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【关键词】 protein
Renal Division, Brigham and Women's Hospital, Boston, Massachusetts
ABSTRACT
The balance between proliferation and apoptosis of mesangial cells is a critical component of proliferative glomerulonephritis. The regulation of cell proliferation and apoptosis is linked at the level of the cell cycle (Shankland SJ. Kidney Int 52: 294308, 199). cPLA2-interacting protein (PLIP), the Tip60 splice variant, interacts with cPLA2 and enhances the susceptibility of renal mesangial cells to serum deprivation-induced apoptosis (Sheridan AM, Force T, Yoon HJ, O'Leary E, Choukroun G, Taheri MR, and Bonventre JV. Mol Cell Biol 21: 44704481, 2001). We report that adenoviral-driven PLIP expression results in enhanced apoptosis of non-serum-deprived mesangial cells associated with a marked decrease in G0/G1 phase cells. The effect of PLIP on the cell cycle may be independent of its interaction with cPLA2 because a mutation of PLIP that does not interact with cPLA2 also causes a decrease in G0/G1 cells. Endogenous PLIP and Tip60 protein levels are increased in cells exposed to injurious stimuli including X-irradiation and H2O2, but the intracellular localization of the splice variants may differ. Whereas PLIP localizes in the nucleus of all mesangial cells, Tip60 localizes in the cytosol of untreated mesangial cells and of cells exposed to low concentrations (50200 μM) of H2O2. Tip60 is targeted to the nucleus of cells exposed to high concentrations (12 mM) of H2O2. We conclude that PLIP may cause cells to exit from the cell cycle after the S phase and may function as part of a G2/M checkpoint mechanism. Tip60 splice variants may function in both cytosolic and nuclear signaling pathways in mesangial cells.
Tip60; mesangial cells; apoptosis; cell cycle
MANY GLOMERULAR DISEASES, including IgA nephropathy, lupus nephritis, and diabetic nephropathy, are characterized by an increase in mesangial cell number (20, 34, 35), which is determined by the balance between cell proliferation and apoptosis (35). As in most cells, mesangial cell proliferation is regulated by the expression and activity of proteins that stringently control the passage of cells at G1/S and G2/M transitions (38). Changes in expression and activity of cyclins, cdks, and cdk inhibitors have been demonstrated in animal models of proliferative glomerulopathies, including the Thy 1 model of proliferative glomerulonephritis (37) and the remnant kidney model (36). The inhibition of cdk-2 activity hastens renal recovery from Thy-1 glomerulonephritis, suggesting that the manipulation of cell cycle regulatory proteins may alter disease progression and offer a potential therapeutic benefit (29).
Increased apoptosis is observed in both animal glomerulonephritis models (1) and human glomerulonephritides (33, 34). Apoptosis occurs in parallel with the increase in cell proliferation in experimental models (1). It is not known whether the increased apoptosis that occurs in proliferative glomerulonephritis hastens recovery or contributes to the glomerulosclerosis that characterizes end-stage renal disease. Apoptosis can be triggered by cell cycle checkpoint machinery. Checkpoints are surveillance mechanisms that detect DNA damage and induce either cell cycle arrest and DNA repair mechanisms or, in the presence of extensive DNA damage, apoptosis (27).
We have identified a protein that may contribute to the G2 checkpoint and modulate mesangial cell apoptosis. The cPLA2-interacting protein, or PLIP, was isolated using two-hybrid cloning by virtue of its interaction with the C2 domain of cPLA2 (39). cPLA2 is a large molecular mass member of the family of phospholipases, which hydrolyze phospholipids to generate free fatty acids and lysophospholipids. cPLA2 shares no homology with secretory isoforms of PLA2 and has been shown to have selectivity for diacylphospholipids containing arachidonic acid at the sn-2 position (9). Activity of this isoform is critical in the production of eicosanoids (2, 43) and has been associated with ROS-mediated cell injury (32) and TNF--induced apoptosis (15) in vitro as well as ischemic injury in vivo (2). The mechanism by which cPLA2 expression enhances apoptosis is not completely understood. To better understand the mechanism by which cPLA2 enhances apoptosis, we searched for interacting proteins and isolated two cPLA2-interacting proteins, including HIV TAT-interacting protein, Tip60 (19), and PLIP (39). The interaction between cPLA2 and Tip60 or PLIP suggests a heretofore unrecognized role for cPLA2 in a novel signaling cascade.
PLIP is a splice variant of Tip60 (19). The Tip60 gene is located on chromosome 11 and consists of 14 exons. The fifth exon, which encodes a 52-amino acid fragment, is expressed in Tip60 but not PLIP (39). Tip60 is structurally characterized by an NH2-terminal chromo domain and a MYST domain that is named for the family of proteins that contain this homologous region (6, 16, 31, 41). The MYST domain includes a histone acetyltransferase (HAT) domain and a C2HC zinc finger. It is not known whether Tip60 and PLIP share common functions in vivo although both splice variants express the MYST domain and recombinant protein comprising regions common to both proteins acetylates core histones H2A, H3, and H, but not H2B, in vitro (45). Tip60 has been identified as part of a nuclear complex of proteins, which includes PAF400, Tip49a, Tip49b, -actin, and BAF49, and plays a role in DNA repair and apoptosis following genotoxic stress (18). Additionally, Tip60 is recruited by E2F1 to promoter regions during late G1, suggesting a potential role in the regulation of G1/S (42). Tip60 associates with, and acetylates, class I nuclear receptors, including the androgen receptor (7, 13, 14, 21), and binds to the amyloid- precursor protein complexed to the nuclear adapter protein Fe65 (8), and increases the transactivation function of both proteins. Tip60 also associates with the Il-9 (40) and endothelin (22) receptors, suggesting that, in addition to its nuclear function(s), Tip60 may play a role in signal transduction initiated by extracellular stimuli.
PLIP increases apoptosis in cultured renal mesangial cells (39). The mechanism by which PLIP achieves its proapoptotic effect in serum-deprived cells is not known. Exogenous PLIP expression enhances the susceptibility to apoptosis of cells derived from cPLA2+/+ but not cPLA2/ mice, suggesting that the proapoptotic effect of PLIP is dependent on expression of cPLA2 (39). However, it has not been demonstrated whether the interaction between PLIP and cPLA2 is necessary for PLIP's proapoptotic effect.
The primary goal of this study was to determine the effect of PLIP on the cell cycle, and to determine whether this effect was related to PLIP's interaction with cPLA2. We show that adenoviral-driven expression of PLIP results in apoptosis and a striking decrease in the percentage of G0/G1 cells. The effect of PLIP appears to be independent of its interaction with cPLA2, because expression of a non-cPLA2-interacting PLIP mutant results in a similar degree of cell cycle exit. A second goal was to determine the effect of DNA-damaging stimuli on the levels and cell localization of PLIP and Tip60. To achieve this goal, we generated a Tip60-specific antibody. We show that full-length Tip60 localizes primarily in the cytosol of mesangial cells and that protein levels are increased in cells exposed to H2O2.
MATERIALS AND METHODS
Cell culture. Mesangial cells were harvested from 6-wk-old Wistar-Kyoto rats. Cortexes of decapsulated, bisected kidneys were minced and forced through a 106-μm sieve (Bellco Glass, Vineland, NJ) followed by passage through a 53-μm sieve. The washed, sieved glomeruli were resuspended in minimal essential medium (MEM) with D-valine, L-glutamine, and Earle's salts (Mediatech, Herndon, VA). After fibroblasts were excluded by growth in D-valine-containing medium, cells were grown in RPMI with 20% FCS (Mediatech). Homogenous cell cultures have been demonstrated using this method (35). Cells were grown in RPMI with 10% FCS. Cells were passaged every 7296 h by trypsinization and used at passage 312. Mesangial cells were plated in 10-cm2 plates at a density of 12 x 106 cells/plate for analysis of protein expression and in 6-cm2 plates at 1 x 105 cells/well for flow cytometry.
Adenoviral constructs. Ad-PLIP and Ad-Lac have been previously described (39). PLIP cDNA was subcloned into the Not1 and Xho1 sites of pADRSV4, which contains adenoviral sequences from the 01.2 and 9.216.1 map units, the Rous sarcoma virus long-terminal repeat promoter, and the SV40 early polyadenylation signal, to generate pAdRSV4-PLIP. Position and orientation of the insert was confirmed by sequencing of the 5'-ends of the constructs using a pADRSV4 primer. pADRSV4-PLIP was cotransfected into 293 cells with pJM17, which contains adenoviral cDNA. Homologous recombinants between pADRSV4-PLIP and pJM17 contain exogenous DNA substituted for E1, which allows for adenoviral-driven expression of the exogenous protein or PLIP. Individual plaques were purified, and PLIP protein expression was confirmed by immunoblotting using an antibody that was generated in rabbits to full-length PLIP and has been previously described (39). The recombinant adenovirus was prepared in high titer by propagation in 293 cells and purification by a CsCl gradient. For all experiments, recombinant adenovirus carrying the Eshcerichia coli LacZ gene encoding -galactosidase was used as a control (Ad-Lac).
Western blot analysis. Mesangial cells were harvested into a buffer containing 10 mM potassium phosphate, pH 7.4, 5 mM EGTA, 50 mM -glycerophosphate, 1 mM sodium vanadate, 1 mM DTT, 2 μM leupeptin, 0.5% NP-40, and 0.1% Brij 35. Protein concentrations were determined using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were separated by gel electrophoresis on either a 7.5 or 10% gel. Proteins were electrophoretically transferred to nitrocellulose membranes (Hybond C, Amersham), and Western blot analysis was performed using the following primary antibodies: anti-PLIP at 1:2,500; anti-Tip60 at 1:2,500; and anti-hemagglutinin (HA) at 1:100. An anti-PLIP antibody was generated in rabbits against full-length recombinant GST-PLIP (39). An anti-Tip60 antibody was raised against Tip60 116134 (FNLPKEREAIPGGEPDQPL), which is contained within the exon 5-encoded region. Anti-mouse and anti-rabbit horseradish peroxidase-conjugated antibodies were obtained from Santa Cruz Biotechnology and used at a dilution of 1:5,000.
Microscopic analysis of apoptosis. Mesangial cells were plated in six-well plates, infected with Ad-Lac or Ad-PLIP as described, and grown in RPMI/10% FCS. After 36 days, cells were gently trypsinized and cells plus supernatant were centrifuged and resuspended in 30 μl of 4% paraformaldehyde. Twenty microliters were spread onto a glass slide and allowed to air dry. Fixed cells were incubated in Hoechst 33342 (10 μg/ml) for 10 min at room temperature and viewed under UV light.
Flow cytometric analysis. Cells were analyzed by flow cytometry according to a method developed and optimized for adherent mesangial cells (11). Confluent monolayers were trypsinized and centrifuged at 190 g for 10 min. Cell pellets were gently resuspended in ice-cold 70% ethanol/30% PBS. Fixed cells were centrifuged at 190 g for 5 min, washed once in 1x PBS, and resuspended in 200 μl of propidium iodide (200 μg/ml) and RNase A (500 μg/ml). Cells were incubated at 37°C for 1 h. Cells were analyzed by flow cytometry using a laser with an excitation beam of 488 nm and a detector for phycoerythrin.
Models of apoptosis. Cells were exposed to 0.58 Gray (Gy) X-irradiation (Siemens Stabilipan 2 X-ray machine operated at 250 kVP, 12 mA, dose rate of 2.08 Gy/min) in the presence of 10% FCS and studied at various times following irradiation. Cells were exposed to 0.12 mM H2O2 for various times in the presence of FCS.
Immunofluorescence. Cells were plated onto coverslips, and experiments were performed when cells were 5080% confluent. Following exposure to experimental or control conditions, cells were fixed with 4% paraformaldehyde followed by 0.1% Triton X-100. Fixed cells were exposed to primary and secondary antibodies over 1 h at room temperature. Antibodies to PLIP and Tip60 were used at 1:100, followed by a goat Cy-3-conjugated anti-rabbit antibody used at 1:500. Mouse anti-HA was used at 1:5, followed by a goat FITC-conjugated anti-mouse antibody at 1:500.
Transfection experiments. COS cells were transfected using DEAE-dextran. For each 10-cm plate, 200 μl of 1x PBS containing DEAE-dextran (10 mg/ml) and chloroquine (2.5 mM) were added to DMEM containing 10% NuSerum (Collaborative Research, Bedford, MA). DNA (20 μg/plate) was added, and the chloroquine/DEAE-dextran/DNA mixture was layered onto cells. After a 4-h incubation at 37°C, the chloroquine/DEAE-dextran/DNA mixture was removed and cells were exposed to 10% DMSO before the addition of fresh medium.
Cell fractionation. Cells were harvested into 1 ml of 1x PBS. Following centrifugation at 4,000 rpm, cells were resuspended into 750 μl of buffer containing (in mM) 150 NaCl, 10 Tris, pH 7.9, 1 EDTA, 5 NaF, 20 -glycerophosphate, and 0.4 PMSF as well as Complete miniprotease cocktail (Roche Diagnostics). NP-40 was added to achieve a final concentration of 0.6%, and tubes were vortexed for 10 s before centrifugation at 4,000 rpm at 4°C. Supernatants containing cytosolic fractions were gently removed, and nuclear pellets were resuspended in 100 μl of buffer containing (in mM) 420 NaCl, 1.5 MgCl2, 10 HEPES, pH 7.9, 0.1 EGTA, 0.1 EDTA, 10 NaF, 0.5 DTT, and 0.5 PMSF as well as 25% glycerol. Proteins were determined by Bio-Rad, and samples were controlled for protein.
Synthesis of PLIP protein. PLIP was ligated into pGEX-Kg using EcoR1 and Xho1 sites. Following transformation with pGEX-Kg-PLIP, the XL-1 Blue E. coli strain (Stratagene) was grown in 50 ml LB to an optical density of 0.6. Following induction of a glutathione-S-transferase (GST) fusion protein with 0.2 mM isothiopropyl-B-D-galactoside (IPTG), cells were centrifuged at 5,000 g and resuspended in 9 mL of 1x PBS containing 10 μl leupeptin, 10 μl PMSF, and 1% Triton X-100. Following sonication, the mixture was centrifuged at 10,000 g for 10 min at 4°C, and the pellet was resuspended in 8 ml sarkosyl/triethanolamine solution (containing 1.5% sarkosyl, 25 mM triethanolamine, and 1 mM EDTA, leupeptin, and PMSF) and mixed for 10 min on ice. After centrifugation at 10,000 g for 10 min at 4°C, Triton X-100 and CaCl2 were added to pellets for final concentrations of 2% and 1 mM, and the supernatants were combined. Five hundred microliters of glutathione agarose were added for 30 min at 4°C. Following centrifugation, 1.5 ml of 5 mM glutathione were added to washed agarose precipitants for 2 min on ice. The supernatant was dialyzed overnight at 4°C against 50% ethylene glycol, 50 mM Tris, pH 7.5, 50 mM NaCl, and 1 mM DTT. The dialyzed protein was incubated with thrombin (2 μg/mg protein) for 20 min at room temperature to cleave GST, which was precipitated with glutathione agarose for 30 min at 4°C. The supernatant, containing PLIP protein, was dialyzed, and the protein was concentration determined by Bio-Rad and analyzed by Western blotting using anti-PLIP antibody.
Statistics. All data are means ± SE. Student's t-test was used for the comparison of two groups of data. ANOVA was used when more than two groups were compared.
RESULTS
Adenoviral-induced PLIP expression results in a decrease in G0/G1 and an increase in sub-G1 cells. Ad-PLIP infection results in PLIP expression in mesangial cells. We used an antibody that was generated to full-length PLIP protein (39) to analyze by Western blotting total cell lysates of mesangial cells infected with Ad-Lac or Ad-PLIP (Fig. 1). The anti-PLIP antibody detected multiple bands in blots of total cell lysates. However, a band at 50 kDa appears in lanes containing lysates of Ad-PLIP-infected but not Ad-Lac-infected cells, suggesting that the band represents adenoviral-driven PLIP. We examined the effect of adenoviral-driven PLIP expression on the regulation of the mesangial cell cycle. Asynchronously growing -galactosidase- and PLIP-expressing cells were stained with propidium iodide, and cellular DNA content was analyzed by flow cytometry. Flow cytometry performed 2 days after infection demonstrated a decrease in the percentage of cells in the G0/G1 phase (21 ± 3 vs. 41 ± 3, P < 0.01) and an increase in the percentage of cells with sub-G1 DNA content among PLIP- compared with -galactosidase-expressing cells (13 ± 1.9 vs. 2 ± 0.4, P < 0.01) (Fig. 1 and Table 1). We observed no difference in the percentage of cells in G2/M between PLIP- and -galactosidase-expressing cells (19 ± 0.4 vs 23 ± 0.2) that were analyzed by flow cytometry 2 days after infection. A similar pattern was observed 4 days after infection. Flow cytometry performed 7 days after infection demonstrated a greater increase in the percentage of PLIP- compared with -galactosidase-expressing cells with sub-G1 DNA content (47 ± 2 vs. 3 ± 0.8, P < 0.01), associated with marked decreases in both G0/G1 (7 ± 0.9 vs. 47 ± 3.9, P < 0.01) and G2/M (5 ± 1 vs. 24 ± 0.5, P < 0.01) phase cells. A sub-G1 DNA content is suggestive of apoptosis (25). To confirm that sub-G1 phase cells are apoptotic, we examined PLIP- and -galactosidase-expressing cells by immunofluorescence microscopy using Hoechst dye to visualize nuclei. Greater numbers of apoptotic cells were counted among PLIP- compared with -galactosidase-expressing cells. (Fig. 2). These data suggest that PLIP induces cells to exit the cell cycle via apoptosis at or after the G2/M transition.
Interaction with cPLA2 is not necessary for PLIP's proapoptotic effect. Because PLIP was originally identified as a cPLA2-interacting protein, and because PLIP failed to cause apoptosis in cells that do not express cPLA2 (39), we wished to determine whether PLIP's proapoptotic effect was dependent on its interaction with cPLA2. We identified a fragment of PLIP that does not interact with cPLA2 (Fig. 3C). PLIP407, which contains PLIP amino acids 1407, expresses both the chromo and MYST domain (Fig. 3A). We have previously shown that PLIP localizes in the nucleus of serum-deprived mesangial cells (39). Like PLIP, PLIP407 localizes to discrete foci within the nucleus (Fig. 3B). To determine the effect of PLIP407 expression in mesangial cells, we generated an adenoviral construct, Ad-PLIP407. Mesangial cells were infected with either Ad-Lac, Ad-PLIP, or Ad-PLIP407, and cells were examined by flow cytometry at 2 and 4 days after infection. The expression of PLIP407 results in an increase in a sub-G1 population of cells and a decrease in G0/G1 phase cells, comparable to results observed among PLIP-expressing cells (Fig. 4). These data suggest that the interaction between PLIP and cPLA2 is not necessary for PLIP to achieve its proapoptotic effect in mesangial cells.
Cellular localization of PLIP and Tip60. To examine the localization and protein levels of PLIP, and/or other members of the Tip60 family, we examined mesangial cells by immunofluorescence microscopy using an anti-PLIP antibody, which was generated in rabbits against full-length PLIP protein (39). PLIP is not present in the nucleus of cells grown in the presence of 10% FCS. However, we observed a striking increase in the nuclear signal in cells subjected to either 100 μM H2O2 or to 8-Gy irradiation (Fig. 5A). The increase in the nuclear signal was detected within 12 h and persisted for 12 h following exposure of cells to H2O2. A nuclear signal was detected by 4 h following X-irradiation. A nuclear signal, detected with the anti-PLIP antibody, was abrogated by preincubation of the anti-PLIP antibody with purified PLIP protein but not with albumin, suggesting that the signal is specific to PLIP or to related proteins in the Tip60 family (Fig. 5B). Because the anti-PLIP antibody recognizes both Tip60 and PLIP, the increase in the nuclear signal may be due to an increase in either protein, or to other splice variants that have been recently described (24). Furthermore, this antibody likely recognizes Tip60 degradation products, which cannot be differentiated from PLIP by immunofluorescence. Because the anti-PLIP antibody produces multiple bands in Western blots that likely represent either other Tip60 splice variants or degradation products, we were unable to identify endogenous PLIP by Western blotting of either total cell lysates or cytosolic fractions of H2O2-treated cells. Furthermore, the presence of anti-PLIP antibody-immunoreactive bands is variable and appears dependent, in part, on the passage number. Western blot analysis of nuclear fractions demonstrates an anti-PLIP antibody-associated band at 50 kDa that migrates in parallel with a band that likely represents adenoviral-driven PLIP and is present in nuclear fractions from H2O2-treated, but not untreated cells (Fig. 5C). The density of the band present in nuclear lysates of H2O2-treated cells is not commensurate with the striking increase in the nuclear signal detected by immunofluorescence, suggesting that the nuclear signal observed via immunofluorescence may represent other degradation products or splice variants within the Tip60 family.
To differentiate between PLIP and Tip60 protein, an anti-Tip60 antibody was raised against a peptide fragment in Tip60, which is contained within the exon 5-encoded region (Fig. 3A). To test the specificity of this antibody, we transfected COS cells with either pMT3, pMT3-Tip60, or pMT3-PLIP, resulting in expression of HA or fusion proteins HA-Tip60 or HA-PLIP (Fig. 6A). Whereas antibodies to HA (anti-HA) and full-length PLIP protein (anti-PLIP) detect bands corresponding to both PLIP and Tip60, the antibody to exon 5 (anti-Tip60) detects a single band at 60 kDa corresponding in size to Tip60. Western blot analysis of lysates of early-passage (between passages 2 and 8), untreated mesangial cells does not show any band that is immunoreactive with anti-Tip60 antibody. However, we observed a 60-kDa band in lysates of H2O2-treated cells from the same passage. A more modest increase in the density of this band was observed in lysates of irradiated cells (Fig. 6B). Western blot analysis of lysates of mesangial cells derived from later passages (passages 812) demonstrated more robust Tip60 protein levels (data not shown). However, we were unable to detect an increase in Tip60-specific protein in the nucleus of irradiated or H2O2-treated cells by immunofluorescence microscopy using the anti-Tip60 antibody (Fig. 6C). A possible explanation for these data is that Tip60 is complexed to other proteins, which shield the antigenic site to which the antibody was generated. An alternative explanation is that PLIP and Tip60 traffic differently within the cell. To determine whether the anti-Tip60 antibody is able to detect nuclear-localized Tip60, we examined HA-Tip60-expressing transfected COS cells by immunofluorescence microscopy using anti-PLIP, anti-Tip60, and anti-HA antibodies (Fig. 7). All three antibodies demonstrated diffuse nuclear staining in the majority of transfected cells. A punctate nuclear pattern that we have previously observed in EGFP-PLIP-transfected COS cells (Fig. 3), as well as in serum-deprived mesangial cells with an anti-PLIP antibody (39), was demonstrated in a significant number of cells. A perinuclear signal was detected in a minority of transfected cells. These data suggest that Tip60 is primarily nuclear but also localizes in the cytosol of transfected COS cells and that the anti-Tip60 antibody is able to detect nuclear Tip60 in COS cells. To directly compare the localization of PLIP and Tip60, we examined COS cells transfected to express either HA-Tip60 or HA-PLIP using the anti-PLIP antibody (Fig. 8). Whereas HA-PLIP was localized to the nucleus of almost all HA-PLIP-transfected cells, HA-Tip60 was localized to the cytosol and nucleus of a large number of cells. These data support the possibility, but do not prove, that Tip60 and PLIP may traffic differently within the cell. It remains possible that Tip60 is complexed to interacting proteins in mesangial cells that prevent its detection by an anti-Tip60 antibody and that the expression levels of these putative interacting proteins are increased by injurious stimuli (as is that of Tip60) and are not present in transfected COS cells to a sufficient degree to sequester Tip60. We thus analyzed nuclear and cytosolic fractions of mesangial cells by Western blotting (Fig. 9). Both anti-PLIP and anti-Tip60 antibodies detect a band at 6070 kDa that appears in cytosolic but not nuclear fractions of untreated mesangial cells. The density of this band increases in cytosolic fractions of cells treated with 100400 μM H2O2 and decreases in the cytosolic fractions of cells exposed to 12 mM H2O2. This band does not appear in the nuclear fractions of untreated mesangial cells or of cells exposed to 0.10.4 mM H2O2 but is present in nuclear fractions of cells exposed to 12 mM H2O2. Immunofluorescent microscopy with the anti-Tip60 antibody clearly demonstrates a nuclear signal in many cells treated with 1 mM H2O2.
These data suggest that Tip60 increases in the cytosol of cells exposed to low concentrations of H2O2 but translocates to the nucleus of cells exposed to high concentrations of H2O2. These data also suggest that either PLIP, or other splice variants and/or degradation products of Tip60, enter the nucleus under conditions in which full-length Tip60 remains in the cytosol. It is possible that Tip60 contains a domain that may either inhibit nuclear import (for example by shielding from exposure an expressed nuclear localization signal that is present in both PLIP and Tip60) or hasten nuclear export via an expressed nuclear export signal (NES). NESs are short stretches of leucine-rich sequences in a particular configuration that direct protein export (44). NES-mediated nuclear export is often, but not always, inhibited by leptomycin-B (10, 12); however, we were unable to alter the localization of Tip60 with leptomycin-B (Fig. 10).
DISCUSSION
We report three observations. First, PLIP expression results in increased apoptosis associated with a decrease in G1 phase in renal mesangial cells. Although this effect is detected by 2 days after adenoviral-mediated gene transfer of PLIP cDNA to cells, it is markedly increased after 47 days of cell growth in serum-containing medium. These data suggest that PLIP expression may regulate apoptosis at or after the G2/M transition, suggesting a potential role for PLIP as part of the G2 checkpoint. Checkpoints are surveillance mechanisms that detect DNA damage and induce either cell cycle arrest and DNA repair mechanisms or, in the presence of extensive DNA damage, apoptosis (27). The G2 checkpoint following DNA damage is incompletely understood, and the mechanism by which cells are ordained to undergo arrest or apoptosis is not known, although current data suggest that the processes are linked as mutations of critical proteins produce defects in both checkpoint activation and apoptosis (27).
These data suggest that PLIP may contribute to the regulation of mesangial cell proliferation and/or apoptosis following glomerular injury. Mesangial cell proliferation and apoptosis have both been observed in various glomerular diseases, and the inhibition of mesangial cell proliferation decreases matrix production and glomerulosclerosis (1, 20). The inability to arrest defective cells or to prevent defective cells from cycling altogether could result in increased proliferation following cell injury. Alternatively, enhanced apoptosis following cell injury could lead to glomerulosclerosis that characterizes the progression of glomerular injury. Although we do not have data to implicate PLIP in glomerulonephritis, other proteins that modulate proliferation and apoptosis have been shown to play roles in glomerular injury. For instance, a decrease in the level of the CDK inhibitor p27Kip1 increases mitogen-induced proliferation of cultured mesangial cells (17, 38). Experimental glomerulonephritis is much more severe in p27Kip1/ mice, as manifested by increased proliferation and production of matrix protein, a greater decline in renal function, and a marked increase in apoptosis (28). In addition, the inhibition of cdk-2 activity by roscovitine decreases mesangial proliferation and extracellular matrix proteins and preserves renal function in the experimental model of Thy-1 glomerulonephritis (29). These data suggest that proteins that modulate cell proliferation and apoptosis may affect the outcome of glomerulonephritis.
PLIP407, which does not interact with cPLA2, causes an increase in apoptosis that is identical to that caused by PLIP. These data suggest that the proapoptotic effect of PLIP may be independent of its interaction with cPLA2. We have shown in a previous study that PLIP expression causes apoptosis in murine mesangial cells derived from cPLA2+/+ but not from cPLA2/ mice, suggesting that cPLA2 is necessary for PLIP's effect on apoptosis (39). It is thus possible that, although cPLA2 expression is necessary for PLIP's proapoptotic effect, interaction between the two proteins is not. PLIP has also been shown to increase serum deprivation-induced cPLA2+/+-dependent PGE2 generation (39). We do not yet know the effect of PLIP407 expression on cPLA2-expecific PGE2 generation, and it remains possible that PLIP's effect on PGE2 generation is independent of its effect on apoptosis. Further studies are necessary to determine which effects of PLIP require its interaction with cPLA2.
We also report the second observation that, under similar conditions, splice variants of Tip60 and PLIP localize to different cellular compartments of mesangial cells. Whereas an antibody generated to full-length PLIP demonstrates an increase in the nuclear signal in cells treated with either irradiation or with 100400 μM H2O2, an antibody that is specific for the exon 5 region of Tip60 demonstrates predominantly cytosolic localization. These data need to be interpreted with caution given the lack of specificity of the anti-PLIP antibody. Whereas the anti-Tip60 antibody recognizes Tip60 but not PLIP, the anti-PLIP antibody recognizes both Tip60 and PLIP, as well as the degradation products of both proteins. Tip60 is subject to ubiquitin-proteosome-mediated degradation (23). The nuclear signal detected with the anti-PLIP antibody could represent any one of the Tip60 degradation products that do not contain the exon 5-encoded fragment and thus would not be detected by the anti-Tip60 antibody. The following data, however, support the hypothesis that PLIP and Tip60 localize to different compartments. First, Western blot analysis with anti-Tip60 demonstrates that Tip60 is in the cytosol of control mesangial cells and of cells exposed to low concentrations of H2O2. The density of the band corresponding to Tip60 increases in the cytosolic fractions of cells exposed to low concentrations of H2O2, whereas, if degradation products of lower molecular weight were entering the nucleus, one would expect the density of this band to be decreased. These data are also consistent with immunofluorescence microscopy (shown in Fig. 6C) that suggests an increase in Tip60 antibody-associated signal in the cytosol of H2O2-treated cells. Although the anti-PLIP antibody detects multiple bands on Western blots of nuclear and cytosolic fractions, Western blot analysis of nuclear lysates of Ad-PLIP-infected cells suggests that adenoviral-driven PLIP is primarily nuclear (Fig. 5C). Anti-PLIP antibody also detects a faint band corresponding to adenoviral-driven PLIP in the nuclear lysates of non-infected cells exposed to 200 μM H2O2 (Fig. 5C). That the anti-PLIP antibody-associated band is of such modest density suggests that the nuclear signal, observed by immunofluorescence microscopy, may represent, in part, degradation products of Tip60 that do not express exon 5, or other splice variants within the Tip60 family. These data support the premise, however, that PLIP localizes primarily in the nucleus of mesangial cells.
PLIP and Tip60 are identical except for a 52-amino acid fragment that is encoded by Tip60 exon 5. It is possible that Tip60 contains a NES that is encoded by exon 5. NESs are short stretches of leucine-rich sequences in a particular configuration [L-X (2-3)-L-X(2-3)-L-X-L] that direct protein export (44). Both proteins express an amino acid sequence that conforms to a classic NES [at Tip60 254262 and PLIP 202210 (LTTLPVLYL)]. Tip60 contains an additional sequence within the exon 5-encoded region that does not conform to a classic NES but does conform to an alternative NES sequence that was first characterized in the Rev proteins of feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) (26) and has also recently been shown to be the operative NES in p27 (10). This putative NES at Tip60 106120 (LPIPVQITLRFNLPK) is characterized by leucine residues that are less tightly clustered compared with the classic NES but which exist in a similar spacing to p27, FIV, and EIAV (10, 26), as well as by a higher concentration of basic residues compared with the classic NES (26). NES-mediated nuclear export involves binding to Crm1 and is typically inhibited by leptomycin-B (12). We were unable to alter the localization of Tip60 with leptomycin-B. The band that appears in some nuclear fractions of leptomycin-B-treated cells in Fig. 10 is inconsistently related to leptomycin-B exposure and may suggest underlying injury to cells, as it occasionally also appears in the nuclear fractions of control cells. Immunofluorescence microscopy of cells exposed to high concentrations of leptomycin-B failed to show nuclear localization of Tip60. However, although we were unable to alter the cellular localization of Tip60 with leptomycin-B, recent data suggest that the nonclassic NES may bind to Crm1 at a non-leptomycin-B-inhibitable site (10). Mutational studies are necessary to confirm or exclude an operative NES in either Tip60 or PLIP.
Finally, we report the observation that Tip60 translocates from the cytosol into the nucleus in response to high concentrations of H2O2. These data suggest that Tip60 may have both cytosolic and nuclear roles in mesangial cells. Although ectopic Tip60 has been localized primarily in the nucleus of nonmesangial cells (8, 30), it has also been shown to colocalize with exogenously expressed IL-9 chain in the cytosolic compartment of HeLa cells (40) and with the endothelin receptor A in a perinuclear region of transfected COS7 cells (22), suggesting cytosolic roles in other nonmesangial cells.
It is not clear, based on these experiments, whether PLIP or Tip60 is the biologically pertinent protein in mesangial cells. Given that PLIP and Tip60 are identical except for the exon 5-encoded fragment, it remains possible that adenoviral-driven expression of Tip60 will achieve the same effects on cell cycling as those observed with adenoviral-driven expression of PLIP. Alternatively, it is possible that differences in the localization of PLIP and Tip60 may allow splice variant-specific roles. That endogenous Tip60 is increased by injurious stimuli supports a role for Tip60 in mesangial cells. Furthermore, because we are unable to demonstrate a marked increase in PLIP-specific protein, it remains possible that the observed effects of adenoviral-driven PLIP on cell cycle events are a result of robust expression of protein. Further investigation is necessary to delineate the specific roles of PLIP vs. Tip60 in mesangial cells and to determine whether these roles are achieved via nuclear or cytosolic signaling.
GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54741 and DK-38452.
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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