Epidermal Growth Factor Activates Hyaluronan Synthase 2 in Epidermal Keratinocytes and Increases Pericellular and Intracellular Hyaluronan

Abstract

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 Synthesis Induces Microvillus-like Cell Surface Protrusions

“…In addition, cell surface protrusions make contacts to extracellular matrix and other cells, sense and interact with the environment, and lead to new attachment sites and cell migration (1). Hyaluronan synthesis increases the migration rate of different cell types (17)(18)(19), but it is currently not known which type of protrusions are involved. The present finding that hyaluronan tethered to plasma membrane strongly promotes cell surface protrusions offers a novel view to a number of previous reports indicating that hyaluronan stimulates cellular locomotion, multidrug resistance, ErbB2 signaling, and other poorly understood impacts of hyaluronan in cellular behavior.…”

Abnormal accumulation of hyaluronan matrix diminishes contact inhibition of cell growth and promotes cell migration

“…Overexpression of the HAS genes enhances the motility of a human melanoma cell line (20), and antisense suppression of HAS2 gene expression reduced keratinocyte migration (21). Important roles for the Ras signaling pathway in HA-dependent cell migration have been demonstrated by disruption of the HAS2 gene (22), and our recent study further indicated that simultaneous activation of Ras-mitogen-activated protein (MAP) kinase and PI3-kinase pathways is required for the activation of HA-dependent cell locomotion (16).…”

Cellular Content of UDP-N-acetylhexosamines Controls Hyaluronan Synthase 2 Expression and Correlates with O-Linked N-Acetylglucosamine Modification of Transcription Factors YY1 and SP1

“…Of the three genes, particularly HAS2 is subject to regulation by growth factors, cytokines, and hormones (4,14,15). In keratinocyte cultures, EGF, keratinocyte growth factor, TNF␣, and retinoic acid induce, whereas TGF␤ inhibits, HAS2 expression (8,10,13,16). Accordingly, the HAS2 promoter has been shown to contain functional response elements (REs) 3 for different transcription factors, including retinoid acid receptor, NF-B, CREB1 (cAMP response elementbinding protein 1), and SP1 (specificity protein 1) (7,11,16).…”

“…Changes in hyaluronan production have been associated mostly with the expression level of HAS genes (3-6), especially in keratinocytes (7)(8)(9)(10)(11)(12)(13). Of the three genes, particularly HAS2 is subject to regulation by growth factors, cytokines, and hormones (4,14,15).…”