Changed lamellipodial extension, adhesion plaques and migration in epidermal keratinocytes containing constitutively expressed sense and antisense hyaluronan synthase 2 (Has2) genes

Abstract

Hyaluronan is a major component of the epidermal extracellular matrix, is actively synthesized by keratinocytes and shows fast matrix turnover in the stratified epithelium. We probed the importance of hyaluronan synthesis in keratinocytes by establishing cell lines carrying the exogenous hyaluronan synthase 2 (Has2) gene in sense and antisense orientations to increase and decrease their hyaluronan synthesis, respectively. Compared with cell lines transfected with the vector only, most clones containing the Has2 sense gene migrated faster in an in vitro wounding assay, whereas Has2 antisense cells migrated more slowly. Has2 antisense clones showed delayed entry into the S phase of cell cycle following plating, smaller lamellipodia and less spreading on the substratum. The decrease of hyaluronan on the undersurface of Has2 antisense cells was associated with an increased area of adhesion plaques containing vinculin. Exogenous hyaluronan added to the keratinocyte cultures had a minor stimulatory effect on migration after wounding but did not restore the reduced migratory ability of Has2 antisense cells. Hyaluronan decasaccharides that displace receptor bound hyaluronan in keratinocytes, and Streptomyces hyaluronidase sufficient to remove most cell surface hyaluronan had little effect on cell migration. The results suggest that the dynamic synthesis of hyaluronan directed by Has2, rather than the abundance of pericellular hyaluronan, controls keratinocyte migration, a cell function vital for the repair of squamous epithelia following wounding.

Chronic Ultraviolet B Irradiation Causes Loss of Hyaluronic Acid from Mouse Dermis Because of Down-Regulation of Hyaluronic Acid Synthases

“…In addition, the HA content governs general functions of the skin such as water content, turgidity, elasticity, and diffusion of nutrients. Detailed information is available on the regulatory pathways of epidermal HA synthesis, 15,35 the function of epidermal HA, 36,37 and the expression of epidermal HA synthases and HA receptors. 12,35 In addition, it has been shown recently that UVA and UVB irradiation decrease epidermal HA and CD44 in mice.…”

Hyaluronan-dependent pericellular matrix☆

“…Studies showing that decreased synthesis of hyaluronan in keratinocytes expressing HAS2 antisense RNA resulted in increased vinculin-positive adhesion plaques and decreased migration provide more evidence that the cell coat may antagonize adhesion to other matrix components [94]. It was proposed that it is the dynamic synthesis of hyaluronan that regulates migration [94].…”

“…It was proposed that it is the dynamic synthesis of hyaluronan that regulates migration [94]. Thus, continuous, active synthesis and/or internalization may be important for the effects of the pericellular matrix on cell movement and internal motor activities related to cell locomotion and mitotic cell rounding.…”

The Human Hyaluronan Synthase 2 Gene Is a Primary Retinoic Acid and Epidermal Growth Factor Responding Gene

“…Interestingly, hyaluronan synthesis rate is stimulated by EGF in epidermal keratinocytes in monolayer (4) and organotypic cultures (5) and by all-trans-RA in human skin organ cultures (3). Direct evidence for the biological role of hyaluronan in epidermal keratinocytes emerged by the finding that Has2-mediated hyaluronan synthesis controls the migration rate of keratinocytes in scratch-wounded monolayer cultures (6). Hyaluronan concentration is closely correlated with the proliferative activity and volume of the vital part of the epidermis and inversely related with the markers of differentiation, suggesting that hyaluronan synthesis regulated by Has2 and Has3 is an important component in the proliferative reactions of the epidermis and is also involved in the epidermal differentiation process (5).…”