Nitric oxide regulates synthesis of gene products involved in keratinocyte differentiation and ceramide metabolism.
During terminal differentiation of keratinocytes the expression of various proteins, which are required for the formation of the epidermal water barrier in the skin of land dwelling animals, is upregulated. Using a cell culture model for the differentiation of human keratinocytes and real-time PCR, we quantified the mRNA levels of several proteins involved in differentiation and ceramide metabolism. A calcium shift (1.1 mM CaCl2, 10 microM linoleic acid) for 8 days triggered an increase in mRNA levels of keratin 10 (75-fold), profilaggrin (55-fold), glucosylceramide synthase (40-fold), beta-glucocerebrosidase (30-fold), prosaposin (15-fold), acid sphingomyelinase (5-fold), and serine palmitoyltransferase (SPTLC2, 4-fold). However, mRNA levels of keratin 14 and acid ceramidase did not change significantly. On the other hand nitric oxide added at concentrations lower than 0.25mM stimulates proliferation of keratinocytes (Krischel et al., J. Invest. Dermatol. 111, 286-291, 1998). Accordingly, the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 0.2 mM) had no effect on the morphology of cultured keratinocytes, whereas in cultured human fibroblasts apoptosis was induced. The expression patterns obtained suggest that keratinocytes remain in a basal proliferative state, with a 3-fold increase in keratin 14 expression, a marked decrease in mRNA levels of differentiation markers and of most ceramide-metabolizing enzymes to negligible levels. The inhibitor of the NO synthase, N(G)-nitro-L-arginine-methyl ester (L-NAME, 10 mM), induced a transient increase in ceramide formation, followed by apoptosis in keratinocytes but not in fibroblasts. Both, SNAP and L-NAME, decreased the mRNA levels of all proteins involved in ceramide metabolism.
Gallala
Hichem
H
Kekulé Institut für Organische Chemie und Biochemie der Universität Bonn, Bonn, Germany.
Macheleidt
Oliver
O
Doering
Thomas
T
Schreiner
Volker
V
Sandhoff
Konrad
K
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology / Q000502:physiology; D002454:Cell Differentiation; D002478:Cells, Cultured; D002518:Ceramides / Q000235:genetics / Q000378:metabolism*; D000091344:Filaggrin Proteins; D002517:Galactosylgalactosylglucosylceramidase / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D005962:Glucosylceramidase / Q000235:genetics / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism; D015603:Keratinocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D007633:Keratins / Q000235:genetics / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D049231:Saposins / Q000235:genetics / Q000378:metabolism; D051102:Serine C-Palmitoyltransferase; D012867:Skin / Q000166:cytology; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics / Q000378:metabolism
D015415:Biomarkers; D002518:Ceramides; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; C483345:PSAP protein, human; D012333:RNA, Messenger; D049231:Saposins; D009569:Nitric Oxide; D007633:Keratins; D019001:Nitric Oxide Synthase; D000217:Acyltransferases; C495089:SPTLC2 protein, human; D051102:Serine C-Palmitoyltransferase; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; D013108:Sphingomyelin Phosphodiesterase; D005962:Glucosylceramidase; D002517:Galactosylgalactosylglucosylceramidase; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester