Regulation of heparan sulfate and chondroitin sulfate glycosaminoglycan biosynthesis by 4-fluoro-glucosamine in murine airway smooth muscle cells.
The importance of the pathological changes in proteoglycans has driven the need to study and design novel chemical tools to control proteoglycan synthesis. Accordingly, we tested the fluorinated analogue of glucosamine (4-fluoro-N-acetyl-glucosamine (4-F-GlcNAc)) on the synthesis of heparan sulfate (HS) and chondroitin sulfate (CS) by murine airway smooth muscle (ASM) cells in the presence of radiolabeled metabolic precursors. Secreted and cell-associated CS and HS were assessed for changes in size by Superose 6 chromatography. Treatment of ASM cells with 4-F-GlcNAc (100 microM) reduced the quantity (by 64.1-76.6%) and decreased the size of HS/CS glycosaminoglycans associated with the cell layer (K(av) shifted from 0.30 to 0.45). The quantity of CS secreted into the medium decreased by 65.7-73.0%, and the size showed a K(av) shift from 0.30 to 0.50. Treatment of ASM cells with 45 microM and 179 microM 4-F-GlcNAc in the presence of a stimulator of CS synthesis, 4-methylumbelliferyl-beta-D-xyloside, reduced the amount of the xyloside-CS chains by 65.4 and 87.0%, respectively. The size of xyloside-CS chains synthesized in the presence of 4-F-GlcNAc were only slightly larger than those with xyloside treatment alone (K(av) of 0.55 compared with that of 0.6). The effects of 4-F-GlcNAc to inhibit CS synthesis were not observed with equimolar concentrations of glucosamine. We propose that 4-F-GlcNAc inhibits CS synthesis by inhibiting 4-epimerization of UDP-GlcNAc to UDP-GalNAc, thereby depleting one of the substrates required, whereas HS elongation is inhibited by truncation when the nonreducing terminus of the growing chain is capped with 4-F-GlcNAc.
Nigro
Julie
J
From the Department of Biomedical Engineering, Lerner Research Institute of The Cleveland Clinic Foundation, Cleveland, Ohio 44195; Commonwealth Scientific and Industrial Research Organization Molecular and Health Technologies, Clayton, Victoria 3168, Australia.
Wang
Aimin
A
From the Department of Biomedical Engineering, Lerner Research Institute of The Cleveland Clinic Foundation, Cleveland, Ohio 44195.
Mukhopadhyay
Durba
D
From the Department of Biomedical Engineering, Lerner Research Institute of The Cleveland Clinic Foundation, Cleveland, Ohio 44195.
Lauer
Mark
M
From the Department of Biomedical Engineering, Lerner Research Institute of The Cleveland Clinic Foundation, Cleveland, Ohio 44195.
Midura
Ronald J
RJ
From the Department of Biomedical Engineering, Lerner Research Institute of The Cleveland Clinic Foundation, Cleveland, Ohio 44195.
Sackstein
Robert
R
Departments of Dermatology and Medicine, Harvard Medical School, Brigham Women's Hospital, Boston, Massachusetts 02115.
Hascall
Vincent C
VC
From the Department of Biomedical Engineering, Lerner Research Institute of The Cleveland Clinic Foundation, Cleveland, Ohio 44195. Electronic address: hascalv@ccf.org.
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000096:biosynthesis*; D006497:Heparan Sulfate / Q000096:biosynthesis*; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism*; D012132:Respiratory Muscles / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014132:Trachea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
D000117:Acetylglucosamine; D002809:Chondroitin Sulfates; D006497:Heparan Sulfate; D006923:Hymecromone; C038142:chondroitin sulfate glycosaminoglycan; C089178:2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoroglucopyranose; C014111:methylumbelliferyl-beta-D-xyloside