Tài liệu Báo cáo khoa học: Multiple promoters regulate tissue-specific alternative splicing of the human kallikrein gene, KLK11⁄hippostasin ppt

Multiple promoters regulate tissue-specific alternative splicing of the human kallikrein gene, KLK11 ⁄ hippostasin Shinichi Mitsui 1, *, Terukazu Nakamura 2 , Akira Okui 3 , Katsuya Kominami 3 , Hidetoshi Uemura 3 and Nozomi Yamaguchi 1 1 Department of Cell Biology, Research Institute for Geriatrics, Kyoto Prefectural University of Medicine, Kyoto, Japan 2 Department of Urology, Kyoto Prefectural University of Medicine, Kyoto, Japan 3 Research and Development Center, Fuso Pharmaceutical Industries, Ltd, Osaka, Japan In humans, tissue kallikrein (KLK) is a subgroup of the serine protease family, which includes 15 members whose genes are located on human chromosome 19q13.4 [1,2]. The expression of each kallikrein mem- ber is regulated in a tissue-specific manner [3]. Mes- senger RNAs of KLK2, KLK3, KLK4, KLK11, and KLK15 are expressed preferentially in the prostate, whereas other subgroups reside in the central nervous system (CNS) (KLK6, KLK7, KLK8, KLK9, KLK14), in the breast (KLK5, KLK6, KLK13), and in the pan- creas (KLK1, KLK6–KLK13). Dysregulation of kallik- rein expression is associated with multiple diseases, including cancers, and many kallikreins are proposed as diagnostic or prognostic biomarkers for several types of cancer, including breast, ovarian, and prostate cancer [4]. Alternative splicing is prevalent in the Keywords hippocampus; oligo cap RACE; prostate; serine protease Correspondence N. Yamaguchi, Cell Biology and Protein Engineering, Environmental Systems Science, Doshisha University, Kyontanabe, Kyoto 610-0394, Japan Fax/Tel: 81 774065 6676 E-mail: nyamaguc@mail.doshisha.ac.jp *Present address Department of Neurobiology and Anatomy, Kochi Medical School, Okou, Nankoku 783- 8505, Japan Database The nucleotide sequence reported in this paper has been entered in the DDBJ ⁄ GenBank ⁄ EMBL databases under accession number AB259014 (Received 27 January 2006, revised 5 June 2006, accepted 12 June 2006) doi:10.1111/j.1742-4658.2006.05372.x The human kallikrein (KLK) family consists of 15 genes located on human chromosome 19q13.4. KLK11 ⁄ hippostasin is a member of the kallikrein family and is expressed in various tissues. Two types of KLK11 isoforms, isoform 1 and isoform 2, have been predicted from cDNA sequences. Iso- form 1 has been isolated from human hippocampus, whereas isoform 2 has been isolated from prostate. However, the regulation and characteristics of these isoforms are unknown. We identified the first three exons (1a, 1b, and 1c) by determining their transcription initiation sites. Exon 1b con- tained the initiation codon of isoform 2, and noncoding exons 1a and 1c contributed to isoform 1 mRNA. The dual luciferase promoter assay revealed three promoter regions, corresponding to the first exon of each isoform. Reverse transcription and PCR showed that exon 1a was expressed in the hippocampus, thalamus, and non-central nervous system (CNS) tissues, whereas exon 1b was detected only in non-CNS tissues. Exon 1c was observed in both CNS and non-CNS tissues, except for saliv- ary glands. In vitro mutagenesis revealed that the initiation codon for iso- form 2 in exon 1b was functional. Isoform 2 had additional hydrophilic amino acids at the amino terminal and was secreted from the neuroblasto- ma cell line Neuro2a. Isoform 1 fused with green fluorescent protein (GFP) was distributed to cellular processes, whereas isoform 2–GFP was retained in the Golgi apparatus. We suggest that not only alternative splicing but also tissue-specific use of multiple promoters regulate the expression and intracellular trafficking of KLK11 ⁄ hippostasin isoforms. Abbreviations CNS, central nervous system; GFP, green fluorescent protein; KLK, kallikrein; PSA, prostate specific antigen. 3678 FEBS Journal 273 (2006) 3678–3686 ª 2006 The Authors Journal compilation ª 2006 FEBS human tissue kallikreins. A recent report estimated that 82 different kallikrein gene transcripts lead to 56 different protein isoforms for KLK1–15 [5]. Over the past several years, we have reported on the tissue-specific expression and clinical application of tissue kallikreins, including KLK6 ⁄ neurosin, KLK8 ⁄ neuropsin, and KLK11⁄ hippostasin [6–16]. KLK11 has three alternative splicing variants, isoforms 1, 2, and 3, which show unique expression patterns. Isoform 1 (GenBank accession number NM006853) is a typical secretory protein with a signal peptide at the amino terminal, whereas isoform 2 (GenBank accession number NM14497) is purported to have 32 hydrophi- lic amino acids at the amino terminal in addition to a hydrophobic region that corresponds to the signal peptide of isoform 1 (Fig. 2A) [8]. The second initi- ation codon is thought to be an actual translation initiation site of isoform 2, instead of the first initi- ation codon followed by 31 amino acids [17]. We recently identified isoform 3 (GenBank accession number AB261897), which contains 25 additional amino acids in the catalytic domain, in the prostate [15]. The expression of such splicing variants is regu- lated in a cell type-specific manner. Only isoform 1 is detected in human hippocampal neurons, whereas both isoforms 1 and 2 are expressed in normal pros- tate epithelial cells [8,14]. This cell type-specific expression of KLK11 isoforms is conserved in mouse tissues [9]. Interestingly, prostate cancer cells express only isoform 1, whereas tissue from normal prostate and benign prostate hypertrophy express both iso- forms. However, the regulation of KLK11 gene expression and differences in the isoform characteris- tics are unknown. To understand the regulation of KLK11 gene expres- sion, we investigated the KLK11 gene promoter regions. Here we report that the KLK11 gene has three promoter regions accompanying the corresponding transcriptional initiation sites. We used reverse tran- scription RT-PCR to study the tissue-specific use of each promoter. We show the translation initiation site of isoform 2 and different intracellular distribution of isoforms 1 and 2, and discuss the regulatory mechan- ism of KLK11 gene expression. Results Three different primary transcripts are derived from the human KLK11 gene We determined the nucleotide sequences of 15 clones of the PCR products from the human prostate by oligo cap RACE in order to determine the transcrip- tion initiation sites of KLK11 because it was already reported that mRNAs for both isoforms 1 and 2 are detected in the organ. All of these nucleotide sequences were identical to the genomic sequence of the human KLK11 gene (GenBank accession number AF164623). We compared the sequences of the PCR products and genomic DNA, and mapped the three exons having unique transcriptional initiation sites on the KLK11 gene (Fig. 1, arrowheads). The most upstream initi- ation site was numbered +1. This site was located 1163 bp downstream from the transcriptional termina- tor signal of KLK12. Two additional initiation sites were mapped at +2 and +4. This exon ended at +49 and spliced to the common second exon, and was des- ignated exon 1a. The next exon, exon 1b, started at +347 or +348 and ended at +536. Exon 1b encoded a Met at +476 followed by 20 amino acids whose sequence was a part of isoform 2 mRNA. The third exon, exon 1c, extended from +1379 to +1454. The noncoding exons of exons 1a and 1c contributed to the 5¢ nontranslated region of isoform 1 mRNA in which the common exon 2 contains an initiation codon. Intronic 5¢ consensus GT was located adjacent to the 3¢ end of each exon. These results suggest that the KLK11 gene has three different promoters, which we designated promoters 1, 2, and 3, and which corres- ponded to transcripts 1, 2, and 3, respectively (sum- marized in Fig. 2A). Therefore, transcripts 1 and 3 encode isoform 1 KLK11, whereas transcript 2 encodes isoform 2. RT-PCR using the primer sets corresponding to the common exons detected KLK11 mRNA in glandular tissues, lung, pancreas, testis, and the CNS (data not shown). To determine whether each transcript is expressed in a tissue-specific manner, RT-PCR using the primer set specific for each transcript was performed. Transcript 1 was detected in some brain regions, including the hippocampus and thalamus, as well as in non-CNS tissues (Fig. 2). Transcript 2 was expressed in only non-CNS tissues, and transcript 3 was detected in both CNS and non-CNS tissues. Determination of KLK11 promoter regions To confirm that each promoter region was functional, we performed a reporter assay. For this purpose, we screened to find a cell lines expressing both isoforms of KLK11, and used human a neuroblastoma cell line, KP-N-YN, because of the transfection efficiency and expression of both KLK11 isoform 1 and isoform 2 in this cell line. Promoter 2 ()15 to +383) and promoter 3 (+524 to +1461) showed transcriptional activity similar to the promoter 1 and 2 regions ()1077 to S. Mitsui et al. Multiple promoters of the human KLK11 gene FEBS Journal 273 (2006) 3678–3686 ª 2006 The Authors Journal compilation ª 2006 FEBS 3679 Multiple promoters of the human KLK11 gene S. Mitsui et al. 3680 FEBS Journal 273 (2006) 3678–3686 ª 2006 The Authors Journal compilation ª 2006 FEBS +383) (Fig. 3). Deletion of the 5¢ region of promoter 2 decreased the activity, however, only a 70 bp frag- ment (+276 to +383) showed significant promoter activity. In contrast, promoter 1 ()1077 to +50) had slight activity. Statistical significance between pro- moter 1 and pGL3 basic was not detected. Some cis- acting elements, including SRY, cdxA, p300, and AP-1, were identified within 2.5 kb of the putative pro- moter regions (Fig. 1A). Neither TATA box nor CAAT box were noted around the transcriptional initi- ation sites. Determining the translation initiation site of isoform 2 KLK11 Messenger RNA for isoform 2 contains two initiation codons near the 5¢ end. To examine whether the first initiation codon is functional, the second initiation codon, which corresponds to the translation initiation site of isoform 1 mRNA, was substituted for a Ser residue (M33S, Fig. 4A). When in vitro synthesized mRNA for M33S was translated in cell-free wheat germ lysate, the translational product detected with an antibody raised against KLK11 was the same size as that of the translational product by isoform 2 mRNA (Fig. 4B). The product by isoform 1 mRNA was smaller than the isoform 2 product. The multiple bands may be explained by incomplete translation from truncated mRNA synthesized in vitro. Other- wise, the artifact may be caused by using a plant translation system, in which mammalian mRNA was inappropriately translated. The transient expression assay indicated that M33S and isoform 1 proteins were secreted into the conditioned media by mouse neuroblastoma, Neuro2a, or COS cells (Fig. 4C and S. Mitsui, unpublished results). The secreted M33S and isoform 2 proteins had the same molecular mass as isoform 1. To investigate the intracellular localization of KLK11 isoforms, each KLK11 isoform was fused with green fluorescent protein (GFP) at the carboxyl terminal and transiently expressed in Neuro2a cells. Isoform 1–GFP was transferred to cellular processes (Fig. 4D, arrows), whereas isoform 2–GFP accumulated near the nucleus (Fig. 4D). Immunocytochemistry using anti-Golgi p58 protein IgG, which is known as a marker protein of Golgi apparatus, showed the localization of isofrom 2– GFP at the Golgi apparatus. Both isoform 1–GFP and A B Fig. 2. Tissue-specific expression of alternative KLK11 transcripts. (A) Schematic structure of alternative KLK11 transcripts and transla- tion products. Coding and noncoding exons are indicated by solid and open boxes, respectively. Three transcripts were generated by their corresponding promoters. Transcript 1 containing noncoding exon 1a and transcript 3 containing noncoding exon 1c encoded isoform 1 KLK11 with a signal peptide (diagonally patterned box). Transcript 2 encoded a longer open reading frame with an addi- tional 32 amino acids because exon 1b contained an initiation codon. The letters H, D, and S show the essential amino acid triads for serine protease. Arrowheads indicate the positions and direc- tions of the PCR primers. Primers F1a, F1b, and F1c were specific for exons 1a, 1b, and 1c, respectively. The sequences of the prim- ers are described in Experimental procedures. (B) Tissue-specific expression of alternative KLK11 transcripts. RT-PCR was performed using primers specific for each alternative transcript, F1a, F1b, or F1c, and the common primer R1. Left, CNS tissues: AB, adult brain; Hi, hippocampus; CN, caudate nucleus; CC, corpus callosum; Th, thalamus; SC, spinal cord. Right, non-CNS tissues: SG, salivary gland; TG, thyroid gland; MG, mammary gland; Pa, pancreas; Lu, lung; Pr, prostate; Te, testis. Fig. 1. Nucleotide sequences of human and mouse KLK11 promoter regions. (A) Promoter sequence and transcription initiation sites of the human KLK11 gene. The most upstream initiation site is numbered +1. Transcription initiation sites are indicated by arrowheads. Exons 1a, 1b, and 1c are boxed. The consensus GT at the 5¢ end of intron is double underlined. Encoded amino acids in exon 1b are indicated under the nucleotide sequence. Underlines indicate cis-acting elements. (B) Comparison of nucleotide sequences of KLK11 promoter region in the human and mouse. Human and mouse sequences of KLK11 promoters are numbered at the most upstream transcription initiation sites at +1. Black arrowheads show the transcription initiation sites of the human and a white arrowhead shows the transcription initiation site of the mouse. Dashes show gaps and asterisks show the same nucleotide in the two species. S. Mitsui et al. Multiple promoters of the human KLK11 gene FEBS Journal 273 (2006) 3678–3686 ª 2006 The Authors Journal compilation ª 2006 FEBS 3681 isoform 2–GFP proteins secreted from the transfected cells were detected in the conditioned media by western blot analysis using anti-GFP IgG (data not shown). Discussion Until recently, only a few proteins such as KLK1 ⁄ glan- dular kallikrein, KLK2, and KLK3 ⁄ prostate specific antigen (PSA) had been classified as tissue kallikreins. Recent studies have identified 15 genes that are classi- fied as kallikrein based on their gene organization on human chromosome 19q13.4 [1,2]. The expression of each gene is regulated in a tissue-specific manner. How- ever, the regulation of KLK gene expression is unknown except for the traditional kallikreins, KLK2 and KLK3 ⁄ PSA, which are expressed in an androgen- dependent manner in the prostate. The androgen- responsive elements on these genes have been well characterized [20,21]. Our data are the first to show the transcriptional and translational regulation of a newly classified kallikrein gene, KLK11. Two isoforms of KLK11, isoforms 1 and 2, were predicted to have different sequences at their 5¢ ends based on their mRNAs [8]. Sequence analysis of the PCR products by oligo cap RACE revealed three first exons, exon 1a, 1b, and 1c, containing the respective transcription initiation sites (Fig. 1). Exons 1a and 1c were noncoding exons, indicating that transcripts 1 and 3 encode isoform 1 KLK11. Exon 1b encoded 21 amino acids following Met, which contributed to iso- form 2 KLK11 (discussed below). The transcriptional initiation sites of exons 1a and 1c are located 1163 bp and 1510 bp, respectively, downstream from the transcription terminator of the KLK12 gene. The promoter region of the KLK11 gene is believed to lie within this region, although we cannot rule out that the regulatory sequence for KLK11 expression may exist within the KLK12 gene. We observed neither the TATA box nor CAAT box within the 1.5 kb region, although some cis-acting elements were located about 200–500 bp upstream from each transcription initiation site (Fig. 1). However, no known cis-acting element was found within the 135 bp region of deleted promoter 1 and the 71 bp of promo- ter 2 which still showed transcriptional activity (Fig. 3). Although steroid hormone treatment enhances Fig. 3. Transcriptional activity of three promoter regions of the KLK11 gene in human neuroblastoma cells. The human KLK11 promoter region was linked to a firefly luciferase reporter gene in a pGL3 basic vector, and transfected into KP-N-YN neuroblastoma with the Renilla luciferase gene in pRL-SV40 as an internal standard. The schematic structure of the KLK11 promoter region, which is the 5¢ portion of Fig. 2A, is indicated on the left upper portion. Schemata at the left side of the bars represent the analyzed promoter regions. The relative activity is indicated as promoter 1+2 ()1077 to +383, a black bar) region and set at 100%. Promoters 2 ()15 to +383, diagonally patterned bars) and 3 (+524 to +1461, vertically patterned bar) showed high transcriptional activity, whereas promoter 1 ()1077 to +50, gray bars) had low activity. Statistical significance was analyzed by the student’s t-test (*, P < 0.01). Values are means ± SD of three individual experiments in triplicates. Multiple promoters of the human KLK11 gene S. Mitsui et al. 3682 FEBS Journal 273 (2006) 3678–3686 ª 2006 The Authors Journal compilation ª 2006 FEBS KLK11 expression [22], we found no steroid hormone- responsive element in any of the KLK11 promoters. Steroid hormone responsive elements may locate on other regions. The determination of the transcription initiation sites and the promoter assay revealed that not only alternative splicing but also alternative promoter con- tributed to the production of KLK11 mRNA iso- forms. Exon 1a in transcript 1 was always spliced to exon 2, which produces isoform 1 protein product, although transcript 1 contains three first exons, exon 1b and 1c as well as exon 1a. Similarly, exon 1b in transcript 2 always spliced to exon 2 despite the exist- ence of exon 1c, which causes isoform 2 KLK11 product. Promoters 1, 2, and 3 regulate transcription from their corresponding first exons, exons 1a, 1b, and 1c, respectively. Hence, the promoter usage affects translational regulation and determines the iso- form of translation products through the determin- ation of the first exon in KLK11 mRNA (summarized in Fig. 2A). Detection of the first exon in each KLK11 mRNA reflects the promoter use in the target organs. All three KLK11 promoters were functional in most non-CNS tissues tested, but the promoter use in the CNS tissues was unexpected (Fig. 2). We found that promoter 1 was functional only in the hippocampus and thalamus, which is consistent with a previous report of the isola- tion of transcript 1 from human hippocampal cDNA [8]. Promoter 3 was functional in all CNS tissues tes- ted, whereas promoter 2 showed no activity in any CNS tissue tested. Such promoter use seems to be con- served in the mouse KLK11 gene. Mouse brain expres- ses only isoform 1 mRNA, whereas mouse prostate expresses both isoforms 1 and 2 [9]. The transcription initiation sites on human promoters 2 and 3 showed 70% and 69% identity to the respective mouse genom- ic DNA sequences (Fig. 1B). The mouse and human forms exhibit similar sequences and tissue specificity of mRNA expression. Mouse KLK11 isoform 1 corres- ponds to human KLK11 transcript 3 and mouse iso- form 2 corresponds to human KLK11 transcript 2. Promoter 1 showed lower similarity (49%) than pro- moter 2 or 3, suggesting that the regulation of pro- moter 1 is different between human and mouse. Alternative transcripts of KLK6 ⁄ neurosin have the same open reading frame with a different 5¢ noncoding exon and are expressed in a tissue-specific manner [23]. Human and mouse 5¢ alternative transcripts of KLK6 ⁄ neurosin display identical genomic organization and tissue-specific expression. We propose that select- ive pressure maintains the variation at the 5¢ end of kallikreins. A B D a d e f b c C Fig. 4. Translation and intracellular localization of KLK11 isoforms. (A) Schematic structure of KLK11 isoforms. Predicted methionines are shown as M. Shaded boxes show the hydrophobic region cor- responding to the signal peptide of isoform 1. In the M33S mutant, the second initiation codon in isoform 2 was substituted for Ser. (B) In vitro translation of KLK11 isoform mRNAs. Messenger RNAs transcribed in vitro were translated in a wheat germ cell-free sys- tem. The translation products were detected by western blot analy- sis using antibody against KLK11. 1, isoform 1; 2, M33S mutant; 3, isoform 2. (C) Secretion of KLK11 isoforms from mouse neuro- blastoma cells. Each mRNA was transiently expressed in Neuro2a mouse neuroblastoma cells after being subcloned in the mamma- lian expression vector, pcDNA3.1 Myc-His. The conditioned media were recovered 48 h after transfection and analysed by western blot analysis using antibody against KLK11. 1, isoform 1; 2, M33S mutant; 3, isoform 2. (D) Intracellular distribution of KLK11. Each isoform of KLK11 was fused with green fluorescent protein at the C-terminal and the fusion proteins expressed in Neuro2a cells (a and d). Golgi apparatus was visualized using anti-Gogi p58 protein IgG 48 h after transfection (b and e). Merged images showed that isofrom 1 was distributed to cellular processes (a and c, arrows), whereas isofrom 2 was still retained in Golgi apparatus (d and f). Scale bar, 20 lm. S. Mitsui et al. Multiple promoters of the human KLK11 gene FEBS Journal 273 (2006) 3678–3686 ª 2006 The Authors Journal compilation ª 2006 FEBS 3683 Whereas KLK11 gene expression is regulated in a tissue-specific manner, recent reports suggest that tumorigenesis disturbs the regulation of the gene’s expression [1–4,24]. Together with our previous report that prostate cancer cells express only isoform 1, but not isoform 2 [14], promoter 2 is possibly inactivated in cancer cells. There may be silencer sequence specific for cancer cells at the promoter 2 region. Such negat- ive regulation of KLK11 expression in prostate cancer cells is compatible with the observation that serum KLK11 concentration is useful in discriminating between prostate cancer and benign prostate hyperpla- sia [16]. We also studied the translational regulation of KLK11 expression. Human KLK11 isoform 2 mRNA has two translational initiation codons at the 5¢ por- tion of the open reading frame. The second initiation codon is predicted to be an actual initiation site for two reasons. First, the second site is identical to the translation initiation site of isoform 1, which is a typ- ical secretory protein. The additional hydrophilic 32-amino acid polypeptide may conceal the signal sequence when translation is started from the first initi- ation codon. Second, when overexpressed in mouse neuroblastoma cells, the translation product of isoform 2 secreted into conditioned medium has the same molecular mass as isoform 1 [17]. Using in vitro trans- lation and transient expression of M33S mutant mRNA, we showed clearly that the first initiation codon is functional (Fig. 4). In addition, the product was secreted into conditioned medium from mouse neuroblastoma cells. These results suggest that isoform 2 mRNA encodes a longer polypeptide of 32 hydrophi- lic amino acids followed by a hydrophobic stretch. The secretion cascade appears to differ in isoforms 1 and 2. The translation product of isoform 2–GFP accumu- lated in the Golgi apparatus, whereas isoform 1–GFP was transported to cellular processes (Fig. 4D). The amino acid sequence around the second Met may affect the efficiency of the intracellular trafficking of KLK11. More experimental evidence is needed to understand the intracellular trafficking of KLK11 iso- forms. Cancer cells are known to express only isoform 1 KLK11 [14], as described above. The efficient secre- tion of isoform 1 may contribute to metastasis of cancer cells through degrading extracellular matrix proteins. The transcripts driven by promoters 1 and 3 have alternative noncoding first exons and common downstream exons, and, thus, the same open reading frame. This suggests that the 5¢ noncoding exon may affect the translational efficiency or mRNA trafficking. In summary, we identified three promoter regions on the human KLK11 gene and showed the tissue-specific use of each promoter. Transcripts 1 and 3 encoded a typical secretory KLK11, isoform 1, although the pri- mary transcript 1 contained exons 1b and 1c. Tran- script 2 encoded another type of KLK11, isoform 2, despite the presence of exon 1c. The secretory path- ways of KLK11 isoforms appeared to differ, although both isoforms were secreted from cells. Hence, the pro- moter use of the KLK11 gene appears to link to the splicing pathway of KLK11 mRNAs and to regulate the trafficking of KLK11 isoforms in the trans-Golgi network. Such complex regulation of KLK11 expres- sion implicates biological significance, because alternat- ive promoters and translation products are conserved between species such as the human and mouse. Experimental procedures Determination of transcription initiation sites by oligo cap RACE We determined the transcription initiation sites of the KLK11 gene by oligo cap RACE, because exon 1a is too small to use the primer-extension method. Oligo cap RACE specifically amplified mRNA has a cap structure at the 5¢ end, which indicates the transcription initiation site of a primary transcript [18]. PolyA + RNA from normal human prostate (Clontech, Mountain View, CA) was used as a template, because this organ abundantly expresses mRNA for both isoforms 1 and 2. Oligo cap RACE was performed using a Gene racer kit (Invitrogen, Carlsbad, CA), accord- ing to the instruction manual. The sequences of gene-speci- fic primers were designed according to the reported sequence (GenBank accession number NM006853 and NM14497): R1, 5¢-ATGGTGTCTGTGATGTTGCCG-3¢ and R2, 5¢-TTCTCACACTTCTGGTGCTC-3¢. DNA sequences of the PCR products were determined using an automatic DNA sequencer (DSQ-1000, Shimadzu, Kyoto, Japan) after cloning into pGEM-T Easy vector (Promega, Madison, WI). Sequence analysis was performed with gen- etyx software (Genetyx, Tokyo, Japan). Potential tran- scription factor binding sites were identified using a motif search program (Bioinfomatics Center, Institute for Chem- ical Research, Kyoto University, Japan; http://motif. genome.jp). RT-PCR PolyA + RNA from various human tissues was purchased from Clontech. One microgram of polyA + RNA was reverse transcribed by Superscript II (Invitrogen) using oligo dT 18 according to the instruction manual. PCR was performed as described elsewhere [14]. Specific amplification was confirmed by determining the nucleotide sequences of PCR products as described above. The PCR primers were: Multiple promoters of the human KLK11 gene S. Mitsui et al. 3684 FEBS Journal 273 (2006) 3678–3686 ª 2006 The Authors Journal compilation ª 2006 FEBS F1a, 5¢-GGACTCAAGAGAGGAACCTG-3¢; F1b, 5¢-CT GCCTTGCTCCACACCTG-3¢; F1c, 5¢-CTGCCGTCTCC GCCGCCACT-3¢; FU, 5¢-TCAAGCCCCGCTACATAG TT-3¢; and R3, 5¢-AGGAACCAAACACCAAGTGG-3¢ (Fig. 2A). Luciferase promoter assay Human genomic DNA was isolated from two volunteers (both male, Japanese, aged 34 and 37 years) after they gave informed consent. A 1460 bp fragment covering promoters 1 and 2 regions (primers 5¢-TCATGCTGGTTGAGACTG GA-3¢ and 5¢-CCAGGTGTGGAGCAAGGCAG-3¢) and a 938 bp fragment of promoter 3 (primers, 5¢-CTCACAG CAGCCAGTAAGTG-3¢ and 5¢-ACTCACAGGCTCTGG GGCTG-3¢) were amplified using a Taq polymerase with high fidelity (Pyrobest DNA Polymerase, Takara Bio Inc., Shiga, Japan). PCR products were cloned into a pTOPO Blunt vector (Invitrogen) and sequenced (pTOPO ⁄ hhipro 1 & 2, pTOPO ⁄ hhipro 3). The DNA fragments were sub- cloned into a pGL3-basic vector (Promega) between the KpnI and BglII sites for luciferase assay (pGL ⁄ hhipro 1 & 2 and pGL ⁄ hhipro 3). To separate promoter 1 from pGL ⁄ hhipro 1 & 2, the 1126 bp fragment was amplified using the primers 5¢-ATAGGTACCAGGAACTCGGGA CCAGCC-3¢ and 5¢-GTAGATCTCTCTTGAGTCCCAG TGG-3¢, and subcloned into a pGL3-basic vector between the KpnI and BglII sites (pGL ⁄ hhipro 1). For promoter 2, a 384 bp fragment between SacI and BglII sites from pGL ⁄ hhipro 1 & 2 was subcloned into pGL3-basic vector (pGL ⁄ hhipro 2). All constructs were confirmed by deter- mining their DNA sequences. The luciferase assay was performed using the human neuroblastoma cell line KP-N-YN, cultured in Ham F12 containing 10% (v ⁄ v) fetal bovine serum [19]. This cell line expressed both KLK11 isoforms and showed higher trans- fection efficiency than other cell lines expressing KLK11 mRNA. Five hundred nanograms of the pGL3 constructs was transfected into 5 · 10 5 cells of KP-N-YN using Lipo- fectAMINE (Invitrogen). The luciferase assay was per- formed using the dual-luciferase reporter assay system (Promega), and the pRL-CMV vector was used as an inter- nal control. Cells were extracted with the lysis buffer sup- plied in the kit 48 h after transfection. Luciferase activity was measured using a luminometer (MicroLumat Plus LB96V, Berthold Technologies GmbH & Co., Wildbad, Germany). In vitro mutagenesis and translation assay To substitute Met33 to Ser (M33S) in isoform 2 hipposta- sin, cDNA fragments were amplified using pcDNA3.1 ⁄ iso- form 2 KLK11 [8] as a template with primer sets, T7 primer and 5¢-AACTGCAGAATTCTAGAGGCCTGG-3¢, or pcDNA3.1 ⁄ BGH reverse primer (Invitrogen) and 5¢-CCAGGCCTCTAGAATTCTGCAGTT-3¢. The synthes- ized primer sequence contained a codon for the Ser instead of the Met. Both PCR fragments were fused at the XbaI site and subcloned into pcDNA3.1 Myc-His (pCDNA3.1 ⁄ M33S KLK11). The sequence was confirmed by an automatic sequencer (DSQ-1000, Shimadzu). The open reading frames of isoform 1, isoform 2, and M33S KLK11 were then subcloned into pEU-3b between the XhoI and KpnI sites (pEU ⁄ isoform 1, isoform 2, and M33S KLK11, respectively). The pcDNA3.1 constructs for iso- form 1 and 2 were described previously [8]. The pEU con- structs were transcribed in vitro using SP6 RNA polymerase after digestion with BglII. The transcribed RNAs were puri- fied by phenol-chloroform extraction and ethanol precipita- tion twice, and translated in vitro for 3 h using a wheat germ translation system (PROTEIOS, Toyobo Co, Osaka, Japan) according to the instruction manual. Transfection study and localization of KLK11 in cultured cells All of the pcDNA3.1 constructs for KLK11 were transfect- ed into mouse neuroblastoma cells, Neuro2a (ATCC CCL- 131), and the transient expression was followed by western blot analysis as described previously [8]. The open reading frames of the KLK11 isoforms were fused with GFP between EcoRI and SmaI sites in the pEGFP-N2 vector (Clontech), pEGFP ⁄ hKLK11 isoform 1 and pEGFP ⁄ hKLK11 isoform 2. These plasmids were transfected in Neuro2a, which were cultured to subconfluence in Dul- becco’s essential medium supplemented with 10% (v ⁄ v) fetal bovine serum. After two days, the cells were fixed in 4% (v ⁄ v) paraformaldehyde in NaCl ⁄ P i containing 0.3% (v ⁄ v) Triton X-100 (NaCl ⁄ P i -Triton), and washed in NaCl ⁄ P i three times. The cells were treated with anti-Gogi p58 protein IgG (clone 58K-9, Sigma, St Louis, MO) dilu- ted 1 : 500 in NaCl ⁄ P i -Triton at 4 °C overnight. After the slices were washed, they were reacted with goat antimouse- IgG ⁄ Alexa Fluor 594 (Molecular Probes Inc., Eugene, OR) for 1 h. Specimens were photographed using a Zeiss Axiop- hot microscope (Carl Zeiss, Jena, Germany) with a VB- 7000 cooled CCD camera (Keyence Co., Osaka, Japan). Acknowledgements We thank Dr Yaeta Endo (Cell-free Science and Tech- nology Research Center, Ehime University, Japan) for supplying pEU-3b and technical suggestions for in vitro translation, and Dr Tohru Sugimoto (Depart- ment of Pediatrics, Kyoto Prefectural University of Medicine) for donating the human neuroblastoma cell line, KP-N-YN. This study was supported in part by Grants-in-Aid for Scientific Research (B), Japan Soci- ety for the Promotion of Science (JSPS). S. Mitsui et al. Multiple promoters of the human KLK11 gene FEBS Journal 273 (2006) 3678–3686 ª 2006 The Authors Journal compilation ª 2006 FEBS 3685 References 1 Borgon ˜ o CA & Diamandis EP (2004) The emerging roles of human tissue kallikreins in cancer. Nat Rev Cancer 4, 876–890. 2 Obiezu CV & Diamandis EP (2005) Human tissue kallikrein gene family: application in cancer. Cancer Lett 244, 1–22. 3 Yousef GM & Diamandis EP (2001) The new human kallikrein gene family: structure, function, and associa- tion to disease. Endocr Rev 22, 184–204. 4 Borgon ˜ o CA, Michael IP & Diamandis EP (2005) Human tissue kallikreins: physiologic roles and applica- tions in cancer. Mol Cancer Res 2, 257–280. 5 Kurlender L, Borgon ˜ o C, Michael IP, Obiezu C, Elliott MB, Yousef GM & Diamandis EP (2005) A survey of alternative transcripts of human tissue kallikrein genes. 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Multiple promoters of the human KLK11 gene S. Mitsui et al. 3686 FEBS Journal 273 (2006) 3678–3686 ª 2006 The Authors Journal compilation ª 2006 FEBS . Multiple promoters regulate tissue-specific alternative splicing of the human kallikrein gene, KLK11 ⁄ hippostasin Shinichi. Hence, the pro- moter use of the KLK11 gene appears to link to the splicing pathway of KLK11 mRNAs and to regulate the trafficking of KLK11 isoforms in the