1: Pol Merkuriusz Lek. 2005 Jul;19(109):94-7. [Development and congenital malformations of the skeleton] [Article in Polish] Burdan F. Pracownia Teratologii Doswiadczalnej Katedry i Zakladu Anatomii Prawidlowej Czlowieka Akademii Medycznej im. prof. Feliksa Skubiszewskiego w Lublinie. fb3@wp.pl The article presents current knowledge on skeleton development, which is initiated by mesenchymal cells condensation. The mesenchymal to epithelial transformation, vascular regression, cartilage formation and ossification are involved in normal skeletogenesis. All the processes are influenced by various genes and regulatory molecules, e.g., N-cadherin, vascular endothelial growth factor, transforming growth factor-beta, syndecan-3 and others. The chondrogenesis depends on the Sox genes expression - Sox5, Sox6 and especially Sox9, which continued action produce persistent cartilage. The essential role in osteogenesis is played by the transcription factor Runx2 that directly activates expression of genes coding typical bone proteins like collagen type I, osteocalcin, osteopontin and osteonectin. Development of fibrous, cartilaginous and synovial joints, bone maturation and remodeling, and mechanisms leading to skeleton dysmorphogenesis are also discussed. PMID: 16194038 [PubMed - in process] --------------------------------------------------------------- 2: Birth Defects Res C Embryo Today. 2005 Sep;75(3):200-12. Transcriptional control of chondrocyte fate and differentiation. Lefebvre V, Smits P. Department of Biomedical Engineering and Orthopaedic Research Center, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA. lefebvv@ccf.org Chondrogenesis is an essential process in vertebrates. It leads to the formation of cartilage growth plates, which drive body growth and have primary roles in endochondral ossification. It also leads to the formation of permanent cartilaginous tissues that provide major structural support in the articular joints and respiratory and auditory tracts throughout life. Defects in chondrogenesis cause chondrodysostoses and chondrodysplasias. These skeletal malformation diseases account for a significant proportion of birth defects in humans and can dramatically affect a person's expectancy and quality of life. Chondrogenesis occurs when pluripotent mesenchymal cells commit to the chondrocyte lineage, and through a series of differentiation steps build and eventually remodel cartilage. This review summarizes and discusses our current knowledge and lack of knowledge about the chondrocyte differentiation pathway, from mesenchymal cells to growth plate and articular chondrocytes, with a main focus on how it is controlled by tissue patterning and cell differentiation transcription factors, such as, but not limited to, Pax1 and Pax9, Nkx3.1 and Nkx3.2, Sox9, Sox5 and Sox6, Runx2 and Runx3, and c-Maf. Copyright (c) 2005 Wiley-Liss, Inc. PMID: 16187326 [PubMed - in process] --------------------------------------------------------------- 3: J Bone Miner Metab. 2005;23(5):337-40. Distinct roles of Sox5, Sox6, and Sox9 in different stages of chondrogenic differentiation. Ikeda T, Kawaguchi H, Kamekura S, Ogata N, Mori Y, Nakamura K, Ikegawa S, Chung UI. Division of Tissue Engineering, University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan. Publication Types: Review Review, Tutorial PMID: 16133682 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Zhonghua Yi Xue Za Zhi. 2005 Apr 27;85(16):1129-32. [Cbfal's effect on chondrogenic gene expression in fibroblast] [Article in Chinese] Zhang W, Zhu YP, Wang J, Deng LF. Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China. OBJECTIVE: Core-binding factor a1, Cbfa1, which belongs to the runt-domain gene family, is an essential transcription factor for osteoblastic differentiation and osteogenesis. To examine the effects of Cbfa1 exhibit on gene expression which involving in the chondrogenesis. METHODS: On the first, using RT-PCR method, we directly cloned Cbfa1/Runx2 full length cDNA from E13.5 days mouse embryos limb buds. Consequently Cbfa1/Runx2 was cloned into pcDNA3.1 plasmid to make a eukaryotic expression vector. All the clones were proved to be correct by enzyme cutting and sequencing analysis. On the second, we used pcDNA3.1-Cbfa1 to tansfect fibroblast. 48 hours later, we contrasted the gene transcription of Sox9, Sox5 and Sox6 between tansfected and untransfected. 72 hours later, type II collagen have also been examined by Western blotting. RESULTS: Cbfa1 overexpression in fibroblast can upregulate the expression of Sox9 and type II collagen, but it have no effect on the expression of Sox5 and Sox6. CONCLUSION: Cbfa1 may involve in the regulation of chondrogenesis. PMID: 16029574 [PubMed - in process] --------------------------------------------------------------- 5: Biochem J. 2005 Aug 1;389(Pt 3):705-16. Highly conserved proximal promoter element harbouring paired Sox9-binding sites contributes to the tissue- and developmental stage-specific activity of the matrilin-1 gene. Rentsendorj O, Nagy A, Sinko I, Daraba A, Barta E, Kiss I. Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Temesvari krt 62, H-6726 Szeged, Hungary. The matrilin-1 gene has the unique feature that it is expressed in chondrocytes in a developmental stage-specific manner. Previously, we found that the chicken matrilin-1 long promoter with or without the intronic enhancer and the short promoter with the intronic enhancer restricted the transgene expression to the columnar proliferative chondroblasts and prehypertrophic chondrocytes of growth-plate cartilage in transgenic mice. To study whether the short promoter shared by these transgenes harbours cartilage-specific control elements, we generated transgenic mice expressing the LacZ reporter gene under the control of the matrilin-1 promoter between -338 and +67. Histological analysis of the founder embryos demonstrated relatively weak transgene activity in the developing chondrocranium, axial and appendicular skeleton with highest level of expression in the columnar proliferating chondroblasts and prehypertrophic chondrocytes. Computer analysis of the matrilin-1 genes of amniotes revealed a highly conserved Pe1 (proximal promoter element 1) and two less-conserved sequence blocks in the distal promoter region. The inverted Sox motifs of the Pe1 element interacted with chondrogenic transcription factors Sox9, L-Sox5 and Sox6 in vitro and another factor bound to the spacer region. Point mutations in the Sox motifs or in the spacer region interfered with or altered the formation of nucleoprotein complexes in vitro and significantly decreased the reporter gene activity in transient expression assays in chondrocytes. In vivo occupancy of the Sox motifs in genomic footprinting in the expressing cell type, but not in fibroblasts, also supported the involvement of Pe1 in the tissue-specific regulation of the gene. Our results indicate that interaction of Pe1 with distal DNA elements is required for the high level, cartilage- and developmental stage-specific transgene expression. PMID: 15804237 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Proc Natl Acad Sci U S A. 2005 Apr 5;102(14):5062-7. Epub 2005 Mar 21. Bmpr1a and Bmpr1b have overlapping functions and are essential for chondrogenesis in vivo. Yoon BS, Ovchinnikov DA, Yoshii I, Mishina Y, Behringer RR, Lyons KM. Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095, USA. Previous studies have demonstrated the ability of bone morphogenetic proteins (BMPs) to promote chondrogenic differentiation in vitro. However, the in vivo role of BMP signaling during chondrogenesis has been unclear. We report here that BMP signaling is essential for multiple aspects of early chondrogenesis. Whereas mice deficient in type 1 receptors Bmpr1a or Bmpr1b in cartilage are able to form intact cartilaginous elements, double mutants develop a severe generalized chondrodysplasia. The majority of skeletal elements that form through endochondral ossification are absent, and the ones that form are rudimentary. The few cartilage condensations that form in double mutants are delayed in the prechondrocytic state and never form an organized growth plate. The reduced size of mutant condensations results from increased apoptosis and decreased proliferation. Moreover, the expression of cartilage-specific extracellular matrix proteins is severely reduced in mutant elements. We demonstrate that this defect in chondrocytic differentiation can be attributed to lack of Sox9, L-Sox5, and Sox6 expression in precartilaginous condensations in double mutants. In summary, our study demonstrates that BMPR1A and BMPR1B are functionally redundant during early chondrogenesis and that BMP signaling is required for chondrocyte proliferation, survival, and differentiation in vivo. PMID: 15781876 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Hypertension. 2005 Apr;45(4):672-80. Epub 2005 Feb 7. Nitric oxide-dependent and nitric oxide-independent transcriptional responses to high shear stress in endothelial cells. Braam B, de Roos R, Bluyssen H, Kemmeren P, Holstege F, Joles JA, Koomans H. Department of Nephrology and Hypertension, University Medical Center, F03.226, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. g.b.braam@azu.nl Shear stress modulates gene expression in endothelial cells (ECs) partly through nitric oxide (NO), acting via enhanced cGMP formation by guanylyl cyclase (GC). We addressed non-cGMP-mediated transcriptional responses to shear stress in human umbilical ECs subjected to high-laminar shear stress (25 dyn/cm2; 150 minutes). RNA was isolated, reverse-transcribed, Cy3/5-labeled, and hybridized to 19 K human microarrays. High shear (n=6), high shear with 100 micromol/L L-NAME (n=3), and high shear with 10 micromol/L ODQ (GC inhibitor) in the perfusate (n=3) was compared with samples not subjected to flow. Among genes responding to high shear were HMOX1 (up) and PPARG (down). A high percentage of gene expression modulation by shear was absent during concomitant L-NAME or ODQ. Several transcriptional modulators were found (up: SOX5, SOX25, ZNF151, HOXD10; down: SOX11); a number of genes were regulated by shear and by shear with ODQ, but not regulated during L-NAME, indicating a nitric oxide synthase (NOS)-dependent, guanylyl cyclase (GC)-independent pathway. Several genes only responded to shear stress during L-NAME, others only responded to shear during ODQ. Upstream binding site analysis indicated shear stress and NO-dependent regulation of transcription via SOX5 and SOX9. Although NO importantly modulated the effect of shear stress on EC transcription, HMOX1 was consistently induced by shear stress, but not dependent on NOS or GC. Using bio-informatics software and databases, a promoter analysis identified SOX5 and SOX9 as potential, novel, shear-sensitive, and NO-dependent transcriptional regulators. The role of HMOX1 as a potential backup for NOS and the downstream role of SOXes should be explored. PMID: 15699468 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Arthritis Rheum. 2004 Nov;50(11):3561-73. The combination of SOX5, SOX6, and SOX9 (the SOX trio) provides signals sufficient for induction of permanent cartilage. Ikeda T, Kamekura S, Mabuchi A, Kou I, Seki S, Takato T, Nakamura K, Kawaguchi H, Ikegawa S, Chung UI. Laboratory for Bone & Joint Diseases, SNP Research Center, RIKEN (The Institute of Physical and Chemical Research), University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. OBJECTIVE: To regenerate permanent cartilage, it is crucial to know not only the necessary conditions for chondrogenesis, but also the sufficient conditions. The objective of this study was to determine the signal sufficient for chondrogenesis. METHODS: Embryonic stem cells that had been engineered to fluoresce upon chondrocyte differentiation were treated with combinations of factors necessary for chondrogenesis, and chondrocyte differentiation was detected as fluorescence. We screened for the combination that could induce fluorescence within 3 days. Then, primary mesenchymal stem cells, nonchondrogenic immortalized cell lines, and primary dermal fibroblasts were treated with the combination, and the induction of chondrocyte differentiation was assessed by detecting the expression of the cartilage marker genes and the accumulation of proteoglycan-rich matrix. The effects of monolayer, spheroid, and 3-dimensional culture systems on induction by combinations of transcription factors were compared. The effects of the combination on hypertrophic and osteoblastic differentiation were evaluated by detecting the expression of the characteristic marker genes. RESULTS: No single factor induced fluorescence. Among various combinations examined, only the SOX5, SOX6, and SOX9 combination (the SOX trio) induced fluorescence within 3 days. The SOX trio successfully induced chondrocyte differentiation in all cell types tested, including nonchondrogenic types, and the induction occurred regardless of the culture system used. Contrary to the conventional chondrogenic techniques, the SOX trio suppressed hypertrophic and osteogenic differentiation at the same time. CONCLUSION: These data strongly suggest that the SOX trio provides signals sufficient for the induction of permanent cartilage. PMID: 15529345 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: J Biol Chem. 2004 Dec 3;279(49):50942-8. Epub 2004 Sep 28. SOX9-dependent and -independent transcriptional regulation of human cartilage link protein. Kou I, Ikegawa S. Laboratory for Bone and Joint Diseases, SNP Research Center, RIKEN, Tokyo 108-8639, Japan. Cartilage link protein is a key component of the cartilage extracellular matrix. The transcriptional regulation of the gene encoding cartilage link protein (CRTL1) is largely unknown, however. Here, we investigated the regulation of CRTL1 by SOX9, a key regulator of cartilage matrix genes and chondrogenesis. Knockdown of SOX9 resulted in decreased CRTL1 expression. SOX9 induced CRTL1 expression effectively in human non-chondrocytic immortalized cell lines as well as in mesenchymal stem cell and adult dermal fibroblast. These results indicate that, like other cartilage matrix genes, SOX9 is a key regulator of CRTL1. Unlike other cartilage matrix genes, however, the activation of CRTL1 by SOX9 and its known transcriptional co-activators L-SOX5 and SOX6 was cell type-dependent. Two cis-acting enhancer elements resided in the 5'-untranslated region of CRTL1. One contained a heptameric SOX binding sequence and showed SOX9-dependent enhancer activity in several cell lines. The other showed cell type-specific SOX9-independent enhancer activity. These findings suggest that the enhancer elements may mediate differential expression of CRTL1 during chondrocyte differentiation and maturation. PMID: 15456769 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Development. 2004 Sep;131(18):4455-65. Epub 2004 Aug 11. LSox5 regulates RhoB expression in the neural tube and promotes generation of the neural crest. Perez-Alcala S, Nieto MA, Barbas JA. Instituto Cajal CSIC, Doctor Arce 37, 28002 Madrid, Spain. Members of the Sox family of transcription factors are involved in a number of crucial developmental processes, including sex determination, neurogenesis and skeletal development. LSox5 is a member of the group D Sox factors that, in conjunction with Sox6 and Sox9, promotes chondrogenesis by activating the expression of cartilage-specific extracellular matrix molecules. We have cloned the chicken homologue of LSox5 and found that it is initially expressed in the premigratory and migratory neural crest after Slug and FoxD3. Subsequently, the expression of LSox5 is maintained in cephalic crest derivatives, and it appears to be required for the development of the glial lineage, the Schwann cells and satellite glia in cranial ganglia. Misexpression of LSox5 in the cephalic neural tube activated RhoB expression throughout the dorsoventral axis. Furthermore, the prolonged forced expression of LSox5 enlarged the dorsal territory in which the neural crest is generated, extended the 'temporal window' of neural crest segregation, and led to an overproduction of neural crest cells in cephalic regions. In addition to HNK-1, the additional neural crest cells expressed putative upstream markers (Slug, FoxD3) indicating that a regulatory feedback mechanism may operate during neural crest generation. Thus, our data show that in addition to the SoxE genes (Sox9 and Sox10) a SoxD gene (Sox5) also participates in neural crest development and that a cooperative interaction may operate during neural crest generation, as seen during the formation of cartilage. PMID: 15306568 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Dev Dyn. 2003 Jul;227(3):402-8. Expression of Sox transcription factors in the developing mouse pancreas. Lioubinski O, Muller M, Wegner M, Sander M. Zentrum fur Molekulare Neurobiologie, Hamburg, Germany. Previous work has identified members of the homeodomain and basic helix-loop-helix families of transcription factors as critical determinants of mammalian pancreatic development. Here, we describe the identification of HMG-box transcription factors of the Sox gene family in the mouse pancreas. We detected transcripts for Sox11, Sox4, Sox13, Sox5, Sox9, Sox8, Sox10, Sox7, Sox17, Sox18, Sox15, and Sox30 in embryonic pancreas and found Sox4, Sox9, and Sox13 in adult pancreatic islets. Expression of seven of these Sox factors was studied in more detail by in situ hybridization from the stage of early pancreatic outgrowth to birth. Expression of Sox11 was found in the mesenchyme surrounding the pancreatic buds, whereas Sox4 and Sox9 were confined to the pancreatic epithelium and later to islets. Sox13 and L-Sox5 showed expression in most of the pancreatic epithelial cells between embryonic days 12.5 and 14.5. Sox8 and Sox10 were detected in a thin layer of cells surrounding the islets. The expression patterns of Sox genes in the embryonic pancreas suggest that they could have important and possibly redundant functions in pancreas development. Copyright 2003 Wiley-Liss, Inc. PMID: 12815626 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Dev Biol. 2003 May 15;257(2):292-301. Analysis of the molecular cascade responsible for mesodermal limb chondrogenesis: Sox genes and BMP signaling. Chimal-Monroy J, Rodriguez-Leon J, Montero JA, Ganan Y, Macias D, Merino R, Hurle JM. Departamento de Biologia Celular y Fisiologia, Instituto de Investigaciones Biomedicas, UNAM, Mexico DF 04510, Mexico. Here, we have studied how Sox genes and BMP signaling are functionally coupled during limb chondrogenesis. Using the experimental model of TGFbeta1-induced interdigital digits, we dissect the sequence of morphological and molecular events during in vivo chondrogenesis. Our results show that Sox8 and Sox9 are the most precocious markers of limb cartilage, and their induction is independent and precedes the activation of BMP signaling. Sox10 appears also to cooperate with Sox9 and Sox8 in the establishment of the digit cartilages. In addition, we show that experimental induction of Sox gene expression in the interdigital mesoderm is accompanied by loss of the apoptotic response to exogenous BMPs. L-Sox5 and Sox6 are respectively induced coincident and after the expression of Bmpr1b in the prechondrogenic aggregate, and their activation correlates with the induction of Type II Collagen and Aggrecan genes in the differentiating cartilages. The expression of Bmpr1b precedes the appearance of morphological changes in the prechondrogenic aggregate and establishes a landmark from which the maintenance of the expression of all Sox genes and the progress of cartilage differentiation becomes dependent on BMPs. Moreover, we show that Ventroptin precedes Noggin in the modulation of BMP activity in the developing cartilages. In summary, our findings suggest that Sox8, Sox9, and Sox10 have a cooperative function conferring chondrogenic competence to limb mesoderm in response to BMP signals. In turn, BMPs in concert with Sox9, Sox6, and L-Sox5 would be responsible for the execution and maintenance of the cartilage differentiation program. PMID: 12729559 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Cytogenet Genome Res. 2002;98(2-3):189-93. Chromosome assignment of eight SOX family genes in chicken. Kuroiwa A, Uchikawa M, Kamachi Y, Kondoh H, Nishida-Umehara C, Masabanda J, Griffin DK, Matsuda Y. Laboratory of Animal Cytogenetics, Center for Advanced Science and Technology, Hokkaido University, Sapporo, Japan. asatok@ees.hokudai.ac.jp Chromosome locations of the eight SOX family genes, SOX1, SOX2, SOX3, SOX5, SOX9, SOX10, SOX14 and SOX21, were determined in the chicken by fluorescence in situ hybridization. The SOX1 and SOX21 genes were localized to chicken chromosome 1q3.1-->q3.2, SOX5 to chromosome 1p1.6-->p1.4, SOX10 to chromosome 1p1.6, and SOX3 to chromosome 4p1.2-->p1.1. The SOX2 and SOX14 genes were shown to be linked to chromosome 9 using two-colored FISH and chromosome painting, and the SOX9 gene was assigned to a pair of microchromosomes. These results suggest that these SOX genes form at least three clusters on chicken chromosomes. The seven SOX genes, SOX1, SOX2, SOX3, SOX5, SOX10, SOX14 and SOX21 were localized to chromosome segments with homologies to human chromosomes, indicating that the chromosome locations of SOX family genes are highly conserved between chicken and human. Copyright 2002 S. Karger AG, Basel PMID: 12698002 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Genes Dev. 2002 Nov 1;16(21):2813-28. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Akiyama H, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B. Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA. 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. PMID: 12414734 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Biochem J. 2003 Jan 1;369(Pt 1):103-15. The TATA-containing core promoter of the type II collagen gene (COL2A1) is the target of interferon-gamma-mediated inhibition in human chondrocytes: requirement for Stat1 alpha, Jak1 and Jak2. Osaki M, Tan L, Choy BK, Yoshida Y, Cheah KS, Auron PE, Goldring MB. Beth Israel Deaconess Medical Center, Rheumatology Division, and New England Baptist Bone & Joint Institute, Harvard Institutes of Medicine, Boston, MA 02115, USA. Interferon-gamma (IFN-gamma) inhibits the synthesis of the cartilage-specific extracellular matrix protein type II collagen, and suppresses the expression of the type II collagen gene ( COL2A1 ) at the transcriptional level. To further examine this mechanism, the responses of COL2A1 regulatory sequences to IFN-gamma and the role of components of the Janus kinase/signal transducer and activators of transcription (JAK/STAT) pathway were examined in the immortalized human chondrocyte cell line, C-28/I2. IFN-gamma inhibited the mRNA levels of COL2A1 and aggrecan, but not Sox9, L-Sox5 and Sox6, all of which were expressed by these cells as markers of the differentiated phenotype. IFN-gamma suppressed the expression of luciferase reporter constructs containing sequences of the COL2A1 promoter spanning -6368 to +125 bp in the absence and presence of the intronic enhancer and stimulated activity of the gamma-interferon-activated site (GAS) luciferase reporter vector, associated with induction of Stat1 alpha-binding activity in nuclear extracts. These responses to IFN-gamma were blocked by overexpression of the JAK inhibitor, JAK-binding protein (JAB), or reversed by dominant-negative Stat1 alpha Y701F containing a mutation at Tyr-701, the JAK phosphorylation site. IFN-gamma had no effect on COL2A1 promoter expression in Jak1 (U4A)-, Jak2 (gamma 2A)- and Stat1 alpha (U3A)-deficient cell lines. In the U3A cell line, the response to IFN-gamma was rescued by overexpression of Stat1 alpha, but not by either Stat1 alpha Y701F or Stat1 beta. Functional analysis using deletion constructs showed that the IFN-gamma response was retained in the COL2A1 core promoter region spanning -45 to +11 bp, containing the TATA-box and GC-rich sequences but no Stat1-binding elements. Inhibition of COL2A1 promoter activity by IFN-gamma persisted in the presence of multiple deletions within the -45/+11 bp region. Our results indicate that repression of COL2A1 gene transcription by IFN-gamma requires Jak1, Jak2 and Stat1 alpha and suggest that this response involves indirect interaction of activated Stat1 alpha with the general transcriptional machinery that drives constitutive COL2A1 expression. PMID: 12223098 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Biochem J. 2001 Dec 1;360(Pt 2):461-70. Regulation of type-II collagen gene expression during human chondrocyte de-differentiation and recovery of chondrocyte-specific phenotype in culture involves Sry-type high-mobility-group box (SOX) transcription factors. Stokes DG, Liu G, Dharmavaram R, Hawkins D, Piera-Velazquez S, Jimenez SA. Department of Medicine, Division of Rheumatology, 233 S. 10th Street, Thomas Jefferson University, Philadelphia, PA 19107, USA. During ex vivo growth as monolayer cultures, chondrocytes proliferate and undergo a process of de-differentiation. This process involves a change in morphology and a change from expression of chondrocyte-specific genes to that of genes that are normally expressed in fibroblasts. Transfer of the monolayer chondrocyte culture to three-dimensional culture systems induces the cells to re-acquire a chondrocyte-specific phenotype and produce a cartilaginous-like tissue in vitro. We investigated mechanisms involved in the control of the de-differentiation and re-differentiation process in vitro. De-differentiated chondrocytes re-acquired their chondrocyte-specific phenotype when cultured on poly-(2-hydroxyethyl methacrylate) (polyHEMA) as assayed by morphology, reverse transcriptase PCR of chondrocyte-specific mRNA, Western-blot analysis and chondrocyte-specific promoter activity. Essentially, full recovery of the chondrocyte-specific phenotype was observed when cells that had been cultured for 4 weeks on plastic were transferred to culture on polyHEMA. However, after subsequent passages on plastic, the phenotype recovery was incomplete or did not occur. The activity of a gene reporter construct containing the promoter and enhancer from the human type-II collagen gene (COL2A1) was modulated by the culture conditions, so that its transcriptional activity was repressed in monolayer cultures and rescued to some extent when the cells were switched to polyHEMA cultures. The binding of Sry-type high-mobility-group box (SOX) transcription factors to the enhancer region was modulated by the culture conditions, as were the mRNA levels for SOX9. A transfected human type-II collagen reporter construct was activated in de-differentiated cells by ectopic expression of SOX transcription factors. These results underscore the overt change in phenotype that occurs when chondrocytes are cultured as monolayers on tissue-culture plastic substrata. PMID: 11716775 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Osteoarthritis Cartilage. 2001;9 Suppl A:S69-75. L-Sox5, Sox6 and Sox9 control essential steps of the chondrocyte differentiation pathway. Lefebvre V, Behringer RR, de Crombrugghe B. Department of Molecular Genetics, The University of Texas, MD Anderson Cancer Center, Houston 77030, USA. lefebvr@bme.ri.ccf.org 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. PMID: 11680692 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: J Bone Miner Res. 2001 Oct;16(10):1837-45. Accelerated up-regulation of L-Sox5, Sox6, and Sox9 by BMP-2 gene transfer during murine fracture healing. Uusitalo H, Hiltunen A, Ahonen M, Gao TJ, Lefebvre V, Harley V, Kahari VM, Vuorio E. Department of Medical Biochemistry and Molecular Biology, University of Turku, Finland. Fracture repair is the best-characterized situation in which activation of chondrogenesis takes place in an adult organism. To better understand the mechanisms that regulate chondrogenic differentiation of mesenchymal progenitor cells during fracture repair, we have investigated the participation of transcription factors L-Sox5, Sox6, and Sox9 in this process. Marked up-regulation of L-Sox5 and Sox9 messenger RNA (mRNA) and smaller changes in Sox6 mRNA levels were observed in RNAse protection assays during early stages of callus formation, followed by up-regulation of type II collagen production. During cartilage expansion, the colocalization of L-Sox5, Sox6, and Sox9 by immunohistochemistry and type II collagen transcripts by in situ hybridization confirmed a close relationship of these transcription factors with the chondrocyte phenotype and cartilage production. On chondrocyte hypertrophy, production of L-Sox5, Sox9 and type II collagen were down-regulated markedly and that of type X collagen was up-regulated. Finally, using adenovirus mediated bone morphogenetic protein 2 (BMP-2) gene transfer into fracture site we showed accelerated up-regulation of the genes for all three Sox proteins and type II collagen in fractures treated with BMP-2 when compared with control fractures. These data suggest that L-Sox5, Sox6, and Sox9 are involved in the activation and maintenance of chondrogenesis during fracture healing and that enhancement of chondrogenesis by BMP-2 is mediated via an L-Sox5/Sox6/Sox9-dependent pathway. PMID: 11585348 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Matrix Biol. 2001 Apr;20(2):123-7. Induction of periosteal callus formation by bone morphogenetic protein-2 employing adenovirus-mediated gene delivery. Uusitalo H, Hiltunen A, Ahonen M, Kahari VM, Aro H, Vuorio E. Skeletal Research Program, Department of Medical Biochemistry and Molecular Biology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. Although the chondrogenic response of periosteum is well established in healing fractures, the mechanisms mediating the proliferation and differentiation of periosteal chondroprogenitor cells are poorly understood. In the present study we demonstrate that bone morphogenetic protein-2 (BMP-2), introduced by adenovirus-mediated gene transfer, alone is capable of inducing callus formation at the site of periosteal injection. Both immunohistochemistry and Northern analysis demonstrated activation of type II collagen production between days 4 and 7 after the injection, followed by activation of type X collagen expression. The activation of chondrogenesis was associated with increased expression of L-Sox5 and Sox9, suggesting that the BMP-2 effect is mediated via Sox proteins. This capacity of adenovirus-mediated overproduction of BMP-2 to induce chondrogenesis (and subsequent endochondral ossification) should be useful for tissue engineering of cartilage and bone. PMID: 11334713 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Matrix Biol. 2000 Sep;19(5):389-94. Transcriptional mechanisms of chondrocyte differentiation. de Crombrugghe B, Lefebvre V, Behringer RR, Bi W, Murakami S, Huang W. Department of Molecular Genetics, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA. bdecromb@mdanderson.org 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. Publication Types: Review Review, Tutorial PMID: 10980415 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: J Mol Evol. 1999 May;48(5):517-27. Diversification pattern of the HMG and SOX family members during evolution. Soullier S, Jay P, Poulat F, Vanacker JM, Berta P, Laudet V. ERS155 du CNRS, Centre de Recherche en Biochimie Macromoleculaire, CNRS, BP5051, Route de Mende, 34293 Montpellier Cedex 5, France. From a database containing the published HMG protein sequences, we constructed an alignment of the HMG box functional domain based on sequence identity. Due to the large number of sequences (more than 250) and the short size of this domain, several data sets were used. This analysis reveals that the HMG box superfamily can be separated into two clearly defined subfamilies: (i) the SOX/MATA/TCF family, which clusters proteins able to bind to specific DNA sequences; and (ii) the HMG/UBF family, which clusters members which bind non specifically to DNA. The appearance and diversification of these subfamilies largely predate the split between the yeast and the metazoan lineages. Particular emphasis was placed on the analysis of the SOX subfamily. For the first time our analysis clearly identified the SOX subfamily as structured in six groups of genes named SOX5/6, SRY, SOX2/3, SOX14, SOX4/22, and SOX9/18. The validity of these gene clusters is confirmed by their functional characteristics and their sequences outside the HMG box. In sharp contrast, there are only a few robust branching patterns inside the UBF/HMG family, probably because of the much more ancient diversification of this family than the diversification of the SOX family. The only consistent groups that can be detected by our analysis are HMG box 1, vertebrate HMG box 2, insect SSRP, and plant HMG. The various UBF boxes cannot be clustered together and their diversification appears to be extremely ancient, probably before the appearance of metazoans. PMID: 10198118 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Nucleic Acids Res. 1999 Mar 1;27(5):1359-64. The DNA-binding specificity of SOX9 and other SOX proteins. Mertin S, McDowall SG, Harley VR. The Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia. SOX (SRY-related HMG box) proteins are transcription factors that have critical roles in the regulation of numerous developmental processes. They share at least 50% homology in their HMG domains, which bind the DNA element AACAAT. How different SOX proteins achieve specific regulation of target genes is not known. We determined the DNA-binding specificity of SOX9 using a random oligonucleotide selection assay. The optimal SOX9 binding sequence, AGAACAATGG, contained a core DNA-binding element AACAAT, flanked by 5' AG and 3' GG nucleotides. The specific interaction between SOX9 and AGAACAATGG was confirmed by mobility shift assays, DNA competition and dissociation studies. The 5' AG and 3' GG flanking nucleotides enhance binding by SOX9 HMG domain, but not by the HMG domain of another SOX factor, SRY. For SRY, different 5' and 3' flanking nucleotides are preferred. Our studies support the notion that SOX proteins achieve DNA sequence specificity through subtle preferences for flanking nucleotides and that this is likely to be dictated by signature amino acids in their HMG domains. Furthermore, the related HMG domains of SOX9 and Sox17 have similar optimal binding sites that differ from those of SRY and Sox5, suggesting that SOX factors may co-evolve with their DNA targets to achieve specificity. PMID: 9973626 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: EMBO J. 1998 Oct 1;17(19):5718-33. A new long form of Sox5 (L-Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively activate the type II collagen gene. Lefebvre V, Li P, de Crombrugghe B. Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 11, Houston, TX 77030, USA. veronique_lefebvre@molgen.tmc.edu Transcripts for a new form of Sox5, called L-Sox5, and Sox6 are coexpressed with Sox9 in all chondrogenic sites of mouse embryos. A coiled-coil domain located in the N-terminal part of L-Sox5, and absent in Sox5, showed >90% identity with a similar domain in Sox6 and mediated homodimerization and heterodimerization with Sox6. Dimerization of L-Sox5/Sox6 greatly increased efficiency of binding of the two Sox proteins to DNA containing adjacent HMG sites. L-Sox5, Sox6 and Sox9 cooperatively activated expression of the chondrocyte differentiation marker Col2a1 in 10T1/2 and MC615 cells. A 48 bp chondrocyte-specific enhancer in this gene, which contains several HMG-like sites that are necessary for enhancer activity, bound the three Sox proteins and was cooperatively activated by the three Sox proteins in non-chondrogenic cells. Our data suggest that L-Sox5/Sox6 and Sox9, which belong to two different classes of Sox transcription factors, cooperate with each other in expression of Col2a1 and possibly other genes of the chondrocytic program. PMID: 9755172 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Mol Med. 1996 Jul;2(4):405-12. SOX genes: architects of development. Prior HM, Walter MA. Ocular Genetics Research Group, University of Alberta, Edmonton, Canada. Development in higher organisms involves complex genetic regulation at the molecular level. The emerging picture of development control includes several families of master regulatory genes which can affect the expression of down-stream target genes in developmental cascade pathways. One new family of such development regulators is the SOX gene family. The SOX genes are named for a shared motif called the SRY box a region homologous to the DNA-binding domain of SRY, the mammalian sex determining gene. Like SRY, SOX genes play important roles in chordate development. At least a dozen human SOX genes have been identified and partially characterized (Tables 1 and 2). Mutations in SOX9 have recently been linked to campomelic dysplasia and autosomal sex reversal, and other SOX genes may also be associated with human disease. Publication Types: Review Review, Tutorial PMID: 8827711 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------