Biomedical subjects

Benoit de Crombrugghe

Publications and source records attributed to Benoit de Crombrugghe.

32 records · Page 2Linked to original sources

PubMed2004

Transgenic mice expressing a ligand-inducible cre recombinase in osteoblasts and odontoblasts: a new tool to examine physiology and disease of postnatal bone and tooth.

The skeleton supports body structures in vertebrates and helps maintain calcium homeostasis throughout life. Disruption of genes involved in mammalian bone formation has often led to embryonic lethality, hence preventing study of these genes' role in adult animals. To develop a usable tool for such study, we generated transgenic mice in which a 2.3-kb mouse Col1a1 proximal promoter, which is active in all osteoblasts, drives a transgene coding for a polypeptide consisting of Cre recombinase fused to a mutated ligand-binding domain of the estrogen receptor. In this Col1a1-CreERT2 mouse line, expression patterns of the transgene and of the resulting Cre-mediated DNA recombination are analyzed by crossing with ROSA26 reporter mice and by measurement of beta-galactosidase activity and X-gal staining. Exposure to 4-hydroxytamoxifen induced Cre-mediated recombination in osteoblasts in virtually all bones and in odontoblasts in teeth of both embryos and postnatal mice. The generation of these transgenic mice provides a new and important tool with which to study the function of specific genes in bone and tooth physiology and diseases in intact animals after birth.

Animals↗

PubMed2003

The B subunit of the CCAAT box binding transcription factor complex (CBF/NF-Y) is essential for early mouse development and cell proliferation.

To understand the physiological function of the mammalian heterotrimeric CCAAT binding factor CBF, also known as NF-Y, we have generated a conditional Cbf-b mouse mutant by introducing loxP sites in the murine Cbf-b/Nf-ya gene. Controlled expression of Cre recombinase deletes the gene in vivo, which leads to a loss of DNA binding by the CBF complex and hence CBF-mediated transcription. Deletion of both Cbf-b alleles causes early embryo lethality, indicating that CBF activity is essential for early mouse development. In primary cultures of mouse embryonic fibroblasts, conditional inactivation of CBF results in a block in cell proliferation and inhibition of S phase or DNA synthesis, which is followed by induction of apoptosis. We conclude that the CBF transcription factor complex is essential for cell proliferation and viability.

Alleles↗

PubMed2003

Sp1 and Sp3 transcription factors mediate interleukin-1 beta down-regulation of human type II collagen gene expression in articular chondrocytes.

Interleukin-1 beta (IL-1 beta) is a pleiotropic cytokine that was shown to inhibit the biosynthesis of articular cartilage components. Here we demonstrate that IL-1 beta inhibits the production of newly synthesized collagens in proliferating rabbit articular chondrocytes and that this effect is accompanied by a decrease in the steady-state levels of type II collagen mRNA. IL-1 beta down-regulates COL2A1 gene transcription through a -41/-33 bp sequence that binds a multimeric complex including Sp1 and Sp3 transcription factors. Specificity of IL-1 beta effects on COL2A1 promoter activity was demonstrated in experiments in which transfection of a wild type -50/+1 sequence of COL2A1 promoter as a decoy oligonucleotide abolished the IL-1 beta inhibition of a -63/+47 COL2A1-mediated transcription. By contrast, transfection of the related oligonucleotide harboring a targeted mutation in the -41/-33 sequence did not modify the negative effect the cytokine. Because we demonstrated previously that Sp1 was a strong activator of COL2A1 gene expression via the -63/+1 promoter region, whereas Sp3 overexpression blocked Sp1-induced promoter activity and inhibited COL2A1 gene transcription, we conclude that IL-1 beta down-regulation of that gene, as we found previously for transforming growth factor-beta 1, is mediated by an increase in the Sp3/Sp1 ratio. Moreover, IL-1 beta increased steady-state levels of Sp1 and Sp3 mRNAs, whereas it enhanced Sp3 protein expression and inhibited Sp1 protein biosynthesis. Nevertheless, IL-1 beta decreased the binding activity of both Sp1 and Sp3 to the 63-bp short COL2A1 promoter, suggesting that the cytokine exerts a post-transcriptional regulatory mechanism on Sp1 and Sp3 gene expressions. Altogether, these data indicate that modulation of Sp3/Sp1 ratio in cartilage could be a potential target to prevent or limit the tissue degradation.

Animals↗

PubMed2003

Sox9 is required for determination of the chondrogenic cell lineage in the cranial neural crest.

Sox9 has essential roles in endochondral bone formation during axial and appendicular skeletogenesis. Sox9 is also expressed in neural crest cells, but its function in neural crest remains largely unknown. Because many craniofacial skeletal elements are derived from cranial neural crest (CNC) cells, we asked whether deletion of Sox9 in CNC cells by using the Cre recombinase/loxP recombination system would affect craniofacial development. Inactivation of Sox9 in neural crest resulted in a complete absence of cartilages and endochondral bones derived from the CNC. In contrast, all of the mesodermal skeletal elements and intramembranous bones were essentially conserved. The migration and the localization of Sox9-null mutant CNC cells were normal. Indeed, the size of branchial arches and the frontonasal mass of mutant embryos was comparable to that of WT embryos, and the pattern of expression of Ap2, a marker of migrating CNC cells, was normal. Moreover, in mouse embryo chimeras Sox9-null mutant cells migrated to their correct location in endochondral skeletal elements; however, Sox9-null CNC cells were unable to contribute chondrogenic mesenchymal condensations. In mutant embryos, ectopic expression of osteoblast marker genes, such as Runx2, Osterix, and Col1a1, was found in the locations where the nasal cartilages exist in WT embryos. These results indicate that inactivation of Sox9 causes CNC cells to lose their chondrogenic potential. We hypothesize that these cells change their cell fate and acquire the ability to differentiate into osteoblasts. We conclude that Sox9 is required for the determination of the chondrogenic lineage in CNC cells.

Animals↗

PubMed2003

Fibroblast-specific expression of a kinase-deficient type II transforming growth factor beta (TGFbeta) receptor leads to paradoxical activation of TGFbeta signaling pathways with fibrosis in transgenic mice.

To better understand the role of disrupted transforming growth factor beta (TGFbeta) signaling in fibrosis, we have selectively expressed a kinase-deficient human type II TGFbeta receptor (TbetaRIIDeltak) in fibroblasts of transgenic mice, using a lineage-specific expression cassette subcloned from the pro-alpha2(I) collagen gene. Surprisingly, despite previous studies that characterized TbetaRIIDeltak as a dominant negative inhibitor of TGFbeta signaling, adult mice expressing this construct demonstrated TGFbeta overactivity and developed dermal and pulmonary fibrosis. Compared with wild type cells, transgenic fibroblasts proliferated more rapidly, produced more extracellular matrix, and showed increased expression of key markers of TGFbeta activation, including plasminogen activator inhibitor-1, connective tissue growth factor, Smad3, Smad4, and Smad7. Smad2/3 phosphorylation was increased in transgenic fibroblasts. Overall, the gene expression profile of explanted transgenic fibroblasts using cDNA microarrays was very similar to that of littermate wild type cells treated with recombinant TGFbeta1. Despite basal up-regulation of TGFbeta signaling pathways, transgenic fibroblasts were relatively refractory to further stimulation with TGFbeta1. Thus, responsiveness of endogenous genes to TGFbeta was reduced, and TGFbeta-regulated promoter-reporter constructs transiently transfected into transgenic fibroblasts showed little activation by recombinant TGFbeta1. Responsiveness was partially restored by overexpression of wild type type II TGFbeta receptors. Activation of MAPK pathways by recombinant TGFbeta1 appeared to be less perturbed than Smad-dependent signaling. Our results show that expression of TbetaRIIDeltak selectively in fibroblasts leads to paradoxical ligand-dependent activation of downstream signaling pathways and causes skin and lung fibrosis. As well as confirming the potential for nonsignaling receptors to regulate TGFbeta activity, these findings support a direct role for perturbed TGFbeta signaling in fibrosis and provide a novel genetically determined animal model of fibrotic disease.

Animals↗

PubMed2003

Adjacent DNA sequences modulate Sox9 transcriptional activation at paired Sox sites in three chondrocyte-specific enhancer elements.

Expression of the type XI collagen gene Col11a2 is directed to cartilage by at least three chondrocyte-specific enhancer elements, two in the 5' region and one in the first intron of the gene. The three enhancers each contain two heptameric sites with homology to the Sox protein-binding consensus sequence. The two sites are separated by 3 or 4 bp and arranged in opposite orientation to each other. Targeted mutational analyses of these three enhancers showed that in the intronic enhancer, as in the other two enhancers, both Sox sites in a pair are essential for enhancer activity. The transcription factor Sox9 binds as a dimer at the paired sites, and the introduction of insertion mutations between the sites demonstrated that physical interactions between the adjacently bound proteins are essential for enhancer activity. Additional mutational analyses demonstrated that although Sox9 binding at the paired Sox sites is necessary for enhancer activity, it alone is not sufficient. Adjacent DNA sequences in each enhancer are also required, and mutation of those sequences can eliminate enhancer activity without preventing Sox9 binding. The data suggest a new model in which adjacently bound proteins affect the DNA bend angle produced by Sox9, which in turn determines whether an active transcriptional enhancer complex is assembled.

3T3 Cells↗

PubMed2003

Downregulation of a rheumatoid arthritis-related antigen (RA-A47) by ra-a47 antisense oligonucleotides induces inflammatory factors in chondrocytes.

Previously we have shown that the expression of RA-A47 (rheumatoid arthritis-related antigen) which is identical to HSP47, a collagen-binding chaperon, is downregulated in chondrocytes by tumor necrosis factor alpha (TNFalpha). RA-A47 was also found on the surface of chondrocytes where it is recognized as an antigen in the serum of rheumatoid arthritis (RA) patients. Its translocation to the cell surface from endoplasmic reticulum membrane where it is normally located was also enhanced by TNFalpha. To understand the significance of RA-A47 downregulation in chondrocytes independent from other effects of TNFalpha, we used an antisense oligonucleotide approach and investigated the effect of this treatment on the expression of molecules related to matrix degradation and production of growth factors for chondrocytic, endothelial, and synovial cells. Here we show that treatment of rabbit chondrocyes and human chondrosarcoma cells HCS-2/8 by ra-a47 antisense S-oligonucleotides significantly reduced the expression of ra-a47 both at mRNA and protein level. Interestingly, this TNFalpha-independent RA-A47 downregulation was associated with a strong induction of matrix metalloproteinase (MMP)-9 mRNA and inducible NO synthase (iNOS) mRNA. The induction of active-type MMP-9 was further detected by gelatin zymography. Under the same conditions, the release of basic fibroblast growth factor (bFGF) and connective tissue growth factor (CTGF) from HCS-2/8 cells into the conditioned medium (CM) was strongly enhanced. These effects were not a result of TNFalpha upregulation, since the ra-a47 antisense oligonucleotide treatment did not enhance TNFalpha synthesis. These observations indicate that downregulation of RA-A47 induces TNFalpha-independent cartilage-degrading pathways involving iNOS and MMP-9. Furthermore, the stimulation of bFGF and CTGF release from chondrocytes may stimulate the proliferation of adjacent endothelial and/or synovial cells.

Animals↗

PubMed2003

Transcriptional mechanisms in osteoblast differentiation and bone formation.

Osteoblasts, the cells responsible for bone formation, differentiate from mesenchymal cells. Here, we discuss transcription factors that are involved in regulating the multistep molecular pathway of osteoblast differentiation. Runx2 and Osx, a newly identified zinc-finger-containing protein, are transcription factors that are expressed selectively and at high levels in osteoblasts. Null mutations of either leads to a complete absence of bone in mice. Runx2 plus its companion subunit Cbf beta are needed for an early step in this pathway, whereas Osx is required for a subsequent step, namely the differentiation of preosteoblasts into fully functioning osteoblasts. The finding that Osx-null cells acquire a chondrocyte phenotype implies that Osx is a negative regulator of Sox9 and of the chondrocyte phenotype. This leads to the hypothesis that Osx might have a role in the segregation of osteoblasts from osteochondroprogenitors. We also discuss recent progress in studies of other transcription factors that affect skeletal patterning and development.

Bone Development↗

PubMed2003

Interferon alfa down-regulates collagen gene transcription and suppresses experimental hepatic fibrosis in mice.

The equilibrium between the production and degradation of collagen is rigorously controlled by a number of growth factors and cytokines. Interferon alfa (IFN-alpha) is now widely used for the treatment of chronic hepatitis C, which can improve serum levels of fibrotic markers and the degree of hepatic fibrosis, not only in patients who responded to therapy but also in those in whom it is ineffective. These findings may suggest that IFN-alpha possesses direct antifibrotic effects in addition to its antiviral activity. However, in contrast to IFN-gamma, which has been shown to suppress collagen gene transcription, little is known about the mechanisms responsible for the antifibrotic effects of IFN-alpha. Here, we report that IFN-alpha, when administered into transgenic mice harboring the alpha2(I) collagen gene (COL1A2) promoter sequence, significantly repressed promoter activation and prevented the progression of hepatic fibrosis induced by carbon tetrachloride injection. Transient transfection assays indicated that IFN-alpha decreased the steady-state levels of COL1A2 messenger RNA (mRNA) and inhibited basal and TGF-beta/Smad3-stimulated COL1A2 transcription in activated hepatic stellate cells (HSC). These inhibitory effects of IFN-alpha on COL1A2 transcription were exerted through the interaction between phosphorylated Stat1 and p300. Blocking of the IFN-alpha signal by overexpressing the intracellular domain-deleted IFN receptor increased basal COL1A2 transcription and abolished the inhibitory effects of IFN-alpha. In conclusion, our results indicate that IFN-alpha antagonizes the TGF-beta/Smad3-stimulated COL1A2 transcription in vitro and suppresses COL1A2 promoter activation in vivo, providing a molecular basis for antifibrotic effects of IFN-alpha.

Animals↗

PubMed2003

SOX9 exerts a bifunctional effect on type II collagen gene (COL2A1) expression in chondrocytes depending on the differentiation state.

As a key transcription factor in cartilage formation, SOX9 is a potent activator of type II collagen expression, a phenotypic marker of articular chondrocytes. This study was designed to determine the potential role of SOX9 on COL2A1 gene transcription during chondrocyte dedifferentiation, a characteristic feature of osteoarthritic cartilage that can be partially mimicked in vitro by subculturing primary chondrocytes. Constructs containing different regions from the promoter and the first intron of human COL2A1 gene were transfected in differentiated (primary) and dedifferentiated (passaged) rabbit articular chondrocytes (RAC), together with an expression vector containing or not the SOX9 cDNA. As expected, low levels of SOX9 overexpression were capable of enhancing COL2A1 gene transcription in both fully differentiated and slightly phenotypically altered chondrocytes, through the specific intronic enhancer. In contrast, when overexpressed at high levels, SOX9 induced an inhibition of COL2A1 gene expression, mediated by the -266 bp promoter region, whatever the differentiation state of chondrocytes. However, in the advanced stages of dedifferentiation, SOX9, independently of its expression level, depressed COL2A1 transcriptional activity through the -63 bp short promoter. Although SOX9 has a crucial role in chondrocyte differentiation, our findings indicate that this factor cannot restore the phenotype of osteoarthritic chondrocytes by itself.

Animals↗

PubMed2002

The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6.

To examine whether the transcription factor Sox9 has an essential role during the sequential steps of chondrocyte differentiation, we have used the Cre/loxP recombination system to generate mouse embryos in which either Sox9 is missing from undifferentiated mesenchymal cells of limb buds or the Sox9 gene is inactivated after chondrogenic mesenchymal condensations. Inactivation of Sox9 in limb buds before mesenchymal condensations resulted in a complete absence of both cartilage and bone, but markers for the different axes of limb development showed a normal pattern of expression. Apoptotic domains within the developing limbs were expanded, suggesting that Sox9 suppresses apoptosis. Expression of Sox5 and Sox6, two other Sox genes involved in chondrogenesis, was no longer detected. Moreover, expression of Runx2, a transcription factor needed for osteoblast differentiation, was also abolished. Embryos, in which Sox9 was deleted after mesenchymal condensations, exhibited a severe generalized chondrodysplasia, similar to that in Sox5; Sox6 double-null mutant mice. Most cells were arrested as condensed mesenchymal cells and did not undergo overt differentiation into chondrocytes. Furthermore, chondrocyte proliferation was severely inhibited and joint formation was defective. Although Indian hedgehog, Patched1, parathyroid hormone-related peptide (Pthrp), and Pth/Pthrp receptor were expressed, their expression was down-regulated. Our experiments further suggested that Sox9 is also needed to prevent conversion of proliferating chondrocytes into hypertrophic chondrocytes. We conclude that Sox9 is required during sequential steps of the chondrocyte differentiation pathway.

Animals↗

PubMed2002

The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation.

We have identified a novel zinc finger-containing transcription factor, called Osterix (Osx), that is specifically expressed in all developing bones. In Osx null mice, no bone formation occurs. In endochondral skeletal elements of Osx null mice, mesenchymal cells, together with osteoclasts and blood vessels, invade the mineralized cartilage matrix. However, the mesenchymal cells do not deposit bone matrix. Similarly, cells in the periosteum and in the condensed mesenchyme of membranous skeletal elements cannot differentiate into osteoblasts. These cells do, however, express Runx2/Cbfa1, another transcription factor required for bone formation. In contrast, Osx is not expressed in Runx2/Cbfa1 null mice. Thus, Osx acts downstream of Runx2/Cbfa1. Because Osx null preosteoblasts express typical chondrocyte marker genes, we propose that Runx2/Cbfa1-expressing preosteoblasts are still bipotential cells.

Animals↗

PubMed2002

Transforming growth factor-beta isoforms differently stimulate proalpha2 (I) collagen gene expression during wound healing process in transgenic mice.

The role of many growth factors and cytokines in the process of wound healing has been intensively investigated in recent two decades. Among them, transforming growth factor-betas (TGF-betas) are well known to have a potent stimulatory effect on collagen synthesis as shown in various in vivo experimental systems. In the present study, we examined the effects of various growth factors on the promoter activity of the proalpha2 (I) collagen gene (COL1A2) during the wound healing process. For this purpose, we utilized transgenic mice harboring the -17 kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase or a bacterial beta-galactosidase gene. These mice exhibited normal phenotypic expression and the wound healing process was not impaired. Full thickness wounds were made by punch biopsy. We examined the effects of TGF-beta1, -beta2, -beta3, basic fibroblast growth factor, platelet-derived growth factor, and connective tissue growth factor by applying them locally to the open wound every 2 days. Among the growth factors examined, all of the three isoforms of TGF- exhibited a more potent stimulatory effect on COL1A2 promoter activity than did other factors. In addition, while TGF-beta1 and -beta2 significantly increased the number of fibroblasts which were positive for X-Gal staining, TGF-beta3 treatment did not change the number of beta-galactosidase expressing cells. Accumulation of collagen fibers was observed to the same extent in the mice treated with TGF-beta1 and those with TGF-beta3. These findings suggest that TGF-beta1 and -beta3 have similar but not identical regulatory mechanisms of COL1A2 expression, and that their pathophysiological roles in wound healing might be different from each other.

Animals↗

PubMed2002

Ligand-dependent genetic recombination in fibroblasts : a potentially powerful technique for investigating gene function in fibrosis.

Strategies for conditional induction of transgene expression in mice are likely to be valuable for testing the role of candidate genes in disease pathogenesis. We have developed a system for lineage-specific, ligand-dependent, induction of sustained transgene expression in fibroblastic cells in mice using a chimeric gene encoding the Cre-ER(T) fusion protein, under the control of a fibroblast-specific regulatory sequence from the pro alpha 2(I)collagen gene. Cre-ER(T) operates as a tamoxifen-dependent DNA recombinase to excise fragments flanked by specific LoxP consensus sequences. To test efficiency and ligand dependency of this strategy, Cre-ER(T)-expressing mice were backcrossed with heterozygous ROSA26-LacZ reporter mice, in which a floxed-STOP cassette has been introduced upstream of a bacterial beta-galactosidase (LacZ) reporter gene at a ubiquitously expressed locus. Constitutive or tamoxifen-induced LacZ expression was examined in embryonic, neonatal, and adult compound-transgenic mice. When pregnant ROSA26-LacZ females received a single dose of tamoxifen, high-level expression of LacZ in the skin was demonstrable from 24 hours after injection in double-transgenic embryos harboring both the Cre-ER(T) transgene and the target ROSA26-LacZ allele. High-level expression of LacZ was also induced postnatally by tamoxifen specifically in dermal and visceral fibroblasts. By allowing efficient embryonic or postnatal modification of alleles that have been targeted to incorporate LoxP sites, or to switch on transgenes cloned downstream of the floxed-STOP cassette, this system will allow fibroblast-specific genetic perturbations to be induced at predetermined embryonic or postnatal time points. This should greatly assist in in vivo functional studies of candidate genes in fibrotic diseases such as systemic sclerosis.

Animals↗