engleski

Expression of membrane peptidases on cultured human keratinocytes

Keratinocytes participate in immune response and inflammation by secreting cytokines and chemokines. Membrane-bound peptidases control local concentrations of signalling peptides and recently have been proposed as an additional mechanism of cell-to-cell interaction and signal transmission. We examined expression of three membrane-bound peptidases: aminopeptidase N (APN ; EC 3.4.11.2 ; CD13), neutral endopeptidase (NEP ; EC 3.4.24.11 ; CD10) and dipeptidyl-peptidase IV (DPPIV ; EC 3.4.14.5 ; CD26) on cultured keratinocytes obtained from normal human skin. Membrane expression of peptidase markers was assessed by means of monoclonal antibodies and FACS and their enzyme activity by measuring hydrolysis of selective substrates. Cultured human skin fibroblasts served as positive controls. CD13 was expressed on 36.0 +/- 19.4% of cultured keratinocytes (n = 25) as compared to fibroblasts which are 99% CD13+ (n = 6). Density of CD13 on keratinocytes was several times lower than on fibroblasts. Expression of CD13 on keratinocytes was associated with significant APN enzyme activity. Inhibitors of APN, actinonin, and substance-P, as well as the APN blocking antibody WM-15, decreased keratinocyte proliferation. The ability of actinonin to inhibit APN enzyme activity was stronger than its ability to suppress keratinocyte proliferation. APN activity was inhibited up to 80% (IC50 = 0.18 microM) and the cell proliferation maximally for 25%. The growth inhibition by actinonin occurred in the range of high (50 to 100 microM) or low concentrations (0.2 to 1.5 microM). That bimodal dose-response probably reflects inhibitory action on cytosolic aminopeptidases (at high concentrations) and on membrane APN (at low concentrations). CD10 and CD26 membrane expression on cultured keratinocytes was negligible (1.6 +/- 1.7% ; n=10 and 2.5 +/- 2.0% ; n=16) as well as their corresponding enzyme activities (NEP and DPPIV). Expression of membrane peptidases was also meassured on freshly isolated epidermal cell samples before culturing. CD13 was found in 5/5 samples tested, but at a lower level (18.9 +/- 10.2%) than on cultured keratinocytes. CD10 was practically absent in two epidermal cell samples tested before culturing (2.2 +/- 1.6%) but surprisingly a high level of CD26 (61.6 +/- 10.5%) was detected. Its expression decreased to negligible values in subsequent passages. In conclusion, functional CD13 with measurable APN activity was found in 36% of cultured, non-stimulated keratinocytes. Its inhibition interfered with keratinocyte proliferation. No significant expression of other two membrane peptidases, CD10/NEP and CD26/DPPIV was found in the same keratinocyte samples. The data suggest a role of APN in regulation of keratinocyte growth.

human keratinocytes; CD13, aminopeptidase N/APN; CD10, neutral endopeptidase/NEP; CD26, dipeptidyl-peptidase IV; human skin fibroblasts; membrane expression; substance-P; growth regulation

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