Kallikrein-related Peptidase Research Articles

Sodium/(calcium + potassium) exchanger NCKX4 optimizes KLK4 activity in the enamel matrix microenvironment to regulate ECM modeling.

Enamel development is a process in which extracellular matrix models from a soft proteinaceous matrix to the most mineralized tissue in vertebrates. Patients with mutant NCKX4, a gene encoding a K+-dependent Na+/Ca2+-exchanger, develop a hypomineralized and hypomature enamel. How NCKX4 regulates enamel protein removal to achieve an almost protein-free enamel is unknown. We characterized the upregulation pattern of Nckx4 in the progressively differentiating enamel-forming ameloblasts by qPCR, and as well as confirmed NCKX4 protein to primarily localize at the apical surface of wild-type ruffle-ended maturation ameloblasts by immunostaining of the continuously growing mouse incisors, posing the entire developmental trajectory of enamel. In contrast to the normal mature enamel, where ECM proteins are hydrolyzed and removed, we found significant protein retention in the maturation stage of Nckx4 -/- mouse enamel. The Nckx4 -/- enamel held less Ca2+ and K+ but more Na+ than the Nckx4 +/+ enamel did, as measured by EDX. The alternating acidic and neutral pH zones at the surface of mineralizing Nckx4 +/+ enamel were replaced by a largely neutral pH matrix in the Nckx4 -/- enamel. In situ zymography revealed a reduced kallikrein-related peptidase 4 (KLK4) activity in the Nckx4 -/- enamel. We showed that KLK4 took on 90% of proteinase activity in the maturation stage of normal enamel, and that recombinant KLK4 as well as native mouse enamel KLK4 both performed less effectively in a buffer with increased [Na+] and pH, conditions found in the Nckx4 -/- developing enamel. This study, for the first time to our knowledge, provides evidence demonstrating the impaired in situ KLK4 activity in Nckx4 -/- enamel and suggests a novel function of NCKX4 in facilitating KLK4-mediated hydrolysis and removal of ECM proteins, warranting the completion of enamel matrix modeling.

Expression and Distribution of Kallikrein-related Peptidases 5, 7, 8 and 10 in Normal Apocrine Gland of Canine Skin

Apocrine glands are sweat glands that are located in the skin of the dog. Anal sac apocrine, circunanal apocrine, and mammary glands are considered modified apocrine structures, and there are about nine possible types of neoplasms and other tumors in the apocrine glands of the dog and cat, including cysts, adenoma, carcinoma, and adenocarcinoma. Thus, it is important to provide new markers to characterize these glands to improve the histopathological diagnosis. In this article, we describe the distribution of kallikrein- related peptidases 5, 7, 8, and 10 in the normal apocrine glands of the dog’s skin. These proteases have been shown to play a fundamental role in the homeostasis of the human skin barrier but have been scarcely studied in canine skin.

The roles of proteases in prostate cancer.

Since the proposition of the pro-invasive activity of proteolytic enzymes over 70 years ago, several roles for proteases in cancer progression have been established. About half of the 473 active human proteases are expressed in the prostate and many of the most well-characterized members of this enzyme family are regulated by androgens, hormones essential for development of prostate cancer. Most notably, several kallikrein-related peptidases, including KLK3 (prostate-specific antigen, PSA), the most well-known prostate cancer marker, and type II transmembrane serine proteases, such as TMPRSS2 and matriptase, have been extensively studied and found to promote prostate cancer progression. Recent findings also suggest a critical role for proteases in the development of advanced and aggressive castration-resistant prostate cancer (CRPC). Perhaps the most intriguing evidence for this role comes from studies showing that the protease-activated transmembrane proteins, Notch and CDCP1, are associated with the development of CRPC. Here, we review the roles of proteases in prostate cancer, with a special focus on their regulation by androgens.

Altered Left Ventricular Rat Gene Expression Induced by the Myosin Activator Omecamtiv Mecarbil.

To explore the impact of omecamtiv mecarbil (OM) on the gene expression profile in adult male rats. Fourteen male Wistar rats were randomly assigned to a single OM (1.2 mg/kg/h; n = 6) or placebo (n = 8) 30-min infusion. Echocardiography was performed before and after OM infusion. Seven days after infusion, rats were euthanized, and left ventricular (LV) tissues were removed for real-time quantitative polymerase chain reaction (RTq-PCR) experiments. After OM infusion, pro-apoptotic Bax-to-Bcl2 ratio was decreased, with increased Bcl2 and similar Bax gene expression. The gene expression of molecules regulating oxidative stress, including glutathione disulfide reductase (Gsr) and superoxide dismutases (Sod1/Sod2), remained unchanged, whereas the expression of antioxidant glutathione peroxidase (Gpx) increased. While LV gene expression of key energy sensors, peroxisome proliferator activator (Ppar) α and γ, AMP-activated protein kinase (Ampk), and carnitine palmitoyltransferase 1 (Cpt1) remained unchanged after OM infusion, and the expression of pyruvate dehydrogenase kinase 4 (Pdk4) increased. The LV expression of the major myocardial glucose transporter Glut1 decreased, with no changes in Glut4 expression, whereas the LV expression of oxidized low-density lipoprotein receptor 1 (Olr1) and arachidonate 15-lipoxygenase (Alox15) increased, with no changes in fatty acid transporter Cd36. An increased LV expression of angiotensin II receptors AT1 and AT2 was observed, with no changes in angiotensin I-converting enzyme expression. The Kalikrein-bradykinin system was upregulated with increased LV expression of kallikrein-related peptidases Klk8, Klk1c2, and Klk1c12 and bradykinin receptors B1 and B2 (Bdkrb1 and Bdkrb2), whereas the LV expression of inducible nitric oxide synthase 2 (Nos2) increased. LV expression in major molecular determinants involved in calcium-dependent myocardial contraction remained unchanged, except for an increased LV expression of calcium/calmodulin-dependent protein kinase II delta (Cacna1c) in response to OM. A single intravenous infusion of OM, in adult healthy rats, resulted in significant changes in the LV expression of genes regulating apoptosis, oxidative stress, metabolism, and cardiac contractility.

KLK6/PAR1 Axis Promotes Tumor Growth and Metastasis by Regulating Cross-Talk between Tumor Cells and Macrophages.

Kallikrein-related peptidase (KLK)6 is associated with inflammatory diseases and neoplastic progression. KLK6 is aberrantly expressed in several solid tumors and regulates cancer development, metastatic progression, and drug resistance. However, the function of KLK6 in the tumor microenvironment remains unclear. This study aimed to determine the role of KLK6 in the tumor microenvironment. Here, we uncovered the mechanism underlying KLK6-mediated cross-talk between cancer cells and macrophages. Compared with wild-type mice, KLK6-/- mice showed less tumor growth and metastasis in the B16F10 melanoma and Lewis lung carcinoma (LLC) xenograft model. Mechanistically, KLK6 promoted the secretion of tumor necrosis factor-alpha (TNF-α) from macrophages via the activation of protease-activated receptor-1 (PAR1) in an autocrine manner. TNF-α secreted from macrophages induced the release of the C-X-C motif chemokine ligand 1 (CXCL1) from melanoma and lung carcinoma cells in a paracrine manner. The introduction of recombinant KLK6 protein in KLK6-/- mice rescued the production of TNF-α and CXCL1, tumor growth, and metastasis. Inhibition of PAR1 activity suppressed these malignant phenotypes rescued by rKLK6 in vitro and in vivo. Our findings suggest that KLK6 functions as an important molecular link between macrophages and cancer cells during malignant progression, thereby providing opportunities for therapeutic intervention.

Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis.

The epidermis is a barrier that prevents water loss while keeping harmful substances from penetrating the host. The impermeable cornified layer of the stratum corneum is maintained by balancing continuous turnover driven by epidermal basal cell proliferation, suprabasal cell differentiation, and corneal shedding. The epidermal desquamation process is tightly regulated by balance of the activities of serine proteases of the Kallikrein-related peptidases (KLK) family and their cognate inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 gene. Imbalance of proteolytic activity caused by a deficiency of LEKTI leads to excessive desquamation due to increased activities of KLK5, KLK7, and KLK14 and results in Netherton syndrome (NS), a debilitating condition with an unmet clinical need. Increased activity of KLKs may also be pathological in other dermatoses such as atopic dermatitis (AD). Here, we describe the discovery of inhibitory antibodies against murine KLK5 and KLK7 that could compensate for the deficiency of LEKTI in NS. These antibodies are protective in mouse models of NS and AD and, when combined, promote improved skin barrier integrity and reduced inflammation. To translate these findings, we engineered a humanized bispecific antibody capable of potent inhibition of human KLK5 and KLK7. A crystal structure of KLK5 bound to the inhibitory Fab revealed that the antibody binds distal to its active site and uses a relatively unappreciated allosteric inhibition mechanism. Treatment with the bispecific anti-KLK5/7 antibody represents a promising therapy for clinical development in NS and other inflammatory dermatoses.

A KLK6 Activity-Based Probe Reveals a Role for KLK6 Activity in Pancreatic Cancer Cell Invasion.

Pancreatic cancer has the lowest survival rate of all common cancers due to late diagnosis and limited treatment options. Serine hydrolases are known to mediate cancer progression and metastasis through initiation of signaling cascades and cleavage of extracellular matrix proteins, and the kallikrein-related peptidase (KLK) family of secreted serine proteases have emerging roles in pancreatic ductal adenocarcinoma (PDAC). However, the lack of reliable activity-based probes (ABPs) to profile KLK activity has hindered progress in validation of these enzymes as potential targets or biomarkers. Here, we developed potent and selective ABPs for KLK6 by using a positional scanning combinatorial substrate library and characterized their binding mode and interactions by X-ray crystallography. The optimized KLK6 probe IMP-2352 (kobs/I = 11,000 M-1 s-1) enabled selective detection of KLK6 activity in a variety of PDAC cell lines, and we observed that KLK6 inhibition reduced the invasiveness of PDAC cells that secrete active KLK6. KLK6 inhibitors were combined with N-terminomics to identify potential secreted protein substrates of KLK6 in PDAC cells, providing insights into KLK6-mediated invasion pathways. These novel KLK6 ABPs offer a toolset to validate KLK6 and associated signaling partners as targets or biomarkers across a range of diseases.

Mast cell stabilization: new mechanism underlying the therapeutic effect of intense pulsed light on rosacea.

Rosacea, a chronic inflammatory disorder of the facial skin, is effectively treated by intense pulsed light (IPL). To explore the potential molecular mechanism underlying the photobiomodulation effect of IPL for rosacea treatment. Skin samples from patients with rosacea were subjected to histological and immunohistological staining. Ten patients were followed up after IPL treatment using the VISIA® skin analysis system, and the severity was assessed. In vivo, skin changes in mice with rosacea-like inflammation induced by intradermal injection of 320μM LL-37 with or without IPL treatment were evaluated using L*a*b colorimetry as well as histological and immunological staining. In vitro, LL-37-stimulated mast cells (MCs) with or without IPL treatment were evaluated for protein expression of matrix metalloproteinase (MMP)-9, kallikrein-related peptidase 5 (KLK5), and cathelicidin using western blotting and qRT-PCR. Profound infiltration of inflammatory cells and evident MC degranulation were found in rosacea skin lesions. The expression of rosacea-related biomarkers and inflammatory cytokines was higher in lesional areas than in non-lesional areas, as demonstrated via immunochemical staining. In all patients, rosacea severity reduced after IPL therapy. In vivo, IPL alleviated inflammation in mice with rosacea-like inflammation, as demonstrated by the significantly decreased MMP-9, KLK5, and cathelicidin expression and reduced percentage of degranulating MCs. In vitro, IPL decreased MMP-9, KLK5, and cathelicidin expression in P815 cells, reducing the release of inflammatory cytokines and inhibiting rosacea-like inflammatory reactions. The photobiomodulation effect of IPL for rosacea treatment may inhibit MC degranulation and alleviate inflammatory reactions.

Amelogenesis imperfecta in a Chinese family resulting from a FAM83H variation and the effect of FAM83H on the secretion of enamel matrix proteins.

To investigate the variant of an amelogenesis imperfecta (AI) family and to explore the function of the FAM83H (family with sequence similarity 83 member H) in the enamel formation. We investigated a five-generation Chinese family diagnosed with AI; clinical data was collected, whole-exome sequencing (WES) was conducted to explore the pathogenic gene and variants and Sanger sequencing was used to verify the variants. The three-dimensional protein structures of wild-type and mutant FAM83H were predicted using alpha fold 2. To study the possible regulatory function of Fam83h on amelogenesis, immunolocalization was performed to observe the expression of Fam83h protein in Sprague-Dawley rat postnatal incisors. The mRNA and protein level of amelogenin, enamelin, kallikrein-related peptidase-4 and ameloblastin were also detected after the Fam83h was knocked down by small interfering RNA (siRNA) in HAT-7 cells. A known nonsense variant (c.973 C > T) in exon 5 of FAM83H gene was found in this family, causing a truncated protein (p.R325X). Immunolocalization of Fam83h in Sprague-Dawley rat postnatal incisors showed that Fam83h protein expression was detected in presecretory and secretory stages. When Fam83h expression was reduced by siRNA, the expression of amelogenin, enamelin, kallikrein-related peptidase-4 decreased. However, the expression of ameloblastin increased. FAM83H gene variant (c.973 C > T) causes AI. FAM83H regulates the secretion of enamel matrix proteins and affects ameloblast differentiation. This study provided that FAM83H variants could influence enamel formation and provided new insights into the pathogenesis of AI.

Accurate diagnosis of prostate cancer with CRISPR-based nucleic acid test strip by simultaneously identifying PCA3 and KLK3 genes

Although serum prostate specific antigen (PSA) testing could decrease the morality of prostate cancer (PCa), its low specificity usually led to misdiagnosis due to prostatitis or benign prostatic hyperplasia (BPH). Prostate cancer antigen 3 (PCA3) as an alternative prostate tumor-specificity biomarker could be used to increase the specificity of PCa diagnosis, however, it usually required sophisticated operation and expensive equipment for routine detection. Herein, we constructed an early detection platform for prostate cancer with reverse transcriptase-recombinase aided amplification (RT-RAA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 based nucleic acid test strip. The amplicons of PCA3 and kallikrein related peptidase 3 (KLK3) gene, which amplified simultaneously by single-amplification unit of RT-RAA were specifically recognized by Cas9-sgRNA and visual on the nucleic acid test strip by naked eyes without instruments. Simultaneously detection of PCA3 and KLK3 gene could improve specificity and accuracy of the diagnosis but avoid mutual interference. In addition, the platform presented a detection limit of 500 fg/μL and 50 fg/μL in PCA3 and KLK3 gene, respectively. Furthermore, the analysis result of signal ratio of PCA3 to KLK3 gene of urine and peripheral blood specimens from 32 men with suspected prostate cancer on test strips illustrated that the area under the curve values of urine and peripheral blood specimens were 0.998 and 1.0 respectively. In summary, our study highlighted a facile strategy to design an accurate prostate cancer gene detection platform which had the potential to conduct prostate cancer early detection in the resource-limited or other point-of-care testing (POCT) environments.

Variants in KLK11, affecting signal peptide cleavage of kallikrein-related peptidase 11, cause an autosomal-dominant cornification disorder.

Mendelian disorders of cornification (MeDOC) are a group of heterogeneous genodermatoses with different genetic bases. The pathogenesis of a substantial group of MeDOC remains to be elucidated. To identify a new causative gene and the pathogenesis of a previously undescribed autosomal-dominant cornification disorder. Whole-exome sequencing was performed in three families with the novel cornification disorder to identify the disease-causing variants. As the variants were located around the signal peptide (SP) cleavage site of a kallikrein-related peptidase, SP cleavage, subcellular localization and extracellular secretion of the variants were evaluated in eukaryotic overexpression systems by Western blotting or immunocytochemistry. Then the trypsin-like and chymotrypsin-like proteolytic activity of the peptidase and degradation of its catalytic substrate were assayed using the patients' stratum corneum (SC) samples. The morphology of the lamellar bodies and corneodesmosomes (CDs) in the patients' SC was ultrastructurally examined. A mouse model harbouring the equivalent variant was constructed and evaluated histologically. We identified two heterozygous variants affecting Gly50 in kallikrein-related peptidase (KLK)11 in a familial case and two sporadic cases with the new disorder, which is characterized by early-onset ichthyosiform erythroderma or erythrokeratoderma. KLK11 belongs to the family of kallikrein-related peptidases participating in skin desquamation by decomposing CDs, a process essential for shedding of the SC. In vitro experiments demonstrated that the variants perturbed the SP cleavage of KLK11, leading to subcellular mislocalization and impaired extracellular secretion of the KLK11 Gly50Glu variant. Both trypsin-like and chymotrypsin-like proteolytic activities were significantly decreased in the patients' SC samples. Reduced proteolysis of desmoglein 1 and delayed degeneration of CDs were detected in patients' SC, indicating delayed skin desquamation. Consistently, the patients showed a thickened, dense SC, indicating abnormal skin desquamation. Mice harbouring the homozygous c.131G>A (p.Gly44Glu) Klk11 variant, which is equivalent to KLK11 c.149G>A (p.Gly50Glu) in humans, exhibited hyperkeratosis and abnormal desquamation, partially recapitulating the phenotype. We provide evidence that variants at Gly50 affecting the SP cleavage of KLK11 cause a new autosomal-dominant cornification disorder with abnormal desquamation. Our findings highlight the essential role of KLKs in maintaining homeostasis of skin keratinization and desquamation.

A Pair of Prognostic Biomarkers in Triple-Negative Breast Cancer: KLK10 and KLK11 mRNA Expression

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with poor patient prognosis and limited therapeutic options. A lack of prognostic biomarkers and therapeutic targets fuels the need for new approaches to tackle this severe disease. Extracellular matrix degradation, release, and modulation of the activity of growth factors/cytokines/chemokines, and the initiation of signaling pathways by extracellular proteolytic networks, have been identified as major processes in the carcinogenesis of breast cancer. Members of the kallikrein-related peptidase (KLK) family contribute to these tumor-relevant processes, and are associated with breast cancer progression and metastasis. In this study, the clinical relevance of mRNA expression of two members of this family, KLK10 and KLK11, has been evaluated in TNBC. For this, their expression levels were quantified in tumor tissue of a large, well-characterized patient cohort (n = 123) via qPCR. Although, in general, the overall expression of both factors are lower in tumor tissue of breast cancer patients (encompassing all subtypes) compared to normal tissue of healthy donors, in the TNBC subtype, expression is even increased. In our cohort, a significant, positive correlation between the expression levels of both KLKs was detected, indicating a coordinate expression mode of these proteases. Elevated KLK10 and KLK11 mRNA levels were associated with poor patient prognosis. Moreover, both factors were found to be independent of other established clinical factors such as age, lymph node status, or residual tumor mass, as determined by multivariable Cox regression analysis. Thus, both proteases, KLK10 and KLK11, may represent unfavorable prognostic factors for TNBC patients and, furthermore, appear as promising potential targets for therapy in TNBC.

KLK6 Functions as an Oncogene and Unfavorable Prognostic Factor in Bladder Urothelial Carcinoma.

Background Kallikrein-related peptidase 6 (KLK6) has been substantiated as a diagnostic, prognostic, and therapeutic molecular in several cancer types. In our study, we attempt to explore the biological functions of KLK6 in bladder urothelial carcinoma (BLCA). Methods KLK6 gene expression prognostic, gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), and immune infiltration were analyzed using The Cancer Genome Atlas (TCGA) database. In vitro and in vivo experimental measurements, including CCK8, transwell migration, TUNEL, and nude mouse transplanted tumor model, were used to evaluate the antineoplastic activities of KLK6 loss-of-function. Results The combination of bioinformatics analyses and experimental measurements demonstrate that KLK6 expression is aberrantly upregulated in human specimens and cell lines of BLCA. GO and GSEA enrichment analyses exhibited that KLK6 is implicated in the inflammatory response and immune infiltration, suggesting that upregulation of KLK6 may be associated with the progression of BLCA. Knockdown of KLK6 is able to inhibit the growth and migration and trigger apoptosis of RT4 and T24 cells. Moreover, the TCGA database indicates that KLK6 high expression in BLCA patients showed a poorer prognosis than those patients with KLK6 low expression. Univariate and multivariate regression analyses suggest KLK6 as an independent prognostic factor to predict unfavorable OS in patients with BLCA. Conclusion KLK6 is an independent prognostic factor and an antitumor target of BLCA. KLK6 expression positively correlates with several immune cells infiltration, indicating that inhibition of KLK6 may contribute to immunotherapy of BLCA.

Anti-inflammatory effects of amarogentin on 2,4-dinitrochlorobenzene-induced atopic dermatitis-like mice and in HaCat cells.

Amarogentin (AMA) is a secoiridoid glycoside extracted from Swertia and Gentiana roots and exhibits many biological effects such as antioxidative, anti-inflammatory, and antitumor activities. Atopic dermatitis (AD) is a chronic inflammatory skin disease caused by disorders in the regulation of multiple inflammatory cytokines. No effective cure has been found for AD now. We constructed the HaCat and splenocyte model and tested the inhibitory effect of AMA on IL-4, IL-6, and IL-13 secretions using enzyme-linked immunosorbent assay (ELISA). The AD mouse model was constructed and treated with AMA, the severity of skin lesions was observed, epidermal tissue was collected, and epidermal thickness and mast cell infiltration were observed using hematoxylin and eosin and toluidine blue staining, respectively. The expression of kallikrein-related peptidase 7 (KLK7) and filaggrin (FLG) was detected using immunostaining and Western blot analysis. The mRNA expression of KLK7 and FLG was detected using quantitative polymerase chain reaction (qPCR). Blood immunoglobulin E (IgE) secretion was detected. AMA inhibited IL-6 secreted by tumor necrosis factor (TNF)-α-induced HaCaT cells and reduced IL-4 and IL-13 secreted by phytohemagglutinin (PHA)-induced primary cells in the mice spleen. It was found that the treatment of AMA with 2,4-dinitrochlorobenzene-induced AD-like mice could promote the recovery of dermatitis, reduce the score of dermatitis severity and the scratching frequency, treat the skin lesions, reduce the epidermal thickness, decrease the infiltration of mast cells, reduce the IgE level in serum, decrease the expression levels of AD-related cytokines, increase protein and mRNA expression of FLG, and reduce the protein and mRNA expression of KLK7 in the skin tissues of AD-like mice. In conclusion, AMA inhibits inflammatory response at the cellular level, and AMA reduces the validation response of specific dermatitis mice, relieves pruritus, and repairs the damaged skin barrier.

The family of kallikrein-related peptidases and kinin peptides as modulators of epidermal homeostasis.

The epidermis is the outermost skin layer and is part of one of the largest organs in the body; it is supported by the dermis, a network of fibrils, blood vessels, pilosebaceous units, sweat glands, nerves, and cells. The skin as a whole is a protective shield against numerous noxious agents, including microorganisms and chemical and physical factors. These functions rely on the activity of multiple growth factors, peptide hormones, proteases, and specific signaling pathways that are triggered by the activation of distinct types of receptors sited in the cell membranes of the various cell types present in the skin. The human kallikrein family comprises a large group of 15 serine proteases synthesized and secreted by different types of epithelial cells throughout the body, including the skin. At this site, they initiate a proteolytic cascade that generates the active forms of the proteases, some of which regulate skin desquamation, activation of cytokines, and antimicrobial peptides. Kinin peptides are formed by the action of plasma and tissue kallikreins on kininogens, two plasma proteins produced in the liver and other organs. Although kinins are well known for their proinflammatory abilities, in the skin they are also considered important modulators of keratinocyte differentiation. In this review, we summarize the contributions of the kallikreins and kallikrein-related peptidases family and those of kinins and their receptors in skin homeostasis, with special emphasis on their pathophysiological role.

Kallikrein-related peptidase-8 (KLK8) aggravated hypoxia-induced right ventricular hypertrophy by targeting P38 MAPK/P53 signaling pathway

Right ventricular (RV) hypertrophy and further heart failure are major co-morbidities, resulting in the premature death of patients with hypoxic pulmonary hypertension (HPH). The regulatory effects of kallikrein-related peptidase (KLK) family members on cardiac function have been extensively studied. However, to the best of the authors’ knowledge, the regulatory effects of KLK8 on RV hypertrophy caused by HPH have yet to be reported. The aim of the present study was to assess KLK8 expression in the RV tissue of HPH-modeled rats, and to further explore the effects and underlying mechanism of KLK8 in regulating the hypertrophy of hypoxia-induced H9c2 cardiomyocytes. In HPH model rats, increases in the right ventricle hypertrophy index, the right ventricular systolic pressure, cardiac output, as well as pulmonary artery wall thickness were observed. Western blot analysis revealed that KLK8 expression and MAPK/p53 signaling activity were enhanced in the RVs of rats in an RV HPH rat model. In hypoxia-induced H9c2 cardiomyocytes, KLK8 overexpression promoted cardiomyocyte hypertrophy, whereas KLK8 silencing showed the opposite results. KLK8 overexpression increased the expression levels of ventricular hypertrophy markers, including atrial natriuretic peptide, brain natriuretic peptide and myosin heavy chain 7, which were blocked upon addition of the p38 MAPK inhibitor, SB202190. Conversely, KLK8 silencing caused a decrease in the expression levels of the ventricular hypertrophy markers, which were further reduced via inhibition of the p38 MAPK/p53 signaling pathway. Taken together, the results of the present study have shown that KLK8 may subtly regulate RV hypertrophy, and therefore KLK8 may be a promising therapeutic target for treating HPH-induced RV hypertrophy.

410 Three stepwise pH zones to form functional stratum corneum

Stratum corneum (SC) is the outermost layer of the epidermis, which consists of nonviable anuclear keratinocytes (corneocytes) and function as a protective skin barrier. The pH distribution of SC has long been considered to impact its homeostasis. However, pH distribution and its biological significance in SC homeostasis remain unclear due to challenges in measuring and visualizing pH at a single-corneocyte resolution and elucidating the biological significance of pH profiles in SC. To tackle these challenges, we generated mice expressing Venus–mCherry, the fusion protein of pH-sensitive probe Venus and pH-insensitive probe mCherry from the uppermost stratum granulosum cells (SASP-Venus-mCherry mice), which enables the observation of relative pH changes and calculation of pH values via simultaneous ratiometric imaging. Confocal microscopic analysis of SASP-Venus-mCherry mice revealed that the SC has three stepwise pH zones, lower-moderately acidic (pH 6.0), middle-acidic (pH 5.4), and upper-nearly neutral (pH 6.7) zones, rather than gradual change over SC layers, in various body parts. Topical application of phosphate buffers with pH 4.1, 5.3, 6.4 and 7.2 did not affect pH of lower and middle SC-pH zones, while it changed pH of upper SC-pH zone according to the applied buffer. Additionally, the lower and middle SC-pH zones were dependent on claudin-1, a tight junction protein, which diminished in claudin-1-deficient mice; thus, the entire SC exhibits neutral pH. Finally, mathematical modeling demonstrated that the stepwise SC-pH profile pH6.0–pH5.4–pH6.7, but not the gradient SC-pH profile pH7–pH5 reproduced the increase of effective catalytic activity of kallikrein-related peptidases toward the SC surface. Collectively, these findings indicate that the three stepwise SC-pH zones are tightly regulated and are necessary to mediate adequate desquamation, which leads to functional SC differentiation.

Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis.

Kallikrein-related peptidases (KLKs) are implicated in many cancer-related processes. KLK6, one of the 15 KLK family members, is a promising biomarker for diagnosis of many cancers and has been associated with poor prognosis of colorectal cancer (CRC) patients. Herein, we evaluated the expression and cellular functions of KLK6 in colon cancer-derived cell lines and in clinical samples from CRC patients. We showed that, although many KLKs transcripts are upregulated in colon cancer-derived cell lines, KLK6, KLK10, and KLK11 are the most highly secreted proteins. KLK6 induced calcium flux in HT29 cells by activation and internalization of protease-activated receptor 2 (PAR2). Furthermore, KLK6 induced extracellular signal–regulated kinases 1 and 2 (ERK1/2) phosphorylation. KLK6 suppression in HCT-116 colon cancer cells decreased the colony formation, increased cell adhesion to extracellular matrix proteins, and reduced spheroid formation and compaction. Immunohistochemistry (IHC) analysis demonstrated ectopic expression of KLK6 in human colon adenocarcinomas but not in normal epithelia. Importantly, high levels of KLK6 protein were detected in the ascites of CRC patients with peritoneal metastasis, but not in benign ascites. These data indicate that KLK6 overexpression is associated with aggressive CRC, and may be applied to differentiate between benign and malignant ascites.

Phage Display Selected Cyclic Peptide Inhibitors of Kallikrein-Related Peptidases 5 and 7 and Their In Vivo Delivery to the Skin.

Kallikrein-related peptidases 5 (KLK5) and 7 (KLK7) are serine proteases with homeostatic functions in the epidermis that play a critical role in Netherton syndrome (NS), a rare yet life-threatening genetic disorder that currently lacks specific treatment. Previous research suggests that controlling KLKs could lead to the development of NS therapies, but existing synthetic inhibitors have limitations. Herein, we used phage display to screen libraries comprising more than 100 billion different cyclic peptides and found selective, high-affinity inhibitors of KLK5 (Ki = 2.2 ± 0.1 nM) and KLK7 (Ki = 16 ± 4 nM). By eliminating protease-prone sites and conjugating the inhibitors to an albumin-binding peptide, we enhanced the inhibitor stability and prolonged the elimination half-life to around 5 h in mice. In tissue sections taken from mice, a fluorescently labeled peptide was detected in the epidermis, suggesting that the inhibitors can reach the KLKs upon systemic delivery and should be suited to control deregulated protease activity in NS.

Phase 1 study of JNJ-69086420, an actinium-225-labeled antibody targeting human kallikrein-2, for advanced prostate cancer.

TPS5104 Background: Radioligand therapy for metastatic castration resistant prostate cancer (mCRPC) has been shown to prolong survival, delay disease progression, and improve quality of life, raising hopes that these gains will be amplified with even more cancer-specific targets and more powerful radioligands. Human kallikrein-related peptidase 2 (hK2) is a tumor-associated member of the kallikrein family that shares significant homology to prostate-specific antigen and is minimally expressed in normal non-prostate tissues. JNJ-69086420 (JNJ-420; 225Ac-DOTA-h11B6 [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]), is a first-in-class radioimmunotherapy targeted to hK2 antigen. In a phase 0 study of [111In]-DOTA-h11B6, patients with mCRPC (progressed on standard therapies), treatment with a single dose of [111In]-DOTA-h11B6 (2 mg) with/without 8 mg h11B6, demonstrated safety, good tumor localization, nominal normal-organ uptake, and no difference in pharmacokinetics (PK) between 2 and 10 mg antibody mass (Morris et al. J Clin Oncol. 2021 39:6 suppl, 122). We have initiated the first-in-human study to assess the safety, PK, pharmacodynamic (PD), and clinical activity of Ac-225 radiolabeled JNJ-420, to determine its recommended phase 2 dose (RP2D) in adults with advanced PC. Methods: This open-label, multicenter, phase 1 study will recruit approximately 50 men (aged ≥18 years) with advanced PC across dose escalation (Part 1) and expansion (Part 2) parts. Key eligibility criteria: mCRPC with histologic confirmation of adenocarcinoma, Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, adequate organ function based on hematology and serum chemistry, and 1 or more prior novel androgen receptor-targeted therapies (prior chemotherapy acceptable). Key exclusion criteria: prior treatment with radium/strontium/samarium/radioconjugate therapy, superscan findings as protocol defined, active central nervous system metastases. In Part 1, men will receive intravenous (IV) injection of 50 μCi/ 2 mg JNJ-420 (once every 8 weeks) with one or multiple doses; escalation of dose levels to be based on dose limiting toxicities (DLTs) evaluation, until RP2D identification. In Part 2, JNJ-420 is to be given at one of the RP2D(s) determined in Part 1. Primary endpoint is safety (incidence and severity [grading per NCI-CTCAE V5.0] of AEs including DLTs). Secondary endpoints include prostate specific antigen response rate, overall response rate (PCWG3 modified RECIST 1.1 criteria), PK, PD, immunogenicity, and biomarker analyses. Enrollment began in Dec 2020; as of 10 Feb 2022, 4 sites have been initiated and 23 patients enrolled; currently, dose escalation is ongoing. Clinical trial information: NCT04644770.