Hyaluronan Bound to CD44 on Keratinocytes Is Displaced by Hyaluronan Decasaccharides and Not Hexasaccharides

Abstract

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...

Plasma Membrane Residence of Hyaluronan Synthase Is Coupled to Its Enzymatic Activity

“…The same finding was observed when GFP-xlHas1 was transfected into oocytes (14). REKs are also known to display cell surface hyaluronan as patches, 100 -200/ cell, perhaps corresponding to the HAS sites (35). The present data, a part of which was obtained from high resolution threedimensional reconstructions of live cells, provided important novel information on this issue, suggesting that these GFP-HAS enrichment sites were actually cell surface protrusions.…”

Oligosaccharides of Hyaluronan Are Potent Activators of Dendritic Cells

“…Specifically, perinuclear CD44 staining of sHA-treated endothelial cells was not different from that of untreated cells (36), indicating that the sHA uptake and specific NF-B-dependent signaling was not mediated by CD44. The notion that sHA fragments interact with cells independent of CD44 is supported by recent findings from Culty et al (31) and Tammi et al (42). On the basis of HMW-HA competition studies, these investigators concluded that only sHA fragments of at least 6-to 10-oligosaccharide length, but not smaller fragments, bind to CD44 on endothelial cells or keratinocytes, respectively.…”

Overexpression of Hyaluronan-binding Protein 1 (HABP1/p32/gC1qR) in HepG2 Cells Leads to Increased Hyaluronan Synthesis and Cell Proliferation by Up-regulation of Cyclin D1 in AKT-dependent Pathway

“…Hyaluronan digestion was carried out using 50 g/ml of bovine testicular hyaluronidase (BTH) for 3 h at 37°C. Equal volumes of both undigested and BTH-digested products derived from two cell lines were loaded onto a 5-20% gradient gel (19), along with BTH-digested and undigested pure polymeric HA from human umbilical cord acting as positive controls. The gel was then stained with 1% Alcian blue in 3% acetic acid, destained, and subsequently stained with silver nitrate (19).…”