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1.
Proc Natl Acad Sci U S A ; 110(31): 12643-8, 2013 Jul 30.
Article in English | MEDLINE | ID: mdl-23858471

ABSTRACT

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.


Subject(s)
Bone and Bones/metabolism , Cartilage/metabolism , Hematopoiesis/physiology , Hematopoietic Stem Cells/metabolism , Signal Transduction/physiology , Stem Cell Niche/physiology , Animals , Antigens, Differentiation/biosynthesis , Antigens, Differentiation/genetics , Bone and Bones/cytology , Cartilage/cytology , Cytokines/genetics , Cytokines/metabolism , Gene Expression Regulation/physiology , Hematopoietic Stem Cells/cytology , Mice , Mice, Transgenic , Stromal Cells/cytology , Stromal Cells/metabolism
2.
Nature ; 457(7228): 490-4, 2009 Jan 22.
Article in English | MEDLINE | ID: mdl-19078959

ABSTRACT

Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.


Subject(s)
Cartilage/cytology , Hematopoietic Stem Cells/cytology , Osteogenesis/physiology , Stem Cell Niche/cytology , Stem Cell Niche/physiology , Animals , Antigens, CD/metabolism , Cartilage/embryology , Choristoma , Fetus/cytology , Hematopoietic Stem Cells/metabolism , Mandible/cytology , Mandible/embryology , Mice , Mice, Inbred C57BL , Skull/cytology , Skull/embryology , Sp7 Transcription Factor , Thy-1 Antigens/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/metabolism
3.
Growth Factors ; 26(4): 226-37, 2008 Aug.
Article in English | MEDLINE | ID: mdl-19021035

ABSTRACT

Pharmacological glucocorticoids (GCs) inhibit bone formation, leading to osteoporosis. GCs inhibit bone morphogenetic protein-2 (Bmp2) expression, and rhBMP-2 restores mineralization in GC-arrested osteoblast cultures. To better understand how GCs regulate BMPs, we investigated Bmp transcription, as well as rhBMP-induced Smad and alkaline phosphatase (ALP) activity. Bmp2 cis-regulatory regions were analyzed by reporter plasmids and LacZ-containing bacterial artificial chromosomes. We found that GCs inhibited Bmp2 via a domain > 50 kb downstream of the coding sequence. Bmp expression was evaluated by RT-PCR; whereas GCs strongly inhibited Bmp2, Bmp4 was abundantly expressed and resistant to GCs. Both rhBMP-2 and rhBMP-4 restored mineralization in GC-arrested cultures; rhBMP-2 was 5-fold more effective when dosing was based on ALP activation, however, the rhBMPs were equipotent when dosing was based on Smad transactivation. In conclusion, GCs regulate Bmp2 via a far-downstream domain, and activation of Smad, not ALP, best predicts the pro-mineralization potential of rhBMPs.


Subject(s)
Bone Morphogenetic Protein 2/metabolism , Bone Morphogenetic Protein 4/metabolism , Calcification, Physiologic/drug effects , Gene Expression/drug effects , Glucocorticoids/pharmacology , Osteoblasts/drug effects , 3T3 Cells , Alkaline Phosphatase/analysis , Alkaline Phosphatase/metabolism , Animals , Bone Morphogenetic Protein 2/genetics , Bone Morphogenetic Protein 4/genetics , Calcification, Physiologic/genetics , Genes, Reporter , Humans , Luciferases/metabolism , Mice , Osteoblasts/metabolism , Recombinant Proteins/metabolism , Signal Transduction/drug effects , Transfection , beta-Galactosidase/analysis , beta-Galactosidase/metabolism
4.
Bone ; 41(1): 39-51, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17459803

ABSTRACT

Integrins link the inside of a cell with its outside environment and in doing so regulate a wide variety of cell behaviors. Integrins are well known for their roles in angiogenesis and cell migration but their functions in bone formation are less clear. The majority of integrin signaling proceeds through focal adhesion kinase (FAK), an essential component of the focal adhesion complex. We generated transgenic mice in which FAK was deleted in osteoblasts and uncovered a previously unknown role in osteoblast differentiation associated with bone healing. FAK mutant cells migrated to the site of skeletal injury and angiogenesis was unaffected yet the transgenic mice still exhibited numerous defects in reparative bone formation. Osteoblast differentiation itself was unperturbed by the loss of FAK, whereas the attachment of osteoclasts to the bone matrix was disrupted in vivo. We postulate that defective bi-directional integrin signaling affects the organization of the collagen matrix. Finally, we present a compensatory candidate molecule, Pyk2, which localized to the focal adhesions in osteoblasts that were lacking FAK.


Subject(s)
Bone Regeneration/physiology , Bone Remodeling/physiology , Focal Adhesion Kinase 1/physiology , Osteoblasts/cytology , Animals , Base Sequence , Bone Matrix/cytology , Bone Regeneration/genetics , Bone Remodeling/genetics , Cell Adhesion , Cell Differentiation/genetics , Cell Differentiation/physiology , DNA Primers/genetics , Female , Fetal Development , Focal Adhesion Kinase 1/deficiency , Focal Adhesion Kinase 1/genetics , Focal Adhesion Kinase 2/physiology , Heterozygote , In Vitro Techniques , Mice , Mice, Knockout , Mice, Transgenic , Osteoclasts/cytology , Pregnancy , Signal Transduction
5.
J Mol Endocrinol ; 33(1): 175-93, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15291752

ABSTRACT

Glucocorticoid (GC) treatment for the management of autoimmune and inflammatory diseases is associated with decreased bone formation and increased risk for fracture. In MC3T3-E1 cell cultures, 0.1-1 microM dexamethasone (DEX) arrests development of the osteoblast phenotype when administration commences at a commitment stage around the time of confluency. To gain new insights into GC-induced osteoporosis, we performed microarray-based gene expression analysis of GC-arrested MC3T3-E1 cultures, 2.5 days after the administration of DEX. Of the >12 000 transcripts interrogated, 74 were up-regulated and 17 were down-regulated by at least 2.5-fold (P < or = 0.05). Some of these genes, such as Mmp13, Serum/GC-regulated kinase and Tieg, have previously been reported as GC-responsive. Others are shown here for the first time to respond to GCs. DEX strongly repressed Krox20/Egr2 at both the mRNA and the protein level. This is especially significant because mice lacking this transcription factor develop osteoporosis. The data also suggest that the bone morphogenetic protein (BMP) pathway, which is involved in regulating bone mass, and other pathways that influence BMP signaling, are abrogated by GCs: (i) DEX increased the mRNA levels of the BMP antagonists Follistatin and Dan; (ii) DEX increased the levels of p21 Rasgap3 and Ptpn16/MKP-1 mRNAs, negative regulators of the MAP kinase pathway; and (iii) DEX decreased Cox mRNA levels. DEX also increased thrombospondin mRNA levels, which negatively regulate bone mass in vivo, as well as the adipocytic marker Fkbp51. These and other observations disclose novel gene targets, whose regulation by GCs in osteoblasts may shed light on and provide new therapeutic approaches to osteoporosis.


Subject(s)
Dexamethasone/pharmacology , Gene Expression Profiling , Osteoblasts/drug effects , 3T3 Cells , Animals , Base Sequence , Blotting, Western , DNA Primers , Mice , Oligonucleotide Array Sequence Analysis , Osteoblasts/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics
6.
J Biol Chem ; 278(45): 44995-5003, 2003 Nov 07.
Article in English | MEDLINE | ID: mdl-12933820

ABSTRACT

Glucocorticoids (GCs) inhibit bone formation in vivo. In MC3T3-E1 osteoblasts, chronic administration of 1 microm dexamethasone (DEX) starting at confluency results in >98% inhibition of bone morphogenetic protein 2 (BMP-2) expression and apatite mineral deposition. Here, it is shown that brief exposure to recombinant human BMP-2 (rhBMP-2), as short as 6 h, is sufficient to induce irreversible commitment to mineralization in DEX-treated cultures. RhBMP-2 dose dependently rescued mineralization but not collagen accumulation in DEX-inhibited cultures. The selective restoration of mineralization was evident in the higher mineral to matrix ratios of DEX/rhBMP-2 co-treated cultures compared with control. We tested the involvement of the runt-related transcription factor 2 (Runx2) in the DEX inhibition and rhBMP-2 rescue of mineralization. Surprisingly, DEX did not decrease Runx2 DNA binding activity, transactivation, or association with the endogenous osteocalcin gene promoter. Furthermore, the rhBMP-2 rescue did not involve Runx2 stimulation, suggesting an important role for factors other than Runx2 in BMP-2 action. Finally, we studied the differentiation-related cell cycle, which persists during commitment to mineralization in untreated cultures, but is inhibited along with mineralization in DEX-treated cultures. Although both rhBMP-2 alone and DEX alone were antimitogenic, rhBMP-2 stimulated this cell cycle in DEX-inhibited cultures. In conclusion, brief rhBMP-2 treatment restores mineralization in DEX-inhibited MC3T3-E1 osteoblasts via a mechanism different from Runx2 stimulation. This restoration may be functionally related to the accompanying rescue of the differentiation-related cell cycle. The efficacy of short term BMP-2 treatment supports the potential of short-lived BMP vectors for skeletal therapy in both traditional and gene therapeutic approaches.


Subject(s)
Bone Morphogenetic Proteins/pharmacology , Calcification, Physiologic/drug effects , Cell Cycle/drug effects , Glucocorticoids/pharmacology , Neoplasm Proteins , Osteoblasts/drug effects , Transforming Growth Factor beta , Alkaline Phosphatase/metabolism , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/genetics , Cell Differentiation , Cell Line , Core Binding Factor Alpha 1 Subunit , DNA/metabolism , Dexamethasone/pharmacology , Humans , Osteoblasts/cytology , Osteoblasts/metabolism , Osteocalcin/genetics , Promoter Regions, Genetic/genetics , Recombinant Proteins/pharmacology , Spectroscopy, Fourier Transform Infrared , Transcription Factors/metabolism , Transfection
7.
J Bone Miner Res ; 18(7): 1186-97, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12854828

ABSTRACT

UNLABELLED: The anti-glucocorticoid potential of BMP-2 in osteoblasts was tested in MC3T3-E1 cells using dexamethasone (1 microM) and rhBMP-2 (10 or 100 ng/ml). rhBMP-2 restored mineralization but not condensation or collagen accumulation. These results demonstrate the potential and limitations of BMPs in counteracting glucocorticoids. INTRODUCTION: Pharmacologic glucocorticoids (GCs) inhibit osteoblast function and induce osteoporosis. Bone morphogenetic proteins (BMPs) stimulate osteoblast differentiation and bone formation. Here we tested the anti-glucocorticoid potential of BMP-2 in cultured osteoblasts. MATERIALS AND METHODS: MC3T3-E1 cells were treated with dexamethasone (DEX; 1 microM) and/or recombinant human BMP-2 (rhBMP-2; 10 or 100 ng/ml). Culture progression was characterized by cell cycle profiling, biochemical assays for DNA, alkaline phosphatase (ALP), collagen, and calcium, and by reverse transcriptase-polymerase chain reaction (RT-PCR) of osteoblast phenotypic mRNAs. Mineralization was characterized by Alizarin red and von Kossa staining and by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). RESULTS: DEX inhibited differentiation-related cell cycle, nodule formation, collagen accumulation, osteocalcin, and BMP-2 gene expression as well as mineralization. Replenishment of GC-inhibited cultures with 10 or 100 ng/ml rhBMP-2 dramatically rescued mineral deposition. The rhBMP-2-rescued mineral was bone-like apatite nearly identical to the mineral of control cultures. The rhBMP-2 rescue was associated with increased mRNA levels for alpha1(I) collagen, osteocalcin, and Cbfa1 types I and II, as well as ALP activity. In contrast, rhBMP-2 did not rescue the GC-inhibited differentiation-related cell cycle, nodule formation, or collagen accumulation. When administered alone, rhBMP-2 also increased the mRNA levels for alpha1(I) collagen, osteocalcin, and Cbfa1 types I and II, as well as ALP activity. However, treatment with rhBMP-2 alone inhibited cell cycle progression, nodule formation, and collagen accumulation. Surprisingly, in contrast to its rescue of mineralization in DEX-treated cultures, rhBMP-2 inhibited mineralization in the absence of DEX. In parallel to its bimodal effect on mineralization, rhBMP-2 stimulated endogenous BMP-2 mRNA in the presence of DEX, but inhibited endogenous BMP-2 mRNA in the absence of DEX. CONCLUSIONS: Suppression of BMP-2 gene expression plays a pivotal role in GC inhibition of osteoblast differentiation. However, the inability of rhBMP-2 to rescue the entire osteoblast phenotype suggests BMP-2-independent inhibitory effects of CCs. BMP-2 exerts both positive and negative effects on osteoblasts, possibly depending on the differentiation stage and/or the existing BMP signaling.


Subject(s)
Bone Morphogenetic Proteins/pharmacology , Calcification, Physiologic/drug effects , Calcium/metabolism , Glucocorticoids/pharmacology , Osteoblasts/drug effects , Transforming Growth Factor beta , Animals , Blotting, Northern , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/genetics , Bone Morphogenetic Proteins/metabolism , Cell Line , Collagen/metabolism , Gene Expression Regulation/drug effects , Humans , Kinetics , Mice , Osteoblasts/metabolism , Osteocalcin/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins
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