Posttranscriptional regulation of TSC-22 (TGF-β -stimulated clone-22) gene by TGF-β 1

BBRC Biochemical and Biophysical Research Communications 305 (2003) 846–854 www.elsevier.com/locate/ybbrc

Posttranscriptional regulation of TSC-22 (TGF-b-stimulated clone-22) gene by TGF-b1q Daisuke Uchida,a,1 Fumie Omotehara,b,1 Koh-ichi Nakashiro,a Yoshihisa Tateishi,a Satoshi Hino,a Nasima-Mila Begum,a Takahiro Fujimori,b and Hitoshi Kawamatab,* a

Second Department of Oral and Maxillofacial Surgery Tokushima University School of Dentistry, 3-18-15 Kuramoto, Tokushima 770-8504, Japan b Department of Surgical and Molecular Pathology, Dokkyo University School of Medicine, 880 Kita-kobayashi, Mibu-machi, Shimo-tsuga-gun, Tochigi-ken 321-0293, Japan Received 25 April 2003

Abstract TSC-22 gene was composed of three exons and its length was approximately 5.5 kb including 2.9 kb promoter region. The transcription starting site was located at 7 and 29 bp downstream from TATA box. Promoter analysis revealed that 2146 bp of TSC22 promoter was activated by several differentiation inducing drugs. Although originally TSC-22 was isolated as a TGF-b-inducible gene, TSC-22 promoter was not activated by the enhanced TGF-b signaling. We found 3 copies of the Shaw–Kamens sequence (AUUUA) in the human TSC-22 mRNA 30 -UTR and identified three proteins (40, 20, and 15 kDa) which bound to this. Only the 40 kDa protein–RNA complex was decreased by treatment with TGF-b1. Moreover, the TSC-22 mRNA 30 -UTR destabilized the heterologous luciferase mRNA, but the destabilization was recovered with TGF-b1. These observations suggest that up-regulation of TSC-22 mRNA by TGF-b1 is achieved by mRNA stabilization, but not by transcriptional activation. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: TSC-22; Salivary gland cancer; mRNA stability; AU-rich-element

We isolated human transforming growth factor (TGF)-b stimulated clone-22 (TSC-22) cDNA as an anti-cancer drug (vesnarinone)-inducible gene in a human salivary gland cancer cell line, TYS [1]. We also reported that TSC-22 negatively regulated the growth of TYS cells [1] and that down-regulation of TSC-22 in TYS cells played a major role in the salivary gland tumorigenesis [2]. More recently, we reported that overexpression of TSC-22 enhanced chemosensitivity and radiation-sensitivity by inducing apoptosis in the cancer cells [3–5]. TSC-22 was originally reported as a TGF-b-inducible gene in mice osteoblastic cells, MC3T3E1 [6]. Then, TSC-22 was shown to encode a putative transcriptional q Sequence data from this paper have been deposited with the DDBJ/EMBL/GenBank Data libraries under Accession No. AF256226. * Corresponding author. Fax: +81-282-86-1681. E-mail address: [email protected] (H. Kawamata). 1 These authors contributed equally to this paper.

regulator containing a leucine zipper-like structure [6]. Subsequently, TSC-22 was demonstrated to be up-regulated by many different stimuli such as TPA, choleratoxin, dexamethasone [6], follicle-stimulating hormone [7], tumor necrosis factor a, interferon-c, interleukin-1b, lipopolysaccharide [8], progesterone [9], and epidermal growth factor (EGF; [10]). TSC-22 did not have a classical DNA-binding domain as bZip or bHLH-Zip families did. Therefore, TSC-22 was hypothesized to act as a transcriptional regulator by binding other leucine zipper containing transcription factors. Ohta et al. [8] reported TSC-22 as a transcription factor for C-type natriuretic peptide gene. However, we and Kester et al. recently reported that TSC-22 acted as a transcriptional enhancer [5] or repressor [11] when fused to the DNA binding domain of yeast transcription factor GAL4. On the other hand, several investigators reported the important role of TSC-22 in the embryonic development. Treisman et al. [12], Kania et al. [13] and Dobens et al. [14] found that Drosophila TSC-22 gene, shortsighted or bunched, was essential for the Drosophila

0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00854-4

D. Uchida et al. / Biochemical and Biophysical Research Communications 305 (2003) 846–854

development, and that Drosophila TSC-22 gene was an effector gene which could integrate multiple extracellular signals. More recently, Dohrmann et al. [15] and Kester et al. [16] demonstrated that, during mouse embryogenesis, TSC-22 was up-regulated at sites of epithelial– mesenchymal interaction and expressed in many neural crest-derived cells. TGF-bs are 25 kDa homodimeric polypeptides belonging to a superfamily of growth regulatory molecules and potent inhibitors of cell proliferation. TGF-bs assemble a receptor complex that activates SMADs, and the SMADs assemble multisubunit complexes that regulate transcription [17]. Moreover disruption of components of the TGF-b signaling pathway leads to tumorigenesis [18–21]. TGF-b1 activates the transcription of mammalian genes that are important for morphogenesis and cellular differentiation, such as p21 [22], p15 [23], a2(type 1) procollagen, fibronectin, and plasminogen activator inhibitor-1 genes [24]. TGF-b1 responsive region-CAGA sequence, so called CAGA-box or SMAD-binding-element (SBE) in plasminogen activator inhibitor-1 gene has been identified in its promoter [25,26]. On the other hand, it has been reported that the mRNA levels of several genes, such as receptor for HAmediated motility (RHAMM; [27]), Indian Hedgehog [28], Type IV collagenase [29], and cyclooxygenase-2 [30], are posttranscriptionally regulated by TGF-b1. As described above, TSC-22 was up-regulated by many different stimuli such as anti-cancer drugs and so many growth regulators, and induced at a specific stage in the embryonic development of mouse and Drosophila. However, the precise mechanism of TSC-22 gene expression was not fully understood. In this study, we isolated human TSC-22 gene and characterized its structure. Furthermore, we examined the transcriptional regulation of TSC-22 gene by several stimuli and the posttranscriptional regulation of TSC-22 gene by TGF-b1.

847

saline citrate–0.5% (w/v) SDS at room temperature and once at 65 °C for 40 min with the same washing buffer. Subsequently, the filter was exposed to an X-ray film with an intensifying screen at )70 °C. Bands that showed positive signal were gel purified and ligated into pUC19 vector for sequence analysis. DNA sequencing was performed by ALOKA DNA sequencer (LIC-4200L, Tokyo, Japan). The TSC-22 promoter sequence and genomic structure were analyzed for putative cis-acting elements and trans-acting factor binding sites utilizing the GENETYX-SV/R sequence analysis software (version9.0, Software Development, Tokyo, Japan), TFSEARCH program (http:// pdap1.trc.rwcp.or.jp/research/db/TFSEARCH.html), PANORAMA program (http://atlas.swmed.edu/panorama_form.html), or CENSOR program (http://www.girinst.org/Censor_Server.html) [32]. Identification of the transcription starting site. Transcription starting site of human TSC-22 gene was identified by Oligo-CAP method using human brain CAP-site cDNA library (Nippon gene, Toyama, Japan), as described previously [33]. In brief, Cap-site cDNAdT was amplified by TSC-22 specific primer TSC-1 (50 -GCTACCACACTTGCACCAGAG-30 ) and r-oligo specific primer 1RDT (50 -CAAGGTACGCCAC AGCGTATG). PCR was performed as follows; the final concentrations of dNTPs and primers in the reaction mixture were 200 and 0.5 lM, respectively. Gene TaqNT (Nippon gene) was added to the mixture at a final concentration of 0.05 U/ll and the reaction was carried out in Takara Thermal Cycler MP (Takara Biomedicals). PCR product was gel purified and ligated into pGEM-TEasy (Promega, Madison, WI). Candidate clones were confirmed by sequencing analysis. Construction of TSC-22 promoter–luciferase reporter plasmids. A 2146 bp of PstI–XbaI fragment (Fig. 1) containing promoter region of human TSC-22 gene was treated with T4 DNA polymerase (Takara Biomedicals), and the fragment was subcloned into SmaI site of the luciferase reporter plasmid, pGL3-Basic (Promega), to generate pGL3TSC22-PRO. Moreover, putative CAGA box (Fig. 1) located on the upstream of PstI site was amplified from P1 plasmid DNA using a pair of primers, TSC-CAGA-UP: 50 -CCGCTCGAGTGTCATGCTTGC

Materials and methods Cell culture. TYS [31], HeLa, and Mv1Lu cells were grown in DMEM (Sigma, St. Louis, MO) supplemented with 10% (v/v) fetal calf serum (FCS; Bio-Whittaker, Walkersville, MD), 100 lg/ml streptomycin, 100 U/ml penicillin (Life Technologies, Gaithersburg, MD), and 0.25 lg/ml amphotericin B (Life Technologies) in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. Isolation of human TSC-22 gene. Human P1 genomic library (Genome System, St. Louis, MO) was screened by human TSC-22 cDNA open reading frame as a probe. Positive P1 plasmids were prepared by QIAGEN maxi-prep (Qiagen, Hilden, Germany), followed by restriction digestion with EcoRI, PstI, and BamHI. All of the restriction endonuclease used in this experiment was purchased from Takara Biomedicals (Kusatsu, Japan). The DNA fragments were electrophoresed and transferred to a nylon filter. The nylon filter was hybridized with 32 P-labeled human TSC-22 open reading frame cDNA probes in 50% (v/v) formamide, 5 saline–sodium phosphate–EDTA, 0.1% (w/v) SDS, 5 DenhardtÕs solution, and 100 lg/ml salmon sperm DNA at 42 °C for 15 h. Extensive washing was done, twice with 0.1 standard

Fig. 1. Genomic structure of human TSC-22. (A) The structure of human TSC-22 gene (GenBank Accession No. AF256226). TSC-22 gene was composed of three exons and two introns. The locations of the predicted CpG island are indicated. (B) Partial promoter sequence of TSC-22 gene ()68 to 240). CCAAT and TATA sequences underlined. White letters and capital letters indicate transcription starting site and initiation codon, respectively.

848

D. Uchida et al. / Biochemical and Biophysical Research Communications 305 (2003) 846–854

TACCAA-30 and TSC-CAGA-DN: 50 -GAAGATCTACTGAATGCC AACTTCATGAA-30 , respectively. The amplified fragment was ligated to the upstream of 2146 bp promoter (pGL3-TSC22-PRO) to generate pGL3-TSC22-2919. Several truncated promoter constructs (Fig. 2) were generated by restriction digestion of pGL3-TSC22-PRO with KpnI and EcoRI for pGL3-TSC22-804, with KpnI and ApaI for pGL3TSC22-289. The digested fragments were treated with T4 DNA polymerase and self-ligated with T4 DNA ligase (Takara Biomedicals). Transcriptional regulation of TSC-22 promoter by several stimuli. TYS cells or HeLa cells (1  105 cells/dish) were seeded in 35 mm culture dish in DMEM supplemented with 10% FCS. Twenty-four hours later, the cells were transfected with 5 lg of several pGL3 luciferase reporter vectors and pCMV-bgal (Clontech, Palo Alto, CA). After transfection, the cells were treated with several agents, such as TGF-b1 (5 ng/ml; R&D, Minneapolis, MN), all-trans-retinoic-acid (ATRA; 107 M; Sigma), dibutyryl-cyclic AMP (dB-cAMP; 1 mM; Sigma), TPA (50 ng/ml; Sigma), EGF (25 ng/ml; Life Technologies), and Vesnarinone (Otsuka Pharmaceutical Company, Tokyo, Japan). Before TGF-b1 treatment, FLAG-tagged Smads (Smad2, Smad3, and Smad4 gifted from Dr. K. Miyazono [34]) or control plasmid (pcDNA1, Invitrogen, Carlsbad, CA) were co-transfected with the reporter plasmid. Twenty hours later, cells were lysed with cell culture lysis reagent (Promega) and luciferase activities were measured by scintillation counter with a Promega luciferase assay kit (Promega) according to the manufacturerÕs instruction. The luciferase activities were normalized by the amount of protein concentration or b-galactosidase activity. Each experiment was repeated at least twice. In vitro transcription. The MAXIscript kit from Ambion (Austin, TX) was used for the synthesis of TSC-22 30 -UTR probes. [a-32 P]CTP was used to label the probes. pUC19-hTSC-22 was digested with EcoRV and HindIII, and TSC-22 30 -UTR fragment (860-bp) was cloned into pBluescript KS (-) (Stratagene, La Jolla, CA) to generate pBS-860UTR. pBS-860UTR was digested with BglII and self-ligated to generate pBS-350UTR, respectively. These plasmids were digested with XhoI and run-off transcription was performed to generate each TSC-22 30 -UTR RNA probe. UV cross-linking assay. TYS cells treated with or without TGF-b1 (5 ng/ml) were suspended in hypotonic buffer [20 mM N -2-hydroxyethylpiperazine-N 0 -2-ethanesulfonic acid (Hepes)–NaOH (pH 7.9, at 25 °C), 1 mM EDTA, and 1 mM dithiothreitol (DTT)] containing 0.2%

(v/v) Nonidet P-40 (NP-40) and protease inhibitors (1 lg/ml each of leupeptin, pepstatin, aprotinin, and 0.5 mM of phenylmethylsulfonyl fluoride) on ice for 10 min. After 16,000g centrifugation at 4 °C for 10 s, supernatants were collected (cytoplasmic fraction). The pellets were suspended in hypertonic buffer containing 420 mM NaCl and 20% (v/v) glycerol. After 16,000g centrifugation at 4 °C for 2 min, supernatants (nuclear-fraction) were collected. Twenty five microgram of extracted nuclear-fraction was incubated with 20,000 cpm of radio-labeled RNA probes (as described above) in 15 mM KCl, 1 mM DTT, 12 mM Hepes (pH 7.8, at 25 °C), 0.24 mM EDTA, 5 mM MgCl2 , 0.4% (v/v) NP-40, and 200 ng/ll of yeast tRNA in a total volume of 25 ll for 30 min at 30 °C. Additionally, 10 lg of yeast tRNA was added to suppress nonspecific binding. After incubation, 145 U RNaseT1 (Ambion) was added and incubated for 30 min at 30 °C. After 3 ll of heparin (5 mg/ ml) was added, the samples were incubated on ice for 10 min and irradiated with 254 nm UV light from a distance of 5 cm for 20 min, using a trans-illuminator T1-100 (TOMY, Tokyo, Japan). Five microgram of RNaseA was then added and RNA was digested for 30 min at 37 °C. The samples were heated for 5 min at 65 °C after addition of sample loading buffer [50 mM Tris–HCl (pH 6.8, at 25 °C), 2% (w/v) SDS, 0.1% (w/v) bromphenol blue, 10% (v/v) glycerol, and 1.25% (v/v) of 2-mercaptoethanol]. The samples were then loaded on a SDS– polyacrylamide (10%) electrophoresis gel. RNA–protein binding was visualized by autoradiography. Northern blotting and RT-PCR. Subconfluent TYS cells were treated by TGF-b1 (5 ng/ml) in the presence of 100 lM of DRB (5,6dichloro-1-b-D -ribofuranosylbenzimideazol, Sigma). After 24 h, RNA isolation was performed using Trizol reagent (Life Technologies) according to manufacturerÕs instructions. Northern blotting and semiquantitative RT-PCR were carried out, as described previously [1]. The probe used was a 1.3 kb PstI fragment containing partial ORF and 30 -UTR of human TSC-22 cDNA. Examination of mRNA stability by 30 -UTR region of TSC-22 gene. Several 30 -UTR reporter vectors were constructed as follows; the luciferase vector pGL3-Control (Promega) was digested at the unique XbaI site located immediately after the stop codon of the luciferase gene. The linearized vector was then filled in with T4 DNA polymerase and dephosphorylated with bacterial alkaline phosphatase (Takara Biomedicals). Two different human TSC-22 cDNA 30 -UTR fragments were excised by the digestion with Eco RV and SphI, or BglII and SphI from pUC19-hTSC-22 for generating pGL3-Cont-860UTR or pGL3-Cont-350UTR, respectively. TYS cells were co-transfected with pGL3-Cont-860UTR or pGL3-Cont-350UTR and pSV2-neo (gifted from Dr. Y. Ebina), and stable transfectants were isolated, as described previously [2]. After treatment of the transfectants with TGF-b1, luciferase activity was measured, as described above.

Results Genomic structure of human TSC-22 gene

Fig. 2. Transcription-regulatory element on TSC-22 promoter region. Various TSC-22 promoter–luciferase reporter plasmids (depicted in the left panel) were co-transfected with pCMV-bgal control vector into TYS and HeLa cells. After 48 h, the cells were collected and luciferase activity was determined, as described under ‘‘Materials and methods.’’ Data were corrected for transfection efficiency (b-galactosidase expression). Transfection was performed in duplicate and the data are representative of three separate experiments with similar results.

The structure of human TSC-22 gene (GenBank Accession No. AF256226) is shown in Fig. 1. Comparison of the genomic and cDNA sequences indicated that the human TSC-22 gene was composed of three exons and two introns. All introns followed the ‘‘GT–AG’’ rule for exon/intron junctions. The sizes of exon 1, exon 2, and exon 3 were 374 (or 352), 52, and 1351 bp, respectively. The first exon contained an initiation codon and nuclear export signal (NES) that we recently identified [5]. The TSC-box, which was conserved between human, mouse, rat, chicken TSC-22, and Drosophila shortsighted or bunched, was distributed over exon 2 to

D. Uchida et al. / Biochemical and Biophysical Research Communications 305 (2003) 846–854

849

exon 3. Exon 3 contained leucine-zipper, stop codon, and 30 -UTR. Moreover, a lot of CpG islands are conserved in TSC-22 gene, indicating its wide expression in various tissues except hematopoietic cells [35]. Transcriptional starting site of human TSC-22 gene We isolated two kinds of independent clones TSCcap4 and TSC-cap6 from more than 30 clones by OligoCAP method. TSC-cap4 was started at 7 bp downstream from TATA-like box, and TSC-cap6 was at 29 bp downstream from TATA-like box, indicating that TSC22 has two major transcription starting sites (Fig. 1B). The TSC-22 gene lacked a canonical TATA box, but a TATAT sequence and a CCAAT box were present at positions )7 or )29 bp and )31 or )53 bp relative to the transcription starting site, respectively. The TATA box and pentanucleotide sequence CCAAT are commonly found 25 and 50–100 bp upstream from transcription starting site of eukaryotic genes, respectively [36]. Therefore, these sequences may represent a functional TATA and CCAAT box leading to transcription from the identified CAP site. Identification of transcription-regulatory element on TSC-22 promoter region When we transiently transfected TYS or HeLa cells with the reporter plasmids, the activities of all reporter constructs were higher than that of control plasmid, pGL3-Basic, indicating that these promoters were biologically active in both cells (Fig. 2). A 2146 bp of human TSC-22 promoter contained various transcription factor binding sites, such as AP1, SP1, MYOD, and so many others, indicating diverse function of TSC-22 (data not shown). The TSC-22 promoter constructs with 804 bp of 50 -flanking DNA showed comparably high luciferase activity (approximately 7- to 8-fold relative to pGL3-Basic). Moreover, the luciferase activity of pGL3289 was still higher than that of pGL3-Basic (approximately 3- to 4-fold), even higher than that of pGL3-2919 or pGL3-TSC22-PRO (2146 bp promoter). These results indicated that positions between )809 and )29 (TATA box) contained cis-acting regulatory elements needed for high level of basal promoter activity in these cancer cells.

Fig. 3. Transcriptional regulation of TSC-22 gene by several stimuli. (A) TYS cells were seeded in 6 well plates and transfected with pGL3TSC22-PRO. After 24 h, the cells were treated with indicated agents. Twenty hours later, cells were collected and luciferase activity was determined, as described under ‘‘Materials and methods.’’ (B,C) HeLa cells were seeded in 6 well plates and transfected with pGL3-TSC22PRO. After 24 h, the cells were treated with indicated agents. Twenty hours later, cells were collected and luciferase activity was determined. Column shows the average of each samples and bars show standard deviation. The experiments were performed at least twice. *, Statistically significant (p < 0:05, one-way ANOVA).

EGF marginally affected this promoter region in HeLa cells (Fig. 3C).

Transcriptional regulation of TSC-22 gene by several stimuli

Transcriptional regulation of TSC-22 gene by TGF-b1

Vesnarinone, a differentiation inducing drug, activated TSC-22 promoter, pGL3-TSC22-PRO (2146 bp) in TYS cells (Fig. 3A). In addition, ATRA and dBcAMP induced the luciferase activity in HeLa cells (4and 3-fold activation, respectively) (Figs. 3B and C). However, TPA did not activate the 2146 bp promoter region of TSC-22 gene in both cells (Figs. 3A and B) and

TSC-22 was first isolated as a TGF-b1 responsive gene [6]. However, there was no significant activation of the 2146 bp of TSC-22 promoter after addition of exogenous TGF-b1 and/or co-transfection of SMAD expression vectors in Mv1Lu cells (Fig. 4). Moreover, neither 804 nor 289 bp promoters were activated by the treatment with TGF-b1 in Mv1Lu cells (data not

850

D. Uchida et al. / Biochemical and Biophysical Research Communications 305 (2003) 846–854

Fig. 4. Transcriptional regulation of TSC-22 gene by TGF-b1. Mv1Lu cells were seeded in 6 well plates and co-transfected with pGL3-TSC22-PRO and Smad expression vectors in DMEM supplemented with 0.2% (v/v) FCS. Nine hours later, TGF-b1 (5 ng/ml) was added to the culture and the cells were incubated for 24 h. Then the luciferase activity in the cell lysate was measured. Column shows the average of three samples and bars show standard deviation. The experiments were performed at least three times.

shown). This lack of promoter activation was not cell line specific because TYS cells also failed to respond to TGF-b1 (data not shown). As a positive control, p3TPLux (gifted from Dr. K. Miyazono) was highly activated by the enhanced TGF-b signaling in Mv1Lu cells (Fig. 4). A 2146 bp of human TSC-22 promoter has one copy of 50 -CAGAC-30 sequence at position )399, however, it may not be enough to be activated by TGF-b1. To inquire into the TGF-b1 responsive element in further upstream of 50 flanking region of the TSC-22 gene, we performed the computer cloning from draft sequence of human genome project. We found the sequence for chromosome 13q14 region (GenBank Accession No. AL139184) corresponding to TSC-22 genomic locus. This sequence almost coincides with our 5.5 kb genomic DNA. Because we found several CAGAC sequence, at the upstream region from PstI site (at position )2146, in Fig. 1), we amplified about 1 kb fragment containing two CAGAC sequences and subcloned into pGL3-Basic. However, contrary to our expectation, this 2919 bp promoter sequence was not activated by the enhanced TGF-b1 signaling (data not shown). Posttranscriptional regulation of TSC-22 gene by TGFb1 We previously demonstrated that TGF-b1 increased the TSC-22 mRNA level in TYS cells by Northern blotting [1]. To further investigate the mechanism of TSC-22 induction by TGF-b1, TYS cells were treated by

TGF-b1 in the presence of the inhibitor for transcription, DRB. TGF-b1 apparently enhanced the mRNA level for TSC-22 even in the presence of DRB (Fig. 5A). These results demonstrate that TGF-b1 regulates TSC22 posttranscriptionally through an increase in mRNA stability. The knowledge that 30 -UTR of other mRNAs have previously been implicated in the regulation of growth factor alterations of message stability [27,37] led us to test the hypothesis that TGF-b1 responsive trans-acting factors exist that can bind to cis elements of TSC-22 mRNA 30 -UTR. In order to clarify the position of cis elements of TSC-22 mRNA, we subcloned three different lengths of TSC-22 30 -UTR under the control of T7 RNA promoter. As shown in Fig. 5B, closed triangle showed the position of the TGF-b1 responsive GCUUGC sequence which was identified in the 30 -UTR of RHAMM mRNA [27]. Open triangles showed the three copies of the Shaw–Kamens sequence (AUUUA) [37]. To provide insight into proteins which bound TSC22 mRNA 30 -UTR, we performed a UV cross-linking experiment. Using the 860 and 350 bp UTR probes with nuclear extract, three proteins (40, 20, and 15 kDa) were identified. Intensity of the band for 40 kDa protein– RNA complex was reduced by the treatment with TGFb1 (Fig. 5C). However, the intensity of the bands for 20 and 15 kDa protein–RNA complex was not affected by the treatment with TGF-b1 (Fig. 5C). By densitometric analysis, the 40 kDa band was reduced down to 72% on 860 bp probe, 53% on 350 bp probe after treatment with TGF-b1 (data not shown). We repeated the experiment

D. Uchida et al. / Biochemical and Biophysical Research Communications 305 (2003) 846–854

851

Fig. 6. Effect of 350 bp 30 -UTR of TSC-22 gene on mRNA stability after treatment with TGF-b1. pGL3-Control based reporter vectors were co-transfected with pSV2-neo and stable transfectants were isolated. Transfectants were seeded in 24 well plates. After starvation in DMEM supplemented with 0.2% (v/v) FCS for 24 h, TGF-b1 (5 ng/ml) was added for 36 h, followed by harvest and measuring the luciferase activity. Each point shows the average of four samples and bars show standard deviation. The experiment was performed twice. *, Statistically significant (p < 0:05, one-way ANOVA).

Fig. 5. Specific RNA–protein interactions on the 30 -UTR of TSC-22 mRNA. (A) Posttranscriptional up-regulation of TSC-22 by TGF-b1. TYS cells were treated with TGF-b1 for 24 h in the presence of an inhibitor for transcription, DRB (100 lM), and total RNA was isolated by TRIzol reagent. Then Northern blotting (left panel) and semiquantitative RT-PCR (right panel) were performed. The probe used for Northern blotting was a 1.3 kb PstI fragment containing partial ORF and 30 -UTR of human TSC-22 cDNA. TGF-b1 apparently enhanced the mRNA level for TSC-22 even in the presence of DRB. (B) Schematic diagrams of the TSC-22 cDNA 30 -UTR constructs. The TSC-22 cDNA 30 -UTR sequences were fused to downstream of the T7 RNA polymerase promoter. Restriction enzyme XhoI was used for run-off transcription. Closed triangle shows the position of the TGFb1 responsive GCUUGC sequence. Open triangles show the 3 copies of the Shaw–Kamens sequence (AUUUA). (C) Nuclear extract was prepared from TYS cells that were treated with 5 ng/ml TGF-b1 for 36 h. The nuclear extract was incubated with radio-labeled 720 and 350 bp of TSC-22 mRNA 30 -UTR probes. Then, UV cross-linking reactions were performed.

twice and confirmed the down-regulation of the 40 kDa band by TGF-b1 in the different extracts. Effect of 350 bp 30 -UTR of TSC-22 gene on mRNA stability after treatment with TGF-b1 To investigate the possible role of the TGF-b1-responsive cis-element within 350 bp of 30 -UTR in the stabilization of TSC-22 mRNA, we assessed the contribution of the 30 -UTR of TSC-22 mRNA to the stability of a heterologous luciferase mRNA. We

transfected TYS cells with luciferase gene fused to 860 or 350 bp of TSC-22 30 -UTR and obtained several stable transfectants. Stable transfectants carrying the same reporter plasmid were combined together and luciferase activity in the bulk-transfectants was measured. Luciferase activity in the transfectants with luciferase gene fused to TSC-22 30 -UTR was significantly lower than that in the cells with control plasmid (p < 0:05, one-way ANOVA). After treatment with TGF-b1 for 36 h, the luciferase activities in the cells with pGL3-Cont860UTR and pGL3-Cont-350UTR were significantly increased when compared with those in the untreated cells (Fig. 6, p < 0:05, one-way ANOVA). Furthermore, the levels of luciferase activity in the cells with pGL3Cont-860UTR and pGL3-Cont-350UTR after treatment with TGF-b1 were almost recovered upto the level in the cells with control plasmid (pGL3-Cont) (Fig. 6).

Discussion In this study, we isolated TSC-22 gene and examined its structure and transcriptional regulation. TSC-22 gene was small (approximately 5.5 kb including 2.9 kb promoter region) and composed of three exons. Moreover, a lot of CpG islands were conserved in TSC-22 gene (Fig. 1). In cancer cells, several CpG islands became hypermethylated, shutting down the expression of the contiguous gene, such as a tumor suppressor gene and thereby enabling malignant growth. Examples of genes

852

D. Uchida et al. / Biochemical and Biophysical Research Communications 305 (2003) 846–854

suffering this aberrant methylation included p16INK4a, p14ARF, p15INK4b, adenomatous polyposis coli, p73, E-cadherin, and Retinoblastoma [38–40]. TSC-22 was demonstrated to be up-regulated by many different stimuli including anti-cancer drug and growth inhibitors. We found that 2146 bp of TSC-22 promoter responded to several drugs such as Vesnarinone, ATRA, and dB-cAMP which showed a differentiation inducing activity [41–44]. The responsive elements of ATRA and dB-cAMP were demonstrated to be AGGTCA and CCAAT sequences, respectively [45,46]. TSC-22 promoter (2146 bp) contained one copy of AGGTtA sequence at position )1299, one copy of CCAAT at positions )55, three copies of CCAAa at position )1898, )1672, and )1589, two copies of CCAAg at positions )1358 and )1072 (small letters showed the different nucleotide relative to the consensus sequences). Concerning a differentiation inducing anticancer drug, Vesnarinone, we previously demonstrated that Vesnarinone induced the growth arrest and the expression of p21waf 1 gene in a human salivary gland cancer cell line, TYS [47]. Recently, we found that the Vesnarinone responsive elements were situated in the p21waf 1 promoter at )124 to )61 relative to the transcription start site, and that SP1 and SP3 transcription factors bound to this region [48]. There were three independent SP1 binding sites in the TSC-22 promoter at positions )413, )120, and )78. These SP1 binding sites may contribute to the induction of TSC-22 gene by Vesnarinone. Although the 2919 bp promoter region of TSC-22 contained 3 copies of CAGAC sequences, treatment with TGF-b1 did not activate both 2146 and 2919 bp promoter regions. The CAGAC sequence was calculated to be present on average once in every 1024 bp in the genome, or about once in the regulatory region of any average size genes [49]. If the binding of SMAD proteins to the SMAD-binding-element (SBE) in the gene was sufficient for SMAD-dependent transcriptional activation, an activated-SMAD protein would lead to the non-selective activation of massive number of genes. However, this was not the case. Therefore, it was understood that by associating with DNA-binding partners, forming complexes of specific composition and geometry, the Smads could achieve high affinity, selective interactions with cognate DNA. Thus, up-regulation of TSC-22 gene by TGF-b1 may be achieved by posttranscriptional regulation, such as mRNA stabilization, but not by transcriptional activation. The 30 -UTR of the human TSC-22 transcript contained 3 copies of the Shaw–Kamens sequence (AUUUA), also known as AU rich elements [37,50]. In our study, 350 bp of the TSC-22 30 -UTR containing only the last AUUUA bound the proteins in nuclear extract. Furthermore, the 40 kDa protein that was cross linked with 350 bp of the TSC-22 30 -UTR was down-regulated

when the cells were treated with TGF-b1. The 40 kDa protein may be a destabilizing protein of mRNA which constitutively bound to the third AUUUA sequence on 30 -UTR region of TSC-22 gene and may be released or degraded by TGF-b1. Further investigations for this 40 kDa molecule will clarify the detailed transcriptional mechanism of TSC-22.

Acknowledgments We are indebted to Kohei Miyazono (Department of Molecular Pathology, Tokyo University Graduate School of Medicine) for Smad expression vectors and p3TP-Lux, and Yusuke Ebina (Division of Molecular Genetics, Department of Gene Information, Tokushima University) for pSV2-neo. We thank Mitsunobu Sato and Hideo Yoshida (Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry) for encouragement on this project. We also thank Chiaki Sato-Matsuyama, Ayako Shimizu, Takako Ohtsuki, and Midori Matsuura (Department of Surgical and Molecular Pathology, Dokkyo University School of Medicine) for their excellent technical assistance. This study was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan.

References [1] H. Kawamata, K. Nakashiro, D. Uchida, S. Hino, F. Omotehara, H. Yoshida, M. Sato, Induction of TSC-22 by treatment with a new anti-cancer drug, vesnarinone, in a human salivary gland cancer cell, Br. J. Cancer 77 (1998) 71–78. [2] K. Nakashiro, H. Kawamata, S. Hino, D. Uchida, Y. Miwa, H. Hamano, F. Omotehara, H. Yoshida, M. Sato, Down-regulation of TSC-22 (transforming growth factor b-stimulated clone 22) markedly enhances the growth of a human salivary gland cancer cell line in vitro and in vivo, Cancer Res. 58 (1998) 549–555. [3] D. Uchida, H. Kawamata, F. Omotehara, Y. Miwa, S. Hino, N.M. Begum, H. Yoshida, M. Sato, Over-expression of TSC-22 (TGF-b stimulated clone-22) markedly enhances 5-fluorouracilinduced apoptosis in a human salivary gland cancer cell line, Lab. Invest. 80 (2000) 955–963. [4] F. Omotehara, D. Uchida, S. Hino, N.M. Begum, H. Yoshida, M. Sato, H. Kawamata, In vivo enhancement of chemosensitivity of human salivary gland cancer cells by overexpression of TGF-b stimulated clone-22, Oncol. Rep. 7 (2000) 737–740. [5] S. Hino, H. Kawamata, D. Uchida, F. Omotehara, Y. Miwa, N.M. Begum, H. Yoshida, M. Sato, Nuclear translocation of TSC-22 (TGF-b-stimulated clone-22) concomitant with apoptosis: TSC-22 as a putative transcriptional regulator, Biochem. Biophys. Res. Commun. 278 (2001) 659–664. [6] M. Shibanuma, T. Kuroki, K. Nose, Isolation of a gene encoding a putative leucine zipper structure that is induced by transforming growth factor b1 and other growth factors, J. Biol. Chem. 267 (1992) 10219–10224. [7] K.G. Hamil, S.H. Hall, Cloning of rat Sertoli cell follicle-stimulating hormone primary response complementary deoxyribonucleic acid: regulation of TSC-22 gene expression, Endocrinology 134 (1994) 1205–1212. [8] S. Ohta, Y. Shimekake, K. Nagata, Molecular cloning and characterization of a transcription factor for the C-type natriuretic peptide gene promoter, Eur. J. Biochem. 242 (1996) 460–466. [9] H.A. Kester, B.-J.M. van der Leede, P.T. van der Saag, B. van der Burg, Novel progesterone target genes identified by an improved differential display technique suggest that progestin-induced

D. Uchida et al. / Biochemical and Biophysical Research Communications 305 (2003) 846–854

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17] [18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

growth inhibition of breast cancer cells coincides with enhancement of differentiation, J. Biol. Chem. 272 (1997) 16637–16643. T. Trenkle, J. Welsh, B. Jung, F. Mathieu-Daude, M. McClelland, Non-stoichiometric reduced complexity probes for cDNA arrays, Nucleic Acids Res. 26 (1998) 3883–3891. H.A. Kester, C. Blanchetot, J. den Hertog, P.T. van der Saag, B. van der Burg, Transforming growth factor-b-stimulated clone-22 is a member of a family of leucine zipper proteins that can homoand heterodimerize and has transcriptional repressor activity, J. Biol. Chem. 274 (1999) 27439–27447. J.E. Treisman, Z.C. Lai, G.M. Rubin, Shortsighted acts in the decapentaplegic pathway in Drosophila eye development and has homology to a mouse TGF-b-responsive gene, Development 121 (1995) 2835–2845. A. Kania, A. Salzberg, M. Bhat, D. DÕEvelyn, Y. He, I. Kiss, H.J. Bellen, P-element mutations affecting embryonic peripheral nervous system development in Drosophila melanogaster, Genetics 139 (1995) 1663–1678. L.L. Dobens, J.S. Peterson, J. Treisman, L.A. Raftery, Drosophila bunched integrates opposing DPP and EGF signals to set the operculum boundary, Development 127 (2000) 754. C.E. Dohrmann, M. Belaoussoff, L.A. Raftery, Dynamic expression of TSC-22 at sites of epithelial–mesenchymal interactions during mouse development, Mech. Dev. 84 (1999) 147–151. H.A. Kester, T.M. Ward-van Oostwaard, M.J. Goumans, M.A. van Rooijen, P.T. van der Saag, B. van der Burg, C.L. Mummery, Expression of TGF-b stimulated clone-22 (TSC-22) in mouse development and TGF-b signalling, Dev. Dyn. 218 (2000) 563–572. J. Massague, TGF-b signal transduction, Annu. Rev. Biochem. 67 (1998) 753–791. S. Markowitz, J. Wang, L. Myeroff, R. Parsons, L. Sun, J. Lutterbaugh, R.S. Fan, E. Zborowska, K.W. Kinzler, B. Vogelstein, M. Brattain, J.K.V. Willson, Inactivation of the type II TGF-b receptor in colon cancer cells with microsatellite instability, Science 268 (1995) 1336–1338. K. Eppert, S.W. Scherer, H. Ozcelik, R. Pirone, P. Hoodless, H. Kim, L.C. Tsui, B. Bapat, S. Gallinger, I.L. Andrulis, G.H. Thomsen, J.L. Wrana, L. Attisano, MADR2 maps to 18q21 and encodes a TGFb-regulated MAD-related protein that is functionally mutated in colorectal carcinoma, Cell 86 (1996) 543–552. A. Hata, R.S. Lo, D. Wotton, G. Lagna, J. Massague, Mutations increasing autoinhibition inactivate tumour suppressors Smad2 and Smad4, Nature 388 (1997) 82–87. J.L. Dai, K.K. Turnacioglu, M. Schutte, A.Y. Sugar, S.E. Kern, Dpc4 transcriptional activation and dysfunction in cancer cells, Cancer Res. 58 (1998) 4592–4597. A. Elbendary, A. Berchuck, P. Davis, L. Havrilesky, R.C. Bast, J.D. Iglehart, J.R. Marks, Transforming growth factor b1 can induce CIP1/WAF1 expression independent of the p53 pathway in ovarian cancer cells, Cell Growth Differ. 5 (1994) 1301–1307. J.-M. Li, M.A. Nichols, S. Chandrasekharan, Y. Xiong, X.-F. Wang, Transforming growth factor b activates the promoter of cyclin-dependent kinase inhibitor p15INK4B through an Sp1 consensus site, J. Biol. Chem. 270 (1995) 26750–26753. J. Keski-Oja, R. Raghow, M. Sawdey, D.J. Loskutoff, A.E. Postlethwaite, A.H. Kang, H.L. Moses, Regulation of mRNAs for type-1 plasminogen activator inhibitor, fibronectin, and type I procollagen by transforming growth factor-b. Divergent responses in lung fibroblasts and carcinoma cells, J. Biol. Chem. 263 (1998) 3111–3115. S. Dennler, S. Itoh, D. Vivien, P. ten Dijke, S. Huet, J.M. Gauthier, Direct binding of Smad3 and Smad4 to critical TGF binducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene, EMBO J. 17 (1998) 3091–3100. Y. Shi, Y.F. Wang, L. Jayaraman, H. Yang, J. Massague, N.P. Pavletich, Crystal structure of a Smad MH1 domain bound to

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36] [37]

[38]

[39] [40]

[41]

[42]

[43]

853

DNA: insights on DNA binding in TGF-b signaling, Cell 94 (1998) 585–594. F.M. Amara, J. Entwistle, T.I. Kuschak, E.A. Turley, J.A. Wright, Transforming growth factor-b1 stimulates multiple protein interactions at a unique cis-element in the 30 -untranslated region of the hyaluronan receptor RHAMM mRNA, J. Biol. Chem. 271 (1996) 15279–15284. S. Murakami, A. Nifuji, M. Noda, Expression of Indian hedgehog in osteoblasts and its posttranscriptional regulation by transforming growth factor-b, Endocrinology 138 (1997) 1972–1978. I. Sehgal, T.C. Thompson, Novel regulation of type IV collagenase (matrix metalloproteinase-9 and -2) activities by transforming growth factor-b1 in human prostate cancer cell lines, Mol. Biol. Cell 10 (1999) 407–416. H. Sheng, J. Shao, D.A. Dixon, C.S. Williams, S.M. Prescott, R.N. DuBois, R.D. Beauchamp, Transforming growth factor-b1 enhances Ha-ras-induced expression of cyclooxygenase-2 in intestinal epithelial cells via stabilization of mRNA, J. Biol. Chem. 275 (2000) 6628–6635. T. Yanagawa, Y. Hayashi, H. Yoshida, Y. Yura, S. Nagamine, T. Bando, M. Sato, An adenoid squamous carcinoma-forming cell line established from an oral keratinizing squamous cell carcinoma expressing carcinoembryonic antigen, Am. J. Pathol. 124 (1986) 496–509. J. Jurka, P. Klonowski, V. Dagman, P. Pelton, CENSOR-a program for identification and elimination of repetitive elements from DNA sequences, Comput. Chem. 20 (1996) 119–122. K. Maruyama, S. Sugano, Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides, Gene 138 (1994) 171–174. A. Nakao, T. Imamura, S. Souchelnytskyi, M. Kawabata, A. Ishisaki, E. Oeda, K. Tamaki, J. Hanai, C.H. Heldin, K. Miyazono, P.T. Dijke, TGF-b receptor-mediated signalling through Smad2, Smad3 and Smad4, EMBO J. 16 (1997) 5353–5362. P. Jay, J.W. Ji, C. Marsollier, S. Taviaux, J.-L. Berge-Lefranc, P. Berta, Cloning of the human homologue of the TGF b-stimulated clone 22 gene, Biochem. Biophys. Res. Commun. 222 (1996) 821–826. P.F. Johnson, S.L. McKnight, Eukaryotic transcriptional regulatory proteins, Annu. Rev. Biochem. 58 (1989) 799–839. G. Shaw, R. Kamen, A conserved AU sequence from the 30 untranslated region of GM-CSF mRNA mediates selective mRNA degradation, Cell 46 (1986) 659–667. M. Esteller, P.G. Corn, S.B. Baylin, J.G. Herman, A gene hypermethylation profile of human cancer, Cancer Res. 61 (2001) 3225–3229. S.B. Baylin, J.G. Herman, DNA hypermethylation in tumorigenesis: epigenetics joins genetics, Trends Genet. 16 (2000) 168–174. M. Esteller, A. Sparks, M. Toyota, M. Sanchez-Cespedes, G. Capella, M.A. Peinado, S. Gonzalez, G. Tarafa, D. Sidransky, S.J. Meltzer, S.B. Baylin, J.G. Herman, Analysis of adenomatous polyposis coli promoter hypermethylation in human cancer, Cancer Res. 60 (2000) 4366–4371. M. Sato, K. Harada, H. Yoshida, Induction of differentiation and apoptosis, and LeY antigen expression by treatment with 3,4dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinoline (vesnarinone) in a human salivary cancer cell line, Acta Histochem. Cytochem. 27 (1994) 591–599. M. Sato, K. Harada, Y. Yura, T. Bando, M. Azuma, H. Kawamata, H. Iga, H. Yoshida, Induction of tumor differentiation and apoptosis, and LeY antigen expression in treatment with differentiation inducing agent, vesnarinone, of a patient with sarivary adenoid cystic carcinoma, Apoptosis 2 (1997) 106–113. L.Z. He, T. Merghoub, P.P. Pandolfi, In vivo analysis of the molecular pathogenesis of acute promyelocytic leukemia in the mouse and its therapeutic implications, Oncogene 18 (1999) 5278–5292.

854

D. Uchida et al. / Biochemical and Biophysical Research Communications 305 (2003) 846–854

[44] K.N. Prasad, P.K. Sinha, Effect of sodium butyrate on mammalian cells in culture: a review, In Vitro 12 (1976) 125–132. [45] M.P. Costa-Giomi, M.P. Gaub, P. Chambon, P. Abarzua, Characterization of a retinoic acid responsive element isolated by whole genome PCR, Nucleic Acids Res. 20 (1992) 3223–3232. [46] P.J. Chilco, J.M. Gerardi, S.J. Kaczmarczyk, S. Chu, V. Leopold, J.D. Zajac, Calcitonin increases transcription of parathyroid hormone-related protein via cAMP, Mol. Cell. Endocrinol. 94 (1993) 1–7. [47] M. Sato, H. Kawamata, K. Harada, K. Nakashiro, Y. Ikeda, H. Gohda, H. Yoshida, T. Nishida, K. Ono, M. Kinoshita, M. Adachi, Induction of cyclin-dependent kinase inhibitor, p21WAF1, by treatment with 3,4-dihydro-6-[4-(3,4)-dimethoxybenzoyl-1-piperazinyl]-2(1H)-quinoline (vesnarinone) in

a human salivary cancer cell line with mutant p53 gene, Cancer Lett. 112 (1997) 181–189. [48] F. Omotehara, H. Kawamata, D. Uchida, S. Hino, K. Nakashiro, T. Fujimori, Vesnarinone, a differentiation inducing drug, directly activates p21waf1 gene promoter via Sp1 sites in a human salivary gland cancer cell line, Br. J. Cancer 87 (2002) 1042–1046. [49] J. Massague, D. Wotton, Transcriptional control by the TGF-b/ Smad signaling system, EMBO J. 19 (2000) 1745–1754. [50] D. Caput, B. Beutler, K. Hartog, R. Thayer, S. Brown-Shimer, A. Cerami, Identification of a common nucleotide sequence in the 30 -untranslated region of mRNA molecules specifying inflammatory mediators, Proc. Natl. Acad. Sci. USA 83 (1986) 1670–1674.