Protease-Activated Receptor 2 in inflammatory skin disease: current evidence and future perspectives

Summary of effects of activation of PAR2 on inflammatory skin diseases.

2.1.1. Expression and activation of PAR2 in skin

PAR2 is expressed in the granular layer of the epidermis, and on eosinophils, neutrophils, monocytes, macrophages, dendritic cells, mast cells, and T cells in the dermis (18–20). PAR2 can be activated by different endogenous or exogenous proteases, and its activation elicits changes in intracellular calcium levels. The main activators of PAR2 in the skin may be kallikreins (KLKs), serine protease channel-activating protein-1 (Cap1/Prss8), Cathepsin S, tryptase, and exogenous proteins such as dust mite and cockroach allergens (20). Stefansson et al. employed four human skin-derived KLKs to stimulate KNRK cells in vitro and conducted an intracellular calcium flux analysis to determine whether KLKs can cleave and activate PAR2. KLK5 and KLK14 were observed to activate PAR2, while KLK7 and KLK8 did not. KLK14 and PAR2 were also discovered to be co-expressed in inflammatory skin disease models, including rosacea and atopic dermatitis. This finding implies that KLKs may be endogenous PAR2 activators in keratinocytes and may be involved in the activation of PAR2 in the epidermis and the inflammatory response (21). Cathepsin S is a cysteine protease found in dendritic cells. It was further demonstrated to activate the inflammatory modulating agent PAR2 by cleaving its N-terminus-like serine proteases (22).

Both cockroach allergen extract and dust mite allergens can activate PAR2 through serine protease activity (23, 24). Kato et al. treat primary human keratinocytes with serine protease-rich extract of whole mite culture (WCE) and recombinant group 1 allergens (rDer f 1 and rDer p 1), which exclusively exhibit cysteine protease activity. They discovered that the protease activity of the WCE induced intracellular Ca²⁺ mobilization, whereas rDer f 1 and rDer p 1 did not. The results suggest that mite-derived serine protease activity may contribute to the pathogenesis of atopic dermatitis by activating keratinocytes via PAR2 activation (24). Jeong et al. further confirmed that allergens increased protease activities in the epidermis and delayed barrier recovery and lamellar body secretion in murine skin when applied topically to barrier-disrupted sites. Yet the barrier recovery was restored by the topical application of a PAR2 antagonist or protease inhibitors, indicating that cockroach allergens may be involved in skin barrier repair by activating PAR2 (23). The findings of Lee et al. demonstrated that supernatants from Cutibacterium acnes (C. acnes) cultures could activate PAR2 in keratinocytes, thereby upregulating the mRNA expression levels of inflammatory cytokines such as IL-1α, IL-8, and TNF-α (25). Ritchie et al. noted that PAR2 expression was increased in human umbilical vein endothelial cells (HUVECs) following treatment with TNF-α or IL-1β. Subsequently, pro-inflammatory factors may induce an increase in PAR2, which may ultimately generate a positive feedback loop in inflammatory skin diseases (26).

2.1.2. Impact of PAR2 on cell differentiation and skin barrier

Previous research has demonstrated that PAR2 is expressed above the basal layer in the human and mouse epidermis, and is most significantly expressed in the granular layer, suggesting that PAR2 may influence epidermal differentiation (27). Derian et al. reported that the activation of PAR2 inhibits the growth and differentiation of keratinocytes. Activation of PAR2 diminished protein expression of the differentiation marker transglutaminase-1 induced by either calcium or IFN-γ (28). Treatment with SLIGKV-NH2 has been shown to decrease the expression of markers of terminal differentiation in keratinocytes, including filaggrin, loricrin, and kallikrein 5 and 7 (29). Nevertheless, the influence of PAR2 on cell differentiation continues to be controversial. Demerjian et al. utilized caspase-14 and TUNEL(+) cells as corneocyte markers and concluded that PAR2 activation was indispensable for corneocyte terminal differentiation (30). Barr discovered that the PAR2 antagonist PZ-235 substantially inhibited the differentiation marker K10 in human keratinocytes (97). Shin et al. found that treated cultured immortalized keratinocyte cell line (SV-HEKs) with PAR2 agonist can increase loricrin and filaggrin expression, a terminal differentiation marker. They also noted that PAR2 expression in apocrine sweat glands is position-dependent. Strong PAR2 immunoreactivity was detected in the granular layer of normal human skin and the acrosyringium of eccrine sweat glands. In contrast, the granular layer of callused skin and the duct and gland cells of eccrine sweat glands exhibited faint PAR2 immunoreactivity, indicating that PAR2 is linked to the terminal differentiation of the epidermis and eccrine sweat glands (31). Lee et al. demonstrated that the small interfering RNA (siRNA)-mediated PAR2 knockdown in sebocytes led to aberrant differentiation and lipogenesis (32). The seemingly controversial effects of PAR2 on keratinocyte differentiation probably rely on the selection of differentiation markers, differences in experimental conditions and models, the complexity of signaling pathways and data analysis methods.

The primary function of the epidermis is to serve as a protective barrier against damage from the external environment and to prevent the leakage of water and electrolytes from the epidermis. Acute barrier disruption transiently elevates the ambient pH of normal SC from its usual acidic level (around 5.0) toward neutrality, subsequently activating serine proteases (SPs) in the outer epidermis. This increase in SP activity, in turn, activates PAR2, resulting in a delay in barrier recovery kinetics (30). Hachem et al. discovered that the permeability barrier’s recuperation is delayed and lamellar body (LB) secretion is inhibited by topical applications of the PAR2 agonist peptide SLIGRL. Conversely, PAR2 knockout mice exhibit accelerated barrier recovery and increased LB secretion, accompanied by enhanced lamellar body formation and elevated caveolin-1 expression. These results show that PAR2 is particularly important for the regulation of the permeability barrier and itch conduction (33). CAP1/Prss8 is a glycosyl-phosphatidylinositol-anchored serine protease mainly expressed in the granular and spinous layers of the epidermis and is crucial for epidermal barrier function. Frateschi et al. reported that transgenic expression of either CAP1/Prss8 (K14-CAP1/Prss8) or protease-activated receptor-2 (PAR2; Grhl3(PAR2/+)) in mouse skin leads to epidermal hyperplasia, ichthyosis, and itching. K14-CAP1/Prss8 ectopic expression impairs epidermal barrier function and causes skin inflammation. Surprisingly, the gross and functional manifestations of ectopic K14-CAP1/Prss8-induced phenotypes are wholly negated when they are introduced into a PAR2-null background, indicating that PAR2 is a critical mediator of epidermal barrier function (34).

The impact of PAR2 on barrier function has been confirmed by various clinical studies, the most notable being Netherton syndrome (NS). 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). The absence of LEKTI leads to the uncontrolled activity of kallikrein‐related peptidase 5 (KLK5), which activates KLK7, KLK14, and elastase 2 (ELA2). This premature degradation of desmosomes leads to the aberrant detachment of the stratum corneum from the granular layer (35, 36). Briot and colleagues provided that unregulated KLK5 directly activates PAR2 and induces NF-κB-mediated overexpression of TSLP, ICAM-1, TNFα, and IL-8, leading to increased permeability of the barrier and exacerbating skin inflammation and barrier defects (37). Briot et al. also generated Spink5/Par2 double knockout (DKO) mice. These mice display a dramatic decrease in TSLP expression, although stratum corneum detachment persists, confirming the role of the KLK5-PAR2 cascade in TSLP-mediated early proallergic signaling (38).

2.1.3. PAR2 involvement in skin inflammatory response and immune response

Inflammation is a defensive response characterized by redness, swelling, heat, pain, and impaired function. Notably, PARs can actively modulate cellular signaling in a variety of inflammatory regions throughout the human body to facilitate or hinder inflammatory responses, suggesting a double-edged sword effect in inflammation (14, 39). For example, PAR2 activation was proven to increase intestinal wall thickness, granulocyte infiltration and bacterial translocation through the PAR2/Akt/mTOR pathway in inflammatory bowel disease (IBD) (40, 41). Whereas, blockage of PAR2 exacerbates inflammation in IBD co-occurring with metabolic syndrome partly through autophagy inhibition (42). This implies that PAR2 may have pleiotropic effects on inflammation depending on the environment.

PAR2 can regulate cytokine responses, induce lymphocyte maturation and assist in the formation of immune organs (43). Thrombin efficiently cleaves PAR2 in a thrombomodulin(TM)-dependent manner, resulting in pro-inflammatory IL-8 release (44). It is worth mentioning that leukocyte migration, as one of the immune responses to inflammation, can be regulated by PAR2 through β-arrestin (45). PAR2 activation can increase vascular permeability and produce cytokines and adhesion molecules in the early phases of skin inflammation, resulting in further leukocyte aggregation (18, 19, 25, 46). Palmer et al. observed that activation of PAR2 on the surface of mast cells leads to degranulation and release of inflammatory mediators (47). Studies have shown that in human keratinocytes, PAR2 activation induces NF-κB activation, resulting in the production of pro-inflammatory cytokines and inducing Th2-associated inflammation ( Figure 2 ) (13, 48). Moreover, wide-type mice had significantly higher levels of pro-inflammatory mediators than PAR2-KO mice during the acute contact dermatitis onset, along with leukocyte recruitment (44, 48, 49). PAR2 could contribute to skin immunity, by enabling ‘non-immune’ cells like keratinocytes to participate in skin protective functions. Sharlow and his colleagues observed that PAR2 activation increased the phagocytic capability of macrophages, fibroblasts and keratinocytes could be particularly significant to processes like wound healing or inflammation. This PAR2-mediated increase in phagocytic capability correlated with an increase in actin polymerization and α-actinin reorganization, cell surface morphological changes and increased soluble protease activity (50).

Figure 3 illustrates the well-established impact of PAR2 on inflammatory skin diseases. Steinhoff et al. first observed positive PAR2 immunoreactivity in epidermal-dermal junction and perivascular areas in the skin of patients with AD and psoriasis, but not in normal skin (51). Giblin et al. generated a fully human PAR2 antibody specifically blocking the protease cleavage site in the N-terminal domain. The antibodies effectively inhibited PAR2-mediated intracellular calcium release and cytokine secretion in various cell types stimulated with trypsin. In a short-term mouse model of inflammation, the trans vivo DTH model, anti-PAR2 antibodies showed inhibition of the inflammatory swelling response, showing a 72% inhibition at a dose of 20 mg/kg (P<0.05), similar to the effect of dexamethasone at 10 mg/kg (52). These research findings indicate that PAR2 is involved in the inflammatory process in vivo and holds potential as a new therapeutic target for inflammatory skin conditions.

2.2.1. PAR2, acne vulgaris and seborrheic dermatitis

Acne vulgaris is a chronic inflammatory disease of the pilosebaceous unit. C. acnes triggers acne inflammation by releasing various enzymes such as lipases, proteases, and hyaluronidases (53). It has been demonstrated that the extracellular proteinase produced by C. acnes is a heterogeneous mixture of at least three proteases, including a neutral protease containing a serine group and two alkaline proteases (54). Lee and colleagues found increased protease activity in acne lesions and enhanced PAR2 immunoreactivity in the vicinity of comedones. The culture supernatant of C. acnes activated calcium signaling in keratinocytes through PAR2 and upregulated the mRNA expression of several inflammatory mediators, including interleukin (IL)-1α, IL-8, tumor necrosis factor (TNF)-α, human beta-defensin (hBD)-2, LL-37, and matrix metalloproteinases (MMP)-1, -2, -3, -9, and -13. This effect was significantly reduced by the presence of a serine protease inhibitor and a selective PAR2 antagonist. These findings suggest that PAR2 is crucial in acne pathogenesis by triggering inflammatory responses to proteases secreted by C. acnes ( 25).

Increased sebum content and altered composition are also important factors contributing to the development and progression of acne. PAR2 expression was detected at both the mRNA and protein levels in SZ95 sebocytes. Detection of intracellular Ca²⁺ mobilization by the C. acnes culture supernatant suggests that it is a potent activator of PAR2 on sebocytes. The mRNA levels of SREBP1 in sebocytes were elevated with the treatment of PAR2 agonist peptide(AP), and the protein levels of both precursor and mature forms of SREBP-1 increased as well. These findings suggest that PAR2 is involved in the differentiation and production of sebum in sebocytes. Moreover, PAR2 AP induced IL-8, TNF-a and hBD-2 transcription in sebocytes (32). These findings indicate that PAR2 probably plays a crucial role in the development and progression of acne, as it mediates differentiation, inflammation, and innate immunity in SZ95 sebocytes in response to C. acnes.

Antibiotics such as macrolides and tetracyclines are frequently employed to treat acne. Their therapeutic effects are not only related to antibacterial properties but also anti-inflammatory activity and immunomodulatory effects. Ishikawa et al. found that tetracycline and its derivatives, such as doxycycline and minocycline, significantly inhibited IL-8 release in normal human epidermal keratinocytes (NHEKs) triggered by the PAR2 agonist peptide SLIGKIV-NH2. This research suggests that tetracyclines can effectively reduce cutaneous inflammation by attenuating the PAR2/IL-8 axis in keratinocytes (55). Another study showed that doxycycline indirectly inhibits the production of KLKs and TSLP by reducing the secretion of MMPs in keratinocytes. This pathway may also be implicated in the anti-inflammatory process of doxycycline, as KLK5 and KLK14 have the ability to activate PAR2. Similarly, studies have found that macrolide antibiotics can modulate inflammatory response by attenuating the PAR2/IL-8 axis (56). Seborrheic dermatitis(SD) is also a skin disorder characterized by inflammatory scaling rush in seborrheic areas of the body. Viodé et al. demonstrated an increase in cathepsin S, PAR2 and histamine protein levels in SD lesions, indicating cathepsin S may be implicated in the pathogenesis of seborrheic dermatitis by activating PAR2 (57).

2.2.2. PAR2 and rosacea

Rosacea is a chronic inflammatory skin disorder, characterized by facial flushing, telangiectasia, inflammatory papules and pustules on the central region of the face (58). Previous studies have reported that serine proteases (SPs) activity and antimicrobial peptides (AMPs) expression are significantly elevated in the skin of rosacea patients (59). The epidermal antimicrobial peptide is degraded by SPs to its active form, cathelicidin LL-37. The latter can mediate vascular action and inflammation, which are features of rosacea. AMPs are important defenders in the innate immune response of the skin and can directly kill invading microorganisms as well as trigger the adaptive immune response, suggesting a potential therapeutic target for rosacea (60). Furthermore, cathelicidin LL-37 and PAR2 show significant positional correlation on immunohistochemistry. Cathelicidin LL-37, PAR2, and VEGF mRNA and protein levels were all elevated after the administration of a PAR2 agonist peptide to keratinocytes (61).

Zhong et al. conducted a two-week randomized, vehicle-controlled, split-face study on 30 rosacea patients using 3% tranexamic acid solution and vehicle-control treatment. Tranexamic acid, acting as a serine protease inhibitor, has demonstrated efficacy in hastening the repair of compromised skin barriers. The nuclei and cytoplasm of the normal epidermis exhibited a mild expression of PAR2, whereas rosacea patients exhibited a more intense staining of PAR2 in both the nuclei and cytoplasm. Tranexamic acid inhibits calcium mobilization induced by PAR2 activation, improves permeability barrier function and relieves clinical manifestations of rosacea patients (62). After 4-6 weeks of topical application of 10% tranexamic acid, Jakhar et al. observed a significant reduction in facial erythema, telangiectasia, and stinging sensation in patients with rosacea (63). We hypothesize that the pathogenesis of rosacea may be influenced by exogenous stimuli (such as heat, spicy food, UV, physical, and microbes) and serine proteases (such as kallikrein 5 and kallikrein 14) that upregulate cathelicidin LL-37 through enhanced PAR2 signaling. This, in turn, modulates innate immune responses and vascular activity.

2.2.3. PAR2 and psoriasis

Psoriasis is a chronic autoimmune skin disease characterized by red, itchy, and scaly skin patches (64). Research has demonstrated that PAR2 expression is elevated in the epidermis of psoriasis patients, and it may contribute to the pathogenesis of psoriasis by raising TNF-α (65). Moreover, the pathogenesis of psoriasis has been linked to the production of IL-8 by keratinocytes. A novel serine protease, human airway trypsin-like protease (HAT), was isolated from mucoid sputa of patients with chronic airway diseases. Iwakiri et al. detected that the immunofluorescence staining of HAT in psoriatic epidermis was more extensive and intense than that in normal skin. HAT treatment resulted in a synergistic increase in IL-8 release, while PAR-2 inhibition led to a decrease in IL-8 release induced by HAT (66). Therefore, HAT may facilitate the accumulation of inflammatory cells in the epidermal layer of psoriasis by promoting PAR2-mediated IL-8 production, thereby contributing to the aggregation of inflammatory cells in the psoriatic epidermis.

Carvalho et al. observed a substantial increase in the percentage of mast cells expressing PAR2 in the lesional skin of psoriasis compared to the non-lesional skin when analyzing the expression of PAR2 immunoreactivity in mast cells isolated from lesional and non-lesional skin of patients with plaque psoriasis. Activation of cord-blood-derived mast cells (CBMC) with the PAR2 agonist peptide (2-furoyl-LIGRLO-NH2) led to an increase in the secretion of IL-8, but no histamine release was observed (67). Abji et al. isolated tryptase-6, a serine protease that can activate PAR2, from the synovial fluid of patients with psoriatic arthritis (PsA) and observed increased levels of monocyte chemoattractant protein-1 (MCP-1) from PsA patient-derived monocytes/macrophages in response to PAR2 activation, highlighting a potential mechanism of PAR2-mediated recruitment of monocytes/macrophages to the PsA joint (68). In the murine psoriasis-like epidermis, KLK6, KLK7, and KLK8 were discovered to be overexpressed (69). Since PAR2 is a downstream target of KLKs, we hypothesized that overexpressed KLKs lead to inflammation and aberrant differentiation of keratinocytes in psoriasis occurrence.

2.2.4. PAR2 and atopic dermatitis

Atopic dermatitis (AD) is a chronic pruritic skin disease characterized by skin hyperreactivity, recurrent erythema, itching, and eczema-like lesions (70). PAR2 expression is significantly increased by keratinocytes throughout the epidermis in skin lesions of patients with AD and expresses at lower levels in normal skin (51). PAR2 is implicated in the development and occurrence of AD, as evidenced by the detection of KLK5, -7, -8, and -14 in the perspiration of AD patients (79). Epidermal serine protease activity is increased in the skin lesions of AD patients due to the overexpression of KLKs, which facilitates the invasion of microorganisms and allergens and further aggravates AD (24, 71). Furthermore, Briot and colleagues have previously reported that unregulated KLK5 directly activates PAR2 and induces NF-kB-mediated overexpression of TSLP, ICAM-1, TNF-α, and IL-8 (37). TSLP is instrumental in the development of AD by promoting the production of Th2 cell-promoting cytokines by activated mast cells in conjunction with TNF-α (72).

Growing evidence indicates that PAR2 is intimately involved in driving itch during atopic dermatitis. Proteases that activate PAR2 in primary sensory neurons stimulate the release of substance P and Calcitonin Gene-Related Peptide (CGRP) in peripheral tissues, leading to neurogenic inflammation and pruritus (73). TRPV3, a warm temperature-sensitive Ca²⁺-permeable cation channel that is abundantly expressed in cutaneous keratinocytes, has been confirmed to play a critical role in the transmission of PAR2-induced itch. Scratching would further activate the PAR2-TRPV3 pathway in the epidermis, leading to the release of TSLP and the exacerbation of the vicious itch-scratch cycle (74). Moreover, antihistamines lack efficacy in treating itch in AD, suggesting the existence of histamine-independent itch pathways in AD (73).

Transgenic mouse models as well as human studies have demonstrated that a strong association exists between PAR2 overexpression and AD. Frateschi et al. found that keratinocyte-specific overexpression of PAR2 results in AD-like lesions characterized by epidermal hyperplasia, ichthyosis, and itching (22). With the application of house dust mites, Meng et al. observed that Grhl3PAR2^/+ mice exhibit atopic-like inflammation, scaly dry skin, epidermal hyperplasia, pruritus behaviors, and increased brain natriuretic peptide transcription in adjacent sensory neurons (75). Buhl et al. established a murine model with epidermal overexpression of PAR2 (PAR2OE) and found that PAR2 is sufficient to drive barrier dysfunction, inflammation, and pruritus, highlighting the importance of serine proteases and PAR2 in AD pathogenesis (76).

Thus, PAR2 antagonism and/or selective protease inhibitors may represent a novel approach for the treatment of AD.Tsujii et al. investigated the involvement of tryptase and proteinase-activated receptor (PAR) subtypes in spontaneous scratching in NC mice. Intravenous administration of an anti-PAR2 antibody significantly reduced spontaneous scratching in mice with dermatitis. Conversely, intradermal injection of the PAR2-activating peptide SLIGRL-NH2 induced scratching behavior in healthy mice, whereas peptides activating PAR1, PAR3 or PAR4 did not produce the same effect (77). IL-27 is a member of the IL-6/IL-12 cytokine family, a group that also includes IL-31, a critical mediator in itch generation and cutaneous AD-associated pruritus. Li et al. discovered that the pruritus response in the cheek model was exacerbated by coinjection SLIGRL or trypsin and IL-27, which in turn increased the expression of Tslp via PAR2 (78). Passive cutaneous anaphylaxis-induced scratching behavior was substantially inhibited by pretreatment of the ICR animals with PAR2-neutralizing antibody and protease inhibitor (leupeptin) (79).

2.2.5. PAR2 and systemic sclerosis

Systemic sclerosis (SSc), also called scleroderma, is an immune-mediated rheumatic disease that is characterized by fibrosis of the skin and internal organs and vasculopathy. Fibroblasts are instrumental in the pathophysiology of fibrotic diseases by coordinating the remodeling of the extracellular matrix (80). Cevikbas et al. observed PAR2 immunoreactivity in fibroblasts of SSc skin, but not in healthy skin. Additionally, PAR2 co-localized with 21 ± 8% a-SMA-positive myofibroblasts. In human dermal cultured fibroblasts (HDF), PAR2-induced intracellular calcium mobilization was only measurable after bleomycin pretreatment, indicating PAR2 may be inducible and upregulated by an unknown stimulus in systemic SSc (81). Dipeptidyl peptidase-4 (DPP4) is a functional requirement for fibroblast activation and tissue fibrosis and may serve as an activation marker (82). The signaling signature induced by sDPP4 suggests that sDPP4 might be an agonist for PAR2. PAR2 silencing prevents sDPP4-induced ERK1/2 activation and NF-κB phosphorylation, as well as IL-6 and IL-8 upregulation in human smooth muscle cells (83). Lee et al. demonstrated that the PAR2 antagonist or knocking down PAR2 can prevent sDPP4–induced fibrotic marker expression via SMAD/NF-κb signaling in fibroblasts. This implies that PAR2 is a receptor for soluble DPP4 and facilitates the development of fibrosis (84).

PAR2 modulators, mechanism, and potential applications.