RESUMO
The role that transduced mouse bone marrow stromal cells (mBMSCs) engineered to overexpress human bone morphogenetic protein 2 (BMP-2) play in healing critical-sized skeletal defects is largely unknown. We evaluated the interaction between host osteoprogenitor cells and donor mBMSCs transduced with either a lentiviral (LV) vector-expressing red fluorescent protein (RFP) with or without BMP-2 that were implanted into a critical-sized femoral defect. Radiographs taken at the time of killing were evaluated using a five-point scaled scoring system. Frozen histologic sections were analyzed to assess both the transduced cells' role in bone repair and the local osteoprogenitor response. There was complete radiographic bridging in 94% of group I (LV-RFPch-BMP-2-cmyc) and 100% of group III (recombinant human BMP-2) specimens. Radiographs demonstrated a lack of healing in group II (LV-RFPch). Mouse BMSCs transduced with an LV-RFPch-BMP-2 vector were able to induce host cells to differentiate down an osteoblastic lineage and heal a critical-sized defect. However, the donor cells appeared to be functioning as a delivery vehicle of BMP-2 rather than actually differentiating into osteoblasts capable of participating in bone repair as evidenced by a lack of colocalization of the transduced cells to the sites of skeletal repair where the host progenitor cells were found.
Assuntos
Células da Medula Óssea/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Regeneração Óssea , Fêmur/citologia , Fêmur/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Cicatrização , Animais , Células Cultivadas , Vetores Genéticos , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes/metabolismo , Células Estromais/metabolismo , Tíbia/citologia , Tíbia/metabolismo , Transdução GenéticaRESUMO
Human mutations and in vitro studies indicate that DLX3 has a crucial function in bone development, however, the in vivo role of DLX3 in endochondral ossification has not been established. Here, we identify DLX3 as a central attenuator of adult bone mass in the appendicular skeleton. Dynamic bone formation, histologic and micro-computed tomography analyses demonstrate that in vivo DLX3 conditional loss of function in mesenchymal cells (Prx1-Cre) and osteoblasts (OCN-Cre) results in increased bone mass accrual observed as early as 2 weeks that remains elevated throughout the lifespan owing to increased osteoblast activity and increased expression of bone matrix genes. Dlx3OCN-conditional knockout mice have more trabeculae that extend deeper in the medullary cavity and thicker cortical bone with an increased mineral apposition rate, decreased bone mineral density and increased cortical porosity. Trabecular TRAP staining and site-specific Q-PCR demonstrated that osteoclastic resorption remained normal on trabecular bone, whereas cortical bone exhibited altered osteoclast patterning on the periosteal surface associated with high Opg/Rankl ratios. Using RNA sequencing and chromatin immunoprecipitation-Seq analyses, we demonstrate that DLX3 regulates transcription factors crucial for bone formation such as Dlx5, Dlx6, Runx2 and Sp7 as well as genes important to mineral deposition (Ibsp, Enpp1, Mepe) and bone turnover (Opg). Furthermore, with the removal of DLX3, we observe increased occupancy of DLX5, as well as increased and earlier occupancy of RUNX2 on the bone-specific osteocalcin promoter. Together, these findings provide novel insight into mechanisms by which DLX3 attenuates bone mass accrual to support bone homeostasis by osteogenic gene pathway regulation.
Assuntos
Densidade Óssea/genética , Diferenciação Celular/genética , Proteínas de Homeodomínio/metabolismo , Homeostase/genética , Osteoblastos/citologia , Osteoblastos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Osso e Ossos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fatores de Transcrição/genéticaRESUMO
Mesenchymal stem cells (MSCs) derived from the adult bone marrow are multipotent stem cells that can give rise to lineages of bone, cartilage, muscle, fat, and others. The rabbit is a common preclinical model used for cardiovascular and orthopaedic applications. MSCs derived from the rabbit whole bone marrow are routinely investigated in these models for regenerative medicine applications. However, rabbit MSCs (rbMSCs) have not been extensively characterized in terms of immunophenotypic characteristics and differentiation potential and more specifically, in comparison to human MSCs (hMSCs). This study examined rbMSCs' surface antigens as well as their multipotent differentiation potential. In addition, the transduction efficiency of rbMSCs using a lentiviral vector with red fluorescent protein (RFP) as a method for labeling the cells for in vitro and in vivo use was also examined. RbMSCs were positive for CD44 and CD29 and negative for CD45 and CD14, which is similar to hMSCs, but rbMSCs did not express CD90. RbMSCs also expressed the pluripotent transcription factor, Sox2. The rbMSCs at early passages differentiated along the osteoblastic, chondrocytic, and adipocytic lineages. However, quantitative analyses demonstrated lower levels of differentiation markers for rabbit cells as compared to human cells. Transduction efficiency of 90.5% was observed for rbMSC transfected with RFP. Transduced cells also retained their osteogenic potential, but proliferation was reduced in comparison to nontransduced cells. This study demonstrates that MSCs isolated from the rabbit bone marrow have differences from human cells and should be considered when using rbMSCs in preclinical models for MSC regenerative medicine or tissue engineering strategies.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Antígenos de Superfície/metabolismo , Técnicas de Cultura de Células , Proliferação de Células , Condrócitos/citologia , Condrócitos/metabolismo , Masculino , Osteócitos/citologia , Osteócitos/metabolismo , Coelhos , Medicina Regenerativa , Engenharia TecidualRESUMO
'Ex vivo' gene therapy using viral vectors to overexpress BMP-2 is shown to heal critical-sized bone defects in experimental animals. To increase its safety, we constructed a dual-expression lentiviral vector to overexpress BMP-2 or luciferase and an HSV1-tk analog, Δtk (LV-Δtk-T2A-BMP-2/Luc). We hypothesized that administering ganciclovir (GCV) will eliminate the transduced cells at the site of implantation. The vector-induced expression of BMP-2 and luciferase in a mouse stromal cell line (W-20-17 cells) and mouse bone marrow cells (MBMCs) was reduced by 50% compared with the single-gene vector. W-20-17 cells were more sensitive to GCV compared with MBMCs (90-95% cell death at 12 days with GCV at 1 µg ml(-1) in MBMCs vs 90-95% cell death at 5 days by 0.1 µg ml(-1) of GCV in W-20-17 cells). Implantation of LV-Δtk-T2A-BMP-2 transduced MBMCs healed a 2 mm femoral defect at 4 weeks. Early GCV treatment (days 0-14) postoperatively blocked bone formation confirming a biologic response. Delayed GCV treatment starting at day 14 for 2 or 4 weeks reduced the luciferase signal from LV-Δtk-T2A-Luc-transduced MBMCs, but the signal was not completely eliminated. These data suggest that this suicide gene strategy has potential for clinical use in the future, but will need to be optimized for increased efficiency.
Assuntos
Células da Medula Óssea/metabolismo , Fraturas do Fêmur/terapia , Genes Transgênicos Suicidas , Terapia Genética/métodos , Simplexvirus/enzimologia , Células Estromais/metabolismo , Timidina Quinase/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/virologia , Transplante de Medula Óssea/métodos , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Linhagem Celular , Terapia Combinada/efeitos adversos , Fraturas do Fêmur/patologia , Ganciclovir/farmacologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Humanos , Lentivirus/efeitos dos fármacos , Lentivirus/genética , Luciferases/metabolismo , Masculino , Camundongos , Células Estromais/efeitos dos fármacos , Células Estromais/virologia , Timidina Quinase/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
During the phase of overt tooth cytodifferentiation that occurs after birth in the mouse and using the 3.6Collagen1a-Cre and the BMP4 floxed and BMP4 knockout mice, the BMP4 gene was deleted in early collagen producing odontoblasts around postnatal day 1. BMP4 expression was reduced over 90% in alveolar osteoblasts and odontoblasts. There was decreased rate of predentin to dentin formation and decreased mature odontoblast differentiation reflected in reduced DMP1 expression and proper dentinal tubule formation, as well as reduced Collagen type I and Osteocalcin expression. We observed mutant dysmorphogenic odontoblasts that failed to properly elongate and differentiate. The consequence of this failed differentiation process leads to permanent loss of dentin thickness, apparent enlarged pulp chambers in the molars and reduced bone supporting the tooth structures in mice as old as 10-12 months. Deletion of the BMP4 gene in odontoblasts also indirectly disrupted the process of enamel formation that persisted throughout life. The mechanism for this altered differentiation program in the absence of the BMP4 gene in odontoblasts is from decreased BMP signaling, and decreased expression of three key transcription factors, Dlx3, Dlx5, and Osterix. BMP signaling, as well as Dlx3 and Amelogenin expression, is also indirectly reduced in the ameloblasts of the odontoblast BMP4 cKO mice. This supports a key paracrine or endocrine postnatal role of odontoblast derived BMP4 on the proper amelogenesis and formation of the enamel.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular/fisiologia , Dente/citologia , Dente/metabolismo , Ameloblastos/citologia , Ameloblastos/metabolismo , Amelogenina/genética , Animais , Proteína Morfogenética Óssea 4/genética , Diferenciação Celular/genética , Esmalte Dentário/metabolismo , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Knockout , Odontoblastos/citologia , Odontoblastos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Fatores de Transcrição/genéticaRESUMO
OBJECTIVE: To determine the specificity and efficiency of the tamoxifen (TM)-induced Cre-recombination in articular chondrocytes of adult Col2a1-CreER(T2) transgenic mice. METHODS: Col2a1-CreER(T2) transgenic mice were bred with Rosa26 reporter mice. Two-week-old Col2a1-CreER(T2);R26R mice were administered TM for 5 days and were sacrificed 1 and 6 months after TM induction. X-Gal staining was performed. RESULTS: Efficient Cre-recombination is achieved in adult articular chondrocytes 1 and 6 months after TM induction. CONCLUSION: Our findings demonstrate that the Col2a1-CreER(T2) transgenic mouse model is a valuable tool to target genes specifically expressed in articular chondrocytes in a temporally controlled manner in adult mice.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Cartilagem Articular/enzimologia , Condrócitos/enzimologia , Integrases/metabolismo , Tamoxifeno/farmacologia , Animais , Células Cultivadas , Camundongos , Camundongos Transgênicos , Reação em Cadeia da PolimeraseRESUMO
Fibroblast growth factor-2 (FGF-2) is made by osteoblasts and modulates their function. There are high molecular weight (HMW) protein isoforms of FGF-2 that have nuclear localization sequences and a low molecular weight (LMW) 18 kDa FGF-2 protein that is exported from cells. Since FGF-2 is a trophic factor and potent mitogen for osteoblasts, the goal of this study was to utilize targeted overexpression of FGF-2 as a novel means of assessing different FGF-2 isoforms on osteoblastic cell viability and proliferation. Either LMW or HMW human Fgf2 cDNAs were cloned downstream of 3.6 kb alpha1(I)-collagen 5' regulatory elements (Col 3.6). A set of expression vectors, called Col3.6-Fgf2 isoforms-IRES-GFPsaph, capable of concurrently overexpressing either LMW or HMW FGF-2 isoforms concomitant with GFPsaph from a single bicistronic mRNA were built. Viable cell number in ROS 17/2.8 cells stably transfected with Vector (Col3.6-IRES-GFPsaph) versus each of the Col3.6-Fgf2-IRES-GFPsaph constructs were compared. In the presence of 1 or 10% serum, DNA synthesis was increased in cells expressing any isoform of FGF-2 compared with vector. However, cells transfected with HMW isoform had augmented DNA synthesis in 1 or 10% serum compared with cells expressing either ALL or LMW FGF-2 isoforms. A neutralizing FGF-2 antibody significantly reduced the mitogenic response in cells harboring ALL or the LMW FGF-2 isoforms but did not block the mitogenic effect of cells harboring the HMW isoforms. In summary, overexpression of any isoform of FGF-2 protein increased viable cell number and OB proliferation in the presence of low or high concentrations of serum. However, the HMW/nuclear isoforms preferentially mediate augmented OB proliferation. We conclude that differential expression of FGF-2 proteins isoforms is important in modulating OB function.
Assuntos
Fator 2 de Crescimento de Fibroblastos/biossíntese , Osteoblastos/metabolismo , Isoformas de Proteínas/biossíntese , Animais , Northern Blotting , Western Blotting , Divisão Celular/genética , Sobrevivência Celular , Clonagem Molecular , Fator 2 de Crescimento de Fibroblastos/genética , Imunofluorescência , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes , Microscopia de Fluorescência , Isoformas de Proteínas/genética , RNA Mensageiro/análise , Ratos , Fatores de Tempo , Transfecção , TransgenesRESUMO
Fibroblast growth factor 2 (FGF2) and noggin are two unrelated ligands of two distinctly different signaling pathways that have a similar inhibitory effect on osteoblast differentiation. Because of their differences, we postulated that they probably acted at a different stage within the osteoprogenitor differentiation pathway. This study was performed on primary murine bone cell cultures under conditions where alkaline phosphatase (AP) and type I collagen expression (Col1a1) were observed by day 7 (preosteoblast stage), followed by bone syaloprotein (BSP) at day 11 (early osteoblast) and osteocalcin (OC) by day 15-18 (mature osteoblast stage). FGF2 completely inhibited expression of AP and the mRNA transcript for Col1a1, while noggin showed only a partial inhibition of these markers of preosteoblast differentiation. However, the markers of differentiated osteoblasts (BSP and OC) were completely inhibited in both the FGF2 and noggin treated cultures, suggesting that noggin acts at later point in the osteoprogenitor differentiation pathway than FGF2. To further verify that the inhibition was occurring at a different stage of osteoblasts development, primary cultures derived from transgenic mice harboring segments of the collagen promoter driving green fluorescent protein (GFP) that activate at different levels of osteoblast differentiation were analyzed. Consistent with the endogenous markers, pOBCol3.6GFP and pOBCOL2.3GFP transgene activity was completely inhibited by continuous addition of FGF2, while noggin showed partial inhibition of pOBCol3.6GFP and complete inhibition of the pOBCol2.3GFP transgene. Upon removal of either agent, endogenous and GFP markers of osteoblast differentiation reappeared although at a different temporal pattern. This work demonstrates that FGF2 and noggin can reversibly modulate osteoblast lineage differentiation at different maturational stages. These agents may be useful to enrich for and maintain a population of osteoprogenitor cells at a defined stage of differentiation.
Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Inibidores do Crescimento/farmacologia , Osteoblastos/fisiologia , Células-Tronco/fisiologia , Animais , Proteínas de Transporte , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/genética , Terapia Genética , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Camundongos , Camundongos Transgênicos , Osteoblastos/efeitos dos fármacos , Proteínas/antagonistas & inibidores , Proteínas/genética , Proteínas/farmacologia , Células-Tronco/efeitos dos fármacos , Transgenes/efeitos dos fármacosRESUMO
Green fluorescent protein (GFP)-expressing transgenic mice were produced containing a 3.6-kilobase (kb; pOBCol3.6GFPtpz) and a 2.3-kb (pOBCol2.3GFPemd) rat type I collagen (Col1a1) promoter fragment. The 3.6-kb promoter directed strong expression of GFP messenger RNA (mRNA) to bone and isolated tail tendon and lower expression in nonosseous tissues. The 2.3-kb promoter expressed the GFP mRNA in the bone and tail tendon with no detectable mRNA elsewhere. The pattern of fluorescence was evaluated in differentiating calvarial cell (mouse calvarial osteoblast cell [mCOB]) and in marrow stromal cell (MSC) cultures derived from the transgenic mice. The pOBCol3.6GFPtpz-positive cells first appeared in spindle-shaped cells before nodule formation and continued to show a strong signal in cells associated with bone nodules. pOBCol2.3GFPemd fluorescence first appeared in nodules undergoing mineralization. Histological analysis showed weaker pOBCol3.6GFPtpz-positive fibroblastic cells in the periosteal layer and strongly positive osteoblastic cells lining endosteal and trabecular surfaces. In contrast, a pOBCol2.3GFPemd signal was limited to osteoblasts and osteocytes without detectable signal in periosteal fibroblasts. These findings suggest that Col1a1GFP transgenes are marking different subpopulations of cells during differentiation of skeletal osteoprogenitors. With the use of other promoters and color isomers of GFP, it should be possible to develop experimental protocols that can reflect the heterogeneity of cell differentiation in intact bone. In primary culture, this approach will afford isolation of subpopulations of these cells for molecular and cellular analysis.
Assuntos
Colágeno Tipo I/genética , Proteínas Luminescentes/genética , Osteoblastos/classificação , Osteoblastos/metabolismo , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Diferenciação Celular , Células Cultivadas , Fêmur/citologia , Fêmur/crescimento & desenvolvimento , Fêmur/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde , Camundongos , Camundongos Transgênicos , Osteoblastos/citologia , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/genética , Tendões/citologia , Tendões/metabolismo , Distribuição TecidualRESUMO
This study tested the transduction efficiency of human bone marrow stromal cells (hBMSCs) with vesicular stomatitis virus (VSV)-pseudotyped retrovectors and their subsequent osteogenic differentiation in vitro. Two different retrovectors encoding beta-galactosidase (beta-gal) or enhanced green fluorescent protein (eGFP) as marker genes were examined for transduction of hBMSCs. hBMSCs were obtained from bone marrow filtrates of normal donors (aged 5-35 years), cultured in alpha-minimal essential medium (alpha-MEM) containing 10% fetal calf serum and infected with retrovectors soon after the adherent cells started to form individual colonies. Transduced hBMSCs were observed to express eGFP protein 4-7 days after infection in primary cultures, and the majority of hBMSCs were eGFP-positive. hBMSCs were also stained for beta-gal in the secondary cultures and virtually all hBMSCs expressed beta-gal activity. Transduced hBMSCs were examined for their osteogenic potential. These cells were found to express markers of osteogenic differentiation, including alkaline phosphatase, type I collagen, bone sialoprotein, decorin, and osteocalcin, as strongly as uninfected control cells. Mineralization was also induced by dexamethasone in transduced cells as well as control cells. These results demonstrate that hBMSCs are highly susceptible to infection with VSV-pseudotyped retrovectors with the majority of cultured cells expressing the viral transgenes without antibiotic selection. Transduced cells retain their osteogenic potential in vitro. hBMSCs are a promising cellular vehicle for systemic human gene therapy and VSV-pseudotyped retrovectors should be effective for their in vitro transduction prior to cellular engraftment.
Assuntos
Células da Medula Óssea/fisiologia , Vetores Genéticos , Osteoblastos/fisiologia , Células Estromais/fisiologia , Vírus da Estomatite Vesicular Indiana , Adolescente , Adulto , Células da Medula Óssea/citologia , Diferenciação Celular/genética , Criança , Pré-Escolar , Proteínas de Fluorescência Verde , Humanos , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/genética , Osteoblastos/citologia , Células Estromais/citologia , Transdução Genética/métodos , beta-Galactosidase/genéticaRESUMO
AIM: To determine whether retrovirally expressed Dlx5, a homeobox-containing transcription factor, can induce a 2.3 kb rat COL1A1 promoter-reporter construct, which is transduced into osteoblastic cells by the use of a retrovirus vector. METHODS: A self-inactivating retrovirus vector containing the rat COL1A1 driving green fluorescent protein (GFP) was transduced into chick calvarial periosteal cells. These cells were then infected with a replication-competent retroviral vector expressing Dlx5, or a control vector. The cells were cultured in the presence of ascorbic acid and beta-glycerol-phosphate, which promotes osteoblastic differentiation. Expression of the COL1A1 promoter was assessed by detecting GFP with fluorescence microscopy. RESULTS: GFP was detected only in cells infected with the Dlx5 expressing retrovirus. The GFP positive cells were observed in regions of the culture that had undergone osteoblastic differentiation, as detected by cell morphology and the presence of a mineralized matrix. CONCLUSION: The 2.3 kb rat COL1A1 promoter fragment contains elements responsive to Dlx5, and the self-inactivating retroviral vector allows these elements to be used appropriately.
Assuntos
Colágeno Tipo I , Colágeno/metabolismo , Proteínas de Homeodomínio/fisiologia , Retroviridae/genética , Animais , Diferenciação Celular , Células Cultivadas , Embrião de Galinha , Cadeia alfa 1 do Colágeno Tipo I , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Ratos , TransfecçãoRESUMO
The modular organization of the type I collagen promoter allows creation of promoter-reporter constructs with preferential activity in different type I collagen-producing tissues that might be useful to mark cells at different stages of osteoblastic differentiation. Primary marrow stromal cell (MSC) and mouse calvarial osteoblast (mCOB) cultures were established from transgenic mice harboring different Col1a1 promoter fragments driving chloramphenicol acetyltransferase (CAT). In these models, Col1a1 messenger RNA (mRNA) and alkaline phosphatase (ALP) are the first markers of differentiation appearing soon after the colonies develop. Bone sialoprotein (BSP) is detected 2-3 days later, followed by osteocalcin (OC) expression and nodule mineralization. A 3.6 Col1a1 fragment (ColCAT3.6) initiated activity concomitant with ALP staining and type I collagen mRNA expression. In contrast, a 2.3 Col1a1 fragment (ColCAT2.3) became active coincident with BSP expression. The pattern of transgene expression assessed by immunostaining was distinctly different. ColCAT3.6 was expressed within and at the periphery of developing nodules whereas the ColCAT2.3 expression was restricted to the differentiated nodules. The feasibility of using green fluorescent protein (GFP) as a marker of osteoblast differentiation was evaluated in ROS17/2.8 cells. A 2.3-kilobase (kb) Col1a1 promoter driving GFP (pOB4Col2.3GLP) was stably transfected into the cell line and positive clones were selected. Subcultures lost and then regained GFP expression that was localized in small clusters of cells throughout the culture. This suggests that expression from the 2.3-kb Col1A1 fragment is determined by the state of differentiation of the ROS17/2.8 cells. Col1a1 transgenes should be useful in appreciating the heterogeneity of a primary or immortalized culture undergoing osteoblastic differentiation.
Assuntos
Linhagem da Célula/genética , Colágeno Tipo I , Colágeno/genética , Osteoblastos/citologia , Regiões Promotoras Genéticas/genética , Transgenes/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Genes Reporter/genética , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Células Estromais/citologia , Células Estromais/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Marrow stromal cells (MSC) and neonatal calvarial cells have the potential to differentiate and express markers of mature osteoblasts. Furthermore, MSCs can generate multiple differentiated connective tissue phenotypes. These properties and their ability to be expanded ex vivo make them good models for ex vivo gene therapy. In this study we examined the ability of vesicular stomatitis virus (VSV-G) pseudotyped retroviral vectors to transduce osteoprogenitor cells derived from bone marrow and from neonatal calvaria. Retrovectors encoding either beta-galactosidase or green fluorescent protein (eGFP) were used for transduction of primary murine marrow stromal and primary neonatal calvarial cell cultures. High infection efficiency was demonstrated by fluorescence-activated cell analysis when GFP was used as a marker or by estimating the number of beta-galactosidase-positive cells. Expression of markers of differentiated bone cells, including Col1a1, bone sialoprotein, and osteocalcin mRNA and alkaline phosphatase activity was not impaired by retroviral transduction. Our data suggest that VSV-G pseudotypes retroviral vectors are suitable for introducing genes into osteoprogenitor cells without affecting osteoprogenitor lineage progression.
Assuntos
Glicoproteínas de Membrana , Osteoblastos/virologia , Retroviridae , Transformação Genética , Proteínas do Envelope Viral/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Vetores Genéticos , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , CamundongosRESUMO
Although the primary function of U1 snRNA is to define the 5' donor site of an intron, it can also block the accumulation of a specific RNA transcript when it binds to a donor sequence within its terminal exon. This work was initiated to investigate if this property of U1 snRNA could be exploited as an effective method for inactivating any target gene. The initial 10-bp segment of U1 snRNA, which is complementary to the 5' donor sequence, was modified to recognize various target mRNAs (chloramphenicol acetyltransferase [CAT], beta-galactosidase, or green fluorescent protein [GFP]). Transient cotransfection of reporter genes and appropriate U1 antitarget vectors resulted in >90% reduction of transgene expression. Numerous sites within the CAT transcript were suitable for targeting. The inhibitory effect of the U1 antitarget vector is directly related to the hybrid formed between the U1 vector and target transcripts and is dependent on an intact 70,000-molecular-weight binding domain within the U1 gene. The effect is long lasting when the target (CAT or GFP) and U1 antitarget construct are inserted into fibroblasts by stable transfection. Clonal cell lines derived from stable transfection with a pOB4GFP target construct and subsequently stably transfected with the U1 anti-GFP construct were selected. The degree to which GFP fluorescence was inhibited by U1 anti-GFP in the various clonal cell lines was assessed by fluorescence-activated cell sorter analysis. RNA analysis demonstrated reduction of the GFP mRNA in the nuclear and cytoplasmic compartment and proper 3' cleavage of the GFP residual transcript. An RNase protection strategy demonstrated that the transfected U1 antitarget RNA level varied between 1 to 8% of the endogenous U1 snRNA level. U1 antitarget vectors were demonstrated to have potential as effective inhibitors of gene expression in intact cells.
Assuntos
Expressão Gênica , Marcação de Genes/métodos , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Células 3T3 , Animais , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , DNA/genética , Genes Reporter , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Camundongos , Transfecção , beta-Galactosidase/genéticaRESUMO
Gene therapy of bone would benefit from the availability of vectors that provide stable, osteoblast-specific expression. This would allow bone-specific expression of Col1a1 cDNAs for treatment of osteogenesis imperfecta. In addition, such a vector would restrict expression of secreted therapeutic proteins to the bone-synthesizing regions of the bone marrow after ex vivo transduction of marrow stromal cells and reintroduction of the cells into patients. Retrovirus vectors stably integrate into target cell genomes; however, long-term regulated expression from internal cellular promoters has not been consistently achieved. In some cases this is due to a stem cell-specific mechanism for transcriptional repression of retroviruses. We evaluated the ability of self-inactivating ROSA-derived vectors containing a bone-directed 2.3-kb rat Col1a1 promoter to display osteoblast-specific expression. In vitro expression was examined in bone marrow stromal cell cultures induced to undergo osteoblastic differentiation. In vivo expression was evaluated in chimeric mice derived from transduced embryonic stem cells. The results indicate that self-inactivating retrovirus vectors containing the Col1a1 promoter are not permanently inactivated in embryonic stem cells and are specifically expressed in osteoblasts in vivo and in vitro. Thus these vectors should be useful for bone-directed gene therapy.
Assuntos
Células da Medula Óssea/citologia , Osso e Ossos/metabolismo , Colágeno Tipo I , Colágeno/genética , Camundongos Transgênicos , Regiões Promotoras Genéticas , Retroviridae/genética , Animais , Linhagem Celular , Células Cultivadas , Cadeia alfa 1 do Colágeno Tipo I , DNA Complementar/metabolismo , Embrião de Mamíferos/citologia , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/biossíntese , Camundongos , Microscopia de Contraste de Fase , Modelos Genéticos , Osteoblastos/metabolismo , Ratos , Células-Tronco/metabolismo , Fatores de Tempo , Transdução Genética , Transfecção , beta-Galactosidase/metabolismoRESUMO
Two transgenic mouse lines were generated with a DNA construct bearing a 2.3-kilobase (kb) fragment of the rat alpha1 type I collagen promoter driving a truncated form of the herpes thymidine kinase gene (Col2.3Atk). Expression of the transgene was found in osteoblasts coincident with other genetic markers of early osteoblast differentiation. Mice treated with ganciclovir (GCV) for 16 days displayed extensive destruction of the bone lining cells and decreased osteoclast number. In addition, a dramatic decrease in bone marrow elements was observed, which was more severe in the primary spongiosum and marrow adjacent to the diaphyseal endosteal bone. Immunostaining for transgene expression within the bone marrow was negative and marrow stromal cell cultures developed normally in the presence of GCV until the point of early osteoblast differentiation. Our findings suggest that the early differentiating osteoblasts are necessary for the maintenance of osteoclasts and hematopoiesis. Termination of GCV treatment produced an exaggerated response of new bone formation in cortical and trabecular bone. The Col2.3deltatk mouse should be a useful model to define the interrelation between bone and marrow elements as well as a model to analyze the molecular and cellular events associated with a defined wave of osteogenesis on termination of GCV treatment.
Assuntos
Osteoblastos/citologia , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores , Diferenciação Celular , Linhagem da Célula , Chlorocebus aethiops , Colágeno Tipo I/genética , Ganciclovir/farmacologia , Expressão Gênica , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Ratos , Sialoglicoproteínas/metabolismo , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/genética , Células VeroRESUMO
We examined the effect of parathyroid hormone and various signaling molecules on collagen synthesis and chloramphenicol acetyltransferase activity in cultured transgenic mouse calvariae carrying fusion genes of the rat Col1a1 promoter and the chloramphenicol acetyltransferase reporter. After 48 h of culture, parathyroid hormone, forskolin, dibutyryl cAMP, 8-bromo cAMP, and phorbol myristate acetate inhibited transgene activity, while the calcium ionophore ionomycin had no effect. Pretreatment of calvariae with the phosphodiesterase inhibitor isobutylmethylxanthine potentiated the inhibitory effect of 1 nM parathyroid hormone on transgene activity and collagen synthesis. Parathyroid hormone further inhibited transgene activity and collagen synthesis in the presence of phorbol myristate acetate. Parathyroid hormone inhibition of transgene activity and collagen synthesis was not affected by indomethacin or interleukin-6. After 48 h of culture, parathyroid hormone inhibited chloramphenicol acetyltransferase activity by 50-85% in cultured calvariae carrying transgenes having progressive 5' upstream deletions of promoter DNA down to -1683 bp. These data show that the inhibitory effect of parathyroid hormone on Col1a1 expression in mouse calvariae is mediated mainly by the cAMP signaling pathway. Prostaglandins and IL-6 are not local mediators of the parathyroid hormone response in this model. Finally, regions of the Col1a1 promoter downstream of -1683 bp are sufficient for parathyroid hormone inhibition of the Col1a1 promoter.
Assuntos
Colágeno/biossíntese , Colágeno/genética , Hormônio Paratireóideo/farmacologia , Crânio/metabolismo , Animais , AMP Cíclico/metabolismo , Camundongos , Camundongos Transgênicos , Pró-Colágeno/genética , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Msx2 is believed to play a role in regulating bone development, particularly in sutures of cranial bone. In this study we investigated the effects of retroviral-mediated overexpression of Msx2 mRNA, in both sense and antisense orientations, on primary cultured chick calvarial osteoblasts. Unregulated overexpression of sense mRNA produced high levels of Msx2 protein throughout the culture period, preventing the expected fall as the cells differentiate. The continued high expression of Msx2 prevented osteoblastic differentiation and mineralization of the extracellular matrix. In contrast, expression of antisense Msx2 RNA decreased proliferation and accelerated differentiation. In other studies, we showed that the Msx2 promoter was widely expressed during the proliferative phase of mouse calvarial osteoblast cultures but was preferentially downregulated in osteoblastic nodules. These results support a model in which Msx2 prevents differentiation and stimulates proliferation of cells at the extreme ends of the osteogenic fronts of the calvariae, facilitating expansion of the skull and closure of the suture.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Osteoblastos/efeitos dos fármacos , RNA Antissenso/genética , Crânio/embriologia , Animais , Calcificação Fisiológica , Divisão Celular , Células Cultivadas , Embrião de Galinha , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos/genética , Proteínas de Homeodomínio , Morfogênese , Osteoblastos/citologia , RNA Mensageiro/genética , Retroviridae/genética , Crânio/citologia , TransfecçãoRESUMO
To identify candidate cis-acting regulatory regions involved in regulation of the Msx2 gene in anterior limb mesenchyme and calvarial osteoblasts, DNase I hypersensitive sites (DHSs) from -6.1 kb to +8.4 kb relative to the translation start site of the chicken Msx2 gene were identified in anterior and posterior limb mesenchyme, calvarial osteoblasts, and embryonic fibroblasts. A total of 12 DHSs were detected. Except for the DHS in the basal promoter region, none of the other DHSs were present in all four tissues, suggesting that the chromatin structure in the Msx2 gene locus is differently organized in these four cell types. One DHS was unique to Msx2 expressing cells and a second site to nonexpressing cells. Anterior and posterior limb mesenchyme had similar patterns of DHSs that were much more complex than observed in calvarial osteoblasts possibly reflecting differences in the complexity of Msx2 regulation in these two tissues.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Desoxirribonuclease I/metabolismo , Fibroblastos/química , Mesoderma/química , Osteoblastos/química , Animais , Células Cultivadas , Embrião de Galinha , Cromatina/genética , Desoxirribonuclease EcoRI , Extremidades/embriologia , Proteínas de Homeodomínio , Regiões Promotoras Genéticas , Mapeamento por Restrição , Crânio/embriologiaRESUMO
We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to -3518, -2297, -1997, -1794, -1763, and -1719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2D3 had a dose-dependent inhibitory effect on the expression of the -3518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2D3, suggesting that the sequences required for 1, 25(OH)2D3 inhibition are downstream of -1719 bp. The inhibitory effect of 1,25(OH)2D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1, 25(OH)2D3 responsive element downstream of -1719 bp. The inhibitory effect does not require new protein synthesis.