Abundant hyaluronan is present between epidermal keratinocytes. However, virtually nothing is known regarding its organization in the limited extracellular space between these cells. We have used metabolic labeling with [ 3 H]glucosamine and [ 35 S]sulfate and a hyaluronan-specific biotinylated probe to study the metabolism of hyaluronan and its localization in monolayer cultures of a rat epidermal keratinocyte cell line. Hyaluronan (ϳ20 fg/cell) was present on the apical and lateral surfaces of the cells in two nearly equal pools, either in patches (ϳ160/cell) or diffusely spread. The hyaluronan in the patches is bound to CD44 as indicated by co-localization with an antibody to CD44, and by displacement with hyaluronan decasaccharides as well as with an antibody that blocks hyaluronan binding to CD44. The inability of hyaluronan oligomers shorter than 10 monosaccharides to displace hyaluronan suggests that CD44 dimerization or cooperative interactions are required for tight binding. The diffuse hyaluronan pool is likely bound to hyaluronan synthase during its biosynthesis.Hyaluronan is well known as a constituent of connective tissue extracellular matrices, but more recent studies have also demonstrated its abundance in stratified squamous epithelia including the epidermis (1-3). In contrast with connective tissue extracellular matrices that contain mixtures of collagens, fibronectins, other multiadhesive glycoproteins, proteoglycans, and hyaluronan, hyaluronan is the only known extracellular matrix macromolecule present in high concentration, ϳ2 mg/ ml, in the small extracellular space between adjacent epithelial cells (keratinocytes) that form the epidermis (4, 5). Additionally, studies of human skin organ cultures have shown that the hyaluronan within the epidermis is rapidly turned over (6), an observation that suggests that the epidermis possesses efficient mechanisms to catabolize hyaluronan that are closely coordinated with its synthesis.Although the coating of keratinocytes by hyaluronan is not generally appreciated, it is widely known that cell types of mesodermal origin (7), including fibroblasts (8), chondrocytes (9, 10), and mesothelial cells (11) display surface coats, often several micrometers in thickness, visualized indirectly as a domain excluding particles such as red blood cells. These coats 1) are removed by digestion with highly specific hyaluronidase, 2) can be stabilized by the serum-derived protein inter-␣-trypsin inhibitor which interacts with hyaluronan (12-14), and 3) can be increased in size and reinforced by proteoglycans that bind specifically to hyaluronan (15, 16).Extracellular hyaluronan is often anchored to CD44, a ubiquitous, abundant, and structurally variable plasma membrane receptor that has a hyaluronan binding domain (17). Smaller amounts of hyaluronan may bind to RHAMM, a receptor involved in cell motility and cell transformation through hyaluronan-dependent signaling involving tyrosine phosphorylation (18). In addition, some cell-surface hyaluronan appears to remain tet...

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.

A continuous rat epidermal cell line (rat epidermal keratinocyte; REK) formed a morphologically well-organized epidermis in the absence of feeder cells when grown for 3 weeks on a collagen gel in culture inserts at an air-liquid interface, and developed a permeability barrier resembling that of human skin. By 2 weeks, an orthokeratinized epidermis evolved with the suprabasal layers exhibiting the differentiation markers keratin 10, involucrin, and filaggrin. Granular cells with keratohyalin granules and lamellar bodies, and corneocytes with cornified envelopes and tightly packed keratin filaments were present. Morphologically, vitamin C supplementation of the culture further enhanced the normal wavy pattern of the stratum corneum, the number of keratohyalin granules present, and the quantity and organization of intercellular lipid lamellae in the interstices of the stratum corneum. The morphological enhancements observed with vitamin C correlated with improved epidermal barrier function, as indicated by reduction of the permeation rates of tritiated corticosterone and mannitol, and transepidermal water loss, with values close to those of human skin. Moreover, filaggrin mRNA was increased by vitamin C, and western blots confirmed higher levels of profilaggrin and filaggrin, suggesting that vitamin C also influences keratinocyte differentiation in aspects other than the synthesis and organization of barrier lipids. The unique REK cell line in organotypic culture thus provides an easily maintained and reproducible model for studies on epidermal differentiation and transepidermal permeation.

Mast cells are suggested to participate in regenerative processes, but their influence on epithelialization and wound healing has not been well studied. Since mast cells can be found in contact with epidermis in chronic inflammatory skin diseases and venous ulcers, the effect of mast cells on keratinocyte growth was studied. Keratinocytes were cultured in serum-free conditions with (complete medium) or without (basal medium) epidermal growth factor (EGF) and bovine pituitary extract (BPE) to reach subconfluence in a 24-well plate, and the cells were treated with different mast cell mediators histamine, heparin and tryptase, or lysate from HMC-1 cells, a human leukemic mast cell line. Whole skin cultures were used as a model for in vitro wounds to study the effect of mast cells on epithelial outgrowth from skin specimens. Histamine inhibited 3H-thymidine incorporation of keratinocytes dose-dependently by 29% at 1 mM, and 89% at 5 mM histamine. In whole skin culture, histamine inhibited epithelial outgrowth dose-dependently by 64% already at 0.1 mM histamine and maximally (91%) at 1 mM histamine. Heparin inhibited 3H-thymidine incorporation dose-dependently by up to 33% at 2 microg/ml in the absence, but not in the presence, of EGF/BPE. In contrast, in whole skin culture, heparin first inhibited the epithelial outgrowth by up to 27% at 2 microg/ml, but then reversed the inhibition to 30% stimulation at 200 microg/ml. Skin tryptase (0.0285 to 2.85 microg/ml) with or without heparin (0.5 to 20 microg/ml) did not affect thymidine incorporation in keratinocytes. Lysate from HMC-1 cells, but not that from control, neuroblastoma cells, inhibited 3H-thymidine incorporation in keratinocytes dose-dependently, and maximal (47%) inhibition was reached with 16,700 lysed HMC-1 cells/ml. In whole skin culture, HMC-1 lysate inhibited the epithelial outgrowth by up to 36% at 67,000 lysed cells/ml. The results show that mast cells and their mediators are inhibitory to keratinocyte 3H-thymidine incorporation and epithelial outgrowth in vitro, although, the inhibitory effect of histamine was seen at high concentrations suggesting a requirement for close morphologic vicinity of mast cells to keratinocytes. Thus, mast cells are assumed to control epidermal regeneration and to impair epithelialization of chronic ulcers.

An ideal application of CD spectroscopy is the study of hyaluronan (HA) interaction with HA binding proteinlreceptor, chiral and non-chiral ligandsldrugs. For a non-chiral ligand, an induced CD (ICD) will be observed only for the bound ligand species whilst no CD will be detected for the free ligand species. For chiral ligands, changes in the CD of the ligand upon binding can still be used to discriminate binding interactions though this is less direct than ICD for non-chiral ligands. The ability to discriminate binding interactions in vdm is important to ascertain the drug-carrier properties of any polymer. For the non-chiral diclofenac anti inflammatory drug, the lack of ICD was indicative of no interaction with HA. However, the binding of diclofenac to human serum albumin (HSA) was found to be affected by HA in a concentration dependent manner. To elucidate this, the effect of pH, temperature and n-octanoate on the diclofenadHSA binding were investigated by CD spectroscopy. Hyaluronan bound to CD44 on keratinocytes is displaced by hyaluronan decasaccharides and not hexasaccharides 34 AN N-TERMINAL LINK PROTEIN PEPTIDE 36 Epidermal growth factor regulates keratinocyte hyaluronan STIMULATES BIOSYNTHESIS OF COLLAGEN AND PROTEOGLYCANS BY EXPLANTS OF HUMAN ARTICULAR CARTILAGE. metabolism We measured the effects of a 16 amino acid peptide, identical in sequence to the N-terminal of link protein, on the synthesis of collagen and proteoglycan by explants of human articular cartilage. Explants !?om normal adult knee cartilage were maintained for periods between 2 to 10 days in Dulbecco's modified Eagle's medium with or without 2% foetal calf serum. Peptides were added during each day of culture. Synthesis of collagen was determined by measuring the incorporation of ['Hlproline into hydroxyproline and proteoglycms by incorporation of [)'S]sulphate. The sizes and types of newly synthesised proteoglycan molecules were measured by gel chromatography and collagen by SDS PAGE. The LN peptide stimulated synthesis of type I1 collagen and aggrecan in cartilage !?om a number of different subjects. Maximum upregulation of both collagen and proteoglycam was attained at an identical concentration of 10 ng/d of peptide. Synthesis was upregulated both m the presence and absence of serum, although stimulation was greater when serum was added. It was concluded that the peptide may act as a growth factor and have an important role in the feed-back control of cartilage matrix synthesis. We wish to thank Action Research and The Home of Rest for Horses for their financial support and The Bone Tumor Service, Royal Orthopaedic Hospital, Birmingham, for human cartilage.