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【关键词】 telangiectasia-mutated
Laboratory of Kidney and Electrolyte Metabolism, National Heart Lung and Blood Institute, and Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland
ABSTRACT
High NaCl activates the transcription factor tonicity-responsive enhancer/osmotic response element binding protein (TonEBP/OREBP) by increasing its abundance and transactivation, the latter signaled by a variety of protein kinases. In addition, high NaCl causes TonEBP/OREBP to translocate into the nucleus, but little is known about the signals directing this translocation. The result is increased transcription of protective genes, including those involved in accumulation of organic osmolytes. High NaCl also damages DNA, and DNA damage activates ataxia telangiectasia-mutated (ATM) kinase through autophosphorylation on serine 1981. We previously found that ATM is involved in the high NaCl-induced increase in TonEBP/OREBP transactivation. The purpose of the present studies was to test whether ATM is also involved in high NaCl-induced TonEBP/OREBP nuclear translocation. We quantified TonEBP/OREBP in nuclear and cytoplasmic extracts from cultured cells by Western blot analysis. In COS-7 cells, wortmannin, an inhibitor of ATM, reduces high NaCl-induced nuclear translocation of TonEBP/OREBP. We used AT cells (in which ATM is inactive) to test the specificity of this effect. Nuclear translocation of native TonEBP/OREBP and of its recombinant NH2-terminal rel homology domain, which contains the nuclear localization signal, is reduced in AT cells and is restored when the cells are reconstituted with functional ATM. In conclusion, activation of ATM contributes to high NaCl-induced nuclear translocation of TonEBP/OREBP.
ataxia telangiectasia-mutated kinase; tonicity-responsive enhancer/osmotic response element binding protein; wortmannin; AT cells
NaClCONCENTRATION IS NORMALLY high in renal inner medullary interstitial fluid, where it energizes the urinary concentrating mechanism, and it varies with concentration of the urine (4). Such high levels of NaCl can be deadly for cells (4, 20, 22), yet the inner medullary cells evidently survive and function in vivo, dependent on a number of protective mechanisms, including accumulation of compatible organic osmolytes (26), which protect cells by normalizing their internal inorganic ion concentration, thus compensating for the hypertonicity caused by high NaCl (26). The renal-compatible organic osmolytes include glycine betaine (betaine), myo-inositol (inositol), and sorbitol (1). Accumulation of these renal organic osmolytes is regulated by a transcription factor, tonicity-responsive enhancer/osmotic response element binding protein (TonEBP/OREBP) (16, 21). TonEBP/OREBP initiates the accumulation of compatible organic osmolytes by increasing transcription of the betaine/-aminobutyric acid transporter (BGT1), the sodium-myo-inositol cotransporter (SMIT), and aldose reductase (AR) (5, 16, 21). BGT1 and SMIT transport betaine and inositol, respectively, into the cells, and AR catalyzes conversion of glucose to sorbitol.
Regulation of TonEBP/OREBP transcriptional activity is complex. Within 30 min of hypertonicity, TonEBP/OREBP becomes phosphorylated and translocates into the nucleus (9, 16, 21). Some hours later, TonEBP/OREBP mRNA and protein abundance increase (16, 21). Also, hypertonicity increases transactivation of TonEBP/OREBP, associated with phosphorylation of its transactivation domain (TAD) (13). Several protein kinases are known to contribute to the hypertonicity-induced increase in TonEBP/OREBP transcriptional activity and transactivation, including p38 (15, 23), Fyn (15), ataxia telangiectasia-mutated kinase (ATM) (14), and protein kinase A (PKAc) (12), none of them alone being sufficient for full activation (12).
Much less is known about how high NaCl signals translocation of TonEBP/OREBP. MG-132 inhibits high NaCl-induced nuclear translocation of TonEBP/OREBP in Madin-Darby canine kidney (MDCK) (25) cells, implicating proteasome activity (25). Cyclosporin A has a similar effect in MDCK cells (24). The proteins affected by MG-132 and cyclosporin A have not yet been identified. Considering the multiplicity of kinases that signal high NaCl-induced increase in TonEBP/OREBP transactivation, it seemed probable to us that there might also be additional pathways that signal its nuclear translocation, possibly including kinases. Therefore, in the present studies we tested the effects of wortmannin on high NaCl-induced translocation of TonEBP/OREBP in COS-7 cells as a start toward identifying them. We found that wortmannin inhibits high NaCl-induced nuclear localization of TonEBP/OREBP. Because wortmannin inhibits ATM, we tested for a specific role of ATM, using AT cells, which lack functional ATM. The results confirm that ATM is involved in signaling nuclear translocation of TonEBP/OREBP but that other targets of wortmannin also play a role.
MATERIALS AND METHODS
Cell culture. COS-7 cells (American Type Culture Collection) were maintained in Dulbecco’s modified Eagle’s medium and AT cells, in which one allele of ATM is truncated at amino acid 1774 of 3056, the other at amino acid 2769 (GM09607 B, Coriell Cell Repositories, Camden, NJ), were maintained in Eagle’s minimum essential medium according to instructions of the suppliers. Osmolality of the basal medium was 300 mosmol/kgH2O. NaCl was added to prepare 500-mosmol/kgH2O medium, and a 200-mosmol/kgH2O medium (low NaCl) was specially prepared by BioFluids (Rockville, MD). Wortmannin (Calbiochem) or MG-132 (Sigma) was solubilized with Me2SO (DMSO), and the same final concentration of DMSO (<0.25%) was added to controls. Cells were preincubated with inhibitors or DMSO for 1 h and subjected to osmotic changes for 30 min, 4 h, or 16 h.
Plasmids and transfection. Human TonEBP/OREBP cDNA clone KIAA0827 was a gift of T. Nagase (Kazusa DNA Research Institute, Chiba, Japan), and pcDNA3 expression vector containing wild-type Flag-ATM (6) was a gift of M. B. Kastan (St. Jude Children’s Research Hospital). A sequence coding for TonEBP/OREBP amino acids 1547 from KIAA0827 was cloned into pcDNA6 V5-His (Invitrogen) expression vector to generate 1547-V5. The construct was generated using standard cloning procedures and verified by restriction enzyme digestion and DNA sequencing. DNA was transfected into cells using Effectene according to the supplier’s instructions (Qiagen).
Western blot analysis. Cells were lysed with Nuclear and Cytoplasmic Extraction Reagents (Pierce) according to the supplier’s instructions, with added protease inhibitor cocktail (Roche Diagnostics) and phosphatase inhibitor cocktails 1 (P2850, Sigma) and 2 (P5726, Sigma). This procedure produces separate nuclear and cytoplasmic fractions. The adequacy of the separation was confirmed by observations that 99.9% of p38 is present in the cytoplasmic fraction and 90.5% of poly(ADP-ribose) polymerase-1 is in the nuclear fraction under all the osmotic conditions that we used. Cytoplasmic protein (20 μg/lane) and nuclear protein (10 μg/lane) were separated on 412% Novex Tris-glycine gels and transferred to nitrocellulose membranes (Invitrogen). Western blot analysis was performed according to instructions for the Odyssey Infrared Imaging System. In brief, nonspecific binding was blocked by incubating membranes overnight at 4°C with blocking buffer (Odyssey) diluted 1:1 in phosphate-buffered saline. Membranes were then incubated with anti-NFAT5 (TonEBP/OREBP) rabbit polyclonal antibody (Affinity BioReagents) or anti-V5 mouse monoclonal antibody (Invitrogen) for 2 h at room temperature. After being washed with 0.1% Tween 20 in phosphate-buffered saline, blots were incubated with Alexa Fluor 680 goat anti-rabbit IgG or Alexa Fluor 780 goat anti-mouse IgG (Molecular Probes) for 1 h in the dark. Blots were visualized and quantitated using an LI-COR Odyssey Infrared Imager. Alternatively, membranes were washed and exposed to secondary antibody [goat anti-rabbit IgG conjugated to horseradish peroxidase (no. 31463, diluted to 1:5,000, Pierce)] for 1 h at room temperature. After being washed, bands were visualized using enhanced chemiluminescence (Amersham Biosciences) with band densities determined by laser densitometry (Personal Densitometer SI).
Calculation of nuclear/cytoplasmic ratios. From the relative quantities of TonEBP/OREBP and the relative volumes of the cytoplasmic or nuclear extract, we calculated the relative total amounts of TonEBP/OREBP in the cytoplasmic and nuclear fractions and the nuclear/cytoplasmic ratio.
Statistical analysis. Data were compared by multiple comparison tests, ANOVA (followed by a Student-Newman-Keuls posttest), or false discovery rate (8). Results are expressed as means ± SE (n = number of independent experiments). Differences were considered significant for P 0.05.
RESULTS
MG-132 does not affect location of TonEBP/OREBP in COS-7 cells but does in Hep G2 cells. High NaCl causes TonEBP/OREBP to translocate from cytoplasm to nucleus (16, 21). Previously, the proteasome inhibitor MG-132 (1 μM) was found to inhibit high NaCl-induced nuclear translocation of TonEBP/OREBP in MDCK cells (25), implicating proteasome activity. We have confirmed this effect in Hep G2 cells (Fig. 1C). However, using COS-7 cells, we do not find any effect of 1 μM MG-132 on nuclear/cytoplasmic ratio of native TonEBP/OREBP at 200, 300, or 500 mosmol/kgH2O (Fig. 1A). We conclude that the effect of MG-132 on high NaCl-induced nuclear translocation of TonEBP/OREBP is cell type dependent.
Wortmannin decreases high NaCl-induced TonEBP/OREBP translocation in COS-7 cells. ATM contributes to a high NaCl-induced increase in TonEBP/OREBP transcriptional activity (14). At least part of this effect is due to increased transactivation (14). In the present experiments we asked whether ATM also contributes to high NaCl-induced nuclear translocation of TonEBP/OREBP. In support of this possibility, 20 μM wortmannin, which inhibits ATM, significantly decreases nuclear/cytoplasmic ratio of native TonEBP/OREBP in COS-7 cells at 500 mosmol/kgH2O (Fig. 1B). However, because wortmannin also inhibits other kinases, including DNA-PK, phosphatidylinositol 3-kinase (PI3-K), and myosin light chain kinase (MLCK) (10), we sought to examine the possible role of ATM more specifically.
Expression of functional ATM contributes to high NaCl-induced nuclear translocation of TonEBP/OREBP. AT cells lack functional ATM, but ATM activity can be reconstituted in these cells by transfection with wild-type ATM (2). We used this system to test for a specific effect of ATM on NaCl-dependent location of TonEBP/OREBP. We transfected AT cells with either empty vector (EV) or functional ATM. In AT cells reconstituted with active ATM, the nuclear/cytoplasmic ratio of native TonEBP/OREBP is significantly greater at 500 mosmol/kgH2O than in AT cells transfected with EV, which lack ATM activity (Fig. 2, AC). The difference exists from 30 min to at least 16 h after osmolality is increased by elevating NaCl. There is no difference at 300 or 200 mosmol/kgH2O. We conclude that ATM specifically contributes to sustained high NaCl-induced nuclear translocation of TonEBP/OREBP.
ATM signals high NaCl-induced nuclear translocation of recombinant TonEBP/OREBP that is truncated to contain only amino acids 1547. High NaCl-induced translocation of TonEBP/OREBP involves its NH2-terminal third, which contains the nuclear localization signal (16, 21). Recombinant TonEBP/OREBP, containing amino acids 77547 of TonEBP/OREBP, was previously shown to translocate into the nucleus when NaCl concentration is increased (16). We tested whether ATM might contribute to high NaCl-induced nuclear translocation of recombinant TonEBP/OREBP 1547-V5. Increasing osmolality from 300 to 500 mosmol/kgH2O by adding NaCl increases the nuclear/cytoplasmic ratio of TonEBP/OREBP 1547-V5 in AT cells, and the increase is greater when the cells are reconstituted with active ATM (Fig. 2D). We conclude that ATM signals increased the nuclear/cytoplasmic ratio of TonEBP/OREBP through sites within amino acids 1547, which does not, however, exclude the possibility of modulation via effects of ATM on TonEBP/OREBP-5481531.
In the absence of functional ATM, wortmannin increases high NaCl-induced translocation of TonEBP/OREBP. For the sake of completeness, we also tested the effect of wortmannin on the nuclear-to-cytoplasmic ratio of TonEBP/OREBP in AT cells that are not reconstituted with functional ATM. To our surprise, wortmannin increases the ratio under those conditions (Fig. 3A), which is the opposite of the effect in AT cells reconstituted with functional ATM (Fig. 3B). We speculate that some wortmannin-inhibitable signaling molecule, other than ATM, acts to inhibit high NaCl-induced nuclear translocation of TonEBP/OREBP. Possible candidates are DNA-PK, PI3-K, and MLCK, all of which are inhibited by wortmannin (10).
DISCUSSION
High NaCl damages DNA in cell culture and in vivo. Acute elevation of NaCl causes DNA double-strand breaks in cell culture (11, 17), and the DNA breaks persist after the cells adapt to high NaCl (11). When NaCl is lowered, however, the breaks are rapidly repaired. Renal inner medullary cells in vivo are normally exposed to a variable, but always high, level of NaCl (4). While NaCl is at its normal high level in the mouse inner medulla in vivo, the cells contain numerous DNA breaks (11), but when NaCl is lowered by the diuretic furosemide, the breaks are rapidly repaired (11). Thus both in cell culture and in vivo, high NaCl causes persistent DNA damage that is not repaired unless the level of NaCl is reduced.
High NaCl activates ATM. ATM protein kinase mediates responses to ionizing radiation-induced DNA damage in mammalian cells (2). Mutations of ATM that inactivate it cause the human disease ataxia-telangiectasia, in which cells are hypersensitive to DNA damaging agents. In unirradiated cells, ATM is held inactive as a dimer or higher-order multimer, with the kinase domain bound to a region surrounding serine 1981. Ionizing radiation induces rapid intermolecular autophosphorylation of serine 1981, resulting in dimer dissociation and initiation of ATM kinase activity (2). High NaCl, which damages DNA, also activates ATM via autophosphorylation on serine 1981 (14). Activated ATM phosphorylates a number of proteins, including p53, murine double minute-2, Csk homologous kinase 2, breast cancer 1, and NBS1, contributing to DNA repair, cell cycle delay, and apoptosis (3, 6, 7, 18, 19).
How does ATM contribute to high NaCl-induced nuclear localization of TonEBP/OREBP ATM contributes to both high NaCl-induced nuclear translocation of TonEBP/OREBP (Fig. 2, AC) and high NaCl-induced increase in TonEBP/OREBP transactivation (14). The effect on translocation is mediated by sites within amino acids 1547 of TonEBP/OREBP (Fig. 2D), whereas the effect on transactivation is mediated by sites within amino acids 5481531 (14). We were able to identify consensus ATM phosphorylation sites at amino acids serine 1197, serine 1247, and serine 1367 of TonEBP/OREBP (14), and transcriptional activity of TonEBP/OREBP is increased less by high NaCl when these sites are mutated to prevent their phosphorylation (14). However, we are unable to identify clear consensus ATM phosphorylation sites within amino acids 1547 of TonEBP/OREBP. We are left with the possibilities that ATM is affecting high NaCl-induced nuclear translocation of TonEBP/OREBP indirectly, through some intermediary signaling protein, or, if directly, through an ATM phosphorylation site that we do not recognize.
Unexpected wortmannin-induced increase in TonEBP/OREBP translocation in cells lacking ATM activity. ATM contributes to high NaCl-induced nuclear translocation of TonEBP/OREBP (Fig. 2, AC). Wortmannin inhibits ATM, which can explain its inhibition of high NaCl-induced nuclear localization of TonEBP/OREBP in cells expressing active ATM (Figs. 1B and 3B). However, it also increases high NaCl-induced nuclear localization of TonEBP/OREBP in AT cells (Fig. 3A), which presumably involves some wortmannin-inhibitable signaling molecule "other" than ATM. Other known targets of wortmannin include DNA-dependent protein kinase catalytic subunit (DNA-PKcs), PI3-K, and MLCK (10). The question is why inhibition by wortmannin of a signal that enhances NaCl-induced nuclear translocation of TonEBP/OREBP (via ATM) does not cancel the effect of a signal that inhibits it (the "other"). We speculate that interaction of full-length ATM with the other normally protects the other from wortmannin, so that this effect of wortmannin is only evident in AT cells.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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