Epidermal growth factor activates hyaluronan synthase 2 in epidermal keratinocytes and increases pericellular and intracellular hyaluronan.

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 approximately 6 copies/cell in cultures before change of fresh medium, up to approximately 54 copies/cell after 6 h in EGF-containing medium. A control medium with 10% serum caused a maximum level of approximately 21 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.

A preformed basal lamina alters the metabolism and distribution of hyaluronan in epidermal keratinocyte "organotypic" cultures grown on collagen matrices.

A rat epidermal keratinocyte (REK) line which exhibits histodifferentiation nearly identical to the native epidermis when cultured at an air-liquid interface was used to study the metabolism of hyaluronan, the major intercellular macromolecule present in basal and spinous cell layers. Two different support matrices were used: reconstituted collagen fibrils with and without a covering basal lamina previously deposited by canine kidney cells. REKs formed a stratified squamous, keratinized epithelium on both support matrices. Hyaluronan and its receptor, CD44, colocalized in the basal and spinous layers similar to their distribution in the native epidermis. Most (approximately 75%) of the hyaluronan was retained in the epithelium when a basal lamina was present while most (approximately 80%) diffused out of the epithelium in its absence. While REKs on the two matrices synthesized hyaluronan at essentially the same rate, catabolism of this macromolecule was much higher in the epithelium on the basal lamina (half-life approximately 1 day, similar to its half-life in native human epidermis). The formation of a true epidermal compartment in culture bounded by the cornified layer on the surface and the basal lamina subjacent to the basal cells provides a good model within which to study epidermal metabolism.

Spreading depression-induced gene expression is regulated by plasma glucose.

BACKGROUND AND PURPOSE: Plasma glucose and spreading depression (SD) are both determinants of brain ischemia. The purpose of this study was to examine whether plasma glucose affects SD-induced gene expression in the cortex. METHODS: SD was induced by topical application of KCl. Hyperglycemia and hypoglycemia were induced by intraperitoneal injection of glucose and insulin, respectively. The expression of c-fos, cyclooxygenase-2 (COX-2), protein kinase C-delta (PKCdelta), and heme oxygenase-1 (HO-1) was determined by in situ hybridization. RESULTS: SD alone induced expression of c-fos (by 340%), COX-2 (210%), HO-1 (470%), and PKCdelta (410%). Hypoglycemia (2.4+/-0.9 mmol/L) alone did not induce gene expression, and hyperglycemia (22.1+/-3.7 mmol/L) alone induced only c-fos by 42%. When hypoglycemia was induced 30 minutes before SD, c-fos induction was enhanced by 145%, but the induction of HO-1 and PKCdelta was reduced to 43% and 64%, respectively. When hyperglycemia was induced 30 minutes before SD, c-fos induction was enhanced by 388% and COX-2 expression by 53%, whereas the induction of PKCdelta and HO-1 was reduced to 54% and 51%, respectively. The frequency, amplitude, and duration of direct current potentials were unaltered in hyperglycemic SD animals, whereas in hypoglycemic animals the duration was increased by 47%. CONCLUSIONS: While SD induces expression of several genes, the availability of glucose regulates the extent of the gene induction. The effect of glucose is different on early-response genes (c-fos and COX-2) compared with late-response genes. Plasma glucose may contribute to neuronal damage partially by regulating gene expression.