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1.
J Dent Res ; 94(12): 1668-75, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26341973

ABSTRACT

For decades, it has been widely accepted that hypertrophic chondrocytes undergo apoptosis prior to endochondral bone formation. However, very recent studies in long bone suggest that chondrocytes can directly transform into bone cells. Our initial in vivo characterization of condylar hypertrophic chondrocytes revealed modest numbers of apoptotic cells but high levels of antiapoptotic Bcl-2 expression, some dividing cells, and clear alkaline phosphatase activity (early bone marker). Ex vivo culture of newborn condylar cartilage on a chick chorioallantoic membrane showed that after 5 d the cells on the periphery of the explants had begun to express Col1 (bone marker). The cartilage-specific cell lineage-tracing approach in triple mice containing Rosa 26(tdTomato) (tracing marker), 2.3 Col1(GFP) (bone cell marker), and aggrecan Cre(ERT2) (onetime tamoxifen induced) or Col10-Cre (activated from E14.5 throughout adult stage) demonstrated the direct transformation of chondrocytes into bone cells in vivo. This transformation was initiated at the inferior portion of the condylar cartilage, in contrast to the initial ossification site in long bone, which is in the center. Quantitative data from the Col10-Cre compound mice showed that hypertrophic chondrocytes contributed to ~80% of bone cells in subchondral bone, ~70% in a somewhat more inferior region, and ~40% in the most inferior part of the condylar neck (n = 4, P < 0.01 for differences among regions). This multipronged approach clearly demonstrates that a majority of chondrocytes in the fibrocartilaginous condylar cartilage, similar to hyaline cartilage in long bones, directly transform into bone cells during endochondral bone formation. Moreover, ossification is initiated from the inferior portion of mandibular condylar cartilage with expansion in one direction.


Subject(s)
Bone Development/physiology , Chondrocytes/physiology , Mandibular Condyle/growth & development , Animals , Apoptosis/physiology , Cartilage/cytology , Cartilage/growth & development , Cell Differentiation/physiology , Cell Lineage/physiology , Mandibular Condyle/cytology , Mice , Mice, Transgenic , Microscopy, Confocal
2.
J Dent Res ; 94(3): 430-8, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25568170

ABSTRACT

Bone and dentin share similar biochemical compositions and physiological properties. Dentin, a major tooth component, is formed by odontoblasts; in contrast, bone is produced by osteoblasts. Osterix (Osx), a zinc finger-containing transcription factor, has been identified as an essential regulator of osteoblast differentiation and bone formation. However, it has been difficult to establish whether Osx functions in odontoblast differentiation and dentin formation. To understand the role of Osx in dentin formation, we analyzed mice in which Osx was subjected to tissue-specific ablation under the control of either the Col1a1 or the OC promoter. Two independent Osx conditional knockout mice exhibited similar molar abnormalities. Although no phenotype was found in the crowns of these teeth, both mutant lines exhibited short molar roots due to impaired root elongation. Furthermore, the interradicular dentin in these mice showed severe hypoplastic features, which were likely caused by disruptions in odontoblast differentiation and dentin formation. These phenotypes were closely related to the temporospatial expression pattern of Osx during tooth development. These findings indicate that Osx is required for root formation by regulating odontoblast differentiation, maturation, and root elongation. Cumulatively, our data strongly indicate that Osx is a site-specific regulator in tooth root formation.


Subject(s)
Odontogenesis/physiology , Tooth Root/growth & development , Transcription Factors/physiology , Zinc Fingers/physiology , Animals , Cell Differentiation/physiology , Cell Proliferation/physiology , Collagen Type I/physiology , Collagen Type I, alpha 1 Chain , Dental Pulp/cytology , Dentin/abnormalities , Dentinogenesis/physiology , Mice , Mice, Knockout , Molar/abnormalities , Odontoblasts/physiology , Osteocalcin/physiology , Sp7 Transcription Factor , Tooth Crown/cytology , Tooth Root/abnormalities , X-Ray Microtomography/methods
3.
Int J Immunopathol Pharmacol ; 24(3): 595-601, 2011.
Article in English | MEDLINE | ID: mdl-21978691

ABSTRACT

Transgenic mice that over-express connective tissue growth factor (CTGF) in fibroblasts under the control of an enhancer/promoter element of the Col1a2 gene (Col1a2-CTGF) recapitulate multiorgan fibrosis similar to fibrosis observed in Scleroderma (SSc). In this study we investigate the regulation of secreted protein acidic and rich in cysteine (Sparc) and Ctgf siRNAs on the expression of several extracellular matrix components in the fibroblasts derived from Col1a2-CTGF transgenic mice. Three fibroblast lines were obtained from each of wide type C57BL/6 and CTGF transgenic C57BL/6, and were transfected with Sparc siRNA or Ctgf siRNA. Real-time quantitative RT-PCR and Western blotting were used to examine the transcription and protein levels of type I collagen, CTGF and SPARC. Student's t-tests were used to determine the significance of the results. Our results showed that Col1a2 and Ctgf increased expression at both transcriptional and translational levels in the fibroblasts from the Col1a2-CTGF transgenic mice compared with those in the fibroblasts from their normal wild-type littermate. The treatment with Sparc siRNA or Ctgf siRNA attenuated the mRNA and/or protein expression of the Col1a2, Ctgf and Sparc in these fibroblasts. Sparc and Ctgf siRNAs also showed a reciprocal inhibition at transcript levels. Therefore, our results indicated that both SPARC and CTGF appeared to be involved in the same biological pathway, and they have the potential to serve as a therapeutic target for fibrotic diseases such as SSc.


Subject(s)
Connective Tissue Growth Factor/genetics , Extracellular Matrix/genetics , Fibroblasts/metabolism , Gene Expression Regulation/drug effects , Osteonectin/genetics , RNA, Small Interfering/pharmacology , Animals , Blotting, Western , Collagen Type I/biosynthesis , Fibroblasts/drug effects , Green Fluorescent Proteins , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Reverse Transcriptase Polymerase Chain Reaction , Scleroderma, Systemic/genetics , Scleroderma, Systemic/pathology , Skin/metabolism , Skin/pathology , Transfection
4.
Osteoarthritis Cartilage ; 11(4): 233-41, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12681949

ABSTRACT

OBJECTIVE: The Sox9 transcription factor has emerged as an important determinant of chondrocyte differentiation, including the regulation of type II collagen (Col2) and aggrecan gene expression. We sought to identify a human cell line model that conserves the Sox9 regulatory pathways identified in the mouse. DESIGN: The SW1353 chondrosarcoma cell line was considered to be a candidate for Sox9 studies. The activity of a Sox9 regulated Col2a1 enhancer reporter gene was analyzed in response to treating cells with known regulators of murine Sox9 expression/activity. The effect of treatment on expression of the endogenous Sox9 gene was analyzed by real-time PCR and Western blot. RESULTS: Col2 enhancer activity was stimulated by fibroblast growth factors (FGF-1 and -2) and repressed by inflammatory cytokines (IL-1beta and TNFalpha) in SW1353 cells. These effects correlated with changes in Sox9 mRNA and protein levels. In addition, FGF-9 was shown to stimulate enhancer activity and Sox9 expression. Cotreatment studies demonstrated that FGFs functionally antagonize the cytokine-mediated repression of Sox9 expression and Col2 enhancer activity. CONCLUSIONS: SW1353 cells represent a useful human cell model as they conserve many Sox9 signaling pathways previously demonstrated in mouse chondrocytes. We identify FGF-9 as a particularly potent Sox9 agonist. The antagonism between FGFs and cytokines on Sox9 expression and Col2 enhancer activity suggests that Sox9 integrates the opposing activities of FGFs and cytokines. We also find that SW1353 cells respond to very low doses of IL-1 with Col2 enhancer activation, while increasing doses lead to repression.


Subject(s)
Chondrosarcoma/genetics , Cytokines/genetics , Enhancer Elements, Genetic/genetics , Fibroblast Growth Factors/genetics , Signal Transduction/genetics , Transcription Factors/genetics , Animals , Blotting, Western/methods , Cells, Cultured , Chondrocytes , Gene Expression/genetics , Humans , Interleukin-1/genetics , Mice , Neoplasm Proteins/analysis , Polymerase Chain Reaction , RNA, Messenger/analysis , Transfection , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/genetics
5.
Dev Cell ; 1(2): 277-90, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11702786

ABSTRACT

L-Sox5 and Sox6 are highly identical Sry-related transcription factors coexpressed in cartilage. Whereas Sox5 and Sox6 single null mice are born with mild skeletal abnormalities, Sox5; Sox6 double null fetuses die with a severe, generalized chondrodysplasia. In these double mutants, chondroblasts poorly differentiate. They express the genes for all essential cartilage extracellular matrix components at low or undetectable levels and initiate proliferation after a long delay. All cartilages are thus extracellular matrix deficient and remain rudimentary. While chondroblasts in the center of cartilages ultimately activate prehypertrophic chondrocyte markers, epiphyseal chondroblasts ectopically activate hypertrophic chondrocyte markers. Thick intramembranous bone collars develop, but the formation of cartilage growth plates and endochondral bones is disrupted. L-Sox5 and Sox6 are thus redundant, potent enhancers of chondroblast functions, thereby essential for endochondral skeleton formation.


Subject(s)
Cartilage/embryology , Cartilage/physiology , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , High Mobility Group Proteins/metabolism , High Mobility Group Proteins/physiology , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Transcription Factors , Animals , Bone Development , Bone and Bones/abnormalities , Cell Differentiation , Chondrocytes/cytology , Chondrocytes/metabolism , Exostoses, Multiple Hereditary/genetics , In Situ Hybridization , Mice , Microscopy, Fluorescence , Models, Biological , Models, Genetic , Mutation , Phenotype , SOXD Transcription Factors
6.
Curr Opin Cell Biol ; 13(6): 721-7, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11698188

ABSTRACT

Three transcription factors of the Sox family have essential roles in different steps of the chondrocyte differentiation pathway. Because the transcription factor Cbfa1, which is needed for osteoblast differentiation, also stimulates hypertrophic chondrocyte maturation, it links the chondrocyte and osteoblast differentiation pathways in endochondral bone formation. Signaling molecules, including Indian Hedgehog, PTHrP and FGFs, also establish essential links either between these pathways, between steps in these pathways or between signaling molecules and transcription factors, so that a more comprehensive view of endochondral bone formation is emerging.


Subject(s)
Bone and Bones/embryology , Cartilage/embryology , Chondrogenesis , Osteogenesis , Animals , Cell Differentiation , Chondrocytes/physiology , Growth Plate/anatomy & histology , Growth Plate/embryology , Humans , Mesoderm/physiology , Mice , Models, Biological , Signal Transduction , Transcription Factors/physiology
7.
Osteoarthritis Cartilage ; 9 Suppl A: S69-75, 2001.
Article in English | MEDLINE | ID: mdl-11680692

ABSTRACT

OBJECTIVE: This work was carried out to identify transcription factors controlling the differentiation of mesenchymal cells into chondrocytes. DESIGN: We delineated a cartilage-specific enhancer in the collagen type 2 gene (Col2a1) and identified transcription factors responsible for the activity of this enhancer in chondrocytes. We then analyzed the ability of these transcription factors to activate specific genes of the chondrocyte differentiation program and control cartilage formation in vivo. RESULTS: A 48-bp sequence in the first intron of Col2a1 drove gene expression specifically in cartilage in transgenic mouse embryos. The transcription factors L-Sox5, Sox6, and Sox9 bound and cooperatively activated this enhancer in vitro. They belong to the Sry-related family of HMG box DNA-binding proteins, which includes many members implicated in cell fate determination in various lineages. L-Sox5, Sox6, and Sox9 were coexpressed in all precartilaginous condensations in mouse embryos and continued to be expressed in chondrocytes until the cells underwent final hypertrophy. Whereas L-Sox5 and Sox6 are highly homologous proteins, they are totally different from Sox9 outside the HMG box domain. The three proteins cooperatively activated the Col2a1- and aggrecan genes in cultured cells. Heterozygous mutations in SOX9 in humans lead to campomelic dysplasia, a severe and generalized skeletal malformation syndrome. Embryonic cells with a homozygous Sox9 mutation were unable to form cartilage in vivo and activate essential chondrocyte marker genes. Preliminary data indicated that the mutation of Sox5 and Sox6 in the mouse led to severe skeletal malformations. CONCLUSIONS: L-Sox5, Sox6, and Sox9 play essential roles in chondrocyte differentiation and, thereby, in cartilage formation. Their discovery will help to understand further the molecular mechanisms controlling chondrogenesis in vivo, uncover genetic mechanisms underlying cartilage diseases, and develop novel strategies for cartilage repair.


Subject(s)
Cell Differentiation/physiology , Chondrocytes/cytology , DNA-Binding Proteins/physiology , High Mobility Group Proteins/physiology , Nuclear Proteins/physiology , Transcription Factors/physiology , Animals , Collagen Type II/genetics , Gene Expression , Mesoderm/cytology , Mice , Mice, Transgenic , SOX9 Transcription Factor , SOXD Transcription Factors
8.
J Biol Chem ; 276(44): 40621-30, 2001 Nov 02.
Article in English | MEDLINE | ID: mdl-11514576

ABSTRACT

To understand the role of CCAAT-binding factor (CBF) in transcription in the context of chromatin-assembled DNA, we used regularly spaced nucleosomal DNA using topoisomerase IIalpha (topo IIalpha) and alpha2(1) collagen promoter templates, which were subsequently reconstituted in an in vitro transcription reaction. Binding of CBF to the nucleosomal wild-type topo IIalpha promoter containing four CBF-binding sites disrupted the regular nucleosomal structure not only in the promoter region containing the CBF-binding sites but also in the downstream region over the transcription start site. In contrast, no nucleosome disruption was observed in a mutant topo IIalpha promoter containing mutations in all CBF-binding sites. Interestingly, CBF also activated transcription from nucleosomal wild-type topo IIalpha promoter. In this experiment, a promoter containing one wild-type CBF-binding site was activated very weakly, whereas the promoter containing mutations in all sites was not activated by CBF. A truncated CBF that lacked the glutamine-rich domains did not activate transcription from nucleosomal wild-type topo IIalpha promoter but disrupted the nucleosomal structure about as much as did the binding of full-length CBF. Two nucleosomal mouse alpha2(1) collagen promoter DNAs, one containing a single and the other containing four CBF- binding sites, were also reconstituted in an in vitro transcription reaction. None of the nucleosomal collagen promoters was activated by CBF. However, both of these collagen promoters were activated by CBF when the transcription reaction was performed using naked DNA templates. Binding of CBF to the nucleosomal collagen promoter containing four binding sites disrupted the nucleosomal structure, similarly as observed in the topo IIalpha promoter. Altogether this study indicates that CBF-mediated nucleosomal disruption occurred independently of transcription activation. It also suggests that specific promoter structure may play a role in the CBF-mediated transcription activation of nucleosomal topo IIalpha promoter template.


Subject(s)
CCAAT-Binding Factor/physiology , DNA Topoisomerases, Type II/genetics , Nucleosomes/metabolism , Promoter Regions, Genetic , Transcriptional Activation/physiology , 3T3 Cells , Animals , Antigens, Neoplasm , Base Sequence , DNA Primers , DNA-Binding Proteins , Mice
9.
Proc Natl Acad Sci U S A ; 98(12): 6698-703, 2001 Jun 05.
Article in English | MEDLINE | ID: mdl-11371614

ABSTRACT

In humans, SOX9 heterozygous mutations cause the severe skeletal dysmorphology syndrome campomelic dysplasia. Except for clinical descriptions, little is known about the pathogenesis of this disease. We have generated heterozygous Sox9 mutant mice that phenocopy most of the skeletal abnormalities of this syndrome. The Sox9(+/-) mice died perinatally with cleft palate, as well as hypoplasia and bending of many skeletal structures derived from cartilage precursors. In embryonic day (E)14.5 heterozygous embryos, bending of radius, ulna, and tibia cartilages was already prominent. In E12.5 heterozygotes, all skeletal elements visualized by using Alcian blue were smaller. In addition, the overall levels of Col2a1 RNA at E10.5 and E12.5 were lower than in wild-type embryos. We propose that the skeletal abnormalities observed at later embryonic stages were caused by delayed or defective precartilaginous condensations. Furthermore, in E18.5 embryos and in newborn heterozygotes, premature mineralization occurred in many bones, including vertebrae and some craniofacial bones. Because Sox9 is not expressed in the mineralized portion of the growth plate, this premature mineralization is very likely the consequence of allele insufficiency existing in cells of the growth plate that express Sox9. Because the hypertrophic zone of the heterozygous Sox9 mutants was larger than that of wild-type mice, we propose that Sox9 also has a role in regulating the transition to hypertrophic chondrocytes in the growth plate. Despite the severe hypoplasia of cartilages, the overall organization and cellular composition of the growth plate were otherwise normal. Our results suggest the hypothesis that two critical steps of the chondrocyte differentiation pathway are sensitive to Sox9 dosage. First, an early step presumably at the stage of mesenchymal condensation of cartilage primordia, and second, a later step preceding the transition of chondrocytes into hypertrophic chondrocytes.


Subject(s)
Bone and Bones/abnormalities , Calcification, Physiologic , Cartilage/abnormalities , High Mobility Group Proteins/genetics , Mutation , Transcription Factors/genetics , Animals , Bone Diseases, Developmental/genetics , Cell Differentiation , Chondrocytes/physiology , High Mobility Group Proteins/physiology , Humans , Mesoderm/physiology , Mice , Mice, Inbred C57BL , SOX9 Transcription Factor , Transcription Factors/physiology
10.
Arthritis Rheum ; 44(3): 712-22, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11263787

ABSTRACT

OBJECTIVE: Reporter transgenes were introduced into the type 1 tight-skin (Tsk1/+) mouse model of scleroderma to test the hypothesis that fibroblast-specific genetic programs are activated in fibrosis. METHODS: Transgenes harboring upstream fragments of the 5' flanking region of the mouse proalpha2(I) collagen gene (Col1a2), linked to a 400-bp minimal Col1a2 promoter driving an Escherichia coli beta-galactosidase (LacZ) reporter gene, were introduced into Tsk1/+ mice by breeding. Expression of these transgenes, which function as lineage-specific markers of fibroblast differentiation, was compared between the Tsk-LacZ mice and non-Tsk littermates. Responsiveness of these constructs to the profibrotic cytokine, transforming growth factor beta1 (TGFbeta1), was investigated by transient transfection of reporter constructs in tissue-culture cells. RESULTS: There was significant activation of reporter genes harboring the upstream enhancer in Tsk1/+ mice starting from 1 week of age. This was maximal at 6 weeks old (mean +/- SD 237 +/- 24% of non-Tsk controls; P= 0.001). Recombinant TGFbeta1 significantly activated reporter genes regulated by the upstream enhancer in transient transfection, and Tsk-LacZ fibroblasts showed elevated LacZ expression in tissue culture. CONCLUSION: These data suggest that activating signals in Tsk1/+ mice may act via fibroblast-specific regulatory elements within the murine Col1a2 gene. Although TGFbeta has been implicated in the pathogenesis of fibrosis, and reporter genes regulated by the upstream enhancer appear to be TGFbeta responsive in vitro, our results suggest that fibroblast-specific pathways may also be involved.


Subject(s)
Fibroblasts/metabolism , Procollagen/genetics , Scleroderma, Systemic/genetics , Animals , Disease Models, Animal , Enhancer Elements, Genetic/genetics , Gene Expression Regulation , Mice , Mice, Transgenic
11.
J Biol Chem ; 276(24): 21754-64, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11279244

ABSTRACT

We have examined the chromatin structure around and upstream of the transcriptional start site of the human alpha2(I) collagen (COL1A2) gene. Four strong DNase I-hypersensitive sites (HS2-5) were only detected in fibroblasts, and a weaker one (HS1) was identified in type I collagen-negative cells. Another hypersensitive site potentially involved in COL1A2 silencing was found in intron 1 (HS(In)). HS1 and HS2 were mapped within conserved promoter sequences and at locations comparable to the mouse gene. HS3, HS4, and HS5 were likewise mapped approximately 20 kilobases upstream of COL1A2 at about the same position as the mouse far-upstream enhancer and within a remarkably homologous genomic segment. DNase I footprinting identified twelve areas of nuclease protection in the far-upstream region (FU1-12) and within stretches nearly identical to the mouse sequence. The region containing HS3-5 was found to confer high and tissue-specific expression in transgenic mice to the otherwise minimally active COL1A2 promoter. Characterization of the human element documented functional differences with the mouse counterpart. Enhancer activity substantially decreased without the segment containing FU1-7 and HS5, and inclusion of AluI repeats located 3' of HS3 augmented position-independent expression of the transgene. Hence, subtle differences may characterize the regulation of mammalian alpha2(I) collagen genes by evolutionarily conserved sequences.


Subject(s)
Collagen/genetics , Enhancer Elements, Genetic , Evolution, Molecular , Gene Expression Regulation, Developmental , Promoter Regions, Genetic , Animals , Base Sequence , Cell Line , Chromatin/genetics , Collagen Type I , Conserved Sequence , Crosses, Genetic , DNA/chemistry , DNA Footprinting , Deoxyribonuclease I , Embryonic and Fetal Development , Fibroblasts/metabolism , Humans , Lung , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Molecular Sequence Data , Restriction Mapping , Sequence Alignment , Sequence Homology, Nucleic Acid
12.
Biotechniques ; 30(1): 94-8, 100, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11196326

ABSTRACT

The SOS recruitment system (SRS), a recently developed method for detecting protein-protein interactions, provides an attractive alternative to identify biologically important protein interactions. In SRS, the protein-protein interactions take place in the cytoplasm instead of the nucleus, as is the case in the conventional two-hybrid system. Although the SRS has overcome some of the disadvantages of the conventional two-hybrid system, it still has several problems and limitations. Here, we describe a new protocol for SRS library screening. A new combination of growth media to avoid the tedious step of replica plating greatly increases the number of independent colonies in a single library screening. Furthermore, we designed a pair of ras-specific primers and a one-step simple PCR to rule out the most abundant false positive, the mammalian ras cDNA, in SRS library screening.


Subject(s)
Gene Library , Son of Sevenless Proteins/metabolism , Animals , DNA, Complementary/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Galactose/pharmacology , Glucose/pharmacology , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Plasmids/genetics , Polymerase Chain Reaction , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SOX9 Transcription Factor , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Son of Sevenless Proteins/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Transformation, Genetic , ras Proteins/metabolism
13.
J Biol Chem ; 276(15): 12212-21, 2001 Apr 13.
Article in English | MEDLINE | ID: mdl-11152695

ABSTRACT

We have discovered a new member of the class I small leucine-rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression of asporin partially overlaps with the expression of decorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22-9q21.3 where asporin is part of a SLRP gene cluster that includes extracellular matrix protein 2, osteoadherin, and osteoglycin. Further analysis shows that, with the exception of biglycan, all known SLRP genes reside in three gene clusters.


Subject(s)
Gene Expression , Glycoproteins/genetics , Leucine/chemistry , Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins , Chromosome Mapping , Chromosomes, Human, Pair 9 , DNA, Complementary , Extracellular Matrix Proteins , Glycoproteins/chemistry , Humans , Leucine-Rich Repeat Proteins , Mice , Molecular Sequence Data , Proteins/chemistry , Sequence Homology, Amino Acid
14.
Matrix Biol ; 19(5): 389-94, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10980415

ABSTRACT

With the goal of identifying master transcription factors that control the genetic program of differentiation of mesenchymal cells into chondrocytes, we first delineated a 48-bp chondrocyte-specific enhancer element in the gene for proalpha1(II) collagen (Col2a1), an early and abundant marker of chondrocytes. Our experiments have demonstrated that the HMG-box-containing transcription factor, Sox9 which binds and activates this enhancer element, is required for chondrocyte differentiation and for expression of a series of chondrocyte-specific marker genes including Col2a1, Col9a2, Col11a2 and Aggrecan. In the absence of Sox9 the block in differentiation occurs at the stage of mesenchymal condensation, suggesting the hypothesis that Sox9 might also control expression of cell surface proteins needed for mesenchymal condensation. Since Sox9 also contains a potent transcription activation domain, it is a typical transcription factor. Two other members of the Sox family, L-Sox5 and Sox6, also bind to the 48-bp Col2a1 enhancer and together with Sox9 activate this enhancer as well as the endogenous Col2a1 and aggrecan genes. L-Sox5 and Sox6 have a high degree of sequence identity to each other and are likely to have redundant functions. Except for the HMG-box, L-Sox5 and Sox6 have no similarity to Sox9 and, hence, are likely to have a complementary function to that of Sox9. Our experiments suggest the hypothesis that, like Sox9, Sox5 and Sox6 might also be needed for chondrocyte differentiation. Other experiments, have provided evidence that the Sox9 polypeptide and the Sox9 gene are targets of signaling molecules that are known to control discrete steps of chondrogenesis in the growth plate of endochondral bones. Protein kinase A (PKA) phosphorylation of Sox9 increases its DNA binding and transcriptional activity. Since PKA-phosphorylated-Sox9 is found in the prehypertrophic zone of the growth plate, the same location where the gene for the receptor of the parathyroid hormone-related peptide (PTHrP) is expressed and since PTHrP signaling is mediated by cyclic AMP, we have hypothesized that Sox9 is a target for PTHrP signaling. Other experiments have also shown that fibroblast growth factors (FGFs) increase the expression of Sox9 in chondrocytes in culture and that this activation is mediated by the mitogen-activated protein kinase pathway. These results favor the hypothesis that in achondroplasia, a disease caused by activating mutations in FGF receptor 3, there might also be an abnormally high Sox9 expression.


Subject(s)
Chondrocytes/cytology , Chondrocytes/metabolism , Animals , Cell Differentiation/genetics , Gene Expression , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Models, Biological , Mutation , SOX9 Transcription Factor , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic
15.
Osteoarthritis Cartilage ; 8(4): 248-57, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10903878

ABSTRACT

OBJECTIVE: The present study was conducted on transgenic Del1 (+/-) mice harboring six copies of a transgene with small deletion mutation engineered into mouse type II collagen gene. Incorporation of transgene into mouse genome was predicted to cause reduced mechanical strength of articular cartilage with deposition of structurally inferior collagen network and consequently to predispose the animal to early-onset joint degeneration. DESIGN: Progression of degenerative chances in the knee joints of Del1 (+/-) and control mice was followed by macroscopic and histologic analyses at 3-5 month intervals between 3 and 22 months of age. Expression and distribution of type II collagen was studied with Northern hybridization, RNase protection assay and immunohistochemistry. RESULTS: Articular cartilage degeneration began with superficial fibrillation at the age of 3 months in Del1 (+/-) mice. These changes coincided with a significant reduction in the expression of both endogenous and transgene-derived type II collagen mRNA. The defects gradually progressed into erosions penetrating the articular cartilage, bony sclerosis, degeneration of menisci, mineralization of various joint structures, cyst formation and exposure of subchondral bone. Nontransgenic controls also developed osteoarthritic lesions, but these appeared significantly later and were less severe. Increased transcription of type IIA procollagen mRNA, typical for chondroprogenitor cells and cartilage repair was also observed at six months in Del1 (+/-) mice. CONCLUSION: These findings suggest that the impact of truncated type II collagen transgene, together with maturation-related reduction in type II collagen production significantly contribute to the early-onset degeneration of knee joints in Del1 (+/-) mice. These mice with osteoarthritis-like phenotype should provide a useful model for studies on the early pathogenic mechanisms involved in articular cartilage degeneration.


Subject(s)
Collagen/genetics , Gene Deletion , Osteoarthritis/genetics , Animals , Blotting, Northern , Female , Knee Joint , Male , Mice , Mice, Transgenic , Osteoarthritis/metabolism , Osteoarthritis/pathology , RNA, Messenger/analysis , Ribonucleases/analysis
16.
Mol Cell Biol ; 20(11): 4149-58, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10805756

ABSTRACT

Sox9 is a high-mobility-group domain-containing transcription factor required for chondrocyte differentiation and cartilage formation. We used a yeast two-hybrid method based on Son of Sevenless (SOS) recruitment to screen a chondrocyte cDNA library and found that the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA-Calpha) interacted specifically with SOX9. Next we found that two consensus PKA phosphorylation sites within SOX9 could be phosphorylated by PKA in vitro and that SOX9 could be phosphorylated by PKA-Calpha in vivo. In COS-7 cells cotransfected with PKA-Calpha and SOX9 expression plasmids, PKA enhanced the phosphorylation of wild-type SOX9 but did not affect phosphorylation of a SOX9 protein in which the two PKA phosphorylation sites (S(64) and S(211)) were mutated. Using a phosphospecific antibody that specifically recognized SOX9 phosphorylated at serine 211, one of the two PKA phosphorylation sites, we demonstrated that addition of cAMP to chondrocytes strongly increased the phosphorylation of endogenous Sox9. In addition, immunohistochemistry of mouse embryo hind legs showed that Sox9 phosphorylated at serine 211 was principally localized in the prehypertrophic zone of the growth plate, corresponding to the major site of expression of the parathyroid hormone-related peptide (PTHrP) receptor. Since cAMP has previously been shown to effectively increase the mRNA levels of Col2a1 and other specific markers of chondrocyte differentiation in culture, we then asked whether PKA phosphorylation could modulate the activity of SOX9. Addition of 8-bromo-cAMP to chondrocytes in culture increased the activity of a transiently transfected SOX9-dependent 48-bp Col2a1 chondrocyte-specific enhancer; similarly, cotransfection of PKA-Calpha increased the activity of this enhancer. Mutations of the two PKA phosphorylation consensus sites of SOX9 markedly decreased the PKA-Calpha activation of this enhancer by SOX9. PKA phosphorylation and the mutations in the consensus PKA phosphorylation sites of SOX9 did not alter its nuclear localization. In vitro phosphorylation of SOX9 by PKA resulted in more efficient DNA binding. We conclude that SOX9 is a target of cAMP signaling and that phosphorylation of SOX9 by PKA enhances its transcriptional and DNA-binding activity. Because PTHrP signaling is mediated by cAMP, our results support the hypothesis that Sox9 is a target of PTHrP signaling in the growth plate and that the increased activity of Sox9 might mediate the effect of PTHrP in maintaining the cells as nonhypertrophic chondrocytes.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Enhancer Elements, Genetic , High Mobility Group Proteins/metabolism , Transcription Factors/metabolism , Transcriptional Activation , 8-Bromo Cyclic Adenosine Monophosphate/metabolism , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Chondrocytes , Chondrosarcoma , Collagen/metabolism , Consensus Sequence , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , DNA/metabolism , Enzyme Activation , High Mobility Group Proteins/genetics , Mice , Molecular Sequence Data , Mutagenesis , Phosphorylation , Rats , SOX9 Transcription Factor , Saccharomyces cerevisiae , Serine/metabolism , Subcellular Fractions , Transcription Factors/genetics , Tumor Cells, Cultured , Two-Hybrid System Techniques
17.
Proc Natl Acad Sci U S A ; 97(3): 1113-8, 2000 Feb 01.
Article in English | MEDLINE | ID: mdl-10655493

ABSTRACT

Recent experiments have established that Sox9 is required for chondrocyte differentiation. Here, we show that fibroblast growth factors (FGFs) markedly enhance Sox9 expression in mouse primary chondrocytes as well as in C3H10T1/2 cells that express low levels of Sox9. FGFs also strongly increase the activity of a Sox9-dependent chondrocyte-specific enhancer in the gene for collagen type II. Transient transfection experiments using constructs encoding FGF receptors strongly suggested that all FGF receptors, FGFR1-R4, can transduce signals that lead to the increase in Sox9 expression. The increase in Sox9 levels induced by FGF2 was inhibited by a specific mitogen-activated protein kinase kinase (MAPKK)/mitogen-activated protein kinase/ERK kinase (MEK) inhibitor U0126 in primary chondrocytes. In addition, coexpression of a dual-specificity phosphatase, CL100/MKP-1, that is able to dephosphorylate and inactivate mitogen-activated protein kinases (MAPKs) inhibited the FGF2-induced increase in activity of the Sox9-dependent enhancer. Furthermore, coexpression of a constitutively active mutant of MEK1 increased the activity of the Sox9-dependent enhancer in primary chondrocytes and C3H10T1/2 cells, mimicking the effects of FGFs. These results indicate that expression of the gene for the master chondrogenic factor Sox9 is stimulated by FGFs in chondrocytes as well as in undifferentiated mesenchymal cells and strongly suggest that this regulation is mediated by the MAPK pathway. Because Sox9 is essential for chondrocyte differentiation, we propose that FGFs and the MAPK pathway play an important role in chondrogenesis.


Subject(s)
Fibroblast Growth Factors/pharmacology , Gene Expression Regulation/drug effects , MAP Kinase Signaling System , Animals , Base Sequence , Butadienes/pharmacology , Cartilage/cytology , Cell Differentiation , Gene Expression Regulation/physiology , Mesoderm/cytology , Mice , Mice, Inbred C3H , Molecular Sequence Data , Nitriles/pharmacology , Recombinant Fusion Proteins/pharmacology , Transfection
18.
J Biol Chem ; 275(5): 3687-92, 2000 Feb 04.
Article in English | MEDLINE | ID: mdl-10652367

ABSTRACT

The inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) strongly inhibit the expression of genes for cartilage extracellular matrix proteins. We have recently obtained genetic evidence indicating that the high mobility group domain containing transcription factor Sox9 is required for cartilage formation and for expression of chondrocyte-specific genes including the gene for type II collagen (Col2a1). We show here that IL-1 and TNF-alpha cause a marked and rapid decrease in the levels of Sox9 mRNA and/or protein in chondrocytes. A role for the transcription factor NFkappaB in Sox9 down-regulation was suggested by the ability of pyrrolidine dithiocarbamate, an inhibitor of the NFkappaB pathway, to block the effects of IL-1 and TNF-alpha. This role was further supported by the ability of a dominant-negative mutant of IkappaBalpha to block the IL-1 and TNF-alpha inhibition of Sox9-dependent Col2a1 enhancer elements. Furthermore, forced expression of the NFkappaB subunits p65 or p50 also inhibited Sox9-dependent Col2a1 enhancer. Because Sox9 is essential for chondrogenesis, the marked down-regulation of the Sox9 gene by IL-1 and TNF-alpha in chondrocytes is sufficient to account for the inhibition of the chondrocyte phenotype by these cytokines. The down-regulation of Sox9 may have a crucial role in inhibiting expression of the cartilage phenotype in inflammatory joint diseases.


Subject(s)
Cartilage/physiology , High Mobility Group Proteins/genetics , Interleukin-1/pharmacology , Transcription Factors/genetics , Tumor Necrosis Factor-alpha/pharmacology , Animals , Cell Line , Down-Regulation/drug effects , Extracellular Matrix/metabolism , High Mobility Group Proteins/biosynthesis , Interleukin-1/genetics , Interleukin-1/metabolism , Mice , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , SOX9 Transcription Factor , Transcription Factors/biosynthesis , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
19.
Bone ; 26(1): 27-32, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10617154

ABSTRACT

Type I collagen is the major extracellular protein in bone, tendons, ligaments, and skin. DNA elements of the mouse pro-alpha1 (I) collagen promoter were shown to drive the bone-selective expression of a luciferase transgene. We examined whether this expression can be used to evaluate the effect of anabolic agents on bone formation in vivo. Treatment of either intact males, intact females, or ovariectomized (ovx) mice with 80 microg/kg/day of human parathyroid hormone (hPTH), for 5 to 11 days increased luciferase levels in tibiae by two- to threefold compared with vehicle-treated mice. The increases were tissue specific, as no changes in skin luciferase expression were observed. Treatment with prostaglandin E2, a potent bone anabolic agent, for 11 days also increased expression of the transgene in bone, but not in skin. Treatment with dihydrotestosterone (DHT) for 11 days increased luciferase activity in skin, but not in bone. Histomorphometric analysis revealed that 28-day treatment with PTH increased bone formation; 60-day treatment of OVX mice with DHT did not. These findings show a correlation between bone formation and the expression of a transgene driven by DNA elements of the mouse pro-alpha1 (I) collagen promoter, suggesting that this expression can be used as an indicator and provide a faster readout for the ability of agents to stimulate bone formation in this mouse strain.


Subject(s)
Bone and Bones/drug effects , Dinoprostone/pharmacology , Luciferases/metabolism , Parathyroid Hormone/pharmacology , Procollagen/genetics , Promoter Regions, Genetic , Transgenes , Animals , Bone and Bones/enzymology , Dihydrotestosterone/pharmacology , Humans , Luciferases/genetics , Male , Mice , Mice, Transgenic , Skin/drug effects , Skin/enzymology
20.
J Cell Biochem ; 76(3): 437-51, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10649441

ABSTRACT

The transcriptional regulation of the fibronectin (FN) gene in hepatoma cells by phorbol myristate acetate (PMA) was investigated. PMA increased the synthesis and mRNA levels of FN and its promoter activity in Hep3B hepatoma cells. The PMA-induced activation of FN expression was blocked by a protein kinase C (PKC) inhibitor and did not require a new protein synthesis. Deletion analysis revealed that the sequence between positions -69 and +136 of the FN gene was responsible for the PMA induction. Two PMA-inducible nuclear protein complexes were found to bind to a putative NF-kappaB site at -41 and were identified as a p65/p50 heterodimer and a p50/50 homodimer of NF-kappaB family. Mutations in the -41 NF-kappaB site, however, did not block the PMA induction of the FN promoter but rather enhanced it. Overexpression of p65 increased the FN promoter activity. While overexpression of p50 alone did not affect the promoter activity, it decreased the p65-induced activation of the FN promoter. Mutations in the -41 NF-kappaB site attenuated the p50-mediated suppression of the p65 transactivation of the FN promoter. Deletion of the sequence between +1 and +136 decreased the basal and PMA-induced activities of the FN promoter. This study shows that PMA induces the transcription of the FN gene in hepatoma cells via the PKC pathway. The DNA sequence between +1 and +136 is responsible, at least in part, for the PMA-induced activation of the FN gene, while the -41 NF-kappaB binding site plays as a negative regulatory element for it. In addition, this study is the first to show a role for NF-kappaB p65 in the transcriptional activation of the FN gene.


Subject(s)
Fibronectins/genetics , NF-kappa B/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Base Sequence , Binding Sites/genetics , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cycloheximide/pharmacology , DNA Primers/genetics , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Fibronectins/biosynthesis , Gene Expression Regulation/drug effects , Genes, Reporter , Humans , Molecular Sequence Data , Promoter Regions, Genetic , Protein Kinase C/metabolism , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Sequence Deletion , Tumor Cells, Cultured
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