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

Abstract

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.

Immunohistochemical localization of proteoglycans in human periodontium.

“…Similar staining has also been observed for human skin (results not shown). The significance of this crossreactivity to the epithelial layer is not known, since the PGs of gingival epithelium are of heparan sulfate type (44) and human keratinocytes in culture synthesize relatively small PGs (45). However, we cannot rule out the possibility that this crossreactivity is due to nonspecific binding or that an epitope exists in the epithelial PGs that is recognized by MAb 5D5.…”

Connective tissue elements as diagnostic aids in periodontology

“…The culture of oral keratinocytes has resulted in the characterization of heparan sulfate proteoglycans with small amounts of chondroitin/dermatan sulfate (130,136).…”

A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin.

“…Heparan sulfate is closely related to heparin sulfate and has also been shown to exert growth-inhibitory effects in some cell types (11). These sulfated GAGs may be covalently linked to protein core molecules in the form of proteoglycans (PGs) and are produced and externalized by mammalian cells (11,23). Underexpression of sulfated PGs has also been correlated with diminished density-dependent growth inhibition and neoplastic transformation (11).…”