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Expression of CD13/aminopeptidase N and CD10/neutral endopeptidase on cultured human keratinocytes

Keratinocytes actively participate in immune response and inflammation by secreting cytokines and chemokines. Membrane-bound peptidases serve as negative loop in controlling concentration of peptide signalling molecules. Recently, they have also been proposed as additional mechanism of cell-to-cell interaction and as signalling molecules. In this study we examined expression of two membrane-bound peptidases: aminopeptidase N (APN ; EC 3.4.11.2 ; CD13) and neutral endopeptidase (NEP ; EC 3.4.24.11 ; CD10) on nonstimulated cultured human keratinocytes obtained from healthy skin. Membrane expression of CD13 and CD10 was analysed by FACS and fluorescent microscope. Functional properties of CD13 and CD10 were examined by testing their enzymatic activity towards selective substrates. The data were compared to those obtained on cultured nonstimulated human skin fibroblasts expressing both CD13/APN and CD10/NEP. Approximately one third (i.e. 31.7 +/- 2.8% ; n=3) of culture keratinocytes express CD13 as compared to fibroblasts which are 100% CD13+ (n = 3). Density of CD13 on keratinocytes is several times lower than on fibroblasts. Membrane CD13 expression on keratinocytes was associated with significant enzyme activity, which on the basis of substrate (L-Ala-betaNA) and inhibitor (bestatin, actinonin) selectivity could be ascribed to aminopeptidase N. Kinetic parameter Vmax revealed lower APN activity expressed on +/-keratinocytes than on fibroblasts (Vmax = 1.49 M/60 min/5x104 cells +/- 0.08 for keratinocytes/n = 3/ vs Vmax = 4.09 +/- 0.76 M/60 min/5x104 cells for fibroblasts /n = 3/). Likewise, Km value of APN on keratinocytes was lower as compared to fibroblasts (Km = 0.307 mM +/- 0.090 for keratinocytes/n = 3/ vs Km = 0.766 mM +/- 0.065 for fibroblasts /n = 3/). CD13 demonstrated on cultured keratinocytes, is at least partly due to its constitutive expression since it was also found on freshly prepared epidermal skin cells. Inhibitors of APN, actinonin, bestatin and substance-P, as well as the APN blocking antibody WM-15, decreased keratinocytes growth. In contrast to membrane CD13 associated with APN enzyme activity, neither membrane CD10, nor its enzyme (NEP) activity could be found on the same keratinocyte samples. In conclusion, functional CD13, associated with APN activity, was found on about one third of cultured, non-stimulated keratinocytes, whereas no CD10/NEP was found on the same keratinocyte samples. Role of APN in regulation of keratinocyte growth is suggested, as its inhibition resulted in decreased keratinocyte growth.

human keratynocytes; CD13; aminopeptidase N/APN; CD10; neutral endopeptidase/NEP; human skin fibroblasts; membrane expression; substance-P; growth regulation

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