Báo cáo khoa học: Induction of translationally controlled tumor protein (TCTP) by transcriptional and post-transcriptional mechanisms pot

Induction of translationally controlled tumor protein (TCTP) by transcriptional and post-transcriptional mechanisms Irina Schmidt, Michael Fa ¨ hling, Benno Nafz, Angela Skalweit and Bernd-Joachim Thiele Charite ´ , Universita ¨ tsmedizin Berlin, Institut fu ¨ r Vegetative Physiologie, Germany Translationally controlled tumor protein (TCTP) is a highly conserved 19 kDa GDP ⁄ GTP exchange factor [1,2] that is involved in control of cell growth and pro- liferation by regulating the activity of Rheb (Ras homolog enriched in brain), a Ras superfamily GTPase [3]. It has been identified in a broad spectrum of eukaryotic organisms, from plants, yeasts, fungi, para- sites, insects, fishes, and birds up to mammals, and is involved in a wide range of cellular processes [4]. TCTP attracted interest mainly as an antiapoptotic factor [5–7], for its role in tumorigenesis [8,9], and as a tubulin-binding [10] or Ca 2+ -binding protein [11,12]. Moreover, it has other, extracellular, functions as a histamine-releasing factor and cytokine [13,14]. TCTP synthesis is extensively regulated at the tran- scriptional and post-transcriptional levels [4,15,16]. It is ubiquitously expressed in all mammalian tissues ana- lyzed so far, at a low rate in postmitotic tissues such as Keywords gene expression; post-transcriptional control; TPT1 gene, transcription; translationally controlled tumor protein (TCTP) Correspondence B J. Thiele, Charite ´ , Universita ¨ tsmedizin Berlin, Institut fu ¨ r Vegetative Physiologie, Tucholskystr. 2, 10117 Berlin, Germany Fax: +49 30 450 528972 Tel: +49 30 450 528184 E-mail: bernd.thiele@charite.de (Received 14 June 2007, revised 23 August 2007, accepted 28 August 2007) doi:10.1111/j.1742-4658.2007.06069.x Expression of the human TPT1 gene coding for translationally controlled tumor protein (TCTP) was investigated in Calu-6 and Cos-7 cells under the influence of 4b-phorbol 12-myristate 13-acetate (PMA), forskolin, dioxin and the heavy metals copper, nickel and cobalt. Transcriptional and post-transcriptional aspects of the mechanism were analyzed by TCTP mRNA ⁄ protein quantification, luciferase reporter gene assays depending on TPT1 promoter sequences or TCTP mRNA 5¢⁄3¢-UTRs and investiga- tion of the interaction of RNA-binding proteins with UTRs by UV-cross- linking. PMA, forskolin, dioxin, cobalt and nickel induced TCTP expression in 24 h in both cell lines about 2.2–3.2-fold at the mRNA level and 1.6–2.2-fold at the protein level. The highest induction rate, 4.5–5.0- fold at the mRNA level and 3.5–4.0-fold at the protein level, was observed with copper. TPT1 promoter assays showed transcriptional activation by PMA, forskolin and dioxin (2.0–3.1-fold) and a 7.0–8.0-fold increase by copper, whereas cobalt and nickel had no effect. Deletion analysis revealed that copper-dependent transcriptional control was transmitted by a metal- responsive element residing in the TPT1 promoter. Post-transcriptional activation of TCTP expression was associated with the action of dioxin, nickel, cobalt (1.8–2.3-fold) and copper (2.5–3.0-fold), whereas stimulation of TCTP synthesis by copper was mediated by the TCTP mRNA 3¢-UTR (3.2-fold) but not by the 5¢-UTR (0.5-fold). mRNA stabilization was found to mediate these effects of cobalt and nickel. Post-transcriptional regulation was associated with qualitative and quantitative changes in the binding of specific RNA-binding proteins to UTRs. Abbreviations ARE, AU-rich element; CREB, cAMP-responsive element-binding protein; HIF, hypoxia inducible factor; MRE, metal-responsive element; PMA, 4b-phorbol 12-myristate 13-acetate; RNA-BP, RNA-binding protein; TCTP, translationally controlled tumor protein; TPT1, gene coding for human TCTP. 5416 FEBS Journal 274 (2007) 5416–5424 ª 2007 The Authors Journal compilation ª 2007 FEBS brain, but at high levels in tissues undergoing active cell division and in tumor cells [8,9,17]. A wide variety of chemicals are able to induce TCTP synthesis, including substances such as phorbol esters and lipopolysaccha- rides [18], cytotoxic drugs [19], Ca 2+ [11], heavy metals [20], and dioxins [21]. However, less is known about the mechanistic background of their action. A first analysis of transcriptional control of the TPT1 gene revealed that phorbol esters and forskolin stimulate TCTP synthesis by cAMP-responsive element-binding protein (CREB) transcription factors via cAMP signal- ing [22]. How other stimulators, including divalent heavy metals and dioxins, act is unknown. In this study we investigated the capability of poten- tial TCTP synthesis-stimulating substances with differ- ent chemical structures to induce TPT1 gene expression at the transcriptional or post-transcriptional level in culture cells. For this purpose, we employed TCTP mRNA and protein quantification as well as luciferase reporter gene expression, using constructs in which the luciferase gene is under control of TPT1 promoter sequences or TCTP mRNA 5¢⁄3¢-UTRs and by study- ing RNA–protein interactions. Results We investigated TCTP expression in human Calu-6 cells, a lung carcinoma cell line, and in monkey Cos-7 cells, a kidney fibroblast cell line, under the influence of 4b-phorbol 12-myristate 13-acetate (PMA), forskolin and dioxin, or the heavy metals nickel, cobalt and copper. To discriminate between transcriptional and post-transcriptional actions, we performed cell transfec- tion experiments and luciferase reporter gene assays in two different ways. To analyze the effect of TCTP induction on transcription, the main promoter of the human TPT1 gene () 312 ⁄ ) 1) [22] was cloned upstream of the promoterless luciferase gene and assayed. For characterization of mRNA-related regula- tive capacity, 5¢- and 3 ¢-UTRs of TCTP mRNA were cloned upstream and downstream of the luciferase gene containing a constitutive promoter. Figures 1 and 2 show TCTP mRNA and protein quantification data, demonstrating the way in which TCTP expression responded to the six selected substances. It is evi- dent that PMA, forskolin, dioxin, cobalt and nickel were able to induce TCTP in both cell lines at the mRNA and protein levels by 2.2–3.2-fold (mRNA) and 1.6–2.2-fold (protein) in 24 h. The highest induction rate was observed with copper, i.e. 4.5–5.0-fold (mRNA) and 3.5–4.0-fold (protein). Figure 3 depicts TPT1 promoter ⁄ luciferase reporter gene assays. It is obvious that the transcriptional activ- ity of the TPT1 promoter has only a partial similarity to TCTP mRNA and TCTP protein expression. In both cell lines, PMA, forskolin and dioxin stimulated transcription about 2.0–3.1-fold. Only copper stimu- lated the luciferase reporter gene at a high rate between 7.0-fold and 8.0-fold, whereas nickel and cobalt did not stimulate transcription; instead, they reduced luciferase expression slightly. In Fig. 4, the influence of potential TCTP stimulators on luciferase expression is shown, with the luciferase gene put under control of TCTP mRNA UTRs. A moderate post- transcriptional stimulatory effect was seen with dioxin, nickel and cobalt (1.8–2.3-fold), and again the highest rate was seen with copper (2.5–3.0-fold). The finding that copper ions are able to stimulate TPT1 gene transcription very effectively prompted us to investigate the mechanistic background in more detail. A comparative theoretical analysis of the human TPT1 promoter led to the discovery of a puta- tive metal-responsive element (MRE) in position ) 81 ⁄ ) 68 (see Discussion). A variant of the TPT1 pro- moter, in which the MRE was deleted, was used in the TCTP mRNA/β-Actin mRNA A B Fig. 1. Influence of various substances on TCTP expression at the mRNA level. Human lung tumor Calu-6 or monkey kidney tumor Cos-7 cells were incubated in the absence or presence of PMA, forskolin (Fors), dioxin (Diox), NiCl 2 (Ni), CoCl 2 (Co) or CuCl 2 (Cu) for 24 h. Total RNA was extracted, and TCTP mRNA was analyzed by northern blotting and quantified by scanning using SCION IMAGE soft- ware. TCTP mRNA levels were normalized to the internal control, b-actin mRNA. (A) Representative northern blot including loading control (18S and 28S rRNA stained by ethidium bromide). (B) Statis- tical analysis of three independent experiments. For quantification, the sum of both TCTP mRNA signals (0.8 and 1.2 kb, differing in the length of the 3¢-UTR [22]) was used. I. Schmidt et al. TCTP induction FEBS Journal 274 (2007) 5416–5424 ª 2007 The Authors Journal compilation ª 2007 FEBS 5417 same type of luciferase reporter gene experiment as described in Fig. 3. The deletion of the putative MRE resulted in a nearly complete loss of the ability of the TPT1 promoter to respond to copper ions. The resid- ual luciferase activity dropped to about 10% as com- pared to the construct with an intact MRE (Fig. 5A). As demonstrated in Fig. 4, UTRs of mRNA are targets mediating post-transcriptional regulation of TCTP expression. To discriminate between 5¢- and 3¢-UTR-mediated actions, we created luciferase con- structs that were separately under control of the TCTP 5¢-or3¢ -UTR and performed copper-depen- dent transfection ⁄ reporter gene assays (Fig. 5B). The results show that the post-transcriptional copper stimulation of TCTP expression was associated exclu- sively with the 3¢-UTR (3.2-fold stimulation). In con- trast, the 5¢-UTR had an inhibitory effect of about 50%, and the 5¢⁄3¢-UTR combination resulted in intermediate values (about 2.5-fold stimulation). Nickel and cobalt seem to stimulate TCTP expres- sion, not by transcriptional activation but solely by mRNA-targeted post-transcriptional processes. The results shown in Fig. 1 demonstrate that the action of cobalt and nickel led to an increase in mRNA concen- tration, but not to activation of the promoter (Fig. 3). This could imply an influence on mRNA stability. To clarify this, we inhibited transcription with actinomy- cin D and followed the kinetics of TCTP mRNA decay (Fig. 6). Interestingly, inhibition of transcription by actinomycin D treatment did not accelerate mRNA decay as expected; instead, it slowed down this process slightly. Nickel and cobalt treatment delayed TCTP mRNA decay further, and clearly increased mRNA stability. To test to what extent RNA-binding proteins (RNA-BPs) are involved in post-transcriptional control of TCTP synthesis, electromobility shift assays and UV-crosslinking assays were performed. For this pur- pose, 32 P-labeled in vitro transcripts of the 5¢-UTR and 3¢-UTR of the main TCTP mRNA were incubated with cytosolic extracts (S10) of Calu-6 cells, which were grown with a selection of inducing agents (PMA, dioxin, cobalt and nickel) or without effector as a con- trol. The 5¢- and 3¢-UTR gave specific signals of TCTP/β-Actin A B Fig. 2. Influence of various substances on TCTP expression at the protein level. Cells were treated with TCTP-inducing substances as described in Fig. 1, cytoplasmic protein extracts were separated on SDS gels, and TCTP was visualized by western blotting. Blots were quantified by scanning, as described in Fig. 1. TCTP levels were normalized to internal b-actin using an antibody to b-actin. (A) Rep- resentative western blot including b-actin control. (B) Statistical analysis of three independent experiments. Fig. 3. Effect of TCTP-inducing substances on TPT1 promoter activ- ity. Three hundred and twelve nucleotides of the human TPT1 pro- moter () 312 ⁄ ) 1) were cloned 5¢- to the promoterless firefly luciferase gene in the vector pGLbasic. Plasmids were transfected into Calu-6 and Cos-7 cells in the presence or absence of TCTP- inducing substances. For normalization of transfection efficiency, cells were cotransfected with Renilla luciferase. Luciferase assays were performed as described in Experimental procedures (n ¼ 6). Fig. 4. Effect of TCTP mRNA 5¢- and 3¢-UTR on luciferase reporter gene expression in the presence of TCTP-inducing substances.The complete 5¢- and 3¢-UTR of human TCTP mRNA were cloned upstream and downstream of the luciferase coding sequence into the pGL3promoter vector containing a constitutive SV40 promoter, thereby replacing luciferase UTRs. Cells were transfected in the presence or absence of TCTP-inducing substances, and luciferase activity was assayed as described in Fig. 3 (n ¼ 6). TCTP induction I. Schmidt et al. 5418 FEBS Journal 274 (2007) 5416–5424 ª 2007 The Authors Journal compilation ª 2007 FEBS RNA–protein complexes with the electromobility shift assay; however, significant differences in the electro- phoretic migration behavior of the complexes were observed neither with control nor with effector-treated cell extracts (not shown). To detect more subtle differ- ences in the composition of RNA–protein complexes, we performed UV-crosslinking experiments (Fig. 7). The results can be summarized as follows: (a) the pat- terns of protein binding to the 5¢-UTR or 3¢-UTR dif- fer significantly, although some proteins seem to bind to both; (b) the pattern of 5¢-UTR and 3¢-UTR protein binding of PMA-treated cell extracts does not differ as compared to the control; (c) dioxin-, nickel- and cobalt-treated extracts show qualitative (appearance of new bands and suppression of control bands) and quantitative (signal intensity) differences in binding properties at the 5¢-UTR as well at the 3¢-UTR as compared to controls. Discussion TCTP is a ubiquitous protein with a basic role in G-protein signaling. It is effectively induced under a wide variety of growth and stress conditions, and by chemical substances with quite different structures. Recently, we demonstrated the transcriptional activa- tion of the human TPT1 promoter by phorbol ester and forskolin in T24 cells, a bladder carcinoma cell line, via cAMP-responsive element–CREB interaction [22]. As shown in the present study, the TPT1 pro- moter responded in a similar way in Calu-6 and Cos-7 cells. Therefore, we investigated whether TCTP expres- sion could also be stimulated by post-transcriptional events. Reporter gene assays demonstrated that the UTRs of TCTP mRNA were able to mediate a stimula- tory effect on luciferase expression under the influence of dioxin, copper, nickel and cobalt. On the other hand, PMA and forskolin seem to act by a pure tran- scriptional mechanism, as suggested in our earlier work [22]. As demonstrated by actinomycin D inhibition experiments (Fig. 6), nickel and cobalt are able to stabilize TCTP mRNA, thereby improving TCTP synthesis. UTRs mediate their controlling properties predominantly by interaction with regulative RNA- BPs. The action of nickel and cobalt was primarily discussed in the context of their capability to interact with transcription factor hypoxia inducible factor (HIF), thereby simulating hypoxic conditions [23–25]. Our experiments demonstrate that cobalt and nickel do not interfere with TPT1 transcription. Moreover, no HIF-responsive element can be detected in the TPT1 promoter. On the other hand, as shown with erythro- poietin or tyrosine hydroxylase, improved expression by hypoxia is partially controlled by UTRs of the appropriate mRNAs in interaction with RNA-BPs [26,27]. In the case of TCTP mRNA, this view is sup- ported by our RNA–protein interaction studies (Fig. 7). Nickel as well as cobalt treatment changes the pattern of protein binding to the 5¢-UTR and 3¢-UTR. This indicates that RNA-BPs may contribute to post-transcriptional control of TCTP mRNA stability. Interestingly, the crosslinking patterns of cobalt- and A B Fig. 5. Transcriptional and post-transcriptional control of TCTP expression by copper. (A) Copper-dependent luciferase expression in Cos-7 cells after transfection with luciferase vector pGL3 pro- moter modified with the TPT1 promoter. The SV40 promoter of pGL3 has been replaced by the TPT1 promoter () 312 ⁄ )1 ) con- taining an MRE at position ) 81 ⁄ ) 68, or using a construct in which the MRE was deleted (– MRE). Copper-related reporter gene expression was dependent on the presence of the MRE (+ MRE) in the TPT1 promoter. (B) Copper-dependent luciferase expression after transfection with vector pGL3 promoter modified with TCTP UTRs. The Luc UTRs have been replaced by TCTP mRNA 5¢- and 3¢-UTRs (5¢⁄3¢), by TCTP mRNA 5¢-UTR (5¢), or by TCTP mRNA 3¢- UTR. The TCTP 3¢-UTR but not the 5¢-UTR stimulated reporter gene expression in the presence of copper. Experiments were repeated three times. I. Schmidt et al. TCTP induction FEBS Journal 274 (2007) 5416–5424 ª 2007 The Authors Journal compilation ª 2007 FEBS 5419 nickel-treated cell extracts are not identical. This sug- gests that mechanistic details may differ between nickel and cobalt action. However, the nature of putative cis-elements in TCTP mRNA and trans-acting factors, as well as the role of nickel and cobalt ions, have yet to be analyzed experimentally. So far, our experiments demonstrate (Fig. 6) that cobalt and nickel act via inhi- bition of TCTP mRNA decay, and that this is associ- ated with differences in the interaction of cytosolic proteins with the TCTP mRNA 5¢-UTR and 3¢-UTR. Dioxin induced TCTP synthesis in a mixed tran- scriptional ⁄ post-transcriptional mode. The high toxic- ity of dioxins is related to their action as ‘endocrine disrupters’. The endocrine disrupter function includes changes of expression of dioxin target genes, one of which is the gene encoding TCTP [21]. Dioxins bind to the aryl hydrocarbon receptor, which then dimerizes with the aryl hydrocarbon receptor nuclear transloca- tor, to interact with a xenobiotic response element motif in the DNA. Oikawa et al. found by computer search a xenobiotic response element motif 5 kb upstream of the mouse and human TPT1 transcription initiation site [21], which could explain the observed transcriptional activation. The TCTP mRNA UTRs, on the other hand, seem to contain additional sequences that could be involved in a xenobiotic response. The crosslinking pattern obtained with dioxin-treated cell extracts differs significantly from controls and from experiments with heavy metal-trea- ted cells (Fig. 7), suggesting the participation of indi- vidual RNA-BPs in this response. Potential candidate proteins and mechanistic details have yet to be investi- gated. The TCTP-inducing capability of copper was known from experiments with earthworms inhabiting copper- contaminated soils [20]. No data, however, exist from mammals or from mammalian cells in culture. In Calu- 6 and Cos-7 cells, copper turned out to be a potent inducing agent, comparable to the situation in earth- worms. From the mechanistic point of view, it is of interest that the action of copper involves both a tran- scriptional and a post-transcriptional effect, although the strong promoter action exceeds the influence of the UTRs by a factor of 3 in reporter gene assays. Proto- types of metal-inducible genes, which play an important role in metal ion homeostasis and which are effectively induced by transitional metals such as copper, cadmium or zinc, are the metallothionein genes. Metal-inducible metallothionein transcription is regulated primarily through the interaction between MREs and metal tran- scription factor-1 [28,29]. The MRE consensus sequence is described as a core motif with the sequence TGC(G ⁄ A)CNC flanked by a short GC-rich domain [30]. This sequence shows a high degree of homology A B TCTPmRNA/rRNA rel.units Fig. 6. Control of TCTP mRNA stability by cobalt and nickel. Cos-7 cells were cultivated for up to 36 h in the presence of actinomycin D with or without 300 l M NiCl 2 (Ni) or 300 lM CoCl 2 (Co). Total RNA was extracted at each time point, and TCTP mRNA was analyzed by northern blotting and quantified by scanning using SCION IMAGE software. TCTP mRNA levels were normalized to internal 18S and 28S rRNA stained with ethidium bromide, which were also quantified by scanning. (A) Representative northern blot and rRNA staining. (B) Statistical analysis of three independent experiments. For mRNA quantification, the sum of both TCTP transcripts of 0.8 and 1.2 kb [22] was used. Inhibition of transcription by actinomycin D increased the TCTP mRNA half-life slightly, but nickel and cobalt gave an additional and significant increase in mRNA stability. TCTP induction I. Schmidt et al. 5420 FEBS Journal 274 (2007) 5416–5424 ª 2007 The Authors Journal compilation ª 2007 FEBS with a sequence in the TPT1 promoter (TGCGtCGCT- TCCGG, ) 81 ⁄ ) 68), which is highly conserved in mammalian TPT1 genes [16,22]. It mismatches the con- sensus core motif in only one nucleotide (printed in lower case), and could be addressed as an MRE-like sequence, as it functions in copper induction of prion protein expression [31]. Of particular interest is the find- ing that copper stimulates TCTP expression at the post- transcriptional level. In earlier work, post-transcrip- tional events were postulated in the context of induc- tion of human metallothionein genes by copper, although no mechanistic details were published [32]. In this study, we have shown that the post-transcriptional copper response at the mRNA level is associated with the 3¢-UTR. With regard to the mRNA sequence, it has been known since the first cloning of human TCTP mRNA that three AU-rich elements (AREs) of the AUUUA type reside in the TCTP 3¢-UTR [33]. In general, AREs are associated with mRNA stabiliza- tion ⁄ destabilization, depending on ARE type and on the interaction with different ARE-binding proteins such as AUF1 or HuR [34]. At present, we can only speculate that these AREs could be targets for TCTP mRNA stabilization, a process possibly triggered by copper ions. The molecular details of the nature of the cis-elements and trans-acting factors involved (RNA- BPs or micro RNAs) and, in particular, the signaling pathway related to the interaction with copper ions remain to be investigated. Recently, the renal transcriptional response of TCTP to another heavy metal, uranium, was investigated. Uranyl ions induced TCTP at the mRNA level about two-fold and at the protein level at least five-fold [35]. This suggests a transcriptional as well as a post-tran- scriptional mechanism in the stress response to ura- nium, as for copper. Experimental procedures Cell culture and RNA ⁄ protein isolation Human fetal lung carcinoma Calu-6 cells were maintained in MEM ⁄ Earle’s (Biochrom AG, Berlin, Germany) con- taining 10% heat-inactivated fetal bovine serum, 1 · MEM nonessential amino acids, 1 · MEM sodium pyruvate, 0.065% sodium hydrogen carbonate, 200 l m l- glutamine, 100 UÆmL )1 penicillin, and 10 lgÆmL )1 strepto- mycin. Monkey kidney fibroblast Cos-7 cells were main- tained in DMEM (high glucose; PAA Laboratories GmbH, Co ¨ lbe, Germany) supplemented with 10% heat- inactivated fetal bovine serum, 200 lm l-glutamine, 100 UÆmL )1 penicillin, 10 lgÆmL )1 streptomycin, and 10 mm Hepes buffer. The cells were cultured in a 5% CO 2 atmosphere at 37 °C. Stimulation experiments were carried out with forskolin, 10 lm final concentration (Sigma, Taufkirchen, Germany; stock solution 10 mm in dimethylsulfoxide), PMA, 100 nm final concentration (Sigma, stock solution 100 lm in dim- ethylsulfoxide), dioxin, 100 nm final concentration (50 lgÆmL )1 stock solution of 2,3,7,8-tetrachlorodibenzo-p- dioxin in dimethylsulfoxide; LGC Promochem, Wesel, Germany), 30 lm CuCl 2 (VWR International, Dresden, Germany), 300 lm CoCl 2 (Sigma) or 300 lm NiCl 2 (Sigma), for the times indicated. For inhibition of transcrip- tion, actinomycin D (MoBiTech, Goettingen, Germany) was used at a concentration of 10 lgÆmL )1 . For RNA and protein isolation, cells were washed with ice-cold NaCl ⁄ P i . RNA was prepared using RNA-Bee (Biozol Diagnostica Vertrieb GmbH, Eching, Germany), in accordance with the manufacturer’s protocol. Protein extracts (10 000 g supernatants, S10, Sigma 3K15 centrifuge with 12154-H rotor, Sigma Laborzentrifugen GmbH, Fig. 7. Interaction of RNA-BPs with TCTP mRNA 5¢- and 3¢-UTR analyzed by UV-crosslinking. The complete 5¢-UTR (134 nucleotides) and 3¢-UTR1 (257 nucleotides) of the main TCTP mRNA were tran- scribed in vitro in the presence of [ 32 P]UTP and incubated with cytoplasmic cell extracts (S10) of Calu-6 cells (control) or cells, in which TCTP was induced for 24 h by PMA, dioxin, NiCl 2 , or CoCl 2 . After UV-crosslinking and RNase digestion, transfer-labeled RNA- BPs were analyzed by SDS ⁄ PAGE and autoradiography. As molecu- lar weight markers, the 14 C-labeled protein marker mixture of Amersham was run in the same gel. I. Schmidt et al. TCTP induction FEBS Journal 274 (2007) 5416–5424 ª 2007 The Authors Journal compilation ª 2007 FEBS 5421 Osterode, Germany) were isolated using lysis buffer (10 mm Tris, pH 7.5, 140 mm NaCl, 1 mm EDTA, 25% glycerol, 0.1% SDS, 0.5% Nonidet P-40, 1 mm dithiothreitol and complete protease inhibitor mix; Roche Diagnostics, Mann- heim, Germany). Cell transfection and luciferase reporter gene assays Calu-6 and Cos-7 cells were cultured in 96-well plates (lClear Platte 96K; Greiner BIO-ONE GmbH, Fricken- hausen, Germany) and cotransfected with the firefly lucif- erase pGL3 promoter vector (Promega, Mannheim, Germany), or with constructs containing TPT1 promoter or TCTP mRNA UTR sequences and the Renilla lucifer- ase phRL-TK vector (ratio 1 : 1.5 or 1 : 2), using the FuGENE 6 Transfection Reagent (Roche Diagnostics) according to the manufacturer’s protocol. Transfection was carried out in the presence or absence of TCTP-stim- ulating substances (see ‘Cell culture and RNA ⁄ protein iso- lation’). After 6 h, the transfection medium was removed, and measurements were started after addition of fresh medium. The luciferase activity was detected using a lumi- nometer (Luminoscan RS, Thermo Electron GmbH, Dreieich, Germany) programmed with individual software (luminoscan rii; R. Mrowka, Institute of Physiology, Charite ´ , Berlin). The transfection with the Renilla lucifer- ase served as control. Molecular cloning procedures For analysis of the TCTP promoter in luciferase reporter gene assays, the promoterless luciferase vector pGL3basic was used. Nucleotides ) 1 ⁄ ) 312 of the human TPT1 pro- moter were amplified from a genomic clone [22] and inserted into the multiple cloning region (KpnI ⁄ NphEI site) of pGL3basic. For analysis of TCTP UTRs, the luciferase vector pGL3 promoter (Promega; constitutive SV40 promoter) was modified. Therefore, the vector-specific 5¢- and 3¢-UTRs of luciferase mRNA were replaced by human TCTP mRNA UTRs. The UTRs (5¢-UTR, 134 nucleo- tides, and 3¢-UTR, 257 nucleotides) [16] were amplified by PCR, and restriction sites were added by primer extension. The 5¢-UTR of TCTP mRNA was cloned using the pGL3p vector-specific HindIII and NcoI restriction sites, and the 3¢-UTR using the XbaI and BamHI restriction sites. The quality of processed vectors was confirmed by sequencing. The resulting vector constructs expressed a constitutively transcribed luciferase transcript with or with- out specific TCTP UTRs. The ) 73 ⁄ ) 80 nucleotide-deleted human TPT1 promoter ) 1 ⁄ ) 312 construct in pGL3p was obtained by PCR for assessment of the influence of the MRE region on luciferase expression under stress. Northern and western blot analysis Total cellular RNA (15 lg) was separated by electrophore- sis on 1% agarose gels containing formaldehyde. The RNA was capillary-transferred to positively charged nylon membranes (Roche Diagnostics), visualized by ethidium bromide staining to document relative levels of 18S ⁄ 28S rRNA, and hybridized to digoxygenin-labeled TCTP anti- sense transcripts. The detection was performed using the digoxygenin RNA Labeling Kit (Roche Diagnostics) according to the manufacturer’s protocol. mRNA levels were normalized to 18S and 28S rRNA. Protein extracts (20 lg per sample) were separated by SDS ⁄ PAGE. After electrophoresis, proteins were trans- ferred to Hybond-P membranes (Amersham Pharmacia Biotech, Freiburg, Germany) using a Bio-Rad (Munich, Germany) Mini Trans-Blot transfer cell. The membranes were blocked for 1 h with 5% Blot-Quick Blocker (Chem- icon, Schwelbach, Germany). After the blocking step, the membranes were incubated in 1% blocking solution containing a primary antibody to TCTP (MoBiTec, Goet- tingen, Germany) at room temperature for 1 h. The mem- branes were washed three times with NaCl ⁄ Tris containing Tween-20 and were then incubated with a secondary antibody (donkey anti-rabbit; Santa Cruz, Heidelberg, Germany) for 1 h. After additional washing steps, bands were detected using the ECL plus Western Blotting Detec- tion System (Amersham Pharmacia Biotech). Membranes were stripped for 5 min with distilled water, for 3–5 min with 0.2 m NaOH, and for 5 min with distilled water, and reprobed with anti-b-actin (Chemicon) or anti-(glyceralde- hyde-3-phosphate dehydrogenase) (Acris Antibodies GmbH, Hiddenhausen, Germany) antibodies, with a sec- ondary antibody (anti-mouse; Santa Cruz) to detect rela- tive b-actin and glyceraldehyde-3-phosphate dehydrogenase levels as loading control. UV-crosslinking UV-crosslinking experiments were performed as described previously [36]. Briefly, the following protocol was used. In vitro transcripts representing the 5¢-or3¢-UTR of TCTP mRNA (cloned in TOPO II vector, transcription with T7 or SP6 polymerase) were radioactively labeled using [ 32 P]UTP[aP] (800 CiÆmmol )1 ; MP Biomedicals GmbH, Heidelberg, Germany). For UV-crosslinking experiments 1– 2ng of [ 32 P]UTP[aP]-labeled in vitro transcripts represent- ing 200 000 c.p.m. were incubated with 40–60 lg of cyto- solic protein extract for 15 min in a total volume of 10 lL at room temperature in 10 mm Hepes, pH 7.2, 3 mm MgCl 2 , 5% glycerol, 1 mm dithiothreitol, 150 mm KCl, and 2UÆlL )1 RNaseOUT (Invitrogen, Karlsruhe, Germany), in the presence of rabbit rRNA (0.5 lgÆlL )1 ). Then, the sam- ples were exposed to UV light for 15 min on ice (255 nm, 1.6 J; UV-Stratalinker, Stratagene, Heidelberg, Germany), TCTP induction I. Schmidt et al. 5422 FEBS Journal 274 (2007) 5416–5424 ª 2007 The Authors Journal compilation ª 2007 FEBS treated with RNase A (30 lgÆmL )1 final concentration) and RNase T1 (750 UÆmL )1 final concentration) for 15 min at 37 °C, and subjected to 12% SDS ⁄ PAGE and autoradiog- raphy. Statistical analysis Data are shown as means ± SD. 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