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:
*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.
Biochim Biophys Acta 1775, 1–14.
6 Yamashiro K, Tsuruoka N, Kodama S, Tsuijimoto M,
Yamamura T, Nakazato H & Yamaguchi N (1997)
Molecular cloning of a novel trypsin-like serine protease
(neurosin) preferentially expressed in brain. Biochim

Biophys Acta 1350, 11–14.
7 Mitsui S, Tsuruoka N, Yamashiro K, Nakazato H &
Yamaguchi N (1999) A novel form of human neuropsin,
a brain-related serine protease, is generated by alterna-
tive splicing and is expressed preferentially in human
adult brain. Eur J Biochem 260, 627–634.
8 Mitsui S, Yamada T, Okui A, Kominami K, Uemura H
& Yamaguchi N (2000) A novel isoform of a kallikrein-
like protease, TLSP ⁄ hippostasin (PRSS20), is expressed
in the human brain and prostate. Biochem Biophys Res
Commun 272, 205–211.
9 Mitsui S, Okui A, Kominami K, Uemura H & Yamagu-
chi N (2000) cDNA cloning and tissue-specific variants
of mouse hippostasin ⁄ TLSP (PRSS20). Biochim Biophys
Acta 1494, 206–210.
10 Mitsui S, Okui A, Uemura H, Mizuno T, Yamada T,
Yamamura Y & Yamaguchi N (2002) Decreased cere-
brospinal fluid levels of neurosin (KLK6), an aging-
related protease, as a possible new risk factor for
Alzheimer’s disease. Ann N Y Acad Sci 977, 216–223.
11 Okui A, Kominami K, Uemura H, Mitsui S & Yamagu-
chi N (2001) Characterization of a brain-related serine
protease, neurosin (human kallikrein 6), in human cere-
brospinal fluid. Neuroreport 12, 1345–1350.
12 Ogawa K, Yamada T, Tsujioka Y, Taguchi J, Takaha-
shi M, Tsuboi Y, Fujino Y, Nakajima M, Yamamoto
T, Akatsu H, Mitsui S & Yamaguchi N (2000) Localiza-
tion of a novel type trypsin-like serine protease, neuro-
sin, in brain tissue of Alzheimer’s disease and
Parkinson’s disease. Psychiatry Clin Neurosci 54,

419–426.
13 Diamandis EP, Okui A, Mitsui S, Luo LY, Soosaipillai
A, Grass L, Nakamura T, Howarth DJ & Yamaguchi
N (2002) Human kallikrein: a new biomarker of pros-
tate and ovarian carcinoma. Cancer Res 62, 295–300.
14 Nakamura T, Mitsui S, Okui A, Kominami K, Nomoto T,
Ukimura O, Kawauchi A, Miki T & Yamaguchi N (2001)
Alternative splicing isoforms of hippostasin (PRSS20 ⁄
KLK11) in prostate cancer cell lines. Prostate 49, 72–78.
15 Nakamura T, Mitsui S, Okui A, Miki T & Yamaguchi
N (2003) Molecular cloning and expression of a variant
form of hippostasin ⁄ KLK11 in prostate. Prostate 54,
299–305.
16 Nakamura T, Scorilas A, Stephan C, Jung K, Soosaipil-
lai AR & Diamandis EP (2003) The usefulness of serum
human kallikrein 11 for discrimination between prostate
cancer and benign prostatic hyperplasia. Cancer Res 63,
6543–6546.
17 Yoshida S, Taniguchi M, Suemoto T, Oka T, He X &
Shiosaka S (1998) cDNA cloning and expression of a
novel serine protease, TLSP. Biochim Biophys Acta
1399, 225–228.
18 Suzuki Y, Taira H, Tsunoda T, Mizushima-Sugano J,
Sese J, Hata H, Ota T, Isogai T, Tanaka T, Morishita
S, Okubo K, Sakaki Y, Nakamura Y, Suyama A &
Sugano S (2001) Diverse transcriptional initiation
revealed by fine, large-scale mapping of mRNA start
sites. EMBO Rep 2, 388–393.
19 Sugimoto T, Ueyama H, Hosoi H, Inazawa J, Kato T,
Kemshead JT, Reynolds CP, Gown AM, Mine H &

Sawada T (1991) Alpha-smooth-muscle actin and des-
min expressions in human neuroblastoma cell lines.
Int J Cancer 48, 277–283.
20 Cleutjens KB, van Eekelen CC, van der Korput HA,
Brinkmann AO & Trapman J (1996) Two androgen
response regions cooperate in steroid hormone regulated
activity of the prostate-specific antigen promoter. J Biol
Chem 271, 6379–6388.
21 Yu DC, Sakamoto GT & Henderson DR (1999) Identi-
fication of the transcriptional regulatory sequences of
human kallikrein 2 and their use in the construction of
calydon virus 764, an attenuated replication competent
adenovirus for prostate cancer therapy. Cancer Res 59,
1498–1504.
22 Yousef GM, Scorilas G & Diamandis EP (2000) Geno-
mic organization, mapping, tissue expression, and hor-
monal regulation of trypsin-like serine protease (TLSP
PRSS20), a new member of the human kallikrein gene
family. Genomics 63, 88–96.
23 Christophi GP, Isackson PJ, Balber S, Balber M,
Rodriguez M & Scarisbrick IA (2004) Distinct promo-
ters regulate tissue-specific and differential expression of
kallikrein 6 in CNS demyelinating disease. J Neurochem
91, 1439–1449.
24 Mitsui S, Okui A, Kominami K, Konishi E, Uemura H
& Yamaguchi N (2005) A novel serine protease highly
expressed in the pancreas is expressed in various kinds
of cancer cells. FEBS J 272, 4911–4923.
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