Regulation of Type II Collagen Expression by Histone Deacetylase in Articular Chondrocytes (2007)

Histone deacetylase (HDAC) regulates various cellular processes by modulating gene expression. Here, we investigated the role of HDAC in the expression of type II collagen, a marker of differentiated chondrocytes. We found that HDAC activity in primary articular chondrocytes decreases during dedifferentiation induced by serial monolayer culture and that the activity recovered during redifferentiation induced by three-dimensional culture in a cell pellet. Inhibition of HDAC with trichostatin A or PXD101 was sufficient to block type II collagen expression in primary culture chondrocytes. HDAC inhibition also blocked the redifferentiation of dedifferentiated chondrocytes by suppressing the synthesis and accumulation of type II collagen. HDAC inhibition promoted the expression of Wnt-5a, which is known to inhibit type II collagen expression, and knockdown of Wnt-5a blocked the ability of HDAC inhibitors to suppress type II collagen expression. In addition, the induction of Wnt-5a expression by HDAC inhibitors was associated with acetylation of the Wnt-5a promoter. Taken together, our results suggest that HDAC promotes type II collagen expression by suppressing the transcription of Wnt-5a. Histone deacetylase (HDAC) regulates various cellular processes by modulating gene expression. Here, we investigated the role of HDAC in the expression of type II collagen, a marker of differentiated chondrocytes. We found that HDAC activity in primary articular chondrocytes decreases during dedifferentiation induced by serial monolayer culture and that the activity recovered during redifferentiation induced by three-dimensional culture in a cell pellet. Inhibition of HDAC with trichostatin A or PXD101 was sufficient to block type II collagen expression in primary culture chondrocytes. HDAC inhibition also blocked the redifferentiation of dedifferentiated chondrocytes by suppressing the synthesis and accumulation of type II collagen. HDAC inhibition promoted the expression of Wnt-5a, which is known to inhibit type II collagen expression, and knockdown of Wnt-5a blocked the ability of HDAC inhibitors to suppress type II collagen expression. In addition, the induction of Wnt-5a expression by HDAC inhibitors was associated with acetylation of the Wnt-5a promoter. Taken together, our results suggest that HDAC promotes type II collagen expression by suppressing the transcription of Wnt-5a. Differentiation of uncommitted mesenchymal cells into chondrocytes is initiated by the proliferation and recruitment of chondrogenic mesenchymal cells into condensations. These precartilage condensations develop into cartilage nodules containing differentiated chondrocytes. Chondrogenesis can be mimicked by micromass culture of mesenchymal cells in vitro. Differentiated chondrocytes are characterized by the ability to synthesize cartilage-specific extracellular matrix molecules including type II collagen and sulfated proteoglycans, which are necessary for normal cartilage development (1Sandell L.J. Adler P. Front. Biosci. 1999; 4: D731-D742Crossref PubMed Google Scholar, 2DeLise A.M. Fischer L. Tuan R.S. Osteoarthritis Cartilage. 2000; 8: 309-334Abstract Full Text PDF PubMed Scopus (649) Google Scholar). The activity of cartilage-specific matrix molecule synthesis by the differentiated chondrocytes is unstable and rapidly lost in response to certain environmental changes. For instance, proinflammatory cytokines such as interleukin (IL) 2The abbreviations used are: IL, interleukin; ChIP, chromatin immunoprecipitation; DMEM, Dulbecco's modified Eagle's medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC, histone deacetylase; MMP, matrix metalloproteinase; TSA, trichostatin A; P, passage; qRT-PCR, quantitative real-time polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; siRNA, small interfering RNA. 2The abbreviations used are: IL, interleukin; ChIP, chromatin immunoprecipitation; DMEM, Dulbecco's modified Eagle's medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC, histone deacetylase; MMP, matrix metalloproteinase; TSA, trichostatin A; P, passage; qRT-PCR, quantitative real-time polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; siRNA, small interfering RNA.-1β cause the loss of differentiated chondrocyte phenotypes (dedifferentiation) by halting the synthesis of cartilage-specific matrix molecules (3Ghosh P. Smith M. Biogerontology. 2002; 3: 85-88Crossref PubMed Scopus (66) Google Scholar, 4Lefebvre V. Peeters-Joris C. Vaes G. Biochim. Biophys. Acta. 1990; 1052: 366-378Crossref PubMed Scopus (225) Google Scholar). Chondrocyte dedifferentiation is also induced by serial monolayer culture and is accompanied by a gradual shift in the expression of type II collagen and aggrecan to type I and III collagen and versican, respectively (5Benya P.D. Padilla S.R. Nimni M.E. Biochemistry. 1977; 16: 865-872Crossref PubMed Scopus (257) Google Scholar, 6Yoon Y.-M. Kim S.-J. Oh C.-D. Ju J.-W. Song W.-K. Yoo Y.-J. Huh T.-L. Chun J.-S. J. Biol. Chem. 2002; 277: 8412-8420Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Dedifferentiated chondrocytes redifferentiate in three-dimensional culture in an alginate gel or cell pellet (6Yoon Y.-M. Kim S.-J. Oh C.-D. Ju J.-W. Song W.-K. Yoo Y.-J. Huh T.-L. Chun J.-S. J. Biol. Chem. 2002; 277: 8412-8420Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 7Bonaventure J. Kadhom N. Cohen-Solal L. Ng K.H. Bourguignon J. Lasselin C. Freisinger P. Exp. Cell Res. 1994; 212: 97-104Crossref PubMed Scopus (449) Google Scholar). Although maintenance of a differentiated chondrocyte phenotype is important for cartilage homeostasis, the detailed mechanisms of the differentiation and dedifferentiation of chondrocytes remain largely unknown. Because chondrocyte differentiation/dedifferentiation is regulated by various soluble factors including growth factors and cytokines, we hypothesized that histone deacetylase (HDAC) may regulate the differentiation status of chondrocytes. HDAC modulates the growth and differentiation of various cell types by governing chromatin structure and repressing the activity of specific transcription factors (8Wade P.A. Hum. Mol. Genet. 2001; 10: 693-698Crossref PubMed Scopus (277) Google Scholar, 9McKinsey T.A. Zhang C.L. Olson E.N. Curr. Opin. Genet. Dev. 2001; 11: 497-504Crossref PubMed Scopus (348) Google Scholar). The major function of HDAC is to remove acetyl groups from histones, which causes the condensation of the chromatin structure. For example, HDAC can reverse the acetylation of histone H3 on Lys9 and Lys14 and histone H4 on Lys8 and Lys12, which are associated with transcriptional activation (10Ogryzko V.V. Schilts R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2376) Google Scholar). Chromatin condensation inhibits the access of transcription factors to the promoter regions of specific target genes and ultimately leads to transcriptional repression. The specific roles of various HDAC family members have been investigated by gene deletion experiments. For instance, knock-out of HDAC4 in mice causes premature ossification of developing bones due to an early onset of chondrocyte hypertrophy (11Vega R.B. Matsuda K. Oh J. Barbosa A.C. Yang X. Meadows E. McAnally J. Pomajzl C. Shelton J.M. Richardson J.A. Karsenty G. Olson E.N. Cell. 2004; 119: 555-566Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, 12Hug B.A. Cell. 2004; 119: 448-449Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Also, HDAC1 is required for the normal formation of the craniofacial cartilage of zebrafish (13Pillai R. Coverdale L.E. Dubey G. Martin C.C. Dev. Dyn. 2004; 231: 647-654Crossref PubMed Scopus (69) Google Scholar). Among the growth factors, leukemia/lymphoma-related factor is known to interact with HDAC1, inhibiting the expression of the cartilage oligomeric matrix protein gene and the differentiation of chondrocytes (14Liu C.J. Prazak L. Fajardo M. Yu S. Tyagi N. Di Cesare P.E. J. Biol. Chem. 2004; 279: 47081-47091Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Furthermore, HDAC4 and 5 are required for the inhibition of Runx2 by transforming growth factor β and are involved in osteoblast differentiation (15Kang J.S. Alliston T. Delston R. Derynck R. EMBO J. 2005; 24: 2543-2555Crossref PubMed Scopus (276) Google Scholar). In addition, inhibiting HDAC suppresses the enhancement of chondrogenesis by inhibitors of retinoic acid receptor-mediated signaling in primary cultures of mouse limb mesenchyme (16Weston A.D. Chandraratna R.A. Torchia J. Underhill T.M. J. Cell Biol. 2002; 158: 39-51Crossref PubMed Scopus (129) Google Scholar). The above findings suggest that HDAC regulates the differentiated phenotypes of chondrocytes by promoting the expression of cartilage-specific matrix molecules, but the evidence so far is indirect. Therefore, in the current study, we examined the role of HDAC in the expression of type II collagen by primary cultures of chondrocytes. We report that HDAC activity is required for the expression of type II collagen, a marker of differentiated chondrocytes. We also show evidence that HDAC acts by blocking the expression of Wnt-5a. Cell Culture—Mesenchymal cells were derived from the distal tips of E11.5 mouse embryo limb buds. The cells were suspended at a density of 2.0 × 107 cells/ml in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing 10% (v/v) fetal bovine serum, 50 μg/ml streptomycin, and 50 units/ml penicillin. Chondrogenesis was induced by placing the cells in 15-μl drops in culture dishes and incubating for 5 day (17Oh C.-D. Chang S.-H. Yoon Y.-M. Lee S.-J. Lee Y.-S. Kang S.-S. Chun J.-S. J. Biol. Chem. 2000; 275: 5613-5619Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Articular chondrocytes were isolated from the knee cartilage of 2-3-week-old New Zealand White rabbits (6Yoon Y.-M. Kim S.-J. Oh C.-D. Ju J.-W. Song W.-K. Yoo Y.-J. Huh T.-L. Chun J.-S. J. Biol. Chem. 2002; 277: 8412-8420Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Briefly, cartilage slices were digested enzymatically in 0.2% collagenase type II, and individual cells were grown in complete DMEM. To induce dedifferentiation, passage (P) 0 chondrocytes were serially subcultured up to P4 by plating cells each time at a density of 3 × 104 cells/cm2. Redifferentiation of dedifferentiated chondrocytes was induced by three-dimensional culture in a cell pellet as described previously (6Yoon Y.-M. Kim S.-J. Oh C.-D. Ju J.-W. Song W.-K. Yoo Y.-J. Huh T.-L. Chun J.-S. J. Biol. Chem. 2002; 277: 8412-8420Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Briefly, a suspension (1 ml) of dedifferentiated cells (2 × 105 cells) in complete DMEM was sedimented by brief centrifugation. Cell pellets were incubated up to 6 day to cause redifferentiation. The differentiation status of chondrocytes was determined by examining the expression of type II collagen. Cartilage explants were obtained from rabbit knee joints by cutting them into 32-mm3 pieces and then culturing the explants for 72 h in complete DMEM with or without 0.3 μM trichostatin A (TSA) with daily changes of the culture medium. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)—Total RNA was isolated using RNA STAT-60 (Tel-Test Inc., Friendswoods, TX) and reverse-transcribed with ImProm-II™ reverse transcriptase (Promega, Madison, WI). The resulting cDNA was amplified by PCR with Taq polymerase (TaKaRa Bio, Shiga, Japan). The PCR primers and conditions are summarized in Table 1. The qRT-PCR was performed using an iCycler™ thermal cycler (Bio-Rad) and SYBR Premix Ex Taq™ (TaKaRa Bio). All qRT-PCR reactions were performed in duplicate, and the amplification signal from the target gene was normalized by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal in the same reaction.TABLE 1Oligonucleotide primers and PCR conditionsGenePrimer sequencesSizeATaAT, annealing temperature.OriginbOrigin, primers are designed from known sequence of indicated species.bp°CCollagen-IIScS, sense primer.5′-GAC CCC ATG CAG TAC ATG CG-3′37062RabbitAsdAs, antisense primer.5′-AGC CGC CAT TGA TGG TCT CC-3′Collagen-IIS5′-GGG TCT CCT GCC TCC TCC TGC TC-3′20454MouseAs5′-TCC TTT CTG CCC CTT TGG CCC TAA TTT TCG GG-3′Collagen-XIS5′-TTTCCTCCATCCCATCTG-3′39656HumanAs5′-GGACTCCCTGGAACTAAG-3′Chondromodulin-IS5′-ACAGTGACCAAGCAGAGCATC-3′50456MouseAs5′-GCGGCATCCTTGGTAATTG-3′HDAC-1S5′-TTACTACTACGACGGGGATGTTG-3′33058HumanAs5′-GCGATGTCCGTCTGCTGCTTAT-3′HDAC-3S5′-CGGGTGCTCTACATTGATATCGACATCCAC-3′390NDeND, not detected under various conditions.MouseAs5′-TACCAGGAGAGGGATATTGAAACTCTTGAC-3′HDAC-6S5′-CGGTAATGGAACTCAGCACA-3′33956HumanAs5′-AGGTGGGTGAGGTGGGCATAAC-3′HDAC-7aS5′-GGACAAGAGCAAGCGAAGT-3′438NDHumanAs5′-CCGTGCTCGCTGTCATTG-3′HDAC-11S5′-TGCACACGAGGCGCTATCTTA-3′35652HumanAs5′-GAAGTCTCGCTCATGCCCA-3′Wnt-5aS5′-ACGCTAAGGGTTCCTATGAGA-3′47660HumanAs5′-CATAGCAGCACCAGTGAAACT-3′β-CateninS5′-GAAAATCCAGCGTGGACAATGGCTACTC-3′54256HumanAs5′-ACCATAACTGCAGCCTTATTAACC-3′PlakoglobinS5′-CTTCCGGCTCAACACCAT-3′38056HumanAs5′-GGCACATCGCTGGAGTACA-3′c-JunS5′-ATGGAGTCCCAGGAGCGGATCAA-3′25156HumanAs5′-GTTTGCAACTGCTGCGTTAG-3′p21S5′-CCGTGGACAGTGAGCAGTTG-3′58756MouseAs5′-TGGGCACTTCAGGGTTTTCT-3′MMP-13S5′-TGATGGACCTTCTGGTCTTCTGGC-3′57355MouseAs5′-CATCCACATGGTTGGGAAGTTCTG-3′GAPDHS5′-TCACCATCTTCCAGGAGCGA-3′29962RabbitAs5′-CACAATGCCGAAGTGGTCGT-3′GAPDHS5′-TCACTGCCACCCAGAAGAC-3′45050MouseAs5′-TGTAGGCCATGAGGTCCAC-3′Wnt-5a (A)S5′-AGTCCCTGGAAGCAGACG-3′64562Mouse~ChIPfChIP, primers are designed for ChIP assay.As5′-CTTCTTCATGGCGAGGG-3′Wnt-5a (B)S5′-AGTCCCTGGAAGCAGACG-3′44962Mouse~ChIPAs5′-AGGAGTTGGTATAGCGTGGA-3′β-actinS5′-GGCCAGCGTTTGCCTTTTATGGTAATAAT-3′16762Mouse~ChIPAs5′-CGAACTATCAAGACACAAAAGAAGGCTATA-3′MMP-13S5′-CATTTCCCTCAGATTCTGCCA-3′13162Mouse~ChIPAs5′-GGGAGTCCAGCTCAACAAGAAG-3′p21S5′-AGAGACATGGAGGAAGGTGGGA-3′22962Mouse~ChIPAs5′-GACCACACTAATGGCTACCTGC-3′a AT, annealing temperature.b Origin, primers are designed from known sequence of indicated species.c S, sense primer.d As, antisense primer.e ND, not detected under various conditions.f ChIP, primers are designed for ChIP assay. Open table in a new tab Immunofluorescence Microscopy—Rabbit articular chondrocytes cultured on coverslips were fixed with 3.5% paraformaldehyde in phosphate-buffered saline for 10 min at room temperature. The cells were permeabilized and blocked for 30 min with 0.1% Triton X-100 and 1% bovine serum albumin in phosphate-buffered saline. The fixed cells were washed and incubated for 1 h with an antibody (10 μg/ml) against type II collagen (Chemicon International Inc., Temecula, CA), followed by rhodamine-conjugated secondary antibody for 30 min. Nuclei were stained with Hoechst dye. Immunohistochemistry—Cartilage explants and chondrocyte pellets were fixed in 4% paraformaldehyde for 3 h at 4 °C, dehydrated with a graded series of ethanol, embedded in paraffin, and cut into 4-μm sections as described previously (18Ryu J.-H. Kim S.-J. Kim S.-H. Oh C.-D. Hwang S.-G. Chun C.-H. Oh S.-H. Seong J.-K. Huh T.-L. Chun J.-S. Development (Camb.). 2002; 129: 5541-5550Crossref PubMed Scopus (130) Google Scholar). The sections were stained using the LSAB2 system-horseradish peroxidase kit (Dako Co., Carpinteria, CA) according to the manufacturer's instructions. Briefly, sections on slides were incubated for 2 h at room temperature with antibodies against type II collagen (Chemicon International Inc.) or acetylated histone H4 (Upstate Biotechnology, Charlottesville, VA) for 2 h at room temperature, followed by biotinylated link reagent, and finally streptavidin-horseradish peroxidase reagent for 10 min. Immunoreactive proteins were then visualized with the 3-amino-9-ethylcarbazole substrate-chromogen solution. In Situ Hybridization—Digoxigenin-conjugated riboprobes for type II collagen and Wnt-5a were synthesized using digoxigenin RNA labeling mix (Roche Diagnostics) as described by Ryu and Chun (19Ryu J.-H. Chun J.-S. J. Biol. Chem. 2006; 281: 22039-22047Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The cDNA fragment of rabbit type II collagen was amplified by PCR using primers 5′-GGG TCT CCT GCC TCC TCC TGC TC-3′ (sense) and 5′-CTC CAT CTC TGC CAC GGG GT-3′ (antisense), and the PCR product was inserted into the pSPT-18 vector. For Wnt-5a, the full-length cDNA encoding rabbit Wnt-5a was cut with PstI and SacI, and the product was inserted into the pSPT-18 vector. The cloned pSPT-18 plasmids containing type II collagen or the Wnt-5a fragment were linearized with EcoRI or HindIII and transcribed with SP6 and T7 RNA polymerases (Roche Diagnostics), respectively. The resulting transcripts were used to synthesize digoxigenin-conjugated antisense and sense riboprobes. For hybridization, sections of cartilage explants were treated with 0.2 n HCl for 10 min and then permeabilized with 20 μg/ml protease K for 10 min at 37 °C. After acetylation with 0.25% acetic anhydride for 10 min, the sections were incubated with hybridization buffer (40% formamide, 10% dextran sulfate, 1 × Denhardt's solution, 4 × SSC, 10 μM dithiothreitol, 1 mg/ml yeast tRNA, and 1 mg/ml salmon sperm DNA) containing denatured sense or antisense digoxigenin-labeled riboprobes. After hybridization overnight at 42 °C, the sections were washed, and nonspecifically bound riboprobes were eliminated by digestion with RNase. The sections were incubated overnight at 4 °C with alkaline phosphatase-conjugated mouse anti-digoxigenin antibody, and hybridization signals were visualized with a solution of 4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-iodolyl-phosphate (Roche Diagnostics). Western Blot Analysis—Whole-cell lysates were prepared as described previously (6Yoon Y.-M. Kim S.-J. Oh C.-D. Ju J.-W. Song W.-K. Yoo Y.-J. Huh T.-L. Chun J.-S. J. Biol. Chem. 2002; 277: 8412-8420Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Proteins were size-fractionated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with 3% nonfat dry milk in Tris-buffered saline and incubated with antibodies against type II collagen, acetylated histone H4 (K8; Upstate Biotechnology) or extracellular signal-regulated protein kinase (Pharmingen). The blots were developed using a peroxidase-conjugated secondary antibody, followed by ECL reagents (Amersham Biosciences, Buckinghamshire, UK). HDAC Activity Assay—HDAC activity was determined using nuclear extracts, which were prepared by homogenizing chondrocytes in lysis buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol) supplemented with protease inhibitors (10 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml aprotinin, and 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride) and phosphatase inhibitors (1 mm NaF and 1 mm Na3VO4). Following addition of 0.6% (v/v) Nonidet P-40, the cells were incubated for 15 min on ice and centrifuged at 13,000 × g for 30 s at 4 °C. The nuclear pellets were resuspended in nuclear extraction buffer B (20 mm HEPES, pH 7.9, 0.4 m NaCl, 1 mm EDTA, 1 mm EGTA, and 1 mm dithiothreitol) containing protease and phosphatase inhibitors and incubated on ice for 15 min. Following centrifugation at 13,000 × g for 5 min at 4 °C, the supernatant (nuclear fraction) was collected and used for the HDAC activity assay. HDAC activity was determined fluorimetrically using a Fluor de Lys HDAC assay system (Biomol, Plymouth Meeting, PA) and 10 μg of nuclear extract (20Myzak M.C. Karplus P.A. Chung F.-L. Dashwood R.H. Cancer Res. 2004; 64: 5767-5774Crossref PubMed Scopus (438) Google Scholar). Transfection of Expression Vectors—The mammalian expression vectors for human HDAC-1, -6, and -11 (pCNS-hHDAC-1, pCMV-SPORT6-hHDAC-6, and pCMV-SPORT6-hHDAC-11) were obtained from Korea Bioinformation Center (Daejun, Korea). Empty and expression vectors were transfected into P1 chondrocytes using Metafectene™ (Biontex, Planegg, Germany) through the procedure recommended by the manufacturer. Transfected cells were maintained in complete DMEM for 48 h and used for further analyses (19Ryu J.-H. Chun J.-S. J. Biol. Chem. 2006; 281: 22039-22047Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Preparation of Wnt-5a-conditioned Media—Wnt-5a-producing mouse fibroblast L929 cells were obtained from the American Type Culture Collection (Manassas, VA). medium was collected from L929 cells h in DMEM. The medium was by centrifugation at × g for 5 min and by in an cell with a membrane as described by Ryu and Chun (19Ryu J.-H. Chun J.-S. J. Biol. Chem. 2006; 281: 22039-22047Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). of Wnt-5a by for Wnt-5a was designed on the sequence of rabbit Wnt-5a The were synthesized by (sense) and GCC GCC After the for 2 min at °C to secondary the sense and antisense were to at 30 °C for 1 were then transfected with the using Chromatin were performed using a ChIP assay kit (Upstate Biotechnology) and chondrocytes differentiated from mesenchymal mesenchymal cells derived from the distal tips of E11.5 mouse embryo limb were maintained as a micromass culture to induce chondrogenic differentiation in the or of 20 or The cells at day 5 were with 1% for 10 min, suspended in of lysis buffer 10 mm EDTA, and 50 mm pH and incubated for 10 min on The lysates were at for using a with on ice for 1 min each The were and the supernatant was with ChIP buffer (Upstate After with and of salmon sperm for 1 h at 4 °C, chromatin was incubated with 3 μg H4 antibody or in a of 2 for h at were isolated by with of salmon sperm After the pellets were treated with prepared buffer and 0.1 m and the was with 20 of 5 m and for 4 h at °C. was by extraction and used as a for The PCR primers for the ChIP assay were designed to to the promoter regions of Wnt-5a, matrix and HDAC Activity in on the Differentiation the role of HDAC in the maintenance of differentiated chondrocyte we examined HDAC during the dedifferentiation and redifferentiation of articular chondrocytes. chondrocytes were serially subcultured as a monolayer to induce was by a in type II collagen expression HDAC activity was in a as type II collagen expression during dedifferentiation The in HDAC activity by dedifferentiation was during the redifferentiation of chondrocytes by three-dimensional culture in a cell pellet These results suggest that HDAC activity in chondrocytes is to the differentiation Inhibition of HDAC Activity Type II Expression in Articular HDAC activity is associated with maintenance of differentiated phenotypes in we examined the role of HDAC in type II collagen expression, a marker of differentiated chondrocytes. For we used HDAC M. M. M. T. J. Biol. Chem. 1990; Full Text PDF PubMed Google and PXD101 J.A. C.J. R. Mol. Cancer Google Scholar). inhibitors have a of activity against II, and with for individual HDAC C. J. Chem. 2005; PubMed Scopus Google Scholar). and PXD101 induced the and of histone H4 in chondrocytes and A HDAC activity assay that HDAC inhibitors HDAC activity B and without cell or proliferation Inhibition of HDAC by or PXD101 the of type II as determined by The of or PXD101 on type II collagen expression were and as determined by RT-PCR, qRT-PCR, and Western A and In to the of HDAC inhibitors on type II collagen expression, and PXD101 not not expression of chondrogenic molecules such as type collagen Res. 2004; PubMed Scopus Google and Res. 1996; PubMed Scopus Google Scholar). Expression of a known HDAC target gene J.S. Kim 2004; PubMed Scopus Google was by HDAC and were detected as The that of type II collagen expression by HDAC is specific in chondrocytes. We also examined the of the HDAC inhibitors in cartilage which in in primary and PXD101 not type II collagen expression and induced the of histone H4 in cartilage Furthermore, inhibition of HDAC by B and or PXD101 not blocked the expression and accumulation of type II collagen during redifferentiation of dedifferentiated chondrocytes in three-dimensional results that HDAC activity is required for type II collagen expression in inhibition suppresses type II collagen expression in cartilage explants and inhibits the redifferentiation of dedifferentiated chondrocytes. A, cartilage explants were or treated with 0.3 μM for 72 The of the type II collagen was detected by in histone H4 was detected by using a peroxidase B and chondrocytes were subcultured up to as a monolayer Dedifferentiated cells were cultured in cell pellets for 3 or 6 day in the or of 0.3 μM The of type II collagen expression was determined by and Western The accumulation of type II collagen and acetylation of histone H4 in the three-dimensional culture were detected by The results from experiments. extracellular signal-regulated Western We examined which of HDAC is involved in the of type II collagen expression. in and the and of HDAC activity during dedifferentiation and not changes in the expression of HDAC-1, -6, or We not and in rabbit articular chondrocytes not In addition, of HDAC-1, -6, or -11 in P1 chondrocytes not type II collagen expression that HDAC are not associated with type II collagen expression. of Wnt-5a in the of Type II Expression by the inhibition of HDAC promotes gene transcription by inhibiting chromatin is that the collagen gene is a target gene of we previously found that expression of type II collagen in chondrocytes is by the of Wnt-5a (19Ryu J.-H. Chun J.-S. J. Biol. Chem. 2006; 281: 22039-22047Abstract Full Text Full Text PDF PubMed Scopus (97) Google and S.-G. Ryu J.-H. Kim Kim K. Kim S.-J. Chun J.-S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). I