Directed Inhibition of Nuclear Import in Cellular Hypertrophy
2001; Elsevier BV; Volume: 276; Issue: 23 Linguagem: Inglês
10.1074/jbc.m101950200
ISSN1083-351X
AutoresCarmen Pérez-Terzic, A. Marquis Gacy, Ryan Bortolon, Petras P. Dzeja, Michel Pucéat, Marisa Jaconi, Franklyn G. Prendergast, André Terzic,
Tópico(s)Trace Elements in Health
ResumoEach nuclear pore is responsible for both nuclear import and export with a finite capacity for bidirectional transport across the nuclear envelope. It remains poorly understood how the nuclear transport pathway responds to increased demands for nucleocytoplasmic communication. A case in point is cellular hypertrophy in which increased amounts of genetic material need to be transported from the nucleus to the cytosol. Here, we report an adaptive down-regulation of nuclear import supporting such an increased demand for nuclear export. The induction of cardiac cell hypertrophy by phenylephrine or angiotensin II inhibited the nuclear translocation of H1 histones. The removal of hypertrophic stimuli reversed the hypertrophic phenotype and restored nuclear import. Moreover, the inhibition of nuclear export by leptomycin B rescued import. Hypertrophic reprogramming increased the intracellular GTP/GDP ratio and promoted the nuclear redistribution of the GTP-binding transport factor Ran, favoring export over import. Further, in hypertrophy, the reduced creatine kinase and adenylate kinase activities limited energy delivery to the nuclear pore. The reduction of activities was associated with the closure of the cytoplasmic phase of the nuclear pore preventing import at the translocation step. Thus, to overcome the limited capacity for nucleocytoplasmic transport, cells requiring increased nuclear export regulate the nuclear transport pathway by undergoing a metabolic and structural restriction of nuclear import. Each nuclear pore is responsible for both nuclear import and export with a finite capacity for bidirectional transport across the nuclear envelope. It remains poorly understood how the nuclear transport pathway responds to increased demands for nucleocytoplasmic communication. A case in point is cellular hypertrophy in which increased amounts of genetic material need to be transported from the nucleus to the cytosol. Here, we report an adaptive down-regulation of nuclear import supporting such an increased demand for nuclear export. The induction of cardiac cell hypertrophy by phenylephrine or angiotensin II inhibited the nuclear translocation of H1 histones. The removal of hypertrophic stimuli reversed the hypertrophic phenotype and restored nuclear import. Moreover, the inhibition of nuclear export by leptomycin B rescued import. Hypertrophic reprogramming increased the intracellular GTP/GDP ratio and promoted the nuclear redistribution of the GTP-binding transport factor Ran, favoring export over import. Further, in hypertrophy, the reduced creatine kinase and adenylate kinase activities limited energy delivery to the nuclear pore. The reduction of activities was associated with the closure of the cytoplasmic phase of the nuclear pore preventing import at the translocation step. Thus, to overcome the limited capacity for nucleocytoplasmic transport, cells requiring increased nuclear export regulate the nuclear transport pathway by undergoing a metabolic and structural restriction of nuclear import. 1,4-piperazinediethanesulfonic acid high pressure liquid chromatography fluorescein-tagged histone 1 field-emission scanning electron microscopy atomic force microscopy Hypertrophy is a fundamental adaptive process that enables heart muscle to accommodate demands for increased workload or to compensate for the loss of cardiac cells (1Hunter J.J. Chien K.R. N. Engl. J. Med. 1999; 341: 1276-1283Crossref PubMed Scopus (740) Google Scholar, 2McKinsey T.A. Olson E.N. Curr. Opin. Gen. Dev. 1999; 9: 267-274Crossref PubMed Scopus (84) Google Scholar, 3Swynghedauw B. Physiol. 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Science. 1995; 270: 1835-1838Crossref PubMed Scopus (173) Google Scholar, 28Rakowska A. Danker T. Schneider S. Oberleithner H. J. Membr. Biol. 1998; 163: 129-136Crossref PubMed Scopus (73) Google Scholar, 29Perez-Terzic C. Gacy A.M. Bortolon R. Dzeja P.P. Puceat M. Jaconi M. Prendergast F.G. Terzic A. Circ. Res. 1999; 84: 1292-1301Crossref PubMed Scopus (50) Google Scholar). Transport can be activated and inactivated during the cell cycle (30Moore J.D. Bioessays. 2001; 23: 77-85Crossref PubMed Google Scholar), indicating that traffic through the nuclear pores is a dynamic process determined by the functional and metabolic state of a cell. We report a down-regulated nuclear import in hypertrophy, which is restored by the removal of the hypertrophic signal or blockade of nuclear export. Thus, cardiac cells suppress nuclear import under conditions of increased demand for nucleocytoplasmic communication to secure the availability of the nuclear transport pathways required for the generation of the hypertrophic phenotype. Hearts were removed from 1–2-day-old rats, and cardiomyocytes were isolated and cultured (29Perez-Terzic C. Gacy A.M. Bortolon R. Dzeja P.P. Puceat M. Jaconi M. Prendergast F.G. Terzic A. Circ. Res. 1999; 84: 1292-1301Crossref PubMed Scopus (50) Google Scholar). Hypertrophy was induced with phenylephrine (100 μm), an α-adrenoreceptor agonist (in the presence of 10 μm propranolol, a β-adrenoreceptor antagonist), or with angiotensin II (100 nm). Myocyte size and sarcomeric α-actin content were used as markers of hypertrophy (4Izumo S. Lompre A.M. Matsuoka R. Koren G. Schwartz K. Nadal-Ginard B. Mahdavi V. J. Clin. Invest. 1987; 79: 970-977Crossref PubMed Scopus (373) Google Scholar). Size was quantified by measuring the cell surface area with laser confocal microscopy (LSM 410 Carl Zeiss) and a × 40 (1.3 NA) objective. The expression of α-actin was determined by phalloidin staining that recognizes sarcomeric actin. To this end, cells fixed with 3% paraformaldehyde were incubated (20 min) with 20 nm phalloidin tagged with fluorescein, washed in 3% Tween in phosphate-buffered saline, and imaged by laser confocal microscopy using a × 63 (1.4 NA) objective. The light source was an argon/krypton laser tuned at 488 nm, and emission light was collected using a 510-nm-long pass dichroic beam splitter and a 515-nm-long pass emission filter. Two-dimensional confocal images were acquired by scanning 512 × 512 pixels per image and processed on a Silicon Graphics Iris Computer with ANALYZE software (Mayo Foundation). Control or hypertrophied cardiomyocytes were transferred to prewarmed Dulbecco's modified Eagle's medium with 0.5% bovine serum albumin, 10 mm HEPES (pH 7.5), and 20 mm 2,3-butanedione monoxime (Sigma). Microinjections into the cytosol were carried out with a nanometer-precision microinjector unit (Eppendorf 5242) coupled to a micromanipulator (Eppendorf 5171) mounted on a fluorescence microscope (Carl Zeiss Axiovert 100) (29Perez-Terzic C. Gacy A.M. Bortolon R. Dzeja P.P. Puceat M. Jaconi M. Prendergast F.G. Terzic A. Circ. Res. 1999; 84: 1292-1301Crossref PubMed Scopus (50) Google Scholar, 31Jaconi M. Bony C. Richards S. Terzic A. Arnaudeau S. Vassort G. Puceat M. Mol. Biol. Cell. 2000; 11: 1845-1858Crossref PubMed Scopus (83) Google Scholar). Pipettes were filled with injection buffer (150 mm KCl, 1 mmPIPES,1 0.1 mmEDTA, 0.025 mm EGTA, pH 7.2) containing fluorescein-coupled histones H1 (0.07 mg/ml) or fluorescein-coupled dextrans (5 mm). Cardiomyocytes were superfused with 116 mm NaCl, 4 mm KCl, 2 mmMgCl2, 2 mm NaH2PO4, 4 mm NaHCO3, 21 mm HEPES, and 1 mm CaCl2 (pH 7.4, 37 °C). Nuclear transport was measured using × 40 (1.3 NA) or × 63 (1.4 NA) objectives on a laser confocal imaging system (LSM 410). The thickness of the optical sections of imaged cells was set at 1–2 μm to discriminate fluorescence emitted from nuclear versusnonnuclear regions. Fluorescent probes were excited (at 488 nm) using an argon/krypton visible laser (Omnichrome), and emission spectra were collected using a 510-nm-long pass dichroic beam splitter and a 515-nm-long pass emission filter. Confocal images were acquired by scanning a field at 16 s/frame. Fluorescence intensity in the nucleusversus the cytosol was determined with ANALYZE on a Silicon Graphics Iris computer. Nuclear accumulation was expressed as the ratio of nuclear over cytosolic fluorescence (27Stehno-Bittel L. Perez-Terzic C. Clapham D.E. Science. 1995; 270: 1835-1838Crossref PubMed Scopus (173) Google Scholar, 29Perez-Terzic C. Gacy A.M. Bortolon R. Dzeja P.P. Puceat M. Jaconi M. Prendergast F.G. Terzic A. Circ. Res. 1999; 84: 1292-1301Crossref PubMed Scopus (50) Google Scholar). To localize the monomeric GTPase Ran, cardiomyocytes were fixed, permeabilized, and labeled with an anti-Ran monoclonal antibody (32Hieda M. Tachibana T. Yokoya F. Kose S. Imamoto N. Yoneda Y. J. Cell Biol. 1999; 144: 645-655Crossref PubMed Scopus (50) Google Scholar). Optical z-sections of cells (0.2 μm) were acquired with a 1300 YHS CCD camera (Princeton Instruments) using an objective mounted on a piezoelectric controller driven by the Metamorph software (Universal Imaging) (33Meyer N. Jaconi M. Landopoulou A. Fort P. Puceat M. FEBS Lett. 2000; 478: 151-158Crossref PubMed Scopus (109) Google Scholar). Images were processed by the Imaris software (Bitplane) using the isosurface module (for three-dimensional reconstruction) following digital deconvolution (Huygens, Scientific Volume Imaging) (33Meyer N. Jaconi M. Landopoulou A. Fort P. Puceat M. FEBS Lett. 2000; 478: 151-158Crossref PubMed Scopus (109) Google Scholar). To determine nucleotide levels, perchloric acid cardiomyocyte extracts were prepared (34Dzeja P.P. Vitkevicius K.T. Redfield M.M. Burnett J.C. Terzic A. Circ. Res. 1999; 84: 1137-1143Crossref PubMed Scopus (177) Google Scholar). Cells, washed with ice-cold phosphate-buffered saline and immersed into liquid nitrogen, were layered with 0.6 m HClO4 and 1 mmEDTA and then centrifuged (12,000 rpm, 4 °C) (Hermline Z230 MA microcentrifuge, Labnet). The supernatant was neutralized with 2m K2HCO3, and the precipitate was removed by centrifugation. ATP was measured in the supernatant by using a coupled enzyme assay in 25 mm Tris-HCl buffer (pH 7.5), 2 mm MgCl2, 2 mm glucose, 1 mm dithiothreitol, 50 μmNADP+, 20 μm diadenosine pentaphosphate, 4 units/ml of hexokinase, and 2 units/ml of glucose-6-phosphate dehydrogenase. NADPH levels, reflecting ATP concentration, were measured using a fluorometer with a minicell kit (Turner TD-700). ATP, GTP, and GDP were also determined by HPLC (System Gold, Beckman) using a QHR5/5 column (Amersham Pharmacia Biotech). Nucleotides were eluted with a linear gradient of triethylammonium bicarbonate buffer. Cells were extracted with 150 mm NaCl, 60 mm Tris-HCl (pH 7.5), 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml aprotinin, and 0.2% Triton X-100 and were then centrifuged (10,000 × g, 4 °C). Creatine kinase activity was measured with a Beckman DU 7400 spectrophotometer in 100 mm Tris acetate (pH 7.5), 20 mm glucose, 2 mm EDTA, 10 mm MgCl2, 2 mm dithiothreitol, 2 mm NADP+, 2 mm ADP, 5 mm AMP, 20 mm creatine phosphate, 20 μm diadenosine pentaphosphate, 4.5 units/ml hexokinase, and 2 units/ml glucose-6-phosphate dehydrogenase (35Dzeja P.P. Pucar D. Redfield M.M. Burnett J.C. Terzic A. Mol. Cell. Biochem. 1999; 201: 33-40Crossref PubMed Google Scholar). Adenylate kinase was measured in 100 mm potassium acetate, 20 mm HEPES (pH 7.5), 20 mm glucose, 4 mm MgCl2, 2 mm NADP+, 1 mm EDTA, 1 mm dithiothreitol, 2 mmADP, 4.5 units/ml hexokinase, and 2 units/ml glucose-6-phosphate dehydrogenase (35Dzeja P.P. Pucar D. Redfield M.M. Burnett J.C. Terzic A. Mol. Cell. Biochem. 1999; 201: 33-40Crossref PubMed Google Scholar). Cardiac nuclei were imaged with transmitted and field-emission scanning electron microscopy (FESEM). Cardiomyocytes were fixed in 0.1 m phosphate-buffered saline containing 1% glutaraldehyde and 4% formaldehyde (pH 7.2). For transmitted scanning electron microscopy, cells were postfixed in phosphate-buffered 1% OsO4, stained en blocwith 2% uranyl acetate, dehydrated in ethanol and propylene oxide, and embedded in low viscosity epoxy resin. Thin (90-nm) sections were cut on an ultramicrotome (Reichert Ultracut E), placed on 200-μm mesh copper grids, and stained with lead citrate. Micrographs were taken on a JEOL 1200 EXII electron microscope operating at 60 kV. For FESEM, cardiomyocytes were stripped of sarcolemma by using a hypotonic solution followed by a 5-min treatment with 1% Triton X-100 (29Perez-Terzic C. Gacy A.M. Bortolon R. Dzeja P.P. Puceat M. Jaconi M. Prendergast F.G. Terzic A. Circ. Res. 1999; 84: 1292-1301Crossref PubMed Scopus (50) Google Scholar). Sarcolemma-stripped cardiomyocytes were fixed in situ with 1% glutaraldehyde and 4% formaldehyde in phosphate-buffered saline (pH 7.2). The specimen was rinsed in 0.1 m phosphate buffer (pH 7.2), and the buffer was supplemented with 1% osmium. Cells, which were dehydrated with ethanol and dried in a critical point dryer, were coated with platinum using an Ion Tech indirect argon ion voltage of 9.5 kV and 4.2 mA and then examined at accelerating voltages (1.0, 2.4, 3.5, and 5.0 kV) on a JEOL JSM 6400 field-emission scanning microscope. Contact-mode atomic force microscopy (AFM) was performed in air with silicon nitride NP-S tips (spring constant, 0.58 newtons/m) using a Digital Instruments Multimode AFM with a Nanoscope III controller (29Perez-Terzic C. Gacy A.M. Bortolon R. Dzeja P.P. Puceat M. Jaconi M. Prendergast F.G. Terzic A. Circ. Res. 1999; 84: 1292-1301Crossref PubMed Scopus (50) Google Scholar). The nuclear envelope of sarcolemma-stripped and fixed cardiomyocytes was scanned with an E-type (15 × 15 μm maximum area) scanner. Images were collected by raster scanning at 512 pixels/line with linear scanning frequencies ranging from 5 to 15 Hz to build 512 × 512 pixel images. AFM images were analyzed using Nanoscope IIIa software, and three-dimensional images were generated from topographical height information. Open and closed states of individual nuclear pore complexes were determined from 3 × 3 μm scans. Results are expressed as mean ± S.E. Statistical analysis was carried out by the Student's ttest. The significant difference was accepted at the p< 0.05 level. Neonatal cardiomyocytes treated with growth factors such as α1-adrenoreceptor agonists are an established cell system of hypertrophy (9Chien K.R. Knowlton K.U. Chien S. FASEB J. 1991; 5: 3037-3046Crossref PubMed Scopus (696) Google Scholar, 12Sadoshima J. Izumo S. Annu. Rev. Physiol. 1997; 59: 551-571Crossref PubMed Scopus (721) Google Scholar). Within 12 h of phenylephrine treatment (100 μm), cardiomyocytes nearly doubled in size from 655 ± 49 μm2 (n = 40) to 1158 ± 91 μm2 (n = 21) and markedly increased their content of actin filaments organized in contractile myofibrils (Fig. 1). Cardiomyocytes further enlarged to 1580 ± 143 μm2 (n = 32) and 1950 ± 177 μm2 (n = 28) at 24 and 48 h following phenylephrine treatment. Histones, major constituents of eukaryotic chromatin, are imported into nuclei by active transport (36Thomas J.O. Curr. Opin. Cell Biol. 1999; 11: 312-317Crossref PubMed Scopus (180) Google Scholar, 37Breeuwer M. Goldfarb D.S. Cell. 1990; 60: 999-1008Abstract Full Text PDF PubMed Scopus (202) Google Scholar, 38Jäkel S. Albig W. Kutay U. Bischoff F.R. Schwamborn K. Doenecke D. Görlich D. EMBO J. 1999; 18: 2411-2423Crossref PubMed Scopus (205) Google Scholar). When microinjected into the cytosol of control cardiomyocytes, fluorescein-tagged histone 1 (fl-H1) were readily transported into the nucleus, resulting in pronounced nuclear fluorescence (Fig. 1). However, early in hypertrophy, the active import of fl-H1 was down-regulated with the nuclear/cytoplasmic ratio, an index of nuclear transport (27Stehno-Bittel L. Perez-Terzic C. Clapham D.E. Science. 1995; 270: 1835-1838Crossref PubMed Scopus (173) Google Scholar, 29Perez-Terzic C. Gacy A.M. Bortolon R. Dzeja P.P. Puceat M. Jaconi M. Prendergast F.G. Terzic A. Circ. Res. 1999; 84: 1292-1301Crossref PubMed Scopus (50) Google Scholar, 39Greber U.F. Gerace L. J. Cell Biol. 1995; 128: 5-14Crossref PubMed Scopus (180) Google Scholar), decreasing by 74% from 3.18 ± 0.23 (n = 54) to 0.82 ± 0.12 (n = 7) within 12 h following the addition of phenylephrine (p < 0.05) (Fig. 1). With prolonged hypertrophy (48 h), the import of fl-H1 remained at reduced levels with the nuclear/cytoplasmic ratio of 0.74 ± 0.20 (n = 59) (Fig. 1). To determine whether the down-regulated transport of fl-H1 was attributable to hypertrophy rather than to a nonhypertrophy-related effect of phenylephrine, we evaluated nuclear transport in cells in which hypertrophy was induced through another receptor system. Angiotensin II (100 nm), which acts via angiotensin receptors (12Sadoshima J. Izumo S. Annu. Rev. Physiol. 1997; 59: 551-571Crossref PubMed Scopus (721) Google Scholar), also induced hypertrophy, and the cell surface increased from 933 ± 57 μm2 (n = 81) to 2176 ± 288 μm2 (n = 24 at 48 h of treatment). In angiotensin II-treated cells, the nuclear import of fl-H1 was also rapidly reduced within 12 h, and the nuclear/cytoplasmic ratio decreased by 57% (from 3.35 ± 0.23,n = 47 to 1.43 ± 0.34, n = 12;p < 0.05). Like phenylephrine and angiotensin II, the purinergic agonist ATP activates the phosphoinositide pathway without, however, inducing hypertrophy (40Post G.R. Goldstein D. Thuerauf D.J. Glembotski C.C. Brown J.H. J. Biol. Chem. 1996; 271: 8452-8457Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Inositol trisphosphate, a product of this pathway, releases Ca2+ from the nuclear cisterna and can inhibit nuclear import (27Stehno-Bittel L. Perez-Terzic C. Clapham D.E. Science. 1995; 270: 1835-1838Crossref PubMed Scopus (173) Google Scholar, 39Greber U.F. Gerace L. J. Cell Biol. 1995; 128: 5-14Crossref PubMed Scopus (180) Google Scholar, 41Perez-Terzic C. Jaconi M. Clapham D.E. Bioessays. 1997; 19: 787-792Crossref PubMed Scopus (63) Google Scholar). To exclude the possibility that impaired transport in hypertrophied cells was attributable to the inositol trisphosphate-induced decrease in cisternal Ca2+, we examined cells treated with ATP (50 μm, 48 h). ATP did not increase the cell size or actin content (40Post G.R. Goldstein D. Thuerauf D.J. Glembotski C.C. Brown J.H. J. Biol. Chem. 1996; 271: 8452-8457Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), but it activated phosphoinositide turnover (42Puceat M. Vassort G. Biochem. J. 1996; 318: 723-728Crossref PubMed Scopus (51) Google Scholar). The cell surface was 582 ± 36 μm2 (n = 81) and 618 ± 43 μm2 (n = 62) in untreated and ATP-treated cells, respectively (p > 0.05). In ATP-treated cells, even following prolonged exposure (48 h) to the purinergic agonist, fl-H1 was readily imported in the nucleus. The nuclear/cytoplasmic ratio for fl-H1 was 3.35 ± 0.23 (n = 47) and 3.12 ± 0.33 (n = 6) in controls and ATP-treated cells (p > 0.05), respectively. Thus, the down-regulation of active nuclear import is concomitant with the development of cell hypertrophy. Small molecular weight molecules such as dextrans commonly lack a nuclear localization signal and passively diffuse into the nucleus (39Greber U.F. Gerace L. J. Cell Biol. 1995; 128: 5-14Crossref PubMed Scopus (180) Google Scholar). When microinjected into the cytosol, fluorescein-conjugated 10-kDa dextrans diffused readily into the nucleus of control cells (Fig. 1). With the development of hypertrophy, passive diffusion appeared unaltered with nuclear/cytoplasmic ratios at 1.66 ± 0.12 (n= 12; 12 h) and 1.78 ± 0.08 (n = 17; 24 h), values not significantly different from the control ratio of 1.58 ± 0.18 (n = 4; p > 0.05) (Fig. 1). The passive diffusion of 10-kDa dextrans decreased only in the advanced stages of hypertrophy. In fact, 48 h was required after the initiation of hypertrophy for a significant reduction in the nucleocytoplasmic ratio (0.65 ± 0.04; n = 5) (Fig. 1). Even smaller molecules such as 3-kDa dextrans demonstrate unregulated diffusion across the nuclear membrane (27Stehno-Bittel L. Perez-Terzic C. Clapham D.E. Science. 1995; 270: 1835-1838Crossref PubMed Scopus (173) Google Scholar, 29Perez-Terzic C. Gacy A.M. Bortolon R. Dzeja P.P. Puceat M. Jaconi M. Prendergast F.G. Terzic A. Circ. Res. 1999; 84: 1292-1301Crossref PubMed Scopus (50) Google Scholar) regardless of the hypertrophic state. The nuclear/cytoplasmic ratio for 3-kDa dextrans was 1.92 ± 0.10 (n = 16) in control and 1.99 ± 0.10 (n = 14) following 48 h of phenylephrine treatment (p > 0.05). With the removal of the α1-adrenoreceptor agonist, cells progressively returned to their original sizes. At 48 and 72 h after the withdrawal of phenylephrine, the cell surface was 910 ± 103 μm2 (n = 11) and 750 ± 40 μm2 (n = 77), respectively, values close to those obtained prior to hypertrophy (Fig.2). With the reversal of the hypertrophic phenotype, active nuclear import was partially restored with a nuclear/cytoplasmic ratio of 1.94 ± 0.21 (n = 11). This represents an increase of 61% of the control, compared with ∼25% of the control at 12 and 48 h of hypertrophy (Fig. 2). With the removal of the hypertrophic stimulus, passive nuclear diffusion promptly returned to prehypertrophy values with a nuclear/cytoplasmic ratio of 2.08 ± 0.10 (n = 9) within 48 h of phenylephrine withdrawal (Fig. 2). Thus, the increase in cell size and the down-regulation of nuclear import are reversed on the removal of the hypertrophic signal. With hypertrophy, a more dense actin network may have impeded transfer to the nuclear surface, thereby precluding nuclear import. Cytochalasin B (20 μm), a disrupter of the cytoskeleton (43Terzic A. Kurachi Y. J. Physiol. 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