Abstract:
In mammals, there are four p38 protein kinases: p38α, p38β, p38γ and p38δ. p38β was identified in 1996 as a closely related protein kinase of p38α, sharing 74% sequence identity and the Thr-Gly-Tyr dual phosphorylation motif characteristic of all p38 MAPKs. p38β is widely distributed in cells and tissues, but less so than p38α; p38β is particularly abundant in endothelial cells. p38β is activated in vivo by dual phosphorylation at Thr180 and Tyr182 by the MAP2K, MKK3 and MKK6 in response to a multitude of stimuli including environmental stressors, cytokines and growth factors. p38β can be dephosphorylated on both its Thr and Tyr residues by Dual-Specificity Phosphatases. p38β, like p38α, is targeted by a class of pyridinyl imidazole drugs that do not target the other two p38 MAPKs. These compounds were invaluable in discovering functions regulated by p38α and p38β. However, they do not permit to distinguish functions mediated by p38β from those regulated by p38α. This distinction has been made possible by the use of genetically engineered mice. p38β-deficient mice are not embryonic lethal such as those lacking p38α. However ectopic expression of p38β can rescue the lethality of p38α-deficiency. This suggests that p38α is the “dominant” form but that functional redundancy exists between the two related protein kinase. p38β has been shown to play specific roles in gene expression, regulation of cell death, cell differentiation and neuropathic pain. However, p38β is not involved in transducing pro-inflammatory signals, myogenesis or cell motility, when p38α is present.Cite
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
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Tumor necrosis factor-α (TNF-α) and lymphotoxin-β receptor (LTβR) signaling both play important roles in inflammatory and immune responses through activation of NF-κB. Using various deficient mouse embryonic fibroblast cells, we have compared the signaling pathways leading to NF-κB induction in response to TNF-α and LTβR activation. We demonstrate that LTβR ligation induces not only RelA/p50 dimers but also RelB/p50 dimers, whereas TNF-α induces only RelA/p50 dimers. LTβR-induced binding of RelB/p50 requires processing of p100 that is mediated by IKKα but is independent of IKKβ, NEMO/IKKγ, and RelA. Moreover, we show that RelB, p50, and p100 can associate in the same complex and that TNF-α but not LTβ signaling increases the association of p100 with RelB/p50 dimers in the nucleus, leading to the specific inhibition of RelB DNA binding. These results suggest that the alternative NF-κB pathway based on p100 processing may account not only for the activation of RelB/p52 dimers but also for that of RelB/p50 dimers and that p100 regulates the binding activity of RelB/p50 dimers via at least two distinct mechanisms depending on the signaling pathway involved. Tumor necrosis factor-α (TNF-α) and lymphotoxin-β receptor (LTβR) signaling both play important roles in inflammatory and immune responses through activation of NF-κB. Using various deficient mouse embryonic fibroblast cells, we have compared the signaling pathways leading to NF-κB induction in response to TNF-α and LTβR activation. We demonstrate that LTβR ligation induces not only RelA/p50 dimers but also RelB/p50 dimers, whereas TNF-α induces only RelA/p50 dimers. LTβR-induced binding of RelB/p50 requires processing of p100 that is mediated by IKKα but is independent of IKKβ, NEMO/IKKγ, and RelA. Moreover, we show that RelB, p50, and p100 can associate in the same complex and that TNF-α but not LTβ signaling increases the association of p100 with RelB/p50 dimers in the nucleus, leading to the specific inhibition of RelB DNA binding. These results suggest that the alternative NF-κB pathway based on p100 processing may account not only for the activation of RelB/p52 dimers but also for that of RelB/p50 dimers and that p100 regulates the binding activity of RelB/p50 dimers via at least two distinct mechanisms depending on the signaling pathway involved. NF-κB transcription factors are key regulators of transcription of a variety of genes involved in inflammatory and immune responses and in the control of cell proliferation, differentiation, and apoptosis (1Barnes P.J. Karin M. N. Engl. J. Med. 1997; 336: 1066-1071Google Scholar, 2Barkett M. Gilmore T.D. Oncogene. 1999; 18: 6910-6924Google Scholar, 3Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Google Scholar, 4Silverman N. Maniatis T. Genes Dev. 2001; 15: 2321-2342Google Scholar, 5Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Google Scholar, 6Karin M. Cao Y. Greten F.R. Li Z.W. Nat. Rev. Cancer. 2002; 2: 301-310Google Scholar, 7Hatada E.N. Krappmann D. Scheidereit C. Curr. Opin. Immunol. 2000; 12: 52-58Google Scholar). In mammalian cells, the NF-κB family is composed of five members, RelA (p65), RelB, c-Rel (Rel), NF-κB1 (p50 and its precursor p105), and NF-κB2 (p52 and its precursor p100), and exists as a heterogeneous collection of homodimers and heterodimers (3Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Google Scholar, 8Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Google Scholar). In resting cells, NF-κB activity is tightly controlled by IκB family members, which include IκBα, IκBβ, IκBϵ, Bcl-3, p100, and p105 (9Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-683Google Scholar, 10Whiteside S.T. Israel A. Semin. Cancer Biol. 1997; 8: 75-82Google Scholar). Phosphorylation of a NF-κB inhibitor protein at specific serine residues by the IKK 1The abbreviations used are: IKK, IκB kinase; LTβR, lymphotoxin-β receptor; TNF-α, tumor necrosis factor-α; TNFR, TNF receptor; mAb, monoclonal antibody; Ab, antibody; MEF, mouse embryonic fibroblasts; EMSA, electrophoretic mobility shift assays; RT, reverse transcription; WT, wild type; NIK, NF-κB-inducing kinase; MCP-1, monocytic chemoattractant protein-1. 1The abbreviations used are: IKK, IκB kinase; LTβR, lymphotoxin-β receptor; TNF-α, tumor necrosis factor-α; TNFR, TNF receptor; mAb, monoclonal antibody; Ab, antibody; MEF, mouse embryonic fibroblasts; EMSA, electrophoretic mobility shift assays; RT, reverse transcription; WT, wild type; NIK, NF-κB-inducing kinase; MCP-1, monocytic chemoattractant protein-1. complex targets it for ubiquitination and subsequent degradation by the proteasome, thus enabling NF-κB dimers to translocate into the nucleus (11Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar). The IKK complex is composed of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ (also known as NEMO) (11Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar). The disruption of genes encoding individual subunits have demonstrated that IKKβ and IKKγ are required for mediating the canonical NF-κB activity (i.e. RelA/p50 dimers) induced by inflammatory signals (12Li Z.W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. Johnson R. Karin M. J. Exp. Med. 1999; 189: 1839-1845Google Scholar, 13Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Google Scholar, 14Schmidt-Supprian M. Bloch W. Courtois G. Addicks K. Israel A. Rajewsky K. Pasparakis M. Mol. Cell. 2000; 5: 981-992Google Scholar, 15Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Google Scholar), whereas IKKα participates in other physiological processes. In particular, IKKα has been shown to be essential for the regulation of keratinocyte differentiation (16Hu Y. Baud V. Delhase M. Zhang P. Deerinck T. Ellisman M. Johnson R. Karin M. Science. 1999; 284: 316-320Google Scholar, 17Hu Y. Baud V. Oga T. Kim K.I. Yoshida K. Karin M. Nature. 2001; 410: 710-714Google Scholar), receptor activator of nuclear factor κB ligand (RANKL) induced IκBα degradation in mammary epithelial cells (18Cao Y. Bonizzi G. Seagroves T.N. Greten F.R. Johnson R. Schmidt E.V. Karin M. Cell. 2001; 107: 763-775Google Scholar), and appropriate basal and inducible processing of NF-κB2 p100 precursor to p52 in B cells and lymphotoxin-β receptor (LTβR)-expressing cells (19Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Google Scholar, 20Claudio E. Brown K. Park S. Wang H. Siebenlist U. Nat. Immunol. 2002; 3: 958-965Google Scholar, 21Dejardin E. Droin N.M. Delhase M. Haas E. Cao Y. Makris C. Li Z.W. Karin M. Ware C.F. Green D.R. Immunity. 2002; 17: 525-535Google Scholar). RelB is the only NF-κB member that cannot homodimerize and only triggers potent transcriptional activation when coupled to p50 or p52 (22Bours V. Azarenko V. Dejardin E. Siebenlist U. Oncogene. 1994; 9: 1699-1702Google Scholar, 23Bours V. Burd P.R. Brown K. Villalobos J. Park S. Ryseck R.P. Bravo R. Kelly K. Siebenlist U. Mol. Cell. Biol. 1992; 12: 685-695Google Scholar, 24Ryseck R.P. Bull P. Takamiya M. Bours V. Siebenlist U. Dobrzanski P. Bravo R. Mol. Cell. Biol. 1992; 12: 674-684Google Scholar, 25Dobrzanski P. Ryseck R.P. Bravo R. Mol. Cell. Biol. 1993; 13: 1572-1582Google Scholar). Analyses of RelB-deficient mice have shown that RelB is essential to the development of medullary epithelium, mature dendritic cell function, and secondary lymphoid tissue organization (26Wu L. D'Amico A. Winkel K.D. Suter M. Lo D. Shortman K. Immunity. 1998; 9: 839-847Google Scholar, 27Weih F. Carrasco D. Durham S.K. Barton D.S. Rizzo C.A. Ryseck R.P. Lira S.A. Bravo R. Cell. 1995; 80: 331-340Google Scholar, 28Weih F. Warr G. Yang H. Bravo R. J. Immunol. 1997; 158: 5211-5218Google Scholar, 29Weih D.S. Yilmaz Z.B. Weih F. J. Immunol. 2001; 167: 1909-1919Google Scholar). Relb–/– mice also spontaneously develop a generalized persistent non-infectious multi-organ inflammatory syndrome (30Weih F. Durham S.K. Barton D.S. Sha W.C. Baltimore D. Bravo R. J. Immunol. 1996; 157: 3974-3979Google Scholar). Until recently, the canonical NF-κB (RelA/p50) was considered to be the predominant inducible κB DNA binding activity in most cell types in response to a broad range of stimuli, whereas RelB represented the major constitutive κB activity in lymphoid cells (31Weih F. Carrasco D. Bravo R. Oncogene. 1994; 9: 3289-3297Google Scholar, 32Dobrzanski P. Ryseck R.P. Bravo R. EMBO J. 1994; 13: 4608-4616Google Scholar). However, in the past few months, an alternative mechanism for inducing NF-κB activity has emerged based on the observation that inducible IKKα-dependent p100 processing allows the resultant p52 to function as transcriptional activator (20Claudio E. Brown K. Park S. Wang H. Siebenlist U. Nat. Immunol. 2002; 3: 958-965Google Scholar, 21Dejardin E. Droin N.M. Delhase M. Haas E. Cao Y. Makris C. Li Z.W. Karin M. Ware C.F. Green D.R. Immunity. 2002; 17: 525-535Google Scholar, 33Coope H.J. Atkinson P.G. Huhse B. Belich M. Janzen J. Holman M.J. Klaus G.G. Johnston L.H. Ley S.C. EMBO J. 2002; 21: 5375-5385Google Scholar, 34Kayagaki N. Yan M. Seshasayee D. Wang H. Lee W. French D.M. Grewal I.S. Cochran A.G. Gordon N.C. Yin J. Starovasnik M.A. Dixit V.M. Immunity. 2002; 17: 515-524Google Scholar). Remarkably, a pathway-dependent specificity in p52 binding partner was demonstrated. Whereas RelA/p52 dimers are the targets of the canonical pathway, nuclear translocation of RelB/p52 is regulated via the alternative NF-κB pathway and leads to the transcription of a specific pool of genes (21Dejardin E. Droin N.M. Delhase M. Haas E. Cao Y. Makris C. Li Z.W. Karin M. Ware C.F. Green D.R. Immunity. 2002; 17: 525-535Google Scholar). Most importantly, all of these studies point to a crucial role for the alternative NF-κB pathway in controlling the development, organization, and function of lymphoid tissue. The participation of RelB in non-lymphoid function is much less well documented. Although RelB was initially identified as an immediate-early gene in fibroblasts (24Ryseck R.P. Bull P. Takamiya M. Bours V. Siebenlist U. Dobrzanski P. Bravo R. Mol. Cell. Biol. 1992; 12: 674-684Google Scholar), it has now been shown to play an essential role in limiting the expression of proinflammatory mediators in lipopolysaccharide-induced fibroblasts (35Xia Y. Pauza M.E. Feng L. Lo D. Am. J. Pathol. 1997; 151: 375-387Google Scholar, 36Xia Y. Chen S. Wang Y. Mackman N. Ku G. Lo D. Feng L. Mol. Cell. Biol. 1999; 19: 7688-7696Google Scholar), thereby playing an important role in the resolution of acute inflammation. Interestingly, in the non-lymphoid cells examined so far (e.g. NIH 3T3, smooth muscle cells), RelB was found mainly in association with p50 but not p52 in the inducible κB DNA binding complexes (24Ryseck R.P. Bull P. Takamiya M. Bours V. Siebenlist U. Dobrzanski P. Bravo R. Mol. Cell. Biol. 1992; 12: 674-684Google Scholar, 37Jiang Y. Woronicz J.D. Liu W. Goeddel D.V. Science. 1999; 283: 543-546Google Scholar, 38Olashaw N.E. J. Biol. Chem. 1996; 271: 30307-30310Google Scholar). In contrast to recent progress in understanding the regulation of RelB/p52 dimers, the mechanisms controlling the inducible RelB/p50 DNA binding activity are still poorly understood. In this study, we have investigated the regulation of RelB/p50 dimers in fibroblasts in response to ligation of TNFR and LTβR, two members of the TNFR superfamily involved in the regulation of inflammatory and immune responses (39Tracey K.J. Cerami A. Annu. Rev. Cell Biol. 1993; 9: 317-343Google Scholar, 40Matsumoto M. J. Med. Invest. 1999; 46: 141-150Google Scholar, 41Baud V. Karin M. Trends Cell Biol. 2001; 11: 372-377Google Scholar, 42Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Google Scholar, 43Shakhov A.N. Nedospasov S.A. Cytokine Growth Factor Rev. 2001; 12: 107-119Google Scholar). We demonstrate that RelB/p50 activation triggered by LTβR ligation requires processing of p100 that is mediated through IKKα but not IKKβ, IKKγ, or RelA. Moreover, we show that RelB, p50, and p100 can associate in the same complex and that TNF-α signaling leads to the inhibition of RelB DNA binding via an increased association of p100 with RelB/p50 dimers in the nucleus. Reagents and Antibodies—Murine recombinant TNF-α was purchased from Sigma, and agonistic anti-LTβR mAb AC.H6 was a kind gift from J. Browning. J. Hiscott and N. Rice generously provided anti-p52/p100 and anti-p50/p105 polyclonal antibodies. The remaining antibodies were purchased from Santa Cruz Biotechnology (IKKα, RelA, RelB, p105/p50, cRel, and phospholipase Cγ-1), Upstate Biotechnology (IKKβ, p100/p52, and p105/p50), and BD Biosciences (IKKγ). Cell Culture and Cell Lines—IKKα-, IKKβ-, NEMO/IKKγ-deficient mouse embryonic fibroblasts (MEFs) were described previously (12Li Z.W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. Johnson R. Karin M. J. Exp. Med. 1999; 189: 1839-1845Google Scholar, 14Schmidt-Supprian M. Bloch W. Courtois G. Addicks K. Israel A. Rajewsky K. Pasparakis M. Mol. Cell. 2000; 5: 981-992Google Scholar, 16Hu Y. Baud V. Delhase M. Zhang P. Deerinck T. Ellisman M. Johnson R. Karin M. Science. 1999; 284: 316-320Google Scholar). RelA-, RelB-, and NF-κB2-deficient MEFs were a kind gift from A. Beg, F. Weih, and J. Caamano, respectively. MEFs were grown in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum, 2 mm l-glutamine, 1 mm sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptavidin. HT29 (ATCC) were cultured in McCoy's 5A medium with the same supplements. Cell Extract Preparation—Whole cell extracts were prepared as reported previously (44Baud V. Liu Z.G. Bennett B. Suzuki N. Xia Y. Karin M. Genes Dev. 1999; 13: 1297-1308Google Scholar). For cytosolic and nuclear proteins, cells were lysed for 5 min on ice in hypotonic buffer (50 mm Tris, pH 8.0, 1 mm EDTA, 0.5 mm dithiothreitol, 0.1% Nonidet P-40, 10% glycerol, 1 μm leupeptin, 1 μm aprotinin, and 1 mm phenylmethylsulfonyl fluoride). The cytosolic fraction was harvested after centrifuging the lysate for 5 min at 4500 × g. The nuclear pellet was washed once with hypotonic buffer and lysed for 30 min on ice in extraction buffer (20 mm Hepes, 500 mm NaCl, 1.5 mm MgCl2, 0.5 mm EDTA, 0.5 mm dithiothreitol, 25% glycerol, 1 μm leupeptin, 1 μm aprotinin, and 1 mm phenylmethylsulfonyl fluoride). Anti-phospholipase C-γ was used as control for cytoplasmic contamination in nuclear fractions. Coimmunoprecipitation and Immunoblotting—For coimmunoprecipitation experiments, 500 μg of nuclear or whole cell extracts were immunoprecipitated for 2 h or overnight at 4 °C using specific antibodies, after which protein A/G-agarose beads (Amersham Biosciences) were added and incubation continued for 90 min at 4 °C. After four washes in lysis buffer, the beads were heat-denatured to release the proteins. Immunoprecipitated proteins were resolved on 8% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Millipore). Immunoblotting was performed with specific antibodies and visualized using the ECL Western blotting detection kit (Amersham Biosciences). For double immunoprecipitation, nuclear or whole cell extracts were incubated with anti-p50 antibody and protein A/G-agarose beads. After five washes in lysis buffer, the antigen-antibody complexes were eluted with a 15-fold excess (w/w) of the specific peptide (Santa Cruz Biotechnology) overnight at 4 °C. The resulting supernatants were immunoprecipitated with anti-RelB antibody and protein A/G-agarose beads. The immune complexes obtained were separated on 8% SDS-polyacrylamide gel and detected by immunoblotting with anti-p100 antibody. Electrophoretic Mobility Shift Assays (EMSA)—Nuclear extracts were prepared and analyzed as previously described using the human immunodeficiency virus long terminal repeat tandem κB oligonucleotide as κB probe (45Feuillard J. Gouy H. Bismuth G. Lee L.M. Debre P. Korner M. Cytokine. 1991; 3: 257-265Google Scholar). For supershift assays, nuclear extracts were incubated with specific antibodies for 30 min on ice before incubation with the labeled probe. RT-PCR—RT-PCR were performed as described previously (46Baud V. Chissoe S.L. Viegas-Pequignot E. Diriong S. N′Guyen V.C. Roe B.A. Lipinski M. Genomics. 1995; 26: 334-344Google Scholar). Linear response ranges were determined for each gene to semiquantify their expression levels. Primer sequences are available upon request. Ligation of LTβR but Not TNFR Activates RelB/p50 DNA Binding—We used EMSA to evaluate the nuclear NF-κB DNA binding activity induced by ligation of LTβR and TNFR. As shown in Fig. 1A, whereas nuclear extracts from untreated WT MEFs contained only low levels of NF-κB DNA binding activity, treatment with either TNF-α or agonistic anti-LTβR mAb AC.H6 both resulted in two phases of NF-κB activation. TNF-α-induced NF-κB binding activity was detected after 30 min of treatment (complex I), decreased to basal levels after 60 min, returned to near maximal levels after 4 h of treatment, and persisted for at least 8 h. Complex I was also induced after 30 min of treatment with anti-LTβR antibody, but a faster migrating κB complex (complex II) was detected after 4–8 h of treatment. The subunit composition of the NF-κB DNA binding complexes was then examined by supershift assays (Fig. 1B). Incubation of the agonistic LTβR Ab-stimulated protein extracts with anti-RelA and anti-p50 antibodies supershifted complex I almost completely, whereas complex II was effectively supershifted with anti-RelB and anti-p50 antibodies. Antibodies to p52 (Fig. 1B) and c-Rel (data not shown) had very little effect on either complex. LTβR-induced Binding of RelB/p50 Dimers Requires IKKα but Not IKKβ nor IKKγ—To determine which subunit of the IKK complex controls the binding of RelB/p50 dimers in response to LTβR ligation, we analyzed the DNA binding activity of the nuclear NF-κB complexes in IKKα-, IKKβ-, and IKKγ-deficient fibroblasts (Fig. 2A) and found that a weak constitutive binding of RelB/p50 (complex II) was present only in MEFs lacking IKKγ. Most importantly, we found that IKKα is absolutely required for the induction of RelB/p50 DNA binding (complex II), whereas IKKβ and IKKγ are not. In contrast, RelA/p50 binding (complex I) was strongly reduced in IKKβ-deficient MEFs and abolished in IKKα- and IKKγ-deficient MEFs. During the preparation of this paper, LTβR-ligation-induced RelB/p50 activation was also reported by others to be independent of IKKγ (47Saitoh T. Nakano H. Yamamoto N. Yamaoka S. FEBS Lett. 2002; 532: 45-51Google Scholar). Because RelB transcription has been reported to be regulated by RelA (48Bren G.D. Solan N.J. Miyoshi H. Pennington K.N. Pobst L.J. Paya C.V. Oncogene. 2001; 20: 7722-7733Google Scholar), we also analyzed LTβR-mediated NF-κB DNA binding activity in MEFs lacking RelA. Complex II was clearly induced in the absence of RelA, albeit at somewhat lower levels. A strong constitutive binding of a third complex (complex III) was also observed in these cells. Super-shift assays revealed that complex III corresponds to a p50-containing complex (data not shown). Given that IKKα exerts a specific function that is not controlled by the two other subunits of the IKK complex, it is possible that some of the IKKα present in cells is not incorporated into the large IKKα/IKKβ/IKKγ-containing complex (49.Rothwarf, D. M., and Karin, M. (1999) Sci Signal Transduction Knowledge Environment RE1, www.sciencemag.org,Google Scholar). To test this possibility, we carried out immunodepletion experiments on whole cell extracts from fibroblasts. Five rounds of depletion were performed using an anti-IKKγ antibody, and the IKK subunit content was analyzed after each round by immunoblotting for IKKα, IKKβ, and IKKγ. IKKβ was almost entirely depleted from the protein extracts after one round of IKKγ depletion (Fig. 2B), showing that most of the IKKβ binds to IKKγ. In contrast, a considerable fraction of IKKα was still detectable after five rounds of IKKγ depletion. This observation suggests the existence of an IKKα-containing complex independent of IKKβ and IKKγ. Whether this alternative complex represents IKKα homodimers or IKKα associated with a different protein(s) remains to be determined. IKKα but Not IKKβ and IKKγ Regulates LTβR-induced p100 Processing and RelB Nuclear Translocation in Mouse Embryonic Fibroblasts—We have previously reported that LTβR-induced processing of p100 generates RelB/p52 dimers that translocate to the nucleus to activate a set of specific target genes (21Dejardin E. Droin N.M. Delhase M. Haas E. Cao Y. Makris C. Li Z.W. Karin M. Ware C.F. Green D.R. Immunity. 2002; 17: 525-535Google Scholar). To determine whether a control mechanism based on p100 processing could also apply to RelB/p50 dimers, we examined p100 and p52 protein levels in WT, IKKα-, IKKβ-, and IKKγ-deficient fibroblasts (Fig. 3A). Treatment with anti-LTβR antibody strongly enhanced processing of p100 to p52 in WT fibroblasts but failed to do so in IKKα-deficient MEFs (Fig. 3A). Importantly, LTβR ligation led to p100 processing with kinetics parallel to those of RelB/p50 binding. Although the steady state level of expression of p100 is low in IKKβ- and even lower in IKKγ-deficient fibroblasts compared with WT fibroblasts, p100 processing still occurred in these cells in response to treatment with the agonistic LTβR Ab. Thus, coincident with the induction of RelB/p50 DNA binding (Fig. 2A), IKKα but not IKKβ and IKKγ is also absolutely required for LTβR-induced processing of p100 (Fig. 3A). These results strongly suggest that the IKKα-dependent p100 processing plays a critical role in the regulation of LTβR-induced activation of RelB/p50 dimers. To further investigate underlying mechanisms, we compared intracellular distributions of RelB in LTβR-stimulated IKKα-, IKKβ-, and IKKγ-deficient MEFs (Fig. 3B). We observed that LTβR ligation-induced RelB nuclear localization was abolished in IKKα- but not β- or γ-deficient MEFs (Fig. 3B). In addition, although similar constitutive RelB protein levels were observed in the cytoplasm of the three IKK-deficient cell lines, constitutive RelB levels in the nucleus were markedly greater in IKKγ-deficient cells, which may explain the constitutive RelB/p50 DNA binding activity observed in these cells (Fig. 2A). Taken together, our data demonstrate that IKKα is required for the LTβR-induced activation of RelB/p50 dimers. Most probably, the processing of p100 and thus the removal of this main inhibitory partner of RelB allows RelB nuclear translocation and DNA binding. Nuclear p100 Inhibits RelB/p50 DNA Binding in Response to TNF-α—To determine whether the failure of TNF-α to induce RelB DNA binding is the result of a lack of nuclear translocation of RelB, we compared RelB protein levels and cellular distributions in response to TNF-α versus agonistic LTβR activation in WT MEFs (Fig. 4). TNF-α induction resulted in a strong increase of RelB in both cytoplasm and nucleus, whereas LTβR stimulation markedly increased only nuclear RelB. Importantly, the levels of induced nuclear RelB were similar in response to TNFR and LTβR ligation. Thus, the increased nuclear RelB expression level observed in TNF-α-induced fibroblasts does not lead to an increased binding activity, suggesting a nuclear control of RelB/p50 activity. The absence of TNF-α-induced RelB DNA binding, despite its accumulation in the nucleus, could also be attributed to a lack of production of its heterodimerization partners p50 and p52 and/or to the absence of p100 degradation. Therefore, we also compared the protein levels and cellular distributions of p105/p50 and p100/p52 in TNF-α- and anti-LTβR-treated WT MEFs (Fig. 4). TNFR and LTβR ligation both had very little effect on p105 and p50 protein levels and cellular distribution. Although no nuclear p105 was detected, a fraction of p50 was constitutively present in the nucleus. Within 4 h after TNFR ligation, p100 levels increased slightly in the cytoplasm but strongly in the nucleus. In contrast, LTβR ligation led to p100 processing accompanied by nuclear accumulation of p52 (Fig. 4) with kinetics parallel to those of RelB/p50 binding. Because the availability of RelB for its DNA binding heterodimerization partner p50 is similar in TNF-α and anti-LTβR mAb AC.H6-stimulated fibroblasts, we hypothesized that the TNF-α-induced increase of nuclear p100 might block RelB/p50 DNA binding. To test this hypothesis, we first examined whether RelB associates with p100 in the nucleus of TNF-α-treated fibroblasts in vivo. p100 was immunoprecipitated using antibody directed against its C-terminal domain to avoid immunoprecipitation of p52. As shown in Fig. 5A, endogenous RelB coimmunoprecipitates with p100 in the nucleus of TNF-α-activated WT fibroblasts. Importantly, the increase of RelB protein levels parallels the increase of p100 protein levels. In contrast, using whole cell extracts as well as nuclear fractions, no association was detected between RelB and p105 (data not shown). We next investigated whether p100 was able to sequester RelB/p50 dimers within the nucleus of TNF-α-treated cells. To first confirm the existence of such an association, we performed double immunoprecipitation using whole cell extracts from untreated fibroblasts. A first immunoprecipitation was performed with an anti-peptide antibody directed against p50 followed by incubation with the corresponding p50 peptide to elute the precipitated proteins. The eluate was then immunoprecipitated with an anti-RelB antibody. An analysis of this second eluate by immunoblotting with an anti-p100 antibody revealed that p100 participates in a multi-protein complex that contains both p50 and RelB (Fig. 5B). The existence of such a p100/RelB/p50 complex is not restricted to fibroblasts. Indeed, we have also observed the association of p100 with RelB/p50 dimers in HT29 cells (see below). To further explore underlying mechanisms, we then used nuclear fractions instead of whole cell extracts in double immunoprecipitation as described above. Most importantly, 8 h of TNF-α stimulation resulted in a strong increase in the association of nuclear RelB/p50 dimers with p100, whereas such an association was not detected in the nucleus of LTβR-activated cells (Fig. 5C). To further elucidate the inhibitory role of p100 on RelB/p50 DNA binding activity downstream of TNFR, we analyzed κB DNA binding activity in NF-κB2-deficient fibroblasts, i.e. lacking p100 (Fig. 6). A constitutive binding of RelB/p50 dimers (complex II) was observed in these cells. Importantly, TNF-α treatment resulted in dramatic increase of RelB/p50 DNA binding (complex II) in the absence of p100, whereas only RelA-containing dimers (complex I) were induced by TNF-α in WT fibroblasts (Fig. 6). Together, these results demonstrate that p100 inhibits TNF-α-induced RelB/p50 DNA binding, most probably via the "trapping" of nuclear RelB/p50 dimers by p100. Ligation of LTβR and TNFR Differentially Regulate Gene Expression—We have shown that LTβR ligation induces both the canonical NF-κB pathway, leading to a rapid and transient activation of RelA/p50 dimers, and the alternative NF-κB pathway, leading to a more delayed and sustained activation of RelB-containing dimers (Ref. 21Dejardin E. Droin N.M. Delhase M. Haas E. Cao Y. Makris C. Li Z.W. Karin M. Ware C.F. Green D.R. Immunity. 2002; 17: 525-535Google Scholar and this report). In contrast, TNF-α only induces the canonical NF-κB pathway, leading primarily to the activation of RelA-containing dimers. To address the physiological relevance of the alternative pathway in the activation of gene expression, WT MEFs were either left untreated or treated with agonistic LTβR mAb or TNF-α for8h and expression of several NF-κB-responsive genes with roles in inflammation was monitored by semiquantitative RT-PCR (Fig. 7). LTβR mAb and TNF-α both induced the genes encoding monocytic chemoattractant protein-1 (MCP-1) and p100, but expression of other target genes including those for the chemokines RANTES (regulated on activation normal T cell expressed and secreted) and interferon-inducible protein-10 was clearly specifically induced by TNF-α (Fig. 7). None of the genes tested thus far was specifically induced in response to LTβR ligation. Nevertheless, our results indicate that there is only partial overlap in the set of genes induced by LTβR mAb and TNF-α, suggesting that the LTβR-induced activation of p100 processing may control a set of specific target genes that remain to be identified. In the study presented here, we have explored the TNFR- and LTβR-mediated signaling events that control NF-κB activity in fibroblasts. We observe that different IKK subunits are required for RelA and RelB regulation. Consistent with previous observations, the data also show that TNF-α-induced RelA/p50 activation requires IKKβ and IKKγ, whereas IKKα is dispensable (12Li Z.W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. Johnson R. Karin M. J. Exp. Med. 1999; 189: 1839-1845Google Scholar, 15Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Google Scholar, 17Hu Y. Baud V. Oga T. Kim K.I. Yoshida K. Karin M. Nature. 2001; 410: 710-714Google Scholar, 50Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Google Scholar). In contrast, no RelB/p50 DNA binding is induced by TNFR ligation, whereas LTβR ligation activates both RelA/p50 and RelB/p50 complexes. In addition, we demonstrate that LTβR-induced binding of RelA/p50 requires IKKα, IKKβ, and IKKγ, whereas LTβR-induced binding of RelB/p50 absolutely requires IKKα but not IKKβ and IKKγ. Recently, it has been reported that IKKα may function as an essential component of the classical IKK complex, being specifically required for RANKL-mediated activation of this complex in mammary epithelial cells (18Cao Y. Bonizzi G. Seagroves T.N. Greten F.R. Johnson R. Schmidt E.V. Karin M. Cell. 2001; 107: 763-775Google Scholar). The data presented here thus provide a second body of evidence for a crucial role of IKKα in the induction of canonical NF-κB DNA binding activity. In contrast to the canonical NF-κB (RelA/p50), we and others have observed that p100 is the only IκB family member that strongly inhibits RelB activity (48Bren G.D. Solan N.J. Miyoshi H. Pennington K.N. Pobst L.J. Paya C.V. Oncogene. 2001; 20: 7722-7733Google Scholar, 51Dobrzanski P. Ryseck R.P. Bravo R. Oncogene. 1995; 10: 1003-1007Google Scholar, 52Solan N.J. Miyoshi H. Carmona E.M. Bren G.D. Paya C.V. J. Biol. Chem. 2002; 277: 1405-1418Google Scholar). 2E. Derudder and M. Körner, unpublished data. Recently, we have shown that LTβR-induced IKKα-dependent p100 processing controls RelB/p52 dimer nuclear translocation and gene regulation (21Dejardin E. Droin N.M. Delhase M. Haas E. Cao Y. Makris C. Li Z.W. Karin M. Ware C.F. Green D.R. Immunity. 2002; 17: 525-535Google Scholar). In this report, we show that the control of p100 processing also plays a critical role in the regulation of LTβR-induced activation of RelB/p50 dimers. Therefore, the newly discovered alternative NF-κB pathway based on p100 processing seems to account not only for the regulation of RelB/p52 dimers but also for that of RelB/p50 dimers. Although RelB/p52 dimers might be expected to result from the processing of RelB/p100 dimers, it was less clear a priori how p100 processing could control RelB/p50 binding activity. Interestingly, we have found that endogenous p100, p50, and RelB can associate in a single multi-protein complex in fibroblasts as well as in HT29 cells. Thus, our data suggest that LTβR ligation releases RelB/p50 dimers from their interaction with full-length p100, allowing RelB nuclear translocation and subsequent DNA binding. Endogenous complexes containing p100 together with RelA/p50 (53Dejardin E. Bonizzi G. Bellahcene A. Castronovo V. Merville M.P. Bours V. Oncogene. 1995; 11: 1835-1841Google Scholar, 54Kanno T. Franzoso G. Siebenlist U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12634-12638Google Scholar) or RelB/p50 (55Dejardin E. Deregowski V. Greimers R. Cai Z. Chouaib S. Merville M.P. Bours V. Oncogene. 1998; 16: 3299-3307Google Scholar) have also been found in human breast and lymphoid cancer cells, suggesting that the release of NF-κB dimers from p100 inhibition could represent a more general mechanism for regulation of NF-κB activity. In IKKβ-deficient fibroblasts, we have observed a clear reduction of LTβR-induced binding of RelB/p50 dimers that does not correlate with an impaired processing of p100 or decreased RelB nuclear translocation. Most probably, the diminished RelB/p50 activity is related to the markedly reduced RelB protein expression in these cells (Fig. 3B). Interestingly, a weak constitutive RelB/p50 DNA binding was detected in MEFs lacking IKKγ, correlating with a high constitutive level of nuclear RelB and a very low level of p100 expression in these cells (Fig. 3). A constitutive RelB/p50 DNA binding was also detected in NF-κB2-deficient fibroblasts (Fig. 6). These observations suggest that there are at least two levels of complexity in the regulation of RelB/p50 activity: 1) the overall expression level of RelB and p100 proteins; and 2) the control of p100 processing. Although TNF-α signaling did not induce RelB/p50 DNA binding in WT fibroblasts, a marked increase of RelB protein level was observed in the nucleus of these cells. This absence of a direct correlation between the nuclear localization of RelB and its DNA binding activity clearly suggested that an additional negative control of RelB activity existed in the nucleus of TNF-α-treated fibroblasts. Here again, p100 was a good candidate, because the level of nuclear p100 was also strongly increased in response to TNF-α. Indeed, we observe that TNF-α signaling strongly induces RelB/p50 activity in NF-κB2-deficient cells, suggesting that it is not the processing of p100 per se but rather the "removal" of p100 that allows RelB/p50 dimers to bind to the DNA. In addition, we demonstrate that the association of p100 with RelB/p50 dimers is dramatically increased in the nucleus of TNF-α-treated cells. In conclusion, TNF-α-induced assembly of the p100/RelB/p50 multimeric complex in the nucleus seems to account for the inhibition of RelB/p50 DNA binding, implying that p100 controls RelB/p50 dimers not only in the cytoplasm but also in the nucleus. How might full-length p100 interact with NF-κB dimers? NF-κB members all contain an N-terminal Rel homology domain responsible for DNA binding, dimerization, and association with the IκBs (3Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Google Scholar, 56Huxford T. Huang D.B. Malek S. Ghosh G. Cell. 1998; 95: 759-770Google Scholar). The C-terminal domain of p100, like the other IκBs, is characterized by an ankyrin-rich domain that interacts with NF-κB via the Rel homology domain. Structures of co-crystals of NF-κB proteins in association with IκBα and IκBβ have been determined previously (56Huxford T. Huang D.B. Malek S. Ghosh G. Cell. 1998; 95: 759-770Google Scholar), and it emerges that the dimerization domain of the NF-κB dimers is the primary region of interaction with IκBs. It seems plausible that p100 might self-associate through its dimerization domain and that its C-terminal ankyrin domain could then serve as a platform for the binding of RelB/p50 dimers. Analyses of NF-κB knock-out mice have revealed that mice lacking p100/p52 have marked defects in splenic microarchitecture very similar to those observed in LTβR-, NIK-, and RelB-deficient mice (29Weih D.S. Yilmaz Z.B. Weih F. J. Immunol. 2001; 167: 1909-1919Google Scholar, 57Futterer A. Mink K. Luz A. Kosco-Vilbois M.H. Pfeffer K. Immunity. 1998; 9: 59-70Google Scholar, 58De Togni P. Goellner J. Ruddle N.H. Streeter P.R. Fick A. Mariathasan S. Smith S.C. Carlson R. Shornick L.P. Strauss-Schoenberger J. Russel J.H. Karr R. Chaplin D. Science. 1994; 264: 703-707Google Scholar). Interestingly, during the revisions of this paper, mice lacking RelB were also reported to be deficient in Peyer's patch organogenesis (59Yilmaz Z.B. Weih D.S. Sivakumar V. Weih F. EMBO J. 2003; 22: 121-130Google Scholar), a phenotype also observed in NF-κB2-, NIK-, and LTβR-deficient animals. Animals lacking p50 do not show those dramatic developmental defects. Nevertheless, their Peyer's patches are reduced in number and size (60Paxian S. Merkle H. Riemann M. Wilda M. Adler G. Hameister H. Liptay S. Pfeffer K. Schmid R.M. Gastroenterology. 2002; 122: 1853-1868Google Scholar). Therefore, although p50-containing dimers are not absolutely required, they seem to contribute to the Peyer's patch developmental program. Therefore, it is tempting to conclude that the processing of p100 downstream of LTβR is critically involved in the functions of stromal cells during secondary lymphoid organ development, most probably through the control of RelB/p52 and, perhaps to a lesser extent, RelB/p50-responsive genes. In addition to the lymphoid organ defects, RelB-deficient mice display a multi-organ inflammatory syndrome that contributes significantly to premature mortality in these mice (28Weih F. Warr G. Yang H. Bravo R. J. Immunol. 1997; 158: 5211-5218Google Scholar). In an effort to better elucidate the physiological relevance of the LTβR-induced alternative NF-κB pathway, we have performed RT-PCR on several known NF-κB target genes with roles in inflammation. We have observed that in WT fibroblasts, p100 and monocytic chemoattractant protein-1 are induced by ligation of both TNFR and LTβR. Interestingly, MCP-1 was previously found to be specifically regulated by NIK in response to LTβR but not TNFR activation (61Yin L. Wu L. Wesche H. Arthur C.D. White J.M. Goeddel D.V. Schreiber R.D. Science. 2001; 291: 2162-2165Google Scholar). Because NIK is required for LTβR-induced p100 processing (21Dejardin E. Droin N.M. Delhase M. Haas E. Cao Y. Makris C. Li Z.W. Karin M. Ware C.F. Green D.R. Immunity. 2002; 17: 525-535Google Scholar), the loss of MCP-1 induction observed in NIK-deficient cells could reflect the lack of activation of RelB-containing dimers. These findings suggest that RelB/p50 dimers control the transcription of inflammatory genes downstream of LTβR. Chromatin immunoprecipitation experiments and microarray analyses designed to determine which genes are specifically regulated by RelB heterodimers will provide a direct test of this hypothesis. We thank M. Karin, M. Pasparakis, A. Beg, F. Weih, J. Caamano, N. Rice, J. Hiscott, and J. Browning for providing valuable cell lines and reagents. We are grateful to A. Israel, G. Courtois, A. Harel-Bellan, and L. Martinez for advice and helpful discussions.
RELB
Lymphotoxin beta receptor
Lymphotoxin alpha
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Amino acid deprivation activates general control nonderepressible 2 (GCN2) kinase and inhibits mammalian target of rapamycin (mTOR), affecting the immune response. In this study, the effects of GCN2 kinase activation or mTOR inhibition on human alloreactive CD4+ T-cells were evaluated. The mixed lymphocyte reaction, as a model of alloreactivity, the GCN2 kinase activator, tryptophanol (TRP), and the mTOR complex 1 inhibitor, rapamycin (RAP), were used. Both TRP and RAP suppressed cell proliferation and induced cell apoptosis. These events were p53-independent in the case of RAP, but were accompanied by an increase in p53 levels in the case of TRP. TRP decreased the levels of the Th2 signature transcription factor, GATA-3, as RAP did, yet the latter also decreased the levels of the Th1 and Th17 signature transcription factors, T-bet and RORγt, whereas it increased the levels of the Treg signature transcription factor, FoxP3. Accordingly, TRP decreased the production of interleukin (IL)-4, as RAP did, but RAP also decreased the levels of interferon-γ (IFN-γ) and IL-17. Both TRP and RAP increased the levels of IL-10. As regards hypoxia-inducible factor-1α (HIF-1α), which upregulates the Th17/Treg ratio, its levels were decreased by RAP. TRP increased the HIF-1α levels, which however, remained inactive. In conclusion, our findings indicate that, in primary human alloreactive CD4+ T-cells, the two systems that sense amino acid deprivation affect cell proliferation, apoptosis and differentiation in different ways or through different mechanisms. Both mTOR inhibition and GCN2 kinase activation exert immunosuppressive effects, since they inhibit cell proliferation and induce apoptosis. As regards CD4+ T-cell differentiation, mTOR inhibition exerted a more profound effect, since it suppressed differentiation into the Th1, Th2 and Th17 lineages, while it induced Treg differentiation. On the contrary, the activation of GCN2 kinase suppressed only Th2 differentiation.
RPTOR
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Epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells is a pathological process that occurs during peritoneal dialysis. EMT leads to peritoneal fibrosis, ultrafiltration failure and eventually to the discontinuation of therapy. Signaling pathways involved in mesothelial EMT are thus of great interest, but are mostly unknown. We used primary mesothelial cells from human omentum to analyze the role of the p38 MAPK signaling pathway in the induction of EMT. The use of specific inhibitors, a dominant-negative p38 mutant and lentiviral silencing of p38α demonstrated that p38 promotes E-cadherin expression both in untreated cells and in cells co-stimulated with the EMT-inducing stimuli transforming growth factor (TGF)-β1 and interleukin (IL)-1β. p38 inhibition also led to disorganization and downregulation of cytokeratin filaments and zonula occludens (ZO)-1, whereas expression of vimentin was increased. Analysis of transcription factors that repress E-cadherin expression showed that p38 blockade inhibited expression of Snail1 while increasing expression of Twist. Nuclear translocation and transcriptional activity of p65 NF-κB, an important inducer of EMT, was increased by p38 inhibition. Moreover, p38 inhibition increased the phosphorylation of TGF-β-activated kinase 1 (TAK1), NF-κB and IκBα. The effect of p38 inhibition on E-cadherin expression was rescued by modulating the TAK1-NF-κB pathway. Our results demonstrate that p38 maintains E-cadherin expression by suppressing TAK1-NF-κB signaling, thus impeding the induction of EMT in human primary mesothelial cells. This represents a novel role of p38 as a brake or 'gatekeeper' of EMT induction by maintaining E-cadherin levels.
Twist transcription factor
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Apoptosis is an important cellular response to UV radiation (UVR), but the corresponding mechanisms remain largely unknown. Here we report that the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI-3K) exerted a proapoptotic role in response to UVR through the induction of tumor necrosis factor alpha (TNF-α) gene expression. This special effect of p85α was unrelated to the PI-3K-dependent signaling pathway. Further evidence demonstrated that the inducible transcription factor NFAT3 was the major downstream target of p85α for the mediation of UVR-induced apoptosis and TNF-α gene transcription. p85α regulated UVR-induced NFAT3 activation by modulation of its nuclear translocation and DNA binding and the relevant transcriptional activities. Gel shift assays and site-directed mutagenesis allowed the identification of two regions in the TNF-α gene promoter that served as the NFAT3 recognition sequences. Chromatin immunoprecipitation assays further confirmed that the recruitment of NFAT3 to the endogenous TNF-α promoter was regulated by p85α upon UVR exposure. Finally, the knockdown of the NFAT3 level by its specific small interfering RNA decreased UVR-induced TNF-α gene transcription and cell apoptosis. The knockdown of endogenous p85α blocked NFAT activity and TNF-α gene transcription, as well as cell apoptosis. Thus, we demonstrated p85α-associated but PI-3K-independent cell death in response to UVR and identified a novel p85α/NFAT3/TNF-α signaling pathway for the mediation of cellular apoptotic responses under certain stress conditions such as UVR.
SIGNAL (programming language)
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Chromatin immunoprecipitation
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Two members of the NF-kappaB (nuclear factor kappaB)/Rel transcription factor family, NF-kappaB1 and NF-kappaB2, are produced as precursor proteins, NF-kappaB1 p105 and NF-kappaB2 p100 respectively. These are proteolytically processed by the proteasome to produce the mature transcription factors NF-kappaB1 p50 and NF-kappaB2 p52. p105 and p100 are known to function additionally as IkappaBs (inhibitors of NF-kappaB), which retain associated NF-kappaB subunits in the cytoplasm of unstimulated cells. The present review focuses on the latest advances in research on the function of NF-kappaB1 and NF-kappaB2 in immune cells. NF-kappaB2 p100 processing has recently been shown to be stimulated by a subset of NF-kappaB inducers, including lymphotoxin-beta, B-cell activating factor and CD40 ligand, via a novel signalling pathway. This promotes the nuclear translocation of p52-containing NF-kappaB dimers, which regulate peripheral lymphoid organogenesis and B-lymphocyte differentiation. Increased p100 processing also contributes to the malignant phenotype of certain T- and B-cell lymphomas. NF-kappaB1 has a distinct function from NF-kappaB2, and is important in controlling lymphocyte and macrophage function in immune and inflammatory responses. In contrast with p100, p105 is constitutively processed to p50. However, after stimulation with agonists, such as tumour necrosis factor-alpha and lipopolysaccharide, p105 is completely degraded by the proteasome. This releases associated p50, which translocates into the nucleus to modulate target gene expression. p105 degradation also liberates the p105-associated MAP kinase (mitogen-activated protein kinase) kinase kinase TPL-2 (tumour progression locus-2), which can then activate the ERK (extracellular-signal-regulated kinase)/MAP kinase cascade. Thus, in addition to its role in NF-kappaB activation, p105 functions as a regulator of MAP kinase signalling.
IκBα
TRAF2
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The non-canonical pathway based on processing of NF-κB2 precursor protein p100 to generate p52 plays a critical role in controlling B cell function and lymphoid organogenesis. Activation of this unique pathway by extracellular stimuli requires NF-κB-inducing kinase (NIK) and de novo protein synthesis. However, how NIK is regulated is largely unknown. Here, we systematically analyzed NIK expression at different levels in the presence or absence of different NF-κB stimuli. We found that NIK mRNA is relatively abundant and undergoes constitutive protein synthesis in resting B cells. However, NIK protein is undetectable. Interestingly, protein expression of NIK is steadily induced by B cell-activating factor or CD40 ligand, two major physiological inducers of p100 processing, but not by mitogen phorbol 12-myristate 13-acetate/ionomycin or cytokine tumor necrosis factor α, two well known inducers of the canonical NF-κB signaling. Remarkably, both B cell-activating factor and CD40 ligand do not significantly induce expression of NIK at translational or transcriptional level but rather rescue the basally translated NIK protein from undergoing degradation. Furthermore, overexpressed or purified NIK protein triggers p100 processing in the presence of protein synthesis inhibitor. Taken together, these studies define one important mechanism of NIK regulation and the central role of NIK stabilization in the induction of p100 processing. These studies also provide the first evidence explaining why activation of the non-canonical NF-κB signaling is delayed and can be inhibited by protein synthesis inhibitor as well as why most classical NF-κB stimuli, including mitogens and tumor necrosis factor α, fail to induce p100 processing.
IκB kinase
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ASK1
Protein kinase R
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