title: Internalization of basic fibroblast growth factor at the mouse blood-brain barrier involves perlecan, a heparan sulfate proteoglycan.
abstract: In this study, the internalization mechanism of basic fibroblast growth factor (bFGF) at the blood-brain barrier (BBB) was investigated using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4 cells) as an in vitro model of the BBB and the corresponding receptor was identified using immunohistochemical analysis. The heparin-resistant binding of [125I]bFGF to TM-BBB4 cells was found to be time-, temperature-, osmolarity- and concentration-dependent. Kinetic analysis of the cell-surface binding of [125I]bFGF to TM-BBB4 cells revealed saturable binding with a half-saturation constant of 76 +/- 24 nm and a maximal binding capacity of 183 +/- 17 pmol/mg protein. The heparin-resistant binding of [125I]bFGF to TM-BBB4 was significantly inhibited by a cationic polypeptide poly-L-lysine (300 micro m), and compounds which contain a sulfate moiety, e.g. heparin and chondroitin sulfate-B (each 10 micro g/mL). Moreover, the heparin-resistant binding of [125I]bFGF in TM-BBB4 cells was significantly reduced by 50% following treatment with sodium chlorate, suggesting the loss of perlecan (a core protein of heparan sulfate proteoglycan, HSPG) from the extracellular matrix of the cells. This type of binding is consistent with the involvement HSPG-mediated endocytosis. RT-PCR analysis revealed that HSPG mRNA and FGFR1 and FGFR2 (tyrosine-kinase receptors for bFGF) mRNA are expressed in TM-BBB4 cells. Moreover, immunohistochemical analysis demonstrated that perlecan is expressed on the abluminal membrane of the mouse brain capillary. These results suggest that bFGF is internalized via HSPG, which is expressed on the abluminal membrane of the BBB. HSPG at the BBB may play a role in maintaining the BBB function due to acceptance of the bFGF secreted from astrocytes.
authors.affiliation: Department of Drug Disposition and Pharmacokinetics, School of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan. deguhi@pharm.teikyo-u.ac.jp
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Deguchi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Okutsu
authors.forename: Takashi
authors.initials: T
authors.lastname: Okura
authors.forename: Shizuo
authors.initials: S
authors.lastname: Yamada
authors.forename: Ryohei
authors.initials: R
authors.lastname: Kimura
authors.forename: Takuro
authors.initials: T
authors.lastname: Yuge
authors.forename: Akihiko
authors.initials: A
authors.lastname: Furukawa
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Morimoto
authors.forename: Masanori
authors.initials: M
authors.lastname: Tachikawa
authors.forename: Sumio
authors.initials: S
authors.lastname: Ohtsuki
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Hosoya
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Terasaki
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D001812:Blood-Brain Barrier / Q000502:physiology*; D001921:Brain / Q000098:blood supply / Q000166:cytology / Q000378:metabolism*; D002196:Capillaries / Q000166:cytology; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism* / Q000493:pharmacokinetics; D019812:Heparan Sulfate Proteoglycans / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D008024:Ligands; D051379:Mice; D012333:RNA, Messenger / Q000096:biosynthesis; D017468:Receptors, Fibroblast Growth Factor / Q000096:biosynthesis / Q000235:genetics
chemical_list: D019812:Heparan Sulfate Proteoglycans; D008024:Ligands; D012333:RNA, Messenger; D017468:Receptors, Fibroblast Growth Factor; D016222:Fibroblast Growth Factor 2; C071927:perlecan