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• W2134213816 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. 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. There are a considerable number of reports showing that the biosynthesis of hyaluronan (HA) 1The abbreviations used are: HA, hyaluronan; MU, 4-methylumbelliferone; pNP, p-nitrophenol; GlcUA, glucuronic acid; GlcNAc, N-acetylglucosamine; Glc, glucose; HA synthase, HAS; Me2SO, dimethyl sulfoxide; PBS, phosphate buffered saline; DTT, dithiothreitol; UGT, UDP-glucuronosyltransferase; HPLC, high pressure liquid chromatography; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcriptase. is elevated in disorders such as fibroses of organs, diseases associated with inflammation, and some types of tumors including mesothelioma and Wilm's tumor (1Laurent T.C. Fraser J.R.E. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2078) Google Scholar, 2Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1241Crossref PubMed Scopus (599) Google Scholar, 3Knudson W. Biswas C. Li X.Q. Nemec R.E. Toole B.P. CIBA Found. Symp. 1989; 143: 150-159PubMed Google Scholar, 4Toole B.P. Wight T.N. Tammi M.I. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 5Laurent T.C. Laurent U.B. Fraser J.R. Ann. Med. 1996; 28: 241-253Crossref PubMed Scopus (167) Google Scholar). For instance, accumulation of HA is associated with the progression of atherosclerosis (6Levesque H. Girard N. Maingonnat C. Delpech A. Chauzy C. Tayot J. Courtois H. Delpech B. Atherosclerosis. 1994; 105: 51-62Abstract Full Text PDF PubMed Scopus (64) Google Scholar). During the progression of hepatitis, HA derived from the Ito cells accumulates in the liver, causing fibrosis and eventually cirrhosis of the liver (7Satoh T. Ichida T. Matsuda Y. Sugiyama M. Yonekura K. Ishikawa T. Asakura H. J. Gastroenterol. Hepatol. 2000; 15: 402-411Crossref PubMed Scopus (34) Google Scholar). Because HA is directly associated with liver fibrosis, it has long been utilized as a marker for the diagnosis of chronic hepatitis (8Plebani M. Burlina A. Clin. Biochem. 1991; 24: 219-239Crossref PubMed Scopus (91) Google Scholar). Also, an exponential increase of HA in the endocervical canal at inappropriate stage of pregnancy can result in miscarriage (9El Maradny E. Kanayama N. Kobayashi H. Hossain B. Khatun S. Liping S. Kobayashi T. Terao T. Hum. Reprod. 1997; 12: 1080-1088Crossref PubMed Scopus (112) Google Scholar). Recent genetic approaches showed that overproduction of HA accelerated tumor growth and is associated with cancer metastasis (10Kosaki R. Watanabe K. Yamaguchi Y. Cancer Res. 1999; 59: 1141-1145PubMed Google Scholar, 11Itano N. Sawai T. Miyaishi O. Kimata K. Cancer Res. 1999; 59: 2499-2504PubMed Google Scholar, 12Liu N. Gao F. Han Z. Xu X. Underhill C.B. Zhang L. Cancer Res. 2001; 61: 5207-5214PubMed Google Scholar, 13Simpson M.A. Wilson C.M. Furcht L.T. Spicer A.P. Oegema Jr., T.R. McCarthy J.B. J. Biol. Chem. 2002; 277: 10050-10057Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 14Jacobson A. Rahmanian M. Rubin K. Heldin P. Int. J. Cancer. 2002; 102: 212-219Crossref PubMed Scopus (112) Google Scholar). HA is a nonsulfated linear glycosaminoglycan composed of thousands of repeating units of GlcNAc-β(1→4)-GlcUA-β(1→3) (15Weissman B. Meyer K. J. Am. Chem. Soc. 1954; 76: 1753-1757Crossref Scopus (216) Google Scholar). In vertebrates, this molecule is a ubiquitous component of the extracellular matrix and plays critical roles in dynamic functions such as embryonic development, tissue regeneration, and cell migration (16Tammi M.I. Day A.J. Turley E.A. J. Biol. Chem. 2002; 277: 4581-4584Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Both eukaryotic and prokaryotic HA synthases (HAS) catalyze the transglycosylation from both UDP-GlcUA and UDP-GlcNAc donors (17Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). Following the first cloning of a HAS gene from Streptococcus in 1993, three distinct mammalian isoforms, HAS1, HAS2, and HAS3, have been identified and characterized from mouse, human, and other species (17Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). Considerable progress in understanding HA biosynthesis and its biological functions has been made in recent years. Identification of a specific inhibitor for HA biosynthesis would not only help elucidate the functions of HA but would also have applications in clinical medicine for the treatment of diseases caused by elevated levels of this glycosaminoglycan. Over the past few decades many researchers have attempted without success to discover specific inhibitors of HA synthesis in mammalian cells (18Goldberg R.L. Toole B.P. J. Biol. Chem. 1983; 258: 7041-7046Abstract Full Text PDF PubMed Google Scholar, 19Smith T.J. J. Clin. Endocrinol. Metab. 1990; 70: 655-660Crossref PubMed Scopus (18) Google Scholar, 20Zaharevitz D.W. Chisena C.A. Duncan K.L. August E.M. Cysyk R.L. Biochem. Mol. Biol. Int. 1993; 31: 627-633PubMed Google Scholar, 21August E.M. Duncan K.L. Malinowski N.M. Cysyk R.L. Oncol. Res. 1993; 5: 415-422PubMed Google Scholar, 22Ueki N. Taguchi T. Takahashi M. Adachi M. Ohkawa T. Amuro Y. Hada T. Higashino K. Biochim. Biophys. Acta. 2000; 1495: 160-167Crossref PubMed Scopus (20) Google Scholar). 4-Methylumbelliferone (MU, 7-hydroxy-4-methyl-2H-1-benzopyran-2-one) was found previously to inhibit HA synthesis in cultured human skin fibroblasts but had no effect on the synthesis of any other glycosaminoglycan (23Nakamura T. Takagaki K. Shibata S. Tanaka K. Higuchi T. Endo M. Biochem. Biophys. Res. Commun. 1995; 208: 470-475Crossref PubMed Scopus (88) Google Scholar, 24Nakamura T. Funahashi M. Takagaki K. Munakata H. Tanaka K. Saito Y. Endo M. Biochem. Mol. Biol. Int. 1997; 43: 263-268PubMed Google Scholar). Since then MU has been used as an inhibitor of HA synthesis in many studies on the functions of HA, although its precise mechanism has not been established in mammalian cells (10Kosaki R. Watanabe K. Yamaguchi Y. Cancer Res. 1999; 59: 1141-1145PubMed Google Scholar, 25Endo Y. Takagaki K. Takahashi G. Kakizaki I. Funahashi M. Yokoyama M. Endo M. Munakata A. Progress in Transplantation. Elsevier Science Publishers B. V., Amsterdam2000: 1-7Google Scholar, 26Sohara Y. Ishiguro N. Machida K. Kurata H. Thant A.A. Senga T. Matsuda S. Kimata K. Iwata H. Hamaguchi M. Mol. Biol. Cell. 2001; 12: 1859-1868Crossref PubMed Scopus (89) Google Scholar). For many years MU has been used safely in human medicine as a cholagogue by oral administration (27Takeda S. Aburada M. J. Pharmacobio-Dyn. 1981; 4: 724-734Crossref PubMed Scopus (57) Google Scholar). The clinical application of MU for controlling HA synthesis could potentially prevent malignant alteration of cancer cells and fibrosis of organs. It would therefore be helpful to clarify the inhibition mechanism of MU in mammalian cells. The information may also be useful in developing new compounds that are more effective inhibitors and/or display lower cytotoxicity than MU. A possible phospholipid-dependent inhibition mechanism of MU was found using group C Streptococcus in our previous paper (28Kakizaki I. Takagaki K. Endo Y. Kudo D. Ikeya H. Miyoshi T. Baggenstoss B.A. Tlapak-Simmons V.L. Kumari K. Nakane A. Weigel P.H. Endo M. Eur. J. Biochem. 2002; 269: 5066-5075Crossref PubMed Scopus (40) Google Scholar). We suggested that MU treatment may inhibit HAS activity by altering the distribution of cardiolipin species surrounding HAS in the plasma membrane. However, the change in the distribution of cardiolipin cannot by itself account for the observed inhibition of HA synthesis. Furthermore, the proposed mechanism of inhibition involving cardiolipin might be specific for Streptococcus, because mammalian cells have low levels of cardiolipin in the plasma membrane. Indeed the effect of cardiolipin on enzymatic activity is distinct between mammalian HAS1 and streptococcal HAS (29Yoshida M. Itano N. Yamada Y. Kimata K. J. Biol. Chem. 2000; 275: 497-506Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), suggesting that the MU-mediated inhibition is quite complex. In this study we demonstrate a UGT-dependent inhibition mechanism for HA synthesis in mammalian cells using transfectants that express mouse HAS2. We propose the inhibition of HA synthesis is due, in part, to a depletion in the pool of UDP-GlcUA, a common substrate of HAS and UGT. Materials and Reagents—MU was purchased from Wako Pure Chemicals (Osaka, Japan). p-Nitrophenol (pNP), pNP-sugars (pNP-Glc, pNPGlcUA, and pNP-GlcNAc), and MU-sugars (MU-Glc, MU-GlcUA, and MU-GlcNAc) were from Sigma. MU and MU-sugars were dissolved in dimethyl sulfoxide (Me2SO); pNP and pNP-sugars were dissolved in ethanol, and the final concentration of these vehicles in the culture medium and the reaction mixtures for the cell-free HA synthesis were adjusted to 0.1%. UDP-[14C]GlcUA (313 mCi/mmol) and [14C]pNP (70 mCi/mmol) were from ICN Biomedicals, Inc. (Irvine, CA) and American Radiolabeled Chemicals Inc. (St. Louis, MO), respectively. UDP-GlcUA, dithiothreitol (DTT), and ATP were from Nakalai Tesque (Kyoto, Japan). UDP-GlcNAc, bovine liver β-glucuronidase, and glutaraldehyde-stabilized sheep erythrocytes were from Sigma. Streptomyces hyaluronidase was obtained from Seikagaku Corp. (Tokyo, Japan). Hyaluronic acid “Chugai” quantitative test kit for the sandwich binding protein assay was purchased from Chugai Pharmaceutical (Tokyo, Japan) (30Chichibu K. Matsuura T. Shichijo S. Yokoyama M.M. Clin. Chim. Acta. 1989; 181: 317-323Crossref PubMed Scopus (128) Google Scholar). Recombinant human UGT1A6 and UGT1A7 proteins were from Calbiochem, and their expression vectors were reported previously (31Ito M. Yamamoto K. Maruo Y. Sato H. Fujiyama Y. Bamba T. Eur. J. Clin. Pharmacol. 2002; 58: 11-14Crossref PubMed Scopus (17) Google Scholar). Cell Culture and Transfection—Stable transfectants were established by transfection of mouse HAS2 and control vector into rat 3Y1 cells as described previously (32Itano N. Sawai T. Yoshida M. Lenas P. Yamada Y. Imagawa M. Shinomura T. Hamaguchi M. Yoshida Y. Ohnuki Y. Miyauchi S. Spicer A.P. McDonald J.A. Kimata K. J. Biol. Chem. 1999; 274: 25085-25092Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). Cells were routinely cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mml-glutamine, and 400 μg/ml G418 at 37 °C. pFLAG-HAS2 and human UGT1A6 expression vector were transiently transfected into COS-1 cells by electroporation as described previously (32Itano N. Sawai T. Yoshida M. Lenas P. Yamada Y. Imagawa M. Shinomura T. Hamaguchi M. Yoshida Y. Ohnuki Y. Miyauchi S. Spicer A.P. McDonald J.A. Kimata K. J. Biol. Chem. 1999; 274: 25085-25092Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). The cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 2 mml-glutamine at 37 °C. Particle Exclusion Assay—3Y1-HAS2 cells plated at 1 × 103 cells in a 35-mm dish were cultured for 2 days and then further cultured for a day with or without 300 μm MU. An aliquot of glutaraldehyde-stabilized sheep erythrocytes (3 × 108) in PBS was then added to the culture medium. After 15 min, the culture was observed using an inverted phase-contrast microscope (Olympus IMT-2) (33Knudson W. Knudson C.B. J. Cell Sci. 1991; 99: 227-235Crossref PubMed Google Scholar). Quantitative Analyses of the HAS Transcripts—The relative level of HAS expression in the HAS2 transfectants was determined by real time quantitative RT-PCR as described previously (11Itano N. Sawai T. Miyaishi O. Kimata K. Cancer Res. 1999; 59: 2499-2504PubMed Google Scholar). The gene-specific PCR primers and probes were designed from the mouse HAS2 and rat HAS sequences using the Primer Express software (Applied Biosystems, Foster City, CA). The sequences of the various oligonucleotides are as follows: the forward primer for mouse HAS2 was 5′-CCTCGGAATCACAGCTGCTTATA-3′, and the reverse primer was 5′-CTGCCCATAACTTCGCTGAATA-3′; the probe for mouse HAS2 was 5′-TCGCATCTCATCATCCAAAGCCTCTTTG-3′; the forward primer for rat HAS1 was 5′-GGAGATGTGAGAATCCTTAACCCTC-3′, and the reverse primer was 5′-TGCTGGCTCAGCCAACGAAGGAA-3′; the probe for rat HAS1 was 5′-CAGAGCTACTTTCACTGTGTGTCCTGCATC-3′; the forward primer for rat HAS2 was 5′-CCTCGGAATCACAGCTGCTTATA-3′, and the reverse primer was 5′-CTGCCCATGACTTCACTGAAGA-3′; the probe for rat HAS2 was 5′-TCACACCTCATCATCCAAAGCCTCTTTG-3′; the forward primer for rat HAS3 was 5′-GGTACCATCAGAAGTTCCTAGGCAGC-3′, and the reverse primer was 5′-GAGGAGAATGTTCCAGATGCG-3′; and the probe for rat HAS3 was 5′-TGGCTACCGGACTAAGTATACAGCACGCTC-3′. Total RNA was isolated from subconfluent rat 3Y1 cells expressing mouse HAS2 using the RNeasy mini kit (Qiagen, Valencia, CA). Two hundred nanograms of total RNA was used for real time RT-PCR and subsequent analysis. The reaction master mix was prepared to give final concentrations of 1× TaqMan EZ buffer, 0.3 mm dATP, 0.3 mm dCTP, 0.3 mm dGTP, 0.6 mm dUTP, 6 mm manganese acetate, 0.01 units/μl uracil N-glycosylase, 0.1 units/μl rTth DNA polymerase, 200 nm primers, and 100 nm TaqMan probe. The conditions for RT-PCR are as follows: 1 cycle at 50 °C for 2 min, 1 cycle at 60 °C for 30 min, 1 cycle at 95 °C for 5 min, 50 cycles at 95 °C for 20 s, and 60 °C for 1 min. Fluorescent signals generated during PCR amplifications were monitored in real time using the 7700 sequence detector system (Applied Biosystems) and analyzed with the sequence detector 1.7 program (Applied Biosystems). The relative amount of glyceraldehyde-3-phosphate dehydrogenase mRNA was measured using the TaqMan rodent glyceraldehyde-3-phosphate dehydrogenase detection reagents (Applied Biosystems). The amount of HAS mRNA was divided by the amount of glyceraldehyde-3-phosphate dehydrogenase mRNA in each sample. The normalized values were designated as the “relative expression coefficient” in this study. Standard curves were generated by serial dilution of total RNA isolated from HAS2 transfectants. Determination of the HA Concentration by ELISA-like Assay—Cells were plated at a density of 1 × 105 and 8 × 105 cells/well in a 6-well plate (defined as exponentially growing phase and confluent phase, respectively). The cells were cultured with various concentrations of MU for 24 h. HA released into the culture medium was quantified by an ELISA-like assay using HA-binding protein according to the manufacturer's instructions for the hyaluronic acid “Chugai” quantitative test kit (30Chichibu K. Matsuura T. Shichijo S. Yokoyama M.M. Clin. Chim. Acta. 1989; 181: 317-323Crossref PubMed Scopus (128) Google Scholar). The quantity of HA was expressed per live cell number. The viability of cells was assessed by trypan blue staining. HA Synthase Assay—HAS activity was monitored in the cell-free HA synthesis system using UDP-[14C]GlcUA and UDP-GlcNAc as donors and a membrane-rich fraction of the transfectants as an enzyme source as described previously (32Itano N. Sawai T. Yoshida M. Lenas P. Yamada Y. Imagawa M. Shinomura T. Hamaguchi M. Yoshida Y. Ohnuki Y. Miyauchi S. Spicer A.P. McDonald J.A. Kimata K. J. Biol. Chem. 1999; 274: 25085-25092Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). Briefly, the HAS transfectants were washed, harvested, and disrupted by sonication in 10 mm Hepes-NaOH, pH 7.1, 0.5 mm dithiothreitol containing 0.25 m sucrose. Suspensions of the disrupted cells were ultracentrifuged in a Beckman TLS rotor at 43,000 rpm for 1 h to give the high speed pellet. The crude membrane fractions prepared from HAS transfectants were resuspended with standard reaction mixture (0.1 ml of 25 mm Hepes-NaOH, pH 7.1, 5 mm DTT, 15 mm MgCl2, 0.1 mm UDP-GlcNAc, 2 μm UDP-GlcUA, and 0.2 μCi of UDP-[14C]GlcUA) and incubated at 37 °C for 1 h. Depending on the type of experiment, MU, pNP, and their sugar derivatives were added to the reaction mixture. Alternatively, the membrane fractions were preincubated at 37 °C for 1 h with 300 μm MU or 300 μm MUGlcUA in the preincubation mixture (0.1 ml of 25 mm Hepes-NaOH, pH 7.1, 5 mm DTT, 15 mm MgCl2, 0.1 mm UDP-GlcUA), and then HA synthesis was initiated by adding 2 mm UDP-GlcNAc and 0.2 μCi of UDP-[14C]GlcUA. The reaction was terminated at the indicated time by addition of SDS to 2% (w/v). The mixtures were then spotted onto Whatman No. 3MM paper, and the paper was developed in 1 m ammonium acetate (pH 5.5) and ethanol (65:35 v/v) for 3 days. The origin, containing the synthesized polymers, was removed, and the amount of radioactivity in the high molecular mass HA was determined by liquid scintillation counting. Agarose Gel Electrophoresis of HA—The size distribution of radiolabeled HA synthesized in the cell-free reaction was analyzed by agarose gel electrophoresis (0.5% gel) as described previously (32Itano N. Sawai T. Yoshida M. Lenas P. Yamada Y. Imagawa M. Shinomura T. Hamaguchi M. Yoshida Y. Ohnuki Y. Miyauchi S. Spicer A.P. McDonald J.A. Kimata K. J. Biol. Chem. 1999; 274: 25085-25092Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). The synthesized HA was incubated at 37 °C for 1 h with or without 1 turbidity reducing unit of Streptomyces hyaluronidase prior to loading on the gel. After drying the gel, the radioactive HA was detected using a BAS 5000 Bio-Imaging Analyzer (Fuji Film Co., Tokyo, Japan). HPLC Analysis of the MU-sugar Derivative—MU-sugar derivatives, from culture conditioned medium and from cell lysate, were analyzed by HPLC using a TSK gel ODS-120T (15 cm × 4.6 mm inner diameter) column. HPLC conditions were identical to those described by Zimmerman et al. (34Zimmerman C.L. Ratna S. Leboeuf E. Pang K.S. J. Chromatogr. 1991; 563: 83-94Crossref PubMed Scopus (21) Google Scholar). Eluted fractions were monitored by detecting fluorescence (excitation 325 nm, emission 380 nm) using a fluorescence spectrophotometer Hitachi F-1050 (Tokyo, Japan). Cells were washed with PBS three times and then disrupted in a solution of 2% SDS-PBS by sonication for 2 min (4 bursts of 30 s) using a sonifier (model UR-20P, Tomy Seiko, Tokyo, Japan). The cell lysate was then obtained by centrifugation at 105,000 × g for 30 min. The supernatants were used for HPLC analysis. For TLC analysis in Fig. 3 and for mass spectrometry, a peak corresponding to MU-GlcUA was collected by HPLC as described below. A fraction containing MU-GlcUA was prefractionated from culture supernatant using a Sep-Pac Plus C18 cartridge (Waters, MA) prior to HPLC. Briefly, the culture supernatant was loaded onto the cartridge, and the cartridge was then washed with water. Bound materials containing MU-GlcUA were eluted with 100% methanol, concentrated by using a vacuum evaporator centrifuge (Iwaki, Tokyo, Japan), and analyzed by HPLC. A peak of MU-GlcUA was collected and de-salted using a Sep-Pac Plus C18 cartridge. After drying, the residue was dissolved into methanol and analyzed by TLC or mass spectrometry. TLC Analysis of the MU-sugar or pNP-sugar Derivative—Radiolabeled MU-sugar or pNP-sugar derivatives produced in the cell-free HA synthesis system was treated with or without 2 units of β-glucuronidase at 37 °C for 1 h and then separated by TLC. TLC was performed on a Silica 60 TLC plate (Merck) using 1-butanol/ethanol/water (5:3:2, v/v/v) as the mobile phase (35Kamst E. Bakkers J. Quaedvlieg N.E. Pilling J. Kijne J.W. Lugtenberg B.J. Spaink H.P. Biochemistry. 1999; 38: 4045-4052Crossref PubMed Scopus (47) Google Scholar). After drying the plate, radioactive spots were detected using a BAS 5000 Bio-Imaging Analyzer. The radiolabeled product derived from the incubation of pNP and UDP-[14C]GlcUA according to the standard assay method of UGT (36Kanou M. Saeki K. Kato T. Takahashi K. Mizutani T. Fundam. Clin. Pharmacol. 2002; 16: 513-517Crossref PubMed Scopus (23) Google Scholar) was used for determining the mobility of pNP-GlcUA. Fluorescent spots containing MU or MU-sugar were detected by ultraviolet irradiation using a transilluminator. Authentic MU-GlcUA was used as a standard. Mass Spectrum Measurements—Mass spectra were obtained on a PE-Sciex API-100 single-quadrupole mass spectrometer (Thornhill, Ontario, Canada) equipped with an atmospheric pressure ionization source described previously (37Takagaki K. Kojima K. Majima M. Nakamura T. Kato I. Endo M. Glycoconj. J. 1992; 9: 174-179Crossref PubMed Scopus (68) Google Scholar). The mass spectrometer was operated in the negative mode. Samples dissolved in 50% 2-propanol were ionized by electrospray ionization and continuously infused into the electrospray ionization chamber at a flow rate of 5 μl min-1. Inhibitory Effect of MU on HA Production and HA Matrix Formation of the HAS2-expressing Cells—MU was originally found to inhibit HA synthesis and HA matrix formation of cultured human skin fibroblasts (23Nakamura T. Takagaki K. Shibata S. Tanaka K. Higuchi T. Endo M. Biochem. Biophys. Res. Commun. 1995; 208: 470-475Crossref PubMed Scopus (88) Google Scholar, 24Nakamura T. Funahashi M. Takagaki K. Munakata H. Tanaka K. Saito Y. Endo M. Biochem. Mol. Biol. Int. 1997; 43: 263-268PubMed Google Scholar). The recent discovery that MU inhibits HA synthesis of group C Streptococcus in a phospholipid-dependent fashion provided new insight into understanding the mechanism of inhibition (28Kakizaki I. Takagaki K. Endo Y. Kudo D. Ikeya H. Miyoshi T. Baggenstoss B.A. Tlapak-Simmons V.L. Kumari K. Nakane A. Weigel P.H. Endo M. Eur. J. Biochem. 2002; 269: 5066-5075Crossref PubMed Scopus (40) Google Scholar). However, the inhibition of HA synthesis by MU cannot be explained by this mechanism alone, particularly in mammalian cells. In this study, we examined the effects of MU on HA synthesis by using HAS2 overexpressing cells in order to clarify which step of HA synthesis is the target for the inhibition. The HAS2 overexpressing cells, named 3Y1-HAS2, were established from rat 3Y1 fibroblasts by transfection with HAS2 cDNA as described previously (32Itano N. Sawai T. Yoshida M. Lenas P. Yamada Y. Imagawa M. Shinomura T. Hamaguchi M. Yoshida Y. Ohnuki Y. Miyauchi S. Spicer A.P. McDonald J.A. Kimata K. J. Biol. Chem. 1999; 274: 25085-25092Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). Formation of pericellular HA matrices and HA production were significantly inhibited in 3Y1-HAS2 cells by treatment with MU as observed previously in cultured human skin fibroblasts (23Nakamura T. Takagaki K. Shibata S. Tanaka K. Higuchi T. Endo M. Biochem. Biophys. Res. Commun. 1995; 208: 470-475Crossref PubMed Scopus (88) Google Scholar, 24Nakamura T. Funahashi M. Takagaki K. Munakata H. Tanaka K. Saito Y. Endo M. Biochem. Mol. Biol. Int. 1997; 43: 263-268PubMed Google Scholar) (Fig. 1). The formation of pericellular HA matrices was inhibited by ∼30% during exposure to 300 μm MU compared with the nontreated control. The level of inhibition was even more apparent at higher concentrations of MU (data not shown). HA accumulation in the conditioned medium was dose-dependently decreased by MU treatment both in the exponentially growing and confluent phases of 3Y1-HAS2 cells (Fig. 1E). Transcription of the mouse HAS2 transgene and the endogenous HAS genes in the transfectants was assessed by real time RT-PCR using mouse- and rat-specific HAS primers and probes. No obvious change in the transcriptional levels of these HAS genes was observed at the low or moderate concentrations of MU, whereas the level of the endogenous HAS2 gene was inhibited during exposure to a high dose of MU (Table I). These results suggest that MU inhibits HAS activity post-transcriptionally at the low or moderate concentrations of MU. In contrast, a high dose of MU inhibits HA synthesis by suppressing HAS functions both transcriptionally and post-transcriptionally.Table IRelative HAS expression in HAS2 transfectants after MU treatmentMUMouse HAS2Rat HAS2Rat HAS3μm%%%010010010010130.7 ± 19.794.7 ± 2598.5 ± 13.930129.1 ± 34.886.1 ± 10.793.1 ± 13.7100137.4 ± 11.6105.0 ± 2893.9 ± 10.330093.3 ± 29.677.1 ± 4.390.4 ± 2.11000104.8 ± 28.448.6 ± 8.297.4 ± 10.6 Open table in a new tab Effect of MU on HA Synthesis and Chain Elongation in a Cell-free HA Synthesis System—Cell-free HA synthesis was examined by using the membrane-rich fraction of 3Y1-HAS2 cells. As shown in Fig. 2A, HAS activity was inhibited by MU in a dose-dependent manner, and the inhibition reached to 50% of the nontreated control at 300 μm MU. The inhibitory effect was observed at an early stage of HA synthesis and reached a plateau 30 min after treatment with MU (Fig. 2B). The size distribution of HA synthesized in the cell-free system was also determined by agarose gel electrophoresis (Fig. 2, C and D). The decrease in the molecular size of HA was caused by MU treatment in both a dose- and time-dependent manner. These data suggest MU inhibits HA synthesis by suppressing HAS function post-transcriptionally. Furthermore, the MU-mediated inhibition of HA synthesis may be caused by direct inhibition of HAS activity. MU has been used as a substrate to measure the activity of UGTs, which are involved in the detoxification of phenolic compounds in mammalian cells, particularly in the liver (38Hanioka N. Jinno H. Tanaka-Kagawa T. Nishimura T. Ando M. J. Pharm. Biomed. Anal. 2001; 25: 65-75Crossref PubMed Scopus (53) Google Scholar). Because HAS possesses a glycosyltransferase activity for UDPGlcUA within its polypeptide (17Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar), we initially hypothesized that the enzyme is able to transfer GlcUA to MU from UDPGlcUA, and the glucuronidation of MU competitively inhibits chain elongation of HA. We therefore tested the effect of pNP, another acceptor for UGTs (39Ciotti M. Marrone A. Potter C. Owens I.S. Pharmacogenetics. 1997; 7: 485-495Crossref PubMed Scopus (175) Google Scholar), on HAS activity. As shown in Fig. 2A, HAS activity was indeed inhibited by pNP. Production of MU-GlcUA in the Culture Medium and in the Reaction Supernatant of the Cell-free HA Synthesis—The effects of both MU and pNP prompted us to investigate whether the glucuronidation of these compounds is involved in the inhibition of HAS activity. The production of MU-GlcUA was analyzed by HPLC by using conditioned medium from 3Y1-HAS2 and 3Y1-Mock cells which were cultured for 24 h in the presence of various concentrations of MU. In the presence of MU," @default.
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• W2134213816 title "A Novel Mechanism for the Inhibition of Hyaluronan Biosynthesis by 4-Methylumbelliferone" @default.
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