Mikko Lammi

Hyaluronan is a polysaccharide found in the extracellular matrix (ECM) of cells from vertebrates. It is synthesized at the plasma membrane by an enzyme which is attached to the inner surface of the plasma membrane. Hyaluronan synthase (HAS) extrudes the growing hyaluronan chain directly into the extracellular space.

   In the course of the presented studies, hyaluronan synthesis rate was experimentally manipulated in keratinocytes to reveal features of the cellular phenotype influenced by hyaluronan. Green fluorescent protein (GFP)-tagged HASs were utilized to study the localization, trafficking and biological effects of HASs by time lapse and 3D confocal imaging.

   Epidermal growth factor (EGF) rapidly increased keratinocyte Has2 mRNA levels, which temporally correlated with an increase in hyaluronan secretion. It also increased pericellular and intracellular hyaluronan, and associated with an elongated cell shape and increased motility in scratched monolayer cultures. Inhibition of hyaluronan synthesis by antisense Has2 gene transfection slowed down the migration of keratinocytes. Correspondingly, 4-methylumbelliferone (4-MU) downregulated the basal and EGF-induced hyaluronan synthesis of keratinocytes and accentuated an epithelial morphology with a flat, round shapem with depleted lamellipodia, and filopodia and a low migration rate. High concentration of 4-MU also caused a reversible block in keratinocyte proliferation, and in the organotypic cultures, prevented the hyaluronan accumulation and epidermal hypertrophy induced by EGF. The induction and inhibition of hyaluronan synthesis showed opposite effects on cell morphology and proliferative and migratory activity, indicating that thesed processes are dependent on active hyaluronan synthesis.

   Confocal microscopy of keratinocytes expressing GFP-HAS fusion proteins showed large amounts of intracellular HAS, mainly in the endoplasmic reticulum (ER), Golgi apparatus and endosomes, and a distinct pool  on the plasma membrane particularly in microvillous extensions. Inhibition of HAS activity through enzyme mutations or substrate starvation excluded HAS from the plasma membrane, keeping it in the intracellular, latent pool. It was rapidly mobilized from ER and Golgi apparatus after release of a 4-MU and brefeldin A (BFA) block, respectively. The turnover time of the GFP-HAS3 protein was 4-5 hours, and the plasma membrane residency approximately 2 h. These results indicate that HAS resides on the plasma membrane only during active hyaluronan synthesis, after which it is endocytosed for degradation or recycling.

   Surpricingly, transffection of the GFP-tagged Has3 was not just expressed on membrane protrusions, but induced the formation of elongated microvilli. These microvilli were rapidly destoyed by Streptomyces hyaluronidase, while hyaluroann oligosaccharides known to replace hyaluronan from cell surface receptors had no influence. This indicates that overexpression of HAS itself causes the extension of plasma membrane into these hyaluronan-dependent microvilli, desribed first time in this work.

   The present studies show that hyaluronan synthesis strongly modulates the morphology of epithelial cells like epidermal keratinocytes, and stimulates their migration and proliferation, resulting in disturbed differentiationof the epidermis. Hyaluronan and HAS are thus potential targets for treatment of epidermal conditions associated with hyperproliferation such as psoriasis and wound healing.