c-Fos activates glucosylceramide synthase and glycolipid synthesis in PC12 cells

Abstract

It has been demonstrated that c-Fos has, in addition to its well recognized AP-1 transcription factor activity, the capacity to associate to the endoplasmic reticulum and activate key enzymes involved in the synthesis of phospholipids required for membrane biogenesis during cell growth and neurite formation. Because membrane genesis requires the coordinated supply of all its integral membrane components, the question emerges as to whether c-Fos also activates the synthesis of glycolipids, another ubiquitous membrane component. We show that c-Fos activates the metabolic labeling of glycolipids in differentiating PC12 cells. Specifically, c-Fos activates the enzyme glucosylceramide synthase (GlcCerS), the product of which, GlcCer, is the first glycosylated intermediate in the pathway of synthesis of glycolipids. By contrast, the activities of GlcCer galactosyltransferase 1 and lactosylceramide sialyltransferase 1 are essentially unaffected by c-Fos. Co-immunoprecipitation experiments in cells co-transfected with c-Fos and a V5-tagged version of GlcCerS evidenced that both proteins participate in a physical association. c-Fos expression is tightly regulated by specific environmental cues. This strict regulation assures that lipid metabolism activation will occur as a response to cell requirements thus pointing to c-Fos as an important regulator of key membrane metabolisms in membrane biogenesis-demanding processes.

FIGURE 1.

Metabolic labeling of glycolipids. Cells cultured 4 days with or without NGF and in the presence of oligonucleotides sense (SO) or antisense (ASO) to c-Fos mRNA, as indicated, were metabolically labeled with [¹⁴C]Gal 8 h before harvesting. Lipids were extracted, purified, and analyzed as described under “Experimental Procedures.” A, radioactivity (∼2000-3000 cpm per lane) of total lipid extracts. Results are the mean ± S.D. of three independent experiments performed in triplicate. ^*, p < 0.005 with respect to -NGF-treated cells as determined by Student's t test. Note that c-Fos ASO abrogates the increase of glycolipid labeling observed in cells induced to differentiate by feeding of NGF. B, Western blot determinations of c-Fos in cells cultured as in A. Nitrocellulose membranes immunostained for c-Fos were stripped and stained for α-tubulin labeling as a gel loading control. C, lipid extracts from cells cultured as in A were purified, chromatographed, and visualized as indicated under “Experimental Procedures.” The positions of co-chromatographed glycolipid standards are indicated. To avoid overexposure of the phosphatidylcholine bands, film exposure time of the middle part of the chromatogram was shorter than that for the rest.

FIGURE 2.

c-Fos-containing homogenates show activated labeling of endogenous glycolipid acceptors in vitro. Homogenates prepared from PC12 cells cultured 4 days under the indicated experimental conditions were incubated in vitro for 2 h with UDP-[³H]Glc. After incubation, labeled glycolipids were purified, and lipid-bound radioactivity was quantified. Results are the mean ± S.D. of three independent experiments performed in triplicate. ^*, p < 0.005 with respect to -NGF-treated cells as determined by Student's t test.

FIGURE 3.

In vitro glycolipid labeling activation by c-Fos or c-Fos deletion mutants. Cells mock transfected (control) or transfected to express c-Fos or the indicated c-Fos deletion mutants (depicted on the left) were cultured for 2 days with NGF, medium was replaced by fresh medium without NGF, and cultures were continued for an additional 2 days. Homogenates prepared from these cells were incubated for 2 h with UDP-[³H]Glc, and glycolipid labeling was determined as in Fig. 2. Results are the mean ± S.D. of three independent experiments performed in triplicate. ^*, p < 0.0001 with respect to control, mock transfected cells as determined by Student's t test.

FIGURE 4.

GlcCerS activity in homogenates determined in the presence or the absence of c-Fos. The in vitro conversion of C₆-NBD-Cer to C₆-NBD-GlcCer was assayed in homogenates obtained from cells cultured in the absence of NGF for 4 days with or without the addition of recombinant c-Fos. A, formation of C₆-NBD-GlcCer with and without addition of c-Fos (1 ng/μg of homogenate protein) to the incubates as determined at the indicated incubation times after HPTLC separation and UV visualization. B, quantification (expressed as arbitrary densitometric units) of C₆-NBD-GlcCer formation in the absence of c-Fos (closed circles) or the presence of c-Fos (opened circles). Results are the mean ± S.D. of four independent experiments.

FIGURE 5.

GlcCerS kinetic parameters in the presence or the absence of c-Fos. Enzyme activity was determined at 15 min of incubation as indicated in Fig. 4 at the indicated increasing concentrations of UDP-Glc and constant saturating concentration of C₆-NBD-Cer (20 μm) (A) or at increasing concentration of C₆-NBD-Cer and constant saturating concentration of UDP-Glc (400 μm) (B) with or without the addition of c-Fos (1 ng/μg of homogenate protein) as indicated. Insets show the Lineweaver-Burk plots for K_m and V_max calculations.

FIGURE 6.

Immunofluorescence examination shows GlcCerS and c-Fos co-localizing at the ER. A, subcellular localization of GlcCerS was examined in PC12 cells co-transfected to express V5-tagged-GlcCerS and the Golgi-resident YFP-tagged SialT2 (upper row) or the ER-resident, YFP-tagged Iip33 (lower row). GlcCerS was evidenced with V5 and Alexa 488 antibodies as described under “Experimental Procedures.” The YFP-tagged markers were detected by intrinsic fluorescence. Note the ER and Golgi distribution of GlcCerS. B, subcellular localization of c-Fos examined in PC12 cells cultured +NGF for 7.5 min that were fixed and immunostained for c-Fos and the GlcCerS tag V5 (top panel) or the Golgi marker TGN-38 (middle panel) or the ER marker calnexin (bottom panel) as described under “Experimental Procedures.” Note co-localization of c-Fos/V5-GlcCerS and of c-Fos/ER. Bar:10 μm.

FIGURE 7.

GlcCerS but not GalT1 or SialT1, physically associates with c-Fos. A, lysates from quiescent cells co-transfected to express c-Fos and V5 tagged-GlcCerS were immunoprecipitated using anti-c-Fos or V5 antibodies as indicated. Immunocomplexes were analyzed by SDS-PAGE followed by Western blotting anti-V5 or c-Fos antibodies as indicated. B, top panel, lysates from quiescent cells transfected to express c-Fos or co-transfected to express c-Fos and V5-tagged-GlcCerS or c-Fos and His-tagged GalT1 or c-Fos and HA-tagged SialT1 were immunoprecipitated using, from left to right, V5 (first two lanes) or His or HA antibodies as indicated. Immunocomplexes were analyzed by SDS-PAGE followed by Western blotting with anti-c-Fos antibody. The lower panel shows the Western blots of c-Fos input (5%) of the corresponding cell lysates of the upper panel.