Báo cáo Y học: Evidence for general stabilization of mRNAs in response to UV light pdf

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Báo cáo Y học: Evidence for general stabilization of mRNAs in response to UV light pdf

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Evidence for general stabilization of mRNAs in response to UV light Frank Bollig*, Reinhard Winzen*, Michael Kracht, Beniam Ghebremedhin, Birgit Ritter, Arno Wilhelm, Klaus Resch and Helmut Holtmann Institute of Pharmacology, Medical School Hannover, Germany mRNA stabilization plays an important role in the changes in protein expression initiated by inducers of inflammation or direct cell stress such as UV light. This study provides evidence that stabilization in response to UV light differs from that induced by proinflammatory stimuli such as bacterial lipopolysaccharide or interleukin (IL)-1. Firstly, UV-induced stabilization is independent of the p38 MAP kinase pathway, which has previously been shown to medi- ate stabilization induced by IL-1 or lipopolysaccharide. UV-induced mRNA stabilization was insensitive to the dominant negative forms of p38 MAP kinase and its sub- strate MAP kinase-activated protein kinase 2 (MK2), or to the p38 MAP kinase inhibitor SB 203580, demonstrating that it occurs through a different signaling mechanism. Secondly, UV-induced stabilization exhibits a different transcript selectivity. Activation of the p38 MAP kinase pathway, by expressing active MAP kinase kinase 6, induced stabilization only of transcripts containing AU-rich elements. UV light also induced stabilization of transcripts lacking AU-rich elements. This effect could not be mimicked by expressing MEKK1, an upstream activator of the p38, JNK, ERK and NF-jB pathways. UV light also stabilized endogenous histone mRNA, which lacks AU-rich elements and a poly(A) tail. This effect was not mimicked by active MAP kinase kinase 6 and not sensitive to a p38 MAP kinase inhibitor. This suggests that UV light induces stabilization through a mechanism that is independent of p38 MAP kinase and affects a broad spectrum of mRNAs. Keywords: AU-rich element; MAPKAP kinase 2; mRNA stability; p38 MAP kinase; UV light. Higher organisms respond to an external insult by switching on the expression of certain genes the products of which are involved in the defense against pathogens and in tissue repair. Pathogen-derived material, direct cell stress and endogenous mediators activate gene expression at multiple levels, including transcriptional activation as well as post- transcriptional mechanisms. The importance of the latter has been demonstrated in gene-targeted mice where over- production of inflammatory proteins due to dysregulation of mRNA degradation or translation caused severe disease of the animals [1–5]. The molecular basis underlying the regulation of mRNA translation and decay is not completely understood. An important type of regulatory mRNA element are AU-rich elements (AREs), which are found in the 3¢-UTRs of many rapidly inducible genes such as oncogenes and cytokine genes [6,7]. By imposing rapid degradation on the transcript, the AREs limit basal expression and allow rapid reversion to basal mRNA levels subsequent to gene induction. Stability and translation of ARE-containing transcripts can be affected by signaling mechanisms activated by the damaging agents directly or by released inflammatory cytokines. Cell stressors, infectious pathogens and inflammatory cytokines activate various signaling pathways simulta- neously. Extensive overlap exists in the sets of pathways activated by the different agents. Pathways activated include NF-jB and the mitogen-activated protein (MAP) kinase cascades. The JNK pathway has been reported to stabilize the short-lived interleukin (IL)-2 mRNA on activation of the T-cell line Jurkat [8] and the IL-3 mRNA in the murine mast cell line PB-3c [9]. Several groups have shown an mRNA- stabilizing effect of protein kinase C activation and/or increased intracellular Ca 2+ concentrations [6,8–12]. The results of others, including our own, show that stabilization of several ARE-containing mRNAs, triggered by IL-1 or bacterial lipopolysaccharide (LPS), involves activation of p38 MAP kinase [13–17] and its substrate MAP kinase- activated protein kinase 2 (MK2) [16–18]. Consistent with these findings, MK2-deficient mice exhibit reduced synthesis of several cytokines in response to LPS [19]. Similarly to LPS and IL-1, UV light is a potent inducer of inflammation and induces expression of numerous genes including cytokines and oncogenes [20,21], which is in part due to the stabilization of mRNAs [22,23]. UV light strongly activates stress signaling pathways, including the p38/MK2 pathway [24]. However, the signaling mecha- nisms involved in mRNA stabilization in response to UV light have not been identified, nor has the transcript selectivity of UV-induced stabilization been defined. In this study, we show that, in HeLa cells, mRNA stabilization induced through the p38/MK2 pathway is Correspondence to H. Holtmann, Institute of Pharmacology, Medical School Hannover, Carl-Neuberg Strasse-1, D-30625 Hannover, Germany. Fax: + 49 511 5324081, Tel.: + 49 511 5322800, E-mail: holtmann.helmut@mh-hannover.de Abbreviations: ARE, AU-rich element; GFP, green fluorescent pro- tein; GM-CSF, granulocyte–macrophage colony-stimulating factor; IL, interleukin; LPS, lipopolysaccharide; MAP, mitogen-activated protein; MK2, MAP kinase-activated protein kinase 2 (also named MAPKAP kinase 2); MKK6, MAP kinase kinase 6. *Note: these two authors contributed equally to this work. (Received 5 July 2002, revised 2 October 2002, accepted 8 October 2002) Eur. J. Biochem. 269, 5830–5839 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03300.x limited to AU-rich transcripts, whereas UV light stabilizes a broad spectrum of mRNAs either containing or lacking AU-rich elements by a mechanism that is independent of the p38/MK2 pathway. MATERIALS AND METHODS Cells and materials HeLa cells constitutively expressing the tet transactivator protein [25] (kindly provided by H. Bujard, Center for Molecular Biology, University of Heidelberg, Germany) were cultured in Dulbecco’s modified Eagle’s medium complemented with 5% fetal bovine serum. Plasmids ptet- BBB-I18 972)1310 and ptet-BBB-GM-CSF ARE encode the rabbit b-globin mRNA with AU-rich regions of the Il-8 and GM-CSF transcripts respectively inserted into the BglII site of the b-globin 3¢-UTR [16,26]. The pUHC13-3 plasmid, kindly donated by H. Bujard, contains the Photinus pyralis luciferase cDNA downstream of a tetracycline-regulated promoter [25]. pUHD10-CAT- TIMP1 was generated by excising the IL-8 fragment of pUHD10-CAT-IL-8 [16] with BamHI and inserting a fragment of human TIMP1 (nucleotide 19–782, accession no. NM_003254) generated by RT-PCR with primers containing BamH1 sites. To obtain pUHD10-GFP a fragment of pEGFP-C1 (Clontech) including the green fluorescent protein (GFP) cDNA and 3¢ adjacent restric- tion sites was amplified with XbaI-flanked primers and inserted into the XbaI site of pUHD10.3 [25]. Expression plasmids for constitutively active MAP kinase kinase 6 (MKK6 2E ), dominant negative p38, dominant negative and constitutively active MK2 have been described [16]. To generate HeLa cells with inducible expression of active MKK6, the MKK6 2E cDNA was placed in-frame downstream of the GFP cDNA in pUHD10-GFP. HeLa cells were cotransfected with this plasmid and a plasmid for puromycin resistance, and stable transfectants selected by culture in 1 lgÆmL )1 puromycin. Myc-tagged HuR was expressed with the plasmid pTet-Myc-over-HuR [27] (a gift from A B. Shyu, University of Texas, Houston, TX, USA). Rabbit antiserum against AUF1 was a gift from G. Brewer, University of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA. Mouse monoclonal antibodies 19F12 against HuR and 9H10 against hnRNP A1 were kindly donated by H. Furneaux, University of Conneticut Health Center, Farmington, CT, USA, and G. Dreyfuss, University of Pennsylvania School of Medicine, Philadelphia, PA, USA, respectively. Transfections and reporter assays for mRNA stability Transient transfections by the calcium phosphate method and RNA degradation kinetics were performed as described [16]. Briefly, cells (5 · 10 6 seeded per 9-cm- diameter dish) were transfected with the indicated plas- mids. Amounts of plasmid DNA within each experiment were kept constant by adding empty vector. For each kinetics of mRNA degradation assay, cells from one dish were trypsinized and distributed into parallel cultures to ensure equal transfection efficiency within the group of samples. The next day transcription from the tetracycline- regulatable promoter was stopped by addition of doxycycline (3 lgÆmL )1 ). At the indicated times thereaf- ter, total RNA was isolated, and Northern-blot analysis was performed using digoxigenin-labeled antisense RNA probes. RNA half lives were determined as in [16], using a video imaging system and the MOLECULAR ANALYST program (Bio-Rad). Preparation of cytoplasmic extracts Cytoplasmic extracts were prepared as described by Wang et al. [22]. All steps were carried out in the cold. The cells (10 6 per sample) were washed once with NaCl/P i ,harvested by scraping, pelleted by centrifugation, and resuspended in 200 lL hypotonic buffer (10 m M Hepes, pH 7.9, 10 m M KCl, 1.5 m M MgCl 2 ,1lgÆmL )1 leupeptin, 1 lgÆmL )1 aprotinin and 0.5 m M phenylmethanesulfonyl fluoride). Then 25 lL of the same buffer including 2.5% (v/v) Nonidet P-40 was added. After centrifugation at 1000 g for 4 min, the supernatants were removed, freeze–thawed five times, and cleared by centrifugation. Aliquots were frozen at )70 °C. In vitro transcription and electrophoretic mobility-shift assays Labeled RNA (typically 10 7 )10 8 c.p.m.Ælg )1 ) was synthes- ized by incubating 1–3 lg linearized plasmid DNA in a mixture of 50 lCi [a- 32 P]UTP (400 CiÆmmol )1 , Hartmann Analytic, Braunschweig, Germany), 18 l M unlabeled UTP, 0.5 m M unlabeled ATP, CTP and GTP, 2 UÆlL )1 T7 RNA polymerase or T3 RNA polymerase as required (both enzymes from Roche) and 2 UÆlL )1 RNase inhibitor (MBI) for 1 h at 37 °C. RNase-free DNase (Roche) was then added to a final concentration of 1 UÆlL )1 . After incuba- tion for 15 min at 37 °C the RNA was passed through a NucTrap push column (Stratagene) to remove free nucle- otides, and stored at )80 °C. Radiolabeled RNA probes (1.5 · 10 5 c.p.m.) were incubated with cytoplasmic extracts (6 lg protein per sample) in 20 lL buffer containing 20 m M Hepes, pH 7.9, 100 m M KCl, 2 m M MgCl 2 , 3% (v/v) glycerol, 0.5 m M dithiothreitol, 0.5 m M phenyl- methanesulfonyl fluoride, 5 lgÆmL )1 pepstatin A and 200 lgÆmL )1 tRNA for 10 min at 30 °C. RNase T1 (30 units/sample) was then added, and incubation continued for 20 min at 37 °C. Where indicated, antibodies were included for the last 10 min. Samples were electrophoresed on a nondenaturing polyacrylamide gel (5% acrylamide in 0.25 · Tris/borate/EDTA buffer). The gels were dried and autoradiographed. Western blot and in vitro kinase assay HeLa cells were lysed in 20 m M Hepes, pH 7.5, containing 50 m M KCl, 2 m M MgCl 2 ,0.5m M dithiothreitol, 0.5 m M phenylmethanesulfonyl fluoride, 5 lgÆmL )1 pepstatin, 5 lgÆmL )1 leupeptin, 30 m M NaF, 15 m M b-glycerophos- phate and 0.2% Nonidet P-40. After 10 min on ice, lysates were centrifuged for 5 min at 10 000 g, and supernatants were saved (cytoplasm). Expression of GFP-MKK6 2E was analyzed by Western blotting as described elsewhere [16]. Briefly, cytoplasmic proteins were separated by SDS/PAGE and electrophoretically transferred to poly(vinylidene difluoride) membranes (Immobilon-P TM ;Milipore).After Ó FEBS 2002 General mRNA stabilization by UV light (Eur. J. Biochem. 269) 5831 blocking with 5% dried milk in Tris-buffered saline, the membranes were incubated with monoclonal antibodies against GFP (Roche Diagnostics) for 16 h, washed and incubated with peroxidase-coupled second antibody. GFP-MKK6 2E was detected by using the SuperSignalÒ chemiluminescence system (Pierce). For in vitro kinase assays, 20 lg cytoplasmic proteins were diluted in kinase buffer (20 m M Tris/HCl, pH 7.4, 5 m M MgCl 2 ,0.2m M dithiothreitol, 0.1 m M EDTA, 0.1 m M EGTA, 20 m M b-glycerophosphate, 1 m M phenylmethanesulfonyl fluoride, 10 l M ATP), and 4 lCi [c- 32 P]ATP and 1 lg recombinant HSP27 (kindly provided by Matthias Gaestel, Medical School Hannover, Germany) were added. After 30 min at 30 °C, SDS/PAGE sample buffer was added, and samples were boiled for 5 min and separated by SDS/PAGE. Phosphorylated HSP27 was detected by autoradiography of the dried gel. RESULTS UV light induces stabilization of AU-rich mRNAs independently of the p38 MAP kinase/MK2 pathway Mechanisms that affect mRNA turnover were studied in HeLa-tTA cells expressing the tetracycline-sensitive trans- activator [25]. The decay of mRNAs expressed with the tet-off system was followed subsequent to inhibition of their transcription by the tetracycline analog doxycycline. The b-globin mRNA exhibits a long half-life under these conditions (> 10 h [16], and additional data, not shown). Decay of ARE-containing transcripts was investigated by expressing b-globin reporter constructs containing the regulatory region of the IL-8 3¢-UTR (BBB-IL-8 972)1310 ) and, to minimize the chance of detecting effects specific only for that region, with the well characterized ARE of GM-CSF (BBB-GMCSF ARE ). In agreement with previous studies [6,16,26], the mRNAs derived from both constructs were rapidly degraded in unstimulated cells (Fig. 1B). Exposure to UV light (UV-B) induced marked and dose- dependent stabilization of both hybrid mRNAs (Fig. 1B,C). According to kinetic studies, the increase in stability persisted for about 14 h after exposure to UV light and gradually disappeared thereafter (not shown). As reported previously [16], activators of the p38 MAP kinase/MK2 pathway induce stabilization of AU-rich mRNAs, including BBB-IL-8 972)1310 and BBB- GMCSF ARE (Fig. 1A). To determine whether mRNA stabilization induced by UV light also involved the p38 MAP kinase pathway, dominant-negative mutants of p38 MAP kinase (p38 AGF ) or MK2 (MK2 K76R )werecoex- pressed (Fig. 2A). As expected, the expression of each of the mutants strongly interfered with the stabilization induced by MKK6 2E , a selective activator of p38 MAP kinase. In contrast, neither the dominant-negative p38 MAP kinase mutant nor the dominant negative MK2 mutant affected stabilization induced by UV light (Fig. 2A). In support of this result, the pyridinyl imidazole SB 203580, a selective p38 MAP kinase inhibitor, inhibited mRNA stabilization induced by MKK6 2E , whereas it did not have a significant effect on stabilization by UV light (Fig. 2B). The data indicate that stabilization in response to UV light occurs independently of the p38 MAP kinase/ MK2 pathway. Fig. 1. UV light induces stabilization of AU-rich mRNAs. (A) Scheme of mRNAs expressed. b-Globin mRNAs with the ARE of GM-CSF (BBB-GMCSF ARE ) (Fig. 1A) or with an ARE-containing region of IL-8 mRNA (BBB-IL-8 972)1310 ) were expressed under the control of a tetracycline-regulatable promoter (for details see Materials and methods). (B) HeLa cells constitutively expressing the tet transactiva- tor protein were transfected with ptetBBB-IL-8 972)1310 and ptetBBB- GM-CSF ARE . At 2 h after UV-B exposure (1200 JÆm )2 ), doxycycline (3 lgÆmL )1 ) was added. Total RNA was isolated at the indicated times and analyzed by Northern blotting. Ethidium bromide staining of 28S rRNA is shown to allow comparison of RNA amounts loaded. (C) Quantification of results for BBB-IL-8 972)1310 mRNA from an experiment performed as in (B), but with different doses of UV light. 5832 F. Bollig et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Fig. 2. mRNA stabilization by UV light is independent of the p38/MK2 pathway. Degradation of BBB-IL-8 972)1310 and BBB-GM-CSF ARE tran- scripts was determined as in Fig. 1 in untreated or UV-exposed HeLa cells cotransfected with empty vector or expression vectors for constitutively active MKK6 (MKK6 2E ). (A) Plasmids encoding dominant negative p38 MAP kinase (p38 AGF )ordominantnegativeMK2(MK2 K76R )were cotransfected as indicated. (B) Cells received SB 203580 (2 l M ) or vehicle 3 h before the assay of RNA stability. Half-lives for BBB-IL-8 972)1310 were quantified as described in Materials and Methods. Ó FEBS 2002 General mRNA stabilization by UV light (Eur. J. Biochem. 269) 5833 UV light but not activation of p38 MAP kinase increases cytoplasmic HuR-binding activity Analysis of protein–RNA binding showed that several complexes were formed when GM-CSF ARE is incubated with cytoplasmic extracts from control cells (Fig. 3A, lane 1). Formation of two of the complexes increased with cytoplasmic extracts from UV-treated cells (Fig. 3A, lanes 1–4). It has been reported recently that UV light induces translocation of the RNA-binding protein HuR to the cytoplasm, an effect that contributes to UV-induced stabi- lization of p21/WAF message [22]. In accordance with that study, the two complexes modulated by UV light were supershifted by antibodies against HuR (Fig. 3A, lanes 5–7). Antibodies against AUF1 induced a supershift of a different complex which was not influenced by UV treat- ment (lane 8), whereas antibodies against hnRNP-A1 did not affect any of the complexes (lane 9). The increase in formation of HuR-containing complexes on exposure to UV light was also observed with the AU-rich IL-8 mRNA fragment (Fig. 3A, lanes 10–12). Expression of Myc-tagged HuR increased the amounts of both UV-inducible com- plexes, and a further increase was observed in response to UV light (Fig. 3B, compare lanes 1 and 2 with lanes 3 and 4). On the other hand, active MKK6 did not result in any detectable change in the amounts of complexes containing coexpressed Myc-tagged HuR (Fig. 3B, lanes 5 and 6), nor in the pattern of other complexes formed with the RNA (additional results, not shown). UV light but not p38 MAP kinase activation induces stabilization of non-AU-rich mRNAs The observation that stabilization of mRNAs on exposure to UV light is independent of the p38 MAP kinase pathway suggested that the mechanism of stabilization itself may be different. To elucidate this, transcript selectivity of the two ways of inducing stabilization was compared. For this purpose, additional mRNAs lacking AU-rich sequences were included in the experiments (Fig. 4). The GFP mRNA contains a short 3¢-UTR that consists of vector-derived sequences. The luciferase mRNA derived from the plasmid pUHC13-3 [25] harbors a long 3¢-UTR with regions of high A + U content, but with no overlapping AUUUA motifs nor UUAUUUA U/A U/A motif suggested to confer regulation of stability [28–30]. The TIMP1 cDNA was cloned downstream of a 196-nucleotide CAT fragment to express a CAT-TIMP1 hybrid RNA that can be distin- guished from endogenous TIMP1 transcript. The 3¢-UTR of TIMP1 is short (96 nucleotides) and devoid of AU-rich regions. The basal half-life of the GFP transcript is long (5 h), whereas that of luciferase and CAT-TIMP1 tran- scripts is rather short (Fig. 4A). Expression of MKK6 2E induced stabilization of the two ARE-containing mRNAs (BBB-GMCSF ARE and BBB-IL8 972-1310 ) but failed to exert any effect on the degradation rate of the GFP, luciferase and CAT-TIMP1 mRNAs (Fig. 4A). In contrast, exposure to UV light resulted in stabilization of all mRNAs investigated, including the short-lived CAT-TIMP1 and luciferasemRNAsaswellasthelong-livedGFPmRNA, Fig. 3. Effects of UV light and activation of p38/MK2 on complex formation between AU-rich mRNAs and cytoplasmic proteins. (A) HeLa cells were left untreated or exposed to UV light (doses 1200 JÆm )2 or as indicated). Cytoplasmic extracts were prepared and incubated with radiolabeled in vitro-transcribed RNAs consisting of the ARE of GM-CSF or the AU-rich region of the IL-8 3¢-UTR (nucleotides 972–1310). Where indicated, antibodies specific for HuR (0.75 lg), AUF1 (1 lL of a 1 : 10 dilution of serum) or hnRNP A1 (1 lLofa 1 : 10 dilution of ascites) were included. Protein–RNA complexes were separated on nondenaturing polyacrylamide gels and detected by autoradiography. Filled arrowheads indicate UV-induced complexes, and open arrowheads indicate complexes supershifted by antibodies. (B) HeLa cells transfected with expression vectors for Myc-tagged HuR or MKK6 2E as indicated were left untreated or exposed to UV light (1200 JÆm )2 ). Interaction of proteins from cytoplasmic extracts with labeled GM-CSF ARE RNA was assayed as described in (A). Fig. 4. UV light and the p38/MK2 pathway induce mRNA stabilization with different transcript selectivities. (A) Degradation of the indicated mRNAs, all expressed under the control of a tetracycline-regulatable promoter, was determined in HeLa cells expressing MKK6 2E or exposed to UV light as described in Fig. 1. (B) Degradation of the indicatedmRNAswascomparedincellstransfectedwithanexpres- sion vector for MEKK1D or with empty vector as control. 5834 F. Bollig et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Ó FEBS 2002 General mRNA stabilization by UV light (Eur. J. Biochem. 269) 5835 the half-life of which was reproducibly found to be extended (Fig. 4A). This indicates that UV light can induce stabi- lization of a much broader spectrum of mRNAs including species containing and lacking AREs in their 3¢-UTR. Of note, the mRNAs devoid of AU-rich sequences did not interact with HuR in electrophoretic mobility-shift assays (data not shown). Expression of a truncated mutant of the MAP triple kinase MEKK1 (MEKK1-D) is known to activate the p38 MAP kinase and also the JNK, ERK and NF-jB signaling pathways ([16,31] and references cited therein). Expression of MEKK1-D, in agreement with previously published results [16], induced stabilization of the two AU-rich mRNAs. However, it did not induce stabilization of the non-AU-rich mRNAs (Fig. 4B). This indicates that the pathways activated by MEKK1-D are not sufficient to induce the UV-activated mechanism of mRNA stabilization. UV light but not p38 MAP kinase activation induces stabilization of endogenous histone mRNA To ensure that the results are not only applicable to mRNAs expressed from transfected plasmids, we investigated the degradation of endogenous histone mRNA. As shown in Fig. 5A, UV light also induced stabilization of the endo- genous mRNA. The histone transcripts do not contain an ARE in their 3¢-UTR, confirming the results in Fig. 4 that this type of regulatory element is not essential for UV-induced stabilization. Unlike most other mRNAs, the histone transcripts are not polyadenylated [32]. Therefore this result indicates that the poly(A) tail is also dispensable Fig. 5. UV light but not activation of the p38/MK2 pathway induces stabilization of endogenous histone mRNA. (A) Degradation of endogenous histone mRNA in untreated and UV-exposed cells was determined by Northern-blot analysis of total RNA isolated at the indicated times after inhibition of transcription with actinomycin D (5 lgÆmL )1 ). (B) HeLa cells stably transfected with a tetracycline-regulated expression vector for GFP-MKK6 2E were incubated for 24 h with tetracycline (100 ngÆmL )1 ) to suppress GFP-MKK6 2E expression, or without tetracycline to allow GFP-MKK6 2E expression, and with SB 203580 (2 l M ) as indicated. Cytoplasmic lysates were assayed for GFP-MKK6 2E protein by Western blot, and for activation of the p38 MAP kinase/MK2 pathway by in vitro kinase assays with recombinant HSP27 as substrate. (C) Stable HeLa cell transfectants were kept with (control, UV) or without (GFP-MKK6 2E ) tetracycline to suppress or to allow GFP-MKK6 2E expression, respectively, as described for (B). SB 203580 (2 l M ) was added where indicated. Exposure to UV light (UV) was performed 2 h before addition of actinomycin D. Degradation of histone mRNA was determined as in (A). 5836 F. Bollig et al.(Eur. J. Biochem. 269) Ó FEBS 2002 for UV-induced stabilization. The effect of the p38 MAP kinase pathway on the stability of endogenous histone mRNAwasassayedincellsstablytransfectedwitha plasmid encoding constitutively active MKK6 2E .This procedure allowed the expression of the active kinase in all cells as opposed to transient transfection which affects only part of the cells. The stable transfectants express GFP- MKK6 2E downstream of the tetracycline-regulatable pro- moter. Removal of tetracycline from the culture medium resulted in GFP-MKK6 2E expression as observed by green fluorescence (not shown) and Western blot (Fig. 5B, upper panel). It also resulted in activation of the p38 MAP kinase pathway, as determined by phosphorylation of recombinant HSP27 in in vitro kinase assays with cytoplasmic lysates (Fig. 5B, lower panel). The half-life of endogenous histone mRNA in the cells kept with tetracycline was similar to that of untransfected HeLa cells assayed in parallel (62 vs. 68 min; not shown). Omission of tetracycline to induce expression of GFP-MKK6 2E did not alter histone mRNA stability (Fig. 5C), indicating that the p38 MAP kinase pathway does not affect its degradation. UV light induced stabilization of the transcript, confirming the result in untransfected cells shown in Fig. 5A. Importantly, the addition of SB 203580, which inhibited p38 MAP kinase activity (Fig. 5B, lower panel), did not impair UV-induced stabilization (Fig. 5C). These results illustrate that UV light can induce stabilization of a non-AU-rich endogenous mRNA in a p38 MAP kinase-independent manner. The results in Figs 4 and 5 provide evidence for two distinct modes of mRNA stabilization: selective, ARE- dependent stabilization of mRNAs by activation of the p38 MAP kinase cascade, as opposed to a general mechanism of stabilization triggered by UV light. DISCUSSION Extracellular stimuli affect gene expression in part by modulating the degradation rate of mRNAs. In the present study we investigated the effect of UV light on the stability of mRNAs expressed using the tet-off system which allows the termination of only the synthesis of the mRNA under study. This enabled us to also investigate transcripts with comparatively long half-lives. The data in Fig. 4 indicate that UV light exerts a strong stabilizing effect on a range of different mRNAs without apparent selectivity. Most no- tably, UV light stabilized mRNAs with a 3¢-UTR that did not contain an ARE, a type of cis-element involved in the control of many cytokine and oncogene mRNAs (e.g. [6,7,16]). Results corresponding to mRNAs derived from trans- fected tet-off plasmids were obtained with endogenous histone mRNA (Fig. 5). Although lacking an ARE, histone mRNA was also markedly stabilized on treatment of cells with UV light. Furthermore, as histone mRNA does not carry a poly(A) tail at its 3¢ end [32], this structural element common to most other mRNAs is apparently also dispen- sable for UV-induced stabilization. The inflammatory stimuli LPS and IL-1 induce stabi- lization of several AU-rich mRNAs by activating the p38 MAP kinase/MK2 pathway [16,18]. However, p38 MAP kinase activation did not affect the degradation of non-ARE- containing transcripts, as shown for the plasmid-derived mRNAs (Fig. 4) as well as for the endogenous histone mRNA (Fig. 5). Thus with respect to selectivity of the mRNAs affected, the effect of UV light differs from stabilization induced by the p38 MAP kinase/MK2 pathway. Although UV light activates p38 MAP kinase and MK2 [24], stabilization of mRNAs in response to UV light occurs mainly through a mechanism independent of these kinases. This is suggested by the different transcript selectivity of stabilization by UV light as opposed to activation of the p38/MK2 pathway (discussed above), as well as by the results obtained when this pathway is inhibited. The p38 MAP kinase inhibitor SB 203580 failed to inhibit UV- induced stabilization of endogenous histone mRNA (Fig. 5). Stabilization of ARE-containing reporter mRNAs was also insensitive to the inhibitor, as well as to coexpres- sion of inhibitory mutants of the p38 and MK2 kinases (Fig. 2). Insensitivity of UV-induced stabilization to each of the two mutants alone would have left the possibility that p38 MAP kinase activates a different downstream compo- nent, or that MK2 is activated by a different upstream component. The lack of inhibition by both kinase mutants together strongly indicates that the effect of UV light on mRNA stability is independent of the p38 MAP kinase/ MK2 pathway. It cannot be excluded, however, that a p38/ MK2-dependent component of UV-induced mRNA stabil- ization may be detected if the predominant mechanism is inhibited. The mechanism of UV-induced stabilization remains unidentified. The lack of stabilization of non-ARE tran- scripts by MEKK1 (Fig. 4B), which in our cells is an effective activator of p38, JNK and NF-jB pathways, renders involvement of these pathways in this effect of UV light highly unlikely. Others have shown that UV-induced stabilization of p21 involves translocation of HuR to the cytoplasm [22]. HuR can bind to AU-rich sequences [33], including those of the GM-CSF and IL-8 mRNAs (Fig. 3). Although we also observed increased binding of HuR to these RNAs in the cytoplasm of cells exposed to UV light, the non-ARE transcripts did not interact with HuR in vitro (not shown). This may be due to technical limitations to detect such interactions in gel shifts, and/or to a more indirect association of HuR with those transcripts. Alternatively, HuR may affect only the fate of a subset (i.e. ARE- containing) of mRNAs in response to UV light. Of note, activation of the p38 pathway did not seem to affect HuR binding to the GM-CSF ARE. This is in agreement with a recent report by Dean and colleagues [34] who did not observe a change in levels of HuR in p100 or nuclear fractions of a macrophage-like cell line in response to LPS. UV light, unlike IL-1, damages cells directly. It induces activation of caspases and cell death. Under the condi- tions applied in this study, about 30% of the cells undergo apoptosis within 24 h of UV exposure. Caspases have been shown to cleave translation initiation factors [35]. This effect is expected to alter mRNA metabolism and could also be involved in the stabilization observed. However, addition of the caspase inhibitor Z-VAD.fmk effectively reduced apoptosis in response to UV light, but did not interfere with the UV-induced mRNA stabiliza- tion (not shown). This argues against the involvement of caspase activation. One of the direct effects of UV light is the site-specific damage of 28S rRNA [36]. This has been suggested to be Ó FEBS 2002 General mRNA stabilization by UV light (Eur. J. Biochem. 269) 5837 involved in triggering of the ribotoxic stress response which includes translational inhibition and activation of stress kinase pathways and gene expression. It is possible that ribosomal damage or its consequences leads to inhibition of mRNA decay mechanisms. As it is known that UV light also targets the transcriptional machinery [37,38], stabiliza- tion of mRNAs may represent an element of the cellular response to direct damage. This may serve to prevent mRNAs from being degraded in conditions where new synthesis is impaired. In summary, the data presented here and in the literature cited point to two different modes of mRNA stabilization triggered by inflammatory stimuli and UV light. The p38/ MK2 pathway, activated by inflammatory stimuli such as IL-1 and LPS, induces stabilization of various ARE- containing mRNAs. Independently of this pathway, UV light activates a more general mechanism of mRNA stabilization, one that does not require an ARE or a poly(A) tail as a cis-element. ACKNOWLEDGEMENTS This work was supported by the Deutsche Forschungsgemeinschaft (grants Ho1116/2, GK-GRK 99/2-98, SFB 566/A10 and Kr1143/2-3, SFB 566/B06). 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The data indicate that stabilization in response to UV light occurs independently of the p38 MAP kinase/ MK2 pathway. Fig. 1. UV light induces stabilization. 2002 General mRNA stabilization by UV light (Eur. J. Biochem. 269) 5833 UV light but not activation of p38 MAP kinase increases cytoplasmic HuR-binding activity Analysis of protein–RNA binding. IL-1, UV light is a potent inducer of in ammation and induces expression of numerous genes including cytokines and oncogenes [20,21], which is in part due to the stabilization of mRNAs [22,23]. UV

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