TonEBP/OREBP Is a Regulator of Nucleus Pulposus Cell Function and Survival in the Intervertebral Disc
2006; Elsevier BV; Volume: 281; Issue: 35 Linguagem: Inglês
10.1074/jbc.m601969200
ISSN1083-351X
AutoresTsung‐Ting Tsai, Keith G. Danielson, Asha Guttapalli, Erbil Oğuz, Todd J. Albert, Irving M. Shapiro, Makarand V. Risbud,
Tópico(s)Spinal Hematomas and Complications
ResumoThe nucleus pulposus is an aggrecan-rich hydrated tissue that permits the intervertebral disc to resist compressive loads. Adaptation to loading is achieved through an elevation in disc osmolarity mediated by the numerous charged glycosoaminoglycan side chains of the aggrecan molecule. The goal of this investigation was to determine the functional role of the osmo-regulatory protein, TonEBP, in cells of the nucleus pulposus. We found that TonEBP and its downstream target genes were robustly expressed in the tissues of the disc. Above 330 mosmol/kg, cultured nucleus pulposus cells up-regulated target genes TauT, BGT-1, and SMIT; above 450 mosmol/kg, there was raised expression of HSP-70. In hypertonic media there was activation of TauT and heat shock protein-70 (HSP-70) reporter activity and increased binding of TonEBP to the TonE motif. When cells were transfected with the dominant-negative form of TonEBP (DN-TonEBP) there was suppression of TauT and HSP-70 reporter gene expression; pTonEBP enhanced reporter gene expression. Moreover, in hypertonic media, forced expression of DN-TonEBP induced apoptosis. We suppressed TonEBP using small interfering RNA technique and noted a decrease in TauT reporter activity in isotonic as well as hyperosmolar media. Finally, we report that the aggrecan promoter contains two conserved TonE motifs. To evaluate the importance of these motifs, we overexpressed DN-TonEBP and partially silenced TonEBP using small interfering RNA. Both approaches resulted in suppression of aggrecan promoter activity. It is concluded that TonEBP permits the disc cells to adapt to the hyperosmotic milieu while autoregulating the expression of molecules that generate the unique extracellular environment. The nucleus pulposus is an aggrecan-rich hydrated tissue that permits the intervertebral disc to resist compressive loads. Adaptation to loading is achieved through an elevation in disc osmolarity mediated by the numerous charged glycosoaminoglycan side chains of the aggrecan molecule. The goal of this investigation was to determine the functional role of the osmo-regulatory protein, TonEBP, in cells of the nucleus pulposus. We found that TonEBP and its downstream target genes were robustly expressed in the tissues of the disc. Above 330 mosmol/kg, cultured nucleus pulposus cells up-regulated target genes TauT, BGT-1, and SMIT; above 450 mosmol/kg, there was raised expression of HSP-70. In hypertonic media there was activation of TauT and heat shock protein-70 (HSP-70) reporter activity and increased binding of TonEBP to the TonE motif. When cells were transfected with the dominant-negative form of TonEBP (DN-TonEBP) there was suppression of TauT and HSP-70 reporter gene expression; pTonEBP enhanced reporter gene expression. Moreover, in hypertonic media, forced expression of DN-TonEBP induced apoptosis. We suppressed TonEBP using small interfering RNA technique and noted a decrease in TauT reporter activity in isotonic as well as hyperosmolar media. Finally, we report that the aggrecan promoter contains two conserved TonE motifs. To evaluate the importance of these motifs, we overexpressed DN-TonEBP and partially silenced TonEBP using small interfering RNA. Both approaches resulted in suppression of aggrecan promoter activity. It is concluded that TonEBP permits the disc cells to adapt to the hyperosmotic milieu while autoregulating the expression of molecules that generate the unique extracellular environment. The intervertebral disc is a specialized biomechanical structure that permits movement between vertebrae and protects the vertebral bone from mechanical forces. It consists of an outer ligament, the annulus fibrosus that encloses a gel-like tissue, the nucleus pulposus. While sparse, cells in the nucleus pulposus secrete a complex extracellular matrix that contains fibrillar collagens and the proteoglycan, aggrecan. The numerous glycosaminoglycan side chains of the aggrecan molecule bind cations thereby raising the osmolarity of the disc tissues (1Maroudas A. Muir H. Wingham J. Biochim. Biophys. Acta. 1969; 177: 492-500Crossref PubMed Scopus (251) Google Scholar, 2Maroudas A. Biophys. J. 1970; 10: 365-379Abstract Full Text PDF PubMed Scopus (248) Google Scholar, 3Urban J.P. Holm S. Maroudas A. Biorheology. 1978; 15: 203-221Crossref PubMed Scopus (160) Google Scholar, 4Kraemer J. Kolditz D. Gowin R. Spine. 1985; 10: 69-71Crossref PubMed Scopus (116) Google Scholar). While the hydration properties of the nucleus pulposus permits dynamic loading and unloading, the mechanism by which these cells adapt to elevated osmotic forces is poorly understood.In a number of tissues, cellular adaptation to hyperosmotic stress is mediated by the tonicity enhancer-binding protein (TonEBP), 2The abbreviations used are: TonEBP, tonicity enhancer-binding protein; DN-TonEBP, dominant-negative form of TonEBP; RT, reverse transcription; PBS, phosphate-buffered saline; Wt, wild type; Mt, mutant; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA, small interfering RNA.2The abbreviations used are: TonEBP, tonicity enhancer-binding protein; DN-TonEBP, dominant-negative form of TonEBP; RT, reverse transcription; PBS, phosphate-buffered saline; Wt, wild type; Mt, mutant; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA, small interfering RNA. also called OREBP (5Miyakawa H. Woo S.K. Dahl S.C. Handler J.S. Kwon H.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2538-2542Crossref PubMed Scopus (470) Google Scholar) or NFAT5, (5Miyakawa H. Woo S.K. Dahl S.C. Handler J.S. Kwon H.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2538-2542Crossref PubMed Scopus (470) Google Scholar, 6Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (312) Google Scholar). Upon activation, TonEBP binds to the tonicity-responsive enhancer element (TonE) of genes required for osmotolerance and cell survival. These genes include the betaine/γ-aminobutyric acid transporter, sodium myo-inositol co-transporter (7Ko B.C. Ruepp B. Bohren K.M. Gabbay K.H. Chung S.S. J. Biol. Chem. 1997; 272: 16431-16437Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 8Miyakawa H. Woo S.K. Chen C.P. Dahl S.C. Handler J.S. Kwon H.M. Am. J. Physiol. 1998; 274: F753-F761PubMed Google Scholar, 9Rim J.S. Atta M.G. Dahl S.C. Berry G.T. Handler J.S. Kwon H.M. J. Biol. Chem. 1998; 273: 20615-20621Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), taurine transporter (10Zhang Z. Ferraris J.D. Brooks H.L. Brisc I. Burg M.B. Am. J. Physiol. 2003; 285: F688-F693Crossref PubMed Scopus (51) Google Scholar, 11Ito T. Fujio Y. Hirata M. Takatani T. Matsuda T. Muraoka S. Takahashi K. Azuma J. Biochem. J. 2004; 382: 177-182Crossref PubMed Scopus (107) Google Scholar), and aldose reductase (6Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (312) Google Scholar). By regulating levels of betaine, myo-inositol, taurine, and sorbitol, these genes control the osmotic properties of the cytosol. HSP-70, a molecular chaperone that maintains cellular function under hypertonic stress is also induced by TonEBP (12Woo S.K. Lee S.D. Na K.Y. Park W.K. Kwon H.M. Mol. Cell. Biol. 2002; 22: 5753-5760Crossref PubMed Scopus (173) Google Scholar, 13Shim E.H. Kim J.I. Bang E.S. Heo J.S. Lee J.S. Kim E.Y. Lee J.E. Park W.Y. Kim S.H. Kim H.S. Smithies O. Jang J.J. Jin D.I. Seo J.S. EMBO Rep. 2002; 3: 857-861Crossref PubMed Scopus (81) Google Scholar).Most homozygous TonEBP knock-out mice evidence midgestational lethality. Of the few that survive, all exhibit severe growth retardation and kidney dysfunction (14Lopez-Rodriguez C. Antos C.L. Shelton J.M. Richardson J.A. Lin F. Novobrantseva T.I. Bronson R.T. Igarashi P. Rao A. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2392-2397Crossref PubMed Scopus (220) Google Scholar). A transgenic mouse expressing a dominant-negative form of TonEBP (DN-TonEBP) in collecting duct epithelial cells demonstrates an absolute requirement of TonEBP for expression of the urea transporter gene and aquaporin-2 (15Lam A.K. Ko B.C. Tam S. Morris R. Yang J.Y. Chung S.K. Chung S.S. J. Biol. Chem. 2004; 279: 48048-48054Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Aside from osmoregulation, TonEBP is required for T cell proliferation and function (16Trama J. Go W.Y. Ho S.N. J. Immunol. 2002; 169: 5477-5488Crossref PubMed Scopus (85) Google Scholar, 17Go W.Y. Liu X. Roti M.A. Liu F. Ho S.N. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10673-10678Crossref PubMed Scopus (226) Google Scholar), and it is implicated in cancer cell migration and metastasis (18Jauliac S. Lopez-Rodriguez C. Shaw L.M. Brown L.F. Rao A. Toker A. Nat. Cell Biol. 2002; 4: 540-544Crossref PubMed Scopus (347) Google Scholar). A recent study by Wang et al. (19Wang Y. Ko B.C. Yang J.Y. Lam T.T. Jiang Z. Zhang J. Chung S.K. Chung S.S. J. Biol. Chem. 2005; 280: 19986-19991Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) showed that expression of DN-TonEBP in lens fiber cells promotes cataract formation by causing defects in their elongation. Since TonEBP is expressed by a number of cell types, it is reasonable to assume that it serves a variety of physiologic functions, especially those that impact on tissue hydration and the osmotic environment (20Maouyo D. Kim J.Y. Lee S.D. Wu Y. Woo S.K. Kwon H.M. Am. J. Physiol. 2002; 282: F802-F809Crossref PubMed Scopus (61) Google Scholar).The goal of the present study is to examine the role of TonEBP in cells of the postnatal intervertebral disc. We show that TonEBP, and its downstream target genes, are expressed in the nucleus pulposus and the annulus fibrosus. Importantly, our data indicate that TonEBP binds to a TonE motif in the aggrecan promoter and regulates its transcriptional activity. This finding lends credence to the view that TonEBP regulates the hydration status of the disc thereby enhancing cell function in the hyperosmotic environment.EXPERIMENTAL PROCEDURESIsolation of Nucleus Pulposus Cells—Rat nucleus pulposus cells were isolated using method reported earlier by Risbud et al. (21Risbud M.V. Guttapalli A. Stokes D.G. Hawkins D. Danielson K.G. Schaer T.P. Albert T.J. Shapiro I.M. J. Cell. Biochem. 2006; 98: 152-159Crossref PubMed Scopus (208) Google Scholar). Briefly, male Wistar rats (250 g) were euthanized with CO2, and lumbar intervertebral discs were removed from the spinal column. The gel-like nucleus pulposus was separated from the annulus fibrosus, using a dissecting microscope, and treated with 0.1% collagenase and 10 units/ml hyaluronidase for 4–6 h. The partially digested tissue was maintained as an explant in Dulbeccos modified Eagle's medium and 10% fetal bovine serum supplemented with antibiotics. Nucleus pulposus cells migrated out of the explant after 1 week. When confluent, the cells were lifted using a trypsin (0.25%) EDTA (1 mm) solution and subcultured in 10-cm dishes.RT-PCR Analysis—RNA was isolated from cells using Trizol reagent (Invitrogen) following the manufacturer's instructions. Briefly, 2 μg of total RNA was reverse-transcribed into cDNA using Superscript II RT enzyme (Invitrogen) and oligo(dT) primers. PCR reactions were performed using cDNA samples (1 μl) with Superscript DNA polymerase (Invitrogen). Primers for rat genes were custom designed and synthesized by Integrated DNA Technologies (Coralville, IA). The PCR product was run on a 1.2% agarose gel and the amplicon visualized on a Kodak 440 imaging station.Immunohistological Studies—Freshly isolated discs were immediately fixed in 4% paraformaldehyde in PBS and then embedded in paraffin. Transverse and coronal sections, 6–8 μm in thickness, were deparaffinized in xylene, rehydrated through graded ethanol, and stained with Alcian blue, and with eosin and propidium iodide. For localizing TonEBP, sections were incubated with the anti-TonEBP antibody (Abcam, Cambridge, MA) in 2% bovine serum albumin in PBS at a dilution of 1:100 at 4 °C overnight. After thoroughly washing the sections, the bound primary antibody was incubated with biotinylated universal secondary antibody, at a dilution of 1:20 (Vector Laboratories, Burlingame, CA) for 10 min at room temperature. Sections were incubated with a streptavidin/peroxidase complex for 5 min and washed with PBS, and color was developed using 3′-3-diaminobenzidine (Vecta Stain Universal Quick Kit, Vector Laboratories).Immunofluorescence Microscopy—Cells were plated in flat bottom 96-well plates and maintained in isotonic or hypertonic medium for 24 h. After incubation, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 10 min, blocked with PBS containing 5% fetal bovine serum, and incubated with anti-TonEBP antibody (1:200) (Abcam) at 4 °C overnight. As a negative control, cells were reacted with mouse isotype IgG under similar conditions. After washing, the cells were incubated with Alexa Fluor-488-conjugated anti-mouse secondary antibody (Molecular Probes, St. Louis, MO) at a dilution of 1:50 and 10 μm propidium iodide for 1 h at room temperature. Quantitative image analysis was performed using nine random fields of cells per experimental group. The 36-bit color images were recorded by confocal microscopy using the green and red channel for TonEBP and propidium iodide (nuclear), respectively. Image Pro-plus software (Media Cybernetics, Silver Spring, MD) was used to calculate the threshold for all cells in the field. The mean density of TonEBP was then plotted as a histogram. Caspase-3 activation was evaluated by visualizing cleavage of the PhiPhiLux-G1D2 substrate (OncoImmune Inc., Gaithersburg, MD) following the manufacturer's protocol. Cells were imaged using a laser scanning confocal microscope (Olympus Fluoview, Tokyo, Japan), and images were analyzed as described above.Nuclear Extracts and Western Blotting—Nuclear extracts were prepared according to the method of Dignam et al. (22Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9143) Google Scholar) using the CellLytic NuCLEAR extraction kit (Sigma). After culture in isotonic or hypertonic medium, cells were immediately placed on ice. Cells were treated with a hypotonic buffer (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, and 0.5 mm dithiothreitol) and incubated on ice for 15 min. Igeapal CA-630 was added to a final concentration of 0.6% and the mixture was vortexed vigorously for 10 s. Nuclei were recovered by centrifugation at 3,300 × g for 30 s at 4 °C and extracted by gentle shaking in buffer containing 20 mm HEPES, pH 7.9, 0.42 m NaCl, 25% glycerol, 1.5 mm MgC12, 0.2 mm EDTA, and 0.5 mm dithiothreitol for 30 min at 4 °C. The extract was then centrifuged for 15 min at 25,000 × g, and the supernatant was snapfrozen at –70 °C. All buffers contained a protease inhibitor mixture.Nuclear extracts were resolved on 10% SDS-polyacrylamide gels. Proteins were transferred by electroblotting to nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% nonfat dry milk in TBST (50 mm Tris, pH 7.6, 150 mm NaCl, 0.1% Tween 20) and incubated overnight at 4 °C in 3% nonfat dry milk in TBST with the anti-TonEBP antibody (Abcam) at a dilution of 1:500. Immunolabeling was detected using the ECL (Amersham Biosciences).Reporter Plasmids—PCR amplification of a 2.5 kb fragment of the upstream mouse aggrecan promoter spanning –2204 to +290 was performed using LA Taq polymerase (Takara Mirus Bio) using the following primers: forward, 5′-GAGGAGCTCCAGCACAATATCGATGCTCCAGCTAGTGTGCGAAAT-3′, SacI site underlined; reverse, 5′-GGTAGATCTGCAGGGTCGATGAAGGGAAGGGACACAGAAAGGTG-3′, BglII site underlined. The resulting DNA fragment was subcloned into pCR2.1 (Invitrogen), isolated by restriction digestion with SacI and BglII, and ligated into the luciferase basic expression vector, pGL3 (Promega). As an internal transfection control, vector pRL-TK (Promega) containing Renilla reniformis luciferase genes was used. The amount of transfected plasmid, the pretransfection period after seeding, and the post-transfection period before harvesting were optimized for rat nucleus pulposus cells using pSV β-galactosidase plasmid (Promega). Reporter plasmids were kindly provided by Dr. Takashi Ito, Osaka University, Osaka, Japan (taurine transporter (TauT) (Wt), TauT (Mt); Ref. 11Ito T. Fujio Y. Hirata M. Takatani T. Matsuda T. Muraoka S. Takahashi K. Azuma J. Biochem. J. 2004; 382: 177-182Crossref PubMed Scopus (107) Google Scholar) and Dr. H. M. Kwon, University of Maryland (HSP-70 (Wt), HSP-70 (Mt); Ref. 12Woo S.K. Lee S.D. Na K.Y. Park W.K. Kwon H.M. Mol. Cell. Biol. 2002; 22: 5753-5760Crossref PubMed Scopus (173) Google Scholar); pTauT (Wt) and pHSP-70 (Wt) contains active TonE, whereas the base mutations in pTauT (Mt) and pHSP-70 (Mt) disrupt TonEBP binding to the TonE site. Dr. Ben C. Ko, University of Hong Kong, Hong Kong, China, kindly provided FLAG-DN-TonEBP (dominant-negative expression vector), FLAG-TonEBP, and FLAG-CMV2 expression plasmids (15Lam A.K. Ko B.C. Tam S. Morris R. Yang J.Y. Chung S.K. Chung S.S. J. Biol. Chem. 2004; 279: 48048-48054Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar).Transfections and Dual Luciferase Assay—Nucleus pulposus cells were transferred to 24-well plates at a density of 7.5 × 104 cells/well 1 day before transfection. Lipofectamine 2000 (Invitrogen) was used as a transfection reagent. For each transfection, 330–500 ng of reporter gene plasmid, and 330–500 ng of control plasmid pRL-TK, and where applicable, 330 ng of either DN-TonEBP or FLAG-TonEBP or FLAG-CMV2 (empty vector) expression plasmid, were premixed with the transfection reagent. Twenty-four hours after transfection, the osmolarity of the medium was increased to 400 or 500 mosmol/kg by addition of NaCl. The next day, the cells were harvested and a Dual-Luciferase™ reporter assay system (Promega) was used for sequential measurements of firefly and Renilla luciferase activities. Quantification of luciferase activities and calculation of relative ratios were carried out using a luminometer (TD-20/20, Turner Designs, Sunnyvale, CA). At least three independent transfections were performed, and all analyses were carried out in triplicate.Electrophoretic Mobility Shift Assay—Electromobility shift assays were performed as previously described with minor modifications (21Risbud M.V. Guttapalli A. Stokes D.G. Hawkins D. Danielson K.G. Schaer T.P. Albert T.J. Shapiro I.M. J. Cell. Biochem. 2006; 98: 152-159Crossref PubMed Scopus (208) Google Scholar). Briefly, the binding reaction was carried out in 12.5 mm HEPES, pH 7.9, 50–100 mm NaCl, 5% glycerol, 0.5 mg/ml BSA, 1–2 μg poly(dI-dC), 0.1 mm EDTA, 0.1 mm dithiothreitol, using 50 fmol of biotin end-labeled double-stranded oligonucleotide (top strand sequence 5′-CAAGCTGGTATTTTTCCACCCAGCA-3′ for TauT and 5′-TGAGAGATTCGGGAGATTTCCACTACACTGCCTGA-3′ for aggrecan) and 10–15 μg of nuclear protein. Following incubation for 45 min at room temperature, extracts were loaded onto 5% acrylamide, 0.5× Tris borate-EDTA gels, electrophoresed at 130 V for 1 h, and transferred onto a positively charged nylon membrane (HybondTM-N+, Pierce) in 0.5× Tris borate-EDTA at 100 V for 45 min. Transferred DNAs were cross-linked to the membrane at 120 mJ/cm2 and detected using horseradish peroxidase-conjugated streptavidin according to the manufacturer's instructions (LightShift™ Chemiluminescent EMSA kit, Pierce).Silencing of TonEBP by siRNA—The motif chosen corresponded to the second codon initiating site for the rat TonEBP/NFAT5 gene (23Na K.Y. Woo S.K. Lee S.D. Kwon H.M. J. Am. Soc. Nephrol. 2003; 14: 283-288Crossref PubMed Scopus (57) Google Scholar). As a negative control, the inverse cDNA sequence of the second motif was utilized (23Na K.Y. Woo S.K. Lee S.D. Kwon H.M. J. Am. Soc. Nephrol. 2003; 14: 283-288Crossref PubMed Scopus (57) Google Scholar). The annealed oligonucleotides were ligated into an expression vector (pSuppressorNeo, Imgenex, San Diego, CA). The plasmid DNA was then linearized with ScaI at a site in the 8-bp loop region and sequenced in both directions as suggested by Ducat et al. (24Ducat D.C. Herrera F.J. Triezenberg S.J. BioTechniques. 2003; 34: 1140-1144Crossref PubMed Scopus (9) Google Scholar). Primary rat nucleus pulposus cells were transfected with circular plasmid DNA containing siRNA sequences using the Trans-Fast transfection reagent (Promega), and clones were selected by culturing in medium containing G418 (400 μg/ml) for 7 days. Following expansion of the clones, total RNA was isolated using RNAeasy columns (Qiagen, Valencia, CA). RT-PCR was used to determine which clones were partially silenced.Measurement of Cell Viability—To measure cell viability, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was carried out as described previously (25Risbud M.V. Fertala J. Vresilovic E.J. Albert T.J. Shapiro I.M. Spine. 2005; 30: 882-889Crossref PubMed Scopus (109) Google Scholar). Briefly, after treatment, MTT diluted in PBS was added to the culture medium to a final concentration of 0.5 mg/ml. At the end of the incubation period (2–4 h at 37 °C), the medium was removed, and the precipitated formazan crystals were solubilized in dimethyl sulfoxide. Product formation was measured by reading the absorbance at 560 nm using a microplate reader (Spectra Flour Plus, Tecan, Durham, NC).Statistical Analysis—All measurements were performed in triplicate, data are presented as mean ± S.D. Differences between groups were analyzed by the Student's t test; *, p < 0.05.RESULTSFig. 1A shows that intervertebral disc tissue of the adult rat robustly express TonEBP mRNA. Cartilage, another aggrecanrich skeletal tissue, also expresses TonEBP mRNA; however, the level of expression is lower than the disc. As might be expected, TonEBP mRNA expression in the kidney is the highest of all the tissues analyzed. The expression of the TauT, HSP-70, and betaine-γ-aminobutyric acid (GABA) transporter-1 (BGT-1) is elevated in the disc tissue. However, the level of expression of sodium myo-inositol transporter (SMIT) is lower than that of heart, kidney, or liver. The level of expression of TauT, HSP-70, SMIT, and BGT-1 in the disc is higher than that of cartilage. Immunohistological analysis of neonatal as well as adult rat disc reveals that TonEBP is expressed in neonatal and mature nucleus pulposus cells (Fig. 1, B and D). In both cases, much of the staining is nuclear. However, some staining is seen in the cytosol of the nucleus pulposus cells of the mature discs (Fig. 1D). There is also a prominent expression of TonEBP protein in the annulus fibrosus cells of the mature rat disc (Fig. 1F), which are localized in a narrow zone of Alcian blue-positive matrix (Fig. 1G).Fig. 2A shows that when rat nucleus pulposus cells are cultured in hyperosmotic media (400 mosmol/kg) there is induction of TonEBP mRNA. However, no further increases in mRNA levels are evident when the medium osmolarity is raised to 550 mosmol/kg. A similar pattern of expression is observed for TauT, SMIT, and BGT-1. In contrast, an increase in HSP-70 mRNA expression is seen when the osmolarity is raised to 450 mosmol/kg. Fig. 2B shows that TonEBP protein is robustly expressed under hyperosmotic culture conditions. In accord with the immunohistochemical observations (see Fig. 1), most of the protein is localized to the cell nucleus.FIGURE 2A, influence of osmolarity on expression of TonEBP and its target genes by rat nucleus pulposus cells. Following isolation from the disc, nucleus pulposus cells were maintained in culture in hyperosmolar media (400–550 mosmol/kg). The expression of TonEBP and target genes was evaluated by RT-PCR. An increase in medium osmolarity from isotonic to 400 mosmol/kg results in an elevation in TonEBP, TauT, SMIT, BGT-1, and HSP-70 mRNA levels. Except for HSP-70, further increases in medium osmolarity did not elevate any of the expressed genes. B, Western blot analysis of the expression of TonEBP protein by nucleus pulposus cells at 330–500 mosmol/kg. Most of the TonEBP protein is present in the nuclear fraction of the nucleus pulposus cells. An increase in medium osmolarity from isotonic to 400 and 500 mosmol/kg results in a robust increase in TonEBP protein in the nuclear fraction. Under hypertonic conditions (400 mosmol/kg), there is also a small increase in the cytosolic level of TonEBP, while at 500 mosmol/kg all the protein is localized to the nucleus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3, A and B, shows TauT and HSP-70 reporter activities under hypertonic conditions. When the medium osmolarity is raised, a significant induction in TauT reporter activity is seen. Thus, at 400 mosmol/kg there is 5–6-fold increase in reporter activity. A further increase in medium osmolarity to 500 mosmol/kg causes an additional doubling of the activity. HSP-70 reporter activity is marginally induced at 400 mosmol/kg; however, when the medium osmolarity is raised to 500 mosmol/kg there is a 5–6-fold increase in activity. To further examine the functional interaction of TonEBP protein with the TauT promoter, a gel mobility shift assay was performed. Fig. 3C shows that when the medium is isotonic (330 mosmol/kg) or hypertonic (400–500 mosmol/kg), there is binding of TonEBP to the oligonuclotide probe containing the TonE site of the TauT promoter. The gel shift assay shows that binding of the TonEBP to the TonE probe is increased with raised medium osmolarity. To examine the specificity of this interaction, we used a probe containing mutation in the TonE site; in addition a competition analysis was performed. As expected, there was no/or extremely low binding to the mutant probe. Likewise, excess mutant probe could not compete the binding of TonEBP to wild type TonE.FIGURE 3A and B, evaluation of TonEBP responsive promoter activity in nucleus pulposus cells cultured in hypertonic medium. TauT (A) or HSP-70 (B) reporter plasmids containing a Wt or a Mt TonE, driving firefly luciferase, were transfected into rat nucleus pulposus cells along with pRL-TK vector. Cells were cultured in isotonic (330 mosmol/kg) or hypertonic (400 or 500 mosmol/kg) media for 24 h, and luciferase reporter activity was measured. When the medium osmolarity is raised to 400 mosmol/kg there is a 5-fold increase in TauT reporter activity. At 500 mosmol/kg there is a further doubling of activity. HSP-70 reporter activity does not change until the osmolarity of the medium is increased to 500 mosmol/kg, it then increases 5–6-fold. Cells transfected with mutant TauT (A) or HSP-70 plasmids (Mt) (B) display a very low luciferase activity. Values shown are mean ± S.D. of three independent experiments; *, p < 0.05. C, electromobility shift assay to examine the functional binding of TonEBP protein to TonE. Nuclear protein was isolated from rat nucleus pulposus cells cultured under isotonic or hypertonic conditions. 10 μg of nuclear protein was incubated with 50 fmol of biotin end-labeled double-stranded oligonucleotide probes containing the TonE motif in the TauT promoter. Binding was resolved on a polyacrylamide gel, transferred to a Nylon membrane, and detected using chemiluminescence. Under hypertonic conditions, there is more pronounced binding of TonEBP protein to the DNA probe. When a probe containing the mutant TonE site was used in the binding reaction, there is no or very little binding. Furthermore, the mutant competitor probe did not compete with the binding of TonEBP to labeled wild type TonE probe, confirming the specificity of the interaction.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm that TonEBP is required for the induction of TauT and HSP-70 promoter activity, nucleus pulposus cells were transiently transfected with plasmids encoding DN-TonEBP and full-length TonEBP. Fig. 4 shows that forced expression of DN-TonEBP significantly suppresses TauT (A) and HSP-70 (C) promoter activity. When the medium is hypertonic, the level of expression in cells transfected with DN-TonEBP is lower than expression under isotonic condition. A significant inhibitory effect of DN-TonEBP expression on TauT reporter activity is seen at a dose of 50 ng, which is further enhanced when the concentration of the DN plasmid is increased to 100 ng. Despite further increases in the DN plasmid dose, the reporter activity is not decreased. On the other hand, overexpression of TonEBP under isotonic conditions, using pFLAG-TonEBP vector, results in a significant increase in both TauT (Fig. 4B) and HSP-70 (Fig. 4D) promoter activities. To ensure that the FLAG fusion protein is expressed in nucleus pulposus cells, Western blot and immunofluorescnce analysis was performed (Fig. 4E). As expected, using both the techniques, anti-FLAG antibody detects FLAG-DN-TonEBP in transfected cells only. Moreover, DN-TonEBP does not alter the level of native TonEBP expression in transfected cells.FIGURE 4A–D, TonEBP regulation of TauT and HSP-70 reporter activity in nucleus pulposus cells. Rat nucleus pulposus cells were co-transfected with TauT (Wt) or HSP-70 (Wt) reporter plasmids along with either pFLAG-DN-TonEBP or pFLAG-TonEBP or pFLAG-CMV2 (empty vector). The cells were cultured in isotonicor hypertonic (500 mosmol/kg) medium for 24 h, and reporter activity was measured. Under hypertonic conditions, cells receiving
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