c-Fos associates with the endoplasmic reticulum and activates phospholipid metabolism.

c-Fos, a transcription factor that constitutes DNA-binding AP-1 complexes, regulates gene expression that promotes long-lasting cellular changes. We show that, in addition to its transcription factor activity, c-Fos regulates the metabolism of phospholipids cytoplasmically by an AP-1-independent activity. Two waves of c-Fos expression that promote subsequent waves of stimulation of 32P-orthophosphate incorporation into phospholipids are evidenced in quiescent cultured fibroblasts induced to re-enter the cell cycle. The first wave of c-Fos expression peaks at 7.5 min and returns to control levels by 15 min. The second wave starts by 30 min and remains elevated at 120 min. In the first wave, the lipids that incorporate 32P are predominantly second-messenger polyphosphoinositides (PIP, PIP2, PIP3); whereas in the second wave, membrane-biogenesis-related lipids (PI, PE, PA), become radioactive. Both waves of phospholipid activation depend on c-Fos expression. It is interesting that a peptide that blocks AP-1 nuclear import does not affect phospholipid activation. Immunocytochemical examination showed c-Fos immunoreactivity associated to the endoplasmic reticulum. We conclude that c-Fos, rapidly induced upon cell stimulation, associates to the endoplasmic reticulum where it first regulates the synthesis/ replenishment of phospholipids required for signal transduction pathways and subsequently regulates enzymes involved in the genesis of new membrane necessary for cell growth.

Light affects c-fos expression and phospholipid synthesis in both retinal ganglion cells and photoreceptor cells in an opposite way for each cell type.

Retina photoreceptor and ganglion cells isolated from chicks that in vivo were exposed to light have a different phospholipid labeling capacity than those from chicks in the dark. In the light exposed animals, the phospholipid labeling in the ganglion cells is higher (Delta% 45, p<0.005) than in those maintained in the dark, whereas in the photoreceptor cells, the opposite occurs, that is, the phospholipid labeling is higher in the dark than in light. The light-dark differences for phospholipid labeling correlate with the expression of c-fos: when c-fos expression increases (both in mRNA and in c-Fos protein content), phospholipid labeling increases concomitantly. That is, in ganglion cells, c-fos expression and the phospholipid synthesis is higher in light with respect to dark, whereas in photoreceptor cells, c-fos expression and phospholipid synthesis is higher in dark with respect to light. Moreover, when an oligonucleotide antisense to c-fos is administered intraocularly prior to separating the animals into light and dark, no differences in c-fos expression and, consequently, no differences in phospholipid synthesis are found between animals in light and dark. Taken together, these results point to a novel mechanism by which rapid genomic responses to cell stimulation are converted to longer lasting changes in the cell components.

High-density lipoprotein from hypercholesterolemic animals has peroxidized lipids and oligomeric apolipoprotein A-I: its putative role in atherogenesis.

Oxidized lipoproteins have been involved in the pathogenesis of atherosclerosis and atherosclerotic lesions contain oxidized low density lipoprotein. Conversely, the presence of oxidized high density lipoprotein (HDL) in vivo has not been clearly established. Oxidation of HDL in vitro models produces an increase in peroxidized lipids and the appearance of apolipoprotein A-I (apo A-I) oligomers. We investigated the oxidative status of HDL in an in vivo model: the hypercholesterolemic chicken. The HDLs from control and hyperlipemic animals were analyzed for the content of lipid peroxides employing spectroscopic and fluorescence techniques, for the level of apo A-I oligomerization, and for susceptibility to in vitro oxidation. HDL from hypercholesterolemic chickens was more peroxidized (as detected by fluorescence), had higher amount of oligomeric apo A-I, and was oxidized to a greater extent by uv irradiation than that of control animals. We speculate that apo A-I oligomerization could be a key step in the atheroma formation.

Identification of an endogenous inhibitor of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyl transferase as apolipoprotein A1.

A previously described inhibitor of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyltransferase (GalNAc-T) (Quiroga et al., 1, 2), was purified from chicken blood serum by a new procedure. When subjected to SDS-PAGE, two major polypeptides of 27 and 70 kDa were observed. When tested in vitro, only the 27 kDa polypeptide inhibited the GalNAc-T. When added to chick cerebral embryonic neurons in culture, both polypeptides inhibited neuritogenesis. Both the 27 kDa and the 70 kDa fractions were present in the cells at 3 h following their addition to the cultures; both polypeptides had aneuritogenic activity and both inhibited the incorporation of [3H]-galactose into the cell gangliosides modifying their labeling pattern to a similar extent. Sequencing of the amino terminal end of the polypeptides showed that 18 and 9 amino acids from, respectively, the 27 and the 70 kDa polypeptides, were 100% homologues with the corresponding region of chick apolipoprotein Al (apo Al). After addition to cells in culture, no interconversion between the two polypeptides was detected after up to 20 h in culture. A monoclonal antibody that recognizes only the 70 kDa polypeptide, blocks its aneuritogenic effect without modifying that of the 27 kDa fraction. It is concluded that the endogenous inhibitor of GalNAc-T is apo Al.

Effects of the N-acetylgalactosaminyltransferase inhibitor on cultured cerebral cells.

The inhibitor preparation of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyltransferase (EC 2.4.1.92) (GalNAc-T) produces effects on the neurons and the glial (astrocytes) cells of the cerebrum in culture. The effect in culture is evidenced by aneuritogenesis, deficiency in the GalNAc-T activity, and decrease in the content of gangliosides, proteins, and lipids. In isolated glial cells the effect is evidenced by cytoplasm vesiculation and premature cessation of proliferation compared with control culture. The pattern of gangliosides in the inhibited culture shows a decrease in the amount of GD1a with respect to GD3; this is compatible with the notion that the effect is due to an inhibitor of the GM2 synthase. The inhibitor effects are reverted when it is eliminated after 24 or 48 hr in the culture medium.