Abstract
We have previously shown that ongoing glucosylceramide (GlcCer) synthesis is required for basic fibroblast growth factor (bFGF) and laminin to stimulate axonal growth in cultured hippocampal neurons (Boldin, S., and Futerman, A. H. (1997) J. Neurochem. 68, 882-885). We now demonstrate that stimulation of axonal growth by bFGF leads to an increase in the rate of GlcCer synthesis. Within minutes of incubation with bFGF, a significant increase in the rate of metabolism of [(14)C]hexanoyl ceramide to [(14)C]hexanoyl GlcCer is detected, but there are no changes in the rate of [(14)C]hexanoyl sphingomyelin synthesis. In vitro analysis of GlcCer synthase activity revealed an approximately 2-fold increase in the rate of [(14)C]hexanoyl GlcCer synthesis upon incubation with either bFGF or laminin; other growth factors, which did not effect the rate of axon growth, had no effect on the rate of [(14)C]hexanoyl GlcCer synthesis. The increased rate of [(14)C]hexanoyl GlcCer synthesis was not affected by preincubation with either cycloheximide or actinomycin, and no elevation of GlcCer synthase mRNA levels was detected, suggesting that GlcCer synthase is up-regulated by a post-translational mechanism. The relevance of these results for understanding the regulation of axonal growth is discussed.
Highlights
Glucosylceramide (GlcCer),1 the simplest glycosphingolipid, is synthesized from ceramide (Cer) on the cytosolic leaflet of the Golgi apparatus [1,2,3,4]
We have previously shown that ongoing glucosylceramide (GlcCer) synthesis is required for basic fibroblast growth factor and laminin to stimulate axonal growth in cultured hippocampal neurons
Two possibilities could explain the increase in the rate of [14C]hexanoyl Cer uptake. (i) basic fibroblast growth factor (bFGF) could directly affect the rate of [14C]hexanoyl Cer uptake into neurons, and, as a consequence, the rate of [14C]hexanoyl GlcCer synthesis is elevated because of increased availability of intracellular [14C]hexanoyl Cer. (ii) bFGF could directly affect the rate of [14C]hexanoyl GlcCer synthesis, and, as a result, more [14C]hexanoyl Cer is taken up by neurons to provide sufficient substrate for increased [14C]hexanoyl GlcCer synthesis
Summary
Materials—n-Hexanoic acid [1-14C]N-hydroxysuccinimide ester (53.2 or 55 mCi/mmol) was from American Radiolabeled Chemicals, Inc. Cell Culture—Hippocampal neurons plated at high-density (180,000 cells/24-mm polylysine-coated glass coverslip) were cultured in serumfree medium as described [14, 16, 17] and grown for 2 days in 100-mm Petri dishes containing a monolayer of astroglia. After resuspension in a small volume of water, lipids were extracted [19] and separated by thin layer chromatography using CHCl3/CH3OH/9.8 mM CaCl2 (60:35:8, v/v/v) as the developing solvent. Thin layer chromatography plates were exposed to a 14Csensitive imaging plate (BAS-TR2040S, Fuji Photo Film Co., Ltd., Japan), lipids were recovered from the plates by scraping, and radioactiv-. RT-PCR products were resolved on 1.5% agarose-gels
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