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Molecular and Cellular Biology, December 2004, p. 10246-10255, Vol. 24, No. 23
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.23.10246-10255.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Proteins Reveals Cellular Proliferation Inhibition and Substrate Specificity
Medical Faculty of the Charité, The Franz Volhard Clinic, HELIOS Klinikum-Berlin, and The Max Delbrueck Center for Molecular Medicine,1 Department of Biology, Chemistry, and Pharmaceutics, Free University of Berlin, Berlin,2 Institute of Biology, University of Lübeck, Lübeck, Germany3
Received 30 March 2004/ Returned for modification 7 May 2004/ Accepted 9 September 2004
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ABSTRACT |
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and ß. Humans have only one importin ß, while six importins have been described. Whether or not distinct importins are essential for specific import pathways in living human cells is unclear. We used RNA interference technology to specifically down-regulate the expression of ubiquitously expressed human importins in HeLa cells. Down-regulation of importins 3, 5, 7, and ß strongly inhibited HeLa cell proliferation, while down-regulation of importins 1 and 4 had only a minor effect or no effect. Nucleoplasmin import was not prevented by down-regulation of any importin, indicating that the importin /ß pathway was generally not affected. In contrast, importin 3 or 5 down-regulation specifically inhibited the nuclear import of the Ran guanine nucleotide exchange factor, RCC1. Coinjection of recombinant importins and RCC1 into down-regulated cells demonstrated that these transport defects were specifically caused by the limited availability of importin 3 in both cases. Thus, importin 3 is the only importin responsible for the classical nuclear import of RCC1 in living cells. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß heterodimer, also known as karyopherin /ß (for reviews, see references 23, 34, and 43). Importin acts as an adapter by binding both the import substrate and importin ß. The trimeric import complex docks to the NPC via importin ß and translocates into the nucleus. Recently, Npap60/Nup50 was identified as an additional mammalian cofactor for importin /ß-dependent nuclear protein import (22). While only one importin ß isoform exists, six human importins have been described (3, 4, 18, 21, 28, 36, 42). In contrast, the yeast Saccharomyces cerevisiae possesses only one gene for importin , which is essential (45).
The importins are grouped into three subfamilies based on sequence homology. The first subfamily consists of importin 1/Rch1. Its most closely related homologue, importin 2, has been found in Xenopus laevis and other vertebrates but not in mammals. Importins 3/Qip1 and 4/hSRP1
are members of the second subfamily. The third subfamily consists of importins 5/hSRP1, 6, and 7 (18). The isoforms of one subfamily are highly homologous, showing about 85% sequence identity and differing mostly in regions outside the NLS binding pockets (18, 21, 26). Although the importins differ in their cell- and tissue-specific expression patterns, most are expressed ubiquitously (16, 18, 20, 21, 28, 31, 40). Only importin 6 expression seems to be restricted to the testis (21). The reason for the importin diversity in higher eukaryotes, especially in humans and other vertebrates, is unknown. Invertebrates such as Caenorhabditis elegans and Drosophila melanogaster have three importin isoforms with about 50% homology to each other. Knockout and knockdown experiments targeting distinct importin genes have resulted in severe phenotypes. These studies suggest that importin 3 is required during development, and it seems to be essential for oogenesis in Drosophila (25) and C. elegans (8). In contrast, Gorjanacz et al. and Mason et al. found that Drosophila importin 2 (homologue to human importin 1) seems to be essential for gametogenesis (10, 24). The Adam and the Mattaj laboratories showed the importance of C. elegans importins IMA-3 (8) and IMA-2 (1, 9) for embryonic development.
Vertebrates as well as humans possess more than three different importins. Several in vitro studies have shown that the individual human importins are able to import the same target proteins. This state of affairs might argue against specialized roles of distinct importins in human cells. Interestingly, many of these studies also provided in vitro evidence that the importins differ in their substrate-specific import efficiency (7, 18, 19, 27, 28, 37, 44). Whether or not these results allow conclusions for living cells is unclear. Protein concentrations in these import assays are different from those in vivo, and probably the most important factor is the lack of competing substrates. Two competing substrates added simultaneously in the in vitro assays can change the import capacity of importins (18). Whether importins can substitute for one another in vivo is still unknown. To address this issue, we conducted a systematic study of the effects of specific importin down-regulation in cultured human cells.
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MATERIALS AND METHODS |
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Preparation of protein lysates and Western blotting.
For preparation of protein lysates, cells were extracted with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris [pH 8.0]-150 mM NaCl-0.1% sodium dodecyl sulfate [SDS]-1% Igepal-0.5% deoxycholic acid) including an EDTA-free protease inhibitor cocktail (Roche) for 10 min on ice. Nuclear and cytosolic extracts were prepared as described previously (35). Protein concentrations were estimated by using the DC protein assay (Bio-Rad) according to the manufacturer's instructions. SDS-polyacrylamide gel electrophoresis with equal amounts of total protein lysates per lane was performed, and proteins were electroblotted onto a Poly-Screen polyvinylidene difluoride transfer membrane (NEN). The membranes were incubated with the previously described affinity-purified polyclonal antibodies against importins 1, 3, 4, 5, 7, and ß (11, 18). Since the antibody directed against the C terminus of importin 7 shows some cross-reactivity with recombinant importins 4 and 5, we confirmed our findings with a second antibody against the N-terminal region. To control for equal loading and protein transfer, we used Coomassie staining of the SDS-polyacrylamide gel and the antibody against trap (17). For detection of apoptosis, we used antibodies against poly(ADP-ribose) polymerase (PARP) (Santa Cruz Biotechnology) and DNA fragmentation factor (DFF) (Affinity Bioreagents). Detection was achieved by chemiluminescence (DuPont). To determine the amounts of the various importins by Western blotting, specific band intensities derived from cellular protein lysates were compared to bands of diluted standard curves. After incubation with specific antibodies, intensities of specific bands were quantified by using a luminescent image analyzer (LAS-1000 CH) from Fuji Photo Film Co.
Immunofluorescence analysis and Hoechst staining.
Cells were grown on coverslips. After a wash with PBS, cells were fixed with 3.7% formaldehyde for 10 min at room temperature and permeabilized with 0.1% Triton X-100 for 10 min. Staining was performed by using the polyclonal anti-trap antibody in a 1:200 solution for 1 h at room temperature, followed by incubation with a secondary antibody in a 1:1,000 dilution for 1 h (Alexa 594-labeled goat-anti-rabbit antibody; Molecular Probes). Hoechst staining in a 1-µg/ml solution was performed for 5 min at room temperature. After extensive washing, cells were mounted and analyzed by using a fluorescence microscope (Axioplan 2; Zeiss) equipped with a charge-coupled device camera (Axiocam; Zeiss).
FACS analysis. Cells were trypsinized, washed, and fixed with ice-cold 70% ethanol for several days at 4°C. After a wash, fixed cells were stained with 20 µg of propidium iodide/ml in a buffer containing PBS, 0,1% Triton, 0,1 mM EDTA, and 200 µg of RNase A/ml for 30 min at room temperature in the dark. Fluorescence-activated cell sorter (FACS) analyses were performed with a FACSCalibur system (Becton Dickinson). Data were analyzed by using ModFit LT software (Verity Software House).
siRNA sequences.
Small interfering RNA molecules (siRNAs) corresponding to importins 1, 3, 4, 5, 7, and ß were designed as recommended (5) and obtained from Dharmacon Research. The siRNA sequences targeting importins 1, 3, 4, 5, 7, and ß corresponded to the following coding regions after the start codons: for 1, nucleotides 581 to 603; for 3, nucleotides 463 to 484 and 588 to 610; for 4, nucleotides 417 to 439; for 5, nucleotides 769 to 791; for 7, nucleotides 619 to 641; and for ß, nucleotides 870 to 892.
siRNA transfection.
Transfections were performed either with Oligofectamine (Invitrogen) or by electroporation; the two methods yielded similar results. For transfection with Oligofectamine, 3.5 x 104 cells were seeded per well into a 24-well plate 1 day before transfection with siRNAs. At day 0, transfection of siRNAs with Oligofectamine was carried out as described previously (5). After 3 days, cells were split, counted, and transferred to a fresh 24-well plate (3.5 x 104 cells per well) for a second transfection at day 4. After another 3 days, cells were harvested and counted, protein lysates were prepared by using RIPA buffer, and Western blotting was performed. Transfection efficiency was determined by using another siRNA targeting an unrelated protein, trap . Immunofluorescence studies were performed using the previously described anti-trap antibody (17) 7 days after transfection; they showed that in more than 95% of the cells exposed, levels of the target protein were strongly reduced, while in less than 5% of the cells, the trap level was comparable to that in untransfected cells.
For electroporation, HeLa cells were trypsinized, washed, and resuspended in medium to a concentration of 2.5 x 106 cells. A 0.5-ml volume of the cell suspension was mixed with 100 µl of siRNA to a final concentration of 2 µM, placed in 4-mm-gap electrode cuvettes, and electroporated with a Bio-Rad Gene Pulser II system at a setting of 240 V and 950 µF capacitance. Electroporated cells were seeded into six 3.5-cm-diameter tissue culture dishes. Seventy-two hours after electroporation, the medium was replaced by fresh medium supplemented with 100 µg of streptomycin/ml and 100 U of penicillin/ml. Twenty-four hours later, cells were used for microinjections. One of the dishes was used for Western blotting to confirm down-regulation of the targeted importin (data not shown).
Microinjection studies.
Recombinant importins 3 and 5, RCC1, and nucleoplasmin were expressed, purified, and assayed for activity by using in vitro nuclear import assays as described previously (21). RCC1 was labeled with fluorescein-maleimide, and nucleoplasmin was labeled with Texas red-maleimide (Molecular Probes) according to the manufacturer's instructions. The concentrations of labeled RCC1 and nucleoplasmin were 25 and 20 µM, respectively. Concentrations of purified proteins in the injection mixtures were 6 µM ( importins) or 18 µM (RCC1). The concentration of Alexa 594-labeled immunoglobulin G (IgG) (Molecular Probes) was 200 µg/µl. All proteins were diluted in injection buffer (20 mM HEPES-KOH [pH 7.4]-200 mM KCl). Microinjections were performed at room temperature with Femtotips (Eppendorf) by using a Nikon Narishige microinjector. Images were captured by using a 40x long-distance objective lens on a Nikon Diaphot 300 inverted microscope equipped with a Bio-Rad MRC1024 confocal system. Each microinjection study was performed at least twice, with a minimum of 10 cells investigated per experiment.
Statistics. Statistical analyses were performed by analysis of variance followed by a post hoc t test.
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RESULTS |
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protein levels in cultured HeLa cells by RNA interference (RNAi).
The sequences used for siRNA design within the cDNAs of the importins are summarized in Fig. 1A. For importin 3, we designed two siRNAs to check for primer-specific effects. Seventy-two hours after transfection with siRNAs, adherent cells were harvested and analyzed or seeded again at the identical cell density. The next day, a second round of transfection with siRNAs was performed for another 72 h. To estimate transfection efficiency, we designed a control siRNA that targeted an unrelated protein, trap . Immunofluorescence studies using our anti-trap antibody revealed a strong signal reduction in more than 95% of the cells, whereas in less than 5% of the cells the signal was similar to that in untransfected cells (Fig. 2). Importin down-regulation was verified by immunoblotting. Importin protein levels were compared with those of dilution series derived from untransfected cells (Fig. 1B and D; also data not shown).
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1 and 5 protein levels were strongly reduced, whereas levels of the other isoforms were only slightly diminished (Fig. 1B and data not shown). However, after 7 days, the expression of all other importins was strongly down-regulated to about 20% of the control values (Fig. 1C). The two different siRNAs directed against importin 3 showed very similar effects (Fig. 1D). In contrast, down-regulation of importin ß was remarkably weaker, albeit reproducible (Fig. 1D and data not shown). Specific down-regulation of a single importin isoform by siRNA transfection did not affect the expression levels of the other isoforms or their subcellular distribution (Fig. 1C and 3).
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1 and 5 were significantly diminished, to about 50%, within 6 h (Fig. 4). This effect was inhibited by addition of the proteasome inhibitor ALLN, demonstrating that the turnover of importins 1 and 5 was proteasome dependent. In contrast to importins 1 and 5, the other importins, as well as importin ß, were stable after 48 h of cycloheximide treatment (Fig. 4). Thus, the reduction in the protein level in siRNA treated cells was mostly dependent on cell division.
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importins.
To analyze the effect of importin down-regulation on cell fate, we counted siRNA-transfected and mock-transfected control cells after 7 days. Down-regulation of importin 3, 5, or 7 resulted in a strong decrease in the number of living HeLa cells (Fig. 5). The decreased cell numbers after importin 3 down-regulation were similar with the two different siRNAs we used. A slight but significant effect was also seen for importin 1 down-regulation. In contrast to that of the other importins, down-regulation of importin 4 had no inhibitory effect on HeLa cell proliferation. Importin ß down-regulation also caused a strong decrease in cell number similar to that caused by importin 3, 5, or 7. Interestingly, the amount of importin ß in surviving cells was much less reduced after siRNA treatment compared to the various importins, suggesting that a small reduction in importin ß levels is more harmful for the cell than a reduction in the level of any importin.
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1 or 4 and control cells. Down-regulation of other importins showed tendencies toward small increases in apoptotic cells. However, only down-regulation of importin 3 and importin ß resulted in statistically significant differences (Fig. 6B). For cell cycle analysis, we transfected cells by using electroporation but the same time schedule as for transfection with Oligofectamine. The reduction in cell numbers after transfection with siRNAs directed against importin 3, 5, or 7 by electroporation was similar to that after transfections with Oligofectamine (data not shown). Additionally, we could not detect any obvious cell cycle defect by FACS analyses of propidium iodide-stained control and siRNA-transfected cells (Fig. 6C). Analysis of the raw data with ModFit software displayed small shifts of maximally 3 to 8% of the cells from S to G1 phase, which reached significant levels only for importin 4, 7, or ß down-regulation.
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3 causes delayed nuclear import of RCC1.
A model that could explain the reduced proliferation is based on the idea that importins have some nonoverlapping substrate specificity. Several in vitro studies have found that the nuclear import of RCC1, the Ran guanine nucleotide exchange factor, is preferentially mediated by importin 3 but not by the other importin isoforms (18, 38), showing that RCC1 is a good candidate for analyzing specific nuclear transport. As a control we analyzed nucleoplasmin, which is imported at least in vitro by various importins. We monitored the distribution of fluorescently labeled proteins between the cytoplasm and nucleus after microinjection of these proteins into the cytoplasm of HeLa cells. In untreated cultured HeLa cells, both RCC1 and nucleoplasmin accumulated in the nucleus in less than 1 min. After 3 min, almost all injected RCC1 and nucleoplasmin were concentrated within the nucleus. IgG stayed exclusively in the cytoplasm, demonstrating that the nuclear envelopes remained intact (Fig. 7A).
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importins. HeLa cells were transfected with siRNAs by electroporation, and microinjections were performed 4 days after the first transfection. At that time point, all importins were down-regulated to 10 to 20% of the control cell levels (data not shown). Protein down-regulation using electroporation was more efficient in our hands than down-regulation by Oligofectamine transfection despite similar transfection efficiencies (Fig. 2 and data not shown).
Analysis of mock-transfected cells showed rapid nuclear accumulation of RCC1 as in untreated cells. Cells transfected with siRNAs directed against importin 1 or 4 also showed nuclear accumulation of RCC1 in less than 1 min, similar to that in untreated cells. Nevertheless, about 10% of the cells showed reduced RCC1 import, indicating nonspecific side effects. However, rapid nuclear accumulation of RCC1 (less than 1 min) was significantly impaired in cells in which importin 3 or 5 was down-regulated (Fig. 7B and C). Even 3 min after injection, RCC1 was not imported into the nucleus in about half of the cells lacking importin 3 or 5. At this time point, in mock-transfected cells and in importin 1 or 4 knockdown cells, RCC1 was transported into the nuclei of most cells. Cells treated with siRNA directed against importin 7 showed a weak RCC1 import defect. However, because of the substantial variability between different sets of experiments, even though the down-regulation was reproducible, we believe that this import defect was not significant. Nucleoplasmin accumulation in the nucleus was never inhibited after down-regulation of any importin. Control injections of IgGs displayed exclusive cytoplasmic staining, showing that nuclear envelopes and pores remained intact in all experiments performed.
If the effects observed are directly linked to the down-regulation of importin 3 or 5, the RCC1 import deficiency should be rescued by the readdition of these proteins. Thus, recombinantly expressed importins were added to the injection mixture. Coinjection of importin 3 completely rescued the transport defect observed after down-regulation of either importin 3 or (surprisingly) importin 5 (Fig. 8). Furthermore, this importin 3 rescue resulted in rapid accumulation of RCC1 in the nucleus, regardless of whether or not importin 3 or 5 had been knocked down before. In contrast, coinjection of importin 5 could not rescue this RCC1 import defect in cells lacking importin 3 or 5. The percentage of cells coinjected with importin 5 that displayed no nuclear RCC1 signal after 1 and 3 min was similar to that for cells injected with RCC1 alone. Thus, readdition of importin 5 to cells lacking importin 5 had no effect on the RCC1 transport defect.
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DISCUSSION |
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importins and their functional interplay in human cells. We specifically focused on the question of whether the importins are able to substitute for each other in vivo. Based on our data, they are not. Even importins 3 and 4, which show a high degree of conservation (about 85% sequence identity), appear to target different import substrates. With our siRNAs we were able to specifically down-regulate each individual importin without affecting the others. All ubiquitously expressed importins were down-regulated to the same relative degree compared with mock-transfected cells. Furthermore, no up-regulation of any importin was observed upon RNAi treatment. This finding indicates that each importin is part of specific cellular pathways. Our conclusion is in agreement with earlier observations that showed different expression responses of the various importins to external signals (17, 20).
The absolute expression levels of the various importins in HeLa cells are remarkably different, with importin 1 being the most abundant isoform (17). Therefore, down-regulation of importin 1 resulted in a higher molar loss of the absolute amount of cellular importin than knockdown of, for instance, importin 3. However, the effect of importin 1 down-regulation on proliferation inhibition was much weaker than the effect detected after down-regulation of importin 3. In contrast, down-regulation of importin 4, which is the least abundant isoform expressed in HeLa cells, had no inhibitory effect on cell proliferation. Taken together, these data show that there is no correlation between the absolute amount of importin down-regulation and the effects observed on inhibition of cellular proliferation. This finding supports the conclusion that importins differ in their functional relevance for distinct import pathways.
Our microinjection studies showed that the classical import pathway was not generally affected in these importin down-regulated cells, since nucleoplasmin was imported in less than 1 min in all knockdown cells. This observation is in line with earlier in vitro findings that nucleoplasmin can be imported by all importin isoforms (18). However, the picture changed completely when we analyzed the import of the human Ran guanine nucleotide exchange factor, RCC1. In untreated cells, RCC1 transport kinetics were similar to those observed for nucleoplasmin. In mock-transfected cells and in cells in which importin 1 or 4 was down-regulated, RCC1 was imported into the nucleus at the same rate. Interestingly, upon importin 3 or 5 down-regulation, we observed a significant inhibition of RCC1 import in more than 50% of the cells analyzed. The specific transport inhibition caused by importin 3 or 5 down-regulation is in contrast to earlier in vitro data (18, 38). To exclude indirect effects caused by the knockdown of any of the importins, we rescued the import deficiencies by adding back the importin that had been removed by siRNA treatment. Coinjection of recombinant importin 3 rescued the RCC1 import inhibition in cells lacking importin 3. Surprisingly, recombinant importin 3 also complemented the import deficiency in importin 5 knockdown cells, while readdition of purified importin 5 had no effect. Thus, RCC1 nuclear import inhibition caused by importin 3 down-regulation was direct, whereas importin 5 down-regulation seemed to have an indirect influence on the importin 3 pathways by limiting the pool of free importin 3 available within the cell. Several lines of evidence argue against nonspecific effects. First, the two different siRNAs targeting importin 3 resulted in similar defects. Second, down-regulation of the different importins resulted in distinct phenotypes. Third, the complementation with recombinant importins reverted the observed import deficiencies.
Since our data suggest that the importins are unable to substitute for each other, we were not surprised to detect a strong reduction in the number of living cells after down-regulation of distinct importins. In principle, there are two different possibilities responsible for this finding, namely, an increase in cell death or a decrease in cell proliferation. One or both of them could be involved. We could not detect a dramatic rise in apoptosis or a remarkable increase in the level of necrotic cells. Nevertheless, the observed mild increase in apoptotic cells after importin down-regulation may contribute to the decrease in cell number. In contrast, we found no evidence that alteration of cell cycle progression may contribute to the decrease in cell number after importin down-regulation. Moreover, the slight shift from S to G1 phase after down-regulation of importin 4 or ß does not explain our findings, since importin ß down-regulation caused a strong decrease in cell number whereas down-regulation of importin 4 did not. For these reasons, we believe that diminished cell proliferation is at least in part responsible for the reduced cell number after down-regulation of distinct importins.
Down-regulation of importin 3, 5, or 7 strongly inhibited HeLa cell proliferation. In contrast, down-regulation of importin 1 or 4 caused only a mild growth defect, or none. This state of affairs may exist in other human cells, since proliferation of cultured human renal mesangial cells was inhibited to a similar extent (data not shown). Down-regulation of importin ß caused similar strong inhibition of cell proliferation. The importin ß level in surviving cells was reduced only to 50 to 70% of that in control cells. Importin ß is an essential factor and is responsible not only for the importin -dependent import pathways but also for additional, importin -independent pathways (15, 30, 33). Therefore, down-regulation of importin ß should cause strong selection against importin ß knockdown cells. Obviously, a small reduction in importin ß expression is more harmful to living cells than reduction of any importin.
RCC1 itself is an important factor for cell viability. RCC1 catalyzes the exchange from RanGDP to RanGTP, which is necessary not only for nucleocytoplasmic transport (14) but also for spindle and nuclear envelope assembly during mitosis (for reviews see references 13 and 32). However, we do not believe that RCC1 import inhibition is the reason for reduced cell proliferation, for two reasons. First, RCC1 nuclear import is never completely blocked, since RCC1 was still imported in importin 3 down-regulated cells after 15 to 20 min (Fig. 3). Second, no RCC1 accumulation in the cytoplasm and no differences in nuclear RCC1 levels were detectable in RNAi-treated cells (data not shown). These findings may result from residual importin 3 activity or from the importin /ß-independent RCC1 import pathway described previously (29). Moreover, other lines of evidence favor cargo specificity of importin proteins. Defects in Drosophila oogenesis were strictly dependent on importin 2, as shown by complementation analysis with every fly importin (24). Binding studies with the transcription factor STAT1 suggested specific nuclear import by importin 5 (26, 37). The dominant-negative approach by injecting antibodies against importins 5 and 1 supported this finding (37).
On the basis of our findings, we assume that receptor-specific import pathways of various substrates that are essential for cellular proliferation are blocked, resulting in proliferation inhibition. Additionally, specific importin -interacting proteins such as the spindle assembly factor TPX2 (12, 39) or the kinesin XCTK2 (6) could be faultily localized and thereby contribute to the proliferation defect. Our findings suggest the presence of an increase in specificity of the importins during vertebrate evolution.
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ACKNOWLEDGMENTS |
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The Deutsche Forschungsgemeinschaft (M.K. and T.S.), the Max Delbrueck Center for Molecular Medicine (C.Q. and M.K.), the ESF Project Berlin (project 20010019) (M.K. and B.F.), and the GIF (E.H.) gave support.
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FOOTNOTES |
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