Endocrine Reviews 22 (2): 184-204
Copyright © 2001 by The Endocrine Society
The New Human Tissue Kallikrein Gene Family: Structure, Function, and Association to Disease1
George M. Yousef and
Eleftherios P. Diamandis
Department of Pathology and Laboratory Medicine, Mount Sinai
Hospital, Toronto, Ontario, Canada M5G 1X5; and Department of
Laboratory Medicine and Pathobiology, University of Toronto, Toronto,
Ontario, Canada M5G 1L5
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Abstract
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The human tissue kallikrein gene family was, until recently, thought to
consist of only three genes. Two of these human kallikreins,
prostate-specific antigen and human glandular kallikrein 2, are
currently used as valuable biomarkers of prostatic carcinoma. More
recently, new kallikrein-like genes have been discovered. It is now
clear that the human tissue kallikrein gene family contains at least 15
genes. All genes share important similarities, including mapping at the
same chromosomal locus (19q13.4), significant homology at both the
nucleotide and protein level, and similar genomic organization. All
genes encode for putative serine proteases and most of them are
regulated by steroid hormones. Recent data suggest that at least a few
of these kallikrein genes are connected to malignancy. In this review,
we summarize the recently accumulated knowledge on the human tissue
kallikrein gene family, including gene and protein structure, predicted
enzymatic activities, tissue expression, hormonal regulation, and
alternative splicing. We further describe the reported associations of
the human kallikreins with various human diseases and identify future
avenues for research.
I. Introduction
II. The Human and Rodent Families of Kallikrein Genes
III. Nomenclature
IV. The Human Kallikrein Gene Locus
A. Locus organization
B. Gene organization
V. Protein Homologies and Predicted Enzymatic Activity
VI. Hormonal Regulation of Kallikrein Genes
VII. Tissue Expression of Kallikreins
VIII. Variants of Kallikrein Transcripts
IX. Association of Kallikreins with Human Diseases
X. Physiological Functions
XI. Future Directions
XII. Conclusions
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I. Introduction
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KALLIKREINS are a group of serine proteases that are found
in diverse tissues and biological fluids. The term "Kallikrein" was
introduced in the 1930s by Werle and colleagues (1, 2) who found high
levels of their original isolates in the pancreas (in Greek, the
"Kallikreas"). The kallikrein enzymes are now divided into two
major categories: plasma kallikrein and tissue kallikrein (3, 4). These
two categories differ significantly in their molecular weight,
substrate specificity, immunological characteristics, gene structure,
and type of kinin released. Plasma kallikrein or Fletcher factor
(official symbol
KLKB1)2 is encoded by
a single gene, which is located on human chromosome 4q35 (5, 6). The
gene is composed of 15 exons and encodes for an enzyme that releases
the bioactive peptide bradykinin from a high molecular weight precursor
molecule (high mol wt kininogen) produced by the liver. Plasma
kallikrein is exclusively expressed by liver cells. The function of
plasma kallikrein includes its participation in the process of blood
clotting and fibrinolysis and, through the release of bradykinin, in
the regulation of vascular tone and inflammatory reactions (7).
Plasma kallikrein will not be discussed further in this review since
the gene encoding for this enzyme has no similarities with the tissue
kallikrein genes and clearly, is not a member of this multigene family.
A historical perspective on the discovery of the kallikrein-kinin
system and bradykinin has recently been published (8).
Tissue kallikreins are members of a large multigene family and
demonstrate considerable similarities at the gene and protein level as
well as in tertiary structure. In this review, we will describe recent
developments, exclusively pertinent to the human family of enzymes.
The term "kallikrein" is usually used to describe an enzyme that
acts upon a precursor molecule (kininogen) for release of a bioactive
peptide (kinin) (7, 8, 9, 10). Another term that is also frequently used to
describe these enzymes is "kininogenases." The term "kininase"
is used to describe other enzymes that can inactivate kinins. Among the
known human and animal tissue kallikreins, only one enzyme has the
ability to release efficiently a bioactive kinin from a kininogen. In
humans, this enzyme is known as pancreatic/renal kallikrein or, with
the new nomenclature, as the KLK1 gene, encoding for human kallikrein 1
(hK1 protein) (9, 10, 11, 12). This enzyme acts upon a liver-derived kininogen
(low mol wt kininogen) to release lysyl-bradykinin (also known as
kallidin), which is involved in the control of blood pressure,
electrolyte balance, inflammation, and other diverse physiological
processes. Tissue kallikrein (hK1) may further enzymatically digest
other substrates, including growth factors, hormones, and cytokines, to
mediate pleiotropic effects (7).
It should be emphasized that the generic term "tissue kallikrein"
is not restricted to the description of enzymes that release
bioactive peptides from precursor molecules. The term is used to
describe a group of enzymes with highly conserved gene and protein
structure, which also share considerable sequence homology and
colocalize in the same chromosomal locus as the KLK1 gene. In this
review, the term "kallikrein" will be used to describe a family of
15 genes that have a number of striking similarities, as outlined in
point format in Table 1
(13). The use of
the term "kallikrein" does not necessarily imply that
any of these family members (with the exception of KLK1) have
kininogenase activity. In fact, for human family members that have been
functionally tested, it was found that they possess very low (hK2) (14, 15) or no kininogenase activity [prostate-specific antigen (PSA)]
(14). These enzymes are grouped together with KLK1, based on the
similarities outlined in Table 1.
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II. The Human and Rodent Families of Kallikrein Genes
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The tissue kallikrein literature can be roughly separated into
various periods as follows. Early in the 1920s and 30s, researchers
discovered the basic components of the kallikrein-kinin system and
identified the molecular structure of bradykinin and kallidin
(lysyl-bradykinin) in the 1960s (8). The molecular biology of the
tissue kallikrein gene family was worked out in detail in both the
human and rodents in the 1980s (16, 17, 18, 19). It was then concluded that the
mouse and rat gene families were composed of many genes, clustered in
the same chromosomal locus. In particular, the mouse tissue kallikrein
gene family is localized on chromosome 7 and consists of 24 genes, of
which at least 14 encode for active proteins (the remaining being
pseudogenes) (16, 20, 21, 22). The area on chromosome 7 encompassing the
mouse kallikreins is homologous to an area on human chromosome 19q13.4
that harbors the human kallikrein gene family. The rat tissue
kallikrein gene family is composed of approximately 20 homologous genes
of which at least 10 are expressed (18, 23, 24, 25, 26, 27, 28, 29, 30).
Most of the rodent tissue kallikreins are expressed in the salivary
glands, but a few, including the prostate, pituitary gland, and
endometrium, have more diverse tissue expression (7, 9, 31, 32, 33). It is
not the purpose of this review to describe in detail the rodent or
other animal tissue kallikrein gene families. Excellent reviews on this
subject already exist (9, 16, 17, 21, 22).
The human tissue kallikrein gene family was also discovered in the
1980s and it was then concluded that the entire family is composed of
only three genes, namely KLK1, encoding for pancreatic/renal kallikrein
(hK1 protein), the KLK2 gene, encoding for human glandular kallikrein 2
(hK2), and the KLK3 gene, encoding for PSA (hK3) (34, 35, 36, 37, 38). The major
interest in human kallikreins lies in the very restricted tissue
expression of hK2 and hK3 in the prostate, which qualifies them as
candidate biomarkers for prostatic diseases (39, 40, 41, 42, 43). hK3 (PSA), in
particular, has gained prominence in recent years as the most valuable
tumor marker ever discovered and is currently used widely for the
diagnosis, monitoring, and population screening for prostate cancer
(44, 45, 46, 47, 48, 49, 50, 51). The introduction of this test has had a major impact on
prostate cancer diagnosis and monitoring and this field is still
evolving (52, 53). More recently, PSA applications have extended beyond
the prostate, including breast and other cancers (54, 55, 56, 57). Over the
last few years, human glandular kallikrein 2 is emerging as an
additional prostatic and breast cancer biomarker, and it is now clear
that it can supplement PSA testing for improved identification and
differential diagnosis of prostate cancer (43, 58, 59, 60, 61, 62, 63, 64, 65, 66). It is thus
logical to exploit the possible applications of other members of this
gene family for cancer and other disease diagnosis and monitoring.
In the last 3 yr, we have witnessed the emergence of new knowledge
related to the human kallikrein gene family (13). Independent
researchers have cloned a number of new serine protease genes that show
significant homologies with the classical human kallikreins; in
addition, when these new protease genes were mapped, they were found to
colocalize in the known human kallikrein gene locus on chromosome
19q13.3-q13.4 (67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90). The recent detailed molecular description of
the human kallikrein gene locus (67, 68) enabled us to construct a
physical map containing 15 genes that share significant structural
similarities (Table 1
). Some of these genes appear to be related to
breast, ovarian, and other human cancers, and a few of them appear to
encode for functional tumor suppressor genes. In view of these very
recent developments, we will describe, in this review, the knowledge
that has accumulated on these genes, with special emphasis on the
structure of the genes and proteins, their tissue expression and
hormonal regulation, and their connection to various human diseases.
Where possible, functional aspects of these enzymes will also be
described. We hope that the summary of these new findings on the human
kallikrein gene family will facilitate further research toward better
understanding their physiological function, their pathophysiology and
connection to human diseases, and their possible applications in the
diagnosis and monitoring of various malignancies and their future
suitability as therapeutic targets.
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III. Nomenclature
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Until 2–3 yr ago, only three human kallikrein genes were
recognized: the pancreatic/renal kallikrein (KLK1), the human
glandular kallikrein 2 (KLK2), and PSA (KLK3). Rittenhouse and
co-workers (43, 49) have recently published the revised nomenclature
for these three genes. New developments led to the identification of 15
different genes exhibiting significant homologies and other
similarities, as described in Table 1 (13). Since many of these genes
were cloned independently by different investigators, various empirical
names were initially used for their description.
The Human Genome Organization (HUGO) has recently proposed guidelines
for human gene nomenclature. Initially, some members of the new
kallikrein gene family were classified by HUGO along with other serine
proteases under the prefix "PRSS", standing for "protease
serine." It is now clear that this designation does not serve well
the needs of the future since members of this multigene family are
classified together with other serine proteases that map in different
locations of the genome.
The construction of the first detailed map of the human kallikrein gene
locus (13, 67, 68) allows for a more rational assignment of official
gene symbols. Since the rodent and other animal species kallikrein
multigene families were known before 1992, an international working
party had reached agreement in 1992 on uniform nomenclature of the
animal kallikreins and the three human kallikreins known at that time
(91). Based on this paradigm and the guidelines of HUGO (for details
please visit the Website: http://www. gene.ucl.ac.uk/nomenclature/), an
international group of scientists working in the field agreed to adopt
a nomenclature for the newer human kallikreins, consistent with that
already defined for KLK1–3, as shown in Table 2 (92). In the same table, we also
include previous symbols based on the PRSS system as well as names
originally proposed by the discoverers of these genes (93, 94, 95, 96, 97, 98, 99, 100). Gene
numbering starts from centromere to telomere on chromosome 19q13.4 with
the exception of the three classical kallikreins for which the existing
nomenclature was retained and one newly discovered gene, which maps
between KLK1 and KLK2 genes (69). It is possible that, in the future,
new members of this gene family may be identified, either centromeric
to KLK1 or telomeric to KLK14 (see below). If new kallikrein genes are
identified in this locus, they will be sequentially numbered, starting
with KLK16.
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IV. The Human Kallikrein Gene Locus
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A. Locus organization
The availability of linear genomic sequences around chromosome
19q13.3–q13.4 from the human genome project (the sequences were
generated by the Lawrence Livermore National Laboratory) allowed the
precise localization of the 15 members of the new human kallikrein gene
family with high accuracy (±1 nucleotide) (68) (Fig. 1). The three classical kallikreins,
KLK1, KLK3, and KLK2, cluster together within a 60-kb region, as
previously described by Riegman et al. (36, 37).
and Richards et al. (35). Another newly discovered gene,
KLK15, maps between KLK1 and KLK2 (69). The remaining kallikrein genes
are aligned within this locus, as shown in Fig. 1, without intervention
by other genes. The direction of transcription is from telomere to
centromere with the exception of KLK3 and KLK2. The genomic lengths of
all these genes are relatively small, ranging from 4–10 kb. It is
unlikely that this locus harbors more kallikrein-like genes either
centromeric from KLK1 or telomeric from KLK14. The next neighboring
gene to KLK1 is testicular acid phosphatase (ACPT; GenBank Accession
no. AF321918), which is not related to kallikreins. The next
neighboring gene from KLK14 is Sigelec 9 (101). Siglecs belong to the
immunoglobulin superfamily and encode for transmembrane receptors that
have the ability to bind sialic acid (102, 103). These genes have no
structural or functional relationship to the human kallikreins.
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Figure 1. An approximate 300-kb region of contiguous genomic
sequence around chromosome 19q13.4. The direction of transcription of
each gene is illustrated by arrows. Boxes
represent genes and contain the gene names and their genomic length, in
base pairs. Other commonly used names for these genes are also
mentioned. Distances between genes in base pairs are shown between
boxes. The Siglec and ACPT (testicular acid phosphatase)
genes do not belong to the tissue kallikrein gene family. Figure is not
drawn to scale. For full gene names, see Table 2 and abbreviation
footnote.
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B. Gene organization
All members of the new human kallikrein multigene family encode
for serine proteases. All genes consist of five coding exons, as shown
in Fig. 2. The organization of all genes
is very similar, with the first coding exon having a short
5'-untranslated region, the second exon harboring the amino acid
histidine of the catalytic triad toward the end of the exon, the third
exon harboring the aspartic acid of the catalytic triad around the
middle, and the fifth exon harboring the serine of the catalytic
triad, at the beginning of the exon. Beyond the stop codon, there is a
3'-untranslated region of variable length.
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Figure 2. Schematic diagram showing the comparison of the
coding regions of the 15 kallikrein genes. Exons are shown by
solid bars and introns by the connecting
lines. Letters above boxes indicate relative
positions of the catalytic triad that was found to be conserved in all
genes; H, histidine; D, aspartic acid; and S, serine. Roman
numbers indicate intron phases. The intron phase refers to the
location of the intron within the codon; I denotes that the intron
occurs after the first nucleotide of the codon; II, the intron occurs
after the second nucleotide; 0, the intron occurs between codons. The
intron phases are conserved in all genes. Numbers inside
boxes indicate exon lengths and numbers outside
boxes indicate intron lengths (in base pairs). The
arrowhead represents the position of the start codon and
the arrow indicates the position of the stop codon.
Question mark denotes that region length is unknown.
Figure is not drawn to scale.
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While it is certain that the classical kallikreins do not have
5'-untranslated exons, most other members of this multigene family have
one or two 5'-untranslated exons, as shown in Fig. 2. It is possible
that some other members of this gene family also harbor 5'-untranslated
exons, which have not as yet been identified. In addition, the
3'-untranslated region of many of these genes is sometimes variable,
giving rise to variants with different mRNA lengths, but encoding for
the same protein (variant kallikrein transcripts are described under a
separate heading). It is thus possible that the actual lengths of these
genes, as shown in Figs. 1 and 2, may change slightly in the future.
Although the intron lengths of these genes vary considerably, the exon
lengths are quite comparable or identical. Additionally, the intron
phases between coding exons of all these genes (and those of the rodent
kallikreins) are completely conserved among all members, with phases
I–II–I–O. The intron phases are defined in Fig. 2.
Although TATA boxes have been identified within the proximal promoter
of the classical kallikrein genes (Table 3), no such elements were found for most
of the other kallikreins. This may be due to the absence of these
elements or to the fact that the proximal promoter of some of these
genes has not been accurately defined due to the presence of as yet
unidentified 5'-untranslated exons. This issue merits further
investigation. Classical (AATAAA) or variant polyadenylation signals
have been identified 10–20 bases away from the poly A tail of all
kallikrein mRNAs (Table 3). With only one exception, all
splice-junction sites are fully conserved among the human kallikrein
genes (Table 3).
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V. Protein Homologies and Predicted Enzymatic Activity
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The 15 members of the new human kallikrein gene family have been
aligned to identify similarities (Fig. 3). Maximum homology between all these
proteins is found around the catalytic amino acids histidine (with the
conserved region WVLTAAHC), aspartic acid
(DLMLL), and serine (GDSGGPL). In general, the
amino acid identity between the various members of this family ranges
from about 40–80%. The number and position of cysteine residues are
highly conserved among the 15 human kallikreins and among other serine
proteases. All members of this family possess between 10–12 cysteine
residues, which are expected to form disulfide bridges. A number of
other invariant amino acids (
25–30), especially those around the
active site of serine proteases, have been described (104). In the case
of the human family of genes, there are 39 amino acids that are
completely conserved among all 15 kallikreins (Fig. 3). Numerous other
conservative amino acid substitutions are shown in Fig. 3. A
phylogenetic tree of all human kallikreins and a few other serine
proteases is shown in Fig. 4.
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Figure 3. Alignment of the deduced amino acid sequence of
the 15 kallikrein proteins. Dashes represent gaps to
bring the sequences to better alignment. The amino acids of the
catalytic triad (H, D, S) are shown in italics.
Identical amino acids are highlighted in black and
similar residues in gray. The 29 invariant serine
protease residues are marked by (•) on the bottom, and
the cysteine residues by (+) on top of each block. The
dotted area represents the kallikrein loop sequence. The
asterisk denotes the position of the amino acid of the
binding pocket that is crucial for substrate specificity (for
trypsin-like enzymes the amino acid is D). For more details, see text.
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Figure 4. Dendrogram of the predicted phylogenetic tree for
the 15 kallikrein proteins and a few other related serine proteases.
The neighbor-joining method was used to align these proteins. The
classical kallikreins (hK1, hK2, and PSA) were grouped together; other
kallikreins and serine proteases were separated in different groups, as
shown. For full protein names, please see Table 2 and abbreviation
footnote.
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All proteins encoded by these genes are initially synthesized as
preproenzymes that are then proteolytically processed to yield
proenzymes by removal of the signal peptide, followed by activation
(also by proteolysis) to the mature, enzymatically active forms. In
Table 4, we present the reported signal
and activation peptides as well as the length of the mature proteins
that are encoded by these genes. It is important to mention that most
of these cleavage sites have been predicted by computer programs and
have been verified experimentally for only a few members.
The data of Table 4 suggest that most of the pro-forms of these enzymes
are activated by cleavage at the carboxy-terminal end of either
arginine (R) or lysine (K) residues (the preferred trypsin cleavage
site). Since most of the human kallikrein enzymes have trypsin-like
activity, they may potentially act as activating enzymes for either
themselves (autoactivation) or other pro-forms of kallikreins.
Kallikreins may participate in cascade pathways similar to those
demonstrated for the digestive enzymes, coagulation, and apoptosis.
These possibilities merit further investigation.
Protein sequence examination (Fig. 3) reveals that the three classical
kallikreins possess an amino acid sequence of approximately 9–11 amino
acids (the kallikrein loop) preceding the aspartic acid residue of
serine proteases, which is not present in its entirety in any of the
other 12 enzymes. This short sequence is thought to confer specificity
for kininogenase activity but, as already mentioned, only hK1 is a
potent kininogenase. KLK15 has a unique 8-amino acid sequence at
positions 148–155, not found in any other kallikrein protein.
Similarly, KLK13 possesses a unique amino-terminal and a unique
carboxy-terminal end.
Serine proteases can be divided into two main evolutionary families,
the trypsin-like serine proteases and the subtilisin-like pro-protein
convertases, which presumably evolved through convergent evolution
(105). The trypsin-like serine proteases are believed to have evolved
from a single ancestral gene that duplicated in the course of evolution
to give rise to other genes that have gradually mutated and evolved to
related proteases and protease subfamilies with new functions. The
various serine proteases can be markedly different in relation to their
substrate specificity (106, 107). The differences are due to very
subtle variations in the substrate binding pocket. Trypsin-like serine
proteases have an aspartic acid in their binding pocket, which can form
strong electrostatic bonds with arginine or lysine residues, which are
usually present at the carboxyl-terminal part of the cleavage site. The
important amino acid of the binding pocket, responsible for substrate
specificity, is usually found six amino acids before the catalytic
serine residue. From the 15 proteins aligned in Fig. 3, 11 have
aspartic acid in this position and are expected to have trypsin-like
activity. The four remaining enzymes, namely hK3 (has serine), hK7 (has
asparagine), hK9 (has glycine), and hK15 (has glutamic acid), are
expected to have chymotrypsin-like or other specific enzymatic activity
(see also Table 4). The cleavage specificity of these enzymes needs to
be established experimentally, with the exception of hK3, which has
already been characterized (50).
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VI. Hormonal Regulation of Kallikrein Genes
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KLK1 expression has been studied in animals, and it was concluded,
by using gene-specific probes, that this enzyme is not directly
regulated by androgens either in the salivary glands or the kidney (31, 108, 109, 110, 111). Similarly, no regulation of the KLK1 gene by thyroid
hormones has been demonstrated (109, 110, 111). Results of KLK1 regulation
by mineralocorticoids are inconclusive (112, 113). Other data support
the transcriptional up-regulation of KLK1 by estrogens (114, 115) and
by dopamine in rat pituitary (116). The demonstration that KLK1
expression in human endometrium is higher during the middle of the
menstrual cycle is also suggestive of KLK1 up-regulation by estrogens
in this tissue (117).
Murray et al. (19) have reported the presence of various
motifs that are reminiscent of consensus estrogen-, progestin-,
glucocorticoid-, or cAMP-response elements in the 5'-flanking sequence
of the human KLK1 gene (19). However, these putative elements have not
been functionally tested. Consequently, no conclusion can be drawn
regarding direct regulation of KLK1 transcription by steroid or other
hormones.
The regulation of the PSA (KLK3) gene by steroid hormones has been
extensively studied. Initially, two androgen-response elements were
identified in the proximal PSA promoter, at positions -170
[ARE1] and -394 (ARE2), respectively (118, 119, 120). These AREs have
been functionally tested and found to be active in LNCaP prostate
cancer cells. More recently, Schuur et al. have identified
various regions of 5'-sequences of the PSA gene around -6 to -4 kb
and demonstrated presence of a putative androgen-response element at
position -4,136 (ARE3), which markedly affects PSA transcription upon
induction by androgens (121). It was also demonstrated that this area
harbors an enhancer that is contained within a 440-bp fragment (121, 122). The upstream enhancer, containing the putative ARE3, has a
dramatic effect on PSA transcription, in comparison to the two AREs in
the proximal promoter (122). The hormonal regulation of the PSA gene is
not tissue specific since PSA has also been found to be regulated by
steroid hormones in vitro and in vivo in breast
tissues and breast carcinoma cell lines (123, 124, 125). Despite this, a
number of investigators have used the PSA promoter and enhancer region
to deliver and express therapeutic vectors to prostate tissue, in
experimental gene therapy protocols (126, 127, 128, 129, 130, 131, 132).
A number of investigations have clearly demonstrated hormonal
regulation of the PSA gene primarily by androgens in the prostatic
carcinoma cell line LNCaP (133) and by androgens and progestins in the
breast carcinoma cell lines BT-474, T-47D, and MFM223 (123, 125, 134).
The 5'-promoter sequences of the KLK2 gene have been studied by Murtha
et al. (135) who have identified functional androgen
response elements in the promoter of this gene. The same group has
subsequently shown that KLK2 is up-regulated by androgens and
progestins in the breast carcinoma cell line T47-D (136) while Riegman
et al. (137) showed up-regulation by androgens. More
recently, it has been demonstrated that, similarly to PSA, a
5'-enhancer region exists about 3–5 kb upstream from the transcription
site of the KLK2 gene (138). The enhancer region contains an androgen
response element that was shown to be functionally active. Consistent
with these data are the findings of hK2 protein secretion and
up-regulation by androgens and progestins in the breast cancer cell
lines BT-474, T47-D, and MFM223 (134). Although the KLK2 gene promoter
is not exclusively functional in the prostate, gene therapy protocols
have used it for prostate cancer therapy (139).
The KLK4 gene was found to be up-regulated by androgens in the
prostatic carcinoma cell line LNCaP (70) and by androgens and
progestins in the breast carcinoma cell line BT-474 (71). The mode of
regulation of KLK2 and KLK4 genes appears to be very similar to
the mode of regulation of PSA (KLK3). Stephenson et al. (72)
have identified putative androgen response elements in the proximal
promoter region of the KLK4 gene (up to 553 bp from the transcription
initiation site). However, such putative AREs have not been
functionally tested, and no data have been published as yet on the
characterization of possible enhancer regions further upstream from the
proximal KLK4 promoter.
For the remaining 11 human kallikrein genes that have been recently
identified, in none of them was the promoter functionally tested for
the presence for hormone response elements (HREs). Most studies
regarding hormonal regulation of these new genes have been performed
with the breast carcinoma cell line BT-474 and, in some cases, with the
prostatic carcinoma cell line LNCaP and other breast carcinoma cell
lines. It is clear that for 10 of 11 genes under discussion
(KLK5-KLK15), transcription is affected by steroid hormones, with the
selectivities and potencies shown in Table 5. Most genes appear to be up-regulated
by estrogens, androgens, and progestins but with different potencies.
It is possible that some of these genes are hormonally regulated
through indirect mechanisms, involving trans-acting elements
(140).
Clearly, there is a need to functionally characterize the promoter and
enhancer regions of these genes to understand better the mechanism of
transcriptional and posttranscriptional regulation by steroid hormones.
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VII. Tissue Expression of Kallikreins
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KLK1 gene expression is highest in the pancreas, kidney, and
salivary glands (9). The other two classical kallikrein genes, KLK3 and
KLK2, were thought, for many years, to be expressed exclusively in the
prostate (39, 40, 41, 42, 46, 141, 142). By using highly sensitive
immunological techniques (143), RT-PCR technology (144) as well as
immunohistochemistry (145), it has now been demonstrated unequivocally
that both KLK3 and KLK2 genes are expressed in diverse tissues but at
relatively much lower concentrations than prostatic tissues
(55–57, 146–148). Especially, hK3 (PSA) and hK2 proteins
and mRNA have been found in significant amounts in the female breast
and at lower levels in many other tissues (Table 6). KLK4 also appears to have
prostatic-restricted expression (70) but by RT-PCR, it was demonstrated
that it is also expressed in breast and other tissues (71, 72). None of
the remaining kallikreins is tissue-specific, although certain genes
are preferentially expressed in breast (e.g., KLK5, KLK6,
KLK10, KLK13), skin (KLK5, KLK7, KLK8), central nervous system (KLK6,
KLK7, KLK8, KLK9,KLK14), salivary glands (almost all kallikreins), etc.
A diagrammatic representation of expression of all these kallikreins in
human tissues is shown in Fig. 5. Most
data have been generated by RT-PCR.
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Figure 5. Schematic representation of tissue kallikrein
expression in various tissues. Higher level of expression is shown in
bold. For more information and discussion, see text and
Table 6.
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It is clear that there is frequent coexpression of many kallikreins in
the same tissues, and this may point to a functional relationship. For
example, it has been shown that hK3 and hK2 are regulated by similar
mechanisms (134) (see also previous section) and that they are
frequently coexpressed in tissues and body fluids (146, 147, 148). In
vitro data have demonstrated that hK2, which has trypsin-like
activity, can activate the proform of PSA (149, 150, 151). Other functional
relationships between members of the kallikrein gene family have not
been demonstrated as yet.
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VIII. Variants of Kallikrein Transcripts
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A relatively large number of variant transcripts have already been
identified for the classic and the new human kallikrein genes (Table 7). The functional and diagnostic
importance of these transcripts has not as yet been studied in detail.
It will be interesting to examine whether any of these transcripts are
specific for certain disease states or tissues. Although other forms of
some kallikreins in serum have already been described (e.g.,
kallikreins bound to proteinase inhibitors, internally clipped
kallikreins, circulating proforms, etc.), these will not be described
in this review. Excellent accounts of these forms and their clinical
significance already exist (43, 47, 48, 49, 152, 153, 154, 155, 156, 157, 158). It should be
emphasized that, in general, the putative proteins encoded by these
variant transcripts have not been isolated. By open reading frame
analysis, it has been predicted that most transcripts will produce
truncated proteins due to frameshifts originating from deleted exons.
More details on these variant transcripts and the predicted encoded
proteins can be found in the literature cited in Table 7 (34, 69, 77, 78, 86, 94, 96, 97, 98, 137, 159, 160, 161, 162, 163, 164, 165).
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IX. Association of Kallikreins with Human Diseases
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As already mentioned, the only enzyme with efficient kininogenase
activity, among the human kallikrein family members, is hK1. The
biological effects of this enzyme, and of plasma kallikrein, are
mediated mainly by kinin release. Kinin binds to specific G
protein-coupled cell surface receptors to mediate diverse biological
functions. The kallikrein-kinin system is involved in many disease
processes, including inflammation (9), hypertension (166), renal
disease (167, 168), pancreatitis (169), and cancer (170, 171, 172, 173, 174). A recent
book summarizes elegantly the physiology, molecular biology, and
pathophysiology of the kallikrein-kinin system and its association to
various disease processes (175).
Among all other kallikreins, the best studied, by far, is PSA (hK3) and
especially, its application to prostate cancer diagnostics. A
comprehensive volume on PSA as a tumor marker has been recently
published (176). The extensive literature on PSA and prostate cancer
does not warrant further discussion in this review.
Although PSA concentration is generally elevated in the serum of
prostate cancer patients, one less known and usually not well
understood finding is PSA down-regulation in prostate cancer tissue, in
comparison to normal or hyperplastic prostatic tissues (177, 178, 179, 180, 181, 182).
Furthermore, it has been demonstrated that lower tissue PSA
concentration is associated with more aggressive forms of prostate
cancer (182, 183). These data agree with those published for breast
cancer, where it was found that PSA is down-regulated in cancerous
breast tissues, in comparison to normal or hyperplastic breast tissues,
and in more aggressive forms of breast cancer. Patients with
PSA-positive tumors usually have earlier disease stage, live longer,
and relapse less frequently (184, 185, 186). Furthermore, it was found that
lower PSA levels in nipple aspirate fluid of women are associated with
higher risk for developing breast cancer (187). Other published data
suggest that PSA may be a tumor suppressor (188), an inducer of
apoptosis (188), a negative regulator of cell growth (189), and an
inhibitor of angiogenesis (190, 191) and bone resorption (192, 193).
These data have recently been reviewed (194).
Another set of investigations suggests that PSA may be associated with
unfavorable prognosis/outcomes in breast, prostate, and other cancers.
More specifically, it was found that breast tumors with higher PSA
content do not respond well to tamoxifen therapy (195). Further,
patients with breast tumors, which produce PSA after stimulation by
medroxyprogesterone acetate (a synthetic progestin/androgen), have a
worse prognosis than patients with tumors that do not produce PSA
(196). A number of reports have indicated that PSA may cleave
insulin-like growth factor binding protein-3, thus liberating
insulin-like growth factor I (IGF-I), which is a mitogen for prostatic
stromal and epithelial cells (197, 198, 199). PSA may activate latent
transforming growth factor-ß (TGFß), stimulate cell detachment and
facilitate tumor spread (200). Like other serine proteases, PSA may
mediate proteolysis of basement membrane, leading to invasion and
metastasis (201).
These confusing clinical data are due to differences in methodology,
purity, and source of PSA preparations used, selection of patients,
etc. Furthermore, the lack of knowledge of the biological pathways in
which PSA is participating poses significant difficulties in
interpreting these clinical observations, as further exemplified in a
recent commentary (194).
Human glandular kallikrein 2 (hK2) appears to be a new, promising
biomarker for prostatic carcinoma (43). It is clear that the diagnostic
value of hK2 measurement in serum is not superior to PSA; hK2 may aid
in the differential diagnosis between prostate cancer and benign
prostatic hyperplasia (57, 58, 59, 60, 61, 62, 63, 64, 65, 66) as well as in the identification of
organ-confined vs. non-organ-confined disease (202).
Immunohistochemical studies have shown that prostate cancer tissue
produces more hK2 than normal or hyperplastic tissue (203, 204).
However, recent quantitative data demonstrate that hK2 concentration,
although to a lesser extent than PSA, is also decreased in cancerous
tissue, in comparison to adjacent normal tissue (181). Although hK2 has
been detected in breast and other tissues (146, 147, 148), no studies have
as yet been performed to examine its biological action or its value as
a breast disease biomarker.
Although it has been shown that KLK4 expression is relatively high in
prostate (70, 71), there are no reports describing association or
usefulness of this kallikrein in prostatic disease. It will be
worthwhile to examine the possible clinical value of this kallikrein as
a biomarker in prostatic and other diseases. Recently, KLK4 was found
to be overexpressed in a subset of ovarian tumors (205).
A single report describes overexpression of KLK5 in ovarian carcinomas
and association with less favorable clinical outcomes (206). Further,
KLK6 appears to be dramatically down-regulated at metastatic breast
cancer sites and up-regulated in a subset of primary breast and ovarian
tumors (73). These data should be interpreted with caution since the
number of patients was small and the techniques used were qualitative.
Additionally, Little et al. (74) suggested that KLK6 may be
amyloidogenic and may play a role in the development of Alzheimer’s
disease by cleaving amyloid precursor proteins. Recently, a number of
newly cloned aspartyl proteinases were also shown to be amyloidogenic
(207). The connection between various types of proteases and this
disease is still ill-defined. The connections of KLK7 with skin
diseases, including pathological keratinization and psoriasis, have
already been reported (75, 208). KLK7 was also found to be
overexpressed in a subset of ovarian carcinomas (107). There are
reports describing connection of KLK8 expression with diseases of the
central nervous system, including epilepsy (209, 210, 211, 212), injury (213, 214), and learning disturbances (215). Another report describes
KLK8 overexpression in a subset of ovarian carcinomas (98). Although
KLK10 has been shown to be a breast cancer tumor suppressor in animal
models (76, 99), there is no report as yet describing prognostic
or diagnostic value of KLK10 in breast carcinomas. Recently, KLK10 was
found to be down-regulated in more aggressive forms of prostate cancer
(216). Preliminary data suggest that KLK12, KLK13, and KLK14 may be
down-regulated in a subset of breast carcinomas (77, 78, 90) while
KLK15 may be overexpressed in more aggressive forms of prostate cancer
(69).
The associations of kallikreins to human diseases are summarized in
Table 8. Clearly, except for hK1, hK2,
and hK3, the literature is quite limited and the value of the new
kallikreins as disease biomarkers is just starting to be examined.
Since most studies thus far used small numbers of clinical samples and
qualitative methodologies, the data should be interpreted with caution.
The knowledge that these kallikreins are secreted proteins supports the
idea that they likely circulate in blood and that their concentration
may be altered in certain human diseases, including cancer. The
experience with hK3 (PSA) and hK2 in prostate cancer may be used to
exploit other cancers, including those of breast, ovarian, lung, etc.
These possibilities deserve further investigation.
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X. Physiological Functions
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Among the 15 new human kallikrein genes, only 3 have been assigned
to a specific biological function (Fig. 6). hK1 exerts its biological activity
mainly through the release of lysyl-bradykinin (kallidin) from low
molecular weight kininogen. However, the diverse expression pattern of
hK1 has led to the suggestion that the functional role of this enzyme
may be specific to different cell types (7, 22). Apart from its
kininogenase activity, tissue kallikrein has been implicated in the
processing of growth factors and peptide hormones (217, 218, 219, 220) in light
of its presence in pituitary, pancreas, and other tissues. As
summarized by Bhoola et al. (7), hK1 has been shown to
cleave pro-insulin, low density lipoprotein, the precursor of atrial
natriuretic factor, prorenin, vasoactive intestinal peptide,
procollagenase, and angiotensinogen. Kallikreins, in each cell type,
may possess single or multiple functions, common or unique, but Bhoola
et al. (7) suggest that the release of kinin should still be
considered the primary effect of hK1 (7).
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Figure 6. Biological functions of the three classical
kallikreins. hK1 cleaves low molecular weight kininogen and releases
lysyl-bradykinin which mediates pleiotropic effects. Human glandular
kallikrein 2 activates the pro-form of PSA. Other possible biological
functions and substrates of hK2 are described in the text. hK3 (PSA)
cleaves semenogelins and fibronectin and mediates seminal clot
liquefaction, which increases the motility of spermatozoa. [Adapted
with permission from H. G. Rittenhouse et al.: Crit
Rev Clin Lab Sci 35:275–368, 1998 (43 ). © CRC Press.]
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The physiological function of hK2 protein has been examined only
recently, with the availability of preparations of recombinant origin,
which are essentially free of hK3 (PSA) or other kallikrein
contaminations (221, 222, 223, 224). Three independent groups have reported
activation of the pro-form of PSA by hK2 (Fig. 6) (149, 150, 151) with a
process that is very similar to the autoactivation of hK2 (removal of 7
amino acids) (225). The study of substrate specificities between hK1
and hK2 reveals important differences (106, 226) suggesting that the
two proteins have different natural substrates, a notion that is
supported by the finding of very low kininogenase activity of hK2 in
comparison to hK1 (14, 15). Seminal plasma hK2 was found to be able to
cleave seminogelin I and seminogelin II but at different cleavage sites
and at a lower efficiency than PSA (227). Since the amount of hK2 in
seminal plasma is much lower than PSA (1–5%), the contribution of hK2
in the process of seminal clot liquefaction is expected to be
relatively small (43).
In any biological fluid thus far studied, hK3 (PSA) and hK2 were found
to coexist (146, 147, 148), suggesting a possible functional relationship
along the lines described above. Furthermore, a role of hK2 in
regulating growth factors, through IGFBP-3 proteolysis, has been
suggested (228).
Recently, hK2 was found to activate the zymogen or single-chain
form of urokinase-type plasminogen activator (uPA) in vitro
(229). Since uPA has been implicated in the promotion of cancer
metastasis, hK2 may be part of this pathway in prostate cancer.
While both hK1 and hK2 have trypsin-like enzymatic activities, hK3 has
chymotrypsin-like substrate specificity (230, 231, 232, 233). Since PSA is
present at very high levels in seminal plasma, most studies focused on
its biological activity within this fluid. Lilja (234) has shown that
PSA hydrolyzes rapidly both seminogelin I and seminogelin II, as well
as fibronectin, resulting in liquefaction of the seminal plasma clot
after ejaculation (234) (Fig. 6). Several other potential substrates
for PSA have been identified, including IGFBP-3 (197, 199), TGFß
(200), basement membrane (201), PTH-related peptide (192,
193), and plasminogen (191). The physiological relevance of these
findings is still not clear.
hK3 is now known to be found at relatively high levels in nipple
aspirate fluid (187, 235), breast cyst fluid (236, 237, 238, 239, 240), milk of
lactating women (241), amniotic fluid (242), and tumor extracts
(184, 185, 186). It is thus very likely that hK3 has biological
extraprostatic functions in breast and other tissues and may also play
a role during fetal development (243). These possibilities merit
further investigation.
Among all other human kallikreins, some have been connected to
physiological processes and pathological conditions (as described in
Section IX) but none has been assigned to cleave a
specific substrate. Human kallikrein enzymes, with the exception of
hK1, hK2, and hK3, are not commercially available and the study of
their biological function has not as yet been published. Below, we will
attempt to formulate some functional hypotheses for the human
kallikreins.
First, all kallikreins are predicted to be secreted proteases, and it
is very likely that their biological function is related to their
ability to digest one or more substrates. The diversity of expression
in human tissues further suggests that they may act on different
substrates in different tissues. Their enzymatic activity may initiate,
by activation, or terminate, by inactivation, events mediated by other
molecules, including hormones, growth factors, receptors, and
cytokines. The parallel expression of many kallikreins in the same
tissues further suggests that they may participate in cascade reactions
similar to those established for the processes of digestion,
fibrinolysis, coagulation, and apoptosis. The role of these enzymes in
tumor metastasis, as suggested for other proteases (244, 245), should
be further investigated.
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XI. Future Directions
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In Table 9, we summarize some areas
that may be fruitful for future kallikrein research. We have already
indicated that it will be important to identify the physiological
substrates of these enzymes in different tissues and the metabolic
pathways in which they participate. The mode of hormonal regulation has
been extensively studied only for KLK3 and KLK2. It will be important
to functionally characterize gene promoters in view of the preliminary
knowledge that the expression of most of these proteases in breast and
prostate cancer cell lines is affected by steroid hormones. In
addition, the details of activation and deactivation of these enzymes
are still obscure. For some of these genes, we already have some
information regarding differential expression between normal and
diseased tissues. More data are needed. The possible mutational
spectrum of these genes in cancer has not been examined.
The most successful clinical application of hK3 (PSA) is currently in
the diagnosis and monitoring of prostate cancer. It is anticipated that
all these serine proteases circulate in the peripheral blood since they
are secreted proteins. It will be important to develop the tools
necessary to allow specific and highly sensitive detection of these
proteins in biological fluids. Once these tools are available, we
should examine whether any of these enzymes have value for diagnosis,
monitoring, prediction of therapeutic response, and population
screening for diseases such as prostate, breast, ovarian, and other
cancers. Applicability to nonmalignant diseases, e.g.,
Alzheimer’s disease, skin pathologies, and inflammatory, autoimmune,
and other chronic diseases of many organs in which kallikreins are
expressed, should also be examined. Some of these enzymes may be useful
targets for tumor localization with specific binding reagents or for
therapeutic interventions. If any of these enzymes are shown to
participate in cancer metastasis, it may be useful to examine
proteinase inhibitors for therapeutic applications. Other possibilities
include the use of some of these genes and their promoters for
tissue-specific delivery of gene therapy or for over- or
underexpression, using exogenously administered modulators
(e.g., hormones or hormone blockers) that are known to
affect their expression.
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XII. Conclusions
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In this review, we attempted to summarize the very latest progress
in research related to the human kallikrein gene family. For many
years, this family was thought to consist of only three genes. We have
Top
Abstract
I. Introduction
