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-

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

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

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

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

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,

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-

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-

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
+

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-

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-

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