RESUMO
OBJECTIVE: To examine age-related efficacy of bone morphogenetic protein (BMP)-2, ascorbate, and dexamethasone as osteogenic inducers in canine marrow-derived stromal cells (MSCs). SAMPLE POPULATION: Samples of femoral bone marrow obtained from 15 skeletally immature (< 1 year old) and 4 skeletally mature (> 1.5 years old) dogs. PROCEDURE: First-passage canine MSC cultures were treated with 100 microg of ascorbate phosphate/mL, 10(-7)M dexamethasone, 100 ng of BMP-2/mL, or a combination of these osteoinducers. On day 6, cultures were harvested for quantitation of alkaline phosphatase (ALP) activity and isolation of RNA to prepare cDNA for real-time polymerase chain reaction analyses of osteoblast markers. RESULTS: Early markers of osteogenesis were induced in canine MSCs by BMP-2 but not dexamethasone. In young dogs, the combination of BMP-2 and ascorbate yielded the highest ALP mRNA concentrations and activity. This combination also induced significant increases in mRNA for osteopontin and runt-domain transcription factor 2. In comparison to MSCs from immature dogs, those from mature dogs had diminished ALP activity in response to BMP and ascorbate. Results for cultures treated with 3,4-dehydroproline suggested that ascorbate-induced production of extracellular matrix was important for maximal BMP-2 response in canine MSCs. CONCLUSIONS AND CLINICAL RELEVANCE: BMP-2 was capable of inducing markers of osteogenesis in short-term cultures of canine MSCs. In MSCs obtained from skeletally immature dogs, ascorbate was required for maximal effects of BMP These results define optimal conditions for stem cell osteogenesis in dogs and will facilitate development of stem cell-based treatments for dogs with fractures.
Assuntos
Ácido Ascórbico/análogos & derivados , Proteínas Morfogenéticas Ósseas/farmacologia , Dexametasona/farmacologia , Cães/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Fatores Etários , Fosfatase Alcalina/metabolismo , Animais , Ácido Ascórbico/farmacologia , Proteína Morfogenética Óssea 2 , Primers do DNA , Células-Tronco Mesenquimais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Adherent bone marrow stromal cells are inducible osteoprogenitors, giving rise to cells expressing osteoblast markers including alkaline phosphatase, osteopontin, osteocalcin, and bone sialoprotein. However, the potency of inducers varies in a species-specific manner. Glucocorticoids such as dexamethasone induce alkaline phosphatase activity in both human and rat mesenchymal stem cells, while mouse bone marrow stromal cells are refractory to dexamethasone-induced alkaline phosphatase activity. In contrast, BMP induces alkaline phosphatase activity in both mouse and rat bone marrow stromal cells, while BMP effects on human bone marrow stromal cells are poorly characterized. METHODS: Bone marrow samples were isolated from patients undergoing hip replacement. Mononuclear marrow cells were cultured and grown to confluence without or with 10 (-7) M dexamethasone. Cells from each isolate were passaged into medium containing 100 micro g/mL ascorbate phosphate and treated with dexamethasone, 100 ng/mL BMP, or no inducer. At day 6, alkaline phosphatase activity was assayed, and RNA was prepared for mRNA analyses by real-time polymerase chain reaction. RESULTS: Bone marrow stromal cells from twenty-four of twenty-six patients showed no significant osteogenic response to BMP-2, 4, or 7 as determined by alkaline phosphatase induction. However, BMPs induced elevated levels of other genes associated with osteogenesis such as bone sialoprotein and osteopontin as well as BMP-2 and noggin. If primary cultures of human bone marrow stromal cells were pretreated with dexamethasone, BMP-2 treatment of first-passage cells induced alkaline phosphatase in approximately half of the isolates, and significantly greater induction was seen in cells from males. Dexamethasone treatment, like BMP treatment, also increased expression of the BMP-binding protein noggin. CONCLUSIONS: Most human femur bone marrow stromal cell samples appear incapable of expressing elevated alkaline phosphatase levels in response to BMPs. Since BMP treatment induced expression of several other BMP-regulated genes, the defect in alkaline phosphatase induction is presumably not due to impaired BMP signaling. We hypothesize that the mechanism by which BMPs modulate alkaline phosphatase expression is indirect, involving a BMP-regulated transcription factor for alkaline phosphatase expression that is controlled differently in humans and rodents.
Assuntos
Células da Medula Óssea/citologia , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular/genética , Proteínas de Neoplasias , Osteogênese/genética , Células-Tronco/citologia , Células Estromais/citologia , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Proteínas de Transporte , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Dexametasona/farmacologia , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Osteogênese/efeitos dos fármacos , Reação em Cadeia da Polimerase , Proteínas/genética , RNA Mensageiro/genética , Células-Tronco/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
Bone morphogenetic proteins (BMPs) promote the differentiation of osteoprogenitor cells, and also induce osteogenesis in bone marrow stromal cells (MSC) from rats and mice. However, compared to results with animal models, BMPs are relatively inefficient in inducing human MSC to undergo osteogenesis, and are much less effective in promoting bone formation in human clinical trials. Previous studies indicated that, while human MSC respond to dexamethasone with elevated levels of the osteoblast marker alkaline phosphatase, most isolates of human MSC fail to show alkaline phosphatase induction in response to BMP-2, BMP-4, or BMP-7. Several other genes known to be induced by BMPs are appropriately regulated; thus, human MSC are capable of some BMP-activated signaling. Analysis of the BMP receptors ALK-3 and ALK-6 indicated that, although ALK-6 mRNA was not expressed in human MSC, overexpressing a constitutively active ALK-6 receptor did not induce elevated alkaline phosphatase. Real-time RT-PCR was used to investigate expression of several osteoblast-related transcription factors in MSC after 6 days' exposure to BMP2 or dexamethasone. Msx-2, a transcription factor that has been reported to inhibit differentiation of osteoprogenitor cells, showed 10-fold elevation in BMP-2-treated human MSC, but not in BMP-2-treated rat MSC. Overexpression of Msx-2 in human and rat MSC, however, did not alter alkaline phosphatase levels, which suggests that absence of BMP-stimulated alkaline phosphatase was not caused by the BMP-2-induced increase in Msx-2. Although Runx2 isoforms have been implicated in control of osteoblast differentiation, levels of this transcription factor were unaffected by BMP treatment. Expression of the FKHR transcription factor, which has been reported to regulate alkaline phosphatase transcription in mouse cells, showed a modest increase in response to BMP-2, but a much greater increase in dexamethasone-treated cells. We propose that BMP regulation of the bone/liver/kidney alkaline phosphatase gene is indirect, requiring expression of new transcription factor(s) that behave differently in rodent and human MSC.
Assuntos
Células da Medula Óssea/metabolismo , Proteínas Morfogenéticas Ósseas/fisiologia , Proteínas do Tecido Nervoso , Células Estromais/metabolismo , Fator de Crescimento Transformador beta , Adulto , Idoso , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Proteína Morfogenética Óssea 2 , Receptores de Proteínas Morfogenéticas Ósseas Tipo I , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/farmacologia , Proteínas de Transporte , Linhagem Celular Tumoral , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core , Proteínas de Ligação a DNA/genética , Dexametasona/farmacologia , Feminino , Fêmur/citologia , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica , Proteínas de Homeodomínio , Humanos , Proteína 1 Inibidora de Diferenciação , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Osteoblastos/química , Osteoblastos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas/genética , Ratos , Receptores de Fatores de Crescimento/genética , Proteínas Repressoras/genética , Células Estromais/efeitos dos fármacos , Fatores de Transcrição/genética , TransfecçãoRESUMO
Bone morphogenetic proteins (BMPs) are well known to induce bone formation in animal models and can promote osteogenesis in cultures of multipotential mesenchymal stem cells (MSC) isolated from rat and mouse bone marrow. However, clinical trials of BMPs suggest that BMPs are relatively ineffective inducers of osteogenesis in humans. Recent studies from our lab indicate that when human bone marrow MSC are placed in primary culture, osteogenesis can be induced by dexamethasone (Dex), but not by BMP-2, -4, or -7. We have therefore investigated components of BMP signaling pathways in human MSC. First passage cells, derived from the bone marrow of patients undergoing hip replacement surgery, were cultured with ascorbate phosphate and treated with 100 nM dexamethasone (Dex), 100 ng/ml BMP, or both. After 6 days, alkaline phosphatase activity of cell extracts was measured, and RNA was extracted for RT-PCR analysis of mRNA levels. Among human MSC samples from more than a dozen patients, only one patient sample showed significantly elevated alkaline phosphatase after exposure to BMP; the rest responded to Dex but not BMP. Analysis of mRNA from cultured human MSC indicated that, while Dex treatment caused increased levels of mRNA for alkaline phosphatase, BMP did not. Noggin is a BMP-binding protein that is upregulated by BMPs. BMP-treated human MSC cultures that did not show increased alkaline phosphatase did express elevated levels of noggin mRNA, indicating that the cells are capable of some BMP response. Our results suggest that BMP signaling in mesenchymal stem cells utilizes more than one system for transcriptional activation. The inability of most human MSC to activate transcription of the alkaline phosphatase gene implies that a defect exists in the system required for induction of the osteoblast phenotype.