title: Transcriptional gene silencing of kallikrein 5 and kallikrein 7 using siRNA prevents epithelial cell detachment induced by alkaline shock in an in vitro model of eczema.
abstract: Eczema is widely considered to be an exacerbation of alkaline stress to the skin. Epidermal barrier dysfunction is a feature of eczema pathology, which predisposes affected individuals to distressing morbid symptoms. At least two serine proteases, stratum corneum chymotryptic enzyme (kallikrein 7 [KLK7]) and stratum corneum tryptic enzyme (kallikrien 5 [KLK5]), have increased activity levels in eczematous lesions and both have been implicated in the destruction of corneodesomosomes, which are crucial to epidermal integrity. The present in vitro study investigated whether transcriptional gene silencing after siRNA transfection could influence the activity of these signature enzymes in an in vitro model of eczema induced by alkaline shock. HaCaT epithelial cells were subjected to alkaline stress by the addition of 1,1,3,3-tetramethyl guanidine "superbase" (TMG) to the culture media. The culture media were subsequently tested for chymotryspin, trypsin, plasmin, and urokinase activity using colorimetric peptide assays and for reactive oxygen species using WST1 cell viability reagent. Cells that had been transfected with small interfering ribonucleic acid (siRNA) against KLK5 and KLK7 for 24 h before alkaline shock did not exhibit the increase in serine protease levels observed in untreated controls. Moreover, an endpoint MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) confirmed that detachment of cells from the culture substrate observed in alkaline-stressed cells did not occur in siRNA-treated cells. This in vitro study has established the proof-of-principle that siRNA therapy appears to mitigate the consequences of alkaline shock to the serine protease-associated fragility of epithelial cells that is characteristic of eczema.
authors.affiliation: School of Pharmacy and Centre for Skin Sciences, University of Bradford, Bradford BD71DP, UK. s.t.britland@brad.ac.uk
authors.forename: Stephen
authors.initials: S
authors.lastname: Britland
authors.forename: Milli
authors.initials: M
authors.lastname: Hoyle
mesh_terms: D000468:Alkalies / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D004485:Eczema / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D007610:Kallikreins / Q000235:genetics* / Q000378:metabolism; D008954:Models, Biological; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000201:enzymology
chemical_list: D000468:Alkalies; D034741:RNA, Small Interfering; C400147:KLK5 protein, human; C082472:KLK7 protein, human; D007610:Kallikreins