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Activation of Genes for Growth Factor and Cytokine Pathways Late In CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Genomics 88 (2006) 52–64 www.elsevier.com/locate/ygeno Activation of genes for growth factor and cytokine pathways late in chondrogenic differentiation of ATDC5 cells ⁎ Aki Osawa a,b,c, , Masaki Kato a,1, Eriko Matsumoto b, Katsuro Iwase b, Takashi Sugimoto d, Tsutomu Matsui e, Hiroshi Ishikura e, Sumio Sugano f,2, Hisashi Kurosawa c, Masaki Takiguchi b, Naohiko Seki a a Department of Functional Genomics, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan b Department of Biochemistry and Genetics, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan c Department of Orthopaedics, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan d Department of Life Science Products, Hitachi Software Engineering Co. Ltd., Yokohama 230-0045, Japan e Department of Molecular Pathology, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan f Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan Received 20 November 2005; accepted 24 February 2006 Available online 4 April 2006 Abstract The mouse embryonal carcinoma cell line ATDC5 provides an excellent model system for chondrogenesis in vitro. To understand better the molecular mechanisms of endochondral bone formation, we investigated gene expression profiles during the differentiation course of ATDC5 cells, using an in-house microarray harboring full-length-enriched cDNAs. For 28 days following chondrogenic induction, 507 genes were up- or down-regulated at least 1.5-fold. These genes were classified into five clusters based on their expression patterns. Genes for growth factor and cytokine pathways were significantly enriched in the cluster characterized by increases in expression during late stages of chondrocyte differentiation. mRNAs for decorin and osteoglycin, which have been shown to bind to transforming growth factors-β and bone morphogenetic proteins, respectively, were found in this cluster and were detected in hypertrophic chondrocytes of developing mouse bones by in situ hybridization analysis. Taken together with assigned functions of individual genes in the cluster, interdigitated interaction between a number of intercellular signaling molecules is likely to take place in the late chondrogenic stage for autocrine and paracrine regulation among chondrocytes, as well as for chemoattraction and stimulation of progenitor cells of other lineages. © 2006 Elsevier Inc. All rights reserved. Keywords: Chondrocyte; Endochondral ossification; Oligo capping; Chemokine; Osteoprogenitor cell; Hematopoietic cell; In situ hybridization In skeletal development, chondrogenesis leads to formation their cell volume to reach the differentiated hypertrophic state. of the cartilage template that is eventually converted into bone The majority of hypertrophic chondrocytes appear to undergo [1]. This course is known as endochondral bone formation, apoptosis and are replaced by bone and marrow with the which is one of the most spectacular events in ontogeny. During invasion of blood vessels, osteoprogenitor cells, and hemato- the process, chondrocytes first proliferate and then increase poietic cells. A number of secreted proteins such as Indian hedgehog, ⁎ Corresponding author. Department of Orthopaedics, Juntendo University parathyroid hormone-related protein, fibroblast growth factors School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan. Fax: +81 3 5689 (FGFs), and bone morphogenetic proteins (BMPs) have been 5635. shown to regulate cooperatively the proliferation and differen- E-mail address: [email protected] (A. Osawa). tiation of chondrocytes [1]. These secreted ligands regulate 1 Present address: Laboratory for Biolinguistics, Brain Science Institute, expression of a large number of target genes, as typically RIKEN, Saitama 351-0918, Japan. 2 Present address: Department of Medical Genome Sciences, Graduate School demonstrated by comprehensive gene expression analysis for of Frontier Sciences, University of Tokyo, 5-1-5, Kashiwanoha, Kashiwa, Chiba the targets of FGF [2] and BMP4 [3]. Recently, several 277-8562, Japan. transcription factors including SOX [4,5] and Runx [6–8] 0888-7543/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2006.02.013 A. Osawa et al. / Genomics 88 (2006) 52–64 53 family members have been shown to be essential for endo- sequences of 3680 cDNA clones in the library and compared chondral bone formation. them with the NCBI nucleotide database using the BLAST To understand better the global molecular basis of programs. The 3680 cDNA clones were assembled into 1920 endochondral bone formation, we investigated genes up- or clusters when overlapping clones were integrated. The library down-regulated during the proliferation and subsequent differ- was judged to be enriched with full-length cDNAs, since 68% entiation of the mouse embryonal carcinoma cell line ATDC5, of the cDNA clones had the initiation ATG codon found in the which provides an excellent model system for chondrogenesis public databases. The average length of the cDNA insert was [9,10]. We first constructed a full-length-enriched cDNA library approximately 2.0 kb. [11] derived from ATDC5 cells as well as mouse bone and To construct an in-house cDNA microarray, 1829 clones cartilage tissues and then prepared an in-house microarray were selected from the 1920 clusters and compared with 1614 harboring these cDNAs. Subsequent array analysis revealed that clones of 1627 clusters derived from 3289 sequenced clones of a number of genes for growth factor and cytokine pathways are the mouse fetus (embryonic day 14 (E14)) cDNA library activated in the late phase of ATDC5 differentiation. Among described previously [14]. Integration of 530 shared genes gave them, genes for decorin and osteoglycin, members of the small 2913 clones, whose cDNA inserts were subjected to preparation leucine-rich proteoglycan (SLRP) family [12,13] for multifac- of the microarray (Supplementary Table 1). The NCBI BLAST eted extracellular matrix (ECM) proteins, were shown to be search revealed that 97.9% (2852 clones) of the 2913 clones expressed in hypertrophic chondrocytes displaying multiple coincided with the known mouse mRNA/cDNA sequences, 29 tasks in bone formation. clones with the mouse genome but not with mRNA/cDNA sequences, and 32 clones did not coincide with known Results sequences, while 22 of the 32 clones were apparently of bad or short sequences. Construction of the cDNA library and microarray Microarray analysis We constructed a full-length-enriched cDNA library using the oligo-capping method [11] with RNA mixtures derived from We used the clonal cell line ATDC5 to track the stages of mouse chondrogenic ATDC5 cells and developing mouse chondrogenic differentiation. Undifferentiated ATDC5 cells cartilage and bone tissues (Fig. 1). We determined 5′-end rapidly proliferated and ceased to grow at confluence. In the differentiation medium containing 10 μg/ml insulin, the cells reentered the growth state, with cellular condensation followed by the formation of cartilage nodules being detected micro- scopically (data not shown). Differentiation of ATDC5 cells into chondrocytes and further into hypertrophic chondrocytes was confirmed by monitoring the onset of expression of mRNAs for procollagens type II α1 (Col2a) and type X α1 (Col10a), respectively, as markers [15,16] (data not shown; the Col10a gene was included also in the microarray, see Supplementary Table 2, No. 200). Poly(A)+ RNAs derived from ATDC5 cells at 7, 14, 21, and 28 days after chondrogenic induction with the differentiation medium, and control ATDC5 cells without the induction, were subjected to cDNA synthesis-coupled Cy3 and Cy5 labeling, respectively, at least in duplicate. Both labeled cDNAs were mixed in equal amounts and hybridized to a microarray. Labeling vice versa with respect to Cy3 and Cy5 was also done to correct a possible difference in incorporation efficiency of these dyes, and in total at least four results of the hybridization analysis at each time point were averaged. At 7, 14, 21, and 28 days, respectively 59, 97, 165, and 272 genes were up- regulated, while 57, 122, 85, and 106 genes were down- regulated 1.5-fold or more. Combining these up- and down- Fig. 1. Schematic representation of construction of the in-house cDNA regulated genes, 507 genes were up- or down-regulated 1.5-fold microarray. Preparation of a mouse fetus (E14) cDNA library was described or more at at least one time point after chondrogenic induction previously [14]. A cDNA library derived from ATDC5 cells and mouse cartilage (Supplementary Table 2). and bone was prepared as described under Materials and methods. Indicated To verify the results of our in-house cDNA microarray numbers of cDNA clones of each library were subjected to sequence determination and further to microarray construction after clustering analysis system, we performed real-time RT-PCR analysis for 9 genes and integrating genes shared by the two libraries. A microarray harboring 2913 arbitrarily selected from 272 genes up-regulated at 28 days cDNAs was constructed. (Fig. 2): adrenomedullin (Adm; Entrez Gene No. 11535), 54 A. Osawa et al. / Genomics 88 (2006) 52–64 Fig. 2. Time courses of changes in mRNA levels for several genes during differentiation of ATDC5 cells. Results of microarray analysis (open squares) were compared with those of real-time RT-PCR analysis (closed circles)
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