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1.
Development ; 144(6): 1035-1044, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28292847

RESUMO

Human umbilical cord blood (CB) has attracted much attention as a reservoir for functional hematopoietic stem and progenitor cells, and, recently, as a source of blood-borne fibroblasts (CB-BFs). Previously, we demonstrated that bone marrow stromal cell (BMSC) and CB-BF pellet cultures make cartilage in vitro Furthermore, upon in vivo transplantation, BMSC pellets remodelled into miniature bone/marrow organoids. Using this in vivo model, we asked whether CB-BF populations that express characteristics of the hematopoietic stem cell (HSC) niche contain precursors that reform the niche. CB ossicles were regularly observed upon transplantation. Compared with BM ossicles, CB ossicles showed a predominance of red marrow over yellow marrow, as demonstrated by histomorphological analyses and the number of hematopoietic cells isolated within ossicles. Marrow cavities from CB and BM ossicles included donor-derived CD146-expressing osteoprogenitors and host-derived mature hematopoietic cells, clonogenic lineage-committed progenitors and HSCs. Furthermore, human CD34+ cells transplanted into ossicle-bearing mice engrafted and maintained human HSCs in the niche. Our data indicate that CB-BFs are able to recapitulate the conditions by which the bone marrow microenvironment is formed and establish complete HSC niches, which are functionally supportive of hematopoietic tissue.


Assuntos
Células da Medula Óssea/citologia , Sangue Fetal/citologia , Fibroblastos/citologia , Células-Tronco Hematopoéticas/citologia , Organoides/citologia , Nicho de Células-Tronco , Adulto , Compartimento Celular , Criança , Fibroblastos/transplante , Transplante de Células-Tronco Hematopoéticas , Homeostase , Humanos , Nicho de Células-Tronco/genética , Células Estromais/citologia
2.
Calcif Tissue Int ; 107(6): 603-610, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32875378

RESUMO

We compared the effects of a nitrogen-containing bisphosphonate (N-BP), zoledronic acid (ZA), and an anti-mouse RANKL antibody (anti-mRANKL Ab) on the bone tissue pathology of a transgenic mouse model of human fibrous dysplasia (FD). For comparison, we also reviewed the histological samples of a child with McCune-Albright syndrome (MAS) treated with Pamidronate for 3 years. EF1α-GsαR201C mice with FD-like lesions in the tail vertebrae were treated with either 0.2 mg/kg of ZA at day 0, 7, and 14 or with 300 µg/mouse of anti-mRANKL Ab at day 0 and 21. All mice were monitored by Faxitron and histological analysis was performed at day 42. ZA did not affect the progression of the radiographic phenotype in EF1α-GsαR201C mice. FD-like lesions in the ZA group showed the persistence of osteoclasts, easily detectable osteoclast apoptotic activity and numerous "giant osteoclasts". In contrast, in the anti-mRANKL Ab-treated mice, osteoclasts were markedly reduced/absent, the radiographic phenotype reverted and the FD-like lesions were extensively replaced by newly formed bone. Numerous "giant osteoclasts" were also detected in the samples of the child with MAS. This study supports the hypothesis that osteoclasts per se, independently of their resorptive activity, are essential for development and expansion of FD lesions.


Assuntos
Displasia Fibrosa Óssea/tratamento farmacológico , Células Gigantes , Osteoclastos , Ácido Zoledrônico/uso terapêutico , Animais , Difosfonatos , Modelos Animais de Doenças , Displasia Fibrosa Óssea/patologia , Camundongos , Camundongos Transgênicos
3.
Blood ; 125(10): 1662-71, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25298037

RESUMO

Neonatal bone marrow transplantation (BMT) could offer a novel therapeutic opportunity for genetic disorders by providing sustainable levels of the missing protein at birth, thus preventing tissue damage. We tested this concept in mucopolysaccharidosis type I (MPS IH; Hurler syndrome), a lysosomal storage disorder caused by deficiency of α-l-iduronidase. MPS IH is characterized by a broad spectrum of clinical manifestations, including severe progressive skeletal abnormalities. Although BMT increases the life span of patients with MPS IH, musculoskeletal manifestations are only minimally responsive if the timing of BMT delays, suggesting already irreversible bone damage. In this study, we tested the hypothesis that transplanting normal BM into newborn MPS I mice soon after birth can prevent skeletal dysplasia. We observed that neonatal BMT was effective at restoring α-l-iduronidase activity and clearing elevated glycosaminoglycans in blood and multiple organs. At 37 weeks of age, we observed an almost complete normalization of all bone tissue parameters, using radiographic, microcomputed tomography, biochemical, and histological analyses. Overall, the magnitude of improvements correlated with the extent of hematopoietic engraftment. We conclude that BMT at a very early stage in life markedly reduces signs and symptoms of MPS I before they appear.


Assuntos
Doenças do Desenvolvimento Ósseo/prevenção & controle , Transplante de Medula Óssea/métodos , Mucopolissacaridose I/patologia , Mucopolissacaridose I/terapia , Fatores Etários , Animais , Animais Recém-Nascidos , Doenças do Desenvolvimento Ósseo/metabolismo , Doenças do Desenvolvimento Ósseo/patologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Modelos Animais de Doenças , Feminino , Glicosaminoglicanos/sangue , Glicosaminoglicanos/metabolismo , Humanos , Iduronidase/genética , Iduronidase/metabolismo , Recém-Nascido , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucopolissacaridose I/genética
4.
Skeletal Radiol ; 46(2): 233-236, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27826699

RESUMO

Multiple non-ossifying fibromas (MNOFs) occur either isolated or in association with other anomalies, are usually localized in the long bones of the lower limbs, may be radiographically confused with other skeletal lesions, and tend to heal spontaneously with the completion of the skeletal growth. Segmental distribution, either monomelic or polymelic and ipsilateral, is rare and commonly observed in the context of developmental diseases known as "RASopathies", which are caused by mutations in genes that encode components or regulators within the Ras/mitogen-activated protein kinase signaling pathway. We describe here the radiographic and pathologic features of an 18-year-old Caucasian boy, whose clinical history started at the age of 3 when the diagnosis of aneurysmal bone cyst was made on a lytic lesion of his left clavicle. Over the following 2 years, the patient developed polyostotic and monomelic lesions within the left humerus, radius, and ulna. No other skeletal and extra-skeletal anomalies were clinically detected. The lesions were interpreted as consistent with polyostotic fibrous dysplasia and MNOFs and showed an unusually aggressive clinical course with progressive increase in size and coalescence. The definitive diagnosis of MNOFs was made after the exclusion of fibrous dysplasia by molecular analysis. The polyostotic and monomelic distribution of the lesions and the unusually aggressive clinical course contribute to make this case of MNOFs unique.


Assuntos
Fibroma/diagnóstico por imagem , Fibroma/patologia , Displasia Fibrosa Poliostótica/diagnóstico por imagem , Displasia Fibrosa Poliostótica/patologia , Adolescente , Humanos , Masculino
5.
Bone Res ; 10(1): 50, 2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35853852

RESUMO

The Gsα/cAMP signaling pathway mediates the effect of a variety of hormones and factors that regulate the homeostasis of the post-natal skeleton. Hence, the dysregulated activity of Gsα due to gain-of-function mutations (R201C/R201H) results in severe architectural and functional derangements of the entire bone/bone marrow organ. While the consequences of gain-of-function mutations of Gsα have been extensively investigated in osteoblasts and in bone marrow osteoprogenitor cells at various differentiation stages, their effect in adipogenically-committed bone marrow stromal cells has remained unaddressed. We generated a mouse model with expression of GsαR201C driven by the Adiponectin (Adq) promoter. Adq-GsαR201C mice developed a complex combination of metaphyseal, diaphyseal and cortical bone changes. In the metaphysis, GsαR201C caused an early phase of bone resorption followed by bone deposition. Metaphyseal bone formation was sustained by cells that were traced by Adq-Cre and eventually resulted in a high trabecular bone mass phenotype. In the diaphysis, GsαR201C, in combination with estrogen, triggered the osteogenic activity of Adq-Cre-targeted perivascular bone marrow stromal cells leading to intramedullary bone formation. Finally, consistent with the previously unnoticed presence of Adq-Cre-marked pericytes in intraosseous blood vessels, GsαR201C caused the development of a lytic phenotype that affected both cortical (increased porosity) and trabecular (tunneling resorption) bone. These results provide the first evidence that the Adq-cell network in the skeleton not only regulates bone resorption but also contributes to bone formation, and that the Gsα/cAMP pathway is a major modulator of both functions.

6.
PLoS One ; 15(1): e0227279, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31999703

RESUMO

Fibrous dysplasia (FD) of bone is a complex disease of the skeleton caused by dominant activating mutations of the GNAS locus encoding for the α subunit of the G protein-coupled receptor complex (Gsα). The mutation involves a substitution of arginine at position 201 by histidine or cysteine (GsαR201H or R201C), which leads to overproduction of cAMP. Several signaling pathways are implicated downstream of excess cAMP in the manifestation of disease. However, the pathogenesis of FD remains largely unknown. The overall FD phenotype can be attributed to alterations of skeletal stem/progenitor cells which normally develop into osteogenic or adipogenic cells (in cis), and are also known to provide support to angiogenesis, hematopoiesis, and osteoclastogenesis (in trans). In order to dissect the molecular pathways rooted in skeletal stem/progenitor cells by FD mutations, we engineered human skeletal stem/progenitor cells with the GsαR201C mutation and performed transcriptomic analysis. Our data suggest that this FD mutation profoundly alters the properties of skeletal stem/progenitor cells by pushing them towards formation of disorganized bone with a concomitant alteration of adipogenic differentiation. In addition, the mutation creates an altered in trans environment that induces neovascularization, cytokine/chemokine changes and osteoclastogenesis. In silico comparison of our data with the signature of FD craniofacial samples highlighted common traits, such as the upregulation of ADAM (A Disintegrin and Metalloprotease) proteins and other matrix-related factors, and of PDE7B (Phosphodiesterase 7B), which can be considered as a buffering process, activated to compensate for excess cAMP. We also observed high levels of CEBPs (CCAAT-Enhancer Binding Proteins) in both data sets, factors related to browning of white fat. This is the first analysis of the reaction of human skeletal stem/progenitor cells to the introduction of the FD mutation and we believe it provides a useful background for further studies on the molecular basis of the disease and for the identification of novel potential therapeutic targets.


Assuntos
Células da Medula Óssea/fisiologia , Diferenciação Celular/genética , Cromograninas/genética , Displasia Fibrosa Óssea/patologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Células-Tronco/fisiologia , Proteínas ADAM/metabolismo , Adipogenia/genética , Tecido Adiposo Branco/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células Cultivadas , Cromograninas/metabolismo , Simulação por Computador , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/metabolismo , Conjuntos de Dados como Assunto , Displasia Fibrosa Óssea/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Mutação com Ganho de Função , Perfilação da Expressão Gênica , Voluntários Saudáveis , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/metabolismo , Osteogênese/genética , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Estromais/fisiologia , Regulação para Cima
7.
J Bone Miner Res ; 34(12): 2171-2182, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31295366

RESUMO

Fibrous dysplasia of bone/McCune-Albright syndrome (Polyostotic FD/MAS; OMIM#174800) is a crippling skeletal disease caused by gain-of-function mutations of Gs α. Enhanced bone resorption is a recurrent histological feature of FD and a major cause of fragility of affected bones. Previous work suggests that increased bone resorption in FD is driven by RANKL and some studies have shown that the anti-RANKL monoclonal antibody, denosumab, reduces bone turnover and bone pain in FD patients. However, the effect of RANKL inhibition on the histopathology of FD and its impact on the natural history of the disease remain to be assessed. In this study, we treated the EF1α-Gs αR201C mice, which develop an FD-like phenotype, with an anti-mouse RANKL monoclonal antibody. We found that the treatment induced marked radiographic and microscopic changes at affected skeletal sites in 2-month-old mice. The involved skeletal segments became sclerotic due to the deposition of new, highly mineralized bone within developing FD lesions and showed a higher mechanical resistance compared to affected segments from untreated transgenic mice. Similar changes were also detected in older mice with a full-blown skeletal phenotype. The administration of anti-mouse RANKL antibody arrested the growth of established lesions and, in young mice, prevented the appearance of new ones. However, after drug withdrawal, the newly formed bone was remodelled into FD tissue and the disease progression resumed in young mice. Taken together, our results show that the anti-RANKL antibody significantly affected the bone pathology and natural history of FD in the mouse. Pending further work on the prevention and management of relapse after treatment discontinuation, our preclinical study suggests that RANKL inhibition may be an effective therapeutic option for FD patients. © 2019 American Society for Bone and Mineral Research.


Assuntos
Displasia Fibrosa Óssea/metabolismo , Ligante RANK/antagonistas & inibidores , Animais , Fenômenos Biomecânicos , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Calcificação Fisiológica , Denosumab/farmacologia , Modelos Animais de Doenças , Progressão da Doença , Displasia Fibrosa Óssea/complicações , Displasia Fibrosa Óssea/diagnóstico por imagem , Displasia Fibrosa Óssea/patologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Camundongos Transgênicos , Osteólise/sangue , Osteólise/complicações , Fator 1 de Elongação de Peptídeos/metabolismo , Fenótipo , Ligante RANK/metabolismo , Ratos
8.
Stem Cell Reports ; 6(6): 897-913, 2016 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-27304917

RESUMO

A widely shared view reads that mesenchymal stem/stromal cells ("MSCs") are ubiquitous in human connective tissues, can be defined by a common in vitro phenotype, share a skeletogenic potential as assessed by in vitro differentiation assays, and coincide with ubiquitous pericytes. Using stringent in vivo differentiation assays and transcriptome analysis, we show that human cell populations from different anatomical sources, regarded as "MSCs" based on these criteria and assumptions, actually differ widely in their transcriptomic signature and in vivo differentiation potential. In contrast, they share the capacity to guide the assembly of functional microvessels in vivo, regardless of their anatomical source, or in situ identity as perivascular or circulating cells. This analysis reveals that muscle pericytes, which are not spontaneously osteochondrogenic as previously claimed, may indeed coincide with an ectopic perivascular subset of committed myogenic cells similar to satellite cells. Cord blood-derived stromal cells, on the other hand, display the unique capacity to form cartilage in vivo spontaneously, in addition to an assayable osteogenic capacity. These data suggest the need to revise current misconceptions on the origin and function of so-called "MSCs," with important applicative implications. The data also support the view that rather than a uniform class of "MSCs," different mesoderm derivatives include distinct classes of tissue-specific committed progenitors, possibly of different developmental origin.


Assuntos
Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Microvasos/citologia , Pericitos/citologia , Células Satélites de Músculo Esquelético/citologia , Transcriptoma , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação Celular , Linhagem da Célula/genética , Condrogênese/genética , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Camundongos , Microvasos/metabolismo , Osteogênese/genética , Pericitos/metabolismo , Fenótipo , Células Satélites de Músculo Esquelético/metabolismo , Transplante Heterólogo
9.
Bone ; 70: 55-61, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25240458

RESUMO

Postnatal skeletal stem cells are a unique class of progenitors with biological properties that extend well beyond the limits of stemness as commonly defined. Skeletal stem cells sustain skeletal tissue homeostasis, organize and maintain the complex architectural structure of the bone marrow microenvironment and provide a niche for hematopoietic progenitor cells. The identification of stem cells in the human post-natal skeleton has profoundly changed our approach to the physiology and pathology of this system. Skeletal diseases have been long interpreted essentially in terms of defective function of differentiated cells and/or abnormal turnover of the matrix that they produce. The notion of a skeletal stem cell has brought forth multiple, novel concepts in skeletal biology that provide potential alternative concepts. At the same time, the recognition of the complex functions played by skeletal progenitors, such as the structural and functional organization of the bone marrow, has provided an innovative, unifying perspective for understanding bone and bone marrow changes simultaneously occurring in many disorders. Finally, the possibility to isolate and highly enrich for skeletal progenitors, enables us to reproduce perfectly normal or pathological organ miniatures. These, in turn, provide suitable models to investigate and manipulate the pathogenetic mechanisms of many genetic and non-genetic skeletal diseases. This article is part of a Special Issue entitled Stem cells and Bone.


Assuntos
Doenças Ósseas/patologia , Células-Tronco/citologia , Doenças Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Neoplasias Hematológicas/patologia , Humanos , Microambiente Tumoral
10.
J Bone Miner Res ; 30(6): 1030-43, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25487351

RESUMO

We recently reported the generation and initial characterization of the first direct model of human fibrous dysplasia (FD; OMIM #174800), obtained through the constitutive systemic expression of one of the disease-causing mutations, Gsα(R201C) , in the mouse. To define the specific pathogenetic role(s) of individual cell types within the stromal/osteogenic system in FD, we generated mice expressing Gsα(R201C) selectively in mature osteoblasts using the 2.3kb Col1a1 promoter. We show here that this results in a striking high bone mass phenotype but not in a mimicry of human FD. The high bone mass phenotype involves specifically a deforming excess of cortical bone and prolonged and ectopic cortical bone remodeling. Expression of genes characteristic of late stages of bone cell differentiation/maturation is profoundly altered as a result of expression of Gsα(R201C) in osteoblasts, and expression of the Wnt inhibitor Sost is reduced. Although high bone mass is, in fact, a feature of some types/stages of FD lesions in humans, it is marrow fibrosis, localized loss of adipocytes and hematopoietic tissue, osteomalacia, and osteolytic changes that together represent the characteristic pathological profile of FD, as well as the sources of specific morbidity. None of these features are reproduced in mice with osteoblast-specific expression of Gsα(R201C) . We further show that hematopoietic progenitor/stem cells, as well as more mature cell compartments, and adipocyte development are normal in these mice. These data demonstrate that effects of Gsα mutations underpinning FD-defining tissue changes and morbidity do not reflect the effects of the mutations on osteoblasts proper.


Assuntos
Osso e Ossos , Displasia Fibrosa Óssea , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Mutação de Sentido Incorreto , Osteoblastos , Substituição de Aminoácidos , Animais , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Cromograninas , Modelos Animais de Doenças , Displasia Fibrosa Óssea/genética , Displasia Fibrosa Óssea/metabolismo , Displasia Fibrosa Óssea/patologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Tamanho do Órgão , Osteoblastos/metabolismo , Osteoblastos/fisiologia
11.
Stem Cell Res ; 12(3): 659-72, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24675053

RESUMO

Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells") can establish the hematopoietic microenvironment within heterotopic ossicles generated by transplantation at non-skeletal sites. Here we show that non-mineralized cartilage pellets formed by hBMSCs ex vivo generate complete ossicles upon heterotopic transplantation in the absence of exogenous scaffolds. These ossicles display a remarkable degree of architectural fidelity, showing that an exogenous conductive scaffold is not an absolute requirement for bone formation by transplanted BMSCs. Marrow cavities within the ossicles include erythroid, myeloid and granulopoietic lineages, clonogenic hematopoietic progenitors and phenotypic HSCs, indicating that complete stem cell niches and hematopoiesis are established. hBMSCs (CD146(+) adventitial reticular cells) are established in the heterotopic chimeric bone marrow through a unique process of endochondral bone marrow formation, distinct from physiological endochondral bone formation. In this process, chondrocytes remain viable and proliferate within the pellet, are released from cartilage, and convert into bone marrow stromal cells. Once explanted in secondary culture, these cells retain phenotype and properties of skeletal stem cells ("MSCs"), including the ability to form secondary cartilage pellets and secondary ossicles upon serial transplantation. Ex vivo, hBMSCs initially induced to form cartilage pellets can be reestablished in adherent culture and can modulate gene expression between cartilage and stromal cell phenotypes. These data show that so-called "cartilage differentiation" of BMSCs in vitro is a reversible phenomenon, which is actually reverted, in vivo, to the effect of generating stromal cells supporting the homing of hematopoietic stem cells and progenitors.


Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Condrócitos/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Mesenquimais/citologia , Nicho de Células-Tronco , Adolescente , Adulto , Animais , Cartilagem/citologia , Células Cultivadas , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos SCID , Adulto Jovem
12.
J Bone Miner Res ; 29(11): 2357-68, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24764158

RESUMO

Fibrous dysplasia of bone (FD) is a crippling skeletal disease associated with postzygotic mutations (R201C, R201H) of the gene encoding the α subunit of the stimulatory G protein, Gs. By causing a characteristic structural subversion of bone and bone marrow, the disease results in deformity, hypomineralization, and fracture of the affected bones, with severe morbidity arising in childhood or adolescence. Lack of inheritance of the disease in humans is thought to reflect embryonic lethality of germline-transmitted activating Gsα mutations, which would only survive through somatic mosaicism. We have generated multiple lines of mice that express Gsα(R201C) constitutively and develop an inherited, histopathologically exact replica of human FD. Robust transgene expression in neonatal and embryonic tissues and embryonic stem (ES) cells were associated with normal development of skeletal tissues and differentiation of skeletal cells. As in humans, FD lesions in mice developed only in the postnatal life; a defined spatial and temporal pattern characterized the onset and progression of lesions across the skeleton. In individual bones, lesions developed through a sequence of three distinct histopathological stages: a primary modeling phase defined by endosteal/medullary excess bone formation and normal resorption; a secondary phase, with excess, inappropriate remodeling; and a tertiary fibrous dysplastic phase, which reproduced a full-blown replica of the human bone pathology in mice of age ≥1 year. Gsα mutations are sufficient to cause FD, and are per se compatible with germline transmission and normal embryonic development in mice. Our novel murine lines constitute the first model of FD.


Assuntos
Modelos Animais de Doenças , Displasia Fibrosa Óssea , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Expressão Gênica , Mutação de Sentido Incorreto , Fatores Etários , Substituição de Aminoácidos , Animais , Remodelação Óssea/genética , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/patologia , Displasia Fibrosa Óssea/enzimologia , Displasia Fibrosa Óssea/genética , Displasia Fibrosa Óssea/patologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Camundongos , Camundongos Transgênicos , Osteogênese/genética
13.
J Bone Miner Res ; 25(5): 1103-16, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-19874199

RESUMO

Human skeletal progenitors were engineered to stably express R201C mutated, constitutively active Gs alpha using lentiviral vectors. Long-term transduced skeletal progenitors were characterized by an enhanced production of cAMP, indicating the transfer of the fundamental cellular phenotype caused by activating mutations of Gs alpha. Like skeletal progenitors isolated from natural fibrous dysplasia (FD) lesions, transduced cells could generate bone but not adipocytes or the hematopoietic microenvironment on in vivo transplantation. In vitro osteogenic differentiation was noted for the lack of mineral deposition, a blunted upregulation of osteocalcin, and enhanced upregulation of other osteogenic markers such as alkaline phosphatase (ALP) and bone sialoprotein (BSP) compared with controls. A very potent upregulation of RANKL expression was observed, which correlates with the pronounced osteoclastogenesis observed in FD lesions in vivo. Stable transduction resulted in a marked upregulation of selected phosphodiesterase (PDE) isoform mRNAs and a prominent increase in total PDE activity. This predicts an adaptive response in skeletal progenitors transduced with constitutively active, mutated Gs alpha. Indeed, like measurable cAMP levels, the differentiative responses of transduced skeletal progenitors were profoundly affected by inhibition of PDEs or lack thereof. Finally, using lentiviral vectors encoding short hairpin (sh) RNA interfering sequences, we demonstrated that selective silencing of the mutated allele is both feasible and effective in reverting the aberrant cAMP production brought about by the constitutively active Gs alpha and some of its effects on in vitro differentiation of skeletal progenitors.


Assuntos
Células da Medula Óssea/metabolismo , Displasia Fibrosa Óssea/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Células-Tronco/fisiologia , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Displasia Fibrosa Óssea/patologia , Displasia Fibrosa Óssea/fisiopatologia , Vetores Genéticos , Humanos , Lentivirus/genética , Osteogênese/genética , Fenótipo , Diester Fosfórico Hidrolases/metabolismo , Células Estromais/metabolismo , Transdução Genética
14.
Virology ; 385(2): 343-50, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19138779

RESUMO

The prion protein is a cell surface glycoprotein whose physiological role remains elusive, while its implication in transmissible spongiform encephalopathies (TSEs) has been demonstrated. Multiple interactions between the prion protein and viruses have been described: viruses can act as co-factors in TSEs and life cycles of different viruses have been found to be controlled by prion modulation. We present data showing that human Adenovirus 5 induces prion expression. Inactivated Adenovirus did not alter prion transcription, while variants encoding for early products did, suggesting that the prion is stimulated by an early adenoviral function. Down-regulation of the prion through RNA interference showed that the prion controls adenovirus replication and expression. These data suggest that the prion protein could play a role in the defense strategy mounted by the host during viral infection, in a cell autonomous manner. These results have implications for the study of the prion protein and of associated TSEs.


Assuntos
Adenovírus Humanos/metabolismo , Regulação da Expressão Gênica , Príons/metabolismo , Replicação Viral/fisiologia , Infecções por Adenoviridae/metabolismo , Adenovírus Humanos/genética , Linhagem Celular , Células Cultivadas , Células HeLa , Humanos , Proteínas PrPSc/metabolismo , Príons/genética , Deleção de Sequência
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