Protease-activated receptor-2 mediates the expression of inflammatory cytokines, antimicrobial peptides, and matrix metalloproteinases in keratinocytes in response to Propionibacterium acnes

Abstract

Propionibacterium acnes (P. acnes) has been known to produce various exogenous proteases, however, their role in acne pathogenesis remains largely unknown. Proteases elicit cellular responses, at least in part, via proteinase-activated receptor-2 (PAR-2), which is known to mediate inflammation and immune response. In this study, we investigated whether proteases from P. acnes could activate PAR-2 on keratinocytes and induce pro-inflammatory cytokines, antimicrobial peptides (AMPs), and matrix metalloproteinases (MMPs) via PAR-2 signaling. We examined PAR-2 expression and protease activity in acne lesions using immunofluorescence staining and in situ zymography. The effect of the culture supernatant of P. acnes on Ca(2+) signaling in immortalized keratinocytes (HaCaT) was measured using a fluorescence method. HaCaT cells were treated with P. acnes strain ATCC 6919 culture supernatant, with or without pretreatment with serine protease inhibitor or selective PAR-2 antagonist and the gene expression of pro-inflammatory cytokines, AMPs, and MMPs was detected using real-time reverse transcription-polymerase chain reaction. We found that the protease activity and PAR-2 expression were increased in acne lesions. The P. acnes culture supernatant induced calcium signaling in keratinocytes via PAR-2 and stimulated the mRNA expression of interleukin (IL)-1α, -8, tumor necrosis factor (TNF)-α, human beta defensin (hBD)-2, LL-37, MMP-1, -2, -3, -9, and -13 in keratinocytes, which was significantly inhibited by serine protease inhibitor as well as selective PAR-2 specific antagonist. These results indicate that PAR-2 plays an important role in the pathogenesis of acne by inducing inflammatory mediators in response to proteases secreted from P. acnes.

Fig. 1

In situ zymography analysis. Total protease activities of the lesions and perilesional normal-appearing skin obtained from acne patients were detected by the degradation of the BODIPY FL casein substrate. Protease activity was observed as the intensity of green fluorescence. Lesions of acne showed higher protease activity compared to the perilesional normal-appearing skin. SC stratum corneum, SG stratum granulosum, SB stratum basale

Fig. 2

PAR-2 expression in the comedonal lesions of patients with acne vulgaris and nevus comedonicus. PAR-2 immunostaining was performed using rabbit polyclonal anti-PAR-2 (H99, Santa Cruz Biotechnology, CA, USA) antibody and FITC-labeled goat anti-rabbit antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Immunoreactivity of PAR-2 was highly expressed in comedone of patients with acne vulgaris (a) in comparison to that of patients with nevus comedonicus (b). (c) and (d) Enlarged views in the white box in a and b, respectively

Fig. 3

PAR-2 activation induced by the culture supernatant of P. acnes ATCC6919. HaCaT cells were stimulated with SLIGKV (a) or P. acnes supernatant (b), and intracellular Ca²⁺ mobilization was measured using a fluorescence method as described in the materals and methods section. Fluorescence was measured over 120 s. ENMD-1068 was incubated with cells 6 h prior to addition of agonist stimulation (c). Desensitization protocol showed that these calcium responses are induced via PAR-2 signaling (d). RFU relative fluorescence units

Fig. 4

Effects of serine protease inhibitor or PAR-2 specific antagonist on P. acnes supernatant-induced mRNA expression of pro-inflammatory cytokines in HaCaT cells. The mRNA expression levels of IL-1α, IL-6, IL-8, and TNF-α in HaCaT cells treated with the culture supernatant of P. acnes for 18 h with or without pretreatment with serine protease inhibitor or selective PAR-2 antagonist were determined using RT-PCR. The mRNA levels were normalized to human β-actin and are presented as fold increases over the control (untreated cells) values. Data are mean ± standard deviation. Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01) versus the control. Double crosses indicate statistically significant differences (^# P ≤ 0.05, ^## P ≤ 0.01) between the P. acnes sup-treated and inhibitor-pretreated group

Fig. 5

Effects of serine protease inhibitor or selective PAR-2 antagonist on P. acnes culture supernatant-induced mRNA expression of antimicrobial peptides in HaCaT cells. The mRNA expression levels of hBD-2 and LL-37 in HaCaT cells treated with the culture supernatant of P. acnes ATCC6919 for 12 h (a) or AP for 3, 6, and 12 h (b) were determined using RT-PCR. The mRNA levels were shown as a fold increase versus 0 h untreated samples (b). Cells were pretreated with serine protease inhibitor or selective PAR-2 antagonist and then stimulated with P. acnes supernatant for 12 h (a). The mRNA levels were normalized to human β-actin and are presented as fold increases over the control (untreated cells) values. Data are mean ± standard deviation. Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01) vs the control. Double crosses indicate statistically significant differences (^# P ≤ 0.05, ^## P ≤ 0.01) between the P. acnes sup-treated and inhibitor-pretreated group

Fig. 6

Effects of serine protease inhibitor or selective PAR-2 antagonist on P. acnes culture supernatant-induced mRNA expression of matrix metalloproteinases in HaCaT cells. The mRNA expression levels of MMP-1, -2, -3, -9, -13, TIMP-1, and -2 in HaCaT cells treated with the culture supernatant of P. acnes ATCC6919 for 12 and 18 h (a, b) or AP for 3, 6, 12, and 18 h (c, d) were determined using RT-PCR. The mRNA levels were shown as a fold increase versus 0 h untreated samples (a–d). Cells were pretreated with serine protease inhibitor or selective PAR-2 antagonist and then stimulated with P. acnes supernatant for 18 h and assessed the mRNA expression levels of MMP-1, -2, -3, -9, and -13 (e). The mRNA levels were normalized to human β-actin and are presented as fold increases over the control (untreated cells) values. Data are mean ± standard deviation. Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01) versus the control. Double crosses indicate statistically significant differences (^# P ≤ 0.05, ^## P ≤ 0.01) between the P. acnes sup-treated and inhibitor-pretreated group