Hyaluronan is an abundant and rapidly turned over matrix molecule between the vital cell layers of the epidermis. In this study, epidermal growth factor (EGF) induced a coat of hyaluronan and a 3-5-fold increase in its rate of synthesis in a rat epidermal keratinocyte cell line that has retained its ability for differentiation. EGF also increased hyaluronan in perinuclear vesicles, suggesting concurrent enhancement in its endocytosis. Cell-associated hyaluronan was most abundant in elongated cells that were stimulated to migrate by EGF, as determined in vitro in a wound healing assay. Large fluctuations in the pool size of UDP-N-acetylglucosamine, the metabolic precursor of hyaluronan, correlated with medium glucose concentrations but not with EGF. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed no increase in hyaluronan synthases 1 and 3 (Has1 and Has3), whereas Has2 mRNA increased 2-3-fold in less than 2 h following the introduction of EGF, as estimated by quantitative RT-PCR with a truncated Has2 mRNA internal standard. The average level of Has2 mRNA increased from ϳ6 copies/ cell in cultures before change of fresh medium, up to ϳ54 copies/cell after 6 h in EGF-containing medium. A control medium with 10% serum caused a maximum level of ϳ21 copies/cell at 6 h. The change in the Has2 mRNA levels and the stimulation of hyaluronan synthesis followed a similar temporal pattern, reaching a maximum level at 6 h and declining toward 24 h, a finding in line with a predominantly Has2-dependent hyaluronan synthesis and its transcriptional regulation.Hyaluronan is a large glycosaminoglycan found in the extracellular space of most animal tissues. It forms a loose, highly hydrated, gel-like matrix that contributes to the maintenance of the extracellular space and facilitates nutrient diffusion. Furthermore, hyaluronan is involved in cell proliferation and differentiation, produces an environment favorable for migration (1), and stimulates cell locomotion (2, 3). Elevated tissue levels of hyaluronan occur during embryonic growth of tissues and organs (1), wound healing (4, 5), inflammation (6), and invasion of certain cancers (7-10).In skin epidermis, the narrow extracellular space surrounding keratinocytes contains a high concentration of hyaluronan (11, 12), as do other stratifying squamous epithelia (13, 14). The half-life of labeled epidermal hyaluronan in human skin organ culture is ϳ1 day (15), indicating fast local turnover by keratinocytes. The importance of the strikingly high concentration and turnover of hyaluronan in the multilayered squamous epithelia is not completely understood, but we have hypothesized that the former is necessary to maintain an extracellular space for the nutritional needs of the more superficial cell layers, whereas the latter allows the dramatic modulation of cell shape that occurs during differentiation and for the high migratory potential of keratinocytes that is activated, e.g. in wound healing (16).Unlike other glycosaminoglycans, hyaluronan is synthesized at the inne...

Hyaluronan, a major extracellular matrix molecule in the vital cell layers of skin epidermis, has been suggested to support proliferation and migration of keratinocytes, during challenges like wounding and inflammation. An organotypic keratinocyte culture originated from continuous rat epidermal keratinocyte cell line was subjected to the proliferative and antiproliferative growth factors epidermal growth factor and transforming growth factor beta, respectively, to study their influence on hyaluronan synthesis and epidermal morphology. Epidermal growth factor induced a 4-fold increase of epidermal hyaluronan concentration. This was associated with upregulation of the hyaluronan synthases Has2 and Has3, and the hyaluronan receptor CD44. 5-Bromo-2'-deoxyuridine labeling, basal cell height, and the thickness of vital epidermis were increased, reflecting the hyperplastic effects of epidermal growth factor. The expression of keratin 10 and the maturation of filaggrin were inhibited, and epidermal permeability barrier became less efficient, indicating compromised terminal differentiation by epidermal growth factor. In contrast, transforming growth factor beta reduced the content of hyaluronan and the mRNA of Has2 and Has3. At the same time, transforming growth factor beta suppressed keratinocyte proliferation and epidermal thickness, but retained intact differentiation. The results suggest that epidermal hyaluronan synthesis, controlled by epidermal growth factor and transforming growth factor beta through changes in the expression of Has2 and Has3, correlates with epidermal proliferation, thickness, and differentiation.

Keratinocyte growth factor (KGF) activates keratinocyte migration and stimulates wound healing. Hyaluronan, an extracellular matrix glycosaminoglycan that accumulates in wounded epidermis, is known to promote cell migration, suggesting that increased synthesis of hyaluronan might be associated with the KGF response in keratinocytes. Treatment of monolayer cultures of rat epidermal keratinocytes led to an elongated and lifted cell shape, increased filopodial protrusions, enhanced cell migration, accumulation of intermediate size hyaluronan in the culture medium and within keratinocytes, and a rapid increase of hyaluronan synthase 2 (Has2) mRNA, suggesting a direct influence on this gene. In stratified, organotypic cultures of the same cell line, both Has2 and Has3 with the hyaluronan receptor CD44 were up-regulated and hyaluronan accumulated in the epidermis, the spinous cell layer in particular. At the same time the expression of the early differentiation marker keratin 10 was inhibited, whereas filaggrin expression and epidermal permeability were less affected. The data indicate that Has2 and Has3 belong to the targets of KGF in keratinocytes, and support the idea that enhanced hyaluronan synthesis acts an effector for the migratory response of keratinocytes in wound healing, whereas it may delay keratinocyte terminal differentiation.

Normal and immortalized keratinocytes demonstrate large aggregates of lipid rafts, detectable by membrane staining with fluorescently tagged cholera toxin (CTx). As lipid rafts are known to regulate the function of many surface receptors, we wished to investigate their impact on the EGFR in HaCaT cells. When rafts were disrupted by cholesterol sequestration with methyl-beta-cyclodextrin (MbetaCD) or filipin III, EGFR rearranged into approximately micrometer large clusters outside the CTx(bright) raft aggregates. These clusters contained high concentrations of activated, tyrosine-phosphorylated EGFR exhibiting greatly reduced mobility in the fluorescence recovery after photobleaching experiments. EGFR activation led to the stimulation of extracellular signal-regulated kinase 2, the phosphorylated form of which translocated to the nucleus and stimulated growth of the MbetaCD-treated cells. Experiments with the specific antagonistic antibody proved that the activation of EGFR by lipid raft disruption occurred without the participation of the ligand. We hypothesize that cholesterol depletion leads to the release of EGFR from the damaged rafts into the small confined areas of the membrane, where the receptor molecules are likely to be spontaneously activated owing to a very high density and/or separation from the inhibitory factors remaining in the surrounding portions of the membrane.