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c-Fos associates with the endoplasmic reticulum and activates phospholipid metabolism ¹

CIQUIBIC–Dpto. Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Pabellón Argentina, Ciudad Universitaria, 5000 Córdoba, Argentina

²Correspondence: Departamento de Química Biológica, Facultad de Ciencias Químicas, Pabellón Argentina, Ciudad Universitaria, 5000 Córdoba, Argentina. E-mail: bcaputto{at}dqbfcq.uncor.edu

SPECIFIC AIMS

We address the hypothesis that the inducible transcription factor c-Fos activates the metabolism of phospholipids in the cytoplasm by means of an AP-1 independent activity. These studies were carried out in quiescent NIH 3T3 fibroblast cultures stimulated with 20% fetal calf serum (FCS) to re-enter growth.

PRINCIPAL FINDINGS

1. c-Fos activates phospholipid metabolism
At each time point in which cells were stimulated to re-enter growth with FCS, phospholipid labeling was determined by pulsing cells with ³²P-orthophosphate during the last 15 min before cell harvesting. Two waves of activated ³²P-phospholipid labeling were found (Fig. 1 ). The first wave starts at 2.5 min of stimulation, peaks at 7.5 min, and returns to control values by 15 min; whereas the second wave starts after 30 min of stimulation and remains elevated at least up to 120 min.

View larger version (19K): [in this window] [in a new window]   Figure 1. Time course of phospholipid labeling and c-fos mRNA expression in quiescent fibroblasts stimulated to re-enter growth with 20% FCS. Quiescent NIH 3T3 cells at confluence were stimulated to re-enter growth by adding 20% FCS to the culture medium. Phospholipid labeling (full line) was determined in cell cultures pulsed with 25 µCi/ml of 32P-orthophosphate during the last 15 min before cell harvesting. At stimulation times shorter than 15 min, labeling started in control conditions and stimulation with FCS was initiated accordingly. c-fos mRNA (dashed line) was quantified by in situ hybridization followed by liquid scintillation spectrometry by using the following 35S-oligoprobe: 5'-ATG-CGT-GAG-AAG-GAG-TCG-GCT-GGG-GAA-TGG-TAG-TAG-GAA-AGG-CGA-3'. Results are the mean of six independent determinations done in duplicate ± SE. *P < 0.025 with respect to zero stimulation time.

2. c-fos transcription precedes phospholipid activation
At the same stimulation times at which phospholipid labeling was determined, c-fos mRNA expression was analyzed by in situ hybridization. As shown with phospholipids, c-fos mRNA expression also exhibits a bimodal induction, although both waves are temporally advanced with respect to phospholipids (Fig. 1) . The first wave peaks at 5 min and returns to control levels by 15 min; whereas the second one starts at 20 min, reaches a maximum at 30 min, and declines thereafter. The half-life of both pools of induced c-fos mRNA was different: 10 min in the first peak and 85 min in the second one.

3. Newly synthesized c-Fos immunolocalizes to the cytoplasm, associated to the endoplasmic reticulum (ER)
The expression of c-Fos protein and its subcellular localization was examined by double immunocytochemistry for c-Fos and for -tubulin in fibroblasts fixed at different stimulation times. While -tubulin immunolabeling was essentially the same at all stimulation times examined, detectable amounts of c-Fos were observed by 2.5 min of stimulation, peaked at 7.5 min, and returned to control values by 20 min of stimulation. This finding was a generalized phenomenon as can be verified when cultures are observed at low magnification (Fig. 2B ). A second wave of c-Fos induction started after 20 min of stimulation and remained elevated up to 120 min poststimuli. Noticeably, c-Fos immunoreactivity observed during the first peak of expression was mainly confined to the cytoplasm whereas in the second peak, c-Fos immunoreactivity was mainly nuclear although cytoplasmic immunoreactivity was still observed (Fig. 2A ).

View larger version (79K): [in this window] [in a new window]   Figure 2. c-Fos immunocytochemistry at different times of cell stimulation. A) Cells at 0 (a, b), 2.5 (c, d), 5 (e, f), 7.5 (g, h), 10 (i, j), 20 (k, l), 30 (m, n), and 60 (o, p) min of stimulation with FCS were double-immunostained with a polyclonal anti c-Fos antibody (a,c,e,g,i,k,m,o) and with anti -tubulin antibody (b,d,f,h,j,i,n,p). Micrographs were obtained at 100x. Bar in a: 10 µm. B) Low magnification field (20 x) of c-Fos immunostaining in non-stimulated cells (q) and in cells stimulated with 20% FCS for 7.5 min (r) or 60 min (s). Bar in q: 50 µm.

To determine whether cytoplasm-confined c-Fos is cytosolic or membrane-bound, 7.5- min-stimulated fibroblast homogenates were centrifuged to obtain a particulate and a soluble fraction and then subjected to Western blot. It was found that cytoplasmic c-Fos is membrane-bound. As the ER is the main site of phospholipid synthesis, a possible interaction between components of the ER and c-Fos was considered. To examine this possibility, 7.5 min-stimulated cell homogenates were fractionated by isopycnic centrifugation in a continuous sucrose gradient. Co-distribution of immunoreactivity of c-Fos and an ER marker (calreticulin) was found in the three densest fractions of the gradient, which indicates that c-Fos associates to the ER. Moreover, immunocytochemical examination showed distinct sites of c-Fos and ER co-localization in 7.5 min-stimulated fibroblasts when rhodamine-ER and fluorescein-c-Fos immunoimages of the same cells were merged.

4. Blocking c-Fos expression blocks phospholipid activation
To establish the dependence of phospholipid activation on c-fos expression, c-fos mRNA translation was blocked by feeding the cells with an oligonucleotide antisense to c-fos mRNA, 30 min before 7.5 min cell stimulation with FCS. When increasing amounts of a c-fos mRNA antisense oligonucleotide are added to the culture medium, c-Fos-immunoreactivity decreases accordingly until, in the stimulated cells, it appears similar to that of nonstimulated, quiescent cells. Coincident with the lack of c-Fos expression, a progressive loss of phospholipid activation in response to cell stimulation is observed as the amount of antisense oligonucleotide added to the cultures increases. (We observed no differences in phospholipid labeling between stimulated and nonstimulated cells in the presence of 1 µg oligonucleotide/ml of culture medium.) We found similar results at 7.5 and 60 min of cell stimulation. The corresponding sense oligonucleotide did not modify either c-Fos expression or phospholipid labeling.

5. Blocking c-Fos nuclear import with a peptide containing the AP-1 nuclear localization sequence does not affect phospholipid activation
The time course of c-Fos expression and its extra-nuclear localization during the first wave of phospholipid activation suggest that this regulatory activity of c-Fos is independent of its nuclear, AP-1 transcription factor activity. To further support the cytoplasmic nature of this new regulatory activity of c-Fos, its nuclear import was blocked by feeding the fibroblasts with a peptide containing an AP-1 nuclear import sequence prior to stimulating the cells for 60 min. Under these experimental conditions, a significant decrease in nuclear c-Fos was verified in the stimulated cells by immunocytochemical techniques; phospholipid activation was not affected (³²P-phospholipid labeling: 4525 and 4452 cpm/well in the presence and absence of peptide, respectively).

6. Phospholipid labeling pattern
The pattern of phospholipid labeling in quiescent cells and in the first and second waves of phospholipid labeling activation was examined. We found that, while in quiescent cells and in the second wave of activated phospholipid synthesis, labeling of phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid was predominant. In the first wave of phospholipid activation, the pattern of labeling was clearly different with the phosphatidylinositol-related second messenger molecules (PIP, PIP₂, PIP₃) accounting for most of the labeling.

CONCLUSIONS

The results presented herein identify a new role for c-Fos as a cytoplasmic regulator of the biosynthesis of phospholipids. That c-Fos associates with components of the ER (together with our previous findings in retina in which the activation of two enzymes of the pathway of synthesis of phospholipids, lysophosphatidic acid acyl transferase, and phosphatidic acid phosphatase depends on the expression of c-Fos) suggests a direct effect of this protein on specific enzymes of the pathway of synthesis of phospholipids. Moreover, we have obtained preliminary evidence that the addition of exogenous c-Fos to nonstimulated fibroblast homogenates activates the incorporation of ³²P into phospholipids and further supports the idea that c-Fos, per se and in the absence of other inducible transcription factors, is capable of activating the synthesis of these lipids.

The kinetics of the first wave of c-Fos induction marks this protein as a suitable candidate to regulate the turnover of phospholipids that participate in signaling-transduction pathways: Its level in nonstimulated cells is low and increases rapidly and transiently in response to serum with a very rapid turnover rate. Thus, this first wave of c-Fos–dependent phospholipid activation affects predominantly the turnover rate of polyphosphoinositides, lipids whose regulated hydrolysis to generate second messengers has been well characterized. That the half-life of c-fos mRNA is different in both waves of induced c-Fos expression may point to one of the mechanisms by which a cell’s response to environmental changes is regulated.

We conclude that c-Fos, rapidly induced upon cell stimulation, associates to the endoplasmic reticulum where it first regulates the synthesis/replenishment of phospholipid molecules required for signal transduction pathways and later on regulates enzymes involved in the genesis of new membrane necessary for cell growth to occur.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic representation of the regulatory activities of c-Fos. Rapidly induced c-fos transcription/translation results in the association of c-Fos with the ER, leading to phospholipid activation (upper half). c-Fos mRNA and protein are subsequently rapidly degraded. In the second wave of induction, in addition to its AP-1 transcription factor activity, c-Fos also activates phospholipid synthesis in the ER lower half).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0446fje ; to cite this article, use FASEB J. (January 5, 2001) 10.1096/fj.00-0446fje

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