KLK7 Human

Basic Research Questions

• What is Human KLK7 and where is it primarily expressed?

KLK7 (Kallikrein 7) is a serine protease belonging to the kallikrein-related peptidase family, which includes 15 members in humans. KLK7 is most abundantly expressed in human skin, where it plays crucial roles in desquamation and skin barrier function. According to transcriptomic analyses, KLK5 and KLK7 are the most highly expressed KLKs in normal human skin, together accounting for approximately two-thirds of total KLK transcripts in healthy skin biopsies . Beyond skin, KLK7 is also expressed in adipose tissue, as demonstrated by research using adipose-specific Klk7 knockout mice .

• What are the primary physiological functions of KLK7?

KLK7 serves multiple physiological functions across different tissues and biological systems:

• Skin physiology: Contributes to skin barrier homeostasis through involvement in desquamation processes

• Metabolic regulation: Participates in body weight and fat mass regulation

• Energy metabolism: Influences energy expenditure and substrate utilization

• Adipose tissue function: Regulates adipose tissue expansion and inflammation

• Insulin processing: May regulate insulin degradation, potentially impacting glucose homeostasis

• Proteolytic activity: Cleaves various endogenous substrates including midkine, CYR61, and tenascin-C

• How is KLK7 implicated in skin pathologies?

KLK7 plays significant roles in multiple skin disorders:

RNA-seq analyses have identified KLK7 as the only kallikrein significantly upregulated in human atopic dermatitis, showing approximately 4-fold expression increase in AD-like skin compared to healthy controls . This upregulation appears to contribute directly to pruritus (itching) associated with the condition.

• What methodologies are used to measure KLK7 activity in experimental settings?

Researchers employ various complementary approaches to analyze KLK7 activity:

• Gelatin zymography: Purified active KLK7 is separated on gelatin gels, followed by incubation with renaturing and developing buffers. Visualization with Coomassie staining reveals protease activity as white bands against a dark blue background .

• Kinetic assays: Synthetic fluorogenic substrates (LLVY-AMC and AAPF-AMC) are used at increasing concentrations to determine kinetic parameters (kcat/Km), providing quantitative measures of enzymatic efficiency .

• Proteomics techniques: Both label-free quantitative proteomics and targeted quantitative proteomics (selected reaction monitoring) are employed for substrate identification .

• Cell-based assays: Treatment of cell lines (such as HaCaT keratinocytes) with purified KLK7 followed by analysis of conditioned media can identify physiologically relevant substrates .

• What molecular mechanisms connect KLK7 to metabolic regulation?

KLK7 influences metabolic homeostasis through several mechanisms:

• Adipose tissue remodeling: KLK7 affects adipose tissue development and expansion

• Immune modulation: Regulates macrophage infiltration and polarization in adipose tissue

• Thermogenic programming: Influences brown adipose tissue activity and white adipose tissue browning

• Insulin processing: In vitro studies show KLK7 can cleave insulin, potentially affecting its bioavailability

• Substrate specificity: Mediates proteolytic regulation of metabolic mediators

Studies in Klk7 knockout mice show increased energy expenditure, altered respiratory exchange ratio (indicating preference for carbohydrate oxidation), and enhanced thermogenic gene expression .

Advanced Research Questions

• How does KLK7 knockout affect adipose tissue function and glucose metabolism?

Genetic deletion of KLK7 produces a notable phenotype characterized by:

• Body composition changes: Klk7−/− mice exhibit a leaner phenotype with aging, showing significantly reduced body fat mass under both chow and high-fat diet conditions, while maintaining normal lean body mass

• Energy balance alterations: Despite lower fat mass, Klk7−/− mice demonstrate increased food intake, compensating for significantly higher energy expenditure as measured in metabolic chamber experiments

• Adipose tissue quality: KLK7 deletion prevents high-fat diet-induced browning of brown adipose tissue (BAT), with corresponding increases in Dio2 and Prdm16 expression

• Inflammatory profile: Adipose tissue from Klk7−/− mice shows significantly reduced macrophage infiltration with a shift toward anti-inflammatory M2 polarization rather than pro-inflammatory M1 polarization

• Glucose homeostasis paradox: Klk7−/− mice exhibit approximately 80% higher fasted and fed glucose concentrations with impaired glucose tolerance despite increased sensitivity to exogenous insulin in insulin tolerance tests

These findings suggest KLK7 functions as a significant metabolic regulator beyond its established role in skin physiology.

• What are the known biological substrates of KLK7 and how have they been identified?

KLK7 cleaves multiple physiologically relevant substrates:

Modern degradomics approaches combining mass spectrometry technologies (cell surface degradomics, TAILS, PROTOMAP) with sequence-based substrate specificity analysis have revolutionized KLK7 substrate identification . For instance, studies using midkine as an example demonstrated that KLK7-mediated cleavage reduced the pro-proliferative and migratory effects normally induced by full-length midkine .

• How does the proteome of adipose tissue change with KLK7 depletion?

Label-free quantitative proteome analyses in epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) from adipose-specific Klk7 knockout mice revealed:

• Depot-specific changes: Significantly affected proteins were primarily identified in iWAT (23 proteins under chow diet and 59 proteins under high-fat diet), with fewer changes in eWAT

• Diet-dependent alterations: Limited overlap between proteins identified under chow versus high-fat diet conditions, suggesting context-dependent KLK7 functions

• Unique protein expression: Higher numbers of proteins exclusively detected in iWAT (52 under chow and 66 under HFD) than in eWAT (8 under chow and 21 under HFD), indicating a more prominent role of KLK7 in subcutaneous adipose tissue

• Functional relevance: Differential expression of proteins previously linked to adipose tissue development and function, including COL3A1, CD163, NCK1, HFE, DNAJC3, and AGT

• Inflammatory signature: Regulators of immune response were upregulated, while proteins promoting macrophage infiltration during obesity-associated inflammation (such as CD5L) were reduced

These findings suggest KLK7 plays a depot-specific role in regulating adipose tissue proteome, particularly affecting subcutaneous fat depots.

• What is the relationship between KLK7 and insulin signaling?

KLK7 has a complex relationship with insulin signaling and glucose homeostasis:

• Direct insulin processing: In vitro studies demonstrate that KLK7 can cleave insulin

• Insulin action modulation: Inhibition of KLK7 activity has been associated with prolonged insulin circulation and action in vivo

• Paradoxical response patterns: Klk7−/− mice display increased sensitivity to exogenous insulin in insulin tolerance tests, but show impaired glucose tolerance with higher fasting and fed glucose levels

• Tissue-specific effects: Primary adipocytes isolated from Klk7−/− mice exhibit significantly lower insulin-induced AKT activation, suggesting cellular insulin resistance despite whole-body response to exogenous insulin

• Administration route differences: The differential response to intraperitoneal versus intravenous insulin administration suggests local effects of KLK7 on insulin degradation

The mechanisms underlying these observations remain incompletely understood, but may involve KLK7-mediated insulin processing affecting insulin bioavailability.

• How do KLK7 inhibitors work and what is their therapeutic potential?

KLK7 inhibitors represent a promising therapeutic approach for several conditions:

• Mechanism of action: These compounds selectively bind to KLK7's active site, preventing substrate access and proteolytic activity

• Clinical development: A KLK7-targeting depsipeptide is currently undergoing clinical trials as a novel therapeutic for skin barrier disruption

• Dermatological applications: Given KLK7's upregulation in atopic dermatitis, psoriasis, and other inflammatory skin conditions, inhibitors may reduce inflammation and restore normal barrier function

• Potential metabolic applications: Based on the phenotype of Klk7−/− mice, inhibition might theoretically be explored for metabolic disorders, though this remains speculative

• Delivery challenges: Tissue-specific targeting remains a challenge for development of systemic KLK7 inhibitors

The most advanced clinical applications focus on dermatological conditions, where topical delivery can achieve effective local inhibition while minimizing systemic effects.

• What experimental models are available for studying KLK7 function?

Researchers employ various experimental systems to investigate KLK7 biology:

• Genetic knockout models:

  • Global Klk7−/− mice: Complete ablation of KLK7 throughout the body

  • Adipose-specific Klk7−/− mice (ATKlk7−/−): Tissue-specific deletion to study adipose-centric functions

• Cell culture systems:

  • HaCaT keratinocytes: Human keratinocyte cell line for studying KLK7 in skin biology

  • Melanoma cell lines (WM35, WM902, WM9): Models for investigating KLK7 in cancer contexts

  • Primary adipocytes: Isolated from different fat depots for metabolic studies

• Recombinant protein systems:

  • Purified active KLK7 for in vitro enzymatic assays

  • Substrate specificity profiling using synthetic peptide libraries

• Disease models:

  • Diet-induced obesity models: High-fat diet feeding to study metabolic effects

  • Murine models of atopic dermatitis-like skin conditions

These complementary approaches enable comprehensive investigation of KLK7 biology across different physiological contexts.

• How does KLK7 expression change in pathological conditions?

KLK7 shows distinct expression patterns across various pathological states:

• Atopic dermatitis: RNA-seq analyses identified KLK7 as the most differentially upregulated KLK in human AD skin. In murine models of AD-like skin, Klk7 showed approximately 4-fold expression increase compared to controls

• Metabolic disorders: While direct human data is limited, mouse models suggest potential alterations in KLK7 expression in metabolic contexts, particularly in adipose tissue under high-fat diet conditions

• Skin disorders: Beyond AD, KLK7 expression is dysregulated in multiple skin pathologies including psoriasis, Netherton syndrome, and rosacea

• Cancer: KLK7 expression has been detected in melanoma cell lines, suggesting potential involvement in malignancy

The regulatory mechanisms controlling these expression changes remain incompletely characterized but represent important targets for therapeutic intervention.

• What role does KLK7 play in adipose tissue browning?

KLK7 appears to regulate adipose tissue thermogenic programming:

• Thermogenic gene expression: In chow-fed Klk7−/− mice, genes and proteins related to brown adipose tissue (BAT) activity, mitochondrial function, and white adipose tissue browning were significantly upregulated compared to controls

• BAT phenotype preservation: Klk7−/− mice showed resistance to high-fat diet-induced whitening of brown adipose tissue, with maintained expression of BAT markers Dio2 and Prdm16

• Metabolic consequences: Enhanced thermogenic programming likely contributes to the higher energy expenditure observed in Klk7−/− mice despite increased food intake

• Respiratory exchange ratio: Klk7−/− mice exhibited higher respiratory exchange ratios, indicating preferential carbohydrate oxidation, often associated with enhanced BAT activity

These findings suggest KLK7 may function as a negative regulator of adipose tissue thermogenic programming, with its absence promoting beneficial metabolic adaptations.