Chymotrypsin activity signals to intestinal epithelium by protease-activated receptor-dependent mechanisms.

BACKGROUND AND PURPOSE: Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to epithelial cells and that chymotrypsin signals to them via protease-activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells. EXPERIMENTAL APPROACH: The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids. KEY RESULTS: We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin-10 (IL-10) up-regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin. CONCLUSION AND IMPLICATIONS: Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.

Involvement of CATSPER 2 mutation in a familial context of unexplained infertility and fertilization failure associated with hearing loss: a case report.

Objective: To explore the functional implications of a homozygous CATSPER 2 (cation channel for sperm) deletion within the acrosome reaction pathway during fertilization in 2 brothers, who have unexplained infertility and hearing loss. Design: Case report. Patients: Two twin brothers aged 30 years with hearing loss and unexplained infertility. Exposure or Intervention: Molecular genetic diagnosis of deafness. Evaluation of the acrosome reaction and calcium mobilization assays after induction by progesterone and ionomycin on spermatozoa of the CATSPER 2-mutated patient and on fertile controls. Main Outcome Measures: Fertilization rate during conventional in vitro fertilization. Molecular genetic test. Percentage of acrosome-reacted spermatozoa with peanut agglutinin lectin staining. Recording of progesterone and ionomycin-induced intracellular calcium signals with a fluorescent probe. Results: Mr. S and his brother have normal, conventional sperm parameters. Both brothers have had repeated intrauterine insemination failures and one fertilization failure after conventional in vitro fertilization. Mr. S obtained 2 healthy babies after intracytoplasmic sperm injection. Genetic analysis found a homozygote deletion of the STRC (stereocilin) gene (NM 153700: c.1-? 5328+?del) that removes the CATSPER 2 gene. Mutation of the STRC gene is known to be associated with hearing loss. Sperm functional tests revealed an inability of progesterone to activate intracellular calcium signaling and to induce acrosome reaction. Conclusion: We demonstrate the absence of a calcium signal and acrosome reaction after progesterone in our patient with a CATSPER 2 mutation. We emphasize the importance of the male medical interview and of the genetic investigation of hearing loss. We show that in vitro fertilization-intracytoplasmic sperm injection is necessary, even where normal sperm parameters are present.

Elafin and its precursor trappin-2: What is their therapeutic potential for intestinal diseases?

Elafin and its precursor trappin-2 are known for their contribution to the physiological mucosal shield against luminal microbes. Such a contribution seems to be particularly relevant in the gut, where the exposure of host tissues to heavy loads of microbes is constant and contributes to mucosa-associated pathologies. The expression of trappin-2/elafin has been shown to be differentially regulated in diseases associated with gut inflammation. Accumulating evidence has demonstrated the protective effects of trappin-2/elafin in gut intestinal disorders associated with acute or chronic inflammation, or with gluten sensitization disorders. The protective effects of trappin-2/elafin in the gut are discussed in terms of their pleiotropic modes of action: acting as protease inhibitors, transglutaminase substrates, antimicrobial peptides or as a regulator of pro-inflammatory transcription factors. Further, the question of the therapeutic potential of trappin-2/elafin delivery at the intestinal mucosa surface is raised. Whether trappin-2/elafin mucosal delivery should be considered to ensure intestinal tissue repair is also discussed.

Epithelial production of elastase is increased in inflammatory bowel disease and causes mucosal inflammation.

Imbalance between proteases and their inhibitors plays a crucial role in the development of Inflammatory Bowel Diseases (IBD). Increased elastolytic activity is observed in the colon of patients suffering from IBD. Here, we aimed at identifying the players involved in elastolytic hyperactivity associated with IBD and their contribution to the disease. We revealed that epithelial cells are a major source of elastolytic activity in healthy human colonic tissues and this activity is greatly increased in IBD patients, both in diseased and distant sites of inflammation. This study identified a previously unrevealed production of elastase 2A (ELA2A) by colonic epithelial cells, which was enhanced in IBD patients. We demonstrated that ELA2A hyperactivity is sufficient to lead to a leaky epithelial barrier. Epithelial ELA2A hyperactivity also modified the cytokine gene expression profile with an increase of pro-inflammatory cytokine transcripts, while reducing the expression of pro-resolving and repair factor genes. ELA2A thus appears as a novel actor produced by intestinal epithelial cells, which can drive inflammation and loss of barrier function, two essentials pathophysiological hallmarks of IBD. Targeting ELA2A hyperactivity should thus be considered as a potential target for IBD treatment.

Colitis Linked to Endoplasmic Reticulum Stress Induces Trypsin Activity Affecting Epithelial Functions.

BACKGROUND AND AIMS: Intestinal epithelial cells [IECs] from inflammatory bowel disease [IBD] patients exhibit an excessive induction of endoplasmic reticulum stress [ER stress] linked to altered intestinal barrier function and inflammation. Colonic tissues and the luminal content of IBD patients are also characterized by increased serine protease activity. The possible link between ER stress and serine protease activity in colitis-associated epithelial dysfunctions is unknown. We aimed to study the association between ER stress and serine protease activity in enterocytes and its impact on intestinal functions. METHODS: The impact of ER stress induced by Thapsigargin on serine protease secretion was studied using either human intestinal cell lines or organoids. Moreover, treating human intestinal cells with protease-activated receptor antagonists allowed us to investigate ER stress-resulting molecular mechanisms that induce proteolytic activity and alter intestinal epithelial cell biology. RESULTS: Colonic biopsies from IBD patients exhibited increased epithelial trypsin-like activity associated with elevated ER stress. Induction of ER stress in human intestinal epithelial cells displayed enhanced apical trypsin-like activity. ER stress-induced increased trypsin activity destabilized intestinal barrier function by increasing permeability and by controlling inflammatory mediators such as C-X-C chemokine ligand 8 [CXCL8]. The deleterious impact of ER stress-associated trypsin activity was specifically dependent on the activation of protease-activated receptors 2 and 4. CONCLUSIONS: Excessive ER stress in IECs caused an increased release of trypsin activity that, in turn, altered intestinal barrier function, promoting the development of inflammatory process.

A role for early oral exposure to house dust mite allergens through breast milk in IgE-mediated food allergy susceptibility.

BACKGROUND: Successful prevention of food allergy requires the identification of the factors adversely affecting the capacity to develop oral tolerance to food antigen in early life. OBJECTIVES: This study sought to determine whether oral exposure to Dermatophagoides pteronyssinus through breast milk affects gut mucosal immunity with long-term effects on IgE-mediated food allergy susceptibility. METHODS: Gut immunity was explored in 2-week-old mice breast-fed by mothers exposed to D pteronyssinus, protease-inactivated D pteronyssinus, or to PBS during lactation. We further analyzed oral tolerance to a bystander food allergen, ovalbumin (OVA). In a proof-of-concept study, Der p 1 and OVA levels were determined in 100 human breast milk samples and the association with prevalence of IgE-mediated egg allergy at 1 year was assessed. RESULTS: Increased permeability, IL-33 levels, type 2 innate lymphoid cell activation, and Th2 cell differentiation were found in gut mucosa of mice nursed by mothers exposed to D pteronyssinus compared with PBS. This pro-Th2 gut mucosal environment inhibited the induction of antigen-specific FoxP3 regulatory T cells and the prevention of food allergy by OVA exposure through breast milk. In contrast, protease-inactivated D pteronyssinus had no effect on offspring gut mucosal immunity. Based on the presence of Der p 1 and/or OVA in human breast milk, we identified groups of lactating mothers, which mirror the ones found in mice to be responsible for different egg allergy risk. CONCLUSIONS: This study highlights an unpredicted potential risk factor for the development of food allergy, that is, D pteronyssinus allergens in breast milk, which disrupt gut immune homeostasis and prevents oral tolerance induction to bystander food antigen through their protease activity.

House dust mites activate nociceptor-mast cell clusters to drive type 2 skin inflammation.

Allergic skin diseases, such as atopic dermatitis, are clinically characterized by severe itching and type 2 immunity-associated hypersensitivity to widely distributed allergens, including those derived from house dust mites (HDMs). Here we found that HDMs with cysteine protease activity directly activated peptidergic nociceptors, which are neuropeptide-producing nociceptive sensory neurons that express the ion channel TRPV1 and Tac1, the gene encoding the precursor for the neuropeptide substance P. Intravital imaging and genetic approaches indicated that HDM-activated nociceptors drive the development of allergic skin inflammation by inducing the degranulation of mast cells contiguous to such nociceptors, through the release of substance P and the activation of the cationic molecule receptor MRGPRB2 on mast cells. These data indicate that, after exposure to HDM allergens, activation of TRPV1+Tac1+ nociceptor-MRGPRB2+ mast cell sensory clusters represents a key early event in the development of allergic skin reactions.

Sexual dimorphism in PAR2-dependent regulation of primitive colonic cells.

BACKGROUND: Sexual dimorphism in biological responses is a critical knowledge for therapeutic proposals. However, gender differences in intestinal stem cell physiology have been poorly studied. Given the important role of the protease-activated receptor PAR2 in the control of colon epithelial primitive cells and cell cycle genes, we have performed a sex-based comparison of its expression and of the effects of PAR2 activation or knockout on cell proliferation and survival functions. METHODS: Epithelial primitive cells isolated from colons from male and female mice were cultured as colonoids, and their number and size were measured. PAR2 activation was triggered by the addition of SLIGRL agonist peptide in the culture medium. PAR2-deficient mice were used to study the impact of PAR2 expression on colon epithelial cell culture and gene expression. RESULTS: Colonoids from female mice were more abundant and larger compared to males, and these differences were further increased after PAR2 activation by specific PAR2 agonist peptide. The proliferation of male epithelial cells was lower compared to females but was specifically increased in PAR2 knockout male cells. PAR2 expression was higher in male colon cells compared to females and controlled the gene expression and activation of key negative signals of the primitive cell proliferation. This PAR2-dependent brake on the proliferation of male colon primitive cells was correlated with stress resistance. CONCLUSIONS: Altogether, these data demonstrate that there is a sexual dimorphism in the PAR2-dependent regulation of primitive cells of the colon crypt.

Active thrombin produced by the intestinal epithelium controls mucosal biofilms.

Proteolytic homeostasis is important at mucosal surfaces, but its actors and their precise role in physiology are poorly understood. Here we report that healthy human and mouse colon epithelia are a major source of active thrombin. We show that mucosal thrombin is directly regulated by the presence of commensal microbiota. Specific inhibition of luminal thrombin activity causes macroscopic and microscopic damage as well as transcriptomic alterations of genes involved in host-microbiota interactions. Further, luminal thrombin inhibition impairs the spatial segregation of microbiota biofilms, allowing bacteria to invade the mucus layer and to translocate across the epithelium. Thrombin cleaves the biofilm matrix of reconstituted mucosa-associated human microbiota. Our results indicate that thrombin constrains biofilms at the intestinal mucosa. Further work is needed to test whether thrombin plays similar roles in other mucosal surfaces, given that lung, bladder and skin epithelia also express thrombin.

Functional Proteomic Profiling of Secreted Serine Proteases in Health and Inflammatory Bowel Disease.

While proteases are essential in gastrointestinal physiology, accumulating evidence indicates that dysregulated proteolysis plays a pivotal role in the pathophysiology of inflammatory bowel disease (IBD). Nonetheless, the identity of overactive proteases released by human colonic mucosa remains largely unknown. Studies of protease abundance have primarily investigated expression profiles, not taking into account their enzymatic activity. Herein we have used serine protease-targeted activity-based probes (ABPs) coupled with mass spectral analysis to identify active forms of proteases secreted by the colonic mucosa of healthy controls and IBD patients. Profiling of (Pro-Lys)-ABP bound proteases revealed that most of hyperactive proteases from IBD secretome are clustered at 28-kDa. We identified seven active proteases: the serine proteases cathepsin G, plasma kallikrein, plasmin, tryptase, chymotrypsin-like elastase 3 A, and thrombin and the aminopeptidase B. Only cathepsin G and thrombin were overactive in supernatants from IBD patient tissues compared to healthy controls. Gene expression analysis highlighted the transcription of genes encoding these proteases into intestinal mucosae. The functional ABP-targeted proteomic approach that we have used to identify active proteases in human colonic samples bears directly on the understanding of the role these enzymes may play in the pathophysiology of IBD.

PAR2-dependent activation of GSK3ß regulates the survival of colon stem/progenitor cells.

Protease-activated receptors PAR1 and PAR2 play an important role in the control of epithelial cell proliferation and migration. However, the survival of normal and tumor intestinal stem/progenitor cells promoted by proinflammatory mediators may be critical in oncogenesis. The glycogen synthase kinase-3ß (GSK3ß) pathway is overactivated in colon cancer cells and promotes their survival and drug resistance. We thus aimed to determine PAR1 and PAR2 effects on normal and tumor intestinal stem/progenitor cells and whether they involved GSK3ß. First, PAR1 and PAR2 were identified in colon stem/progenitor cells by immunofluorescence. In three-dimensional cultures of murine crypt units or single tumor Caco-2 cells, PAR2 activation decreased numbers and size of normal or cancerous spheroids, and PAR2-deficient spheroids showed increased proliferation, indicating that PAR2 represses proliferation. PAR2-stimulated normal cells were more resistant to stress (serum starvation or spheroid passaging), suggesting prosurvival effects of PAR2 Accordingly, active caspase-3 was strongly increased in PAR2-deficient normal spheroids. PAR2 but not PAR1 triggered GSK3ß activation through serine-9 dephosphorylation in normal and tumor cells. The PAR2-triggered GSK3ß activation implicates an arrestin/PP2A/GSK3ß complex that is dependent on the Rho kinase activity. Loss of PAR2 was associated with high levels of GSK3ß nonactive form, strengthening the role of PAR2 in GSK3ß activation. GSK3 pharmacological inhibition impaired the survival of PAR2-stimulated spheroids and serum-starved cells. Altogether our data identify PAR2/GSK3ß as a novel pathway that plays a critical role in the regulation of stem/progenitor cell survival and proliferation in normal colon crypts and colon cancer.

Presence of commensal house dust mite allergen in human gastrointestinal tract: a potential contributor to intestinal barrier dysfunction.

BACKGROUND: Abnormal gut barrier function is the basis of gut inflammatory disease. It is known that house dust mite (HDM) aero-allergens induce inflammation in respiratory mucosa. We have recently reported allergen from Dermatophagoides pteronyssinus (Der p1) to be present in rodent gut. OBJECTIVE: To examine whether Der p1 is present in human gut and to assess its effect on gut barrier function and inflammation. DESIGN: Colonic biopsies, gut fluid, serum and stool were collected from healthy adults during endoscopy. Der p1 was measured by ELISA. Effect of HDM was assessed on gut permeability, tight-junction and mucin expression, and cytokine production, in presence or absence of cysteine protease inhibitors or serine protease inhibitors. In vivo effect of HDM was examined in mice given oral HDM or protease-neutralised HDM. Role of HDM in low-grade inflammation was studied in patients with IBS. RESULTS: HDM Der p1 was detected in the human gut. In colonic biopsies from healthy patients, HDM increased epithelial permeability (p<0.001), reduced expression of tight-junction proteins and mucus barrier. These effects were associated with increased tumour necrosis factor (TNF)-α and interleukin (IL)-10 production and were abolished by cysteine-protease inhibitor (p<0.01). HDM effects did not require Th2 immunity. Results were confirmed in vivo in mice. In patients with IBS, HDM further deteriorated gut barrier function, induced TNF-α but failed to induce IL-10 secretion (p<0.001). CONCLUSIONS: HDM, a ubiquitous environmental factor, is present in the human gut where it directly affects gut function through its proteolytic activity. HDM may be an important trigger of gut dysfunction and warrants further investigation.

Mutations in TFIIH causing trichothiodystrophy are responsible for defects in ribosomal RNA production and processing.

The basal transcription/repair factor II H (TFIIH), found mutated in cancer-prone or premature aging diseases, plays a still unclear role in RNA polymerase I transcription. Furthermore, the impact of this function on TFIIH-related diseases, such as trichothiodystrophy (TTD), remains to be explored. Here, we studied the involvement of TFIIH during the whole process of ribosome biogenesis, from RNAP1 transcription to maturation steps of the ribosomal RNAs. Our results show that TFIIH is recruited to the ribosomal DNA in an active transcription-dependent manner and functions in RNAP1 transcription elongation through ATP hydrolysis of the XPB subunit. Remarkably, we found a TFIIH allele-specific effect, affecting RNAP1 transcription and/or the pre-rRNA maturation process. Interestingly, this effect was observed in mutant TFIIH-TTD cells and also in the brains of TFIIH-TTD mice. Our findings provide evidence that defective ribosome synthesis represents a new faulty mechanism involved in the pathophysiology of TFIIH-related diseases.

Mammalian HCA66 protein is required for both ribosome synthesis and centriole duplication.

Ribosome production, one of the most energy-consuming biosynthetic activities in living cells, is adjusted to growth conditions and coordinated with the cell cycle. Connections between ribosome synthesis and cell cycle progression have been described, but the underlying mechanisms remain only partially understood. The human HCA66 protein was recently characterized as a component of the centrosome, the major microtubule-organizing center (MTOC) in mammalian cells, and was shown to be required for centriole duplication and assembly of the mitotic spindle. We show here that HCA66 is also required for nucleolar steps of the maturation of the 40S ribosomal subunit and therefore displays a dual function. Overexpression of a dominant negative version of HCA66, accumulating at the centrosome but absent from the nucleoli, alters centrosome function but has no effect on pre-rRNA processing, suggesting that HCA66 acts independently in each process. In yeast and HeLa cells, depletion of MTOC components does not impair ribosome synthesis. Hence our results suggest that both in yeast and human cells, assembly of a functional MTOC and ribosome synthesis are not closely connected processes.

Elastase 2 is expressed in human and mouse epidermis and impairs skin barrier function in Netherton syndrome through filaggrin and lipid misprocessing.

The human epidermis serves 2 crucial barrier functions: it protects against water loss and prevents penetration of infectious agents and allergens. The physiology of the epidermis is maintained by a balance of protease and antiprotease activities, as illustrated by the rare genetic skin disease Netherton syndrome (NS), in which impaired inhibition of serine proteases causes severe skin erythema and scaling. Here, utilizing mass spectrometry, we have identified elastase 2 (ELA2), which we believe to be a new epidermal protease that is specifically expressed in the most differentiated layer of living human and mouse epidermis. ELA2 localized to keratohyalin granules, where it was found to directly participate in (pro-)filaggrin processing. Consistent with the observation that ELA2 was hyperactive in skin from NS patients, transgenic mice overexpressing ELA2 in the granular layer of the epidermis displayed abnormal (pro-)filaggrin processing and impaired lipid lamellae structure, which are both observed in NS patients. These anomalies led to dehydration, implicating ELA2 in the skin barrier defect seen in NS patients. Thus, our work identifies ELA2 as a major new epidermal protease involved in essential pathways for skin barrier function. These results highlight the importance of the control of epidermal protease activity in skin homeostasis and designate ELA2 as a major protease driving the pathogenesis of NS.

Evolutionarily conserved function of RRP36 in early cleavages of the pre-rRNA and production of the 40S ribosomal subunit.

Ribosome biogenesis in eukaryotes is a major cellular activity mobilizing the products of over 200 transcriptionally coregulated genes referred to as the rRNA and ribosome biosynthesis regulon. We investigated the function of an essential, uncharacterized gene of this regulon, renamed RRP36. We show that the Rrp36p protein is nucleolar and interacts with 90S and pre-40S preribosomal particles. Its depletion affects early cleavages of the 35S pre-rRNA and results in a rapid decrease in mature 18S rRNA levels. Rrp36p is a novel component of the 90S preribosome, the assembly of which has been suggested to result from the stepwise incorporation of several modules, including the tUTP/UTP-A, PWP2/UTP-B, and UTP-C subcomplexes. We show that Rrp36p depletion does not impair the incorporation of these subcomplexes and the U3 small nucleolar RNP into preribosomes. In contrast, depletion of components of the UTP-A or UTP-B modules, but not Rrp5p, prevents Rrp36p recruitment and reduces its accumulation levels. In parallel, we studied the human orthologue of Rrp36p in HeLa cells, and we show that the function of this protein in early cleavages of the pre-rRNA has been conserved through evolution in eukaryotes.

Kallikrein 5 induces atopic dermatitis-like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome.

Netherton syndrome (NS) is a severe genetic skin disease with constant atopic manifestations that is caused by mutations in the serine protease inhibitor Kazal-type 5 (SPINK5) gene, which encodes the protease inhibitor lymphoepithelial Kazal-type-related inhibitor (LEKTI). Lack of LEKTI causes stratum corneum detachment secondary to epidermal proteases hyperactivity. This skin barrier defect favors allergen absorption and is generally regarded as the underlying cause for atopy in NS. We show for the first time that the pro-Th2 cytokine thymic stromal lymphopoietin (TSLP), the thymus and activation-regulated chemokine, and the macrophage-derived chemokine are overexpressed in LEKTI-deficient epidermis. This is part of an original biological cascade in which unregulated kallikrein (KLK) 5 directly activates proteinase-activated receptor 2 and induces nuclear factor kappaB-mediated overexpression of TSLP, intercellular adhesion molecule 1, tumor necrosis factor alpha, and IL8. This proinflammatory and proallergic pathway is independent of the primary epithelial failure and is activated under basal conditions in NS keratinocytes. This cell-autonomous process is already established in the epidermis of Spink5(-/-) embryos, and the resulting proinflammatory microenvironment leads to eosinophilic and mast cell infiltration in a skin graft model in nude mice. Collectively, these data establish that uncontrolled KLK5 activity in NS epidermis can trigger atopic dermatitis (AD)-like lesions, independently of the environment and the adaptive immune system. They illustrate the crucial role of protease signaling in skin inflammation and point to new therapeutic targets for NS as well as candidate genes for AD and atopy.

Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea.

Acne rosacea is an inflammatory skin disease that affects 3% of the US population over 30 years of age and is characterized by erythema, papulopustules and telangiectasia. The etiology of this disorder is unknown, although symptoms are exacerbated by factors that trigger innate immune responses, such as the release of cathelicidin antimicrobial peptides. Here we show that individuals with rosacea express abnormally high levels of cathelicidin in their facial skin and that the proteolytically processed forms of cathelicidin peptides found in rosacea are different from those present in normal individuals. These cathelicidin peptides are a result of a post-translational processing abnormality associated with an increase in stratum corneum tryptic enzyme (SCTE) in the epidermis. In mice, injection of the cathelicidin peptides found in rosacea, addition of SCTE, and increasing protease activity by targeted deletion of the serine protease inhibitor gene Spink5 each increases inflammation in mouse skin. The role of cathelicidin in enabling SCTE-mediated inflammation is verified in mice with a targeted deletion of Camp, the gene encoding cathelicidin. These findings confirm the role of cathelicidin in skin inflammatory responses and suggest an explanation for the pathogenesis of rosacea by demonstrating that an exacerbated innate immune response can reproduce elements of this disease.

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.