Human tissue kallikreins: A road under construction

doi:10.1016/j.cca.2007.02.023

Clinica Chimica Acta

Volume 381, Issue 1, 2 May 2007, Pages 78-84

https://doi.org/10.1016/j.cca.2007.02.023 Get rights and content

Abstract

Background

The human tissue kallikrein gene family, located at chromosome 19q13.4, is the largest contiguous family of proteases in the human genome. The locus encodes all 15 members of the family, 13 of which have been reported as potential biomarkers for several carcinomas and other non-neoplastic diseases. Kallikreins are expressed by a wide range of tissues and implicated in a number of physiological functions, including skin desquamation, semen liquefaction, neural plasticity and the regulation of blood pressure. Kallikrein function is regulated at various levels, including transcription, translation and post-translation. The proteolytic activity of kallikreins is believed to be cascade mediated and may cross-talk with other proteases. These cascades are highly regulated through a series of feedback loops, inhibitors, (auto) degradation and internal cleavage. Uncontrolled proteolytic activity of kallikreins is implicated in a large number of neoplastic and non-neoplastic pathological conditions.

Conclusions

As our understanding of their regulatory and functional mechanisms continues to expand, kallikreins are expected to become novel targets for the design of new therapeutics.

Introduction

Human tissue kallikreins belong to a subgroup of secreted serine proteases within the S1 family of the clan SA [1]. Until recently, the major interest was given to the three members of the family known as the “classic kallikreins” (KLK1, 2 and 3). In the past decade however, work from our lab and others led to remarkable breakthroughs in the characterization of 12 novel members of the family. According to the new nomenclature system recommended by the Kallikrein subcommittee of HGNC (HUGO Nomenclature Committee), kallikreins 1–15 are denoted as KLKs [2]. To distinguish between proteins and genes, kallikrein proteins are written in standard font, e.g., KLK2, while genes are in italics, e.g., KLK2.

Section snippets

Gene structure

Human tissue kallikreins are encoded by the largest contiguous cluster of protease genes in the human genome [3]. So far, fifteen kallikrein genes have been identified as a cluster of approximately 300 kbp on human chromosome 19q13.4 [3], [4]. Kallikrein genes range between 4.4 and 10.5 kbp and share common features, including exon/intron organization, conserved intronic intervals and exon length [5], [6]. In addition, with the exception of the classic KLKs, kallikrein genes contain both 5′ and

Expression profile

The expression patterns of tissue kallikreins have extensively been studied using various techniques, including northern blot, reverse transcription-PCR (RT-PCR), EST and SAGE analyses, ELISA and immunohistochemical studies. Kallikreins are expressed in varying amounts in different cell types and tissues, both at the mRNA and protein levels [3]. Interestingly, many kallikreins display a striking overlapping pattern of expression. Furthermore, they confer a coordinated pattern of up- or

Transcriptional regulation

Kallikrein gene expression is believed to be regulated through hormonal, as well as epigenetic factors such as methylation and histone modification. A large body of evidence indicates that almost all KLKs are regulated by steroid hormones [2]. Promoter studies of several kallikrein genes have revealed hormonal response elements (HREs) believed to be involved in the cis-regulation of transcription [4]. Given the co-expression pattern of kallikreins, a possible regulatory mechanism through a

Substrate specificity

As a subgroup of serine proteases, kallikreins hydrolyze their target substrates through a nucleophilic attack directed by their active serine residues [24]. Analogous to other serine proteases, substrate preference can be predicted based on the amino acid side chain surrounding the active site of the enzyme [5]. The majority of kallikreins contain aspartic or glutamic acid at their S1 position, suggesting their trypsin-like substrate specificity. Kallikreins 3, 7 and 9, with serine, asparagine

Signaling pathways

Kallikreins are believed to function partly through cross-talks with various signaling pathways. Signaling through kinins is by far the most studied signaling pathway in kallikreins. KLKs 1, 2 and more recently KLK12 were shown to release active kinins (bradykinin and kallidin) from the kininogens [37], [38]. Kinin peptides, in turn, mediate signaling through a number of downstream targets such as prostacyclin-cAMP, nitric oxide cGMP and mitogen-activated protein (MAP) kinases [37]. The

Physiological function

Kallikreins have been implicated in various physiological processes ranging from cellular homeostasis to tissue remodeling. As discussed previously, KLKs 2, 3, 5 and 11 are involved in a proteolytic cascade in seminal plasma. These kallikreins are critical in semen liquefaction through a cascade-mediated processing of semenogelins I and II [1]. In addition, KLK5 has been implicated in the proteolytic cascade in skin, mediating desquamation and possibly certain antimicrobial effects through

Pathobiology

Uncontrolled proteolyses due to over-expression or over-activation of kallikreins have been implicated in a number of pathological conditions in cancer as well as non-cancer disease states.

Biomarkers

KLK3/PSA has received by far the most attention as a valuable tumor marker for screening, diagnosis and monitoring of prostate cancer. However, with the recent development of sensitive immunoassays, there is strong evidence that other kallikreins may serve as alternative or complementary diagnostic and/or prognostic biomarkers.

Studies from our lab and other groups have shown the potential of many kallikreins as biomarkers in a large number of carcinomas, as well as in several non-malignant

Conclusion

Even though the value of kallikreins as biomarkers is well established, the functional significance of this family of enzymes has just begun to unravel. Analogous to the majority of proteases, kallikreins are believed to function through highly regulated proteolytic cascades. Dysregulated proteolyses of kallikreins, due to altered expression or uncontrolled activation, have been implicated in various steps of neoplastic development in several carcinomas, as well as many other non-cancer

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Polymorphisms in human KLK1 and 3 gene promoters were found in LN thus KLKs were defined as lupus-susceptibility genes [9]. miRNAs could be potential regulators for post-transcriptional KLKs expression [10] because of the coincidence of dysregulated expression of many KLKs when different miRNAs are dysregulated along with their co-expression in the same tissues [11,12]. Also, multiple miRNA binding sites were identified in the 3′UTR region of some KLK genes [13].
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90 systemic lupus erythematosus (SLE) patients (44 with active non-renal disease and 46 with LN) were examined for relative expression levels of circulating miR-146a, assessed by reverse transcription quantitative real-time PCR (RT-qPCR) and serum KLK1 levels, measured by ELISA. SLE disease activity was assessed using SLEDAI-2 k. Receiver operating characteristic curve was employed to examine the diagnostic performance of both biomarkers and established conventional laboratory parameters of renal function with specific renal histologic features seen in LN. As well, 40 apparently healthy control subjects were enrolled.
Circulating miR-146a levels were significantly lower (p < 0.01), while serum KLK1 levels were significantly higher (p < 0.01) in active LN patients compared to active non-renal and controls. miR-146a levels negatively correlated with serum creatinine, Albumin/Creatinine and SLEDAI-2 k (p ≤ 0.01), while serum KLK1 significantly correlated with serum creatinine, Albumin/Creatinine and C3 (p ≤ 0.05) and inversely with miR-146a (p = 0.002). Both circulating miR-146a and KLK1 revealed an outstanding performance in predicting renal activity among SLE patients with an AUC of 0.949 and 0.947 respectively. In active LN patients, KLK1 and miR-146a exhibited good ability in differentiating proliferative from non-proliferative LN (AUC 0.73, 0.65 respectively), and in predicting biopsy activity index ≥7, surpassing conventional disease measures.
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It has a molecular weight of 1.8 kDa with a conserved disulfide loop between Cys6 and Cys16. On the other hand, the kallikrein-related peptidase (KLK) family has different members and they can be considered as prognostic and diagnostic factors in various cancers [207–210]. It has been found that PE-BBI exerts its anti-tumor activity by binding to KLK and suppressing its expression [211].
Based on the high incidence and mortality rates of cancer, its therapy remains one of the most vital challenges in the field of medicine. Consequently, enhancing the efficacy of currently applied treatments and finding novel strategies are of great importance for cancer treatment. Venoms are important sources of a variety of bioactive compounds including salts, small molecules, macromolecules, proteins, and peptides that are defined as toxins. They can exhibit different pharmacological effects, and in recent years, their anti-tumor activities have gained significant attention. Several different compounds are responsible for the anti-tumor activity of venoms, and peptides are one of them. In the present review, we discuss the possible anti-tumor activities of venom peptides by highlighting molecular pathways and mechanisms through which these molecules can act effectively. Venom peptides can induce cell death in cancer cells and can substantially enhance the efficacy of chemotherapy and radiotherapy. Also, the venom peptides can mitigate the migration of cancer cells via suppression of angiogenesis and epithelial-to-mesenchymal transition. Notably, nanoparticles have been applied in enhancing the bioavailability of venom peptides and providing targeted delivery, thereby leading to their elevated anti-tumor activity and potential application for cancer therapy.
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Metastatic colon cancer is a major cause of deaths among colorectal cancer (CRC) patients. Elevated expression of kallikrein 6 (KLK6), a member of a kallikrein subfamily of peptidase S1 family serine proteases, has been reported in CRC and is associated with low patient survival rates and poor disease prognosis. We knocked down KLK6 expression in HCT116 colon cancer cells to determine the significance of KLK6 expression for metastatic dissemination and to identify the KLK6-associated microRNAs (miRNAs) signaling networks in metastatic colon cancer. KLK6 suppression resulted in decreased cells invasion in vitro with a minimal effect on the cell growth and viability. In vivo, animals with orthotopic colon tumors deficient in KLK6 expression had the statistically significant increase in survival rates (P = .005) and decrease in incidence of distant metastases. We further performed the integrated miRNA and messenger RNA (mRNA) expression profiling to identify functional miRNA-mRNA interactions associated with KLK6-mediated invasiveness of colon cancer.
Through bioinformatics analysis we identified and functionally validated the top two up-regulated miRNAs, miR-182 and miR-203, and one down-regulated miRNA, miRNA-181d, and their seven mRNA effectors. The established miRNA-mRNA interactions modulate cellular proliferation, differentiation and epithelial–mesenchymal transition (EMT) in KLK6-expressing colon cancer cells via the TGF-β signaling pathway and RAS-related GTP-binding proteins. We confirmed the potential tumor suppressive properties of miR-181d and miR-203 in KLK6-expressing HCT116 cells using Matrigel invasion assay. Our data provide experimental evidence that KLK6 controls metastasis formation in colon cancer via specific downstream network of miRNA-mRNA effectors.
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Several putative roles have been proposed for these enzymes in skin pathophysiology, among which skin desquamation remains the most established [32]. It is now clear that a group of skin KLKs is co-expressed in human skin, where they participate in enzymatic cascades, through which each KLK can downstream activate (via a forward feedback mechanism) more members of the KLK family, as seen with the blood coagulation cascade [33,34]. The established roles of these skin enzymes, include skin desquamation, regulation of innate immunity and maintenance of skin barrier function [19].
Sweat is produced by eccrine and apocrine glands and represents a biological fluid with established roles in thermo-regulation and host infection defense. The composition of sweat is highly dynamic and alters significantly in various skin and other disorders. Therefore, in-depth profiling of sweat protein composition is expected to augment our understanding of the pathobiology of several skin diseases and may lead to the identification of useful sweat-based disease biomarkers. We here reported an in-depth analysis of the human sweat proteome and peptidome. Sweat was collected from healthy males and healthy females of ages 20–60 years, following strenuous exercise. Two sweat pools were prepared (1 for males and 1 for females) and were subjected to sample preparation for mass spectrometric analysis. We identified a total of 861 unique proteins during our proteomic analysis and 32,818 endogenous peptides (corresponding to additional 1067 proteins) from our peptidomics workflow. As expected, the human skin was identified as the most abundant source of sweat proteins and peptides. Several skin proteases and protease inhibitors were identified in human sweat, highlighting the intense proteolytic activity of human skin. The presence of several antimicrobial peptides supports the functional roles of sweat in host defense and innate immunity.
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Invited critical reviewHuman tissue kallikreins: A road under construction

Abstract

Background

Conclusions

Introduction

Section snippets

Gene structure

Expression profile

Transcriptional regulation

Substrate specificity

Signaling pathways

Physiological function

Pathobiology

Biomarkers

Conclusion

Cancer Lett

Biochim Biophys Acta

Biochem Biophys Res Commun

J Mol Biol

J Biol Chem

J Biol Chem

J Biol Chem

J Invest Dermatol

J Invest Dermatol

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Neurosci Lett

Int J Biochem Cell Biol

J Invest Dermatol

Clin Chim Acta

Clin Biochem

J Biol Chem

FEBS Lett

Human tissue kallikreins: physiologic roles and applications in cancer

Mol Cancer Res

A comprehensive nomenclature for serine proteases with homology to tissue kallikreins

Biol Chem

The new human tissue kallikrein gene family: structure, function, and association to disease

Endocr Rev

The emerging roles of human tissue kallikreins in cancer

Nat Rev Cancer

Evolution of kallikrein-related peptidases in mammals and identification of a genetic locus encoding potential regulatory inhibitors

Biol Chem

Intron retention: a common splicing event within the human kallikrein gene family

Clin Chem

Differential splicing of KLK5 and KLK7 in epithelial ovarian cancer produces novel variants with potential as cancer biomarkers

Clin Cancer Res

Multiple promoters regulate tissue-specific alternative splicing of the human kallikrein gene, KLK11/hippostasin

FEBS J

Molecular cloning and expression of an alternative hKLK3 transcript coding for a variant protein of prostate-specific antigen

Cancer Res

A novel form of prostate-specific antigen transcript produced by alternative splicing

Cancer Res

Compartmentalized expression of kallikrein 4 (KLK4/hK4) isoforms in prostate cancer: nuclear, cytoplasmic and secreted forms

Endocr Relat Cancer

Kallikrein-related peptidase (KLK) family mRNA variants and protein isoforms in hormone-related cancers: do they have a function?

Biol Chem

Invited critical review
Human tissue kallikreins: A road under construction