Stimulation of α7 nicotinic acetylcholine receptor regulates glutamate transporter GLAST via basic fibroblast growth factor production in cultured cortical microglia.

The α7 nicotinic acetylcholine (nACh) receptor expressed in microglia has a crucial role in neuroprotection. Simulation of α7 nACh receptor leads to increased expression of glutamate/aspartate transporter (GLAST), which in turn decreases synaptic glutamate levels. However, the upregulation of GLAST in cultured rat cortical microglia appears long after (over 18 h) stimulation of the α7 nACh receptor with nicotine. Thus, the current study elucidated the pathway responsible for the induction of GLAST expression in cultured cortical microglia. Nicotine-induced GLAST mRNA expression was significantly inhibited by cycloheximide pretreatment, indicating that a protein intermediary, such as a growth factor, is required for GLAST expression. The expression of fibroblast growth factor-2 (FGF-2) mRNA in cortical microglia was significantly increased 6 and 12h after treatment with nicotine, and this increase was potently inhibited by pretreatment with methyllycaconitine, a selective α7 nACh receptor antagonist. The treatment with nicotine also significantly increased FGF-2 protein expression. Furthermore, treatment with recombinant FGF-2 increased GLAST mRNA, protein expression and (14)C-glutamate uptake, a functional measurement of GLAST activity. Conversely, pretreatment with PD173074, an inhibitor of FGF receptor (FGFR) tyrosine kinase, significantly prevented the nicotine-induced expression of GLAST mRNA, its protein and (14)C-glutamate uptake. Reverse transcription polymerase chain reaction confirmed FGFR1 mRNA expression was confined to cultured cortical microglia. Together, the current findings demonstrate that the neuroprotective effect of activation of microglial α7 nACh receptors could be due to the expression of FGF-2, which in turn increases GLAST expression, thereby clearing glutamate from synapse and decreasing glutamate neurotransmission.

Isolation and characterisation of a 17-kDa staphylococcal heparin-binding protein with broad specificity.

A previous study reported the ability of staphylococci to bind heparin and heparin-dependent host growth factors. The present study isolated and identified heparin- and basic fibroblast growth factor (bFGF)-binding surface components of S. epidermidis strain RP12 and S. haemolyticus strain SM 131. The staphylococcal heparin-binding component(s) were purified by affinity chromatography on heparin-Sepharose and a major heparin-binding protein, here designated HBP, was identified by immunoblot in these two coagulase-negative staphylococcal (CNS) species. The HBP was shown to be acidic with an approximate pI of 4.6 and a molecular mass around 17 kDa. The binding of heparin to HBP was inhibited by heparin, fucoidan, pentosan polysulphate and various other sulphated polysaccharides, but not by non-sulphated compounds. However, the purified HBP from both S. epidermidis and S. haemolyticus revealed broad specificity, and also bound bFGF, thrombospondin, von Willebrand factor and, weakly, fibrinogen. The N-terminal sequences of the 17-kDa HBP from S. epidermidis and S. haemolyticus showed only limited identity. Comparison of the first 15 amino acid residues derived from either strain with known sequences in the protein databases revealed no close similarities. Taken together, these results suggest that the adhesion of at least some CNS to host sulphated glycosaminoglycans may be mediated by a previously uncharacterised group of surface proteins.

High throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry.

We have used intramolecular cross-linking, MS, and sequence threading to rapidly identify the fold of a model protein, bovine basic fibroblast growth factor (FGF)-2. Its tertiary structure was probed with a lysine-specific cross-linking agent, bis(sulfosuccinimidyl) suberate (BS(3)). Sites of cross-linking were determined by tryptic peptide mapping by using time-of-flight MS. Eighteen unique intramolecular lysine (Lys-Lys) cross-links were identified. The assignments for eight cross-linked peptides were confirmed by using post source decay MS. The interatomic distance constraints were all consistent with the tertiary structure of FGF-2. These relatively few constraints, in conjunction with threading, correctly identified FGF-2 as a member of the beta-trefoil fold family. To further demonstrate utility, we used the top-scoring homolog, IL-1beta, to build an FGF-2 homology model with a backbone error of 4.8 A (rms deviation). This method is fast, is general, uses small amounts of material, and is amenable to automation.

Distribution and putative roles of fibroblast growth factor-2 isoforms in corneal endothelial modulation.

PURPOSE: Corneal endothelial modulation factor (CEMF) released by inflammatory cells induces de novo synthesis of fibroblast growth factor (FGF)-2, which is a morphogen and a potent mitogen of corneal endothelial cells (CECs). Four isoforms of FGF-2 have been found in the nucleus, cytoplasm, or extracellular matrix (ECM) in different cell lines. In the present study, the profiles of the isoforms of FGF-2 that are induced by CEMF were investigated, and whether the differential localization of the isoforms of FGF-2 plays a role in CECs proliferation and subsequent modulation was examined. METHODS: Nuclear, cytoplasmic, and ECM fractions of normal and modulated CECs were separated, and FGF-2 isoforms were further purified by heparin-Sepharose column. The molecular sizes of the isoforms were determined by immunoblot analysis, using a specific antibody directed against FGF-2. Cell proliferation was determined by cell counting. Cellular localization of FGF-2 was determined by immunofluorescence staining during different stages of cell growth. RESULTS: To confirm that CEMF modulated CECs under the conditions used in this study, its effect on cell proliferation and cell shape was determined: CEMF-treated cells showed enhanced cell proliferation profiles and fibroblastlike morphology. In rapidly growing normal CECs, FGF-2 was predominantly present in the nucleus. As the cells reached confluence, the staining potential in the nucleus was markedly reduced. Cytoplasmic staining of FGF-2 was barely detectable, regardless of cell stages. In CEMF-modulated cells, the rapidly growing cells showed strong staining of FGF-2 in the nucleus, whereas cytoplasmic and ECM staining was weak. When modulated cells reached confluence, the staining of FGF-2 in the nuclei remained strong, whereas ECM staining was significantly increased. Immunoblot analysis of the subcellular fraction showed that the 24-kDa FGF-2 was predominantly present in the nucleus, whereas the 18-kDa form was the major molecule in cytoplasmic and ECM fractions in normal and modulated cells. CONCLUSIONS: These findings indicate that 24-kDa nuclear FGF-2 may be involved in cell proliferation in growing CECs. The persistent nuclear localization and simultaneous ECM localization of FGF-2 are induced by CEMF, and these FGF-2 isoforms seem to play a role in cell proliferation and modulation.