Human Tissue Kallikrein Gene Family Research Articles

Inhibition of Transcription Factor Specificity Protein 1 Alters the Gene Expression Profile of Keratinocytes Leading to Upregulation of Kallikrein-Related Peptidases and Thymic Stromal Lymphopoietin

Transcription factor specificity protein 1 (Sp1) is involved in diverse cellular functions. We recently found that Sp1 was significantly decreased in skin biopsies from patients with atopic dermatitis (AD) and had an even greater reduction in AD patients with a history of eczema herpeticum. In the current study, we sought to better understand the role of Sp1 in skin biological processes by using a small interfering RNA (siRNA) technique to knock down Sp1 gene expression in normal human keratinocytes (NHK) and investigated the genome-wide gene expression profiling of Sp1- silenced NHK. The gene arrays revealed that 53 genes had more than three-fold changes in expression in Sp1-silenced NHK as compared to scrambled siRNA silenced cells. Strikingly, six kallikrein-related peptidase genes, KLK5, KLK6, KLK7, KLK8, KLK10, and KLK12 were up-regulated in NHK following Sp1 silencing. Functionally, protease activity was significantly enhanced in Sp1-silenced keratinocytes as compared to scrambled siRNA silenced keratinocytes. Moreover, thymic stromal lymphopoietin (TSLP), an epithelial derived TH2 promoting cytokine, was induced in Sp1-silenced keratinocytes due to elevated kallikrein activity. These results indicate that Sp1 expression deficiency leads to abnormally increased kallikrein protease activity in keratinocytes and may contribute to TH2 immune responses in the skin by inducing TSLP.

Differential N-glycosylation of Kallikrein 6 Derived from Ovarian Cancer Cells or the Central Nervous System

Ovarian cancer causes more deaths than any other gynecological disorder. Perturbed glycosylation is one of the hallmarks of this malignancy. Kallikrein 6 (KLK6) elevation in serum is a diagnostic and prognostic indicator in ovarian cancer. The majority of ovarian carcinomas express high levels of KLK6, which diffuses into the circulation. Under physiological conditions, KLK6 is expressed highly in the central nervous system and found at high levels in cerebrospinal fluid from where it enters the circulation. Our aim was to characterize and compare the N-glycosylation status of this protein in ovarian cancer ascites fluid and cerebrospinal fluid. Anion-exchange chromatography was used to reveal different post-translational modifications on the two isoforms. Mobility gel shift Western blot analysis coupled with glycosidase digestion showed that the molecular weight difference between the two isoforms was because of differential glycosylation patterns. The presence of a single N-glycosylation site on KLK6 was confirmed by site-directed mutagenesis. Using a Sambucus nigra agglutinin-monoclonal antibody sandwich enzyme-linked immunosorbent assay approach, it was shown that ovarian cancer-derived KLK6 was modified with alpha2-6-linked sialic acid. The structure and composition of glycans of both KLK6 isoforms was elucidated by glycopeptide monitoring with electrospray ionization-Orbitrap tandem mass spectrometry. Therefore, the extensive and almost exclusive sialylation of KLK6 from ovarian cancer cells could lead to the development of an improved biomarker for the early diagnosis of ovarian carcinoma.

MicroRNAs: a new frontier in kallikrein research

microRNAs (miRNAs) are a recently discovered class of small non-coding RNAs that regulate gene expression. Rapidly accumulating evidence has revealed that miRNAs are associated with cancer. The human tissue kallikrein gene family is the largest contiguous family of proteases in the human genome, containing 15 genes. Many kallikreins have been reported as potential tumor markers. In this review, recent bioinformatics and experimental evidence is presented indicating that kallikreins are potential miRNA targets. The available experimental approaches to investigate these interactions and the potential diagnostic and therapeutic applications are also discussed. miRNAs represent a possible regulatory mechanism for controlling kallikrein expression at the post-transcriptional level. Many miRNAs were predicted to target kallikreins and a single miRNA can target more than one kallikrein. Recent evidence suggests that miRNAs can also exert 'quantitative' control of kallikreins by utilizing multiple targeting sites in the kallikrein mRNA. More research is needed to experimentally verify the in silico predictions and to investigate the possible role in tumor initiation and/or progression.

Human Kallikrein 8 Protease Confers a Favorable Clinical Outcome in Non–Small Cell Lung Cancer by Suppressing Tumor Cell Invasiveness

The human kallikrein 8 (KLK8) gene, a member of the human tissue kallikrein gene family, encodes a serine protease. The KLK8 protein (hK8) is known to be a favorable prognostic marker in ovarian cancer, but the biological basis of this is not understood. We found that overexpressing the KLK8 gene in highly invasive lung cancer cell lines suppresses their invasiveness. This role in invasiveness was further confirmed by the fact that inhibition of endogenous KLK8 expression with a specific short hairpin RNA reduced cancer cell invasiveness. In situ degradation and cell adhesion assays showed that proteins produced from KLK8 splice variants modify the extracellular microenvironment by cleaving fibronectin. DNA microarray experiments and staining of cells for actin filaments revealed that the degradation of fibronectin by hK8 suppresses integrin signaling and retards cancer cell motility by inhibiting actin polymerization. In addition, studies in a mouse model coupled with the detection of circulating tumor cells by quantitative PCR for the human Alu sequence showed that KLK8 suppresses tumor growth and invasion in vivo. Finally, studies of clinical specimens from patients with non-small cell lung cancer showed that the time to postoperative recurrence was longer for early-stage patients (stages I and II) with high KLK8 expression (mean, 49.9 months) than for patients with low KLK8 expression (mean, 22.9 months). Collectively, these findings show that KLK8 expression confers a favorable clinical outcome in non-small cell lung cancer by suppressing tumor cell invasiveness.

Detection and Quantification of Protein Biomarkers from Fewer than 10 Cells

The use of antibody microarrays continues to grow rapidly due to the recent advances in proteomics and automation and the opportunity this combination creates for high throughput multiplexed analysis of protein biomarkers. However, a primary limitation of this technology is the lack of PCR-like amplification methods for proteins. Therefore, to realize the full potential of array-based protein biomarker screening it is necessary to construct assays that can detect and quantify protein biomarkers with very high sensitivity, in the femtomolar range, and from limited sample quantities. We describe here the construction of ultramicroarrays, combining the advantages of microarraying including multiplexing capabilities, higher throughput, and cost savings with the ability to screen very small sample volumes. Antibody ultramicroarrays for the detection of interleukin-6 and prostate-specific antigen (PSA), a widely used biomarker for prostate cancer screening, were constructed. These ultramicroarrays were found to have a high specificity and sensitivity with detection levels using purified proteins in the attomole range. Using these ultramicroarrays, we were able to detect PSA secreted from 100 LNCaP cells in 3 h and from just four LNCaP cells in 24 h. Cellular PSA could also be detected from the lysate of an average of just six cells. This strategy should enable proteomic analysis of materials that are available in very limited quantities such as those collected by laser capture microdissection, neonatal biopsy microspecimens, and forensic samples.

Human tissue kallikrein gene family: applications in cancer

Human tissue kallikrein genes, located on the long arm of chromosome 19, are a subgroup of the serine protease family of proteolytic enzymes. Initially thought to consist of three members, the human kallikrein locus has now been extended and includes 15 tandemly located genes. These genes, and their protein products, share a high degree of homology and are expressed in a wide array of tissues, mainly those that are under steroid hormone control. PSA (hK3) is one of the human kallikreins, and is the most useful tumor marker for prostate cancer screening, diagnosis, prognosis and monitoring. hK2, another prostate-specific kallikrein, has also been proposed as a complementary prostate cancer biomarker. In the past 5 years, the newly discovered kallikreins (KLK4–KLK15) have been associated with several types of cancer. For example, hK4, hK5, hK6, hK7, hK8, hK10, hK11, hK13 and hK14 are emerging biomarkers for ovarian, breast, prostate and testicular cancer. New evidence raises the possibility that some kallikreins are directly involved with cancer progression. We here review the evidence linking kallikreins and cancer and their applicability as novel biomarkers for cancer diagnosis and management.

Steroid hormone regulation of the human kallikrein 10 (KLK10) gene in cancer cell lines and functional characterization of the KLK10 gene promoter

Background: The human kallikrein 10 (KLK10) gene is a new member of the human tissue kallikrein gene family. It encodes for a secreted serine protease (hK10) with predicted trypsin-like enzymatic activity. KLK10 is highly expressed in the sex organs and its expression level changes in malignancy. Methods: To determine the role of steroid hormones in KLK10 gene expression, we investigated its modulation by 17β-estradiol, 5α-dihydrotestosterone, norgestrel, dexamethasone and aldosterone, at both the transcription and translation level, in a panel of cancer cell lines. After steroid hormone stimulation, the change of KLK10 mRNA was monitored with reverse transcriptase polymerase chain reaction and hK10 protein levels in the culture supernatant were quantified with an hK10-specific immunoassay. The presence of hormone response elements in the KLK10 gene promoter was examined with the chloramphenicol acetyltransferase reporter gene system. Results: The KLK10 expression was mainly up-regulated by estrogens, androgens and progestins, and to a lesser extent by dexamethasone and aldosterone in the breast cancer cell lines BT-474, MCF-7 and T-47D, both at the mRNA and protein levels. The effect of stimulation of these steroids on KLK10 expression varied among the cell lines. Estrogens, androgens and progestins were most potent in the BT-474, T-47D and MCF-7 cells, respectively. The up-regulation effect of estrogens, androgens, and progestins on KLK10 expression can be blocked by their antagonists ICI-182, 780, RU-56,187, and mifepristone, respectively. Time course studies showed that hK10 protein started to increase 1 day after steroid hormone stimulation and this increase persisted for 7 days. These data suggest that steroid hormones up-regulate KLK10 gene expression through direct interaction between hormone-receptor complexes and their cognate hormone response elements. To search for hormone response elements, we functionally characterized the KLK10 promoter by placing it upstream of the chloramphenicol acetyltransferase reporter gene. We found that KLK10 promoter activity did not rely on the presence of functional estrogen and androgen receptors. Also, the presence of functional estrogen and androgen receptors did not increase its constitutive activity. We suggest that the hormone response elements that mediate the transcriptional regulation of KLK10 are unlikely to locate in the KLK10 promoter. Conclusions: Estrogens, androgens and progestins modulate KLK10 expression through their own receptors but this regulation is not mediated by steroid hormone response elements in the promoter of the KLK10 gene.

Tissue kallikreins: new players in normal and abnormal cell growth?

Serine proteases are proteolytic enzymes with an active serine residue in their catalytic site. Kallikreins are a subgroup of the serine protease family and are known to have diverse physiological functions. The human tissue kallikrein gene family has now been fully characterized and includes 15 members, clustered in a 300 kb region on chromosome 19q13.4. In this review, we discuss the common structural features of kallikreins at the DNA, mRNA and protein levels. Kallikreins are secreted as inactive zymogens and are activated by cleavage of an N-terminal peptide. Some kallikreins can undergo autoactivation while others may be activated by other kallikreins or other proteases. Most kallikreins are predicted to have trypsin-like enzymatic activity except for three members which may have chymotrypsin-like activity. Circumstantial evidence suggests that at least some kallikreins may be part of an enzymatic cascade pathway which is activated in aggressive forms of ovarian and probably other cancers. Accumulating evidence suggests potential diagnostic and/or prognostic roles of kallikreins in diverse malignancies. In addition to PSA, many other kallikreins show differential expression in malignancy. For example, hK6, 10 and 11 are promising serological markers for ovarian cancer diagnosis. KLK10 may act as a tumor suppressor. In addition to their diagnostic and prognostic values, kallikreins may also be good therapeutic targets.

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

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.

The New Human Tissue Kallikrein Gene Family: Structure, Function, and Association to Disease

The New Human Tissue Kallikrein Gene Family: Structure, Function, and Association to Disease

New Nomenclature for the Human Tissue Kallikrein Gene Family

The human kallikrein gene family is important to the discipline of clinical chemistry because it contains genes that encode for valuable cancer biomarkers, including the best tumor marker available today, prostate-specific antigen (PSA). Despite reports of numerous kallikrein-like genes in the mouse (1), until 2–3 years ago, only three human kallikrein genes were recognized: pancreatic/renal kallikrein (KLK1) , human glandular kallikrein 2 (KLK2) , and prostate-specific antigen (KLK3) (1)(2). The proteins encoded by the three kallikrein genes are now known as hK1, hK2, and hK3 (PSA). These three genes encode for serine proteases with either trypsin-like (hK1, hK2) or chymotrypsin-like (hK3) activity. Traditionally, kallikreins have been defined as enzymes that can act on high-molecular weight substrates and release bioactive peptides, known as kinins (3). Among the known kallikrein enzymes, only plasma kallikrein (encoded by a single gene localized on human chromosome 4q35; official symbol KLKB1 ) and pancreatic/renal kallikrein (hK1) have significant kininogenase activity. The proteins encoded by the KLK2 and the KLK3 genes have minimal or no kininogenase activity (4)(5). Why then are the KLK1 , KLK2 , and KLK3 genes classified together into one gene family (tissue kallikrein gene family), when two of the three enzymes have no significant kallikrein enzymatic activity? This grouping is justified, based on the extensive homologies between the three genes at both the DNA …