Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110
Submitted 3 June 2003 ; accepted in final form 2 September 2003
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ABSTRACT |
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TAT proteins; p38 MAPK; inducible nitric oxide synthase; mesangial cell; interleukin-1
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METHODS |
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Cell culture. Rat primary mesangial cell cultures were prepared from male Harlan Sprague-Dawley rats as previously described (12). Cells were grown in RPMI 1640 medium supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 0.6% (vol/vol) insulin, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 µg/ml amphotericin B, and 15 mM HEPES. Where indicated, mesangial cells were stimulated with IL-1 (100 U/ml). RAW 264.7 cells were obtained from ATCC (American Type Culture Collection) and cultured in 15 mM HEPES, pH 7.4, in DMEM and 10% FCS with added penicillin and streptomycin.
Mutagenesis of p38MAPK isoforms. The cDNA for each of the p38MAPK isoforms was obtained from Roche Pharmaceuticals (generous gift from Dr. Phyllis Whitely). The coding region of each p38 isoform was amplified by PCR and subcloned into the mammalian expression vector pcDNA3 (Invitrogen). Dominant negative mutants for these isoforms were made by mutating the Thr-Gly-Tyr dual phosphorylation motif to Ala-Gly-Phe by site-directed mutagenesis. Mutagenesis was performed with the Transformer Site-Directed Mutagenesis Kit (second version) from Clontech according to the instructions provided by the manufacturer. The oligonucleotides used for mutagenesis are as follows: 5'-GATGATGAAATGGCAGGCTTCGTGGCCACTAG-3' for p38, 5'-GACGAGGA-GATGGCCGGCTTTGTGGCCACG-3' for p38
2, and 5'-GACAGTGAG-ATGGCTGGGTTCGTGGTGACC-3' for p38
, where the mutated bases are underlined. p38
2 wild type and p38
wild type, as well as a p38
kinase-dead mutant, in which Lys54 is mutated to Met54, were obtained from Dr. J. Han from the Scripps Research Institute (La Jolla, CA). The cDNA was mutated in the ATP-binding site, thus making it catalytically inactive. The DNA sequence of the mutated genes was verified by DNA sequencing.
Transient transfections. Mesangial cells were transiently transfected using SuperFect transfection reagent (Qiagen Corp, Valencia, CA). Cells were plated into six-well cluster plates at a density of 2 x 105 cells per well and incubated overnight. Mixtures of 2.5 µg of plasmid DNA in 75 µl of serum-free medium and 15 µl of SuperFect reagent were incubated 510 min at room temperature, followed by dilution to 0.5 ml with complete medium. The DNA-SuperFect complex was layered onto mesangial cells (2.5 µg DNA per well), and after a 2- to 3-h incubation, the medium was changed and cells were incubated overnight for gene expression.
Preparation of TAT-p38 isoforms. The vector pTAT-HA was a generous gift from Dr. Steven Dowdy (Howard Hughes Medical Institute, Washington University, St. Louis, MO). The vector shown in Fig. 3A carries an NH2-terminal 6-histidine leader followed by the 11-amino acid TAT protein transduction domain flanked by glycine residues, a hemaglutinin tag (HA) followed by a polylinker (21, 29). We ligated the cDNA for human ,
,
, and
isoforms of p38MAPK, both wild type (wt) and mutants (mt), into the polylinker for the expression of full-length TAT chimeric proteins. Recombinant proteins were then expressed in Escherichia coli, strain DH5
, denatured, and solubilized in 8 M urea and isolated on a Ni2+ affinity column. Rapid dialysis was then carried out using a Slide-A-Lyser dialysis cassette with a 10,000 MW cutoff (Pierce, Rockford, IL) against 20 mM HEPES, pH 7.4. The protein concentration was determined and recombinant proteins stored in 10% glycerol at 80°C until required for experiments. The TAT chimeric proteins were added to the media at the appropriate concentrations and incubated for 60 min to allow for equilibration with cytosolic concentration. Once inside the cells, transduced denatured proteins may be correctly refolded by chaperones such as HSP90 (21, 28). Transduction across the cell membrane occurred through an unidentified mechanism that is independent of receptors, transporters, and endocytosis(34) and transduced proteins are found in the cytoplasm and nucleus. The internalization occurred at 4°C, and it is therefore unlikely that uptake requires any cell-mediated process or physical arrangement (14).
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Western blot analysis. At the time of harvest, cells were washed with ice-cold phosphate buffer and lysed in whole cell extract (WCE) buffer [25 mM HEPES-NaOH (pH 7.7), 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM dithiothreitol, 20 mM -glycerophosphate, 100 µM NaVO4, 2 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride] to which x6 sample buffer was added before heating. After boiling for 5 min, equal amounts of protein were run on 10% SDS-PAGE. Proteins were then transferred to polyvinylidene difluoride membranes (Immobilon BP; Millipore, Bedford, MA). The membranes were saturated with 5% fat-free dry milk in Tris-buffered saline (50 mM Tris·HCl, pH 8.0, 150 mM NaCl) with 0.05% Tween 20 (TBS-T) for 1 h at room temperature. Blots were then incubated overnight with primary antibodies at 1:1,000 dilution in 5% BSA TBS-T. After being washed with 5% milk TBS-T solution, blots were further incubated for 1 h at room temperature with goat anti-rabbit or mouse IgG antibody coupled to horseradish peroxidase (Amersham, Arlington Heights, IL) at 1:3,000 dilution in TBS-T. Blots were then washed five times in TBS-T before visualization. Enhanced chemiluminescence kit (ECL; Amersham) was used for detection. Blots were quantitated on a densitometer using Quantity One software from PDI. The densitometer was calibrated to an external standard.
RT-PCR and TaqMan real-time PCR. To determine the expression of the message for the p38 isoforms in rat renal mesangial cells, total RNA was isolated from cells using RNA STAT-60 reagent, DNase treated twice, and reverse-transcribed by Omniscript reverse transcriptase (Qiagen) using oligo (dT)15 primers. The PCR amplifications were carried out using rat p38MAPK amplification primers unique for each isoform. For p38, the primers were 5'-AACCTGTCCCCGGTGGGCTCG-3' and 5'-CGATGTCCCGT-CTTTGTATGA-3'; for p38
, primers were 5'-CGCCCAGTCCTGAGGTTCT-3' and 5'-AGACACTGCTGAGGTCCTTCT-3'; for p38
, the primers were 5'-CGCCCCCTCCT-GAGTTT-3' and 5'-GCTTGCGTTGGTCAGGACAGA-3'; and for p38
, the primers were 5'-TGCTCGGCCATCGACAA-3' and 5'-TGGCAAAGATCTCCGACTGG-3'. To quantify p38 isoform mRNA levels, TaqMan real-time PCR was performed using the gene-specific primers above and the double-stranded DNA-binding dye SYBR green I. Fluorescence was detected with an ABI Prism 5700 sequence detection system (PE Biosystems). Amplification primers for GAPDH were 5'-GAAGGTGAAGGTCGGAGTC-3' and 5'-GAAGATGGTGATGGGATTTC-3'. Primer pairs were tested to ensure a robust amplification signal of the expected size with no additional bands. The amount of p38MAPK message in each RNA sample was quantified and normalized to GAPDH content. Relative amounts of p38MAPK cDNA were calculated by the comparative CT method.
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RESULTS |
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Effect of transfection of p38 and -
2 into mesangial cells. For these experiments, p38
(wt and mt) and p38
2 (wt and mt) were transiently transfected into rat mesangial cells and the effects on the expression of Il-1
-induced iNOS expression were assessed. The mammalian expression vector pcDNA3 was used and carried a FLAG epitope tag, which enabled determination of expressed protein in mesangial cells by Western blotting. Figure 2 shows an example of one of our more striking experiments that illustrates what we expected for p38
based on previous published data (11). It shows that transfection of p38
wt had virtually no effect on IL-1
-induced iNOS expression, whereas the mutant p38
inhibited IL-1
-induced iNOS expression. However, the results seen for p38
2 were unexpected. In this instance, the p38
2 mt was without effect, whereas the wt p38
2 inhibited IL-1
-induced iNOS expression. Because the transfection efficiency for mesangial cells was variable, the results obtained with this approach were not statistically significant, although qualitatively and consistently similar to the results as seen in Fig. 2. We therefore chose the alternative strategy of transduction of TAT chimeric proteins to assess the function of the p38 isoforms on IL-1
-induced iNOS expression in renal mesangial cells.
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Concentration-dependent transduction into cells. The cDNA for wild-type and mutant p38 isoforms were ligated in the correct orientation into the prokaryotic expression vector pTAT-HA (Fig. 3A). Bacterial expressed TAT-proteins were purified and isolated as described in METHODS. A Coomassie blue-stained gel (Fig. 3B) shows the p38 wt and mt isoforms eluted from the Ni2+ affinity column with 0.1 M imidazole. This fraction was rapidly dialyzed, and protein concentrations were determined and stored at 70°C in 10% glycerol. TAT-p38 proteins (wt or mt) were added to 25-cm2 flasks with 5 ml of medium to achieve increasing concentrations up to a maximum of 100 µg/ml. The mesangial cells were then allowed to incubate at 37°C for 60 min. The medium was then harvested for Western blotting; the cells were washed twice with ice-cold medium (without TAT proteins), and cell lysates were prepared with WCE buffer. Equal volumes of medium and equivalent amounts of cell lysate were then analyzed by Western blotting using anti-HA antibodies. Densitometric quantitation of the blots was carried out. Figure 4A shows that increasing concentrations of TAT p38 proteins in the medium was associated with a linear increase in protein transduction, which appeared to reach a plateau intracellularly at concentrations of protein that exceeded 25 µg/ml in the medium. Figure 4A, inset, shows the linear relationship of increasing the concentration of TAT protein in the medium over a range of 0.1100 µg/ml of protein. Because of this, we did not exceed 25 µg/ml of TAT protein in our transduction experiments. Figure 4B shows a Western blot of cell lysates from control mesangial cells (lane 1) and cells transduced with 10 (lane 2) and 25 (lane 3) µg/ml of TAT-p38. It can be seen that increasing transduction of TAT proteins had no effect on endogenous p38
protein expression. In data not shown, it was also demonstrated that the efficiency of transduction of a TAT-
-Gal protein was 95100%. Hence, we believe transduction of TAT-p38 was also 95100%.
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Effects of p38 wild-type and mutant isoforms on IL-1-induced iNOS expression. Each of the isoforms of p38MAPK (wt and mt) was transduced into renal mesangial cells in a concentration-dependent manner. Two hours after the addition of p38MAPK to the medium, cells were stimulated with IL-1
at 100 units/ml for 24 h. Mesangial cell lysates were harvested and analyzed by Western blotting for iNOS protein expression. Control lysates from cell cultures transduced with similar concentrations of TAT p38 proteins but not stimulated with IL-1
were harvested over the same time course as IL-1
-stimulated cells. Figure 5 shows a representative Western blot of the effects of increasing concentrations of p38 isoforms on Il-1
-induced iNOS expression. The cumulative results of these experiments are shown in Fig. 6. TAT-p38
wt produced a 3540% increase in IL-1
-induced iNOS expression at the highest concentration of 20 µg/ml of TAT protein but at lower concentrations had no effect. TAT-p38
wt and mt was virtually ineffective over the full concentration range used (not statistically significant). In contrast, p38
2 wt produced a concentration-dependent inhibition of IL-1
-induced iNOS expression in rat renal mesangial cells with an IC50 of 20 µg/ml (
400 nmol). Figure 6 also shows the effect of increasing concentrations of mutant p38 isoforms on IL-1
-induced iNOS expression. TAT-p38
and -
mutant isoforms produced no statistically significant change in iNOS expression over the entire concentration range used. p38
2 mutant was ineffective over the range of concentrations used. As expected, p38
mutant produced a concentration-dependent inhibition of IL-1
-induced iNOS expression with an IC50 of 10 µg/ml (
200 nmol). The data obtained for the p38
and
2 isoforms (wt and mt), shown in Fig. 6, represent means ± SE from five experiments. For the p38
and -
, the data presented are means ±SE of three experiments in duplicate.
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Expression of endogenous p38 isoforms by rat renal mesangial cell. To determine the expression patterns of the various isoforms of p38MAPK in the rat renal mesangial cell, we assessed the mRNA expression by real-time PCR. Amplimers were designed as indicated in METHODS, and GAPDH was used to standardize mRNA expression. Figure 7A shows a representative tracing of the amplification plot obtained. The plot suggests that all four isoforms are expressed, albeit at differing levels. Figure 7B shows the relative abundance of mRNA of the different isoforms expressed using p38 as 100%. The scale used on the ordinate is a log scale, and the figure represents the mean of two PCR runs carried out in triplicate.
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DISCUSSION |
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The quantitative PCR also suggest that all the isoforms are expressed at the mRNA level, however, at markedly different levels. Of some surprise was the observation that p38 was the second most abundant isoform expressed and suggests that this isoform is not restricted in expression to skeletal muscle as was originally suggested (16). What is not known is whether the four isoforms are expressed in unique subcellular compartments that allow unique functions within the cells. Thus, even though p38 and -
may be inhibited equally well by SB-203580, the functional consequences to inhibition could be different depending on where the proteins are expressed and what their respective downstream targets are. Indeed, p38
and -
appear to have opposing effects on AP-1-dependent transcription in two breast cancer cell lines (24). Thus there is a precedent for opposing biology exhibited by certain p38 isoforms, and there is some evidence to support this possibility (31). Another possibility is that the two p38 isoforms (
and
) exert their functions at different sites in the transcriptional and posttrancriptional machinery of the cell. The iNOS gene can be regulated both at a transcriptional (18, 20) and posttranscriptional level, exhibiting both 3' instability determinants in the mRNA (26) and regulation of RNA-binding proteins, which could influence mRNA stability and translation (32). p38MAPK has also been implicated in kinase-dependent and -independent signaling of mRNA stability of AU-rich element-containing transcripts (9). Furthermore, tristetraprolin has been recognized as an mRNA-interacting protein that can be phosphorylated and functionally regulated by p38MAPK (1). Which of the relevant isoforms is capable of carrying out this cellular function remains to be determined. Recently, it has been suggested that p38
can phosphorylate eEF2 kinase and inhibit its activity, thus having an effect on protein translation (17). The evidence would support, therefore, unique functions for differing isoforms of p38MAPK.
We have demonstrated that p38 and -
appear to have opposing function in the rat renal mesangial cell. The subcellular localization where these functions occur and the sites (transcriptional and/or posttranscriptional) within the protein synthetic machinery where these interactions occur remain to be determined. We believe these observations have important implications for drug design, targeting the inhibition of p38MAPK.
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ACKNOWLEDGMENTS |
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This work is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-09976 and DK-59932 (to A. R. Morrison).
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FOOTNOTES |
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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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