Up-regulation of glucosylceramide synthase expression and activity during human keratinocyte differentiation.
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.
Watanabe
R
R
Department of Biochemistry and Molecular Biology, Thoracic Disease Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Wu
K
K
Paul
P
P
Marks
D L
DL
Kobayashi
T
T
Pittelkow
M R
MR
Pagano
R E
RE
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
D000906:Antibodies; D010446:Peptide Fragments; D012333:RNA, Messenger; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; D002118:Calcium