Investigating microRNA Profiles in Prostate Cancer Bone Metastases and Functional Effects of microRNA-23c and microRNA-4328.

MicroRNAs (miRNAs) are aberrantly expressed in prostate cancer (PC), but comprehensive knowledge about their levels and function in metastatic PC is lacking. Here, we explored the differential expression of miRNA profiles during PC progression to bone metastasis, and further focused on the downregulation of miRNA-23c and -4328 and their impact on PC growth in experimental models. Using microarray screening, the levels of 1510 miRNAs were compared between bone metastases (n = 14), localized PC (n = 7) and benign prostate tissue (n = 7). Differentially expressed miRNAs (n = 4 increased and n = 75 decreased, p < 0.05) were identified, of which miRNA-1, -23c, -143-3p, -143-5p, -145-3p, -205-5p, -221-3p, -222-3p and -4328 showed consistent downregulation during disease progression (benign > localized PC > bone metastases). The downregulation of miRNA-23c and -4328 was confirmed by reverse transcription and quantitative polymerase chain reaction analysis of 67 metastasis, 12 localized PC and 12 benign prostate tissue samples. The stable overexpression of miRNA-23c and -4328 in the 22Rv1 and PC-3 cell lines resulted in reduced PC cell growth in vitro, and in the secretion of high levels of miRNA-23c (but not -4328) in extracellular vesicles. However, no tumor suppressive effects were observed from miRNA-23c overexpression in PC-3 cells subcutaneously grown in mice. In conclusion, bone metastases display a profound reduction of miRNA levels compared to localized PC and benign disease. The downregulation of those miRNAs, including miRNA-23c and -4328, may lead to a loss of tumor suppressive effects and provide biomarker and therapeutic possibilities that deserve to be further explored.

KLK4T2 Is a Hormonally Regulated Transcript from the KLK4 Locus.

The human kallikrein-related peptidase 4 (KLK4) and the transcribed pseudogene KLKP1 are reported to be highly expressed in the prostate. When trying to clone transcripts of KLKP1, we partly failed. Instead, we identified an androgen-regulated transcript, KLK4T2, which appeared to be a splice variant of KLK4 that also contained exons of KLKP1. Expression analysis of KLK4, KLK4T2, and KLKP1 transcripts in prostate cancer cell lines showed high levels of KLKP1 transcripts in the nucleus and in unfractionated cell extract, whereas it was almost completely absent in the cytoplasmatic fraction. This was in contrast to KLK4 and KLK4T2, which displayed high to moderate levels in the cytoplasm. In patient cohorts we found significantly higher expression of both KLK4T2 and KLK4 in benign prostatic hyperplasia compared to both primary prostate cancer and bone metastasis. Analysis of tissue panels demonstrated the highest expression of KLK4T2 in the prostate, but in contrast to the classical KLK4, relatively high levels were also found in placenta. So far, the function of KLK4T2 is still to be explored, but the structure of the translation product indicated that it generates a 17.4 kDa intracellular protein with possible regulatory function.

A novel DNA methylation signature is associated with androgen receptor activity and patient prognosis in bone metastatic prostate cancer.

BACKGROUND: Patients with metastatic prostate cancer (PC) are treated with androgen deprivation therapy (ADT) that initially reduces metastasis growth, but after some time lethal castration-resistant PC (CRPC) develops. A better understanding of the tumor biology in bone metastases is needed to guide further treatment developments. Subgroups of PC bone metastases based on transcriptome profiling have been previously identified by our research team, and specifically, heterogeneities related to androgen receptor (AR) activity have been described. Epigenetic alterations during PC progression remain elusive and this study aims to explore promoter gene methylation signatures in relation to gene expression and tumor AR activity. MATERIALS AND METHODS: Genome-wide promoter-associated CpG methylation signatures of a total of 94 tumor samples, including paired non-malignant and malignant primary tumor areas originating from radical prostatectomy samples (n = 12), and bone metastasis samples of separate patients with hormone-naive (n = 14), short-term castrated (n = 4) or CRPC (n = 52) disease were analyzed using the Infinium Methylation EPIC arrays, along with gene expression analysis by Illumina Bead Chip arrays (n = 90). AR activity was defined from expression levels of genes associated with canonical AR activity. RESULTS: Integrated epigenome and transcriptome analysis identified pronounced hypermethylation in malignant compared to non-malignant areas of localized prostate tumors. Metastases showed an overall hypomethylation in relation to primary PC, including CpGs in the AR promoter accompanied with induction of AR mRNA levels. We identified a Methylation Classifier for Androgen receptor activity (MCA) signature, which separated metastases into two clusters (MCA positive/negative) related to tumor characteristics and patient prognosis. The MCA positive metastases showed low methylation levels of genes associated with canonical AR signaling and patients had a more favorable prognosis after ADT. In contrast, MCA negative patients had low AR activity associated with hypermethylation of AR-associated genes, and a worse prognosis after ADT. CONCLUSIONS: A promoter methylation signature classifies PC bone metastases into two groups and predicts tumor AR activity and patient prognosis after ADT. The explanation for the methylation diversities observed during PC progression and their biological and clinical relevance need further exploration.

Marked response to cabazitaxel in prostate cancer xenografts expressing androgen receptor variant 7 and reversion of acquired resistance by anti-androgens.

BACKGROUND: Taxane treatment may be a suitable therapeutic option for patients with castration-resistant prostate cancer and high expression of constitutively active androgen receptor variants (AR-Vs). The aim of the study was to compare the effects of cabazitaxel and androgen deprivation treatments in a prostate tumor xenograft model expressing high levels of constitutively active AR-V7. Furthermore, mechanisms behind acquired cabazitaxel resistance were explored. METHODS: Mice were subcutaneously inoculated with 22Rv1 cells and treated with surgical castration (n = 7), abiraterone (n = 9), cabazitaxel (n = 6), castration plus abiraterone (n = 8), castration plus cabazitaxel (n = 11), or vehicle and/or sham operation (n = 23). Tumor growth was followed for about 2 months or to a volume of approximately 1000 mm3 . Two cabazitaxel resistant cell lines; 22Rv1-CabR1 and 22Rv1-CabR2, were established from xenografts relapsing during cabazitaxel treatment. Differential gene expression between the cabazitaxel resistant and control 22Rv1 cells was examined by whole-genome expression array analysis followed by immunoblotting, immunohistochemistry, and functional pathway analysis. RESULTS: Abiraterone treatment alone or in combination with surgical castration had no major effect on 22Rv1 tumor growth, while cabazitaxel significantly delayed and in some cases totally abolished 22Rv1 tumor growth on its own and in combination with surgical castration. The cabazitaxel resistant cell lines; 22Rv1-CabR1 and 22Rv1-CabR2, both showed upregulation of the ATP-binding cassette sub-family B member 1 (ABCB1) efflux pump. Treatment with ABCB1 inhibitor elacridar completely restored susceptibility to cabazitaxel, while treatment with AR-antagonists bicalutamide and enzalutamide partly restored susceptibility to cabazitaxel in both cell lines. The cholesterol biosynthesis pathway was induced in the 22Rv1-CabR2 cell line, which was confirmed by reduced sensitivity to simvastatin treatment. CONCLUSIONS: Cabazitaxel efficiently inhibits prostate cancer growth despite the high expression of constitutively active AR-V7. Acquired cabazitaxel resistance involving overexpression of efflux transporter ABCB1 can be reverted by bicalutamide or enzalutamide treatment, indicating the great clinical potential for combined treatment with cabazitaxel and anti-androgens.

A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa.

Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite divergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified individual residue contributions to the inhibitor's stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular diversity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain.

Potent, multi-target serine protease inhibition achieved by a simplified ß-sheet motif.

Engagement of an extended ß-sheet is a common substrate/inhibitor interaction at the active site of serine proteases and is an important feature of Laskowski mechanism inhibitors that present a substrate-like loop to a target protease. This loop is cleaved but subsequently relegated forming a stable inhibitor/protease complex. Laskowski inhibitors are ubiquitous in nature and are used extensively in serine protease inhibitor design. However, most studies concentrate on introducing new sidechain interactions rather than the direct contributions of the substrate-like ß-sheet to enzyme inhibition. Here we report the crystal structure of an simplified ß-sheet inhibitory motif within the Sunflower Trypsin Inhibitor (SFTI) in complex with trypsin. We show that the intramolecular hydrogen bond network of this SFTI variant (SFTI-TCTR) engages the inhibitor sidechains that would normally interact with a target protease, giving mainchain interactions a more prominent role in complex formation. Despite having reduced sidechain interactions, this SFTI variant is remarkably potent and inhibits a diverse range of serine proteases. Crystal structural analysis and molecular modelling of SFTI-TCTR complexes again indicates an interface dominated by ß-sheet interactions, highlighting the importance of this motif and the adaptability of SFTI as a scaffold for inhibitor design.

Kallikrein-related peptidase 7 overexpression in melanoma cells modulates cell adhesion leading to a malignant phenotype.

We recently reported that human melanoma cells, but not benign melanocytes, aberrantly express kallikrein-related peptidase 7 (KLK7). Here, we show a KLK7 overexpression-mediated decrease of cell adhesion to extracellular matrix binding proteins, associated with downregulation of α5/ß1/αv/ß3 integrin expression. We also report an up-regulation of MCAM/CD146 and an increase in spheroid formation of these cells. Our results demonstrate that aberrant KLK7 expression leads to a switch to a more malignant phenotype suggesting a potential role of KLK7 in melanoma invasion. Thus, KLK7 may represent a biomarker for melanoma progression and may be a potential therapeutic target for melanoma.

Aberrant expression of kallikrein-related peptidase 7 is correlated with human melanoma aggressiveness by stimulating cell migration and invasion.

Members of the tissue kallikrein-related peptidase (KLK) family not only regulate several important physiological functions, but aberrant expression has also been associated with various malignancies. Clinically, KLKs have been suggested as promising biomarkers for diagnosis and prognosis in many types of cancer. As of yet, expression of KLKs and their role in skin cancers are, however, poorly addressed. Malignant melanoma is an aggressive disease associated with poor prognosis. Hence, diagnostic biomarkers to monitor melanoma progression are needed. Herein, we demonstrate that although mRNA of several KLKs are aberrantly expressed in melanoma cell lines, only the KLK7 protein is highly secreted in vitro. In line with these findings, ectopic expression of KLK7 in human melanomas and its absence in benign nevi were demonstrated by immunohistochemistry in vivo. Interestingly, overexpression of KLK7 induced a significant reduction in melanoma cell proliferation and colony formation. Moreover, KLK7 overexpression triggered an increase in cell motility and invasion associated with decreased expression of E-cadherin and an upregulation of MCAM/CD146. Our results demonstrate, for the first time, that aberrant KLK7 expression leads to a switch from proliferative to invasive phenotype, suggesting a potential role of KLK7 in melanoma progression. Thus, we hypothesize that KLK7 may represent a potential biomarker for melanoma progression.

High levels of the AR-V7 Splice Variant and Co-Amplification of the Golgi Protein Coding YIPF6 in AR Amplified Prostate Cancer Bone Metastases.

BACKGROUND: The relation between androgen receptor (AR) gene amplification and other mechanisms behind castration-resistant prostate cancer (CRPC), such as expression of constitutively active AR variants and steroid-converting enzymes has been poorly examined. Specific aim was to examine AR amplification in PC bone metastases and to explore molecular and functional consequences of this, with the long-term goal of identifying novel molecular targets for treatment. METHODS: Gene amplification was assessed by fluorescence in situ hybridization in cryo-sections of clinical PC bone metastases (n = 40) and by PCR-based copy number variation analysis. Whole genome mRNA expression was analyzed using H12 Illumina Beadchip arrays and specific transcript levels were quantified by qRT-PCR. Protein localization was analyzed using immunohistochemistry and confocal microscopy. The YIPF6 mRNA expression was transiently knocked down and stably overexpressed in the 22Rv1 cell line as representative for CRPC, and effects on cell proliferation, colony formation, migration, and invasion were determined in vitro. Extracellular vesicles (EVs) were isolated from cell cultures using size-exclusion chromatography and enumerated by nanoparticle tracking analysis. Protein content was identified by LC-MS/MS analysis. Blood coagulation was measured as activated partial thromboplastin time (APTT). Functional enrichment analysis was performed using the MetaCore software. RESULTS: AR amplification was detected in 16 (53%) of the bone metastases examined from CRPC patients (n = 30), and in none from the untreated patients (n = 10). Metastases with AR amplification showed high AR and AR-V7 mRNA levels, increased nuclear AR immunostaining, and co-amplification of genes such as YIPF6 in the AR proximity at Xq12. The YIPF6 protein was localized to the Golgi apparatus. YIPF6 overexpression in 22Rv1 cells resulted in reduced cell proliferation and colony formation, and in enhanced EV secretion. EVs from YIPF6 overproducing 22Rv1 cells were enriched for proteins involved in blood coagulation and, accordingly, decreased the APTT in a dose-dependent fashion. CONCLUSIONS: AR amplified CRPC bone metastases show high AR-V7 expression that probably gives resistance to AR-targeting drugs. Co-amplification of the Golgi protein coding YIPF6 gene with the AR may enhance the secretion of pro-coagulative EVs from cancer cells and thereby stimulate tumor progression and increase the coagulopathy risk in CRPC patients. Prostate 77: 625-638, 2017. © 2017 Wiley Periodicals, Inc.

KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype.

Netherton syndrome (NS) is a severe skin disease caused by the loss of protease inhibitor LEKTI, which leads to the dysregulation of epidermal proteases and severe skin-barrier defects. KLK5 was proposed as a major protease in NS pathology, however its inactivation is not sufficient to rescue the lethal phenotype of LEKTI-deficient mice. In this study, we further elucidated the in vivo roles of the epidermal proteases in NS using a set of mouse models individually or simultaneously deficient for KLK5 and KLK7 on the genetic background of a novel NS-mouse model. We show that although the ablation of KLK5 or KLK7 is not sufficient to rescue the lethal effect of LEKTI-deficiency simultaneous deficiency of both KLKs completely rescues the epidermal barrier and the postnatal lethality allowing mice to reach adulthood with fully functional skin and normal hair growth. We report that not only KLK5 but also KLK7 plays an important role in the inflammation and defective differentiation in NS and KLK7 activity is not solely dependent on activation by KLK5. Altogether, these findings show that unregulated activities of KLK5 and KLK7 are responsible for NS development and both proteases should become targets for NS therapy.

Selective Substrates and Inhibitors for Kallikrein-Related Peptidase 7 (KLK7) Shed Light on KLK Proteolytic Activity in the Stratum Corneum.

Proteases have pivotal roles in the skin's outermost layer, the epidermis. In the stratum corneum, serine proteases from the kallikrein-related peptidase (KLK) family have been implicated in several key homeostatic processes, including desquamation. However, the precise contribution of specific KLKs to each process remains unclear. To address this, we used a chemical biology approach and designed selective substrates and inhibitors for KLK7, the most abundant KLK protease in the stratum corneum. The resulting KLK7 inhibitor is the most potent inhibitor of this protease reported to date (Ki = 140 pM), and displays at least 1,000-fold selectivity over several proteases that are related by function (KLK5 and KLK14) or specificity (chymotrypsin). We then used substrates and inhibitors for KLK5, KLK7, and KLK14 to explore the activity of each protease in the stratum corneum using casein zymography and an ex vivo desquamation assay. These experiments provide the most detailed assessment of each KLK's contribution to corneocyte shedding in the plantar stratum corneum, revealing that inhibition of KLK7 alone is sufficient to block shedding, whereas KLK5 is also a major contributor. Collectively, these findings unveil chemical tools for studying KLK activity and demonstrate their potential for characterizing KLK biological functions in epidermal homeostasis.

Exploring the active site binding specificity of kallikrein-related peptidase 5 (KLK5) guides the design of new peptide substrates and inhibitors.

Kallikrein-related peptidase 5 (KLK5) is a promising therapeutic target in several skin diseases, including Netherton syndrome, and is emerging as a potential target in various cancers. In this study, we used a sparse matrix library of 125 individually synthesized peptide substrates to characterize the binding specificity of KLK5. The sequences most favored by KLK5 were GRSR, YRSR and GRNR, and we identified sequence-specific interactions involving the peptide N-terminus by analyzing kinetic constants (kcat and KM) and performing molecular dynamics simulations. KLK5 inhibitors were subsequently engineered by substituting substrate sequences into the binding loop (P1, P2 and P4 residues) of sunflower trypsin inhibitor-1 (SFTI-1). These inhibitors were effective against KLK5 but showed limited selectivity, and performing a further substitution at P2' led to the design of a new variant that displayed improved activity against KLK5 (Ki=4.2±0.2 nm), weak activity against KLK7 and 12-fold selectivity over KLK14. Collectively, these findings provide new insight into the design of highly favored binding sequences for KLK5 and reveal several opportunities for modulating inhibitor selectivity over closely related proteases that will be useful for future studies aiming to develop therapeutic molecules targeting KLK5.

Engineered protease inhibitors based on sunflower trypsin inhibitor-1 (SFTI-1) provide insights into the role of sequence and conformation in Laskowski mechanism inhibition.

Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesizing a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor's resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor's potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor's sequence and structure relate to its binding kinetics and function. Using enzyme assays, MD simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show that the geometry of the binding loop mainly influences the inhibitor's potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor's sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and the dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin and two kallikrein-related peptidases (KLK5 and KLK14) that are implicated in various cancers and skin diseases.

Characterization of SPINK9, a KLK5-specific inhibitor expressed in palmo-plantar epidermis.

SPINK9, a Kazal-type serine protease inhibitor, is almost exclusively expressed in the palmo-plantar epidermis. SPINK9 selectively inhibits kallikrein-related peptidase 5 (KLK5), no other target enzyme is known at present. In this study, we defined the reactive loop to residues 48 and 49 of SPINK9 and characterized the inhibition and binding of different SPINK9 variants towards KLK5, KLK7, KLK8 and KLK14. Substitutions of single amino acids in the reactive loop had a large impact on both inhibitory efficiency and specificity. Binding studies showed that it is mainly the dissociation rate that is affected by the amino acid substitutions. The inhibitory effect of wild-type SPINK9 was clearly pH-dependent with an improved effect at a pH similar to that of the outer layers of the skin. Modeling of the enzyme-inhibitor complexes showed that the reactive loop of SPINK9 fits very well into the deep negatively charged binding pocket of KLK5. A decrease in pH protonates His48 of the wild-type protein resulting in a positively charged residue, thereby explaining the observed decreased dissociation rate. Interestingly, substitution with a positively charged amino acid at position 48 resulted in a more efficient inhibitor at higher pH.

SPINK9: a selective, skin-specific Kazal-type serine protease inhibitor.

A previously unreported Kazal-type serine protease inhibitor, serine protease inhibitor Kazal type 9 (SPINK9), was identified in human skin. SPINK9 expression was strong in palmar epidermis, but not detectable or very low in non palmoplantar skin. Analysis of a human cDNA panel showed intermediate expression in thymus, pancreas, liver, and brain, and low or undetectable expression in other tissues. Using kallikrein-related peptidases (KLKs) 5, 7, 8, and 14, thrombin, trypsin, and chymotrypsin, inhibition with recombinant SPINK9 was seen only for KLK5 using low molecular weight substrates, with an apparent K(i) of 65 nM. Also KLK5 degradation of fibrinogen was totally inhibited by SPINK9. Slight inhibition of KLK8 using fibrinogen substrate could be observed using high concentrations of SPINK9. Analyses by surface plasmon resonance showed heterogeneous binding to SPINK9 of KLK5 and KLK8, but no binding of KLK7 or KLK14. KLK5 has been suggested to play a central role in skin desquamation as an initiating activating enzyme in proteolytic cascades formed by KLKs. An apparently KLK5-specific inhibitor, such as SPINK9, may play a significant regulatory role in such cascades. We suggest a possible role for SPINK9 in the site-specific epidermal differentiation of palms and soles.

The Schistosoma mansoni protein Sm16/SmSLP/SmSPO-1 assembles into a nine-subunit oligomer with potential To inhibit Toll-like receptor signaling.

The Sm16/SmSLP/SmSPO-1 (Sm16) protein is secreted by the parasite Schistosoma mansoni during skin penetration and has been ascribed immunosuppressive activities. Here we describe the strategy behind the design of a modified Sm16 protein with a decreased aggregation propensity, thus facilitating the expression and purification of an Sm16 protein that is soluble in physiological buffers. The Stokes radii and sedimentation coefficients of recombinant and native proteins indicate that Sm16 is an approximately nine-subunit oligomer. Analysis of truncated Sm16 derivatives showed that both oligomerization and binding to the plasma membrane of human cells depend on multiple C-terminal regions. For analysis of immunomodulatory activities, Sm16 was expressed in Pichia pastoris to facilitate the preparation of a pyrogen/endotoxin-free purified protein. Recombinant Sm16 was found to have no effect on T-lymphocyte activation, cell proliferation, or the basal level of cytokine production by whole human blood or monocytic cells. However, Sm16 exerts potent inhibition of the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and poly(I:C) while being less efficient at inhibiting the response to the TLR ligand peptidoglycan or a synthetic lipopeptide. Since Sm16 specifically inhibits the degradation of the IRAK1 signaling protein in LPS-stimulated monocytes, our findings indicate that inhibition is exerted proximal to the TLR complex.

Activation of proteinase-activated receptor-2 by human kallikrein-related peptidases.

Proteinase-activated receptor-2 (PAR2) is a seven transmembrane spanning, G-protein-coupled receptor, present on the membrane of many cell types including keratinocytes. In skin, PAR2 is suggested to play a regulatory role during inflammation, epidermal barrier function, and pruritus. PAR2 is activated by trypsin-like proteases by a unique mechanism where cleavage of the receptor leads to the release of a small peptide, which activates the receptor as a tethered ligand. The endogenous activators of PAR2 on keratinocytes have not been identified as of yet. Potential candidates are kallikrein-related peptidases (KLKs) expressed by epidermal cells. Therefore, the ability of four human skin-derived KLKs was examined with regard to their capacity to activate PAR2 in vitro. PAR2 cleavage was followed by immunofluorescence analysis and functional activation by measurements of changes in intracellular calcium levels. We found that KLK5 and KLK14, but neither KLK7 nor KLK8, induced PAR2 signalling. We conclude that certain, but not all, epidermal KLKs are capable of activating PAR2. We could also show the coexpression of KLK14 and PAR2 receptor in inflammatory skin disorders. These in vitro results suggest that KLKs may take part in PAR2 activation in the epidermis and thereby in PAR2-mediated inflammatory responses, including epidermal barrier repair and pruritus. The role of KLKs in PAR2 activation in vivo remains to be elucidated.

LEKTI fragments specifically inhibit KLK5, KLK7, and KLK14 and control desquamation through a pH-dependent interaction.

LEKTI is a 15-domain serine proteinase inhibitor whose defective expression underlies the severe autosomal recessive ichthyosiform skin disease, Netherton syndrome. Here, we show that LEKTI is produced as a precursor rapidly cleaved by furin, generating a variety of single or multidomain LEKTI fragments secreted in cultured keratinocytes and in the epidermis. The identity of these biological fragments (D1, D5, D6, D8-D11, and D9-D15) was inferred from biochemical analysis, using a panel of LEKTI antibodies. The functional inhibitory capacity of each fragment was tested on a panel of serine proteases. All LEKTI fragments, except D1, showed specific and differential inhibition of human kallikreins 5, 7, and 14. The strongest inhibition was observed with D8-D11, toward KLK5. Kinetics analysis revealed that this interaction is rapid and irreversible, reflecting an extremely tight binding complex. We demonstrated that pH variations govern this interaction, leading to the release of active KLK5 from the complex at acidic pH. These results identify KLK5, a key actor of the desquamation process, as the major target of LEKTI. They disclose a new mechanism of skin homeostasis by which the epidermal pH gradient allows precisely regulated KLK5 activity and corneodesmosomal cleavage in the most superficial layers of the stratum corneum.

Kallikrein-related peptidase 14 may be a major contributor to trypsin-like proteolytic activity in human stratum corneum.

We have previously presented evidence that two human kallikrein-related peptidases, KLK5 (hK5, stratum corneum tryptic enzyme, SCTE) and KLK7 (hK7, stratum corneum chymotryptic enzyme, SCCE), which are abundant in the stratum corneum, may be involved in desquamation. Since we had noted that not all trypsin-like activity in the plantar stratum corneum could be ascribed to KLK5, we set out to identify other skin proteases with similar primary substrate specificity. Here we describe purification of a protease identified as KLK14 from plantar stratum corneum, and show that this enzyme may be responsible for as much as 50% of the total trypsin-like activity in this tissue, measured as activity towards a chromogenic substrate cleaved by a wide variety of enzymes with trypsin-like specificity. This was in spite of very low levels of KLK14 protein compared to KLK5 and KLK7. KLK14 could be detected by immunoblotting in normal superficial stratum corneum of all individuals examined. The majority of KLK14 in the plantar stratum corneum is present in its catalytically active form. KLK14 could be immunohistochemically detected in sweat ducts, preferentially in the intraepidermal parts (the acrosyringium), and in sweat glands. The role played by this very efficient protease under normal and disease conditions in the skin remains to be elucidated.