CD44-dependent Intracellular and Extracellular Catabolism of Hyaluronic Acid by Hyaluronidase-1 and -2

Abstract

Hyaluronic acid (HA) is a high molecular weight glycosaminoglycan involved in a wide variety of cellular functions. However, its turnover in living cells remains largely unknown. In this study, CD44, a receptor for HA, and hyaluronidase-1, -2, and -3 (Hyal-1, -2 and -3) were stably expressed in HEK 293 cells and the mechanism of HA catabolism was systematically investigated using fluorescein-labeled HA. CD44 was essential for HA degradation by both endogenous and exogenously expressed hyaluronidases. Hyal-1 was not able to cleave HA in living cells in the absence of CD44. Intracellular HA degradation was predominantly mediated by Hyal-1 after incorporation of HA by CD44. Although Hyal-1 was active only in intracellular space in vivo, a certain amount of the enzyme was secreted to extracellular space. This extracellular Hyal-1 was found to be incorporated by cells and such uptake of Hyal-1 was, in part, involved in the intracellular degradation of HA. Hyal-2 was involved in the extracellular degradation of HA. Hyal-2 activity was also dependent on the expression of CD44 in both living cells and enzyme assays. Immunofluorescent microscopy demonstrated that both Hyal-2 and CD44 are present on the cell surface. Without CD44 expression, Hyal-2 existed in a granular pattern, and did not show hyaluronidase activity, suggesting that localization change could contribute to Hyal-2 function. A convenient and quantitative enzyme assay was established for the measurement of Hyal-2 activity. Hyal-2 activity was detected in the membrane fraction of cells co-expressing Hyal-2 and CD44. The pH optimum for Hyal-2 was 6.0 -7.0. The membrane fraction of cells expressing Hyal-2 alone did not show hyaluronidase activity. Hyal-3 did not show any hyaluronidase activity in our experimental conditions. Based on these findings, Hyal-1 and -2 contribute to intracellular and extracellular catabolism of HA, respectively, in a CD44-dependent manner, and their HA degradation occurs independently from one another.Hyaluronic acid (HA) 3 is a negatively charged, high molecular weight glycosaminoglycan found predominantly in the extracellular matrix. It is the simplest of the glycosaminoglycans, the only one not covalently linked to core protein, and is unbranched and composed of repeating alternating units of glucuronic acid and N-acetylglucosamine. Despite the simplicity of its composition, HA has a great number of biological functions. It not only functions as a biological glue that participates in lubricating joints or holding together gel-like connective tissues, but also functions as a microenvironmental cue that co-regulates cell behavior during embryonic development and morphogenesis (1, 2), wound healing (3, 4), repair and regeneration, inflammation (5-7), and tumor progression and invasion (8, 9).There are 15 g of HA in a 70-kg individual, of which 5 g is replaced daily. In the skin, which contains 50% of the total body HA, the half-life of HA is about 1 day, and even in as seemingly inert a tissue as cartilage, HA turns over with a ha...

F-19848 A, a Novel Inhibitor of Hyaluronic Acid Binding to Cellular Receptor CD44

“…It has been suggested that HA is catabolized in a CD44-dependent manner [13ϳ15]. Our recent observation also revealed that CD44 has critical roles in the intracellular and extracellular catabolism of HA mediated by hyaluronidase-1 (Hyal-1) and hyaluronidase-2 (Hyal-2), respectively [16]. In addition, it has been shown that the signaling functions of HA are mediated by CD44 [17].…”

“…The membrane fractions from a cell line stably expressing human CD44 in HEK293 (CD44/293) [16,18] was used to determine the inhibitory activity of 1 for CD44-HA binding. The dose-dependent inhibition of CD44-HA binding by 1 is shown in Fig.…”

“…5A. The IC 50 value of 1 for CD44-HA binding was 23.5 m M. Then, the effect of the compound in living cells was examined by Hyal- 2/CD44/293 cells which stably express both human Hyal-2 and CD44 [16]. Hyal-2 is the somatically expressed hyaluronidase which degrades HA pericellularly, and its enzymatic activity depends on the expression of CD44 [16].…”

“…The IC 50 value of 1 for CD44-HA binding was 23.5 m M. Then, the effect of the compound in living cells was examined by Hyal- 2/CD44/293 cells which stably express both human Hyal-2 and CD44 [16]. Hyal-2 is the somatically expressed hyaluronidase which degrades HA pericellularly, and its enzymatic activity depends on the expression of CD44 [16]. As the interaction between CD44 and fl-HA is a crucial step to initiate extracellular degradation of fl-HA by Hyal-2/CD44/293 cells [16], inhibitors for CD44-HA binding are able to interfere with the degradation of fl-HA in the conditioned medium of the cells.…”

“…Hyal-2 is the somatically expressed hyaluronidase which degrades HA pericellularly, and its enzymatic activity depends on the expression of CD44 [16]. As the interaction between CD44 and fl-HA is a crucial step to initiate extracellular degradation of fl-HA by Hyal-2/CD44/293 cells [16], inhibitors for CD44-HA binding are able to interfere with the degradation of fl-HA in the conditioned medium of the cells. Fig.…”