Title: hsp27 as a Switch between Differentiation and Apoptosis in Murine Embryonic Stem Cells
Abstract: Small stress proteins are developmentally regulated and linked to cell growth and differentiation. The early phase of murine embryonic stem (ES) cell differentiation, characterized by a gradual growth arrest, is accompanied with hsp27 transient accumulation. This differentiation process also correlated with changes in hsp27 phosphorylation and oligomerization. The role of hsp27 was investigated in ES clones stably transfected with murine or human hsp27 genes, placed in sense or antisense orientation. Several clones were obtained that either underexpressed endogenous murine hsp27 or overexpressed murine or human hsp27. Maintained undifferentiated, these clones showed similar growth rates. We report here that hsp27 constitutive overexpression enhanced the differentiation-mediated decreased rate of ES cell proliferation but did not alter morphological changes. In contrast, hsp27 underexpression, which attenuated cell growth arrest, induced differentiation abortion because of an overall cell death by apoptosis. Recently, we showed that hsp27 interfered with cell death probably because of its ability to modulate intracellular glutathione. hsp27 accumulation during ES cell differentiation was also correlated with an increase in glutathione, which was attenuated by hsp27 down-expression. Hence, hsp27 transient expression seems essential for preventing differentiating ES cells from undergoing apoptosis, a switch that may be redox regulated. Small stress proteins are developmentally regulated and linked to cell growth and differentiation. The early phase of murine embryonic stem (ES) cell differentiation, characterized by a gradual growth arrest, is accompanied with hsp27 transient accumulation. This differentiation process also correlated with changes in hsp27 phosphorylation and oligomerization. The role of hsp27 was investigated in ES clones stably transfected with murine or human hsp27 genes, placed in sense or antisense orientation. Several clones were obtained that either underexpressed endogenous murine hsp27 or overexpressed murine or human hsp27. Maintained undifferentiated, these clones showed similar growth rates. We report here that hsp27 constitutive overexpression enhanced the differentiation-mediated decreased rate of ES cell proliferation but did not alter morphological changes. In contrast, hsp27 underexpression, which attenuated cell growth arrest, induced differentiation abortion because of an overall cell death by apoptosis. Recently, we showed that hsp27 interfered with cell death probably because of its ability to modulate intracellular glutathione. hsp27 accumulation during ES cell differentiation was also correlated with an increase in glutathione, which was attenuated by hsp27 down-expression. Hence, hsp27 transient expression seems essential for preventing differentiating ES cells from undergoing apoptosis, a switch that may be redox regulated. Mammalian hsp27 belongs to the family of small heat shock proteins (SHSP) 1The abbreviations used are: SHSP, small heat shock proteins; ES, embryonic stem; ROS, reactive oxygen species; LIF, leukemia inhibitory factor; RT-PCR, reverse transcriptase-polymerase chain reaction; HPRT, hypoxanthine phosphoribosyl transferase. that are characterized by a strong homology to lens α-crystallin (reviewed in Ref. 1Arrigo A.-P. Landry J. Morimoto R. Tissières A. Georgopoulos C. Heat Shock Proteins: Structure, Function and Regulation. Cold Spring Harbor Press, New York1994: 335-373Google Scholar). SHSP share the ability to form oligomeric structures (2Arrigo A.-P. Suhan J.P. Welch W.J. Mol. Cell. Biol. 1988; 8: 5059-5071Crossref PubMed Scopus (302) Google Scholar, 3Mehlen P. Arrigo A.-P. Eur. J. Biochem. 1994; 221: 321-334Crossref Scopus (112) Google Scholar) and are often detected as phosphoproteins (4Welch W.J. J. Biol. Chem. 1985; 260: 3058-3062Abstract Full Text PDF PubMed Google Scholar, 5Arrigo A.-P. Mol. Cell. Biol. 1990; 10: 1276-1280Crossref PubMed Scopus (107) Google Scholar). Many stimuli, such as serum, oxidative injury, thermal stress, inflammatory cytokines (tumor necrosis factor-α, interleukin-1), and retinoic acid have been described as potent modulators of mammalian hsp27 phosphorylation and oligomerization (reviewed in Ref. 1Arrigo A.-P. Landry J. Morimoto R. Tissières A. Georgopoulos C. Heat Shock Proteins: Structure, Function and Regulation. Cold Spring Harbor Press, New York1994: 335-373Google Scholar). SHSP expression was shown to protect against cell necrosis induced by stimuli such as hyperthermia (6Landry J. Chrétien P. Lambert H. Hickey E. Weber L.A. J. Cell. Biol. 1989; 109: 7-15Crossref PubMed Scopus (582) Google Scholar, 7Mehlen P. Briolay J. Smith L. Diaz-Latoud C. Fabre N. Pauli D. Arrigo A.-P. Eur. J. Biochem. 1993; 215: 277-284Crossref PubMed Scopus (115) Google Scholar), anti-cancerous drugs, oxidative stress (7Mehlen P. Briolay J. Smith L. Diaz-Latoud C. Fabre N. Pauli D. Arrigo A.-P. Eur. J. Biochem. 1993; 215: 277-284Crossref PubMed Scopus (115) Google Scholar, 8Huot J. Roy G. Lambert H. Chrétien P. Landry J. Cancer Res. 1991; 51: 5245-5252PubMed Google Scholar, 9Mehlen P. Preville X. Chareyron P. Briolay J. Klemenz R. Arrigo A.-P. J. Immunol. 1995; 215: 363-374Google Scholar, 10Mehlen P. Kretz-Remy C. Preville X. Arrigo A.P. EMBO J. 1996; 15: 2695-2706Crossref PubMed Scopus (519) Google Scholar), and inflammatory cytokines (9Mehlen P. Preville X. Chareyron P. Briolay J. Klemenz R. Arrigo A.-P. J. Immunol. 1995; 215: 363-374Google Scholar, 11Mehlen P. Mehlen A. Guillet D. Preville X. Arrigo A.-P. J. Cell. Biochem. 1995; 58: 248-259Crossref PubMed Scopus (100) Google Scholar). Recently, we reported that SHSP are also negative regulators of apoptosis that counteract Fas/APO-1 or staurosporine-induced programmed cell death (12Mehlen P. Schulze-Osthoff K. Arrigo A.-P. J. Biol. Chem. 1996; 271: 16510-16514Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar). To explain the protective activity of SHSP, it has been proposed that these proteins act as molecular chaperones (13Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar) or actin capping/decapping enzymes (14Miron T. Vancompernolle K. Vanderkerckhove J. Wilchek M. Geiger B. J. Cell. Biol. 1991; 11: 255-261Crossref Scopus (389) Google Scholar, 15Lavoie J.N. Gingras-Breton G. Tanguay R.M. Landry J. J. Biol. Chem. 1993; 268: 3420-3429Abstract Full Text PDF PubMed Google Scholar). In addition, we recently reported that the expression of SHSP from different species induced an increase in glutathione that resulted in a decreased level of intracellular reactive oxygen species (ROS) (10Mehlen P. Kretz-Remy C. Preville X. Arrigo A.P. EMBO J. 1996; 15: 2695-2706Crossref PubMed Scopus (519) Google Scholar). This conserved property was found to be essential for the protective activity of SHSP against oxidative stress- or tumor necrosis factor-α-induced cell death. An interesting feature of SHSP concerns their transient expression during development and cell differentiation. This was first observed inDrosophila (reviewed in Ref. 16Arrigo A.-P. Tanguay R.M. Hightower L. Nover L. Heat Shock and Development. Springer-Verlag, Berlin1991: 106-119Google Scholar), and studies performed in other organisms revealed the ubiquitous nature of this phenomenon (reviewed in Refs. 1Arrigo A.-P. Landry J. Morimoto R. Tissières A. Georgopoulos C. Heat Shock Proteins: Structure, Function and Regulation. Cold Spring Harbor Press, New York1994: 335-373Google Scholar and 17Arrigo A.-P. Neuropathol. Appl. Neurobiol. 1995; 21: 488-491Crossref PubMed Scopus (20) Google Scholar). Remarkably, during Drosophiladevelopment, Dhsp27 accumulates during the differentiation of imaginal discs, suggesting that this protein plays a role in this process (18Pauli D. Tonka C.-H. Tissières A. Arrigo A.-P. J. Cell Biol. 1990; 111: 817-828Crossref PubMed Scopus (87) Google Scholar). Recent studies have strengthened the hypothesis that the mammalian small stress protein hsp27 is linked to the differentiation process. Indeed, this protein is transiently expressed and/or phosphorylated during the early differentiation of several mammalian cells including embryonal carcinoma and stem cells (19Stahl J. Wobus A.M. Ihrig S. Lutsch G. Bielka H. Differentiation. 1992; 51: 33-37Crossref PubMed Scopus (44) Google Scholar), Ehrlich ascites cells (20Benndorf R. Kraft R. Otto A. Stahl J. Bohm H. Bielka H. Biochem. Int. 1988; 17: 225-234PubMed Google Scholar), normal B and B lymphoma cells (21Spector N.L. Samson W. Ryan C. Gribben J. Urba W. Welch W.J. Nadler L.M. J. Immunol. 1992; 148: 1668-1673PubMed Google Scholar), osteoblasts, promyelocytic leukemia cells (22Shakoori A.R. Oberdorf A.M. Owen T.A. Weber L.A. Hickey E. Stein J.L. Lian J.B. Stein G.S. J. Cell Biochem. 1992; 48: 277-287Crossref PubMed Scopus (131) Google Scholar, 23Spector N.L. Ryan C. Samson W. Levine H. Nadler L.M. Arrigo A.-P. J. Cell. Physiol. 1993; 156: 619-625Crossref PubMed Scopus (59) Google Scholar, 24Spector N.L. Mehlen P. Ryan C. Hardy L. Samson W. Levine H. Nadler L.M. Fabre N. Arrigo A.-P. FEBS Lett. 1994; 337: 184-188Crossref PubMed Scopus (41) Google Scholar, 25Chaufour S. Mehlen P. Arrigo A.-P. Cell Stress Chaperones. 1996; 1: 225-235Crossref PubMed Scopus (41) Google Scholar), and normal T cells (26Hanash S.M. Strahler J.R. Chan Y. Kuick R. Teichroew D. Neel J.V. Hailat N. Keim D.R. Gratiot-Deans J. Ungar D. Melhem R. Zhu X.X. Andrews P. Loottspeich F. Eckerskorn C. Chu E. Ali I. Fox D.A. Richardson B.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3314-3318Crossref PubMed Scopus (35) Google Scholar). hsp27 accumulation usually occurs concomitantly with the differentiation-mediated decrease of cellular proliferation (23Spector N.L. Ryan C. Samson W. Levine H. Nadler L.M. Arrigo A.-P. J. Cell. Physiol. 1993; 156: 619-625Crossref PubMed Scopus (59) Google Scholar, 24Spector N.L. Mehlen P. Ryan C. Hardy L. Samson W. Levine H. Nadler L.M. Fabre N. Arrigo A.-P. FEBS Lett. 1994; 337: 184-188Crossref PubMed Scopus (41) Google Scholar, 25Chaufour S. Mehlen P. Arrigo A.-P. Cell Stress Chaperones. 1996; 1: 225-235Crossref PubMed Scopus (41) Google Scholar). In this study, we have analyzed the expression and function of hsp27 during the in vitro pluripotential differentiation of embryonic CGR8 embryonic stem cells obtained through leukemia inhibitory factor (LIF) withdrawal. We show that the early phase of the differentiation process is accompanied by a transient accumulation of hsp27, which occurs concomitantly with a decreased rate of cellular proliferation. hsp27 accumulation was preceded by an increase in the level of the mRNA encoding this protein. The transient increase in hsp27 level was also time correlated with an increased oligomerization of this protein, a phenomenon that was preceded by hsp27 dephosphorylation. As an approach toward understanding the role of hsp27 during cell differentiation, we have analyzed CGR8 cells that either under- or overexpressed endogenous murine hsp27 or overexpressed human hsp27. The different cell lines obtained showed similar growth rates in the presence of LIF. However, following LIF withdrawal, the differentiation-mediated decreased rate of cell proliferation was inversely proportional to the level of hsp27 present within the cell. Although murine or human hsp27 overexpression did not seem to modify the differentiation-mediated morphological changes, the underexpression of endogenous hsp27 provoked an abortion of the differentiation process because of an overall cell death by apoptosis. Remarkably, during CGR8 cell differentiation, the raise in hsp27 level correlated with an increase in glutathione, a redox modulator described as being essential for the differentiation process (27Esposito F. Agosti V. Morrone G. Morra F. Cuomo C. Russo T. Venuta S. Cimino F. Biochem. J. 1994; 301: 649-653Crossref PubMed Scopus (42) Google Scholar). The raise in glutathione was less intense in cells that underexpress hsp27. These results are discussed in view of a role of hsp27 as switch that allows differentiating cells to escape from apoptosis through a redox-dependent mechanism. The murine embryonic stem cell CGR8 was obtained from A. Smith (Center of Genome Research, University of Edinburgh, UK). Undifferentiated CGR8 cells were grown on gelatinized flasks in BHK21 medium (Life Technologies, Inc.) supplemented with LIF (1/1000 conditioned medium from p10-6R DIA-LIF transfected COS cell line) (28Smith A.G. Heath J.K. Donalson D.D. Wong G.G. Moreau J. Stahl M. Rogers D. Nature. 1988; 336: 688-690Crossref PubMed Scopus (1483) Google Scholar), 0.05 mmβ-mercaptoethanol, 2 mml-glutamine, 1 mm sodium pyruvate, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 1 × minimal essential medium, and 10% fetal calf serum (Life Technologies, Inc.). To induce differentiation, CGR8 cells were dissociated by trypsinization and seeded at 3 × 105 cells/ml on bacterial grade Petri dishes (Bibby Sterilin Ltd., Stone, UK) in LIF-devoid growth medium. The mammalian expression vector psvK3 (Pharmacia, Uppsala, Sweden) bearing or not human (psvhsp27; Ref. 9Mehlen P. Preville X. Chareyron P. Briolay J. Klemenz R. Arrigo A.-P. J. Immunol. 1995; 215: 363-374Google Scholar) or murine (psvWT; Ref. 29Knauf U. Jakob U. Engel K. Buchner J. Gaestel M. EMBO J. 1994; 13: 54-60Crossref PubMed Scopus (124) Google Scholar) hsp27 cDNAs under the control of the early promoter of SV40 virus were used. To construct antisense expression vectors EcoRI-EcoRI orSacI-SacI, DNA fragments containing the entire human or murine hsp27 coding sequences, respectively, were subcloned in reverse orientation in the corresponding site of psvK3 polylinker; these expression vectors were denoted psvant-hhsp27 (human hsp27) and psvant-mhsp27 (murine hsp27). Anti-hsp70 serum was from Amersham International (Buckinghamshire, UK). The specificity of anti-human hsp27 and anti-murine hsp27 antibodies was as previously described (2Arrigo A.-P. Suhan J.P. Welch W.J. Mol. Cell. Biol. 1988; 8: 5059-5071Crossref PubMed Scopus (302) Google Scholar,9Mehlen P. Preville X. Chareyron P. Briolay J. Klemenz R. Arrigo A.-P. J. Immunol. 1995; 215: 363-374Google Scholar). DNA transfection was performed by electroporation using a Bio-Rad gene pulser (Bio-Rad). 5 × 106 CGR8 cells were resuspended in Opti-MEM medium (Boehringer, Mannheim, Germany) and incubated 10 min at room temperature in the presence of DNA made of 10 μg of pMC1neopoly(A) plasmid (30Bernet A. Sabatier S. Picketts D.J. Ouazana R. Morle F. Higgs D.R. Godet J. Blood. 1995; 86: 1202-1211Crossref PubMed Google Scholar) and 50 μg of psvK3, psvant-hhsp27, psvant-mhsp27, psvhsp27, or psvWT vector. After being electroporated (500 microfarads, 250 V), cells were incubated for 30 min at room temperature and then reseeded on gelatin-treated flasks in BHK-21 medium. 250 μg/ml G418 were added 48 h after electroporation, and resistant clones were isolated 10 days later. Cellular proliferation was monitored by counting cells using a hemocytometer chamber and a Nikon TMS inverted photomicroscope equipped with phase-contrast equipment. Cells were also labeled for 1 h with 1 μCi of [3H]thymidine (Amersham International) as described by Mehlen and Arrigo (3Mehlen P. Arrigo A.-P. Eur. J. Biochem. 1994; 221: 321-334Crossref Scopus (112) Google Scholar). Cell cycle analysis was performed essentially as described by Susuki et al. (31Susuki K. Watanabe M. Miyoshi J. Radiat. Res. 1992; 129: 157-162Crossref PubMed Scopus (33) Google Scholar). At different times before and following LIF removal, cells were washed, fixed with ethanol, resuspended in phosphate-buffered saline, and treated with RNase I. 50 μg/ml propidium iodide were added, and cells were analyzed by flow cytometry (Facscalibur, Becton Dickinson, Belgium). Total RNA from undifferentiated CGR8 cells or CGR8 cells derived from embryoid bodies were prepared with RNAzolTMB (Bioprobe-Interchim, Montluçon, France) according to the manufacturer's instructions. For RT-PCR analyses, 1 μg of total RNA was denatured for 10 min at 65 °C and reverse-transcribed for 1 h at 37 °C in a medium containing 50 mm Tris-HCl, pH 8.3, 10 mm dithiothreitol (Life Technologies, Inc.), 1 unit/μl RNasin (Promega), 0.5 μm random hexamers (Pharmacia, St-Quentin-Yvelines, France), 0.5 μm of each dNTP (Pharmacia), 75 mm KCl, 5 mm MgCl2, and 20 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). Reactions were stopped by incubating the mixtures 5 min at 95 °C. For PCR reactions, 5 μl of the RT reactions were transferred in a medium containing 20 mmTris-HCl, pH 8.3, 50 mm KCl, 1.5 mmMgCl2, 0.2 mm of each dNTP, 0.2 μm of each primer, and 1 unit of Taq DNA polymerase (Life Technologies, Inc.). HPRT-, collagen IV-, or β-major globin-specific primers were identical to those described by Kelleret al. (32Keller G. Kennedy M. Papayannopoulou T. Wiles M.V. Mol. Cell. Biol. 1993; 13: 473-486Crossref PubMed Scopus (778) Google Scholar). PCR reactions were carried out with a Perkin-Elmer thermal cycler for 30 cycles with a regimen of 94 °C for 1.5 min, 50 °C (for HPRT) or 55 °C (for collagen IV and β-major globin) for 1.5 min, and 72 °C for 2 min, followed by 72 °C for 10 min. Aliquots (10 μl) of each PCR reaction were analyzed by gel electrophoresis (2% agarose Nu sieve; Tebu, France) in Tris borate-EDTA buffer. RNA (10 μg) was analyzed in 1% agarose/formaldehyde gel and transferred to Hybond C extra membrane (Amersham International); hybridization was then performed at 65 °C (33Yang H. McLeese J. Weisbart M. Dionne J.L. Lemaire I. Aubin R.A. Nucleic Acids Res. 1993; 21: 3337-3338Crossref PubMed Scopus (59) Google Scholar). The murine hsp27 probe was a 0.6-kilobase SacI cDNA fragment of the psvWT plasmid (29Knauf U. Jakob U. Engel K. Buchner J. Gaestel M. EMBO J. 1994; 13: 54-60Crossref PubMed Scopus (124) Google Scholar). At different times before and following LIF removal, CGR8 cells were washed in phosphate-buffered saline and lysed at 4 °C in a buffer containing 20 mmTris, pH 7.4, 20 mm NaCl, 5 mmMgCl2, 0.1 mm EDTA, and 0.1% Triton X-100. The 20,000 × g supernatants were then applied to a Sepharose 6B gel filtration column (1 × 100 cm) (Pharmacia, Sweden), equilibrated, and developed in the lysis buffer devoid of Triton X-100. The presence of hsp27 in the fraction eluted of the column was detected by one-dimensional immunoblot analysis using anti-hsp27 serum. Molecular mass markers used to calibrate the gel filtration column included blue dextran (>2,000,000 Da), thyroglobulin (669,000 Da), apoferritin (440,000 Da), β-amylase (200,000 Da), and carbonic anhydrase (29,000 Da). One- or two-dimensional gel electrophoresis and immunoblots using hsp27 or hsp70 antisera were performed as already described (9Mehlen P. Preville X. Chareyron P. Briolay J. Klemenz R. Arrigo A.-P. J. Immunol. 1995; 215: 363-374Google Scholar, 11Mehlen P. Mehlen A. Guillet D. Preville X. Arrigo A.-P. J. Cell. Biochem. 1995; 58: 248-259Crossref PubMed Scopus (100) Google Scholar, 34Mehlen P. Kretz-Remy C. Briolay J. Fostan P. Mirault M.E. Arrigo A.-P. Biochem. J. 1995; 312: 367-375Crossref PubMed Scopus (80) Google Scholar) and revealed with the ECL kit from Amersham Corp. Autoradiographs were recorded onto X-Omat AR films (Eastman Kodak Co.). A Bioprofil system (Vilber Lourmat, France) was used for quantification. The analysis was performed within the range of proportionality of the film. The level of hsp27 expressed in CGR8 cells was compared with serial dilutions of the purified protein (StressGen Corp., Victoria, British Columbia, Canada). The vital dye Trypan blue (Sigma, St-Quentin, France) was used to monitor cell death (9Mehlen P. Preville X. Chareyron P. Briolay J. Klemenz R. Arrigo A.-P. J. Immunol. 1995; 215: 363-374Google Scholar). DNA fragmentation was analyzed essentially as described by Hockenberyet al. (35Hockenbery D.M. Nunez G. Milliman C. Schreiber R.D. Korsmeyer S.J. Nature. 1990; 348: 334-336Crossref PubMed Scopus (3544) Google Scholar). Briefly, cells were lysed for 20 min at 4 °C in a medium containing 5 mm Tris buffer, pH 7.4, 0.5% Triton X-100, 20 mm EDTA. After centrifugation at 20,000 × g for 15 min, the supernatants were extracted with phenol-chloroform, and nucleic acids were precipitated in ethanol before being analyzed by gel electrophoresis (1.5% agarose; Nu sieve, Tebu, France). Thereafter, the gel was incubated for at least 3 h at 37 °C in the presence of 20 μg/ml RNase A before being stained with ethidium bromide. Total cellular content glutathione from undifferentiated CGR8 cells (6 × 106 cells) or CGR8 cells derived from embryoid bodies was determined by the Bioxytech GSH-400 enzymatic method from OXIS International (Bonneuil-sur-Marne, France) according to the manufacturer's instructions. Immediately following LIF removal, CGR8 cells gradually decreased their growth rate and underwent pluripotential differentiation. The accumulation of markers, which are specific for different tissues, was used to follow the differentiation of these cells (32Keller G. Kennedy M. Papayannopoulou T. Wiles M.V. Mol. Cell. Biol. 1993; 13: 473-486Crossref PubMed Scopus (778) Google Scholar). This was assessed by specific RT-PCR analysis that was performed before and at different times following LIF withdrawal. As seen in Fig.1 A, the presence of collagen IV mRNA, a marker specific of endodermic cells, began to be detectable 4 days after LIF removal. 1 day later, the mRNA encoding the β-major globin precursor, a marker of hematopoietic cells, was observed. As a control, the level of the ubiquitous HPRT mRNA remained detectable during the whole differentiation process (Fig.1 A). Cell growth inhibition was monitored by [3H]thymidine incorporation and cell numeration. Fig.1 B demonstrates that, 12 h after LIF removal, [3H]thymidine incorporation decreased by a factor of almost 2 compared with the value determined in cells kept in the presence of LIF. After 24 h of differentiation, a 6-fold decreased [3H]thymidine incorporation was observed. This phenomenon reflected the gradual cell division inhibition of differentiating CGR8 cells, which is illustrated in Fig. 1 C by cell numeration. FAC-scan analysis of the different phases of the cell cycle revealed that the early differentiation of CGR8 cells is accompanied with an increased number of cells in G1 phase (not shown and Ref.36Savatier P. Lapillonne H. Van Grunsven L.A. Rudkin B.B. Samarut J. Oncogene. 1996; 12: 309-322PubMed Google Scholar). Since hsp27 has been increasingly linked to cell differentiation, we investigated the level of this protein during the differentiation of CGR8 cells. This was assessed by analyzing the level of this protein before and at different times following LIF withdrawal. The immunoblot analysis presented in Fig. 2 Ashows that hsp27, which is already expressed in undifferentiated CGR8 cells, displayed a transient increased level in cells incubated in the absence of LIF. The maximal increase of hsp27 level (2.5-fold) was observed 24 h after LIF removal. By 72 h of differentiation, the level of this protein was below that observed in control cells (see Fig. 4 C for a quantitative analysis of this phenomenon). A similar observation has been previously reported when ES cells were launched to differentiate as a consequence of retinoic acid treatment (19Stahl J. Wobus A.M. Ihrig S. Lutsch G. Bielka H. Differentiation. 1992; 51: 33-37Crossref PubMed Scopus (44) Google Scholar). Moreover, the accumulation of hsp27 in response to LIF withdrawal did not result in a stress response because the level of the major stress protein hsp70 was not significantly altered (not shown). Analysis performed at the mRNA level showed, already 6 h after LIF removal, an increased accumulation of the mRNA encoding hsp27 (Fig. 2, B and C). This phenomenon lasted for about 12 h; thereafter, the level of hsp27 mRNA rapidly declined.Figure 4Analysis of hsp27 levels in the different CGR8 cell lines. A, immunoblot analysis of the different clones. CGR8 cells were transfected with either the control (cont.) or hsp27 expression vectors. Vectors carrying either antisense (ant-hsp27) or sense (hsp27) hsp27 gene (murine or human) constructions were used (see "Experimental Procedures"). Stable cell lines were obtained, and the level of hsp27 was determined in immunoblot analysis of total cellular proteins probed with an antiserum that recognizes both human and murine hsp27. B, control immunoblot probed with anti-hsp70 antisera. Autoradiographs of ECL-revealed immunoblots are presented. Note the drastic decreased level of the endogenous murine hsp27 in clone ES-ant-hsp27-2 and ES-ant-hsp27-11. Murine hsp27 is slightly increased in clone ES-hsp27-11 and strongly increased in clone ES-hsp27-5. Both resulted in a transfection with the murine sense construct. A slight increase in ES-hsp27-2 and ES-hsp27-3 clones is also observed, but in this case, the transfection was performed with the human sense construct.C, kinetics of endogenous hsp27 accumulation during differentiation of either control CGR8 cells or CGR8 cells that underexpress or overexpress murine hsp27. Total cellular proteins were isolated from ES-control-1, ES-hsp27-5, or ES-ant-hsp27-2 cells following LIF withdrawal and analyzed in immunoblots probed with an antiserum specific for murine hsp27. In each case, the level of hsp27 was estimated by densitometry as described under "Experimental Procedures." Results are in the form of percentage of hsp27 level calculated as the ratio between the level of hsp27 determined for the different samples to that obtained in ES-control-1 cells maintained in the presence of LIF. Percentage of hsp27 level is presented as a function of the duration of cell culture in the absence of LIF.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because changes in hsp27 phosphorylation and oligomerization have been reported to occur during HL60 cell differentiation (24Spector N.L. Mehlen P. Ryan C. Hardy L. Samson W. Levine H. Nadler L.M. Fabre N. Arrigo A.-P. FEBS Lett. 1994; 337: 184-188Crossref PubMed Scopus (41) Google Scholar, 25Chaufour S. Mehlen P. Arrigo A.-P. Cell Stress Chaperones. 1996; 1: 225-235Crossref PubMed Scopus (41) Google Scholar), we have investigated these different properties of hsp27 during the differentiation of CGR8 cells. Phosphorylation analysis was assessed by two-dimensional immunoblots as already described (11Mehlen P. Mehlen A. Guillet D. Preville X. Arrigo A.-P. J. Cell. Biochem. 1995; 58: 248-259Crossref PubMed Scopus (100) Google Scholar, 34Mehlen P. Kretz-Remy C. Briolay J. Fostan P. Mirault M.E. Arrigo A.-P. Biochem. J. 1995; 312: 367-375Crossref PubMed Scopus (80) Google Scholar) and was performed before and at different times following LIF withdrawal. As seen in Fig. 3 A, in cells kept in the presence of LIF, hsp27 was resolved as two major isoforms: "a" and "b." The "a" isoform is the non-phosphorylated form, whereas the "b" isoform is the major phospho-isoform of the protein (1Arrigo A.-P. Landry J. Morimoto R. Tissières A. Georgopoulos C. Heat Shock Proteins: Structure, Function and Regulation. Cold Spring Harbor Press, New York1994: 335-373Google Scholar, 11Mehlen P. Mehlen A. Guillet D. Preville X. Arrigo A.-P. J. Cell. Biochem. 1995; 58: 248-259Crossref PubMed Scopus (100) Google Scholar, 34Mehlen P. Kretz-Remy C. Briolay J. Fostan P. Mirault M.E. Arrigo A.-P. Biochem. J. 1995; 312: 367-375Crossref PubMed Scopus (80) Google Scholar). Because similar levels of the "a" and "b" isoforms are detected in cells kept in the presence of LIF, this means that hsp27 is strongly phosphorylated in undifferentiated CGR8 cells. 1 h after LIF withdrawal, the level of the "b" isoform had declined, and this isoform was no more detectable later, particularly during the transient accumulation of this protein (between 12 and 48 h). These results therefore suggest that ES differentiation is characterized by a drastic dephosphorylation of hsp27. In addition of being a phosphoprotein, hsp27 also undergoes changes in its oligomerization state (1Arrigo A.-P. Landry J. Morimoto R. Tissières A. Georgopoulos C. Heat Shock Proteins: Structure, Function and Regulation. Cold Spring Harbor Press, New York1994: 335-373Google Scholar, 11Mehlen P. Mehlen A. Guillet D. Preville X. Arrigo A.-P. J. Cell. Biochem. 1995; 58: 248-259Crossref PubMed Scopus (100) Google Scholar, 37Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (570) Google Scholar). To study this particular property of hsp27 during ES differentiation, CGR8 cells were harvested and lysed at various time points before and at different times following LIF withdrawal. Lysates were subjected to sizing chromatography on a Sepharose 6B column, and the proteins present in the different fractions were analyzed in immunoblot probed with anti-hsp27 antibody as described under "Experimental Procedures." It is seen in Fig.3 B that in undifferentiated CGR8 cells, hsp27 is in the form of small oligomers whose molecular masses are comprised between 30 and 150 kDa. Remarkably, 24 h after LIF withdrawal, and concomitantly with hsp27 accumulation, hsp27 oligomers drastically shifted toward high molecular masses (100–600 kDa). By 48 h, the reverse phenomenon occurred because the distribution of hsp27 oligomers was again observed in the range of 30–150 kDa. These results therefore indicate that a drastic dephosphorylation and a transient increase in the oligomerization state of hsp27 occur during ES differentiation. As a