Increased expression of cathelicidin by direct activation of protease-activated receptor 2: possible implications on the pathogenesis of rosacea

Abstract

Purpose: Recent findings of increased cathelicidin protein and its proteolytic fragments in rosacea suggest a pathogenic role for cathelicidin in this disease. The relationship between cathelicidin and protease-activated receptor 2 (PAR-2) is therefore of interest, as PAR-2, expressed principally in keratinocytes, regulates pro-inflammatory cytokine expression in the skin. The purpose of this study was to determine the relationship between expression of PAR-2 and cathelicidin in rosacea and to test the effect of direct PAR-2 activation on cathelicidin expression in keratinocytes.

Materials and methods: Samples from 40 patients with clinicopathologic diagnosis of rosacea and facial skin tissue samples from 20 patients with no specific findings or milium without inflammation were retrieved. Intensities of immunohistochemical staining for PAR-2 and cathelicidin were compared between normal and rosacea-affected skin tissues. Additionally, correlations between PAR-2 and cathelicidin staining intensities within rosacea patients were analyzed. In cultured keratinocytes, changes in PAR-2, cathelicidin, and vascular endothelial growth factor (VEGF) mRNA and protein were analyzed after treatment with PAR-2 activating peptide (AP).

Results: Cathelicidin expression was significantly higher in rosacea skin tissues than in normal tissues (p<0.001), while PAR-2 expression was not significantly higher in rosacea tissues than in normal skin tissues. A positive correlation between PAR-2 and cathelicidin within rosacea samples was observed (R=0.330, p=0.037). After treatment of PAR-2 AP, both mRNA and protein levels for PAR-2, cathelicidin, and VEGF significantly increased in cultured keratinocytes, compared with PAR-2 control peptide treatment.

Conclusion: PAR-2 may participate in the pathogenesis of rosacea through activation of cathelicidin LL-37, a mediator of innate immune responses in the skin.

Keywords: Cathelicidin; LL-37; PAR-2; VEGF; rosacea.

Fig. 1

Significant correlation was observed between PAR-2 and cathelicidin expression in rosacea (R=0.330, p=0.037). PAR-2, protease-activated receptor-2.

Fig. 2

Clinical picture of a 38-year-old male patient with papulopustular rosacea (A) and immunohistochemical findings of PAR-2 (B) and cathelicidin (C) (H&E, ×400). Staining intensity of PAR-2, 2.5; cathelicidin, 2.5. Cathelicidin staining on normal skin tissue (staining intensity, 0) (D). PAR-2, protease-activated receptor-2.

Fig. 3

Clinical picture of a 69-year-old female patient with papulopustular rosacea (A) and immunohistochemical findings of PAR-2 (B) and cathelicidin (C) (H&E, ×400). Staining intensity of PAR-2, 2.5; cathelicidin 3. Cathelicidin staining on normal skin tissue (staining intensity, 0) (D). PAR-2, protease-activated receptor-2.

Fig. 4

Effect of PAR-2 AP on the mRNA expression of PAR-2, cathelicidin and VEGF in HaCaT cells. Real time RT-PCR of PAR-2, cathelicidin and VEGF in HaCaT cells after PAR-2 activating peptide and PAR-2 control peptide treatment. Each data point represents the mean (±SEM) result from three independent experiments. ^*p<0.05. AP, activating peptide; CP, control peptide; VEGF, vascular endothelial growth factor; PAR-2, protease-activated receptor-2; SEM, standard error of the mean.

Fig. 5

Effect of PAR-2 AP on the expression of PAR-2, cathelicidin and VEGF proteins in HaCaT cells. Western blotting against PAR-2, cathelicidin and VEGF in HaCaT cells after PAR-2 activating peptide and PAR-2 control peptide treatment. AP, activating peptide; CP, control peptide; GAPDH, glyceraldehyde phosphate dehydrogenase; PAR-2, protease-activated receptor-2; VEGF, vascular endothelial growth factor.