title: Up-regulation of glucosylceramide synthase expression and activity during human keratinocyte differentiation.
abstract: During keratinocyte differentiation, the glycolipid, glucosylceramide (GlcCer), is thought to be synthesized, stored in intracellular lamellar granules and eventually extruded into the intercellular space where GlcCer is hydrolyzed to ceramide, a major component of the epidermal permeability barrier. Previous studies showed that GlcCer synthase (GCS) activity increases during keratinocyte differentiation; however, the mechanism by which GCS activity is regulated was not established. In the present study, we prepared anti-peptide antibodies and amplified cDNA probes based on the cDNA sequence for human GCS (Ichikawa, S., Sakiyama, H., Suzuki, G., Hidari, K. I.-P. J., and Hirabayashi, Y. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4638-4643) in order to study GCS expression during keratinocyte differentiation. Confluent human keratinocytes in culture were induced to terminally differentiate by elevation of Ca+2 in the medium without exogenous hormones or growth factors. GlcCer synthesis assayed in situ using a fluorescent ceramide analog increased approximately 5-fold during keratinocyte differentiation, peaking at day 6. Fluorescence microscopy studies of living keratinocytes showed that fluorescent ceramide and/or its metabolites accumulated in the Golgi in undifferentiated cells but targeted to unique vesicular structures that may be derived from the trans-Golgi region. Expression of both GCS mRNA, a approximately 3. 8-kilobase transcript on Northern blots, and GCS protein, a approximately 38-kDa polypeptide detected by Western blotting, increased dramatically (approximately 5-fold) during differentiation, reaching a maximum at about day 8. These results suggest that GCS is up-regulated at the transcriptional level during keratinocyte differentiation and provide the first direct evidence for GCS up-regulation in any cell type.
authors.affiliation: Department of Biochemistry and Molecular Biology, Thoracic Disease Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
authors.forename: R
authors.initials: R
authors.lastname: Watanabe
authors.forename: K
authors.initials: K
authors.lastname: Wu
authors.forename: P
authors.initials: P
authors.lastname: Paul
authors.forename: D L
authors.initials: DL
authors.lastname: Marks
authors.forename: T
authors.initials: T
authors.lastname: Kobayashi
authors.forename: M R
authors.initials: MR
authors.lastname: Pittelkow
authors.forename: R E
authors.initials: RE
authors.lastname: Pagano
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies; D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic; D005964:Glucosyltransferases / Q000032:analysis / Q000096:biosynthesis*; D006801:Humans; D007231:Infant, Newborn; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D012867:Skin / Q000166:cytology* / Q000201:enzymology; D013997:Time Factors; D014158:Transcription, Genetic
chemical_list: D000906:Antibodies; D010446:Peptide Fragments; D012333:RNA, Messenger; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; D002118:Calcium