Biosynthesis of proteoglycans by proliferating and differentiating normal human keratinocytes cultured in serum-free medium

Normal human keratinocytes (NHK) were cultured in serum-free medium, containing low (0.1 mM) or high (2 mM) calcium, to obtain proliferating and differentiating cultures, respectively. Proteoglycan (PG) synthesis of proliferating and differentiating NHK was investigated. Cultures were labeled with 3...

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Bibliographic Details
Published inJournal of cellular physiology Vol. 140; no. 1; p. 98
Main Authors Rahemtulla, F, Moorer, C M, Wille, Jr, J J
Format Journal Article
LanguageEnglish
Published United States 01.07.1989
Subjects
Calcium - metabolism
Cell Differentiation
Cell Division
Cells, Cultured
Chondroitin Sulfates - analysis
Chromatography, Gel
Chromatography, Ion Exchange
Dermatan Sulfate - analysis
Epidermis - metabolism
Glycosaminoglycans - analysis
Heparitin Sulfate - analysis
Humans
Keratins - metabolism
Male
Proteoglycans - biosynthesis
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Summary:Normal human keratinocytes (NHK) were cultured in serum-free medium, containing low (0.1 mM) or high (2 mM) calcium, to obtain proliferating and differentiating cultures, respectively. Proteoglycan (PG) synthesis of proliferating and differentiating NHK was investigated. Cultures were labeled with 35S-sulfate, and the PGs were extracted from medium and cell layer. The newly synthesized PGs were isolated by ion-exchange chromatography on a column of DEAE-Sephacel. The molecular properties of the PGs and the size and composition of glycosaminoglycans (GAGs) were determined. In general, the PGs are relatively small size (Mr 70,000-120,000). The PGs of proliferating cultures are larger in molecular size than the PGs of differentiating cultures, and this is due to the degradation of the GAG chains. The molecular weight of the GAG chains of proliferating NHK ranged from 4,800 to 22,000, and the range for GAGs from differentiating cultures varied from 2,800 to 9,600. By compositional analysis, these PGs proved to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate as determined by nitrous acid degradation, and chondroitinase ACII and ABC digestion. No significant differences were found in the overall GAG composition of the medium secreted PGs of proliferating and differentiating cultures. In contrast, cell-associated PGs of differentiating cells had higher levels of heparan sulfate than those of proliferating cells.
ISSN:0021-9541
DOI:10.1002/jcp.1041400113