Biomedical subjects

G Karsenty

Publications and source records attributed to G Karsenty.

At least 19 recordsLinked to original sources

PubMed1997

Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation.

The osteoblast is the bone-forming cell. The molecular basis of osteoblast-specific gene expression and differentiation is unknown. We previously identified an osteoblast-specific cis-acting element, termed OSE2, in the Osteocalcin promoter. We have now cloned the cDNA encoding Osf2/Cbfa1, the protein that binds to OSE2. Osf2/Cbfa1 expression is initiated in the mesenchymal condensations of the developing skeleton, is strictly restricted to cells of the osteoblast lineage thereafter, and is regulated by BMP7 and vitamin D3. Osf2/Cbfa1 binds to and regulates the expression of multiple genes expressed in osteoblasts. Finally, forced expression of Osf2/Cbfa1 in nonosteoblastic cells induces the expression of the principal osteoblast-specific genes. This study identifies Osf2/Cbfa1 as an osteoblast-specific transcription factor and as a regulator of osteoblast differentiation.

Amino Acid Sequence↗

PubMed1997

Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein.

Calcification of the extracellular matrix (ECM) can be physiological or pathological. Physiological calcification occurs in bone when the soft ECM is converted into a rigid material capable of sustaining mechanical force; pathological calcification can occur in arteries and cartilage and other soft tissues. No molecular determinant regulating ECM calcification has yet been identified. A candidate molecule is matrix GLA protein (Mgp), a mineral-binding ECM protein synthesized by vascular smooth-muscle cells and chondrocytes, two cell types that produce an uncalcified ECM. Mice that lack Mgp develop to term but die within two months as a result of arterial calcification which leads to blood-vessel rupture. Chondrocytes that elaborate a typical cartilage matrix can be seen in the affected arteries. Mgp-deficient mice additionally exhibit inappropriate calcification of various cartilages, including the growth plate, which eventually leads to short stature, osteopenia and fractures. These results indicate that ECM calcification must be actively inhibited in soft tissues. To our knowledge, Mgp is the first inhibitor of calcification of arteries and cartilage to be characterized in vivo.

Animals↗

PubMed1997

1,25-dihydroxyvitamin D3 inhibits Osteocalcin expression in mouse through an indirect mechanism.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), a key regulator of mineral metabolism, regulates the expression of several genes that are expressed in osteoblasts. In particular, in rat and human osteoblasts, 1,25-(OH)2D3 increases the expression of Osteocalcin by interacting, through a hormone-receptor complex, with a vitamin D-responsive element present in the promoter of the genes. Here we show that in mouse, 1,25-(OH)2D3 inhibits the expression of both osteocalcin genes, OG1 and OG2. This inhibition was observed in primary osteoblast cultures and in the whole animal. From sequence inspection, DNA transfection experiments, and DNA binding assays, we could not identify a functional vitamin D-responsive element in the promoter of OG2 or in the first 3.3 kilobases of the OG1 promoter. However, we show that 1,25-(OH)2D3 treatment of primary osteoblasts abolishes the binding of OSF2, an osteoblast-specific activator of transcription that binds to OSE2, a critical osteoblast-specific cis-acting element present in OG1 and OG2 promoters. Consistent with these DNA binding data, a mutation in OSE2 in the OG2 promoter abrogated the inhibitory effect of 1,25-(OH)2D3 treatment on this promoter activity. This study illustrates that 1,25-(OH)2D3 can play different roles in the expression of the same gene in various species and indicates that this regulation in mouse occurs through an indirect mechanism, 1,25-(OH)2D3 acting on a gene genetically located upstream of Osteocalcin.

Animals↗

PubMed1997

Missense mutations abolishing DNA binding of the osteoblast-specific transcription factor OSF2/CBFA1 in cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder characterized by hypoplastic or absent clavicles, large fontanelles, dental anomalies and delayed skeletal development. The phenotype is suggestive of a generalized defect in ossification and is one of the most common skeletal dysplasias not associated with disproportionate stature. To date, no genetic determinants of ossification have been identified. CCD has been mapped to chromosome 6p21, where CBFA1, a gene encoding OSF2/CBFA1, a transcriptional activator of osteoblast differentiation, has been localized. Here, we describe two de novo missense mutations, Met175Arg and Ser191Asn, in the OSF2/CBFA1 gene in two patients with CCD. These two mutations result in substitution of highly conserved amino acids in the DNA-binding domain. DNA-binding studies with the mutant polypeptides show that these amino acid substitutions abolish the DNA-binding ability of OSF2/CBFA1 to its known target sequence. Concurrent studies show that heterozygous nonsense mutations in OSF2/CBFA1 also result in CCD, while mice homozygous for the osf2/cbfa1 mull allele exhibit a more severe lethal phenotype. Thus, these results together suggest that CCD is produced by haploinsufficiency of OSF2/CBFA1 and provide direct genetic evidence that the phenotype is secondary to an alteration of osteoblast differentiation.

Amino Acid Sequence↗

PubMed1997

Identification of an RNA sequence within an intracisternal-A particle element able to replace Rev-mediated posttranscriptional regulation of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) replication depends on the posttranscriptional regulation by the viral Rev protein and can be replaced with the posttranscriptional RNA control element (CTE) of the type D simian retroviruses. We have identified a sequence which shares only nucleotide sequences of the internal loops and secondary structure with the CTE and which is part of a novel murine intracisternal-A particle (IAP) retroelement, inserted within the transcribed mouse osteocalcin-related gene. This sequence, named CTE(IAP), can replace the Rev-mediated regulation of HIV-1, hence it is a posttranscriptional regulatory element. Related elements have been identified in other IAPs. These results suggest that insertional mutagenesis can affect gene expression by providing a functional posttranscriptional control element. The CTE(IAP) and CTEs of the type D simian retroviruses represent a novel class of RNA elements characterized by unique sequences within the internal loops which are predicted to represent the interaction site with cellular factor(s). These findings suggest that such elements may be involved in posttranscriptional regulation of cellular mRNAs.

Amino Acid Sequence↗

PubMed1997

Ascorbic acid-dependent activation of the osteocalcin promoter in MC3T3-E1 preosteoblasts: requirement for collagen matrix synthesis and the presence of an intact OSE2 sequence.

Osteocalcin is a hormonally regulated calcium-binding protein made almost exclusively by osteoblasts. In normal cells, osteocalcin expression requires ascorbic acid (AA), an essential cofactor for osteoblast differentiation both in vivo and in vitro. To determine the mechanism of this regulation, subclones of MC3T3-E1 preosteoblasts were transiently transfected with 1.3 kb of the mouse osteocalcin gene 2 promoter driving expression of firefly luciferase. AA stimulated luciferase activity 20-fold after 4-5 days. This response was stereospecific to L-ascorbic acid and was only detected in MC3T3-E1 subclones showing strong AA induction of the endogenous osteocalcin gene. Similar results were also obtained in MC3T3-E1 cells stably transfected with the osteocalcin promoter. A specific inhibitor of collagen synthesis, 3,4-dehydroproline, blocked AA-dependent induction of promoter activity, indicating that regulation of the osteocalcin gene requires collagen matrix synthesis. Deletion analysis of the mOG2 promoter identified an essential region for AA responsiveness between -147 and -116 bp. This region contains a single copy of the previously described osteoblast-specific element, OSE2. Deletion and mutation of OSE2 in DNA transfection assays established the requirement for this element in the AA response. Furthermore, DNA-binding assays revealed that MC3T3-E1 cells contain OSF2, the nuclear factor binding to OSE2, and that binding of OSF2 to OSE2 is up-regulated by AA treatment. Taken collectively, our results indicate that an intact OSE2 sequence is required for the induction of osteocalcin expression by AA.

Animals↗

PubMed1996

Osteocalcin promoter-based toxic gene therapy for the treatment of osteosarcoma in experimental models.

Osteocalcin (OC), a noncollagenous bone matrix protein, is expressed in high levels by osteoblasts. To determine whether the OC promoter mediates cell-specific gene expression in cells of osteoblast lineage, we constructed a recombinant adenovirus, Ad-OC-TK, which contains the OC promoter that drives the expression of herpes simplex virus thymidine kinase (TK). We tested the expression of TK by this virus in osteoblast cell lines as well as in non-osteoblastic cell lines by assessing the enzyme activity of TK in vitro. Whereas the OC promoter failed to drive the expression of the TK gene in several non-osteoblastic cell lines such as WH, a human bladder transitional carcinoma, and NIH 3T3, an embryonic mouse fibroblast cell line, the OC promoter mediated high levels of expression in osteoblast cell lines including murine ROS and human MG-63 cells. The addition of acyclovir (ACV), a pro-drug for the inhibition of cell proliferation, resulted in the induction of osteoblast-specific cell death in vitro. Intratumoral injection of Ad-OC-TK into murine ROS osteosarcoma abolished tumor growth in a host treated with subsequent i.p. ACV injection in vivo. The Ad-OC-TK virus plus ACV treatment appears to be highly selective in blocking the growth of both murine and human osteosarcoma cell lines in vitro and murine osteosarcoma in vivo.

3T3 Cells↗

PubMed1996

c-Krox binds to several sites in the promoter of both mouse type I collagen genes. Structure/function study and developmental expression analysis.

We have previously shown that c-Krox is a zinc finger protein that can increases the transcriptional activity of the mouse alpha1(I) collagen promoter through its binding to two GC-rich sequences (Galéra, P., Musso, M., Ducy, P., and Karsenty, G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9372-9376). In this report we show that c-Krox can bind to an additional site in the promoter of the alpha1(I) collagen gene and to three sites in the promoter of the alpha2(I) collagen gene, the other gene coding for type I collagen. One of the binding sites present in both promoters is adjacent to the CCAAT box. We have performed a structure/function analysis of c-Krox locating the transactivation domain in the zinc finger and C-terminal domains and the dimerization domain in the C-terminal end of the protein. We also demonstrate that c-Krox is an early response gene, whose expression is detectable as early as 9.5-day postcoitum in mouse embryos. Whole-mount in situ hybridization shows that c-Krox is expressed in dermatomes, the somite derivatives that generate dermis, and section in situ hybridization shows that c-Krox and alpha1(I) collagen mRNAs colocalized in skin but not in bone during development. This result is consistent with the predominant expression of c-Krox in skin in postnatal life. Thus, our findings suggest that c-Krox is one transcription factor controlling the coordinated expression of the two type I collagen genes in skin.

3T3 Cells↗

PubMed1996

Increased bone formation in osteocalcin-deficient mice.

Vertebrates constantly remodel bone. The resorption of preexisting bone by osteoclasts and the formation of new bone by osteoblasts is strictly coordinated to maintain bone mass within defined limits. A few molecular determinants of bone remodelling that affect osteoclast activity have been characterized, but the molecular determinants of osteoblast activity are unknown. To investigate the role of osteocalcin, the most abundant osteoblast-specific non-collagenous protein, we have generated osteocalcin-deficient mice. These mice develop a phenotype marked by higher bone mass and bones of improved functional quality. Histomorphometric studies done before and after ovariectomy showed that the absence of osteocalcin leads to an increase in bone formation without impairing bone resorption. To our knowledge, this study provides the first evidence that osteocalcin is a determinant of bone formation.

Animals↗

PubMed1996

BMP 7 is required for nephrogenesis, eye development, and skeletal patterning.

In summary, the generation of BMP-7-deficient mice has provided additional evidence that this family of growth factors regulate many morphogenetic processes including but not limited to skeletal development. In particular our experiments demonstrate that BMP 7 acts as an early inducer of glomeruli formation, and that it is required for skeletal patterning and lens formation. Our results not only demonstrate that BMP 7 is involved in the differentiation of several organs during development, but also raise the hypothesis that mutations in the Bmp 7 gene itself or in the genetic pathway could be responsible for several human genetic diseases in which glomerulus formation is impaired.

Animals↗

PubMed1996

Analysis of limb patterning in BMP-7-deficient mice.

Bone morphogenetic proteins (BMPs) are polypeptide signaling molecules, belonging to the TGF-beta superfamily. They were originally identified by their ability to induce ectopic bone formation, but their expression patterns in embryos suggest multiple functions. BMP-7-deficient mice show among other mesodermal and skeletal patterning defects, polydactyly in the hindlimbs [Luo G, Hofmann C, Bronckers ALJJ, Sohocki M, Bradley A, Karsenty G (1995): Genes Dev 9:2808-2820; Dudley AT, Lyons KM, Robertson EJ (1995): Genes Dev 9:2795-2807]. Here we report a more detailed analysis of the limb phenotype in BMP-7-deficient mice using in situ hybridization to monitor expression of molecules implicated in patterning processes of the developing vertebrate limb. In previous studies we showed that Sonic hedgehog (Shh) was expressed normally, but Hoxd-13 expression in limb mesenchyme was lower in BMP-7 mutant limbs. Here we show that Hoxd-11 expression domains are also contracted and decreased in intensity in mutant limbs, suggesting that 5' genes of the Hoxd cluster are coordinately downregulated, while another Bmp, Bmp-2, which can be activated by Shh, is similarly expressed. The mutant limb buds are broader than normal buds, and fibroblast growth factor Fgf-8 is expressed throughout the extended ridge. However, expression of the homeobox gene Msx-1, which has been shown to be involved in epithelial-mesenchymal interactions during limb development, was decreased in the mesenchyme of BMP-7 mutant limbs. Taken together, our data suggest that BMP-7 is involved in regulating proliferation and/or epithelial-mesenchymal interactions in the developing limb.

Animals↗

PubMed1996

DNA fragmentation during bone formation in neonatal rodents assessed by transferase-mediated end labeling.

To study the fate of bone cells, we used the transferase-mediated, biotin-dUTP nick end-labeling (TUNEL) assay to detect DNA fragmentation during the formation of intramembranous and endochondral bone in newly born hamsters, mice, and rats. In alveolar bone forming around the developing tooth crowns, DNA fragmentation was found in three cell types: TRAP-negative mononuclear cells at the bone surface, osteocytes, and some but not all nuclei of TRAP-positive osteoclasts. Osteoblasts did not undergo DNA fragmentation. A strong positive correlation was found between contacts of TUNEL-positive osteocytes and osteoclasts. Extracellular bone matrix also stained occasionally for the presence of DNA fragments. During endochondral bone formation, TUNEL staining was detected in late hypertrophic chondrocytes of the epiphyseal growth plate. During rapid longitudinal growth of long bones, TUNEL-positive hypertrophic chondrocytes were found coincident with or slightly after invasion of blood vessels from the diaphysis. However, during slow longitudinal growth and in secondary ossification centers, DNA fragmentation was seen in hypertrophic chondrocytes still located within their lacunae. We conclude that some of the osteocytes in deeper layers of bone die within their lacuna and disperse nuclear fragments over the extracellular matrix, that a majority of the osteocytes are phagocytosed and degraded by osteoclasts at sites of intense bone resorption, and that during endochondral ossification, substantial numbers of late hypertrophic chondrocyte cells undergo cell death.

Acid Phosphatase↗

PubMed1996

Nuclear DNA fragmentation during postnatal tooth development of mouse and hamster and during dentin repair in the rat.

The TUNEL (transferase-mediated, dUTP-biotin nick end labeling) method for in situ labeling of DNA strands was utilized to localize DNA fragmentation in cells involved in tooth formation in the neonatal mouse and hamster. Positive reactions for the presence of DNA fragments were obtained in some epithelial cells of the cervical loop region of incisors, late secretory, transitional and early maturation stage ameloblasts, stratum intermedium cells and in shortened ameloblasts just before eruption. Also, cells of the periodontal ligament of the continuously erupting incisors stained positive shortly before eruption. Odontoblasts were negative but became strongly positive during the formation of physiological osteodentin at the tip of developing incisors. Osteodentin matrix and the surfaces of unerupted enamel and cementum just prior to eruption stained for DNA fragments as well. DNA fragmentation could be elicited in odontoblasts and underlying pulpal tissues of mature erupted molars after mechanical injury to the odontoblast processes during cavity preparation. We conclude that, in rodents, DNA fragmentation and cell death are biological processes which take place in a variety of cells involved in formation of teeth. The TUNEL staining technique is a simple but powerful tool to examine the fate of cells and tissues undergoing either programmed cell death (apoptosis) or fragmentation of nuclear DNA induced by external factors leading to pathological changes.

Amelogenesis↗

PubMed1996

Study of osteoblast-specific expression of one mouse osteocalcin gene: characterization of the factor binding to OSE2.

In an attempt to understand the mechanisms of osteoblast-specific expression, we analyzed the promoter of the mouse osteocalcin gene 2, an osteoblast-specific gene. In this promoter, using a combination of DNA transfection experiments and DNA binding assays, we have identified two osteoblast-specific cis-acting elements called OSE1 and OSE2. Inspection of OSE2 DNA sequence and site-specific mutagenesis allowed us to define a core sequence for OSE2. This core sequence is identical to the DNA binding site of the PEBP2 alpha transcription factors, the mouse homologues of the Drosophila Runt protein. Here we show that OSF2, the factor present in osteoblast nuclear extracts and binding to OSE2, is immunologically related to the PEBP2 alpha transcription factors. In DNA cotransfection experiment, a human homologue of PEBP2 alpha transcription factor increases the activity of a short osteocalcin promoter through its binding to OSE2. Thus, this study presents evidence that OSF2 is a member of the PEBP2 alpha family of transcription factors.

Animals↗

PubMed1995

A PEBP2 alpha/AML-1-related factor increases osteocalcin promoter activity through its binding to an osteoblast-specific cis-acting element.

To identify osteoblast-specific cis-acting elements and trans-acting factors, we initiated an analysis of the promoter of a mouse osteocalcin gene, an osteoblast-specific gene. In this promoter, we identified two osteoblast-specific cis-acting elements (Ducy, P. and Karsenty, G. (1995) Mol. Cell. Biol. 15, 1858-1869). The sequence of one of these elements, OSE2, is identical to the DNA-binding site of the PEBP2 alpha/AML-1 transcription factors, the mammalian homologues of the Drosophila Runt protein. Here we show, using nuclear extracts, recombinant protein, and a specific antiserum against AML-1 proteins in DNA-binding assays, that one member of this family, AML-1B, binds specifically to OSE2 and is immunologically related to OSF2, the factor present in osteoblast nuclear extracts that binds to OSE2. By DNA cotransfection experiments, we also demonstrate that AML-1B can increase the activity of a short osteocalcin promoter through its binding to OSE2. Lastly, the different mobilities of osteoblast nuclear extract-DNA complexes compared with T-cell nuclear extract-DNA complexes, along with the inability of OSF2 to be upregulated by retinoic acid, unlike the other PEBP2 alpha factors, suggest that OSF2 is a new member of this family of transcription factors. Thus, this study demonstrates that AML-1B can increase gene expression of an osteoblast-specific gene through its binding to an osteoblast-specific cis-acting element and presents evidence that OSF2 is a member of the PEBP2 alpha/AML-1 family of transcription factors.

Animals↗

PubMed1995

BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning.

Bone morphogenetic proteins (BMPs) are multifunctional growth factors originally identified by their ability to induce ectopic bone formation. To investigate the function of one of the BMPs, BMP-7, we have generated BMP-7-deficient mice using embryonic stem cell technology. BMP-7-deficient mice die shortly after birth because of poor kidney development. Histological analysis of mutant embryos at several stages of development revealed that metanephric mesenchymal cells fail to differentiate, resulting in a virtual absence of glomerulus in newborn kidneys. In situ hybridization analysis showed that the absence of BMP-7 affects the expression of molecular markers of nephrogenesis, such as Pax-2 and Wnt-4 between 12.5 and 14.5 days postcoitum (dpc). This identifies BMP-7 as an inducer of nephrogenesis. In addition, BMP-7-deficient mice have eye defects that appear to originate during lens induction. Finally, BMP-7-deficient mice also have skeletal patterning defects restricted to the rib cage, the skull, and the hindlimbs.

Animals↗

PubMed1995

The matrix Gla protein gene is a marker of the chondrogenesis cell lineage during mouse development.

Matrix Gla protein (MGP) is, along with osteocalcin, a skeletal member of the family of extracellular mineral-binding Gla proteins. Although the precise function of these proteins remains obscure, circumstantial evidence suggests that they play a role in endochondral ossification. As a first step toward understanding MGP function we have performed a preliminary characterization of its promoter element and studied the developmental pattern of expression of this gene. DNA transfection experiments indicate that the mouse MGP promoter functions better in cells expressing the MGP gene than in cells that do not express the gene. During mouse development, MGP gene expression is detectable as early as day 10.5 of embryonic development (E10.5), before any skeletal structures are identifiable. In situ hybridization analysis shows that MGP mRNA is initially present at the mesenchymal epithelial interphase in lung and limb buds. As development proceeds, MGP gene is predominantly expressed in cells of the chondrocytic lineage in areas that will undergo endochondral ossification as well as in areas that will remain cartilaginous, such as the trachea and bronchi. In growth plate cartilage, MGP mRNA is present in resting, proliferative, and late hypertrophic chondrocytes. Surprisingly, MGP mRNA is absent from the early hypertrophic chondrocytes and from the osteoblasts. Finally, the MGP gene is expressed at a lower level in kidney medulla and uterus smooth muscle but not in brain, spleen, or heart during development. This study demonstrates that during development MGP gene expression occurs early and is predominant at the epithelial mesenchymal interfaces, principally of lung and limb buds, and in cells of the chondrocytic lineage. This finding raises the intriguing possibility that MGP may play distinct roles during embryogenesis and in the adult organism.

Animals↗

PubMed1995

Osteocalcin cluster: implications for functional studies.

Osteocalcin is a skeletal member of the family of extracellular mineral binding Gla protein. Osteocalcin is synthesized only by the osteoblast and it is secreted into the bone matrix at the time of bone mineralization. The mineral binding properties of osteocalcin as well as its spatial and temporal pattern of expression suggest that it plays a role during bone mineralization, however until now its biological function is unclear. To understand osteocalcin function during skeletogenesis we mutated the two osteocalcin genes by homologous recombination in embryonic stem (ES) cells. Eight targeted clones were identified by Southern analysis using external probes. One of these clones contributed to the germ line of mouse chimera. Interbreeding of heterozygotes is currently in progress. Mutant mice will be useful to understand osteocalcin function in vivo.

Animals↗