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• W2073603121 abstract "Adhesive signaling plays a key role in cellular differentiation, including in chondrogenesis. Herein, we probe the contribution to early chondrogenesis of two key modulators of adhesion, namely focal adhesion kinase (FAK)/Src and CCN2 (connective tissue growth factor, CTGF). We use the micromass model of chondrogenesis to show that FAK/Src signaling, which mediates cell/matrix attachment, suppresses early chondrogenesis, including the induction of Ccn2, Agc, and Sox6. The FAK/Src inhibitor PP2 elevates Ccn2, Agc, and Sox6 expression in wild-type mesenchymal cells in micromass culture, but not in cells lacking CCN2. Our results suggest a reduction in FAK/Src signaling is a critical feature permitting chondrogenic differentiation and that CCN2 operates downstream of this loss to promote chondrogenesis. Adhesive signaling plays a key role in cellular differentiation, including in chondrogenesis. Herein, we probe the contribution to early chondrogenesis of two key modulators of adhesion, namely focal adhesion kinase (FAK)/Src and CCN2 (connective tissue growth factor, CTGF). We use the micromass model of chondrogenesis to show that FAK/Src signaling, which mediates cell/matrix attachment, suppresses early chondrogenesis, including the induction of Ccn2, Agc, and Sox6. The FAK/Src inhibitor PP2 elevates Ccn2, Agc, and Sox6 expression in wild-type mesenchymal cells in micromass culture, but not in cells lacking CCN2. Our results suggest a reduction in FAK/Src signaling is a critical feature permitting chondrogenic differentiation and that CCN2 operates downstream of this loss to promote chondrogenesis. Cartilage is a connective tissue that possesses a wide array of functions, including establishing the skeletal framework during embryogenesis and cushioning joints in adulthood. Chondrocytes, the cell type found in cartilage, generate and maintain the cartilaginous extracellular matrix (ECM), 8The abbreviations used are: ECMextracellular matrixdpcdays post-coitumFAKfocal adhesion kinaseRT-PCRreverse transcription PCRGAPDHglyceraldehyde-3-phosphate-dehydrogenasePBSphosphate-buffered salinePNApeanut agglutininBMPbone morphogenetic proteinWTwild-typeKOknock-out. which is crucial for chondrogenic development and homeostasis (1Karsenty G. Wagner E.F. Dev. Cell. 2002; 2: 389-406Abstract Full Text Full Text PDF PubMed Scopus (1204) Google Scholar). Formation of the adult skeleton is achieved by intramembranous and endochondral ossification (2Kronenberg H.M. Nature. 2003; 423: 332-336Crossref PubMed Scopus (2177) Google Scholar, 3Zelzer E. Olsen B.R. Nature. 2003; 423: 343-348Crossref PubMed Scopus (221) Google Scholar). The endochondral skeleton constitutes most skeletal elements of the body and is formed in two main steps. First, cartilage is formed by chondrogenesis. Chondrogenesis is initiated when mesenchymal cells aggregate to form condensations that ultimately determine the shape and location of future bones (2Kronenberg H.M. Nature. 2003; 423: 332-336Crossref PubMed Scopus (2177) Google Scholar, 3Zelzer E. Olsen B.R. Nature. 2003; 423: 343-348Crossref PubMed Scopus (221) Google Scholar, 4Karsenty G. Nature. 2003; 423: 316-318Crossref PubMed Scopus (343) Google Scholar). During this process, a high cell density is achieved that promotes cell-cell interactions resulting in the propagation of signal transduction events necessary for the initiation of chondrogenesis (5Cohn M.J. Tickle C. Trends Genet. 1996; 12: 253-257Abstract Full Text PDF PubMed Scopus (138) Google Scholar). Cells within these condensations begin to express markers typical of early chondrogenic cells. These markers include such proteins as the “Sox trio” of transcription factors (Sox9 and the related factors L-Sox5 and Sox6), type II collagen, and aggrecan (6Stanton L.-A. Underhill T.M. Beier F. Dev. Biol. 2003; 263: 165-175Crossref PubMed Scopus (141) Google Scholar, 7Seghatoleslami M.R. Roman-Blas J.A. Rainville A.M. Modaressi R. Danielson K.G. Tuan R.S. J. Cell. Biochem. 2003; 88: 1129-1144Crossref PubMed Scopus (29) Google Scholar). Moreover, cells change in morphology from a fibroblast-like appearance to a spheroidal shape (8Wagner E.F. Karsenty G. Curr. Opin. Genet. Dev. 2001; 11: 527-532Crossref PubMed Scopus (190) Google Scholar). Endochondral ossification, a process involving the creation of bone tissue utilizing the cartilage as template, follows (4Karsenty G. Nature. 2003; 423: 316-318Crossref PubMed Scopus (343) Google Scholar). extracellular matrix days post-coitum focal adhesion kinase reverse transcription PCR glyceraldehyde-3-phosphate-dehydrogenase phosphate-buffered saline peanut agglutinin bone morphogenetic protein wild-type knock-out. In developing limbs in vivo, cells originating from the lateral plate mesoderm condense and form aggregations (5Cohn M.J. Tickle C. Trends Genet. 1996; 12: 253-257Abstract Full Text PDF PubMed Scopus (138) Google Scholar). In mouse development, formation of these aggregations occurs 10.5–12.5 days post-coitum (dpc) (9Karsenty G. Genes Dev. 1999; 13: 3037-3051Crossref PubMed Scopus (273) Google Scholar). The level and efficiency of overall chondrogenic differentiation, and hence subsequent bone development, is directly correlated to the density of the initial condensation (10Ahrens P. Solursh M. Reiter R. Dev. Biol. 1977; 60: 69-82Crossref PubMed Scopus (541) Google Scholar). The Sox trio appear to cooperate with each other to co-regulate the expression of chondrogenic markers such as aggrecan and type II collagen. However, much remains unknown about the additional requirements for chondrogenesis. Recently, it was shown that the formation of condensations and cartilage nodules requires adhesive signaling and remodeling of the actin cytoskeleton, via Rho and Rac (11Wang G. Woods A. Agoston H. Ulici V. Glogauer M. Beier F. Dev. Biol. 2007; 306: 612-623Crossref PubMed Scopus (83) Google Scholar, 12Bang O.-S. Kim E.-J. Chung J.G. Lee S.-R. Park T.K. Kang S.-S. Biochem. Biophys. Res. Comm. 2000; 278: 522-529Crossref PubMed Scopus (54) Google Scholar, 13Woods A. Beier F. J. Biol. Chem. 2006; 281: 13134-13140Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 14Woods A. Wang G. Dupuis H. Shao Z. Beier F. J. Biol. Chem. 2007; 282: 23500-23508Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). At later stages, adhesive signaling through integrins is involved (15Aszodi A. Hunziker E.B. Brakebusch C. Fassler R. Genes Dev. 2003; 17: 2465-2479Crossref PubMed Scopus (251) Google Scholar). However, the contribution of cell-ECM interactions in early chondrogenesis is poorly understood. For example, the role of focal adhesion kinase (FAK)/Src, which mediates cell-ECM interactions, in chondrogenesis has not been thoroughly investigated. Moreover, the role of CCN2, a member of the CCN (CYR61, CTGF, NOV) family of pro-adhesive matricellular signaling modulators (16Leask A. Abraham D.J. J. Cell Sci. 2006; 119: 4803-4810Crossref PubMed Scopus (576) Google Scholar), in early chondrogenesis is unclear. Culture systems have been developed that promote chondrogenesis. The three-dimensional micromass system is an excellent culture model to promote chondrogenic differentiation of a wide range of mesenchymal cells by providing sufficiently high cell density to promote the cell-cell interactions required for chondrogenesis (10Ahrens P. Solursh M. Reiter R. Dev. Biol. 1977; 60: 69-82Crossref PubMed Scopus (541) Google Scholar). In this report we use the micromass cell culture system to probe the role of FAK/Src and CCN2 in early chondrogenesis in vitro. Monolayer Cell Culture—Fak+/+ and Fak-/- fibroblast cells (17Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1591) Google Scholar) (8.5 dpc) were purchased (ATCC). Both cell types lack p53 and have been thereby immortalized. Ccn2+/+ and Ccn2-/- mesenchymal cells (13.5 dpc) were isolated and cultured as previously described (18Chen Y. Abraham D.J. Shi-wen X. Pearson J.D. Black C.M. Lyons K.M. Leask A. Mol. Biol. Cell. 2004; 15: 5635-5646Crossref PubMed Scopus (151) Google Scholar, 19Ivkovic S. Yoon B.S. Popoff S.N. Safadi F.F. Libuda D.E. Stephenson R.C. Daluiski A. Lyons K.M. Development. 2003; 130: 2779-2791Crossref PubMed Scopus (594) Google Scholar). Monolayer cultures were plated at 2.5 × 105 cells/well in a 6-well dish (Nunc). Cells were cultured in Dulbecco's modified Eagle's medium, 10% fetal bovine serum (Invitrogen), 1% penicillin/streptomycin/amphotericin B (Invitrogen). For CCN2 rescue experiments, plates were treated overnight with Dulbecco's modified Eagle's medium, 0.5% fetal bovine serum, 100 ng/ml CCN2 (EMP; Genetech). Micromass Cell Culture—Mesenchymal cells were cultured in Dulbecco's modified Eagle's medium, 10% fetal bovine serum (Invitrogen), 1% penicillin/streptomycin/amphotericin B (Invitrogen). Cells were plated at a density of 1 × 105 per 10-μl droplet into each well of a 24-well tissue culture plate (Nunc) and left to adhere for an hour. Once adhered, micromass cultures were given 1 ml of medium. Micromass cultures were grown for a period of 6 days. In experiments involving inhibitor treatment, starting on Day 0, the day of plating, medium was supplemented with the FAK/Src inhibitor PP2 (10 μm in dimethyl sulfoxide (Me2SO) (Calbiochem)), whereas control cultures were supplemented with the vehicle Me2SO only. Medium was replenished daily. The high density culture system allows for the recapitulation of the in vivo high density environment during chondrogenesis that allows for a high degree of cell-cell contact (11Wang G. Woods A. Agoston H. Ulici V. Glogauer M. Beier F. Dev. Biol. 2007; 306: 612-623Crossref PubMed Scopus (83) Google Scholar, 12Bang O.-S. Kim E.-J. Chung J.G. Lee S.-R. Park T.K. Kang S.-S. Biochem. Biophys. Res. Comm. 2000; 278: 522-529Crossref PubMed Scopus (54) Google Scholar, 20Stanton L.A. Sabari S. Sampaio A.V. Underhil T.M. Beier F. Biochem. J. 2004; 378: 53-62Crossref PubMed Scopus (123) Google Scholar, 21Woods A. Wang G. Beier F. J. Biol. Chem. 2005; 280: 11626-11634Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 22James C.G. Appleton C.T. Ulici V. Underhill T.M. Beier F. Mol. Biol. Cell. 2005; 16: 5316-5333Crossref PubMed Scopus (115) Google Scholar). This culture system works to effectively promote chondrogenesis in a variety of mesenchymal cells, including embryonic and adult fibroblasts (22James C.G. Appleton C.T. Ulici V. Underhill T.M. Beier F. Mol. Biol. Cell. 2005; 16: 5316-5333Crossref PubMed Scopus (115) Google Scholar, 23Carlberg A.L. Pucci B. Rallapalli R. Tuan R.S. Hall D.J. Differentiation. 2001; 67: 128-138Crossref PubMed Scopus (96) Google Scholar, 24Nicoll S.B. Barak O. Csoka A.B. Bhatnagar R.S. Stern R. Biochem. Biophys. Res. Commun. 2002; 292: 819-825Crossref PubMed Scopus (42) Google Scholar, 25Gomes Jr., R.R. Farach Carson M.C. Carson D.D. Connect. Tissue Res. 2003; 44: 196-201Crossref PubMed Google Scholar). RNA Isolation and Real-time RT (Reverse Transcription) PCR— RNA extraction was performed with the Qiagen RNeasy kit according to the manufacturer's protocol (Qiagen Inc.). RNA was collected from micromass cultures on days 1, 3, 6 of differentiation. RNA concentration was determined using a spectrophotometer (Beckman Coulter). 25 ng of RNA/reaction was used for real-time RT-PCR according to established protocols (20Stanton L.A. Sabari S. Sampaio A.V. Underhil T.M. Beier F. Biochem. J. 2004; 378: 53-62Crossref PubMed Scopus (123) Google Scholar, 21Woods A. Wang G. Beier F. J. Biol. Chem. 2005; 280: 11626-11634Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 22James C.G. Appleton C.T. Ulici V. Underhill T.M. Beier F. Mol. Biol. Cell. 2005; 16: 5316-5333Crossref PubMed Scopus (115) Google Scholar). A total 15-μl reaction volume was used containing the TaqMan one-step master mix kit (Applied Biosystems) and gene-specific target primers (Assays-on-demand; Applied Biosystems) and probes (FAM (6-carboxyfluorescein) dye layer) and endogenous reference primers and probes (VIC dye layer). The FAM dye layer yields quantification of the target genes, whereas VIC yields simultaneous quantification of glyceraldehyde-3-phosphate-dehydrogenase (Gapdh) as an internal control. Relative gene expression was determined by measuring Col2a1, Agc, Dcn, Hapln, L-Sox5, Sox6, Sox-9, Ccn1, Ccn2, and Ccn5, using 40 cycles on the ABI Prism 7900 HT sequence detector (PerkinElmer Life Sciences). All samples were amplified in three parallel reactions per trial, and three independent trials were performed. RNA Quality Assessment, Probe Preparation, and Gene Chip Hybridization and Analysis—Microarrays and analyses were performed essentially as previously described (19Ivkovic S. Yoon B.S. Popoff S.N. Safadi F.F. Libuda D.E. Stephenson R.C. Daluiski A. Lyons K.M. Development. 2003; 130: 2779-2791Crossref PubMed Scopus (594) Google Scholar, 26Kennedy L. Liu S. Shi-Wen X. Chen Y. Eastwood M. Carter D.E. Lyons K.M. Black C.M. Abraham D.J. Leask A. Exp. Cell Res. 2007; 313: 952-964Crossref PubMed Scopus (85) Google Scholar). Gene Chips were processed at the London Regional Genomics Centre (Robarts Research Institute, London, ON). RNA was harvested (TRIzol; Invitrogen) and quantified. Quality was assessed using the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA), the RNA 6000 Nano kit (Caliper Life Sciences, Mountain View, CA), and the Degradometer. Biotinylated complementary RNA (cRNA) was prepared from 10 μg of total RNA (Affymetrix, Santa Clara, CA). Double-stranded cDNA was synthesized using SuperScript II (Invitrogen) and oligo(dT) 24 primers. Biotin-labeled cRNA was prepared by in vitro transcribing cDNA (Enzo Brioche, New York, NY). Fifteen μg of labeled cRNA was hybridized to Mouse Genome 430 2.0 Gene Chips for 16 h at 45 °C (Affymetrix). Gene Chips were stained with streptavidin-phycoerythrin, followed by an antibody solution and a second streptavidin-phycoerythrin solution (GeneChip Fluidics Station 450; Affymetrix). Gene Chips were scanned with the GeneChip Scanner 3000 (Affymetrix). Signal intensities for genes were generated using GCOS1.2 (Affymetrix) using default values for the statistical expression algorithm parameters and a target signal of 150 for all probe sets and a normalization value of 1. Normalization was performed in GeneSpring 7.2 (Agilent Technologies Inc.). The RMA preprocessor was used to import data from the .cel files. Data were transformed, (measurements <0.01 set to 0.01), normalized per chip to the 50th percentile and per gene to wild-type control samples. Experiments were performed twice, and -fold changes were identified using the GeneSpring filter. Data presented in Table 1 are an average of these independent studies. The -fold change between treated and untreated samples had to be at least 2-fold, in both sets of experiments, to identify a transcript as being altered.TABLE 1Microarray of Ccn2+/+ and –/– mesenchymal cells shows transcripts down-regulated greater than 2-fold CCN family members and early chondrogenic genes are shown.GeneAffy ID-Fold ChangeFull nameCtgf, Ccn21416953_at29.35Connective tissue growth factor (CCN2)Wisp2, Ccn51419015_at3.73Wnt-induced secreted protein-2 (CCN5)Cyr61, Ccn1144234_x_at2.12Cysteine-rich 61 (CCN1)Agc1449827_at4.40AggrecanHapln1438020_at3.45Link proteinDcn1449368_at4.52DecorinSox6142767_a_at2.81Sex-determining region Y (SRY)-box 6 Open table in a new tab Western Blot Analysis—Protein was harvested using radioimmunoprecipitation assay lysis buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.5, 1% Triton-X, 1% deoxycholate, 0.1% SDS, 2 mm EDTA), 50 mm NaF, and 1 mm Na3VO3 (Sigma), supplemented with a protease inhibitor mini complete tablet (Roche Applied Science). Protein concentration was quantified by a kit (BCA; Sigma) as described by the manufacturer's instructions. Equal amounts of cell lysate (40 μg) and of concentrated medium (20 μl) were subjected to SDS/PAGE and transferred to nitrocellulose (Bio-Rad). Membranes were blocked for 1 h in 5% bovine serum albumin in Tris-buffered saline with 0.01% Tween 20 and incubated with anti-Sox6 (1:200; Sigma), anti-Sox5 (1:1000; Santa Cruz Biotechnology), anti-aggrecan (1:500; R&D Systems), anti-CCN2 antibody (1:2000; Abcam), anti-type II collagen (1:2000; Santa Cruz), or anti-FAK antibody (1:2000; Cell Signaling) overnight at 4 °C. Blots were washed three times for 5 min with Tris-buffered saline with 0.01% Tween 20, followed by the application of appropriate horseradish peroxidase-conjugated secondary antibodies and detection of proteins using ECL™ Western blot detection reagents (Amersham Biosciences) according to the manufacturer's instructions and visualized using Chemi-Imager™5500 (Alpha Innotech Inc.). Immunofluorescence—Ccn2+/+ and Ccn2-/- cells were plated in monolayer at a density of 12,000 cells/well in a 24-well dish (Falcon) on glass coverslips. Cells were harvested and fixed in 4% paraformaldehyde for 30 min at 4 °C. Cells were washed in phosphate-buffered saline (PBS), incubated for 5 min with 0.1% Triton-X in PBS, and rinsed again in PBS. Cells were then incubated in blocking solution containing goat serum (Sigma) in PBS, 1:20, for 30 min at room temperature. Primary antibodies directed to Sox6 were diluted in blocking solution at a concentration of 1:200 and incubated with coverslips for 1 h at room temperature. Coverslips were rinsed in PBS and then incubated with an AlexaFluor® 488-conjugated secondary antibody, diluted 1:300 in PBS, for 1 h at room temperature in the dark. Following another wash in PBS, coverslips were mounted in Vecta-Shield anti-fade mounting medium containing 4′,6-diamidino-2-phenylindole. Images were taken with a Zeiss Axiophot microscope using Northern Eclipse software (Empix) and exported into Adobe Photoshop. Peanut Agglutinin (PNA) Staining—Micromass cultures were performed as above and were fixed on day 6 of culture in 4% paraformaldehyde at 4 °C for 30 min. Cells were rinsed with PBS and then incubated for 2 h in 50 μg/ml PNA diluted in PBS. Cultures were washed again with PBS, and PNA was detected colorimetrically by diaminobenzidine (Dako Cytomation). Images were captured with a Nikon SMZ1500 microscope at ×5.6 magnification using a Photometrics coolSNAP-cf color digital camera (Photon Technology International). Alcian Blue Staining—Alcian Blue staining was carried out on day 6 of micromass culture as follows. Cultures were washed twice with cold PBS, fixed in 100% ethanol for 20 min at -20 °C, and incubated with 0.1% HCl-Alcian blue for 2 h (20Stanton L.A. Sabari S. Sampaio A.V. Underhil T.M. Beier F. Biochem. J. 2004; 378: 53-62Crossref PubMed Scopus (123) Google Scholar). Excess stain was washed off with double distilled water and pictures were taken as described above. Stain was quantified by solubilizing the stain in 6.0 m guanidine hydrochloride for 8 h at room temperature. Absorbance was measured using a spectrophotometer at 620 nm. Statistical Analysis—Data collected from real-time RT-PCR are an average of three trials of samples, from completely independent experiments, run in triplicate. Means were quantified relative to Gapdh, and then data were normalized to day 1 of control per trial or in the case where there is only 1 day being examined, data were normalized to the control sample. Statistical significance was determined by Student's paired t test or two-way analysis of variance, with a level of significance defined as p < 0.05 using the Bonferroni post-test and GraphPad Prism version 4.00 for Windows. Loss of FAK Promotes Features of Chondrogenesis, Including Nodule Formation and CCN2 Expression—The earliest stage of chondrogenesis, namely condensation, involves the migration of mesenchymal cells via cell/ECM interactions and is associated with increased tyrosine phosphorylation of FAK (12Bang O.-S. Kim E.-J. Chung J.G. Lee S.-R. Park T.K. Kang S.-S. Biochem. Biophys. Res. Comm. 2000; 278: 522-529Crossref PubMed Scopus (54) Google Scholar). However, the role of FAK in subsequent phases of chondrogenesis, including nodule formation and the induction of chondrogenic genes, is wholly unknown. To specifically assess the effect of loss of FAK on early chondrogenesis, we used the micromass culture system. Micromass cell culture is an established method of inducing chondrogenesis in vitro in a variety of mesenchymal cell types, including embryonic and adult fibroblasts (11Wang G. Woods A. Agoston H. Ulici V. Glogauer M. Beier F. Dev. Biol. 2007; 306: 612-623Crossref PubMed Scopus (83) Google Scholar, 22James C.G. Appleton C.T. Ulici V. Underhill T.M. Beier F. Mol. Biol. Cell. 2005; 16: 5316-5333Crossref PubMed Scopus (115) Google Scholar, 23Carlberg A.L. Pucci B. Rallapalli R. Tuan R.S. Hall D.J. Differentiation. 2001; 67: 128-138Crossref PubMed Scopus (96) Google Scholar, 24Nicoll S.B. Barak O. Csoka A.B. Bhatnagar R.S. Stern R. Biochem. Biophys. Res. Commun. 2002; 292: 819-825Crossref PubMed Scopus (42) Google Scholar, 25Gomes Jr., R.R. Farach Carson M.C. Carson D.D. Connect. Tissue Res. 2003; 44: 196-201Crossref PubMed Google Scholar). To test the contribution of FAK to this process, we subjected Fak+/+ and Fak-/- fibroblasts (Fig. 1a) to high density micromass-induced chondrogenesis. Fak-/- animals die at embryonic day 8.5 (17Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1591) Google Scholar, 27Furuta Y.D. Ilic S. Kanazawa N. Takeda T. Yamamoto S. Aizawa S. Oncogene. 1995; 11: 1989-1995PubMed Google Scholar), precluding use of this type at later embryonic stages. Fibroblasts at 8.5 dpc represent “naïve” cells that have not begun to differentiate (9Karsenty G. Genes Dev. 1999; 13: 3037-3051Crossref PubMed Scopus (273) Google Scholar). Fak+/+ and -/- fibroblasts were plated in micromass cultures for 6 days. On day 6 of culture, PNA-stained images (Fig. 1b) were taken of the cultures. To our surprise, loss of FAK expression, even in naïve E8.5 fibroblasts, resulted in increased PNA staining, a hallmark of chondrogenic differentiation, in micromass culture as compared with wild-type cells (Fig. 1b). In the Fak-/- cells, L-Sox5 and Col2a1 mRNA expression was significantly higher than in the wild-type controls (Fig. 1c). Conversely, Sox9 expression was unaltered (Fig. 1c). Intriguingly, Agc and Hapln mRNAs were not detected either in Fak+/+ and -/- fibroblasts (data not shown). Similarly, Sox6 was not induced in Fak-/- E8.5 cells (Fig. 1c). These results surprisingly suggest that the loss of FAK in these naïve cells was sufficient to induce some, but not all, features of chondrogenesis. CCN2 is a key modulator of adhesive signaling and promotes chondrogenic gene expression in vivo and in vitro (16Leask A. Abraham D.J. J. Cell Sci. 2006; 119: 4803-4810Crossref PubMed Scopus (576) Google Scholar, 18Chen Y. Abraham D.J. Shi-wen X. Pearson J.D. Black C.M. Lyons K.M. Leask A. Mol. Biol. Cell. 2004; 15: 5635-5646Crossref PubMed Scopus (151) Google Scholar). To probe a possible connection between loss of FAK and CCN2 expression, real-time RT-PCR and Western blot analysis of Fak+/+ and -/- fibroblasts in micromass culture for 6 days were used. This analysis revealed that CCN2 protein and mRNA were significantly elevated in the absence of FAK, whereas CCN2 protein and mRNA were not induced in Fak+/+ cells (Fig. 1, a and c). Collectively, these data suggested the intriguing notion that, in cells competent to undergo bone fide chondrogenic differentiation, FAK signaling may suppress chondrogenesis by preventing the induction of CCN2. Loss of Ccn2 Disrupts Expression of Early Chondrogenic Genes in Monolayer Culture—To test the hypothesis that CCN2 acted downstream of the loss of FAK to promote chondrogenesis, it was necessary to employ cells capable of undergoing full chondrogenic differentiation. To investigate this question, we therefore used E13.5 mouse mesenchymal cells, as chondrogenesis occurs in vivo at this stage (9Karsenty G. Genes Dev. 1999; 13: 3037-3051Crossref PubMed Scopus (273) Google Scholar). Microarray analysis of mRNAs isolated from Ccn2+/+ and -/- mesenchymal cells growing in monolayer culture was performed. This analysis revealed that many early chondrogenic genes, such as Agc, Dcn, Hapln (the gene encoding link protein), and Sox6, showed a >2-fold reduction in expression in the absence of CCN2 (Table 1). Moreover, loss of Ccn2 resulted in reduced expression of the CCN family members Ccn1 and Ccn5 (Table 1), both of which have been implicated in bone formation (16Leask A. Abraham D.J. J. Cell Sci. 2006; 119: 4803-4810Crossref PubMed Scopus (576) Google Scholar). Differences in gene expression between wild-type and knock-out cells revealed by microarray analysis were verified using real-time RT-PCR analysis. As expected, real-time PCR analysis revealed that Ccn2 mRNA expression was observed in wild-type, but not in Ccn2-/-, cells (Fig. 2). Confirming our microarray data, real-time PCR analysis also showed that Ccn2-/- mesenchymal cells possessed a significant decrease in the mRNAs encoded by Ccn1 and Ccn5, Sox6, Agc, Dcn, and the link protein gene Hapln (Fig. 2). We used Western blot analysis to confirm reduced expression of Sox6 and aggrecan protein in the absence of CCN2 (Fig. 3A). Moreover, down-regulation of Sox6 in the Ccn2-/- cells was confirmed by immunofluorescence analyses of Ccn2+/+ and -/- mesenchymal cells. Sox6 was localized around the nuclei of Ccn2+/+ cells but was markedly reduced in Ccn2-/- cells (Fig. 3B). In contrast to these results, here was a significant increase in L-Sox5 gene expression and no change in Sox9 (Fig. 2). Furthermore, no change in mRNA transcript levels for Col2a1 or Col10a1 (Fig. 2) was observed in the CCN2-deficient mesenchymal cells. Western blot analysis confirmed that loss of CCN2 did not result in reduced L-Sox5 and type II collagen protein expression (Fig. 3A). These data demonstrate that CCN2 is required for the expression of some, but not all, chondrogenic genes in monolayer cell culture.FIGURE 3In the absence of Ccn2, levels of early chondrogenic matrix-associated proteins Sox6 and aggrecan are decreased. A, protein isolated from Ccn2 +/+ (WT) and -/- (KO) mouse mesenchymal cells was cultured in monolayer for 48 h. Conditioned medium and protein extracts were examined for aggrecan and Sox6, respectively, by Western blot analysis. Both Sox6 and aggrecan protein expression were decreased in Ccn2-/- mouse mesenchymal cells. β-actin was used as a loading control. Densitometry analysis of aggrecan and Sox6, relative to β-actin, shows a significant difference between samples. No difference was seen in the expression of type II collagen and Sox5. Data shown represent means + S.E. from three trials. *, p <0.05 using Student's paired t test. B, for immunofluorescence analysis, cells were fixed in paraformaldehyde and stained with fluorescein isothiocyanate-labeled (FITC) antibody for Sox6 and with 4′,6-diamidino-2-phenylindole (DAPI) for nuclei. Sox6 was localized around the nuclei in the Ccn2 +/+ mouse mesenchymal cells, whereas expression was markedly decreased in the Ccn2-/- mouse mesenchymal cells. Six different fields were examined. A representative field is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Loss of Ccn2 Results in Impaired Chondrogenesis in Micromass Culture—Based on our mRNA and protein expression data indicating a role for CCN2 in early chondrogenesis, we assessed the effect of loss of CCN2 in the micromass culture system (11Wang G. Woods A. Agoston H. Ulici V. Glogauer M. Beier F. Dev. Biol. 2007; 306: 612-623Crossref PubMed Scopus (83) Google Scholar). Ccn2+/+ and -/- mesenchymal cells were plated in high density micromass cultures and cultured for up to 6 days. PNA staining of the resultant cultures revealed that loss of CCN2 expression impaired the overall ability of mesenchymal cells to undergo condensation (Fig. 4a). Similarly, Alcian Blue staining revealed that loss of CCN2 expression resulted in reduced glycosaminoglycan production (Fig. 4b). RNAs were harvested on days 1, 3, and 6 of micromass culture, and transcription of chondrogenic genes was analyzed by real-time RT-PCR. In wild-type mesenchymal cells, CCN2 was expressed at constant levels throughout the time course, and, as expected, CCN2 was undetectable in Ccn2-/- cells (Fig. 5). Confirming our experiments using monolayer culture, loss of CCN2 expression resulted in decreased expression of Agc and Hapln (encoding link protein) mRNAs throughout the 6 days of culture (Fig. 5). In the absence of CCn2, Col2a1 mRNA expression was significantly increased throughout the 6 days of culture (Fig. 5). These data suggest that the absence of CCN2 alters the balance of collagen and proteoglycan gene expression.FIGURE 5Loss of Ccn2 affects proteoglycan mRNA expression. Ccn2+/+ (WT) and -/- (KO) mouse mesenchymal cells were subjected to micromass cultures for 6 days. For real-time RT-PCR, RNA was harvested on days 1, 3, and 6. Agc, Sox6, and Hapln mRNA expression was significantly reduced in Ccn2-/- mouse mesenchymal cells on all 6 days examined. Conversely, Col2a1, L-Sox5, and Sox9 expression was significantly increased in Ccn2-/- mouse mesenchymal cells. Data shown are relative to GAPDH and represent means + S.E. from three independent experiments (each performed in triplicate). *, p < 0.05 using a two-way analysis of variance. Expression in WT cells on day 1 was taken to represent 1.View Large Image" @default.
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