mmol/L). Proliferation was detected with CCK-8 assay at days 1, 3, and 7. Real-time PCR and Western blot were performed to compare the expression of collagen I (Col I), osteocalcin (OCN), osteoprotegerin (OPG), receptor activator for NF- κB ligand (RANKL), and metal matrix proteinases 1 and 2 (MMP1, MMP2). Alkaline phosphatase (ALP) activity was also detected at days 6, 12, and 18. RESULTS. Exposure to high glucose inhibited the proliferation of osteoblasts (P < 0.05), with suppressed OCN and OPG. Meanwhile, Col I, RANKL, MMP1, and MMP2 were unaffected. Metformin attenuated the suppression on proliferation with increased expression of Col I, OCN, and OPG, meanwhile suppressing MMP1 and MMP2. High glucose lowered the intracellular ALP, while metformin raised it. Metformin attenuated the downregulation of ALP completely at day 6, partly at day 12, but not at day 18. CONCLUSIONS. Metformin attenuated the suppression effect of high glucose to the osteoblast proliferation and gene expression, more prominently in earlier stage.
Asunto(s)
Hiperglucemia/patología , Hipoglucemiantes/farmacología , Metformina/farmacología , Osteoblastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Sustancias Protectoras/farmacología , Regulación hacia Arriba/efectos de los fármacos , Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Biomarcadores/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Colágeno Tipo I/agonistas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Represión Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/metabolismo , Cinética , Metaloproteinasa 1 de la Matriz/química , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/química , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Osteoblastos/patología , Osteocalcina/agonistas , Osteocalcina/genética , Osteocalcina/metabolismo , Osteoprotegerina/agonistas , Osteoprotegerina/genética , Osteoprotegerina/metabolismoRESUMEN
Fibrodysplasia ossificans progressiva (FOP) is a skeletal disorder with progressive heterotopic ossification in skeletal muscle. A mutation causing constitutive activation in a bone morphogenetic protein (BMP) type 1 receptor [ALK2(R206H)] is found in most patients with FOP. However, the details in the heterotopic ossification of muscle in FOP and the role of matrix metalloproteinase-10 (MMP-10) in bone remain to be fully elucidated. In the present study, we investigated the role of MMP-10 in the differentiation of mouse myoblastic C2C12 cells into osteoblasts. MMP-10 was extracted as a factor, whose expression was most extensively enhanced by ALK2 (R206H) transfection in C2C12 cells. MMP-10 significantly augmented the levels of Osterix, type 1 collagen, alkaline phosphatase (ALP) and osteocalcin mRNA as well as ALP activity enhanced by BMP-2 in C2C12 cells. Moreover, a reduction in endogenous MMP-10 levels by siRNA significantly decreased the levels of Runx2, Osterix, type 1 collagen, ALP and osteocalcin mRNA enhanced by BMP-2 in these cells. In addition, MMP-10 increased the phosphorylation of Smad1/5/8 as well as enhanced the levels of Smad6 and Smad7 mRNA induced by BMP-2. In conclusion, the present study first demonstrated that MMP-10 promotes the differentiation of myoblasts into osteoblasts by interacting with the BMP signaling pathway. MMP-10 may play some important role in the heterotopic ossification of muscle in FOP.
Asunto(s)
Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular , Regulación Enzimológica de la Expresión Génica , Metaloproteinasa 10 de la Matriz/metabolismo , Osteoblastos/citología , Transducción de Señal , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Sustitución de Aminoácidos , Animales , Proteína Morfogenética Ósea 2/agonistas , Proteína Morfogenética Ósea 2/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Línea Celular , Colágeno Tipo I/agonistas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Humanos , Metaloproteinasa 10 de la Matriz/química , Metaloproteinasa 10 de la Matriz/genética , Ratones , Proteínas Mutantes/agonistas , Proteínas Mutantes/metabolismo , Mioblastos/citología , Mioblastos/enzimología , Mioblastos/metabolismo , Osteoblastos/enzimología , Osteoblastos/metabolismo , Osteocalcina/agonistas , Osteocalcina/genética , Osteocalcina/metabolismo , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína smad6/agonistas , Proteína smad6/genética , Proteína smad6/metabolismo , Proteína smad7/agonistas , Proteína smad7/genética , Proteína smad7/metabolismo , Factor de Transcripción Sp7 , Factores de Transcripción/agonistas , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Calcium, in combination with vitamin D, is an effective treatment for osteoporosis. Since bone mineralisation occurs concurrently with osteoblast to osteocyte transition, we hypothesised that calcium would stimulate this process. The effect of calcium (1.8-11.8mM) was tested on human primary osteoblast (NHBC) differentiation in vitro. Cultures were assayed for cell-associated mineral and gene expression associated with osteoblast differentiation and mineralisation. Treatment with calcium resulted in a striking dose- and time-dependent increase in cell-associated mineralisation. Calcium appeared to promote osteoblast to osteocyte differentiation, as indicated by increased expression of osteocalcin (OCN), E11, dentin matrix protein 1 (DMP1) and SOST mRNA. The expression of the osteoclast inhibitor, osteoprotegerin, was dramatically enhanced by calcium. Calcium also increased the ratio of PHEX mRNA expression relative to that of MEPE, suggesting a mechanism for the pro-anabolic effect. Consistent with this, calcium-dependent mineralisation was reversed in the presence of MEPE-ASARM peptides. This study suggests that calcium promotes osteoblast to osteocyte transition and concurrent matrix mineralisation, at least in part through the PHEX-MEPE axis.
Asunto(s)
Calcio/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteocitos/efectos de los fármacos , ARN Mensajero/genética , Proteínas Adaptadoras Transductoras de Señales , Biomarcadores/metabolismo , Proteínas Morfogenéticas Óseas/agonistas , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Calcio/metabolismo , Diferenciación Celular , Relación Dosis-Respuesta a Droga , Proteínas de la Matriz Extracelular/agonistas , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Marcadores Genéticos/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/agonistas , Osteocalcina/genética , Osteocalcina/metabolismo , Osteocitos/citología , Osteocitos/metabolismo , Osteoprotegerina/agonistas , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Endopeptidasa Neutra Reguladora de Fosfato PHEX/genética , Endopeptidasa Neutra Reguladora de Fosfato PHEX/metabolismo , Fosfoproteínas/agonistas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Cultivo Primario de Células , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
El GPRC6A es un miembro recientemente identificado de la familia C de receptores acoplados a proteínas G (GPCRs) que está estrechamente emparentado con el receptor sensor de calcio (CASR). Se ha demostrado que este receptor es capaz de sensar cationes extracelulares y aminoácidos y que requiere tanto de los cationes extracelulares y de los aminoácidos para su óptima estimulación in vitro. El estudio del perfil de ligandos ha mostrado que la l-ornithine es el más potente eficaz l-aminoácido agonista seguido de varios otros aminoácidos alifáticos, neutros, y básicos. Algunos estudios han mostrado la activación por cationes del GPRC6A, pero comparado con el CASR, se necesitan concentraciones extracelulares más altas de calcio para activar este receptor. Es más, el Mg(2+) ha mostrado ser un modulador positivo de la respuesta a la l-ornithine. Se lo ha propuesto como el candidato para el elusivo mecanismo de sensado de calcio extracelular del osteoblasto, que se sabe responde a altas concentraciones locales de Ca²+. También se ha propuesto al GPRC6A como candidato a receptor de la osteocalcina, regulando el metabolismo energético y como blanco molecular para la acción del estroncio sobre el hueso.
GPRC6A is a recently identified member of family C of G protein-coupled receptors (GPCRs) that is closely related to the calcium-sensing receptor CASR. It has recently been shown that GPRC6A extracellular cations and amino acids and requires both extracellular cations and amino acids for optimal stimulation in vitro. The study of the ligand profile of GPRC6A has shown that l-ornithine is the most potent and efficacious l-amino acid agonist, followed by several other aliphatic, neutral, and basic amino acids. Some studies show cation-dependent activation of GPRC6A, but compared to CASR, much higher extracellular calcium concentrations are needed to activate this receptor. Furthermore, the divalent cation Mg(2+) was found to be a positive modulator of the l-ornithine response. GPRC6A may be a candidate for the elusive extracellular calcium-sensing mechanism known to be present in osteoblasts, which respond to high local Ca²+ concentrations. GPRC6A has also been proposed as a candidate receptor for ostocalcin, regulating energy metabolism and as a molecular target for the action of strontium on bone.