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作者：Qi Cai, Natalia I. Dmitrieva, Joan D. Ferraris, Luis F. Michea, Jesus M. Salvador, M. Christine Hollander, Albert J. Fornace, Jr., Robert A. Fenton, and Maurice B. Burg作者单位：1 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, and 2 Gene Response Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland / J4 \* q2 a0 ^' U x
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4 \5 F. ^4 Y% l 【摘要】
6 S: \9 v5 d5 l- W The response of renal inner medullary (IM) collecting duct cells (mIMCD3) to high NaCl involves increased expression of Gadd45 and p53, both of which have important effects on growth and survival of the cells. However, mIMCD3 cells, being immortalized by SV40, proliferate rapidly, which is known to sensitize cells to high NaCl, whereas IM cells in situ proliferate very slowly and survive much higher levels of NaCl. In the present studies, we have examined the importance of Gadd45 and p53 for survival of normal IM cells in their usual high-NaCl environment by using more slowly proliferating second-passage mouse inner medullary epithelial (p2mIME) cells and comparing cells from wild-type and gene knockout mice. Acutely elevating NaCl (and/or urea) reduces Gadd45a, but increases Gadd45b and Gadd45g mRNA, depending on the mix of NaCl and urea and the rate of increase of osmolality. Nevertheless, p2mIME cells from Gadd45b -/-, Gadd45g -/-, and Gadd45bg -/- mice survive elevation of NaCl (or urea) essentially the same as do wild-type cells. p53 -/- Cells do not tolerate as high a concentration of NaCl (or urea) as p53 / cells, but urinary concentrating ability of p53 -/- mice is normal, as is the histology of inner medullas from p53 -/- and Gadd45abg -/- mice. Thus although Gadd45 and p53 may play roles in osmotically stressed mIMCD3 cells, we do not find that their expression makes an important difference, either for Gadd45 in slower proliferating p2mIME cells or for Gadd45 or p53 in normal inner medullary epithelial cells in situ. 1 r* C/ x, k# G; a, P5 N8 U2 R
【关键词】 high NaCl high urea cell survival
) }1 \7 w* K- N2 L- ?+ Q THE CELLULAR STRESS CAUSED by high NaCl includes an increased number of DNA strand breaks ( 11, 19 ) and impairment of DNA repair ( 10, 11 ). Acute elevation of NaCl in the medium bathing mouse inner medullary collecting duct (mIMCD3) cells increases abundance of the growth arrest- and DNA damage-inducible protein Gadd45 ( 7, 20 ) and increases the protein abundance, phosphorylation, and transcriptional activity of the tumor suppressor p53 ( 8 ). Both are stress-response proteins. Cells eventually adapt to high NaCl, but even after they adapt, the number of DNA breaks remains high and repair of DNA breaks remains inhibited ( 11 ). This is of obvious relevance to the renal medulla, where the interstitial level of NaCl normally is high, varying with urine concentration from 600 to 1,000 mosmol/kgH 2 O or more ( 3 ). The increased expression of Gadd45 and p53 in mIMCD3 cells in response to high NaCl has led us to consider their possible functions in the renal inner medulla, where NaCl normally is high.' j) U; N# e6 H/ G
1 E3 V) e2 v. T& AGadd45 was originally identified as a transcript that increases following stresses such as serum reduction, medium depletion, contact inhibition, hydroxyurea, UV, and methanesulfonate ( 14 ). Later, two additional Gadd45 genes were identified in a yeast 2 hybrid screen of proteins associated with MEKK4 ( 26, 33 ). The three Gadd45 genes are now called Gadd45a (the original Gadd45 ), Gadd45b (originally Myd118 ) ( 1 ), and Gadd45g (originally CR6 ) ( 39 ). The three Gadd45 proteins have similar amino acid sequence (55-57% identity) ( 21, 33 ). They are multifunctional molecules that have been implicated in cell differentiation, apoptosis, negative growth control, DNA repair, genome stability, immunity, and cell survival ( 6, 16 - 18, 29 - 31, 34, 35, 37, 38 ).
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$ C* l, A1 w- |: I; ]/ EAdding NaCl to raise osmolality from 300 to 600-700 mosmol/kgH 2 O increases protein expression of Gadd45 in mIMCD3 cells ( 20 ) and mRNA expression of Gadd45a, Gadd45b, and Gadd45g in both mIMCD3 and passage 2 mouse inner medullary epithelial (p2mIME) cells ( 7 ). Possible functional consequences were examined by overexpressing the Gadd45s in mIMCD3 cells ( 22 ). Overexpression of recombinant Gadd45a, Gadd45b, or Gadd45g inhibits mitosis and promotes G2/M arrest during acute increase of NaCl to 540 mosmol/kgH 2 O, but not in controls at 300 mosmol/kgH 2 O. Under these conditions Gadd45g overexpression strongly potentiates apoptosis. When osmolality bathing mIMCD3 cells is increased acutely to an even higher level (620 mosmol/kgH 2 O) by adding NaCl, which produces apoptosis, Gadd45a/b overexpression transiently increases caspase 3/7 and annexin V binding but inhibits later stages of apoptosis. The conclusion was that Gadd45 isoforms function in common but also in distinct pathways during high NaCl and that their increased abundance contributes to low mitotic index and protection of genomic integrity in cells of the mammalian renal inner medulla ( 22 ).4 C% R5 ^2 }9 c7 h6 P0 k* b6 v
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Best known as a tumor suppressor, p53 ( 24 ) has numerous additional functions. DNA-damaging agents promote the accumulation and activation of p53, which results in cell cycle delay and DNA repair, enhancing cell survival. However, when the DNA is not successfully repaired, p53 activity also can cause apoptosis ( 24 ). In mIMCD3 cells, high NaCl rapidly increases p53 protein abundance, phosphorylation on S15, and transcriptional activity ( 8 ). That restricts DNA replication and reduces hypertonicity-induced apoptosis ( 9 ). These findings suggested to us that, although no renal phenotype was previously noted in p53 -/- mice ( 12 ), more specific examination might identify effects of p53 on the function of renal inner medullary cells in their normal high-NaCl environment.( d+ v9 e! E: Y P- a4 c
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In the present studies, we have examined the importance of Gadd45 and p53 for the osmotic tolerance of renal inner medullary cells at the levels of NaCl that are normal for them, using as models p2mIME cells from wild-type and gene knockout mice and the renal medullas of the mice themselves. We find that elevating NaCl increases expression of Gadd45b and Gadd45g in p2mIME cells, but not Gadd45a. Although lack of p53 reduces the osmotic tolerance of p2mIME cells, lack of Gadd45b and/or Gadd45g does not. Furthermore, we find no morphological changes in renal inner medullas of p53 -/- or Gadd45abg -/- mice, nor deficits in urine concentrating ability. Thus, despite the fact that high NaCl may increase their abundance and activate them, we do not find an important role of Gadd45b, Gadd45g, or p53 in normal renal inner medullary cells under conditions like those in vivo.1 R* X d5 k K" l7 Y3 \
/ Y I& p3 e4 y. j& m; F) pMATERIALS AND METHODS: y# E. D2 F/ s2 X! @2 C
, ^5 ]4 d; \& A( u5 A ]* S7 K7 rMice and tissue culture. Mouse inner medullary epithelial (mIME) cells ( 40 ) were cultured from the renal inner medullas of p53 -/-, Gadd45a -/-, Gadd45b -/-, Gadd45g -/-, and Gadd45bg -/- mice. p53 -/- Mice were purchased from Taconic Farms (Germantown, NY). The derivation of the Gadd45a -/- and Gadd45b -/- mice was previously described ( 2, 17 ). Gadd45g -/- mice were generated by Jesús Salvador and Christine Hollander (Salvador JM and Hollander MC, unpublished observations). The Gadd45bg -/- mice were generated by mating Gadd45b -/- mice with Gadd45g -/- mice, then intercrossing the F1 double heterozygotes to obtain double null mice. The wild-type mice used as controls were obtained by intercrossing F1 heterozygotes. Gadd45ab -/- mice were mated with Gadd45g -/- mice. Resulting mice heterozygous for all three alleles were then crossed. Gadd45abg -/- mice were generated from crosses of Gadd45ab -/- and Gadd45g /- mice. The genotype of each mouse was established by PCR of genomic DNA and confirmed by absence of the corresponding mRNA in RT-PCR analyses. All mouse studies were done under an approved National Institutes of Health animal study protocol (2-KE-32) and mice were housed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility.
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# R/ ?$ J; P8 ^4 C7 G* xTo prepare mIME cultures ( 40 ), renal inner medullas and papillas were dissected, minced (1-2 mm), digested in 25 ml of 640 mosmol/kgH 2 O (80 mM urea and 130 mM NaCl added) DMEM/F12, containing 25 mg collagenase B (Roche, Indianapolis, IN) and 9 mg hyaluronidase (Worthington Biochemical, Lakewood, NJ). Cells were washed with and resuspended in 640 mosmol/kgH 2 O growth medium (10% fetal bovine serum, 45% DMEM, 45% Coon's improved medium F12, 10 mM HEPES, pH 7.5, 5 mg/l transferrin, 10 nM selenium, 50 nM hydrocortisone, 5 pM 3,3,5-triiodo- L -thyronine, 2 mM L -glutamine, 100 U/ml penicillin G, 100 U/ml streptomycin sulfate with 80 mM urea and 130 mM NaCl added) and maintained at 37°C in 5% CO 2. For experiments, cells were plated in the same medium on 8-well slides (Nalge Nunc International, Naperville, IL) or 26-mm cell culture inserts (Becton-Dickinson Labware, Bedford, MA) for 3 days, then in the same medium without serum for 48 h before experimental change in osmolality. Osmolality was increased by increasing NaCl or urea concentration either in a single step or linearly over 19 h ( 5 ).6 e( T( w: i* a7 U$ d
, D1 K! o4 N/ n3 gLaser-scanning cytometry. The number of viable cells was determined by laser-scanning cytometry (LSC; CompuCyte, Cambridge, MA), as previously described ( 4, 40 ). Briefly, cells that had been fixed and stained with propidium iodide (PI) were counted by LSC and the maximal brightness (PI Max Pixel) within each nucleus was determined. Cells acutely exposed to hypertonicity may undergo apoptosis ( 25, 32 ) characterized by nuclear condensation that results in increased brightness of PI staining ( 4 ). Cells whose maximal brightness was not elevated above the level observed at 640 mosmol/kgH 2 O were considered to be viable.
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4 b% X& N: T4 E! A5 c$ ?) xPreparation of total RNA and cDNA. Total RNA was isolated from p2mIME cells or mouse kidney with RNeasy spin columns (Qiagen, Valencia, CA), as recommended by the manufacturer. To eliminate DNA contamination, the RNA was treated for 15 min with RNase-free DNase, using RNeasy columns (Qiagen). Two micrograms of total RNA were reverse transcribed with random hexamers, using a Taq Man Kit (Applied Biosystems, Foster City, CA).; w4 m7 l/ M% g) {, _
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Real-time PCR. cDNA was quantitated, as previously described ( 9 ), with the ABI Prism 7900 Sequence Detection System (Applied Biosystems), following the manufacturer's recommendations. This system utilizes the 5' nuclease activity of Taq DNA polymerase to generate the signal. Briefly, gene-specific primers and oligonucleotide probes containing a 5' fluorescent dye, 6-FAM, and a 3' quencher, TAMRA ( Taq Man probes), were designed using Primer Express software (Applied Biosystems). The primers all span introns. Primer pairs and probe sequences are listed for forward primers, reverse primers, and Taq Man probes ("F," "R," and "P," respectively, in Table 1 ). Multiplex PCR was performed using Taq Man PCR Master Mix (Applied Biosystems) to which was added both the specific primers and probes and 18S rRNA primers and an 18S probe, labeled with the fluorescent dye VIC. The coamplified 18S cDNA serves as an internal control for reverse transcription and cDNA loading. Triplicates of each sample were analyzed in each PCR run. The PCR sequence was 2 min at 50°C for optimal AmpErase UNG enzyme activity; 10 min at 95°C to activate Ampli Taq Gold DNA polymerase; then 40 cycles of 95°C for 15 s and 60°C for 1 min. The PCR products were verified by sequencing (Amplicon Express, Pullman, WA).4 @/ o1 L: |7 R A; S& V4 |
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Table 1. Sequence of primer and probe sets for real-time PCR
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' `( ?: i- {+ A1 K, S( l1 fAnalysis of real-time PCR data. The ABI Prism 7900 system records the number of PCR cycles (Ct) required to produce an amount of product equal to a constant threshold value reached during the exponential phase of the PCR reaction. As previously described ( 13 ), relative mRNA abundance was calculated from the real-time PCR data using the following principles. 1 ) By definition, the number of specific cDNA molecules at the threshold ( N Ct ) is constant for a given cDNA, independent of the number of cycles that it takes to reach it. 2 ) For a specific cDNA, the ratio N (exp) I / N (cont) I is independent of I, assuming only that the efficiency ( E ) of PCR for a specific template is constant, where I is the cycle number, and N ( X ) I is the number of specific cDNA molecules in a sample ( X = control or experimental) at cycle I. 3 ) The ratio of the number of specific cDNA molecules at a cycle, Ct, to the number at another cycle, I, is NI / N Ct = 1/ E (Ct- I ). To compare control and experimental results, we normalized both to the number of specific molecules at an arbitrary cycle I, chosen for convenience to be the largest whole number that is less than any of the experimental values of Ct. Then, we calculated N ( X ) I / N Ct for each sample. Experimental results are presented as a percentage of the corresponding control value.
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Bromodeoxyuridine labeling, immunostaining, and analysis by LSC. Confluent p2mIME cells on eight-well slides were incubated for 48 h in 640 mosmol/kgH 2 O serum-free growth medium, labeled with 10 µM bromodeoxyuridine (BrdU) for 18 h, then processed with BrdU Labeling and Detection Kit I (Roche), according to the manufacturer's instructions. The number of nuclei containing BrdU was counted by LSC as previously described ( 9 ).! j, H, [" w' L/ q9 v5 R* Z
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Western blot immunodetection. Proteins were separated by SDS-PAGE. Equal amounts of protein (5 µg) were loaded in each lane of 12% Tris/glycine polyacrylamide gels. Samples were electrophoresed, blotted onto Immobilon P membrane (Millipore, Bedford, MA) with a Trans-Blot SD semidry transfer cell (Bio-Rad, Hercules, CA). Blots were blocked with 5% fat-free milk for 1 h at room temperature and incubated with mouse anti-p53 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-phospho-p53 (Ser15, Cell Signaling Technology, Beverly, MA) overnight at 4°C. The immune complexes were detected with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:2,000 dilution, Cell Signaling Technology), using enhanced chemiluminescence (ECL plus, Amersham Biosciences, Piscataway, NJ) with exposure to X-ray film (Kodak, Rochester, NY).
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Kidney morphology of p53 -/- and Gadd45abg -/- mice and immunohistological detection of proliferating-cell nuclear antigen. Mouse kidneys were fixed in 4% paraformaldehyde and paraffin embedded, and then sections were cut and mounted on silanized slides (American Histolabs, Gaithersburg, MD). Sections were deparaffinized, hydrated to deionized water, and stained with periodic acid-Schiff reagents (catalogue no. 395-1, Sigma-Aldrich). For proliferating-cell nuclear antigen (PCNA) detection, endogenous peroxidase was quenched by placing the slides in 3% hydrogen peroxide in methanol for 10 min. Heat-induced epitope retrieval was performed by boiling the slides for 6 min in citrate buffer solution, pH 6.0 (catalogue no. 00-5000, Zymed Laboratories). Slides were washed with PBS and stained with anti-PCNA antibody using a PCNA detection Kit (Zymed Laboratories, San Francisco, CA), according to the manufacturer's instructions. Briefly, sections were blocked with serum-blocking solution, then incubated successively with biotinylated primary antibody and streptavidin-peroxidase conjugate. Peroxidase was visualized by addition of 3,3'-diaminobenzidine tetrahydrochloride substrate, which reacts with peroxidase to produce a brown-colored deposit.5 h0 J; b$ j1 F! s+ U7 L
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Studies of urine concentrating ability of p53 -/- mice. Six wild-type and six p53 -/- mice were maintained in mouse metabolic cages (Hatteras Instruments, Cary, NC) for the duration of the study, under controlled temperature and light conditions (12:12-h light-dark cycle). Initially, all mice received a fixed daily ration of 6.5 g of gelled diet·20 g body wt -1 ·day -1. The gelled diet (per 6.5 g total) was made up of 2.5 ml of deionized water, 4 g of special low-salt (NaCl) synthetic food [0.001% Na (wt/wt); Research Diets (New Brunswick, NJ)], 15 mg NaCl, and 65 mg of agar. Drinking water was provided ad libitum during the initial period of the study. After 3 days of adaptation to the cages and diet, urine was collected under mineral oil in collection vials for two successive 24-h periods. After the initial collection period, each mouse received a fixed daily ration of 5.7 g of gelled diet·20 g body wt -1 ·day -1, which contained 1.7 ml of deionized water. Mice did not have access to supplemental drinking water during this period. Urine was collected under mineral oil in collection vials for two successive 24-h periods. Osmolality of the urine was measured with a Vapor Pressure Osmometer (Wescor, Logan, UT).3 R5 |+ z1 k1 g8 W/ n5 H
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Statistical methods. All values were normalized to those in 640 mosmol/kgH 2 O medium (100%) and expressed as means ± SE ( n = no. of experiments). Statistical significance was determined on transformed data, using a t -test (paired test) for single comparisons or ANOVA (with Student-Newman-Keuls multiple comparison test) for multiple comparisons (GraphPad Instat Software). P 2 i8 T/ [5 s9 O1 M2 y
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Effect of high NaCl and/or high urea on expression of Gadd45a, Gadd45b, and Gadd45g mRNA. Osmolality bathing p2mIME cells was increased from 640 mosmol/kgH 2 O (which approximates the lowest normal level in the renal inner medulla in vivo) ( 3 ) to higher levels. Gadd45a mRNA decreases when osmolality is increased in a single step to 1,040 mosmol/kgH 2 O by adding either NaCl or urea ( Fig. 1 A ). In contrast, Gadd45b mRNA rises sixfold with the step addition of either NaCl or urea, and Gadd45g mRNA also increases, although more with added NaCl than with added urea ( Fig. 1 A ). When osmolality is increased by adding NaCl gradually (linear increase), which is more physiological ( 5 ), Gadd45a mRNA still decreases, but both Gadd45b and Gadd45g mRNA fail to change significantly ( Fig. 1 B ). Thus at the high levels of osmolality normally found in the renal inner medulla, a step increase in either NaCl or urea elevates Gadd45b and Gadd45g mRNA, but a more physiological, gradual increase of NaCl does not.
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Fig. 1. Effect of high NaCl and urea on Gadd45 mRNA expression in 2nd-passage mouse inner medullary epithelial (p2mIME) cells and in vivo. A - C : p2mIME cells were grown at 640 mosmol/kgH 2 O. When confluent, they were switched to serum-free medium at 640 mosmol/kgH 2 O for 48 h. A : osmolality was increased from 640 to 1,040 mosmol/kgH 2 O in a single step for 19 h by adding NaCl or urea. B : same as A, except osmolality was increased linearly over 19 h. mRNA was measured at the end of the linear increase and 19 h later. C : osmolality was linearly increased from 640 to 1,640 mosmol/kgH 2 O for 20 h by adding NaCl and urea in combination. D : total RNA was extracted from mouse kidney cortex (CTX) and inner medulla (IM). mRNA was measured by RT-real-time PCR. Values are means ± SE; n = 3 or 4. * P
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* n/ E- S& c6 L6 MDuring antidiuresis, both NaCl and urea increase in renal inner medullary interstitial fluid. Therefore, we tested a combined increase in NaCl and urea. A step increase in NaCl and urea in combination decreases Gadd45a mRNA ( Fig. 1 C ), similar to the result of adding either alone ( Fig. 1 A ), but a linear increase of the combination has no effect on Gadd45a mRNA ( Fig. 1 C ). Step increase in NaCl and urea in combination increases Gadd45b mRNA ( Fig. 1 C ) but much less than when either NaCl or urea is increased alone in a single step ( Fig. 1 A ) or the combination is increased gradually ( Fig. 1 C ). A step increase in NaCl and urea in combination also increases Gadd45g mRNA ( Fig. 1 C ), similar to the effect of adding urea alone ( Fig. 1 A ), but much less than the effect of adding NaCl alone ( Fig. 1 A ) or gradually adding the combination ( Fig. 1 C ). Decreasing the osmolality from 640 to 300 mosmol/kgH 2 O for 48 h does not affect Gadd45a or Gadd45g mRNA but causes a modest increase in Gadd45b mRNA ( Fig. 1 C ).
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We conclude that osmotically induced changes in abundance of the Gadd45 isoforms in p2mIME cells depend on whether NaCl and urea are changed individually or in combination and on the rapidity of the change. However, in general, increasing NaCl and/or urea reduces Gadd45a mRNA abundance and increases Gadd45b and Gadd45g mRNA abundance. Furthermore, the addition of NaCl in a single step raises Gadd45b and Gadd45g mRNA abundance more than when it is added gradually, but the opposite is true when NaCl and urea are added in combination.) `; J, j0 @7 l& e- [% l9 }9 A& Y
; C2 H$ O/ G z: F# Z6 J) `Interstitial NaCl and urea concentrations are much higher in the renal inner medulla than in the cortex. Therefore, it was of interest to compare the mRNA abundance of the Gadd45 isoforms in the two regions of the kidney. There is little difference in Gadd45g mRNA between the cortex and medulla, but Gadd45a and Gadd45b mRNAs are higher in the inner medulla than in the cortex ( Fig. 1 D ). We conclude that Gadd45g mRNA expression is not elevated in association with the high interstitial NaCl and urea concentrations in the inner medulla in vivo, but expression of Gadd45a and of Gadd45b is.
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Effect of Gadd45b and Gadd45g expression on osmotic tolerance of p2mIME cells. Confluent Gadd45b -/- p2mIME cells survive increases in osmolality in a range up to 1,440 mosmol/kgH 2 O, with the addition of either NaCl or urea for 24 h, as well as do Gadd45b / p2mIME cells ( Fig. 2 ). The results are essentially the same after 48 and 72 h (data not shown). The survival of Gadd45g -/- p2mIME cells is significantly less than that of Gadd45g / p2mIME stressed by high NaCl or urea at 1,240 mosmol/kgH 2 O for 16 h, but the difference is small, and there is no difference at other osmolalities ( Fig. 2 ). Because Gadd45b and Gadd45g have some similar functions, including cell cycle control and activation of MEKK4 ( 33, 35 ), it seemed possible that the lack of one of them might be compensated for by the one that remained. Therefore, we tested p2mIME cells from Gadd45bg -/- mice ( Fig. 2 ). Osmotic tolerance of the doubly mutant cells is the same as that of wild-type cells 24 h after osmolality is increased by the addition of either NaCl or urea. Because high urea and/or high NaCl decreases Gadd45a expression ( Fig. 1 ), we did not investigate osmotic tolerance of Gadd45a -/- cells further after finding in a preliminary experiment that it was essentially the same as that of wild-type cells (data not shown).
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Fig. 2. Effect of expression of Gadd45b and Gadd45g on osmotic tolerance of p2mIME exposed to high NaCl or urea. p2mIME cells from Gadd45b -/-, Gadd45g -/-, and Gadd45bg -/- mice were grown at 640 mosmol/kgH 2 O. When confluent, they were switched to serum-free medium at the same osmolality for 48 h. Osmolality was increased from 640 to 840, 1,040, 1,240, or 1,440 mosmol/kgH 2 O for 24 h by addition of NaCl or urea in a single step, as indicated. The number of the viable cells was counted by laser-scanning cytometry (LSC). Values are means ± SE; n = 3.0 W* Q# J- h1 _
. F W: J' G* Y# q8 {- XEffect of p53 expression on tolerance of p2mIME cells for high NaCl and for high urea. In mIMCD3 cells, high NaCl results in a rapid increase in p53 protein abundance, phosphorylation on Ser15, and activation of its transcriptional activity, which protects the cells by restricting DNA replication ( 8 ). mIMCD3 cells are immortalized by expression of SV40 ( 28 ). They proliferate rapidly and continue to proliferate even when confluent. Inner medullary cells normally are exposed to high NaCl in vivo ( 23 ), but their rate of proliferation is very slow ( 40 ), so it is unclear how important p53 might be for their osmotic tolerance. In the present experiments, to investigate whether p53 expression enhances osmotic tolerance of slowly as well as rapidly growing cells, we utilized p2mIME cells, which become contact inhibited when confluent ( 40 ). Osmolality of the medium bathing confluent p2mIME cells was increased above 640 mosmol/kgH 2 O by adding NaCl or urea, and the number of viable cells was counted 16 h later by LSC. When NaCl is added, survival of p53 -/- cells is significantly less than that of wild-type cells at 1,040 and 1,240 mosmol/kgH 2 O ( Fig. 3 ), which is within the normal range in vivo. When urea is added, survival of p53 -/- cells is significantly less than that of wild-type cells at 840 mosmol/kgH 2 O and above ( Fig. 3 ), also within the normal range in vivo. Thus expression of p53 enhances the tolerance of confluent p2mIME cells both to high NaCl and to high urea.
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1 k( N# K( x5 g- ]Fig. 3. Effect of expression of p53 on tolerance of p2mIME cells for high NaCl or urea. p2mIME cells from p53 / or p53 -/- mice were grown at 640 mosmol/kgH 2 O. When confluent, they were switched to serum-free medium at the same osmolality for 48 h. Osmolality was increased from 640 to 840, 1,040, 1,240, or 1,440 mosmol/kgH 2 O by adding NaCl or urea in a single step for 16 h. The number of the viable cells was counted by LSC. Values are means ± SE; n = 3. * P 6 R0 _: K. c' U* E
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Cells that are replicating DNA in the early S phase of the cell cycle are most sensitive to the stress of high NaCl ( 9 ). p53 activity can arrest the cell cycle at the G1/S checkpoint, preventing DNA replication ( 24 ), a protective mechanism that p53 -/- cells would lack. To test for differences in DNA replication between wild-type and p53 -/- cells, we measured BrdU incorporation. BrdU is incorporated in place of thymidine during DNA replication, providing a measure of DNA replication. Only 4% of confluent wild-type p2mIME cells incorporate BrdU ( Fig. 4, A and C ) compared with 22.6% of p53 -/- cells ( Fig. 4, B and C ). Thus at least part of the reason that expression of p53 protects p2mIME cells from high NaCl ( Fig. 3 ) is that p53 expression reduces DNA replication.
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Fig. 4. p53 Expression decreases the percentage of p2mIME cells that are replicating DNA; high NaCl increases the amount of p53 and its phosphorylation on Ser15. A and B : p2mIME cells from p53 -/- or wild-type mice were grown at 640 mosmol/kgH 2 O. When the cells were confluent, bromodeoxyuridine (BrdU) was added for 18 h. BrdU was visualized by immunostaining with anti-BrdU antibody. C : quantification of BrdU-positive cells by LSC. D : subconfluent p1mIME cells were synchronized with aphidicolin, and then osmolality was increased from 640 to 940 mosmol/kgH 2 O by adding NaCl for 1 h, followed by protein extraction and Western blot analysis.( R2 f- @4 u5 l" N- l( r" d) u: [
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High NaCl increases the amount of p53 protein and its phosphorylation on Ser15 in mIMDC3 cells ( 8 ) and NIH 3T3 cells ( 15 ). We were unable to test this directly in confluent p2mIME cells because the level of p53 protein was too low to measure reproducibly by Western blot analysis (data not shown). However, p53 expression is highest during the S phase of the cell cycle ( 27 ), and subconfluent p1mIME (first passage) cells proliferate more rapidly than do p2mIME cells. Therefore, to test for regulation of p53 expression in early-passage IME cells, we synchronized subconfluent p1mIME cells in the S phase with aphidicolin, then added NaCl to increase osmolality from 640 to 940 mosmol/kgH 2 O for 1 h. Under these conditions, p53 protein is measurable, and high NaCl increases expression of total and Ser15-phosphorylated p53 ( Fig. 4 D ).( @* i4 b: o+ j/ g4 n0 Q
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Lack of effect of p53 expression on urinary concentrating ability. Because p53 expression contributes to osmotic tolerance of early-passage inner medullary epithelial cells in tissue culture, it seemed possible that expression of p53 might enhance the function of inner medullary cells in vivo. To test for this, we compared urinary concentrating ability between wild-type and p53 -/- mice by reducing their water intake for 48 h, then measuring urinary osmolality. There is no difference in urinary concentrating ability between wild-type and p53 -/- mice ( Fig. 5 A )., t' F2 R% B- q" `6 F q8 i) J, t
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Fig. 5. p53 Expression does not affect the number of proliferating [proliferating-cell nuclear antigen (PCNA)-positive] cells in mouse renal inner medulla nor does it affect urinary concentrating ability. A : urinary concentrating ability of p53 -/- mice. Wild-type and p53 -/- mice were maintained in metabolic cages. After 5 days of adaptation to the cages and gelled diet, water was restricted for 48 h. Urine was collected for measurement of osmolality for 24 h before water restrictions and for last 24 h of water restriction. Values are means ± SE; n = 6. B : immunostaining for PCNA of kidney sections from wild-type and p53 -/- mice. Intestine section is shown as positive control.6 |+ @' i+ E) H+ D9 z; j
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Cellular proliferation and morphology in kidneys from p53 -/- and Gadd45abg -/- mice. The rate of cellular proliferation is very low in inner medullas of normal mice, resulting in very few PCNA-positive cells ( Fig. 6 A ). A possible explanation came from finding that overexpression of the Gadd45s inhibits mitosis and promotes G2/M arrest in osmotically stressed mIMCD3 cells ( 22 ), which was interpreted to indicate that expression of the Gadd45s contributes to the low mitotic index of cells in the mammalian renal inner medulla ( 22 ). In view of our own results, the same argument can be made for expression of p53. However, the number of PCNA-positive cells is not increased in inner medullas from p53 -/- ( Fig. 5 B ) or Gadd45abg -/- ( Fig. 6 A ) mice. Also, renal inner medullary morphology appears normal in both Gadd45abg -/- ( Fig. 6 B ) and p53 -/- (not shown) mice. In contrast, the number of PCNA-positive cells is increased in the outer medullas of Gadd45abg -/- mice, suggesting that Gadd45 expression might be important for limiting cellular proliferation in that part of the kidney, even though not in the renal inner medulla.
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) ^* v( u+ o2 EFig. 6. Effect of expression of Gadd45 on the number of proliferating (PCNA-positive) cells in mouse renal outer and inner medulla and on morphology of the renal papilla. A : immunostaining for PCNA of kidney sections from wild-type and Gadd45abg -/- mice. B : periodic acid-Schiff staining of sections from renal papillas of wild-type and Gadd45abg -/- mice." }7 g9 U5 x/ _$ ~0 w+ Z1 O
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Because it is difficult to breed Gadd45abg -/- mice, we were unable to obtain enough of them to conduct a formal test of their urinary concentrating ability. However, on ad libitum fluid intake, urine osmolality was high in both the wild-type (1,078 mosmol/kgH 2 O) and Gadd45abg -/- (1,654 mosmol/kgH 2 O) mouse from which the kidney sections in Fig. 6 were prepared, ruling out any severe impairment of urine concentrating ability in the knockout mouse.: F" _; [4 I$ B2 r8 v* H, X; z
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& ~5 I$ u( {' r4 D" B" c5 b5 EEffect of high NaCl and urea on Gadd45 expression. In previous studies of mIMCD3 cells, osmolality was increased acutely above a baseline level of 300 mosmol/kgH 2 O by the addition of NaCl, and then Gadd45 protein ( 20 ) or mRNA ( 7 ) levels were measured. The first study, using an antibody that does not distinguish between the Gadd45 isoforms, demonstrated that raising osmolality to 600 mosmol/kgH 2 O by the addition of NaCl increases Gadd45 protein expression after a delay of 24 h ( 20 ). A second study of mIMCD3 cells found increased expression of Gadd45a, Gadd45b, and Gadd45g mRNA and increased expression of Gadd45a and Gadd45b protein within 6 h ( 7 ). With p2mIME cells, raising osmolality from 300 to 700 mosmol/kgH 2 O by adding NaCl increases Gadd45g mRNA to a peak of sevenfold at 16 h and also increases Gadd45a and Gadd45b mRNA approximately twofold ( 7 ).
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, ~; M, p$ M" @* e0 h8 v( F) [; `However, the lowest normal osmolality of renal medullary interstitial fluid is 600 mosmol/kgH 2 O ( 3 ), which is far higher than the baseline in the studies just mentioned, and the osmolality in the inner medulla increases well above that during antidiuresis. In the present studies, to more closely approximate these physiological conditions, we used a baseline osmolality of 640 mosmol/kgH 2 O and studied normal p2mIME cells, which are not immortalized and withstand much higher osmolalities than do mIMCD3 cells ( 40 ). Raising osmolality to 1,040 mosmol/kgH 2 O by adding either NaCl or urea in a single step increases both Gadd45b and Gadd45g mRNA, but lowers Gadd45a mRNA ( Fig. 1 ). In contrast, a gradual, linear increase in NaCl, which is more physiological ( 5 ), does not alter Gadd45b or Gadd45g mRNA abundance. Because NaCl and urea generally increase together in inner medullas, we also tested the combination. Gradually increasing NaCl and urea in combination to 1,640 mosmol/kgH 2 O increases both Gadd45b and Gadd45g mRNAs ( Fig. 1 ). Thus under conditions similar to those in the inner medulla, Gadd45a does not respond to elevations of NaCl and urea, but Gadd45b and Gadd45g do. We supposed that the increased expression of Gadd45b and Gadd45g might be protective, so we compared the osmotic tolerance of p2mIME cells from wild-type and knockout mice.
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Effect of knockout of Gadd45b and/or Gadd45g on osmotic tolerance of p2mIME cells. We prepared p2mIME cells from Gadd45b -/-, Gadd45g -/-, and Gadd45bg -/- mice and compared their survival, following increases in NaCl and/or urea, to that of cells from wild-type mice. We grew the cells at 640 mosmol/kgH 2 O and used this as the baseline. p2mIME cells from Gadd45b -/- mice tolerate high NaCl or urea essentially as well as do cells from wild-type mice ( Fig. 2 ). Although fewer cells from Gadd45g -/- mice than from wild-type mice survive the increase to 1,240 mosmol/kgH 2 O with the addition of NaCl or urea ( Fig. 2 ), the difference is small and it occurs only at this one osmolality. Because it was conceivable that Gadd45b and Gadd45g might be redundant in this respect, one compensating for the absence of the other, we also tested p2mIME cells from Gadd45bg -/- mice. Loss of Gadd45b and Gadd45g in combination also does not affect the osmotic tolerance of p2mIME cells ( Fig. 2 ), making it unlikely that expression of either is necessary for tolerance of normal cells for changes in NaCl and urea concentrations within the range of that occurring in the renal inner medulla in vivo.! z2 _& n0 Y& ~* l2 p
) |5 R' j$ _' T# m& F$ VGadd45a expression and high NaCl-induced DNA breaks. High NaCl causes double-strand breaks in DNA ( 19 ) and inhibits DNA repair ( 10, 11 ). Gadd45a protein recognizes damaged DNA and modulates DNA accessibility on damaged chromatin ( 6 ). DNA damage generally causes a large, rapid, and sustained increase in Gadd45a mRNA. The increase is more than 25-fold within 4 h of exposure to MMS, 5-fold within 4 h exposure to anisomycin, and 5-fold within 2 h of exposure to UV radiation ( 36 ). In contrast, Gadd45a mRNA does not change or decreases in p2mIME cells when NaCl is increased. Furthermore, Gadd45a mRNA is only twice as high in mouse renal inner medullary cells in vivo, which contain numerous DNA breaks ( 11 ), as in renal cortical cells ( Fig. 1 D ), which contain far fewer ( 11 ). The lack of a more marked increase in expression of Gadd45a in the face of DNA damage caused by high NaCl is consistent with other indications that high NaCl impairs DNA repair, including that most Mre11 exits the nucleus and H2AX becomes phosphorylated to a much lesser extent ( 10 )./ {' m* Z' C: I' `
+ {2 R, ?, v/ l* e* H$ x" l. |2 WLack of p53 expression decreases osmotic tolerance of p2mIME cells. Although we did not detect p53 protein by Western blot analysis in confluent p2mIME (second passage) cells (see RESULTS ), we did in p1mIME (first passage) cells. When p1mIME cells are synchronized in S, p53 protein is readily detectable and both its abundance and its phosphorylation on Ser15 increase when NaCl is elevated ( Fig. 4 D ). Nevertheless, p2mIME cells from p53 -/- mice are less tolerant of high NaCl and urea ( Fig. 3 ) than are wild-type cells. Thus even a very low level of p53 is sufficient to enhance osmotic tolerance in slowly proliferating cells. The lesser tolerance for high urea of cells from p53 -/- mice is unexpected. In our previous studies of mIMCD3 cells, we found that high NaCl, but not high urea increases p53 protein abundance ( 8 ), and that, when p53 is reduced by specific antisense RNA, mIMCD3 cells are less tolerant of high NaCl. A possible role of p53 in tolerance for high urea was not examined. However, based on the present results, we conclude that expression of p53, even at low levels, enhances tolerance for high urea.$ T8 C! a- S' y9 R. O0 K
3 I8 P) J, b/ U, O% i" ~0 N( X9 E2 DHigh NaCl increases DNA double-strand breaks in mIMCD3 cells and increases p53 activity ( 8 ). Such accumulation and activation of p53 in response to DNA-damaging agents generally promotes cell cycle delay and DNA repair, enhancing cell survival ( 24 ). In agreement, accumulation and activation of p53 in mIMCD3 cells restrict DNA replication and reduces apoptosis ( 8, 9 ). High NaCl is most harmful to cells when they are replicating DNA in the S phase of their cell cycle ( 9 ). mIMCD3 cells, being immortalized by SV40, proliferate at a rapid rate, even after they become confluent. In contrast, proliferation of wild-type p2mIME cells is greatly reduced when they become confluent, minimizing the number of cells that are in S phase of the cell cycle, replicating DNA ( 40 ) ( Fig. 4 ). In the absence of p53, i.e., p2mIME cells from p53 -/- mice, cells continue proliferating after they become confluent, resulting in DNA replication in many cells ( Fig. 4 ), which provides an explanation for their decreased tolerance for high NaCl ( Fig. 3 ).* a% u; L" x( s, z3 b
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Perspective. When we began these studies, we knew that elevating NaCl increases Gadd45 expression in mIMCD3 cells ( 7, 20 ), which led us to hypothesize that Gadd45 expression might be important for the osmotic tolerance of renal inner medullary cells. This assumption was reinforced by the report that overexpression of the Gadd45s substantially affects cell cycle and apoptosis in mIMCD3 cells ( 22 ). Similarly, we knew that the increase in p53 activity that occurs in mIMCD3 cells, when NaCl is elevated, protects the cells from apoptosis ( 8 ), which led us to hypothesize that p53 might also be important for osmotic tolerance of renal inner medullary cells in vivo. However, we do not find substantially altered osmotic tolerance in Gadd45b -/- or Gadd45g -/- cells nor morphological or functional abnormalities in the renal medullas of p53 -/- or Gadd45abg -/- mice. Why this unexpected result? L0 o3 S' K. h. L8 x
" s' W! m# e' s4 O% `) q/ ?With reference to Gadd45, considering the possibility of redundancy between the various Gadd45s, we tested combined knockouts. However, we did not find an osmotic phenotype with combining multiple Gadd45 knockouts either in the p2mIME cells or in the renal medullas of the mice from which they were derived. Other genes might compensate for the lack of the Gadd45s, but, if so, they remain to be identified. The lack of effect of knocking out the Gadd45 genes on normal inner medullary cells under the conditions to which they are exposed in vivo is contrary to our initial expectations. Those expectations were based on the large increase in their expression in mIMCD3 cells exposed to high NaCl ( 7, 20 ) and were later reinforced by the effects of overexpressing these genes in mIMCD3 cells exposed to high NaCl ( 22 ). Perhaps, overexpression of the Gadd45s has effects beyond those that occur at normal levels of expression and, in any event, does not directly address the function of normal levels of Gadd45 expression.
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Most important, with reference to both Gadd45 and p53, it is significant that the pertinent earlier studies utilized mIMCD3 cells that are immortalized by expression of SV40 ( 28 ). While these cells are very useful, they have the limitation that they proliferate rapidly and continue to proliferate, replicating DNA, even after they become confluent. In contrast, proliferation of renal inner medullary cells is rare in vivo ( 40 ). We propose that, because DNA replication reduces the tolerance of cells for high NaCl ( 9, 40 ), although expression of Gadd45s and p53 is important for mIMCD3 cells exposed to high NaCl, it is not for normal, nonproliferating inner medullary cells exposed to high NaCl in situ.
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This research was supported by the Intramural Research Programs of the National Institutes of Health, National Heart, Lung, and Blood Institute, and National Cancer Institute.
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, l& b/ b- g( U% ^0 OACKNOWLEDGMENTS
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0 _$ G* i: s' \) I. J+ WWe acknowledge the professional skills and advice of Dr. Christian A. Combs and Daniela Malide (Light Microscopy Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health) regarding microscopy-related experiments performed in this paper.
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" o- r" r7 z8 v, a* p* Y# h2 WPresent addresses: Q. Cai: Rm. 4111 AHSC, Dept. of Physiology, College of Medicine, 1501N Campbell Ave., University of Arizona, Tucson, AR 85724-5051 (e-mail: caiq{at}email.arizona.edu 5 j$ N$ r5 C: g* b8 E
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发表于 2009-4-22 08:39
