Functional Gene Screening System Identified TRPV4 as a Regulator of Chondrogenic Differentiation
2007; Elsevier BV; Volume: 282; Issue: 44 Linguagem: Inglês
10.1074/jbc.m706158200
ISSN1083-351X
AutoresShuji Muramatsu, M. Wakabayashi, Takeshi Ohno, Katsuhiko Amano, Rika Ooishi, Toshinori Sugahara, Satoshi Shiojiri, Kosuke Tashiro, Yutaka Suzuki, Riko Nishimura, Satoru Kuhara, Sumio Sugano, Toshiyuki Yoneda, Akio Matsuda,
Tópico(s)Silk-based biomaterials and applications
ResumoSox9 is a transcription factor that is essential for chondrocyte differentiation and chondrocyte-specific gene expression. However, the precise mechanism of Sox9 activation during chondrogenesis is not fully understood. To investigate this mechanism, we performed functional gene screening to identify genes that activate SOX9-dependent transcription, using full-length cDNA libraries generated from a murine chondrogenic cell line, ATDC5. Screening revealed that TRPV4 (transient receptor potential vanilloid 4), a cation channel molecule, significantly elevates SOX9-dependent reporter activity. Microarray and quantitative real time PCR analyses demonstrated that during chondrogenesis in ATDC5 and C3H10T1/2 (a murine mesenchymal stem cell line), the expression pattern of TRPV4 was similar to the expression patterns of chondrogenic marker genes, such as type II collagen and aggrecan. Activation of TRPV4 by a pharmacological activator induced SOX9-dependent reporter activity, and this effect was abolished by the addition of the TRPV antagonist ruthenium red or by using a small interfering RNA for TRPV4. The SOX9-dependent reporter activity due to TRPV4 activation was abrogated by both EGTA and a calmodulin inhibitor, suggesting that the Ca2+/calmodulin signal is essential in this process. Furthermore, activation of TRPV4 in concert with insulin activity in ATDC5 cells or in concert with bone morphogenetic protein-2 in C3H10T1/2 cells promoted synthesis of sulfated glycosaminoglycan, but activation of TRPV4 had no effect alone. We showed that activation of TRPV4 increased the steady-state levels of SOX9 mRNA and protein and SOX6 mRNA. Taken together, our results suggest that TRPV4 regulates the SOX9 pathway and contributes to the process of chondrogenesis. Sox9 is a transcription factor that is essential for chondrocyte differentiation and chondrocyte-specific gene expression. However, the precise mechanism of Sox9 activation during chondrogenesis is not fully understood. To investigate this mechanism, we performed functional gene screening to identify genes that activate SOX9-dependent transcription, using full-length cDNA libraries generated from a murine chondrogenic cell line, ATDC5. Screening revealed that TRPV4 (transient receptor potential vanilloid 4), a cation channel molecule, significantly elevates SOX9-dependent reporter activity. Microarray and quantitative real time PCR analyses demonstrated that during chondrogenesis in ATDC5 and C3H10T1/2 (a murine mesenchymal stem cell line), the expression pattern of TRPV4 was similar to the expression patterns of chondrogenic marker genes, such as type II collagen and aggrecan. Activation of TRPV4 by a pharmacological activator induced SOX9-dependent reporter activity, and this effect was abolished by the addition of the TRPV antagonist ruthenium red or by using a small interfering RNA for TRPV4. The SOX9-dependent reporter activity due to TRPV4 activation was abrogated by both EGTA and a calmodulin inhibitor, suggesting that the Ca2+/calmodulin signal is essential in this process. Furthermore, activation of TRPV4 in concert with insulin activity in ATDC5 cells or in concert with bone morphogenetic protein-2 in C3H10T1/2 cells promoted synthesis of sulfated glycosaminoglycan, but activation of TRPV4 had no effect alone. We showed that activation of TRPV4 increased the steady-state levels of SOX9 mRNA and protein and SOX6 mRNA. Taken together, our results suggest that TRPV4 regulates the SOX9 pathway and contributes to the process of chondrogenesis. Chondrogenesis is an important biological event for endochondral bone development, skeletogenesis, and tissue patterning (1Kronenberg H.M. Nature. 2003; 423: 332-336Crossref PubMed Scopus (2151) Google Scholar, 2de Crombrugghe B. Kim V. Behringer R.R. Bi W. Murakami S. Huang W. Matrix Biol. 2000; 19: 389-394Crossref PubMed Scopus (402) Google Scholar). The first step in chondrogenesis is the aggregation of mesenchymal cells into prechondrogenic condensations. These condensations start to express cartilage-specific genes and further differentiate into mature chondrocytes. In the growth plate, chondrocytes proliferate and further differentiate into hypertrophic chondrocytes. The control of chondrogenic differentiation and hypertrophy plays a pivotal role in the process. Dysregulation of either step leads to severe skeletal dysplasia in both mice and humans (3Zelzer E. Kim B.R. Nature. 2003; 423: 343-348Crossref PubMed Scopus (221) Google Scholar). The transcription factor Sox9 (SRY (sex-related Y)-type high mobility group box), which contains a SRY-related high mobility group box, has an essential role in the chondrocyte differentiation pathway (4Akiyama H. Kim M.C. Martin J.F. Schedl A. de Crombrugghe B. Genes Dev. 2002; 16: 2813-2828Crossref PubMed Scopus (1372) Google Scholar, 5Bi W. Kim J.M. Zhang Z. Behringer R.R. de Crombrugghe B. Nat. Genet. 1999; 22: 85-89Crossref PubMed Scopus (1403) Google Scholar). Sox9 regulates the transcription of cartilage-specific extracellular matrix molecules, such as collagen type II (6Lefebvre V. Kim W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar), IX (7Zhang P. Kim S.A. Stokes D.G. J. Biol. Chem. 2003; 278: 117-123Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), and XI (8Bridgewater L.C. Kim V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar) and aggrecan (9Sekiya I. Kim K. Koopman P. Watanabe H. Yamada Y. Shinomiya K. Nifuji A. Noda M. J. Biol. Chem. 2000; 275: 10738-10744Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). Heterozygous mutations in the SOX9 gene cause campomelic dysplasia characterized by severe chondrodysplasia (10Giordano J. Kim H.M. Bamforth J.S. Walter M.A. Am. J. Med. Genet. 2001; 98: 176-181Crossref PubMed Scopus (34) Google Scholar). Sox9 heterozygous mutant mice and mice lacking SOX9 function show impaired endochondral bone formation (4Akiyama H. Kim M.C. Martin J.F. Schedl A. de Crombrugghe B. Genes Dev. 2002; 16: 2813-2828Crossref PubMed Scopus (1372) Google Scholar, 5Bi W. Kim J.M. Zhang Z. Behringer R.R. de Crombrugghe B. Nat. Genet. 1999; 22: 85-89Crossref PubMed Scopus (1403) Google Scholar). Sox9 is also involved in the expression of Sox5 and Sox6, both of which form the transcriptional complex with Sox9 and control the expression of type II collagen and aggrecan (4Akiyama H. Kim M.C. Martin J.F. Schedl A. de Crombrugghe B. Genes Dev. 2002; 16: 2813-2828Crossref PubMed Scopus (1372) Google Scholar, 11Lefebvre V. Kim P. de Crombrugghe B. EMBO J. 1998; 17: 5718-5733Crossref PubMed Scopus (677) Google Scholar). These findings indicate that Sox9 plays essential roles in chondrogenesis. Although several molecules involved in chondrocyte differentiation have been identified, the mechanism of chondrogenesis is not fully understood. Identification of the mechanisms that control expression and activity of Sox9 would provide important insights into the regulation of chondrogenesis. We recently established a powerful functional cDNA screening system to identify molecules involved in NF-κB 2The abbreviations used are: NF-κB, nuclear factor-κB; FBS, fetal bovine serum; shRNA, short hairpin RNA; MOI, multiplicity of infection; 4α-PDD, 4α-phorbol 12,13-didecanoate; RR, ruthenium red; GAG, glycosaminoglycan; MAPK, mitogen-activated protein kinase; RT, reverse transcription; BMP, bone morphogenetic protein; Ad-shVR4, adenovirus expressing an shRNA against the TRPV4 gene; FAM, 6-carboxyfluorescein; TAMRA, 6-carboxy tetramethyl rhodamine. and MAPK signaling pathways, and we successfully identified many potential activators of these signals (12Matsuda A. Kim Y. Honda G. Muramatsu S. Matsuzaki O. Nagano Y. Doi T. Shimotohno K. Harada T. Nishida E. Hayashi H. Sugano S. Oncogene. 2003; 22: 3307-3318Crossref PubMed Scopus (334) Google Scholar). That study and others provided a genome-wide screening method based on large scale cDNA transfection and showed that, as a functional genomics method, large scale transfection linked to functional screening is an effective approach for searching for genes related to specific functions (12Matsuda A. Kim Y. Honda G. Muramatsu S. Matsuzaki O. Nagano Y. Doi T. Shimotohno K. Harada T. Nishida E. Hayashi H. Sugano S. Oncogene. 2003; 22: 3307-3318Crossref PubMed Scopus (334) Google Scholar, 13Iourgenko V. Kim W. Mickanin C. Daly I. Jiang C. Hexham J.M. Orth A.P. Miraglia L. Meltzer J. Garza D. Chirn G.W. McWhinnie E. Cohen D. Skelton J. Terry R. Yu Y. Bodian D. Buxton F.P. Zhu J. Song C. Labow M.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12147-12152Crossref PubMed Scopus (314) Google Scholar, 14Chanda S.K. Kim S. Orth A.P. Reisdorph R. Miraglia L. Thomas R.S. DeJesus P. Mason D.E. Huang Q. Vega R. Yu D.H. Nelson C.G. Smith B.M. Terry R. Linford A.S. Yu Y. Chirn G.W. Song C. Labow M.A. Cohen D. King F.J. Peters E.C. Schultz P.G. Vogt P.K. Hogenesch J.B. Caldwell J.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12153-12158Crossref PubMed Scopus (95) Google Scholar, 15Huang Q. Kim A. DeJesus P. Chao S.H. Quon K.C. Caldwell J.S. Chanda S.K. Izpisua-Belmonte J.C. Schultz P.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3456-3461Crossref PubMed Scopus (133) Google Scholar, 16Liu J. Kim A.G. Kintner C. Orth A.P. Chanda S.K. Ding S. Schultz P.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1927-1932Crossref PubMed Scopus (66) Google Scholar). In the present study, we used a similar approach for identifying additional components and modulators that are involved in the regulation of Sox9 and chondrocyte differentiation. We constructed full-length cDNA libraries derived from ATDC5 cells using the oligo-capping method (17Maruyama K. Kim S. Gene (Amst.). 1994; 138: 171-174Crossref PubMed Scopus (532) Google Scholar, 18Suzuki Y. Kim K. Maruyama K. Suyama A. Sugano S. Gene (Amst.). 1997; 200: 149-156Crossref PubMed Scopus (232) Google Scholar) and screened the libraries by performing a luciferase reporter assay using the SOX9-dependent type II collagen gene promoter. We isolated several positive cDNA clones that clearly stimulated the reporter gene and identified TRPV4 (transient receptor potential vanilloid 4) acting as a SOX9 regulator during chondrogenesis. Thus, our findings reveal novel functional roles for TRPV4 in chondrocyte differentiation. Cell Culture and Analysis of Chondrocytic Differentiation—ATDC5, a murine chondrogenic cell line, was obtained from the RIKEN cell bank (Tsukuba, Japan) and cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium (Invitrogen) containing 5% FBS, 10 μg/ml human transferrin (Sigma), and 3 × 10-8 m sodium selenite (Wako) (19Atsumi T. Kim Y. Kimata K. Ikawa Y. Cell Differ. Dev. 1990; 30: 109-116Crossref PubMed Scopus (337) Google Scholar). A murine mesenchymal stem cell line, C3H10T1/2, was obtained from the RIKEN cell bank (Tsukuba, Japan) and cultured in Dulbecco's modified Eagle's medium containing 10% FBS. To induce chondrogenic differentiation, bovine insulin (Sigma) was added to the medium at a concentration of 10 μg/ml for ATDC5 cells, and an appropriate concentration of human recombinant bone morphogenetic protein-2 (BMP-2) (R & D Systems) was added to C3H10T1/2 cells. The medium was replaced once every 2 days. To evaluate chondrocyte differentiation, cells were fixed with 95% methanol and stained with 0.1% Alcian blue 8GS (Sigma) in 0.1 n HCl overnight. Alcian blue-stained cultures were extracted with 6 m guanidine HCl for 2 h at room temperature. Optical density of the extracted dye was measured at 620 nm. Construction of Full-length cDNA Library and Arrayed cDNA Pool—ATDC5 cells were cultured with or without 10 μg/ml bovine insulin for 4 days. RNA samples were prepared from both samples and used for the construction of full-length cDNA libraries in the pME18S-FL3 mammalian expression vector (GenBank™ accession number AB009864). The procedure for constructing the full-length cDNA library using the oligo-capping method is described elsewhere (17Maruyama K. Kim S. Gene (Amst.). 1994; 138: 171-174Crossref PubMed Scopus (532) Google Scholar, 18Suzuki Y. Kim K. Maruyama K. Suyama A. Sugano S. Gene (Amst.). 1997; 200: 149-156Crossref PubMed Scopus (232) Google Scholar). We randomly isolated 40,000 cDNA clones from the cDNA library of ATDC5 cells stimulated with insulin and 80,000 cDNA clones from the library without insulin. In total, 120,000 cDNA clones were isolated and used to construct an arrayed cDNA pool in 96-well microtiter plates. Plasmid DNAs were purified using QIAwell 96 Ultra Plasmid Kits (Qiagen) according to the manufacturer's instructions. Screening of the Full-length cDNA Library—SOX9-dependent transcription was measured by using a reporter construct, tentatively named 4Col2E-Luc, which contained four tandem 48-bp chondrocyte-specific enhancer segments of type II collagen α1(Col2a1) in the pGL3 Basic vector (Promega), as previously reported (6Lefebvre V. Kim W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar, 20Lefebvre V. Kim G. Mukhopadhyay K. Smith C.N. Zhang Z. Eberspaecher H. Zhou X. Sinha S. Maity S.N. de Crombrugghe B. Mol. Cell. Biol. 1996; 16: 4512-4523Crossref PubMed Google Scholar). Reporter assays were performed using transient transfection. In each well of a 96-well microtiter plate, ATDC5 cells were inoculated at a density of 7.5 × 103 cells/well and cultured overnight prior to transfection. Cells were transfected with 50 ng of each cDNA clone and 100 ng of reporter plasmid using 0.3 μl of FuGENE 6 (Roche Applied Science). Immediately after transfection, ATDC5 cells were stimulated with 5 ng/ml insulin-like growth factor-1 (Roche Applied Science). At 48 h after transfection, cells were harvested, and luciferase activity was measured using PicaGeneLT2.0 (Toyo B-Net). cDNAs that produced more than 2-fold induction of luciferase activity relative to the parental plasmid (pME18S-FL3), the mock control, were defined as positive clones. Preparation of Tissue Samples—Primary murine chondrocytes were prepared from the rib cages of 4-week-old DDY mice (Nihon SLC) by collagenase digestion (0.2% collagenase (Wako) in phosphate-buffered saline) after adherent connective tissue and muscle was thoroughly removed by trypsin and collagenase pretreatment. Then cells were subjected to RNA extraction. Articular cartilage tissues were surgically prepared from femoral condyles and the tibial plateaus of 12-week-old ICR mice (Clea Japan). Embryonic hind limb buds were surgically prepared from embryonic day 12 embryos of DDY mice. Both tissues were then subjected to RNA extraction. Quantitative and Conventional Reverse Transcription-PCR Analysis—Total RNA was extracted from cells using an RNeasy kit (Qiagen) with DNase I (Qiagen) treatment, and 0.5 μg of total RNA was used to synthesize cDNA using SuperScript III reverse transcriptase (Invitrogen). The cDNAs were then used for quantitative RT-PCR, the products of which were analyzed using an ABI PRISM 7000 sequence detection system. Expression values were normalized to ribosomal protein RPL19. The following oligonucleotides were used. The dual fluorophore-labeled sequence for mouse TRPV4 was 5′-FAM-TCAGCCACTGGAGGGCACGC-TAMRA-3′, and the PCR primers were 5′-TCTTCACCCTCACCGCCTACT-3′ and 5′-TCCACTGTGGTCCGGTAAG-3′; for mouse COL2A1, the dual fluorophore-labeled primer was 5′-FAM-ACTGAGGGCTCCCAGAACATCACCTA-TAMRA-3′, and the PCR primers were 5′-TCCAGATGACTTTCCTCCGTCTA-3′ and 5′-AGGTAGGCGATGCTGTTCTTACA-3′; for mouse aggrecan, the dual fluorophore-labeled primer was 5′-FAM-CGTGTAAAAAGGGCACCGTGGCC-TAMRA-3′, and the PCR primers were 5′-GCATGAGAGAGGCGAATGGA-3′ and 5′-CTGATCTCGTAGCGATCTTTCTTCT-3′; for mouse SOX6, the dual fluorophore-labeled primer was 5′-FAM-CTTACTATGAAGAACAGGCCCGGCT-TAMRA-3′, and the PCR primers were 5′-CATGTCCAACCAGGAGAAGCA-3′ and 5′-GGGTACTTCTCTAGGTGGATTTTGC-3′; for mouse SOX9, the dual fluorophore-labeled primer was 5′-FAM-TGCCTGCTCAGACTATCACCTGTACCTCC-TAMRA-3′, and the PCR primers were 5′-CCATGTGGCCAGCAGATG-3′ and 5′-TTTTAGCACATGGGATGTCTTGAA-3′; for mouse RPL19, the dual fluorophore-labeled primer was 5′-FAM-CATTCCCGGGCTCGTTGC-TAMRA-3′, and the PCR primers were 5′-ATCCGCAAGCCTGTGACTGT-3′ and 5′-TCGGGCCAGGGTGTTTTT-3′; for mouse alkaline phosphatase, the dual fluorophore-labeled primer was 5′-FAM-CGCTGGGCCAAGGATGCTGG-TAMRA-3′, and the PCR primers were 5′-TCAGGGCAATGAGGTCACATC-3′ and 5′-TCACAATGCCCACGGACTT-3′; for mouse osteocalcin, the dual fluorophore-labeled primer was 5′-FAM-TTCATGTCCAAGCAGGAGGGCA-TAMRA-3′, and the PCR primers were 5′-GGCCCTGAGTCTGACAAAGC-3′ and 5′-GCCGGAGTCTGTTCACTACCTT-3′. Reaction conditions were 60 °C for 2 min and then 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. For analysis of TRPV4 gene expression in cartilaginous tissues, conventional RT-PCR was employed. For amplification of the TRPV4 gene, the PCR primers used were 5′-GTGCACCAACATGAAGGTCTGT-3′ and 5′-CCCAAGTTCTGGTTCCAGTGAG-3′. For the β-actin gene, the PCR primers were 5′-CTAGACTTCGAGCAGGAGATG-3′ and 5′-GACTCATCGTACTCCTGCTTG-3′. Reaction conditions were 94 °C for 3 min and then 35 cycles for TRPV4 and 30 cycles for β-actin of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The PCR products were resolved on 1.2% agarose gels and visualized by ethidium bromide staining. Measurement of Intracellular Ca2+—Cellular Ca2+ was estimated using the ratiometric fluorescence Ca2+ indicator Fura-2. ATDC5 cells were incubated at 37 °C for 30 min in assay buffer (20 mm HEPES (pH 7.4), 115 mm NaCl, 5.4 mm KCl, 0.8 mm MgSO4, 1.8 mm CaCl2, 13.8 mm glucose, and 0.1% bovine serum albumin), containing 5 μm Fura-2 AM (Dojindo) and 0.2% Pluronic F-127 (Molecular Probes). The cells were then washed and resuspended in assay buffer. Cellular Ca2+ was measured by ratio imaging of Fura-2 fluorescence (emission at 510 nm with excitation at 340 and 380 nm) using the Functional Drug Screening System 3000 (Hamamatsu Photonics). Reporter Gene Assay—ATDC5 cells were inoculated at a density of 7.5 × 103 cells/well in 96-well microtiter plates and cultured overnight prior to transfection. Cells were transfected with 100 ng of the 4Col2E-Luc reporter plasmid and 10 ng of phRL-TK (Promega) using 0.3 μl of FuGENE 6 (Roche Applied Science). Six hours after transfection, the medium was replaced by Dulbecco's modified Eagle's medium/F-12 containing 0.5% or 2% FBS, and cells were cultured overnight. On the following day, cells were treated with various concentrations of EGTA or the calmodulin inhibitor W-7 (Calbiochem) for 0.5 to 1 h and then treated with an appropriate concentration of a pharmacological activator of TRPV4, 4α-phorbol 12,13-didecanoate (4α-PDD; Calbiochem) overnight. Reporter activity originating from 4Col2E-Luc and the internal control, phRL-TK, was measured using the dual luciferase reporter assay system according to the manufacturer's instructions (Promega). Preparation of the Adenovirus Vector and RNA Interference—A short hairpin RNA (shRNA) expression vector was constructed as described previously (21Brummelkamp T.R. Kim R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3968) Google Scholar). The shRNA expression cassette was then transferred into the SwaI site of the pAxcw cosmid vector (TaKaRa Bio Inc.). A control adenovirus was constructed using an shRNA expression cassette without the RNA interference sequence. Propagation and generation of recombinant adenoviruses were performed according to the manufacturer's instructions (TaKaRa Bio Inc.). The RNA interference target sequence for mouse TRPV4 mRNA was 5′-CTGGCAAGAGTGAAATCTACCAGTA-3′. For the RNA interference experiments, ATDC5 cells were infected with the adenovirus construct at a multiplicity of infection (MOI) of 300. The transfection experiment was carried out 3 days after adenovirus infection, and luciferase activity was measured the following day. Western Blot Analysis—Cells were lysed with Tris-SDS sample buffer, and cell lysates were electrophoretically separated on a 4–20% SDS-polyacrylamide gel (Daiichi Pure Chemical) and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked with Immuno Block (Dainippon Sumitomo Pharmaceutical) for 1 h at room temperature and incubated with anti-SOX9 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-α-tubulin primary antibody for 1 h at room temperature. The membrane was then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody for 1 h at room temperature. Immunoreactive bands were visualized by ECL (Amersham Biosciences). Genome-wide Identification of Full-length cDNA Involved in Chondrogenesis—In order to identify genes that are involved in chondrocyte differentiation, we screened full-length cDNA libraries using luciferase reporter assays. In these assays, we used a murine chondrogenic cell line, ATDC5, which mimics chondrogenic differentiation by insulin stimulation, as a host cell and 4Col2E-Luc as a SOX9-dependent Col2a1 reporter construct (Fig. 1). We prepared RNAs from ATDC5 cells treated with and without 10 μg/ml bovine insulin and constructed full-length cDNA libraries on the mammalian expression vector, pME18S-FL3. We randomly isolated 40,000 cDNA clones from the cDNA library of ATDC5 cells stimulated with insulin and 80,000 clones from the library without insulin. Each of the cDNAs in the arrayed format was co-transfected with the reporter gene into ATDC5 cells, and luciferase reporter activity was measured. In total, 120,000 clones were assayed, and each clone with reporter activity that was more than 2-fold higher than that of the parental control plasmid was selected. The selected clones were sequenced from both ends, and BLAST searches were performed against the GenBank™ nonredundant data base. We clustered redundant clones and finally identified 46 genes that activated SOX9-dependent luciferase activity (Table 1). SOX5 and SOX6 were identified among this set of clones. The association of both Sox5 and Sox6 with Sox9 has been previously reported (11Lefebvre V. Kim P. de Crombrugghe B. EMBO J. 1998; 17: 5718-5733Crossref PubMed Scopus (677) Google Scholar). Moreover, it has been demonstrated that both molecules act as transcriptional co-activators for Sox9 and are essential for chondrogenic differentiation (22Smits P. Kim P. Mandel J. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Lefebvre V. Dev. Cell. 2001; 1: 277-290Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar). Although many of the genes shown in Table 1 have not been previously reported as being related to chondrogenesis, our result suggests that some genes identified here might be involved in the course of chondrocyte differentiation.TABLE 1Complete list of genes that activated the SOX9-dependent Col2a1 promoter A gene with a more than 2-fold higher level of luciferase activity relative to the mock control was defined as a positive clone. -Fold induction is a ratio of the luciferase activity of each gene to that of the parental plasmid, pME18S-FL3, as a mock control. The clone number is the number of individual clones identified as positive in our screening.Gene IDGene nameInductionClone number-fold18747Protein kinase A50563873TrpV442116653Ki-ras17118176N-ras16273835Interferon-induced transmembrane protein 514120462Splicing factor, arginine/serine-rich 10141020779Src121209446Transcription factor E312221415TCF-310514158fer (fms/fps-related) protein kinase, testis-specific 28115162Hck7.212262914-3-3 η6.84192662Rho GDI α614330171Potassium channel tetramerization domain-containing 105.8193834Peli25.3218596PDGFR β5.1112005Axin15126397MKK34.7120678Sox54.42232334Vestigial-like 44.31226591TOR signaling pathway regulator-like4.21224619TRAF74.1116601Kruppel-like factor 94120679Sox64112045B-cell leukemia/lymphoma 2-related protein A1b3.9212609CCAAT/enhancer binding protein, δ3.8126406MEKK33.8112321Calumenin3.7117210Myeloid cell leukemia sequence 13.6117872Myd1163.3220204Paired related homeobox 23.21110157Raf-13.2173181NFATc43167201RIKEN cDNA 2700085E05 gene2.9114182FGFR12.8280912Pumilio 12.8174168Zinc finger, DHHC domain-containing 162.71192176Filamin, α2.5165247Ankyrin repeat and SOCS box-containing protein 12.5154601AFX/FOXO42.4171966NF-κB inhibitor-interacting Ras-like protein 22.3113682Eukaryotic translation initiation factor 4A22.21234594CCR4-NOT transcription complex, subunit 12.21229589Prune homolog2.2112387β-Catenin2.1120853Staufen (RNA-binding protein) homolog 12.11 Open table in a new tab Gene Expression Analysis of the Identified Genes during Chondrogenic Differentiation—We next examined the expression profiles of genes listed in Table 1 during chondrogenic differentiation of ATDC5 cells stimulated with insulin. For these experiments, we employed cDNA microarray analysis and found that the mRNA levels of TRPV4, interferon-induced transmembrane protein 5, SOX9, and MYD116 were increased during the differentiation of ATDC5 cells (data not shown). In this report, we investigated the TRPV4 cation channel molecule in detail, since it had a strong effect on SOX9-dependent reporter activity (42-fold increase) (Table 1) and its relevance to chondrogenesis remains poorly understood. To confirm the increase in TRPV4 mRNA observed using microarray analysis, we performed quantitative RT-PCR and compared the expression pattern of TRPV4 with that of other chondrogenic marker genes. The expression levels of well known marker genes, such as COL2A1 and aggrecan, were elevated on day 12 after insulin stimulation (Fig. 2A) (19Atsumi T. Kim Y. Kimata K. Ikawa Y. Cell Differ. Dev. 1990; 30: 109-116Crossref PubMed Scopus (337) Google Scholar, 23Shukunami C. Kim C. Atsumi T. Ishizeki K. Suzuki F. Hiraki Y. J. Cell Biol. 1996; 133: 457-468Crossref PubMed Scopus (345) Google Scholar). The elevation in the levels of the two mRNAs reached about 45-fold for COL2A1 and 110-fold for aggrecan on day 21. TRPV4 mRNA was expressed at significantly high levels on day 12 and peaked on day 14 (Fig. 2A). The maximum induction level of TRPV4 was about 7-fold on day 14. We further examined the expression pattern of the TRPV4 gene using another cell line, C3H10T1/2, a murine mesenchymal stem cell line that is known to differentiate into chondrocytes when stimulated with BMP-2. Expression of the COL2A1 gene was elevated by BMP-2 stimulation and reached about 30-fold on day 7 (Fig. 2B). Although expression of the aggrecan gene was not detected on day 0, it was evident on day 3 after BMP-2 stimulation and increased to about 24-fold on day 7 when compared with day 3 (Fig. 2B). We also observed that TRPV4 gene expression had increased about 30-fold by day 3. TRPV4 and COL2A1 mRNAs had increased significantly on day 1, and an increase in aggrecan mRNA was evident on day 3 after BMP-2 stimulation, suggesting that chondrocyte differentiation in C3H10T1/2 cells proceeds faster than in ATDC5 cells. These results indicate that TRPV4 mRNA is elevated during chondrocyte differentiation. Gene Expression of TRPV4 in Cartilage Tissues—The expression of TRPV4 in several murine tissues has been previously reported; however, its expression in cartilage tissues is still uncertain (24Liedtke W. Kim Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar). To determine the importance of TRPV4 in chondrogenesis in vivo, we examined the expression of TRPV4 in murine cartilage tissues using RT-PCR. As shown in Fig. 2C, amplified DNA fragments of predictable size were detected in cartilage tissue of the hind limb in embryonic day 12 embryos and in cartilage tissues of knee joints and primary chondrocytes prepared from the rib cage in adults (Fig. 2C). From these observations, we concluded that the TRPV4 gene is expressed in cartilage tissues as well as in chondrogenic cell lines. These results prompted us to investigate the function of TRPV4 in chondrogenesis. Activation of TRPV4 Promotes SOX9-dependent Transcription—TRPV4 was identified by its ability to elevate SOX9-responsive reporter activity in an ectopic expression experiment. We next examined whether the activation of endogenous TRPV4 resulted in the elevation of SOX9-dependent luciferase activity. For this purpose, we used a pharmacological activator of TRPV4, 4α-PDD, which is a non-protein kinase C-activating phorbol ester derivative, in the following experiments (25Watanabe H. Kim J.B. Smart D. Jerman J.C. Smith G.D. Hayes P. Vriens J. Cairns W. Wissenbach U. Prenen J. Flockerzi V. Droogmans G. Benham C.D. Nilius B. J. Biol. Chem. 2002; 277: 13569-13577Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar). We examined the efficacy of 4α-PDD on 4Col2E-Luc reporter activity. As shown in Fig. 3A, 4α-PDD strongly increased 4Col2E-Luc reporter activity in a dose-dependent manner in ATDC5 cells, and this effect was abolished by the addition of 10 μm ruthenium red (RR), a TRPV antagonist (25Watanabe H. Kim J.B. Smart D. Jerman J.C. Smith G.D. Hayes P. Vriens J. Cairns W. Wissenbach U. Prenen J. Flockerzi V. Droogmans G. Benham C.D. Nilius B. J. Biol. Chem. 2002; 277: 13569-13577Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar). Similar results were also obtained when C3H10T1/2 cells were examined (Fig. 3B). To further confirm that the effect of 4α-PDD on 4Col2E-Luc reporter activity was mediated by TRPV4, we generated an adenovirus expressing an shRNA against the TRPV4 gene (Ad-shVR4) and used it to attempt to inhibit TRPV4 expression. When ATDC5 cells were infected with Ad-shVR4 at an MOI of 300, expres
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