Temporal pattern of Fos and Jun families expression after mitogenic stimulation with FGF-2 in rat neural stem cells and fibroblasts

Intense stimulation of most living cells triggers the activation of immediate early genes, such as Fos and Jun families. These genes are important in cellular and biochemical processes, such as mitosis and cell death. The present study focused on determining the temporal expression pattern of Fos and Jun families in fibroblasts and neural stem cells of cerebellum, hippocampus, and subventricular zone (SVZ) of rats of different ages at 0, 0.5, 1, 3, and 6 h after stimulation with fibroblast growth factor (FGF)-2. In neonates, a similar expression pattern was observed in all cells analyzed, with lower expression in basal condition, peak expression at 0.5 h after stimulation, returning to baseline values between 1 and 3 h after stimulation. On the other hand, cells from adult animals only showed Fra1 and JunD expression after stimulation. In fibroblasts and hippocampus, Fra1 reached peak expression at 0.5 h after stimulation, while in the SVZ, peak level was observed at 6 h after stimulation. JunD in fibroblasts presented two peak expressions, at 0.5 and 6 h after stimulation. Between these periods, the expression observed was at a basal level. Nevertheless, JunD expression in SVZ and hippocampus was low and without significant changes after stimulation. Differences in mRNA expression in neonate and adult animals characterize the significant differences in neurogenesis and cell response to stimulation at different stages of development. Characterizing these differences might be important for the development of cell cultures, replacement therapy, and the understanding of the physiological response profile of different cell types.


Introduction
It is well known that neurogenesis in neonate and adult rodents differs in several manners such as in the processes of proliferation and differentiation, the duration of which is extended in adult cells (1).Such particularities of neonate and adult neurogenesis are directly influenced by gene expression.During development and learning processes, neuronal activation triggers the expression of immediate early genes (IEGs) responsible for coding transcription factors such as members of the Jun and Fos families (2).
The Fos and Jun protein families can work individually or through numerous combinations of dimers to generate the transcription factor activating protein-1 (AP-1) complex.The transcription factor AP-1, which activates the transcription of a range of numerous and diverse genes, is related to cell differentiation and proliferation, learning, motor control, and cognition (3,4).It is known that Fos and Jun expression is regulated by the activation of mitogenactivated protein kinase (MAPK) and phosphorylation of cAMP response element-binding protein (CREB) (5,6).Several growth factors, including fibroblast growth factor (FGF), activate MAPK signaling pathway.
Numerous studies using mice and rats show Fos and Jun gene expression in several regions of the central nervous system when animals are exposed to a wide range of stimuli like trauma (7), water stress (8), fear (9), odors, intraparenchymal injections of various compounds (10,11), and visual stimuli (12).Basal expression of c-Fos has been studied in several species, including mice, rats, cats, monkeys, and humans.Although c-Fos basal levels are relatively low due to its mRNA instability, transcription occurs rapidly (5 min) and continues for up to 15-20 min (13).IEGs have been used as markers of neural activity during memory formation.However, the lack of a temporal comparison between them hinders the interpretation of some results (14).In addition, little is known regarding the comparative expression of these genes in neural stem cells after mitogenic stimulation in different cell lines and at different developmental stages.For this purpose, we chose fibroblasts as the standard, likely one of the best characterized cells in culture, and neural stem cells, due to their relevance to replacement therapy as well as their intrinsic proliferative restriction in adults.
Knowing that FGF-2 is a critical factor for both neural stem cells and fibroblasts, we decided to compare the temporal expression patterns of Fos and Jun gene families in these cell types at different developmental stages to understand some of the changes that are essential for memory formation.Despite the remaining proliferative capacity of both fibroblasts and some neural cells, we hypothesized that there are marked differences in gene expression responses when comparing these two different age groups.

Animals
To determine the temporal pattern of gene expression of Fos and Jun families in neural stem cells and fibroblasts from neonates and adult rodents, we assessed their expression after in vitro mitogenic stimulus with FGF-2.For that, we used 70 neonate (6-day-old; P6) and 130 adult (8-week-old) rats (Wistar).The protocols used here were approved by the UNIFESP Ethics Committee on the Use and Care of Animals (CEP No. 5165260716).Neonate rats were euthanized with scissors and adults were anesthetized with ketamine (60 mg/kg) and xylazine (15 mg/kg) intraperitoneally followed by decapitation with a guillotine.

Cell isolation and culture
Fibroblasts.The primary cell culture of fibroblasts was established using dermal fragments from the abdominal region of neonate and adult rats.Dermal fragments were stored in a plastic tube with DMEM/F-12 (GibcoBRL, USA).Inside the laminar flow, the dermal fragments from adults and neonates were placed in Petri dishes with a 5 mL of 0.5% trypsin (Gibco) solution in PBS (1Â) for 30 min at room temperature.After the incubation time, the fragments were dissociated with scissors and incubated again in an enzymatic solution (5 mL of HEPES/RPMI (Gibco) supplemented with 1 mM sodium pyruvate, 0.1 mg/mL DNAse I (Thermo Fisher, USA), 2.75 mg/mL collagenase (Gibco), and 1.25 mg/mL hyaluronidase (Sigma Aldrich Corp., USA) for 3 h at room temperature.The samples were then dissociated with a glass pipette, passed through a 40-mm mesh filter (Corning, USA), centrifuged for 10 min at 272 g at room temperature, and the pellet was resuspended in 10 mL of supplemented medium containing DMEM/F-12 1:1 (Gibco), 10% of fetal bovine serum (FBS; Cultilab, Brazil), 1% penicillin (Gibco), and 1% streptomycin (Gibco).The cells were then incubated in 75-cm 2 bottles in 5% CO 2 at 37°C.After 24 h of plating, the entire culture medium was withdrawn, and 10 mL of fresh medium was added.Every four days the culture medium was changed.

Stimulus with FGF-2
The established period for cell growth was 15 days for neonatal cells and 45 days for adult cells.After this period, the culture medium was completely changed to a supplemented culture medium without EGF and FGF-2 for 24 h.After this period, cells were stimulated by adding 20 ng/mL of FGF-2 to the medium for different time intervals: 0 h (without stimulus); 0.5, 1, 3, and 6 h.

RNA extraction, reverse transcription, and quantitative PCR
A total RNA sample from all samples was extracted by Trizol s Reagent (Invitrogen, USA) according to the manufacturer's instructions.The RNA concentration was measured on the NanoDrop ND-2000 Spectrophotometer (NanoDrop Technologies, USA), and RNA integrity was verified by electrophoresis in 1% agarose gel.Reverse transcription was performed using 2 mg total RNA and The reaction was carried out in 10 mL final volume using a mixture of 50 ng/mL of cDNA, 5 mL Fast SYBR s Green Master Mix, Forward Primer (10 mM), and Reverse Primer (10 mM).The thermal cycling used for qPCR was 95°C for 10 min, 40Â 95°C for 15 s, 60°C for 1 min, and 72°C for 30 s.The dissociation curve was done at 95°C for 1 min, 60°C for 30 s, and 95°C for 30 s. Relative expression levels were calculated using the 2 -DDCt method and normalized using reference genes that show more stability in geNorm analysis.

Statistical analysis
Data were analyzed using GraphPad Prism s software version 5.0.3 (USA).Comparisons were made with ANOVA followed by the Tukey test.The established levels of statistical significance were Po0.05 and Po0.001.

Expression pattern of neonatal cells
Neonatal fibroblasts.Neonatal dermal fibroblasts had upregulated expression of all genes from the Fos and Jun families, peaking at 0.5 h after stimulation with FGF-2 (Figure 1), followed by a decrease at 1 h.For FosB and Fra2 (Figure 1B and D), the peak expression at 0.5 h was 150,000 times greater than at baseline.Likewise, JunB and JunD (Figure 1F and G) showed an increase in expression of 2,200 and 1,200 times, respectively, that of basal at the same time point.The expression of Fra1 (Figure 1C) increased about 3,500-fold, while c-Fos and c-Jun (Figure 1A and E) showed 150 and 220-fold greater expressions at 0.5 h compared to basal expression levels.Expression of all genes analyzed returned to baseline levels 3 h after the stimulus.All genes except JunD presented significant differences in expression at 1, 3, and 6 h compared to time 0.5 h.For JunD, there was a significant difference only at 1 h compared with the expression at 0.5 h after the stimulus.
Neonatal cerebellar neural stem cells.Similar to fibroblasts, the gene expression of the Fos and Jun families in neural stem cells extracted from the neonatal rodent cerebellum increased and peaked at 0.5 h (Figure 2) after mitogenic stimulation with FGF-2.The expression returned to baseline values at 1 h (Figure 2B-E), except for the c-Fos, JunB, and JunD genes (Figure 2A and F-G), which returned to baseline only at 3 h after the stimulus.Regarding the magnitude of expression at 0.5 h, Fra2 (Figure 2D) was the gene that presented the greatest increase in expression in this cell type, reaching 3,500-fold of the basal condition.Likewise, the expression of FosB and Fra1 (Figure 2B and C) was 2,000 times greater at the same time point.For c-Fos and c-Jun (Figure 2A-E), the increase was respectively 120-350 times greater than the basal condition.Finally, JunB and JunD (Figure 2F and G) were the genes that presented the smallest increases, albeit significant (respectively 30 and 60 times), in expression at 0.5 h.For all genes, there was a significant difference between time points 1, 3, and 6 h in relation to time point 0.5 h, except for JunB, where the difference in relation to time point 0.5 h only existed for time points 3 and 6 h.In contrast, JunD was the only gene that had a significant difference in expression between time point 1 h compared with time points 3 and 6 h.

Expression pattern of adult cells
Fibroblasts and neural stem cells from hippocampus and SVZ of adult animals showed a pattern of expression markedly different from that of neonate animals.Surprisingly, there was no expression of several genes of the Fos (c-Fos, FosB, and Fra2) and Jun (c-Jun and JunB) families.The only genes for which we found differences in the expression pattern were Fra1 and JunD.The expression pattern of those genes, however, varied extensively between the different cell types, and a standard common expression profile was not present.
Fra1 in both fibroblasts and hippocampus neural stem cells (Figure 3A and B) showed a peak expression at 0.5 h following FGF-2 stimulation.In fibroblasts, this increase in expression was 4 times higher than basal expression, while in the hippocampus this increase reached 1,600 times.In both cell types, expression of Fra1 returned to baseline levels at 1 h.For neural stem cells isolated from the SVZ, expression of Fra1 reached statistical significance only 6 h after stimulation (Figure 3C), which was also the peak expression for this gene in this cell type (by 6-fold).
For JunD in both fibroblasts and hippocampus stem cells (Figure 3D and E), peak expression was also at 0.5 h after stimulation.For the subsequent time points, however, there was a markedly different gene expression profile in these two cell types.In fibroblasts, there was a significant subsequent downregulation of gene expression followed by further increases (by 3.5-fold) up to 6 h after stimulation when expression was at a similar level to that seen at 0.5 h (Figure 3D).In adult neural stem cells from the hippocampus, there was a significant decrease in JunD expression at 1, 3, and 6 h after stimulation compared both to basal and 0.5 h levels (Figure 3E).JunD expression in the SVZ did not achieve statistical significance at any of the time points.

Discussion
The Fos gene family is involved in many physiological functions, such as cell cycle control and proliferation, while the Jun gene family is related to differentiation and cell death.When combined, they form the AP-1 complex (4).Fibroblasts and cerebellar neural stem cells from neonates had a similar pattern of gene expression.We observed an increase in mRNA expression for all investigated genes of the Fos and Jun families in neonatal cells after 0.5 h of mitogenic stimulus induced by FGF-2, returning to basal levels after 1 h.As for fibroblasts and neural stem cells from SVZ and hippocampus from adult animals, however, our results showed an expression of only two genes, Fra1 and JunD.
It is known that neonate and adult neurogenesis differ in many aspects, including the proliferation and differentiation processes (15).These particularities are directly influenced by gene expression.Although we did not quantify cell proliferation in the cultures, we speculated that the reported upregulation of gene expression may contribute to the intense neurogenesis and cell proliferation of neonates.
Adepoju et al. ( 16) observed that FGF-2 activated the expression of c-Fos and c-Jun protein at 1 h post-stimulation, whereas c-Fos levels peaked between 1 and 3 h and declined markedly at 6 h after stimulation and c-Jun expression was sustained for more than 12 h.In contrast, we observed that neural stem cells derived from cerebellum of neonate rats expressed all genes of the Fos and Jun families after 0.5 h of mitogenic stimulus, returning to basal levels after 1 h.These differences can be due to the type of material analyzed, the species, and the neurodevelopmental phase.
Kang et al. (17) analyzed the c-Fos expression (gene and protein) in response to FGF-2 in the human embryonic stem cell line MizhES1.About 1 h after stimulation, c-Fos gene mRNA was clearly induced and expression of c-Fos protein reached its highest expression levels at 2 h after stimulation.These results for c-Fos mRNA corroborate with our data that showed an increase of c-Fos after 0.5 h of mitogenic stimulus in neonatal fibroblasts and neural stem cells.
In contrast to the extensive evidence of c-Fos expression at 0.5 h after stimulation in adult rats (for a review, see 18), we did not observe c-Fos expression at this period in neural stem cell culture from adult animals.According to Salehi et al. (19), the inducibility of c-Fos declines with increasing age, supporting the notion that c-Fos expression widely reflects the cellular responsiveness at the transcriptional level.
Several studies indicate that Fos and Jun mRNA expression may in fact be above baseline levels already after 20 min of stimulation of the visual cortex of rats subjected to various periods of light exposure after 24 h of dark rearing (20).c-Fos transcriptional activation occurs within a few minutes of growth factor stimulation (13) and, according to Sheng and Greenberg (21), c-Fos transcription is undetectable at 0.5 h after growth factor treatment (such as FGF-2) in adult neuronal cells, corroborating our study.
Rosenfeldt et al. (22) showed that the level of c-Fos mRNA increased dramatically in fibroblasts from human foreskin specimens and peaked 50 to 60 min after contraction was initiated in stressed collagen matrices.Additionally, Deguchi et al. (23) showed that the level of c-Fos mRNA expression reached a maximum at about 0.5 to 1 h after oxygen reperfusion and declined to basal levels after 2 h in cultured human fibroblasts.Similarly, our data from neonatal fibroblasts showed a rapid Fos and Jun expression, peaking at 0.5 h after FGF-2 stimulation Table 1.Increase in gene expression in all the regions studied after 0.5 h of the stimulus in relation to basal condition.

Genes
Neonates Adults Fibroblasts Cerebellum Subventricular zone Hippocampus Fibroblasts The dash indicates genes that did not express.and returning to basal levels after 1 h.In further agreement with others, Oshima et al. (24) showed that c-Fos mRNA was readily inducible in young cells at 1 h after stimulation with serum, while this response was ablated in senescent cultures.
In addition, many articles show c-Fos expression in astrocytes.In an ischemic model in primary culture of cortical astrocytes, an increased expression in the range of 0.5-2 h (peak at 1 h) is observed.Bradykinin, forskolin, and IL-1 exposure also promotes an increase in c-Fos expression after 1 h in cell culture (2).
Our results showed that JunD expression in fibroblasts and the SVZ was sustained and at a stable level in adult animals.Expression in the hippocampus was lower but similarly stable.FosB, Fra1, c-Jun, and JunB were not expressed at any time after stimulation in any of these cells.Our findings corroborate those of Kallunki et al. (25), who demonstrated that JunD is expressed in neurons and glial cells and displays a basal expression in most cells in the nervous system.Kovary and Bravo (26) demonstrated in Swiss 3T3 cells that there is a variation in the AP-1 complex composition, in which initial dimers are formed by c-Fos/JunB and later on, by Fra1/c-Jun and Fra2/JunD.Therefore, we believe that the Fra1/JunD response encountered here in adult cells might also be associated to a similar specificity of response.Among other processes, the AP-1 complex is involved in cell cycle regulation.For a review on this topic, see (27).
The restricted expression of only Fra1 and JunD in our cell cultures derived from adult animals might also be related to the expression of FGF receptors in these cells.
Indeed, it has been reported that there is a progressive diminution in the expression of FGF receptors in various regions of the central nervous system of adults (28,29).
Another relevant aspect to be discussed is the difference in gene expression between neonate and adult animals regarding the increase in expression between baseline and 0.5 h (Table 1).In both fibroblasts and neural stem cells from neonate animals, all genes of the Fos and Jun families presented a significant increase in expression at 0.5 h after stimulation with FGF-2.Despite differences in expression values, Fra1, Fra2, c-Jun, and JunD showed a similar decrease in expression in the order of 4-5-fold at 1 h after stimulation (compared to 0.5 h).However, for c-Fos, FosB, and JunB, the decrease was 10-15-fold, emphasizing the peak expression at 0.5 h, while the Jun family genes decrease ranged between 0-8-fold at the same time point.We speculate that this difference in the intensity of gene expression over time might be related with the functions associated with those genes.
Here, we showed that expression of the Fos and Jun gene families might change according to age of the animal and type of cell and tissue, indicating that neurogenesis of neonates and adults has many fundamental differences at the molecular level, and we speculate that this may even influence behavioral and cognitive outcomes.