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Here we present the use of surface nanopatterning of covalently immobilized BMP-2 and integrin selective ligands to determine the specificity of their interactions in regulating cell adhesion and focal adhesion assembly. Gold nanoparticle arrays carrying single BMP-2 dimers are prepared by block-copolymer micellar nanolithography and azide-functionalized integrin ligands (cyclic-RGD peptides or α5ß1 integrin peptidomimetics) are immobilized on the surrounding polyethylene glycol alkyne by click chemistry. Compared to BMP-2 added to the media, surface immobilized BMP-2 (iBMP-2) favors the spatial segregation of adhesion clusters and enhances focal adhesion (FA) size in cells adhering to α5ß1 integrin selective ligands. Moreover, iBMP-2 copresented with α5ß1 integrin ligands induces the recruitment of αvß3 integrins in FAs. When copresented with RGD, iBMP-2 induces the assembly of a higher number of FAs, which are not affected by α5ß1 integrin blocking. Our dual-functionalized platforms offer the possibility to study the crosstalk between integrins and BMP receptors, and more in general they could be used to address the spatial regulation of growth factors and adhesion receptors crosstalk on biomimetic surfaces.
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Oro , Nanopartículas del Metal , Adhesión Celular , Integrina alfa5beta1 , Integrina alfaVbeta3 , LigandosRESUMEN
Diseases determining bone tissue loss have a high impact on people of any age. Bone healing can be improved using a therapeutic approach based on tissue engineering. Scientific research is demonstrating that among bone regeneration techniques, interesting results, in filling of bone lesions and dehiscence have been obtained using adult mesenchymal stem cells (MSCs) integrated with biocompatible scaffolds. The geometry of the scaffold has critical effects on cell adhesion, proliferation and differentiation. Many cytokines and compounds have been demonstrated to be effective in promoting MSCs osteogenic differentiation. Oligostilbenes, such as Resveratrol (Res) and Polydatin (Pol), can increase MSCs osteoblastic features. 3D printing is an excellent technique to create scaffolds customized for the lesion and thus optimized for the patient. In this work we analyze osteoblastic features of adult MSCs integrated with 3D-printed polycarbonate scaffolds differentiated in the presence of oligostilbenes.
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Hard tissue regeneration represents a challenge for the Regenerative Medicine and Mesenchymal stem cells (MSCs) could be a successful therapeutic strategy. T-LysYal® (T-Lys), a new derivative of Hyaluronic Acid (HA) possessing a superior stability, has already been proved efficient in repairing corneal epithelial cells damaged by dry conditions in vitro. We investigated the regenerative potential of T-Lys in the hard tissues bone and cartilage. We have previously demonstrated that cells isolated from the tooth germ, Dental Bud Stem Cells (DBSCs), differentiate into osteoblast-like cells, representing a promising source of MSCs for bone regeneration. Herewith, we show that T-Lys treatment stimulates the expression of typical osteoblastic markers, such as Runx-2, Collagen I (Col1) and Alkaline Phosphatase (ALP), determining a higher production of mineralized matrix nodules. In addition, we found that T-Lys treatment positively affects αVß3 integrin expression, key integrin in the osteoblastic commitment, leading to the formation of focal adhesions (FAs). The efficacy of T-Lys was also tested on chondrogenic differentiation starting from human articular chondrocytes (HACs) resulting in an increase of differentiation markers and cell number.
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Cartílago Articular/citología , Diferenciación Celular , Condrocitos/citología , Condrogénesis , Lisina/farmacología , Células Madre Mesenquimatosas/citología , Osteogénesis , Cloruro de Sodio/farmacología , Timina/farmacología , Cartílago Articular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Humanos , Ácido Hialurónico/química , Lisina/química , Células Madre Mesenquimatosas/efectos de los fármacos , Cloruro de Sodio/química , Timina/química , Ingeniería de TejidosRESUMEN
[This corrects the article DOI: 10.1155/2018/6958713.].
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Bone loss and fractures are consequences of aging, diseases or traumas. Furthermore the increased number of aged people, due to the rise of life expectancy, needs more strategies to limit the bone loss and regenerate the lost tissue, ameliorating the life quality of patients. A great interest for non-pharmacological therapies based on natural compounds is emerging and focusing on the oligostilbene Polydatin, present in many kinds of fruits and vegetables, when resveratrol particularly in red wines. These molecules have been extensively studied due to their antioxidant and anti-inflammatory effects, showing more recently Resveratrol the ability to enhance osteogenic differentiation and bone formation. However, the clinical applications of Resveratrol are limited due to its low bioavailability and rapid metabolism, while its natural glycosilated precursor Polydatin shows better metabolic stability and major abundance in fresh fruits and vegetables. Nevertheless the role of Polydatin on osteogenic differentiation is still unexplored. Mesenchymal stem cells (MSCs) from dental tissues, such as dental bud stem cells (DBSCs), are able to differentiate toward osteogenic lineage: thus we investigated how Resveratrol and Polydatin influence the differentiation of DBSCs, eventually affecting bone formation. Our results showed that Polydatin increases MSCs osteogenic differentiation sharing similar properties with Resveratrol. These results encourage to deepen the effects of this molecule on bone health and its associated mechanisms of action, wishing for the future a successful use in bone loss prevention and therapy.
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Diferenciación Celular/efectos de los fármacos , Glucósidos/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Estilbenos/farmacología , Células Cultivadas , Niño , Humanos , Masculino , ResveratrolRESUMEN
Vitamin D (Vit D) by means of its biological active form, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), has a protective effect on the skeleton by acting on calcium homeostasis and bone formation. Furthermore, Vit D has a direct effect on mesenchymal stem cells (MSCs) in stimulating their osteogenic differentiation. In this work, we present for the first time the effect of 1,25(OH)2D3 on MSC adhesion. Considering that cell adhesion to the substrate is fundamental for cell commitment and differentiation, we focused on the expression of αVß3 integrin, which has a key role in the commitment of MSCs to the osteoblastic lineage. Our data indicate that Vit D increases αVß3 integrin expression inducing the formation of focal adhesions (FAs). Moreover, we assayed MSC commitment in the presence of the extracellular matrix (ECM) glycoprotein fibronectin (FN), which is able to favor cell adhesion on surfaces and also to induce osteopontin (OPN) expression: this suggests that Vit D and FN synergize in supporting cell adhesion. Taken together, our findings provide evidence that Vit D can promote osteogenic differentiation of MSCs through the modulation of αVß3 integrin expression and its subcellular organization, thus favoring binding with the matrix protein (FN).
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Cells from dental tissues have a mesenchymal stem cell (MSC) phenotype, are multipotent and can differentiate into osteoblastic cells, as we have previously found. MSCs, due to their tumorhoming ability, are currently being used as cellbased delivery systems for cancer protein therapeutics, such as the TNFrelated apoptosisinducing ligand (TRAIL). In the present study we revealed that dental pulp stem cells (DPSCs) expressed TRAIL to a greater extent when they were differentiated into the osteoblastic lineage. TRAIL affected the viability of undifferentiated DPSCs, while osteoblastic differentiated DPSCs were not sensitive to TRAIL. The expression trend of TRAIL receptors underwent changes during the osteoblastic differentiation of DPSCs exhibiting low DcR2 and high DR5 levels in the undifferentiated DPSCs and an opposite scenario was presented in the differentiated cells. The sensitivity of the undifferentiated DPSCs to the TRAILapoptotic effect was also associated with low levels of intracellular antiapoptotic proteins, such as cFLIP, XIAP and the activation of caspase8 and 3. DPSCdifferentiated osteoblasts expressing high TRAIL levels were capable to affect the cell viability of the human myeloma cell line H929, thus representing an effective anticancer therapeutic method.
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Diferenciación Celular/genética , Mieloma Múltiple/genética , Osteoblastos/metabolismo , Osteogénesis/genética , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Apoptosis/genética , Caspasas/genética , Proliferación Celular/genética , Supervivencia Celular/genética , Pulpa Dental/citología , Pulpa Dental/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/metabolismo , Mieloma Múltiple/patología , Mieloma Múltiple/terapia , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores Señuelo del Factor de Necrosis Tumoral/genética , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
Extracellular vesicles (EVs), based on their origin or size, can be classified as apoptotic bodies, microvesicles (MVs)/microparticles (MPs), and exosomes. EVs are one of the new emerging modes of communication between cells that are providing new insights into the pathophysiology of several diseases. EVs released from activated or apoptotic cells contain specific proteins (signaling molecules, receptors, integrins, cytokines), bioactive lipids, nucleic acids (mRNA, miRNA, small non coding RNAs, DNA) from their progenitor cells. In the brain, EVs contribute to intercellular communication through their basal release and uptake by surrounding cells, or release into the cerebrospinal fluid (CSF) and blood. In the central nervous system (CNS), EVs have been suggested as potential carriers in the intercellular delivery of misfolded proteins associated to neurodegenerative disorders, such as tau and amyloid ß in Alzheimer's Disease (AD), α-synuclein in Parkinson's Disease (PD), superoxide dismutase (SOD)1 in amyotrophic lateral sclerosis and huntingtin in Huntington's Disease. Multiple studies indicate that EVs are involved in the pathogenesis of AD, although their role has not been completely elucidated. The focus of this review is to analyze the new emerging role of EVs in AD progression, paying particular attention to microglia EVs. Recent data show that microglia are the first myeloid cells to be activated during neuroinflammation. Microglial EVs in fact, could have both a beneficial and a detrimental action in AD. The study of EVs may provide specific, precise information regarding the AD transition stage that may offer possibilities to intervene in order to retain cognition. In chronic neurodegenerative diseases EVs could be a novel biomarker to monitor the progression of the pathology and also represent a new therapeutical approach to CNS diseases.
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Enfermedad de Alzheimer/patología , Vesículas Extracelulares/patología , Microglía/patología , Encéfalo/patología , HumanosRESUMEN
Multiple cytokines produced by immune cells induce remodeling and aid in maintaining bone homeostasis through differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. Here, we investigate bone remodeling controlled by the tumor necrosis factor (TNF) superfamily cytokine LIGHT. LIGHT-deficient mice (Tnfsf14-/- ) exhibit spine deformity and reduced femoral cancellous bone mass associated with an increase in the osteoclast number and a slight decrease of osteoblasts compared with WT mice. The effect of LIGHT in bone cells can be direct or indirect, mediated by both the low expression of the anti-osteoclastogenic osteoprotegerin (OPG) in B and T cells and reduced levels of the pro-osteoblastogenic Wnt10b in CD8+ T cells in Tnfsf14-/- mice. LIGHT stimulation increases OPG levels in B, CD8+ T, and osteoblastic cells, as well as Wnt10b expression in CD8+ T cells. The high bone mass in Light and T- and B-cell-deficient mice (Rag- /Tnfsf14- ) supports the cooperative role of the immune system in bone homeostasis. These results implicate LIGHT as a potential target in bone disease. © 2017 American Society for Bone and Mineral Research.
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Remodelación Ósea/inmunología , Hueso Esponjoso/inmunología , Fémur/inmunología , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/deficiencia , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Remodelación Ósea/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Hueso Esponjoso/patología , Fémur/fisiología , Ratones , Ratones Noqueados , Osteoblastos/inmunología , Osteoclastos/inmunología , Osteoclastos/patología , Osteoprotegerina/genética , Osteoprotegerina/inmunología , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunología , Proteínas Wnt/genética , Proteínas Wnt/inmunologíaRESUMEN
Mesenchymal stem cells (MSCs) have been identified in human dental tissues. Dental pulp stem cells (DPSCs) were classified within MSC family, are multipotent, can be isolated from adult teeth, and have been shown to differentiate, under particular conditions, into various cell types including osteoblasts. In this work, we investigated how the differentiation process of DPSCs toward osteoblasts is controlled. Recent literature data attributed to the nuclear receptor related 1 (NURR1), a still unclarified role in osteoblast differentiation, while NURR1 is primarily involved in dopaminergic neuron differentiation and activity. Thus, in order to verify if NURR1 had a role in DPSC osteoblastic differentiation, we silenced it during all the processes and compared the expression of the main osteoblastic markers with control cultures. Our results showed that the inhibition of NURR1 significantly increased the expression of osteoblast markers collagen I and alkaline phosphatase. Further, in long time cultures, the mineral matrix deposition was strongly enhanced in NURR1-silenced cultures. These results suggest that NURR1 plays a key role in switching DPSC differentiation toward osteoblasts rather than neuronal or even other cell lines. In conclusion, DPSCs represent a source of osteoblast-like cells and downregulation of NURR1 strongly prompted their differentiation toward the osteoblastogenesis process.
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Introduction. Adding stem cells to biodegradable scaffolds to enhance bone regeneration is a valuable option. Different kinds of stem cells with osteoblastic activity were tested, such as bone marrow stromal stem cells (BMSSCs). Aim. To assess a correct protocol for osteogenic stem cell differentiation, so BMSSCs were seeded on a bone porcine block (BPB). Materials and Methods. Bone marrow from six minipigs was extracted from tibiae and humeri and treated to isolate BMSSCs. After seeding on BPB, critical-size defects were created on each mandible of the minipigs and implanted with BPB and BPB/BMSSCs. After three months, histomorphometric analysis was performed. Results. Histomorphometric analysis provided percentages of the three groups. Tissues present in control defects were 23 ± 2% lamellar bone, 28 ± 1% woven bone, and 56 ± 4% marrow spaces; in BPB defects were 20 ± 5% BPB, 32 ± 2% lamellar bone, 24 ± 1% woven bone, and 28 ± 2% marrow spaces; in BPB/BMSSCs defects were 17 ± 4% BPB/BMSSCs, 42 ± 2% lamellar bone, 12 ± 1% woven bone, and 22 ± 3% marrow spaces. Conclusion. BPB used as a scaffold to induce bone regeneration may benefit from the addition of BDPSCs in the tissue-engineered constructs.
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1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D (Vit D), increases intestinal absorption of calcium and phosphate, maintaining a correct balance of bone remodeling. Vit D has an anabolic effect on the skeletal system and is key in promoting osteoblastic differentiation of human Mesenchymal Stem Cells (hMSCs) from bone marrow. MSCs can be also isolated from the immature form of the tooth, the dental bud: Dental Bud Stem Cells (DBSCs) are adult stem cells that can effectively undergo osteoblastic differentiation. In this work we investigated the effect of Vit D on DBSCs differentiation into osteoblasts. Our data demonstrate that DBSCs, cultured in an opportune osteogenic medium, differentiate into osteoblast-like cells; Vit D treatment stimulates their osteoblastic features, increasing the expression of typical markers of osteoblastogenesis like RUNX2 and Collagen I (Coll I) and, in a more important way, determining a higher production of mineralized matrix nodules.
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Prior studies show that oxytocin (Oxt) and vasopressin (Avp) have opposing actions on the skeleton exerted through high-affinity G protein-coupled receptors. We explored whether Avp and Oxtr can share their receptors in the regulation of bone formation by osteoblasts. We show that the Avp receptor 1α (Avpr1α) and the Oxt receptor (Oxtr) have opposing effects on bone mass: Oxtr(-/-) mice have osteopenia, and Avpr1α(-/-) mice display a high bone mass phenotype. More notably, this high bone mass phenotype is reversed by the deletion of Oxtr in Oxtr(-/-):Avpr1α(-/-) double-mutant mice. However, although Oxtr is not indispensable for Avp action in inhibiting osteoblastogenesis and gene expression, Avp-stimulated gene expression is inhibited when the Oxtr is deleted in Avpr1α(-/-) cells. In contrast, Oxt does not interact with Avprs in vivo in a model of lactation-induced bone loss in which Oxt levels are high. Immunofluorescence microscopy of isolated nucleoplasts and Western blotting and MALDI-TOF of nuclear extracts show that Avp triggers Avpr1α localization to the nucleus. Finally, a specific Avpr2 inhibitor, tolvaptan, does not affect bone formation or bone mass, suggesting that Avpr2, which primarily functions in the kidney, does not have a significant role in bone remodeling.
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Arginina Vasopresina/fisiología , Densidad Ósea/fisiología , Remodelación Ósea/fisiología , Osteogénesis/fisiología , Oxitocina/fisiología , Receptores de Vasopresinas/metabolismo , Secuencia de Aminoácidos , Animales , Arginina Vasopresina/farmacología , Western Blotting , Densidad Ósea/efectos de los fármacos , Densidad Ósea/genética , Enfermedades Óseas Metabólicas/genética , Remodelación Ósea/efectos de los fármacos , Remodelación Ósea/genética , Eliminación de Gen , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Osteoblastos/metabolismo , Osteoblastos/fisiología , Osteogénesis/genética , Oxitocina/farmacología , Receptores de Oxitocina/genética , Receptores de Oxitocina/metabolismo , Receptores de Vasopresinas/genéticaRESUMEN
Several studies have reported the beneficial effects of mesenchymal stem cells (MSCs) in tissue repair and regeneration. New sources of stem cells in adult organisms are continuously emerging; dental tissues have been identified as a source of postnatal MSCs. Dental bud is the immature precursor of the tooth, is easy to access and we show in this study that it can yield a high number of cells with ≥95% expression of mesenchymal stemness makers and osteogenic capacity. Thus, these cells can be defined as Dental Bud Stem Cells (DBSCs) representing a promising source for bone regeneration of stomatognathic as well as other systems. Cell interactions with the extracellular matrix (ECM) and neighboring cells are critical for tissue morphogenesis and architecture; such interactions are mediated by integrins and cadherins respectively. We characterized DBSCs for the expression of these adhesion receptors and examined their pattern during osteogenic differentiation. Our data indicate that N-cadherin and cadherin-11 were expressed in undifferentiated DBSCs and their expression underwent changes during the osteogenic process (decreasing and increasing respectively), while expression of E-cadherin and P-cadherin was very low in DBSCs and did not change during the differentiation steps. Such expression pattern reflected the mesenchymal origin of DBSCs and confirmed their osteoblast-like features. On the other hand, osteogenic stimulation induced the upregulation of single subunits, αV, ß3, α5, and the formation of integrin receptors α5ß1 and αVß3. DBSCs differentiation toward osteoblastic lineage was enhanced when cells were grown on fibronectin (FN), vitronectin (VTN), and osteopontin (OPN), ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. In addition we established that integrin αVß3 plays a crucial role during the commitment of MSCs to osteoblast lineage, whereas integrin α5ß1 seems to be dispensable. These data suggest that functionalization of biomaterials with such ECM proteins would improve bone reconstruction therapies starting from dental stem cells.
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Cadherinas/metabolismo , Pulpa Dental/citología , Integrinas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Diferenciación Celular , Pulpa Dental/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Osteogénesis , RegeneraciónRESUMEN
It is unclear how physical activity stimulates new bone synthesis. We explored whether irisin, a newly discovered myokine released upon physical activity, displays anabolic actions on the skeleton. Young male mice were injected with vehicle or recombinant irisin (r-irisin) at a low cumulative weekly dose of 100 µg kg(-1). We observed significant increases in cortical bone mass and strength, notably in cortical tissue mineral density, periosteal circumference, polar moment of inertia, and bending strength. This anabolic action was mediated primarily through the stimulation of bone formation, but with parallel notable reductions in osteoclast numbers. The trabecular compartment of the same bones was spared, as were vertebrae from the same mice. Higher irisin doses (3,500 µg kg(-1) per week) cause browning of adipose tissue; this was not seen with low-dose r-irisin. Expectedly, low-dose r-irisin modulated the skeletal genes, Opn and Sost, but not Ucp1 or Pparγ expression in white adipose tissue. In bone marrow stromal cell cultures, r-irisin rapidly phosphorylated Erk, and up-regulated Atf4, Runx2, Osx, Lrp5, ß-catenin, Alp, and Col1a1; this is consistent with a direct receptor-mediated action to stimulate osteogenesis. We also noted that, although the irisin precursor Fndc5 was expressed abundantly in skeletal muscle, other sites, such as bone and brain, also expressed Fndc5, albeit at low levels. Furthermore, muscle fibers from r-irisin-injected mice displayed enhanced Fndc5 positivity, and irisin induced Fdnc5 mRNA expression in cultured myoblasts. Our data therefore highlight a previously unknown action of the myokine irisin, which may be the molecular entity responsible for muscle-bone connectivity.
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Fibronectinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Proteínas Recombinantes/farmacología , Tejido Adiposo/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Fibronectinas/genética , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/genéticaRESUMEN
CD99 is a transmembrane glycoprotein expressed in physiological conditions by cells of different tissues, including osteoblasts (OBs). High or low CD99 levels have been detected in various pathological conditions, and the supernatant of some carcinoma cell lines can modulate CD99 expression in OB-like cells. In the present work we demonstrate for the first time that two different human myeloma cell lines (H929 and U266) and, in a less degree, their conditioned media significantly downregulate CD99 expression in normal human OBs during the differentiation process. In the same experimental conditions the OBs display a less differentiated phenotype as demonstrated by the decreased expression of RUNX2 and Collagen I. On the contrary, when CD99 was activated by using a specific agonist antibody, the OBs become more active as demonstrated by the upregulation of Alkaline Phosphatase, Collagen I, RUNX2, and JUND expression. Furthermore, we demonstrate that the activation of CD99 is able to induce the phosphorylation of ERK 1/2 and AKT intracellular signal transduction molecules in the OBs.
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Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Mieloma Múltiple/patología , Osteoblastos/citología , Osteogénesis/fisiología , Antígeno 12E7 , Fosfatasa Alcalina/biosíntesis , Antígenos CD/biosíntesis , Moléculas de Adhesión Celular/biosíntesis , Diferenciación Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular , Colágeno Tipo I/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Medios de Cultivo Condicionados/farmacología , Regulación hacia Abajo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Osteoblastos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-jun/biosíntesisRESUMEN
LIGHT, a TNF superfamily member, is involved in T-cell homeostasis and erosive bone disease associated with rheumatoid arthritis. Herein, we investigated whether LIGHT has a role in Multiple Myeloma (MM)-bone disease. We found that LIGHT was overproduced by CD14+ monocytes, CD8+ T-cells and neutrophils of peripheral blood and bone marrow (BM) from MM-bone disease patients. We also found that LIGHT induced osteoclastogenesis and inhibited osteoblastogenesis. In cultures from healthy-donors, LIGHT induced osteoclastogenesis in RANKL-dependent and -independent manners. In the presence of a sub-optimal RANKL concentration, LIGHT and RANKL synergically stimulated osteoclast formation, through the phosphorylation of Akt, NFκB and JNK pathways. In cultures of BM samples from patients with bone disease, LIGHT inhibited the formation of CFU-F and CFU-OB as well as the expression of osteoblastic markers including collagen-I, osteocalcin and bone sialoprotein-II. LIGHT indirectly inhibited osteoblastogenesis in part through sclerostin expressed by monocytes. In conclusion, our findings for the first time provide evidence for a role of LIGHT in MM-bone disease development.
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Enfermedades Óseas/patología , Mieloma Múltiple/patología , Osteoblastos/patología , Osteoclastos/patología , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Enfermedades Óseas/metabolismo , Estudios de Casos y Controles , Diferenciación Celular/fisiología , Línea Celular Tumoral , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genéticaRESUMEN
We report that oxytocin (Oxt) receptors (Oxtrs), on stimulation by the ligand Oxt, translocate into the nucleus of osteoblasts, implicating this process in the action of Oxt on osteoblast maturation. Sequential immunocytochemistry of intact cells or isolated nucleoplasts stripped of the outer nuclear membrane showed progressive nuclear localization of the Oxtr; this nuclear translocation was confirmed by monitoring the movement of Oxtr-EGFP as well as by immunogold labeling. Nuclear Oxtr localization was conclusively shown by Western immunoblotting and MS of nuclear lysate proteins. We found that the passage of Oxtrs into the nucleus was facilitated by successive interactions with ß-arrestins (Arrbs), the small GTPase Rab5, importin-ß (Kpnb1), and transportin-1 (Tnpo1). siRNA-mediated knockdown of Arrb1, Arrb2, or Tnpo1 abrogated Oxt-induced expression of the osteoblast differentiation genes osterix (Sp7), Atf4, bone sialoprotein (Ibsp), and osteocalcin (Bglap) without affecting Erk phosphorylation. Likewise and again, without affecting pErk, inhibiting Arrb recruitment by mutating Ser rich clusters of the nuclear localization signal to Ala abolished nuclear import and Oxtr-induced gene expression. These studies define a previously unidentified mechanism for Oxtr action on bone and open possibilities for direct transcriptional modulation by nuclear G protein-coupled receptors.
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Transporte Activo de Núcleo Celular/fisiología , Membrana Nuclear/metabolismo , Osteoblastos/metabolismo , Osteogénesis/fisiología , Oxitocina/fisiología , Receptores de Oxitocina/metabolismo , beta Carioferinas/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Arrestinas/antagonistas & inhibidores , Arrestinas/genética , Arrestinas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica/fisiología , Ligandos , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteogénesis/genética , Fosforilación , Mutación Puntual , Conformación Proteica , Procesamiento Proteico-Postraduccional , ARN Interferente Pequeño/farmacología , Receptores de Oxitocina/química , Receptores de Oxitocina/deficiencia , Proteínas Recombinantes de Fusión/metabolismo , Serina/química , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/genética , beta-Arrestina 1 , Arrestina beta 2 , beta-Arrestinas , Proteínas de Unión al GTP rab5/antagonistas & inhibidores , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Adult stem cells therapy can be an efficacious treatment for many diseases and disabilities. New sources of stem cells in adult organisms are continuously emerging. Dental tissues that are easily accessible by a tooth extraction have been identified as a source of postnatal mesenchymal stem cells capable of self-renewal and multipotency. Here, we describe accurately the technical procedure to isolate mesenchymal stem cells from dental pulp (DPSCs), characterize their immunophenotype, and assay their osteogenic capacity.
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Diferenciación Celular , Pulpa Dental/citología , Osteogénesis , Células Madre/citología , Técnicas de Cultivo de Célula , Separación Celular/métodos , Humanos , Inmunohistoquímica , Cultivo Primario de Células , Medicina Regenerativa , Células Madre/metabolismo , Ingeniería de Tejidos , Adulto JovenRESUMEN
Although hyponatremia is known to be associated with osteoporosis and a high fracture risk, the mechanism through which bone loss ensues has remained unclear. As hyponatremic patients have elevated circulating arginine-vasopressin (AVP) levels, we examined whether AVP can affect the skeleton directly as yet another component of the pituitary-bone axis. Here, we report that the two Avp receptors, Avpr1α and Avpr2, coupled to Erk activation, are expressed in osteoblasts and osteoclasts. AVP injected into wild-type mice enhanced and reduced, respectively, the formation of bone-resorbing osteoclasts and bone-forming osteoblasts. Conversely, the exposure of osteoblast precursors to Avpr1α or Avpr2 antagonists, namely SR49059 or ADAM, increased osteoblastogenesis, as did the genetic deletion of Avpr1α. In contrast, osteoclast formation and bone resorption were both reduced in Avpr1α(-/-) cultures. This process increased bone formation and reduced resorption resulted in a profound enhancement of bone mass in Avpr1α(-/-) mice and in wild-type mice injected with SR49059. Collectively, the data not only establish a primary role for Avp signaling in bone mass regulation, but also call for further studies on the skeletal actions of Avpr inhibitors used commonly in hyponatremic patients.