Serine Protease Signaling of Epidermal Permeability Barrier Homeostasis

Abstract

Evidence is growing that protease-activated receptor-2 (PAR-2) plays a key role in epithelial inflammation. We hypothesized here that PAR-2 plays a central role in epidermal permeability barrier homeostasis by mediating signaling from serine proteases (SP) in the stratum corneum (SC). Since the SC contains tryptic- and chymotryptic-like activity, we assessed the influence of SP activation/inhibition on barrier function. Acute barrier disruption increases SP activity and blockade by topical SP inhibitors (SPI) accelerates barrier recovery after acute abrogation. This improvement in barrier function is due to accelerated lamellar body (LB) secretion. Since tryptic SP signal certain downstream responses through PAR-2, we assessed its potential role in mediating the negative effects of SP on permeability barrier. Firstly, PAR-2 is expressed in the outer nucleated layers of the epidermis and most specifically under basal condition to the lipid raft (LR) domains. Secondly, tape stripping-induced barrier abrogation provokes PAR-2 activation, as shown by receptor internalization (i.e. receptor movement from LR to cytolpasmic domains). Thirdly, topical applications of PAR-2 agonist peptide, SLIGRL, delay permeability barrier recovery and inhibit LB secretion, while, conversely, PAR-2 knockout mice display accelerated barrier recovery kinetics and enhanced LB secretion, paralleled by increased LR formation and caveolin-1 expression. These results demonstrate first, the importance of SP/SPI balance for normal permeability barrier homeostasis, and second, they identify PAR-2 as a novel signaling mechanism of permeability barrier, that is, of response linked to LB secretion.

Acute Modulations in Permeability Barrier Function Regulate Epidermal Cornification

“…9 Two signaling mechanisms are known to regulate the lamellar body secretory response in the epidermis underlying disrupted skin sites: an abrupt decline in epidermal calcium levels stimulates secretion of preformed lamellar bodies from the outer granular layer; 10 -14 and acute barrier disruption also raises the ambient pH of normal SC transiently from its usual acidic levels (ϳ5.0) toward neutrality, which, in turn, activates serine proteases (SPs) in the outer epidermis, a sequence that retards barrier recovery kinetics. [15][16][17] Conversely, either immediate reacidification of SC, 18 or applications of SP inhibitors accelerates barrier recovery. 15,19 The increase in SP activity that results from either barrier disruption or a discrete increase in the pH of SC, in turn, activates the proteaseactivated receptor type 2 (PAR2), which is expressed on the plasma membranes of cells of the stratum granulosum.…”

Lipid Rafts and Caveolae in the Terminal Differentiation of Epidermal Keratinocytes

“…PAR-2 ko demonstrate higher levels of proliferation (PCNA assay), suggesting enhanced epidermal proliferation. In addition, recent data from our group [18] and others [36] suggest that PAR-2 is localized to the LR membrane domains and thus could be implicated in the spatio-temporal distribution of raft domains by organizing the cytoskeletal proteins of keratinocytes. Using the cholera toxin assay to assess LR, PAR-2 -/-mice demonstrate abnormally increased LR formation both under basal and barrier abrogation conditions.…”

“…Thus, SP from the SC activate PAR-2, which may signal terminal differentiation together with LB secretion arrest [18]. PAR-2 ko demonstrate higher levels of proliferation (PCNA assay), suggesting enhanced epidermal proliferation.…”

“…Caveolae represent a subclass of those rafts and are enriched in caveolin proteins, a family of three (cav-1 to -3) small molecular weight (18)(19)(20)(21)(22)(23)(24) proteins, that cycle between the trans-Golgi network and the plasma membrane [9] (i.e. the natural flow of LB).…”

“…We performed western immunoblotting for both caveolin (2 and 3) proteins on protein extracts (lipid rafts -LR-and cytoplasmic -Cfractions) from both cav-1 -/-and +/+ littermates prior and following permeability barrier abrogation. The later was performed by cellophane tape stripping of the SC as described elsewhere [18]. Cav-2 proteins were undetectable by western immunoblotting of the cytoplasmic fraction ( Fig.…”