The kinase c-Src and the phosphatase TC45 coordinately regulate c-Fos tyrosine phosphorylation and c-Fos phospholipid synthesis activation capacity.

Our previous work showed that in T98G cells, a human glioblastoma multiforme-derived cell line, the association of c-Fos to the endoplasmic reticulum (ER) and consequently, the capacity of c-Fos to activate phospholipid synthesis, is regulated by the phosphorylation state of tyrosine (tyr) residues #10 and #30 of c-Fos. The small amount of c-Fos present in quiescent cells is tyr-phosphorylated, is dissociated from the ER membranes and does not activate phospholipid synthesis. However, on induction of the cell to re-enter growth, c-Fos expression is rapidly induced, it is found dephosphorylated, associated to ER membranes and activating phospholipid synthesis (Portal et al., 2007). Herein, using in vivo and in vitro experimental strategies, we show that the kinase c-Src is capable of phosphorylating tyr residues of c-Fos whereas the phosphatase TC45 T-cell protein-tyr phosphatase (TC-PTP) dephosphorylates them, thus enabling c-Fos/ER association and activation of phospholipid synthesis. Results also suggest that the regulation of the phosphorylation/dephosphorylation cycle of c-Fos occurs at the TC-PTP level: induction of cells to re-enter growth promotes the translocation of TC45 from a nuclear to a cytoplasmic location concomitant with its activation. Activated TC45 in its turn promotes dephosphorylation of pre-formed c-Fos, enabling cells to rapidly activate phospholipid synthesis to respond to its growth demands.

N-Terminal c-Fos tyrosine phosphorylation regulates c-Fos/ER association and c-Fos-dependent phospholipid synthesis activation.

c-Fos dephosphorylated on tyrosine (c-Fos), a component of the activator protein-1 (AP-1) family of transcription factors, is expressed at very low levels in resting cells. However, its expression is rapidly upregulated in cells undergoing G(0) to S phase transition leading to AP-1-dependent gene transcription responses. In addition, cytoplasmic c-Fos associates to the endoplasmic reticulum (ER) membranes and activates phospholipid synthesis during cell growth and differentiation. Herein, it is shown that in T98G cells, c-Fos/ER association and consequently phospholipid synthesis activation is regulated by the phosphorylated state of c-Fos tyrosine (tyr) residues. The small amount of c-Fos present in quiescent T98G cells is tyr-phosphorylated and not ER-membrane bound. In growing cells, it is dephosphorylated, associated to ER membranes and promotes phospholipid synthesis activation. Impairing tyr-dephosphorylation abrogates phospholipid synthesis activation and reduces proliferation rates to those of quiescent cells. Substitution of tyr residues 10, 30, 106 and 337 evidence tyr 10 and 30 as relevant for this regulatory phenomenon. It is concluded that phosphorylation of tyr residues 10 and 30 of c-Fos regulate the rate of synthesis of phospholipids by regulating c-Fos/ER association.

Synthesis of retinal ganglion cell phospholipids is under control of an endogenous circadian clock: daily variations in phospholipid-synthesizing enzyme activities.

Retinal ganglion cells (RGCs) are major components of the vertebrate circadian system. They send information to the brain, synchronizing the entire organism to the light-dark cycles. We recently reported that chicken RGCs display daily variations in the biosynthesis of glycerophospholipids in constant darkness (DD). It was unclear whether this rhythmicity was driven by this population itself or by other retinal cells. Here we show that RGCs present circadian oscillations in the labeling of [32P]phospholipids both in vivo in constant light (LL) and in cultures of immunopurified embryonic cells. In vivo, there was greater [32P]orthophosphate incorporation into total phospholipids during the subjective day. Phosphatidylinositol (PI) was the most 32P-labeled lipid at all times examined, displaying maximal levels during the subjective day and dusk. In addition, a significant daily variation was found in the activity of distinct enzymes of the pathway of phospholipid biosynthesis and degradation, such as lysophospholipid acyltransferases (AT II), phosphatidate phosphohydrolase (PAP), and diacylglycerol lipase (DGL) in cell preparations obtained in DD, exhibiting differential but coordinated temporal profiles. Furthermore, cultures of immunopurified RGCs synchronized by medium exchange displayed a circadian fluctuation in the phospholipid labeling. The results demonstrate that chicken RGCs contain circadian oscillators capable of generating metabolic oscillations in the biosynthesis of phospholipids autonomously.

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.

c-Fos is surface active and interacts differentially with phospholipid monolayers.

The transcription factor c-Fos forms stable Gibbs and Langmuir monolayers at the air-buffer interface. Its marked surface activity is enhanced by penetration into phospholipid films above the protein's own maximum adsorption surface pressure to a lipid-free interface. The protein-phospholipid stabilizing interactions at the interface depend on the lipid polar head group and the increases of lateral surface pressure generated are comparable to those of membrane-active proteins. The surface activity of c-Fos is strong enough to thermodynamically drive and retain c-Fos at the membrane interface where it may exert direct or indirect effects.

Circadian regulation of phospholipid metabolism in retinal photoreceptors and ganglion cells.

The neural retina is a key component of the vertebrate circadian system that is responsible for synchronizing the central circadian pacemaker to external light-dark (LD) cycles. The retina is itself rhythmic, showing circadian cycles in melatonin levels and gene expression. We assessed the in vivo incorporation of 32P-phosphate and 3H-glycerol into phospholipids of photoreceptor cells (PRCs) and retina ganglion cells (GCs) from chicks in constant illumination conditions (dark: DD or light: LL) over a 24-h period. Our findings showed that in DD there was a daily oscillation in 32P-labeling of total phospholipids synthesized in GCs and axonally transported to the brain. This metabolic fluctuation peaked during the subjective night (zeitgeber time [ZT] 20), persisted for several hours well into the subjective day and declined at subjective dusk (ZT 10-12). PRCs also exhibited an in vivo rhythm of 32P-phospholipid synthesis in DD. This rhythm peaked around ZT 22, continued a few hours into the day and declined by the end of subjective dusk. The major individual species labeled 1 h after 32P administration was phosphatidylinositol (PI) in both PRCs and GCs. Rhythmic phospholipid biosynthesis was also observed in DD after 3H-glycerol administration, with levels in GCs elevated from midday to early night. PRCs exhibited a similar rhythmic profile with the lowest levels of labeling during midnight. Phosphatidylcholine (PC) accounted for the individual species with the highest ratio of 3H-glycerol incorporation in both cell populations at all phases examined. By contrast, in LL the rhythm of 3H-glycerol labeling of phospholipids damped out in both cell layers. Our findings support the idea that, in constant darkness, the metabolism of retinal phospholipids, including their de novo biosynthesis, is regulated by an endogenous circadian clock.

Immediate early gene expression within the visual system: light and circadian regulation in the retina and the suprachiasmatic nucleus.

Immediate early genes are a family of genes that share the characteristic of having their expression rapidly and transiently induced upon stimulation of neuronal and non-neuronal cells. In this review, first a short description of the IEGs is given, then it is discussed the stimulus-induced and circadian-induced variations in the expression of IEGs in the visual system, mainly in the retina and the suprachiasmatic nucleus. The possible physiological consequences of these variations in IEG expression are also considered. Finally, we refer to two aspects of our recent studies and those of other laboratories involving light-driven IEG expression. The first is the finding that in the chick retina, the expression of c-fos is differentially modulated in the different cell types and that c-fos regulates the synthesis of the quantitatively most important lipids of all cells, the phospholipids, by a non-genomic mechanism. The second is the occurrence of differential waves of IEG expression in the mammalian suprachiasmatic nucleus regarding light induction or spontaneous oscillations.

A simple method to obtain retinal cell preparations highly enriched in specific cell types. Suitability for lipid metabolism studies.

The neural retina is a highly complex tissue composed of excitatory and inhibitory neurons and of glial cells. The biosynthesis of lipids that occurs in the retina may be distinctly regulated in one neuronal type of cells with respect to another. To study the cell-type-specific aspects of lipid metabolism, a method for the separation of different retinal cell populations is needed. Herein, we describe a very simple procedure to isolate preparations highly enriched in specific retinal cell types that are suitable for in vitro biochemical assays. The method consists of selectively obtaining photoreceptors (PRC) and retina ganglion cells (RGC) from lyophilized chicken retinas using Scotch tape to assess, then, the in vitro incorporation of labeled precursors into phospholipid moieties. When their metabolic capability was assayed, it was found that these cell preparations maintain their enzyme activities intact to incorporate (32)P-phosphate into phospholipids in vitro at a similar rate as observed in fresh tissue after 1 h incubation. The highest proportion of labeling was observed in phosphatidylethanolamine (PE), followed by phosphoinositides (PIPs), phosphatidylcholine (PC) and phosphatidic acid (PA). Phosphatidate-phosphohydrolase (PAPase), a key enzyme of glycerolipid metabolism, exhibits similar levels of activity when assessed in fresh or frozen cell preparations, indicating that the lyophilization procedure does not significantly affect this activity. It is concluded that different cell populations obtained by the experimental procedure described herein, are useful to study the cellular metabolism and its regulation.

Light exposure activates retina ganglion cell lysophosphatidic acid acyl transferase and phosphatidic acid phosphatase by a c-fos-dependent mechanism.

We previously reported that the biosynthesis of phospholipids in the avian retina is altered by light stimulation, increasing significantly in ganglion cells in light and in photoreceptor cells in dark. In the present work, we have determined that light significantly increases the incorporation of [3H]glycerol into retina ganglion cell glycerophospholipids in vivo by a Fos-dependent mechanism because an oligonucleotide antisense to c-fos mRNA substantially blocked the light-dark differences. We also studied in vitro the enzyme activities of phosphatidate phosphohydrolase (PAPase), lysophosphatidate acyl transferase (AT II), and phosphatidylserine synthase from retinas of chickens exposed to light or dark. Higher PAPase I and AT II activities were found in incubations of retinal ganglion cells from animals exposed to light; no increase was observed in preparations obtained from light-exposed animals treated with the c-fos antisense oligonucleotide. No light-dark differences were found in phosphatidylserine synthase activity. These findings support the idea that a coordinated photic regulation of PAPase I and AT II is taking place in retina ganglion cells. This constitutes a reasonable mechanism to obtain an overall increased synthesis of glycerophospholipids in stimulated cells that is mediated by the expression of Fos-like proteins.

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.

Light exposure stimulates the activity of ganglioside glycosyltransferases of retina ganglion cells.

In chicks submitted to light stimulation, the synthesis of gangliosides of the retina ganglion cell increases with respect to chicks maintained in the dark. In an attempt to elucidate if the activation of glycosyltransferases participates in the establishment of these light-dark differences detected in vivo, we examined the activity of a key ganglioside glycosyltransferase, the GalNAc-T (N-acetylgalactosaminyltransferase) that converts GM3 to GM2, in the retina ganglion cells isolated from light and dark exposed chicks. We found that GalNAc-T and other glycosyltransferases are active in these ganglion cell preparations; the kinetic parameters for GalNAc-T were similar to those previously reported for chick retina. The other glycosyltransferase activities assayed were the galactosyltransferase (Gal-T2) that converts GM2 to GM1 and the N-acetylneuraminyltransferase (Sialyl-T1) that converts lactosylceramide to GM3. The three glycosyltransferase activities were higher in the ganglion cell preparations obtained from chicks exposed to light compared to those maintained in the dark. For the GalNAc-T activity, the differences disappear when the cell preparations are sonicated or if the assays are carried out in the presence of detergents or if the end product of the reaction is added to the incubates. The results indicate that the activation of the glycosyltransferases is part of the phenomenon required for cells to achieve the precise rate of synthesis of gangliosides needed in vivo.

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.

Immediate early gene c-fos regulates the synthesis of phospholipids but not of gangliosides.

Retinal ganglion cells isolated from chicks that in vivo were exposed to light have a higher phospholipid labeling capacity than those obtained from animals in the dark. Actinomycin D or a mixture of protein synthesis inhibitors or of antisense oligonucleotides to c-fos plus c-jun injected intraocularly 1 hr prior to the stimulation period, abolished the light-dark differences for phospholipids but not for gangliosides. Light stimulation induced the formation (and/or stabilization) of c-fos mRNA and of the protein c-Fos, indicating that immediate early gene induction, and consequently the synthesis of the protein(s) encoded, is essential to increase the synthesis of phospholipids but not of gangliosides. These results suggest a novel mechanism by which immediate early genes engram neural cells, modifying specifically the metabolism of cell constituents producing long-lasting changes in the cells.

Labeling of retina and optic tectum phospholipids in chickens exposed to light or dark.

The labeling of retina ganglion cell and optic tectum phospholipids was determined in chickens given an intraocular injection of 32P and then either exposed to light or maintained in the dark. Significantly higher labeling was found in the optic tectum phospholipids of light-exposed compared with dark-maintained animals after 3-24 h of labeling. In the ganglion cells, the labeling of phospholipids increased in dark with respect to light at 15 and 30 min of labeling; from 60 min to 24 h, the labeling of phospholipids was significantly higher in light with respect to dark, even if the precursor pool showed a higher labeling in dark at all times studied. When labeling was allowed to proceed in the dark for 30 min and then half of the animals were exposed to light for 15 min, the labeling of ganglion cell phospholipids of light-exposed animals was significantly higher than those of animals kept in the dark. No individual phospholipid accounted for the differences observed in the labeling of the total phospholipid pool. These results are interpreted as an increase in the biosynthesis of phospholipids in the ganglion cell somas in light with respect to dark.

Synthesis and translocation of gangliosides and glycoproteins during urethane anesthesia.

In this work, we have studied (a) the contents of gangliosides, glycoproteins, and phospholipids of the vesicle and plasma membrane fractions from brains of anesthetized and control rats and chickens and (b) the labeling of gangliosides and glycoproteins in the retina ganglion cell layer and optic tectum of urethane-anesthetized and control chickens after intraocular injection of a labeled N-acetylneuraminic acid precursor and the distribution of the label after subcellular fractionation. We found an increase in the content of gangliosides relative to protein in the vesicle fraction of both anesthetized rats and chickens relative to their controls. Other values were not affected by anesthesia. These results do not reflect a faster synthesis of gangliosides stimulated by urethane, because their rate of labeling was diminished in anesthetized animals. During the 4-h period after the animals were injected intraocularly with the radioactive precursor, the highest values of ganglioside-specific radioactivity were found in the vesicle fraction of control and anesthetized animals; at longer intervals, the specific radioactivity of the vesicle and plasma membrane fractions became rather similar. These data are in accordance with previous studies from this laboratory suggesting that the synthesis of the carbohydrate chain of gangliosides is regulated by the physiological demands made by the neurotransmitting system.

Optic nerve integrity is required for light to affect retina ganglion cell gangliosides.

The labeling of retina ganglion cell and optic tectum gangliosides after an intraocular injection of N-[3H]acetylmannosamine ([3H]ManNAc) is higher in chickens exposed to light than in those maintained in darkness. In the present work we studied whether the signal for the higher labeling of ganglion cells in light originates in the photoreceptor layer or comes from the nerve terminal. For this purpose the labeling of ganglion cell gangliosides was determined in light and dark in chickens with one optic nerve severed. The results showed that the effect of light occurred only in the eye normally connected to the optic tectum. In the eye with its optic nerve severed, no difference was observed between the labeling of gangliosides in animals in light and dark, having both groups the labeling values of the normal eyes exposed to light. The results indicate that the information that decreases labeling in darkness or accelerates it in light originates in the nerve terminal.

Inhibition of the chicken retinal UDP-GaINAc:GM3, N-acetylgalactosaminyl-transferase by blood serum and by pineal gland extracts.

The effects of the pineal gland extract and blood serum on the activity of the UDP-GalNAc:GM3, N-acetylgalactosaminyltransferase (GM3:GalNAc-T) from chicken retina were studied. Both preparations have inhibitory capability on the enzyme activity. Two types of inhibitory capabilities were found: one is heat labile and decomposes UDP-GalNAc and another is heat stable. When the pineal gland extracts were prepared from light-exposed chickens, the inhibitory capability increased with respect to the extracts from dark-maintained animals; vice versa, blood serum from dark-maintained animals had higher heat-labile and heat-stable capabilities than that from light-exposed chickens. In in vitro experiments, no difference was found in the inhibitory capability of blood serum extracts from pinealectomized animals compared to control animals. In vivo labeling experiments with pinealectomized animals in either light or dark showed similar differences in the labeling of the optic tectum gangliosides as the normal animals.

Short term labeling of proteins, gangliosides and glycoproteins of the optic tract of chickens exposed to light or darkness.

In contrast with previous findings of the labeling of the glycosidic moieties of the gangliosides and glycoproteins in chickens injected with N-[ (3)H ] acetylmannosamine , the labeling of the ganglion cell layer and optic tectum proteins of chicks exposed to light after an intraocular injection of [(3)H]proline showed no differences with those of their counterpart chickens that remained in darkness. The same failure in finding a difference was met when the cytosolic or the particulate proteins or the acid soluble fraction in the retina were compared. Cycloheximide and puromycin inhibited the labeling of retina and optic tectum proteins, gangliosides and glycoproteins in both illumination conditions. Since the labelings in the optic tectum appeared more inhibited than those in the retina ganglion cell layer it was concluded that cycloheximide and puromycin, besides the synthesis of those compounds, also inhibit their axonal transport. On the basis of these contrasting results the working hypothesis is advanced that light stimulation enhances the activity of the Golgi apparatus but not (or less) that of the polyribosomes.

The effect of light exposure following an intraocular injection of [3H]N-acetylmannosamine on the labeling of gangliosides and glycoproteins of retina ganglion cells and optic tectum of singly caged chickens.

Ten-day-old chickens that after a 2-day-period of adaptation to dark received an intraocular injection of [3H]N-acetylamannosamine ([3H]ManNAc) and were exposed, individually housed, to light, have more labeling in the gangliosides and glycoproteins of the ganglion cell layer of retina and in the contralateral optic tectum compared to their counterparts that remained in darkness. No differences were found in the labeling of the acid soluble fraction of the ganglion cell layer between the animals in dark and light at 0.5 and 5 h after the injection of [3H]ManNAc. No differences could be observed in the quality or storage of the gangliosides labeled in light with respect to those labeled in dark, but those labeled in light had a higher percent of labeling released by neuraminidase at 5 h after the intraocular injection of the labelled precursor. In animals exposed to intermittent light, the increased labeling with respect to dark was smaller than that found in animals exposed continuously to light.