Biomedical subjects

G Karsenty

Publications and source records attributed to G Karsenty.

At least 73 records · Page 4Linked to original sources

PubMed1998

Transcriptional regulation of osteoblast differentiation during development.

The osteoblast is the bone-forming cell. The molecular basis of osteoblast-specific gene expression and differentiation begin to be understood. Following the characterization of OSE2, an osteoblast-specific cis-acting element present in the Osteocalcin promoter Osf2/Cbfa1, the protein that binds to OSE2, was identified. Osf2/Cbfa1 is a member of the runt family of transcription factors. Its expression is initiated in the mesenchymal condensations of the developing skeleton and is strictly restricted to cells of the osteoblast lineage thereafter. Osf2/Cbfa1 binds to and regulates the expression of multiple genes expressed in osteoblasts, and forced expression of Osf2/Cbfa1 in nonosteoblastic cells induces the expression of the principal osteoblast-specific genes. Osf2/Cbfa1 gene inactivation in mice leads to failure of mesenchymal progenitor cells to differentiate into osteoblasts. Lastly, heterozygous mutations in the Osf2/Cbfa1 gene cause Cleidocranial dysplasia in human and mice, a condition marked by generalized bone defects. These result demonstrate that Osf2/Cbfa1 is an osteoblast-specific transcriptional activator of osteoblast differentiation whose function is non redundant with the function of other gene products during development.

Animals↗

PubMed1998

Differential roles for bone morphogenetic protein (BMP) receptor type IB and IA in differentiation and specification of mesenchymal precursor cells to osteoblast and adipocyte lineages.

Cumulative evidence indicates that osteoblasts and adipocytes share a common mesenchymal precursor and that bone morphogenetic proteins (BMPs) can induce both osteoblast and adipocyte differentiation of this precursor. In the present study, we investigated the roles of BMP receptors in differentiation along these separate lineages using a well-characterized clonal cell line, 2T3, derived from the mouse calvariae. BMP-2 induced 2T3 cells to differentiate into mature osteoblasts or adipocytes depending upon culture conditions. To test the specific roles of the type IA and IB BMP receptor components, truncated and constitutively active type IA and IB BMP receptor cDNAs were stably expressed in these cells. Overexpression of truncated type IB BMP receptor (trBMPR-IB) in 2T3 cells completely blocked BMP-2-induced osteoblast differentiation and mineralized bone matrix formation. Expression of trBMPR-IB also blocked mRNA expression of the osteoblast specific transcription factor, Osf2/ Cbfa1, and the osteoblast differentiation-related genes, alkaline phosphatase (ALP) and osteocalcin (OC). BMP-2-induced ALP activity could be rescued by transfection of wild-type (wt) BMPR-IB into 2T3 clones containing trBMPR-IB. Expression of a constitutively active BMPR-IB (caBMPR-IB) induced formation of mineralized bone matrix by 2T3 cells without addition of BMP-2. In contrast, overexpression of trBMPR-IA blocked adipocyte differentiation and expression of caBMPR-IA induced adipocyte formation in 2T3 cells. Expression of the adipocyte differentiation-related genes, adipsin and PPARgamma, correlated with the distinct phenotypic changes found after overexpression of the appropriate mutant receptors. These results demonstrate that type IB and IA BMP receptors transmit different signals to bone-derived mesenchymal progenitors and play critical roles in both the specification and differentiation of osteoblasts and adipocytes.

Adipocytes↗

PubMed1998

Genetics of skeletogenesis.

Major advances have been made in the last 10 years in the genetics of skeletogenesis. This has followed the general progress in our understanding of the genetic control of development in chicken and mouse and more recent advances in human genetics. This large field now encompasses three smaller but distinct fields of investigation. Those are skeleton patterning, cell differentiation in the skeleton, and cell function in the skeleton. This review focuses primarily on advances in understanding cell differentiation and cell function in the skeleton at the genetic level.

Animals↗

PubMed1998

Skeletal abnormalities in doubly heterozygous Bmp4 and Bmp7 mice.

Analysis of the skeletal phenotypes caused by the genetic inactivation of individual Bmps, along with the study of their expression patterns, suggest possible functional redundancy of these molecules. To investigate the effect on skeleton development of the combined absence of some Bmp genes expressed in the same areas, we have intercrossed heterozygous Bmp7 mice with Bmp2 +/-, Bmp4 +/-, or Bmp5 +/- animals. Bmp2/7 and Bmp5/7 double heterozygous animals do not present with any abnormalities. In contrast, Bmp4/7 double heterozygotes develop minor defects in two restricted areas of the skeleton, the rib cage, and the distal part of the limbs. In the ribs, Bmp4 and Bmp7 seem to act in the same pathway to assure proper guidance of mesenchymal condensations of the ribs extending toward the sternum. In the limbs, these molecules appear to play a similar role in controlling digit number, possibly through induction of apoptosis in the interdigital and anterior mesenchyme.

Animals↗

PubMed1998

Genomic organization, expression of the human CBFA1 gene, and evidence for an alternative splicing event affecting protein function.

The Cbfa1 gene, which encodes the transcription factor Osf2/Cbfa1 required for osteoblast differentiation in mouse and human, is mutated in cleidocranial dysplasia, a skeletal dysplasia. We describe here the isolation of the full-length human OSF2/CBFA1 cDNAs, the genomic organization of the entire CBFA1 gene, its expression, and the existence of an alternative splicing event. Nucleotide sequence analysis of the human and mouse OSF2/CBFA1 cDNAs showed a 98% homology in the coding sequence and 96% in the 5' untranslated (UTR) sequence. Analysis of CBFA1 genomic clones revealed that the 5' UTR sequence of the human OSF2/CBFA1 cDNA lies 75 kb upstream of the originally described 5' end of the gene. The existence of two OSF2/CBFA1 cDNAs is due to an alternative splicing event around exon 8 that affects the transcriptional activity of the protein. Northern blot analysis demonstrates that the expression of the human OSF2/CBFA1 gene is restricted to osteoblastic cells.

Alternative Splicing↗

PubMed1998

Genetic control of cell differentiation in the skeleton.

The mechanisms of cell differentiation in the skeleton are just beginning to be unraveled. In the past year classical gene expression studies, genetic manipulation in mice and human genetic approaches have led to the identification of Osf2/Cbfa1 as a major regulator of osteoblast differentiation. Important progress was also made in the understanding of the control of osteoclast differentiation through the identification of osteoprotegerin and its ligand. These studies, as well as others of chondrocyte differentiation, provide a better understanding of skeletogenesis.

Animals↗

PubMed1998

Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin.

Osteocalcin, the gamma-carboxyglutamic acid-containing protein, which in most species is the predominant noncollagenous protein of bone and dentin, has been postulated to play roles in bone formation and remodeling. Recently, genetic studies showed that osteocalcin acts as an inhibitor of osteoblast function. Based on von Kossa staining and measurement of mineral apposition rates in tetracycline-labeled bones, osteocalcin knockout animals were reported to have no detectable alterations in bone mineralization. To test the hypothesis that, in addition to regulating osteoblastic activity, osteocalcin is involved in regulating mineral properties, a more sensitive assay of mineralization, Fourier transform infrared microspectroscopy (FT-IRM) was used to study thin sections of femora of 4-week-, 6-month- (intact and ovariectomized), and 9-month-old wild-type and osteocalcin-knockout mice. FT-IRM spectra provided spatially resolved measures of relative mineral and carbonate contents, and parameters indicative of apatite crystal size and perfection. No differences were detected in the mineral properties of the 4-week-old knockout and wild-type mice indicating that the mineralization process was not altered at this time point. Six-month-old wild-type animals had higher mineral contents (mineral:matrix ratios) in cortical as compared with trabecular bones; mineral contents in knockout and wild-type bones were not different. At each age studied, carbonate:phosphate ratios tended to be greater in the wild-type as compared with knockout animals. Detailed analysis of the phosphate nu1,nu3 vibrations in the spectra from 6-month-old wild-type animals indicated that the crystals were larger/more perfect in the cortical as opposed to the trabecular bones. In contrast, in the knockout animals' bones at 6 months, there were no differences between trabecular and cortical bone in terms of carbonate content or crystallite size and perfection. Spectral parameters of the cortical and trabecular bone of the knockout animals resembled those in the wild-type trabecular bone and differed from wild-type cortical bone. In ovariectomized 6-month-old animals, the mineral content (mineral:matrix ratio) in the wild-type cortices increased from periosteum to endosteum, whereas, in the knockout animals' bones, the mineral:matrix ratio was constant. Ovariectomized knockout cortices had lower carbonate:phosphate ratios than wild-type, and crystallite size and perfection resembled that in wild-type trabeculae, and did not increase from periosteum to endosteum. These spatially resolved data provide evidence that osteocalcin is required to stimulate bone mineral maturation.

Age Factors↗

PubMed1998

Studies of osteocalcin function in dentin formation in rodent teeth.

Osteocalcin (OC) is a major non-collagenous protein synthesized by osteoblasts, odontoblasts and cementoblasts. We examined the function of OC in dentinogenesis by exposing rat and hamster tooth organ cultures to 1,25(OH)2vit D3 or to bovine OC added to the culture medium. We furthermore examined dentinogenesis in tooth explants cultured in the presence of warfarin (an inhibitor of gamma-carboxylation of OC). Finally, we analyzed dentin from osteocalcin null mutant mice. Exposure to 1,25(OH)2vit D3 increased OC synthesis by odontoblasts in vitro at the transcriptional and protein levels but had no apparent effect on matrix formation or 45Ca uptake. High levels of bovine OC temporarily suppressed the initial formation of dentin and enamel and uptake of 45Ca. This effect was not seen when tooth explants were exposed to thermally decarboxylated OC. Exposure of tooth explants to warfarin had no significant effect on dentinogenesis. Dentin obtained from two-month-old OC null mutants looked structurally normal and did not show marked differences in dentin matrix thickness and mineral content compared to wild type. We concluded that, although OC at supraphysiological levels has the potential to affect dentin mineralization probably through its Gla-residues, the locally produced levels of OC are not sufficient to markedly influence dentinogenesis.

Amelogenesis↗

PubMed1998

New developments in bone formation.

Two independent strategies have established that the transcription factor, Cbfa1, is a key regulator of both osteoblast differentiation and osteoblast-specific gene expression. Gene targeting experiments in mice have also shown that haploinsufficiency of Cbfa1 expression causes symptoms reminiscent of the Cleidocranial dysplasia syndrome (CCD), a heritable disorder of the skeleton. Direct analysis of the Cbfa1 gene in CCD families has revealed a direct correlation between mutations in this gene and disease phenotype.

Animals↗

PubMed1998

Two domains unique to osteoblast-specific transcription factor Osf2/Cbfa1 contribute to its transactivation function and its inability to heterodimerize with Cbfbeta.

Osf2/Cbfa1, hereafter called Osf2, is a member of the Runt-related family of transcription factors that plays a critical role during osteoblast differentiation. Like all Runt-related proteins, it contains a runt domain, which is the DNA-binding domain, and a C-terminal proline-serine-threonine-rich (PST) domain thought to be the transcription activation domain. Additionally, Osf2 has two amino-terminal domains distinct from any other Runt-related protein. To understand the mechanisms of osteoblast gene regulation by Osf2, we performed an extensive structure-function analysis. After defining a short Myc-related nuclear localization signal, a deletion analysis revealed the existence of three transcription activation domains and one repression domain. AD1 (for activation domain 1) comprises the first 19 amino acids of the molecule, which form the first domain unique to Osf2, AD2 is formed by the glutamine-alanine (QA) domain, the second domain unique to Osf2, and AD3 is located in the N-terminal half of the PST domain and also contains sequences unique to Osf2. The transcription repression domain comprises the C-terminal 154 amino acids of Osf2. DNA-binding, domain-swapping, and protein interaction experiments demonstrated that full-length Osf2 does not interact with Cbfbeta, a known partner of Runt-related proteins, whereas a deletion mutant of Osf2 containing only the runt and PST domains does. The QA domain appears to be responsible for preventing this heterodimerization. Thus, our results uncover the unique functional organization of Osf2 by identifying functional domains not shared with other Runt-related proteins that largely control its transactivation and heterodimerization abilities.

3T3 Cells↗

PubMed1998

Bone morphogenetic protein-7 (osteogenic protein-1, OP-1) and tooth development.

Bone morphogenetic proteins (BMPs) form a family of growth factors originally isolated from extracellular bone matrix that are capable of inducing bone formation ectopically. We studied the expression, tissue localization, and function of BMP-7 (OP-1) during tooth development in rodents. Patterns of BMP-7 gene expression and peptide distribution indicated that BMP-7 was present in dental epithelium during the dental lamina, bud, and cap stages. During the bell stage, BMP-7 mRNA expression and protein distribution shifted from dental epithelium toward the dental mesenchyme. With advancing differentiation of odontoblasts, BMP-7 protein staining in the dental papilla became restricted to the layer of fully functional odontoblasts in the process of depositing (pre)dentin. Secretory-stage ameloblasts exhibited weak immunostaining for BMP-7. A restricted pattern of staining in ameloblasts became apparent in post-secretory stages of amelogenesis. Also, cells of the forming periodontal ligament were immunopositive. Histological analysis of tooth development in neonatal BMP-7-deficient mice did not reveal obvious changes compared with wild-type mice. We conclude that, in developing dental tissues, BMP-7 has distribution and expression patterns similar to those of other BMP members but is not an essential growth factor for tooth development, possibly because of functional redundancy with other BMP members or related growth factors.

Ameloblasts↗

PubMed1998

Molecular determinants of arterial calcification.

Calcification of extracellular matrix (ECM) can be either physiological or pathological. Physiological calcification (or mineralization) of ECM is restricted to bones, teeth and, to a lesser extent, growth plate cartilages. Pathological calcification appears often in the ECM of arteries where it is a frequent complication of atherosclerosis. However, calcification of the ECM of arteries is not restricted to atherosclerosis. Indeed, human diseases have been described that are characterized by calcification of the aortic media in the absence of any atherosclerotic lesions. The existence of these rare diseases, along with several mouse models recently generated and discussed below, indicates that the formation of atherosclerotic lesions and the calcification of the artery ECM are controlled by different genetic pathways. This emerging knowledge has implications for our understanding of ECM calcification beyond atherosclerosis.

Animals↗

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↗