Mikko Lammi

HYALURONAN SYNTHASE 2 (HAS2) REGULATES MIGRATION OF EPIDERMAL KERATINOCYTES

ABSTRACT Hyaluronan (HA) is a linear polysaccharide abundant in the extracellular space between epidermal keratinocytes. It is synthesized at the inner face of the plasma membrane by hyaluronan synthases (Has). We probed the importance of hyaluronan in keratinocytes by establishing cell lines carrying exogenous hyaluronan synthase 2 (Has2) gene(s) in sense and antisense orientations in order to increase and decrease their hyaluronan synthesis, respectively. The cell lines with the sense Has2 cDNA showed increased HA synthesis, while most cell lines with Has2 antisense cDNA contained less HA. Has2 antisense cells differed from control cell lines; they spread at a slower rate and retained a rounded morphology for a longer time. Further, during the first 24 hours after plating, proliferation was delayed in the antisense cell lines. In an in vitro wounding assay the antisense cells showed a significantly decreased migration rate as compared to controls. Cell lines with the Has2 sense cDNA were similar to the control cell lines in spreading and proliferation rates. However, they migrated faster than control cell lines.

Cytoskeleton and Proteoglycan Synthesis in Chondrocytes Under Hydrostatic Pressure

Articular cartilage is normally subject to various magnitudes of pressures which control the content of matrix proteoglycans (PGs). Short-term cyclic hydrostatic pressurization (5MPa, 1.5 h) stimulated PG synthesis in chondrocytes embedded in their natural environment in explants, but inhibited it when maintained in monolayer cultures. In long-term cyclic pressurization (5MPa, 20h), the inhibiting response of the cell cultures was reversed to stimulation. Continuous 30MPa pressure reduced total PG synthesis and the steady-state mRNA level of aggrecan, while continuous 5MPa pressure had no effect. Increased size of aggrecans, secreted under continuous 30MPa pressure, was observed. Continuous 30MPa hydrostatic pressure led to a reversible disorganization of microtubules, and the Golgi apparatus was packed into a perinuclear clump of vesicles. After depolymerization of the microtubules, pressurization did not influence the appearance of the Golgi apparatus or PG secretion. High hydrostatic pressure inhibited the organization of microfilaments and induced heat-shock protein 70 expression both at the mRNA and protein level. The results demonstrate that hydrostatic pressure controls the synthesis and structure of PGs in cultured chondrocytes. The simultaneous cytoskeletal changes may participate in the regulation of PG synthesis and secretion. The results further suggest that microtubules are important for PG metabolism. Hydrostatic pressure may control PG synthesis both at transcriptional and translational/posttranslational levels of biosynthesis.

Hyaluronan stimulates keratinocyte migration and activates the transcription factor AP-1 in keratinocytes through the JNK pathway

Hyaluronan (HA) has been considered a passive extracellular matrix (ECM) polysaccharide, but recent studies have shown its importance in controlling many cell functions including motility, proliferation and adhesion, which imply signaling from ECM to cytosol. Hyaluronan is a major ECM component in stratified epithelia such as skin epidermis. We found that hyaluronan added to the growth medium of newly plated human skin keratinocytes increased cell migration in an in vitro wound-healing assay. Hyaluronan also increased the transcription factor AP-1, as determined by gel shift assays. The kinase signals that apperently led to the increased AP-1 level were associated with the activation of c-Jun, mainly via the JNK pathway as early as 10 min after the addition of hyaluronan, and with the minimum concentration of 10 ng/ml. ERK1 was also slightly activated, while p38 MAPkinase was not affected.

EGF REGULATES HAS2 EXPRESSION, CONTROLS EPIDERMAL THICKNESS AND STIMULATES KERATINOCYTE MIGRATION

High concentrations of hyaluronan reside in the small space between the vital kertinocyte layers of human and animal epidermis and influence keratinocyte interactions, including growth, mobility and differentiation. We have previously found that the content of epidermal hyaluronan in human skin organ cultures is decreased and increased by cortisol and retinoic acid, and associated with enhanced and retarded terminal differentiation, respectively. To further substantiate this idea, we incubated epidermal keratinocytes with epidermal growth factor (EGF), and found a marked increase in hyaluronan synthesis which correlated with faster migration in an in vitro wounding assay of keratinocyte monolayers. EGF increased hyaluronan also in stratified, differentiated organotypic cultures, and increased the height of vital epidermis and reduced the thickness of the cornified layers, findings in line with an inhibition of terminal differentiation of keratinocytes. The stimulation of hyaluronan synthesis by EGF was due to upregulation of hyaluronan synthase 2 (HAS2) but not HAS1 or HAS3. A part of the EGF influence on the structure of epidermis, and on skin wound healing, is thus mediated through its control of HAS2 expression.