c-Fos Activated Phospholipid Synthesis Is Required for Neurite Elongation in Differentiating PC12 Cells

Abstract

We have previously shown that c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. Herein, using PC12 cells induced to differentiate by nerve growth factor, the genomic effect of c-Fos in initiating neurite outgrowth is shown as distinct from its nongenomic effect of activating phospholipid synthesis and sustaining neurite elongation. Blocking c-Fos expression inhibited differentiation, phospholipid synthesis activation, and neuritogenesis. In cells primed to grow, blocking c-Fos expression determined neurite retraction. However, transfected cells expressing c-Fos or c-Fos deletion mutants with capacity to activate phospholipid synthesis sustain neurite outgrowth and elongation in the absence of nerve growth factor. Results disclose a dual function of c-Fos: it first releases the genomic program for differentiation and then associates to the endoplasmic reticulum and activates phospholipid synthesis. Because phospholipids are key membrane components, we hypothesize this latter phenomenon as crucial to support membrane genesis demands required for cell growth and neurite elongation.

The Catalytic Efficiency of Lipin 1β Increases by Physically Interacting with the Proto-oncoprotein c-Fos

“…In differentiating PC12 cells, blocking c-Fos expression impairs both neuritogenesis and the activation of phospholipid and glycolipid synthesis. TLC analysis of total radioactivity in lipid extracts after metabolic labeling of these cells with [ 14 C]Gal showed a 50 -60% increase in the labeling of all 14 Clabeled lipids, including glycolipids and phosphatidylcholine (13). At the same time, similar experiments carried out with [ 32 P]orthophosphate acid show a similar increase on all 32 Plabeled phospholipids (14).…”

“…L-␣-Dipalmitoyl-[glycerol- 14 C]PA (specific activity 100 -200 mCi/mmol) was purchased from PerkinElmer Life Sciences. [2-3 H]Glycerol (specific activity 200 mCi/mmol) and Omnifluor were obtained from PerkinElmer Life Sciences.…”

“…Western Blot Analysis-Total cell lysates and immunoprecipitates were subjected to SDS-PAGE under reducing conditions on 12% polyacrylamide gels and transferred to nitrocellulose membrane as described previously (14). Blocked membranes were incubated with the specified primary antibody, washed twice with PBS/Tween 0.1%, and incubated using the appropriate secondary antibodies (LI-COR) (1:25,000).…”

“…The only difference between these c-Fos deletion mutants is that NB contains the 21 aa of the BD domain of c-Fos, whereas NA lacks them. It is worth mentioning that the relevance of BD has already been proved for phospholipid synthesis activation capacity (13,14,18). Next, we point-mutated basic residues within this domain in full-length c-Fos.…”

“…TLC analysis of total radioactivity in lipid extracts after metabolic labeling of these cells with [ 14 C]Gal showed a 50 -60% increase in the labeling of all 14 Clabeled lipids, including glycolipids and phosphatidylcholine (13). At the same time, similar experiments carried out with [ 32 P]orthophosphate acid show a similar increase on all 32 Plabeled phospholipids (14). These results corroborate a global stimulation of the lipid-synthesizing machinery in the c-Fosmediated response to a differentiation stimulus of these cells.…”