title: A novel mechanism for the inhibition of hyaluronan biosynthesis by 4-methylumbelliferone.
abstract: Specific inhibitors of hyaluronan (HA) biosynthesis can be valuable therapeutic agents to prevent cancer invasion and metastasis. We have found previously that 4-methylumbelliferone (MU) inhibits HA synthesis in human skin fibroblasts and in group C Streptococcus. In this paper, the inhibition mechanism in mammalian cells was investigated using rat 3Y1 fibroblasts stably expressing HA synthase (HAS) 2. Exposure of the transfectants to the inhibitor resulted in significant reduction of HA biosynthesis and matrix formation. The evaluation of HAS transcripts and analysis of cell-free HA synthesis demonstrated the post-transcriptional suppression of HAS activity by MU. Most interesting, the post-transcriptional suppression of HAS activity was also observed using p-nitrophenol, a well known substrate for UDP-glucuronyltransferases (UGT). We investigated whether the inhibition was exerted by the glucuronidation of MU using both high pressure liquid chromatography and TLC analyses. The production of MU-glucuronic acid (GlcUA) was consistent with the inhibition of HA synthesis in HAS transfectants. MU-GlcUA was also detected at a similar level in control cells, suggesting that the glucuronidation was mediated by an endogenous UGT. Elevated levels of UGT significantly enhanced the inhibitory effects of MU. In contrast, the inhibition by MU was diminished to the control level when an excess of UDP-GlcUA was added to the cell-free HA synthesis system. We propose a novel mechanism for the MU-mediated inhibition of HA synthesis involving the glucuronidation of MU by endogenous UGT resulting in a depletion of UDP-GlcUA.
authors.affiliation: Department of Biochemistry, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.
authors.forename: Ikuko
authors.initials: I
authors.lastname: Kakizaki
authors.forename: Kaoru
authors.initials: K
authors.lastname: Kojima
authors.forename: Keiichi
authors.initials: K
authors.lastname: Takagaki
authors.forename: Masahiko
authors.initials: M
authors.lastname: Endo
authors.forename: Reiji
authors.initials: R
authors.lastname: Kannagi
authors.forename: Masaki
authors.initials: M
authors.lastname: Ito
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Maruo
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sato
authors.forename: Tadashi
authors.initials: T
authors.lastname: Yasuda
authors.forename: Satoka
authors.initials: S
authors.lastname: Mita
authors.forename: Koji
authors.initials: K
authors.lastname: Kimata
authors.forename: Naoki
authors.initials: N
authors.lastname: Itano
mesh_terms: D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D015870:Gene Expression; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D006923:Hymecromone / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014166:Transferases / Q000235:genetics
chemical_list: D004791:Enzyme Inhibitors; D020719:Glucuronides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D006923:Hymecromone; D006820:Hyaluronic Acid; D014166:Transferases; D014453:Glucuronosyltransferase; C491097:Has2 protein, rat; D000076002:Hyaluronan Synthases