KLK10 Human

What is KLK10 and which protein family does it belong to?

KLK10 (kallikrein-related peptidase 10) is a secreted serine protease initially identified as normal epithelial cell-specific 1 (NES1). It belongs to the kallikrein-related peptidase 'KLK' family, which consists of 15 secreted serine proteases found as a gene cluster on human chromosome 19q13.4 . This family is distinct from plasma kallikrein, which is encoded on chromosome 4q35 . KLK10 is typically produced as an inactive full-length prepropeptide that is secreted and activated through a complex process to yield an active extracellular enzyme . The mature form of human KLK10 spans from Ala34 to Asn276 .

How is KLK10 regulated in different tissue types?

KLK10 expression shows notable tissue-specific regulation patterns. In vascular endothelial cells, KLK10 expression is regulated by mechanical forces, specifically blood flow patterns. Stable flow (s-flow) upregulates KLK10, while disturbed flow (d-flow) downregulates it . This regulation occurs at both epigenomic and transcriptional levels, as demonstrated by single-cell RNA sequencing (scRNAseq) and scATAC sequencing studies . In cancer tissues, KLK10 regulation varies significantly - it is downregulated in breast, prostate, testicular, and lung cancers but overexpressed in ovarian, pancreatic, and uterine cancers . These opposing patterns suggest context-dependent regulatory mechanisms that may involve tissue-specific transcription factors, epigenetic modifications, or post-transcriptional regulators.

What are the primary physiological functions of KLK10?

In vascular endothelium, KLK10 functions as an anti-inflammatory, barrier-protective, and anti-atherogenic factor . It protects against disturbed flow-induced permeability dysfunction and inflammation in human artery endothelial cells (HAECs), as determined by NFκB activation, expression of vascular cell adhesion molecule 1 and intracellular adhesion molecule 1, and monocyte adhesion . Treatment of mice with recombinant KLK10 (rKLK10) decreases arterial endothelial inflammation in disturbed flow regions, and rKLK10 injection or ultrasound-mediated transfection of Klk10-expressing plasmids inhibits atherosclerosis in Apoe−/− mice . KLK10 expression is significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques, further supporting its protective role in vascular health .

How is KLK10 expression measured in laboratory settings?

Several methodologies are employed to measure KLK10 expression:

• mRNA quantification: Quantitative PCR (qPCR) using specific primers for KLK10 is commonly used . RNA isolation protocols typically involve commercial kits such as the RNeasy Mini Kit, followed by reverse transcription to cDNA using kits like the High-Capacity cDNA Reverse Transcription Kit . qPCR is then performed using SYBR Green-based master mixes with house-keeping genes such as 18S, GAPDH, or ACTB as controls .

• Protein detection: Immunostaining techniques including immunohistochemistry and en face immunostaining are used for tissue localization . For quantification, enzyme-linked immunosorbent assay (ELISA) is employed to measure KLK10 in biological fluids like plasma or cell culture media .

• Single-cell approaches: scRNAseq and scATACseq provide higher resolution analysis of KLK10 expression patterns at cellular and epigenomic levels .

How does blood flow influence KLK10 expression in endothelial cells?

Blood flow patterns significantly influence KLK10 expression in endothelial cells. Stable flow (s-flow), characterized by unidirectional laminar shear stress (typically 15 dynes/cm²), upregulates KLK10 expression at both mRNA and protein levels . In contrast, disturbed flow (d-flow), characterized by oscillatory shear stress (typically ±5 dynes/cm² at 1 Hz), downregulates KLK10 expression .

This flow-dependent regulation has been demonstrated both in vivo using mouse models and in vitro using cultured human aortic endothelial cells (HAECs) . In partial carotid ligation mouse models, KLK10 expression is higher in the stable flow right carotid artery (RCA) compared to the disturbed flow left carotid artery (LCA) . Similarly, in the aortic arch, KLK10 expression is greater in the greater curvature (GC) region exposed to s-flow compared to the lesser curvature (LC) region exposed to d-flow .

What molecular mechanisms mediate KLK10's anti-inflammatory effects in vascular tissue?

KLK10 exerts anti-inflammatory effects in vascular tissue through several molecular mechanisms:

• NFκB inhibition: KLK10 protects against disturbed flow-induced endothelial dysfunction by inhibiting NFκB activation, a master regulator of inflammatory responses .

• Adhesion molecule suppression: This inhibition leads to reduced expression of vascular cell adhesion molecule 1 (VCAM-1) and intracellular adhesion molecule 1 (ICAM-1), which are critical for leukocyte recruitment and adhesion to the endothelium .

• Reduced monocyte adhesion: Consequently, KLK10 reduces monocyte adhesion to endothelial cells, a key early step in atherosclerosis development .

• Proteolytic activity: As a secreted serine protease, KLK10 likely acts through proteolytic mechanisms, potentially cleaving specific substrates involved in inflammatory signaling pathways .

How does KLK10 protect against endothelial dysfunction?

KLK10 protects against endothelial dysfunction through multiple mechanisms:

• Barrier function maintenance: It prevents disturbed flow-induced permeability dysfunction, maintaining the integrity of the endothelial barrier that separates the blood from the vessel wall .

• Inflammation suppression: KLK10 suppresses endothelial inflammation by inhibiting NFκB activation and subsequent expression of adhesion molecules like VCAM-1 and ICAM-1, thereby reducing monocyte adhesion to the endothelium .

• In vivo protection: Treatment with recombinant KLK10 (rKLK10) decreases arterial endothelial inflammation in disturbed flow regions in vivo, demonstrating its potential as a therapeutic agent .

• Atherosclerosis inhibition: rKLK10 injection or ultrasound-mediated transfection of Klk10-expressing plasmids inhibits atherosclerosis in Apoe−/− mice, further confirming its protective role .

What experimental models are most effective for studying KLK10 in atherosclerosis?

Several experimental models have proven effective for studying KLK10 in atherosclerosis:

• Partial carotid ligation (PCL) mouse model: This model creates disturbed flow in the left carotid artery (LCA) while maintaining stable flow in the right carotid artery (RCA), allowing for internal control comparisons .

• Apolipoprotein E-deficient (Apoe−/−) mice: These mice, especially when fed a high-fat diet, develop atherosclerotic lesions that resemble human plaques and serve as a key model for testing KLK10 interventions .

• Aortic arch regions: Examining naturally occurring regions of disturbed flow (lesser curvature) versus stable flow (greater curvature) provides insights into chronic flow-regulated gene expression .

• In vitro flow models: Human aortic endothelial cells (HAECs) exposed to controlled flow conditions using cone-and-plate viscometers allow for precise manipulation of shear stress parameters, mimicking stable flow or disturbed flow conditions .

How can researchers manipulate KLK10 expression in vivo?

Researchers have several strategies available for manipulating KLK10 expression in vivo:

• Recombinant protein administration: Recombinant KLK10 (rKLK10) protein can be administered via injection, with effective doses in mouse studies reported at 0.6 mg/kg .

• Ultrasound-mediated gene delivery: Ultrasound-mediated transfection of Klk10-expressing plasmids has been successfully used to inhibit atherosclerosis in Apoe−/− mice .

• Production considerations: When producing recombinant KLK10, researchers should consider the expression system carefully. Production in mammalian cells (such as CHO-K1) may yield more physiologically relevant rKLK10 compared to bacterial systems, as noted in studies where rKLK10 produced in CHO cells was more effective than E. coli-derived protein .

Why does KLK10 show opposite expression patterns in different cancer types?

The opposing expression patterns of KLK10 in different cancer types reflect its context-dependent functions and regulation. KLK10 is downregulated in breast, prostate, testicular, and lung cancers, suggesting a potential tumor suppressor role in these tissues . In contrast, it is overexpressed in ovarian, pancreatic, and uterine cancers, indicating possible oncogenic functions in these contexts .

This dichotomy likely stems from tissue-specific regulatory mechanisms, including different transcription factors, epigenetic modifications, or microRNA networks that control KLK10 expression. Additionally, the cellular microenvironment, including inflammatory mediators and growth factors, may differently influence KLK10 expression across tissue types. KLK10 was initially discovered as a potential tumor suppressor, but further studies revealed a more complex picture across different cancer types .

How reliable is KLK10 as a biomarker for specific cancer types?

The reliability of KLK10 as a cancer biomarker varies substantially by cancer type. For cancers where KLK10 is consistently downregulated (breast, prostate, testicular, and lung), decreased expression may serve as a potential diagnostic indicator . Conversely, for cancers showing KLK10 overexpression (ovarian, pancreatic, and uterine), elevated levels might indicate disease presence or progression .

In evaluating KLK10 as a biomarker, researchers should conduct receiver operating characteristic (ROC) analysis to determine sensitivity, specificity, and optimal diagnostic cut-off points . The area under the curve (AUC) value provides a measure of the test's discriminatory power, with values approaching 1 indicating stronger diagnostic utility . For optimal reliability, KLK10 should be evaluated as part of a panel of biomarkers rather than in isolation, and researchers should account for potential confounding factors such as age, sex, and comorbidities.

What methodologies are recommended for studying KLK10 in cancer tissues?

A comprehensive methodology for studying KLK10 in cancer tissues should integrate multiple analytical approaches:

• mRNA expression analysis: Quantitative PCR (qPCR) using well-validated primers is recommended . RNA should be isolated using standardized methods (e.g., RNeasy Mini Kit) followed by reverse transcription with high-quality kits (e.g., High-Capacity cDNA Reverse Transcription Kit) . Reference genes such as GAPDH or ACTB should be carefully selected based on their stability in the specific cancer type being studied .

• Protein detection: Immunohistochemistry provides spatial information about KLK10 expression within the tumor microenvironment, while Western blotting offers quantitative analysis of protein levels. ELISA methods can quantify KLK10 in patient serum or plasma .

• Statistical analysis: For clinical samples, proper statistical analysis is crucial; non-parametric tests like the Mann-Whitney U-test or Kruskal-Wallis test are often appropriate as KLK10 expression frequently shows non-Gaussian distribution . Correlations with clinical parameters should be evaluated using Spearman's correlation analysis .

How does KLK10 interact with other cancer-related pathways?

Understanding KLK10's interactions with cancer-related pathways requires integrated analysis of proteolytic networks and signaling cascades:

• Proteolytic activity: As a secreted serine protease, KLK10 likely influences cancer progression through proteolytic activation or inactivation of key substrates involved in cell proliferation, apoptosis, migration, invasion, or angiogenesis .

• KLK family interactions: KLK10 may participate in proteolytic cascades with other KLK family members, matrix metalloproteinases, or components of the coagulation and complement systems, collectively remodeling the tumor microenvironment .

• Context-dependent roles: In contexts where KLK10 acts as a tumor suppressor (breast, prostate, testicular, and lung cancers), it may inhibit pro-oncogenic signaling pathways . Conversely, in cancers where KLK10 is overexpressed (ovarian, pancreatic, and uterine), it might promote oncogenic signaling .

• Inflammatory modulation: KLK10's role in modulating inflammatory responses, as observed in vascular endothelium where it inhibits NF-κB activation, may be relevant to its cancer-related functions given the important role of inflammation in cancer development .

What experimental approaches can resolve contradictory data about KLK10 in cancer?

Resolving contradictory data about KLK10 in cancer requires systematic experimental approaches:

• Standardized detection methods: Researchers should standardize KLK10 detection methods across studies, using validated antibodies for protein detection and consistent primer sets for mRNA quantification .

• Cancer subtype stratification: Study designs should account for cancer subtype heterogeneity, as KLK10's role may differ among molecular or histological subtypes within a given cancer type.

• Tissue-specific analysis: Comparing data from primary tumors, metastatic sites, and adjacent normal tissue can reveal context-dependent functions.

• Temporal dynamics: Analyzing KLK10 expression across disease stages and in response to treatments can provide insights into its dynamic roles during cancer progression.

• Functional validation: Both gain- and loss-of-function approaches in multiple cell lines can confirm causal relationships between KLK10 and cancer phenotypes.

How should researchers design single-cell analyses to study KLK10 expression?

Designing effective single-cell analyses for KLK10 expression requires careful methodological consideration:

• Platform selection: For scRNAseq studies, platforms such as 10x Genomics Chromium or Drop-seq can provide high-throughput analysis, while Smart-seq2 may offer greater sensitivity for detecting low-abundance transcripts like KLK10.

• Epigenomic profiling: For epigenomic profiling, scATACseq can reveal chromatin accessibility at the KLK10 locus, providing insights into its transcriptional regulation across cell populations .

• Quality control: Quality control metrics should include assessments of cell viability, RNA integrity, sequencing depth, and doublet rate.

• Computational analysis: Analysis pipelines should incorporate specialized tools for normalization, dimensional reduction, clustering, and differential expression analysis appropriate for sparse single-cell data.

• Flow-dependent contexts: For experimental designs studying KLK10 in flow-dependent contexts (as in vascular studies), researchers can use approaches similar to those in the partial carotid ligation mouse model, which successfully characterized flow-regulated Klk10 expression at the epigenomic and transcription levels .

What are the best approaches for studying KLK10 proteolytic activity?

Studying KLK10 proteolytic activity requires specialized approaches:

• Substrate identification: Researchers should establish assays using fluorogenic or chromogenic peptide substrates designed based on known or predicted cleavage specificity of KLK10.

• Expression systems: For in vitro studies, recombinant KLK10 (rKLK10) produced in mammalian cells like CHO-K1 is preferable to E. coli-derived protein, as it undergoes proper post-translational modifications essential for activity .

• Activation consideration: Researchers should consider the activation status of KLK10, as it is produced as an inactive prepropeptide that requires processing to yield the active enzyme (Ala34-Asn276 in human KLK10) .

• Activity assays: Mass spectrometry-based proteomic approaches, particularly N-terminomics, can identify protein substrates cleaved by KLK10 in complex biological samples.

• Physiological relevance: Activity assays should be performed under physiologically relevant conditions, considering optimal pH, temperature, and cofactors that may influence enzyme kinetics.

How can researchers distinguish between active and inactive forms of KLK10?

Distinguishing between active and inactive forms of KLK10 is crucial for understanding its biological functions:

• Western blotting: Western blotting with antibodies recognizing different epitopes can detect both pro-KLK10 and mature KLK10 based on molecular weight differences.

• Activity-based probes: Probes containing serine protease-reactive groups can selectively label catalytically active KLK10.

• Enzyme activity assays: Functional assessment using specific substrates can identify active KLK10, while inactive forms show no catalytic activity.

• Mass spectrometry: Mass spectrometry-based approaches, particularly N-terminal sequencing, can precisely identify the cleavage sites that generate active KLK10 from its precursor.

• Expression system selection: When producing recombinant KLK10, mammalian cell-derived KLK10 (e.g., from CHO-K1 cells) is more likely to undergo proper processing than bacterial systems .

What are the recommended statistical approaches for analyzing KLK10 expression data?

Statistical analysis of KLK10 expression data should be tailored to the specific experimental design and data characteristics:

• Distribution assessment: Researchers should assess data distribution, as KLK10 expression often shows non-Gaussian patterns requiring non-parametric methods .

• Group comparisons: For comparing KLK10 expression between two independent groups, the Mann-Whitney U-test is appropriate when normality cannot be assumed . For multiple group comparisons, the Kruskal-Wallis test is recommended .

• Paired analyses: When analyzing paired samples (e.g., before and after treatment), the Wilcoxon Signed-Rank test is appropriate .

• Correlation analysis: Correlations between KLK10 expression and continuous variables should be evaluated using Spearman's correlation coefficient (r) rather than Pearson's if the data are not normally distributed .

• Diagnostic applications: For diagnostic applications, receiver operating characteristic (ROC) analysis can assess KLK10's potential as a biomarker, yielding sensitivity, specificity, and area under the curve (AUC) values .

How can researchers create and validate KLK10 knockdown or knockout models?

Creating and validating KLK10 knockdown or knockout models requires systematic approaches:

• RNA interference: For transient knockdown, small interfering RNA (siRNA) transfection offers a straightforward approach. For stable knockdown, short hairpin RNA (shRNA) delivered via lentiviral or retroviral vectors provides longer-term suppression.

• CRISPR/Cas9: CRISPR/Cas9 technology offers a definitive approach for generating knockout models, requiring careful guide RNA design to avoid off-target effects.

• Conditional approaches: For in vivo models, conditional knockout strategies using Cre-loxP systems allow tissue-specific or inducible deletion, which is particularly valuable for studying KLK10 in specific contexts like vascular endothelium.

• Multi-level validation: Validation should confirm knockdown/knockout at genomic, transcriptomic, and protein levels using PCR, qPCR, and Western blot or ELISA methods similar to those described earlier .

• Functional assessment: Functional validation should assess phenotypes related to known KLK10 functions such as flow-mediated endothelial responses or cancer-relevant processes.

How should KLK10 be evaluated as a potential therapeutic target?

Evaluating KLK10 as a therapeutic target requires a comprehensive translational research approach:

• Context determination: Researchers should establish clear disease contexts where KLK10 modulation is beneficial, distinguishing between conditions requiring KLK10 supplementation (e.g., atherosclerosis, where KLK10 is protective) versus inhibition (potentially in cancers where KLK10 is overexpressed) .

• Delivery strategies: For KLK10 supplementation, recombinant KLK10 (rKLK10) protein therapy has shown promise; studies in mouse models demonstrated that rKLK10 injection at 0.6 mg/kg inhibited atherosclerosis in Apoe−/− mice . Ultrasound-mediated transfection of Klk10-expressing plasmids has also been effective in preclinical models .

• Dosing considerations: Researchers should determine optimal dosing, considering that normal human plasma KLK10 levels are approximately 0.5 ng/ml, and effective concentrations for inhibiting permeability and inflammation in HAECs range from 1-10 ng/ml .

• Target validation: Validation should include both genetic and pharmacological approaches to confirm on-target effects.

• Biomarker development: Biomarkers to monitor therapeutic response should be developed, potentially including measurements of downstream targets affected by KLK10 activity.

What are the key considerations for developing KLK10-based diagnostic tests?

Developing KLK10-based diagnostic tests requires attention to analytical validity, clinical validity, and clinical utility:

• Assay optimization: ELISA methods are commonly used for quantifying KLK10 in clinical specimens, with commercial kits available (e.g., MyBioSource, MBS009286 for human KLK10; NovateinBio, BG-MUS11429 for mouse KLK10) .

• Sample type selection: Plasma KLK10 levels in humans are approximately 0.5 ng/ml, but may vary across different biological fluids and disease states .

• Reference range establishment: Reference ranges must be established in relevant populations, accounting for potential confounding factors such as age, sex, and comorbidities.

• Performance assessment: Diagnostic performance should be assessed using receiver operating characteristic (ROC) analysis to determine sensitivity, specificity, and optimal cut-off values for specific clinical applications .

• Context-specific thresholds: Diagnostic thresholds may differ by disease context, as KLK10 shows opposing expression patterns across different cancer types .

How can researchers address the tissue-specific effects of KLK10 in clinical studies?

Addressing tissue-specific effects of KLK10 in clinical studies requires tailored approaches:

• Targeted sampling: Researchers should design tissue-specific sampling strategies, collecting multiple tissue types when feasible. In vascular research, this might involve examining both atheroprone regions (exposed to disturbed flow) and atheroprotected regions (exposed to stable flow) .

• High-resolution analysis: Single-cell approaches can reveal cell type-specific expression within heterogeneous tissues, providing higher resolution than bulk tissue analysis .

• Patient stratification: Stratification based on tissue-specific KLK10 expression patterns may identify subgroups more likely to benefit from targeted interventions.

• Targeted delivery: For therapeutic development, tissue-targeted delivery strategies should be considered, such as ultrasound-mediated delivery to specific sites .

• Heterogeneity accounting: Clinical trial designs should account for potential heterogeneity in treatment effects based on tissue-specific KLK10 expression or activity.

What sample handling protocols optimize KLK10 stability in clinical specimens?

Optimizing KLK10 stability in clinical specimens requires standardization of pre-analytical procedures:

• Collection methods: Blood samples should be collected in appropriate anticoagulants and processed within 1-2 hours of collection to minimize ex vivo proteolytic activity.

• Processing protocols: Rapid separation of plasma or serum by centrifugation helps preserve protein integrity. For RNA isolation, procedures similar to those described using the RNeasy Mini Kit would be appropriate .

• Inhibitor addition: Addition of protease inhibitor cocktails to samples is recommended, particularly for functional studies of KLK10 activity.

• Storage conditions: Samples should be aliquoted to avoid freeze-thaw cycles and stored at -80°C for long-term preservation.

• Quality control: Quality control measures should include assessment of sample integrity markers and documentation of collection-to-processing times.

How should researchers interpret changes in KLK10 expression in response to treatments?

Interpreting changes in KLK10 expression in response to treatments requires nuanced analysis:

• Baseline establishment: Researchers should first establish baseline KLK10 expression patterns in the tissue or cell type of interest, recognizing that normal levels vary considerably across contexts .

• Temporal assessment: KLK10 responses should be assessed at multiple time points to distinguish between transient and sustained effects.

• Magnitude contextualization: The magnitude of change should be interpreted in the context of physiologically relevant ranges; in endothelial cells, for example, KLK10 levels in anti-inflammatory conditions (0.3 ng/ml) versus pro-inflammatory conditions (0.13 ng/ml) represent a significant but not dramatic fold change .

• Transcription-translation correlation: Researchers should distinguish between changes in KLK10 mRNA versus protein levels, as post-transcriptional regulation may lead to discordant patterns.

• Functional correlation: Functional consequences of altered KLK10 expression should be directly assessed through relevant biological assays, such as endothelial permeability, inflammatory marker expression, or cell adhesion in vascular studies .