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. 2014 Oct 30;35(3):363–376. doi: 10.1007/s10571-014-0132-z

Up-Regulation of c-Fos Associated with Neuronal Apoptosis Following Intracerebral Hemorrhage

Xiaomei Chen 1, Jiabing Shen 2, Yang Wang 1, Xiaojing Chen 1, Shi Yu 1, Huili Shi 1, Keke Huo 1,✉
PMCID: PMC11486182  PMID: 25354492

Abstract

The proto-oncogene c-Fos is an important member of the activating protein 1 (AP-1) transcription complex involved in major cellular functions such as transformation, proliferation, differentiation, and apoptosis. The expression of c-Fos is very tightly regulated and responses rapidly and transiently to a plethora of apoptotic stimuli. However, it is still unclear how c-Fos functions on neuronal activities following intracerebral hemorrhage (ICH). In the present studies, we uncovered that the up-regulation of c-Fos is related to neuronal apoptosis following ICH probably via FasL/Fas apoptotic pathway. From the results of Western blot and immunohistochemistry, we obtained that c-Fos is significantly up-regulated surrounding the hematoma following ICH and co-locates with active caspase-3 in the neurons. Besides, electrophoretic mobility shift assay exhibits high AP-1 DNA-binding activities in ICH groups due to the increase of c-Fos expression. In addition, there are concomitant up-regulation of Fas ligand (FasL), which is the target protein of AP-1, Fas, active caspase-8, and active caspase-3 in vivo and in vitro studies. What is more, our in vitro study showed that using c-Fos-specific RNA interference in primary cortical neurons, the expression of FasL and active caspase-3 are suppressed. Thus, our results indicated that c-Fos might exert its pro-apoptotic function on neuronal apoptosis following ICH.

Keywords: C-Fos, Intracerebral hemorrhage, FasL, Neuronal apoptosis, Hemin, Rat

Introduction

Intracerebral hemorrhage (ICH) is the deadliest and least treatable type of stroke (Hwang et al. 2011; Kuramatsu et al. 2013). Although multiple resources have been invested into clinical and basic researches, this high rate of mortality seems still not changed (Aronowski and Zhao 2011; Bacigaluppi et al. 2013; Creutzfeldt et al. 2009; Gomes and Manno 2013). Pathologic changes of ICH include neuronal apoptosis, astrocyte proliferation, and oligodendrocyte death, among which neuronal death is regarded as the most crucial event that composed of perplexing pro-apoptotic activations and subtle anti-apoptotic accommodation (Li et al. 2013).

The proto-oncogene c-Fos encodes a transcription factor that plays a pivotal role in cell proliferation, differentiation, and apoptosis (Guller et al. 2008; Habib 2010; Herrera and Robertson 1996). Previous research has shown that c-Fos is an immediate early gene that expresses rapidly and transiently, and is transcriptionally up-regulated by different factors, but the mechanisms by which c-Fos activation is triggered remain unclear (Ferrero et al. 2012; Niu et al. 2012). Multiple studies have shown that different stimuli can induce c-Fos expression, and transient c-Fos expression in the central nervous system (CNS) was first observed in hippocampal (da Silva et al. 2014; Herrera and Robertson 1996). After that, rapid activation of c-Fos has also been detected throughout the cortex after injury from mechanical trauma and ischaemic stroke (Hata et al. 2000; Nagy et al. 2002; Niu et al. 2012; Sharma et al. 2000). However, the functions of the c-Fos activation after brain injury seem not to be in a completely agreement. In majority conditions, c-Fos is thought to be required during the phases of the cell cycle in exponentially growing cells, and to be a potent inducer of cell proliferation (Pai and Bird 1994; Shaulian and Karin 2001). On the contrary, some studies have suggested that c-Fos is poorly contributed to proliferation, and is totally dispensable for or even down-regulated cell growth (Brusselbach et al. 1995; Kovary and Bravo 1991; Mikula et al. 2003). What is more, there is also a report revealing that continuous c-Fos expression precedes and exacerbates neuronal cell death in c-Fos-lacZ transgenic mice after treatment of kainic acid, a potent activator of glutamate receptors (Smeyne et al. 1993).

Several papers demonstrated that in neurons, c-Fos is the main constitution of activating protein 1 (AP-1) transcription complex, which can promote neuronal apoptosis (Caputto et al. 2014; Laderoute 2005; Tsai et al. 2011; Yamamura et al. 2000). The molecular mechanism that c-Fos mediated neuronal apoptosis remains elusive but might relate to FasL/Fas apoptotic pathway, since c-Fos could directly band to the FasL promoter through a single AP-1 binding site in transient transfection experiments (Lauricella et al. 2006; Matsumoto et al. 2007). In our study, we have disclosed that c-Fos is probably associated with neuronal apoptosis in rat ICH models, and FasL/Fas-mediating apoptotic pathway might be involved in this process in vivo and in vitro experiments. Thus, we for the first time investigated the expression and distribution of c-Fos in rat basal ganglia around the hematoma after ICH and lay the foundation on clinical treatment regarding ICH.

Materials and Methods

Animals and the ICH Model

All animal care and surgical procedures were carried out based on Guide for the Care and Use of Laboratory Animals promulgated by National Research Council in 1996, and supported by the Chinese National Committee to Use of Experimental Animals for Medical Purposes, Shanghai Branch. Male Sprague–Dawley rats with an average body weigh t of 250 g (ranging from 220 to 275 g) were used. All animals were maintained in a temperature-controlled room (22 ± 1 °C) on a 12-h light–dark cycle and the food and water were available adlibitum. The number of animals studied was the minimum to obtain significant results, and all efforts were made to minimize their discomfort caused by the experimental procedures. For ICH model, the rats were anesthetized intraperitoneally with sodium pentobarbital (50 mg/kg) and then positioned in a stereotaxic frame. Autologous whole blood was collected by putting the tail tip in warm water for 3–4 min, cleaning the skin with 75 % alcohol, cutting the tail tip, and drawing 50 μL of freely dripping blood into a sterile syringe. The syringe was secured in the frame, and the needle was quickly introduced into right basal ganglia (coordinates: 0.2 mm anterior, 5.5 mm ventral, and 3.5 mm lateral to the bregma), while the sham group only had a needle insertion. After injection, the sterile syringe was left in situ for over 10 min, and then the skin incision was closed. All of the animals were allowed to recover from surgery.

Behavioral Testing Procedures

Forelimb Placing Test

The rats were held by torsos, thus the forelimb could hang free. Independent testing of each forelimb was elicited by brushing the respective vibrissae on the corner edge of a countertop. Intact rats put the forelimb quickly onto the countertop. According to the extent of injury, placing of the forelimb contralateral to the injury may be impaired. During the experiments, each rat (6 rats for per time point) was tested ten times for each forelimb, and the percentage of trials in which the rat placed the left forelimb was calculated (Karabiyikoglu et al. 2004).

Corner Turn Test

The rats were allowed to proceed into a corner, whose angle was 30°. To exit the corner, the rat should turn either to the left or the right, and only the turns involving full rearing along either wall were involved (a total of eight per animal, and 6 rats for per time point). According to the extent of injury, rats may show a tendency to turn to the side of the injury, and the percentage of right turns was used as the corner turn score. Rats were not picked up immediately after each turn so that they would not develop an aversion for their prepotent turning response (Hua et al. 2002).

Western Blot Analysis

Rats were sacrificed at different time points (3, 6, and 12 h; 1, 2, 3, 5, and 7 days; n = 6 per time point) by injecting overdose of chloral hydrate. Tissues surrounding the hematoma (2 mm) from the ICH or corresponding areas from the sham rats were dissected and flash-frozen at −80 °C. For preparation of brain tissue proteins, the samples were weighted, homogenized in modified RIPA lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1 % Nonidet P-40, 1 % sodium dodecyl sulfate (SDS), 1 % sodium deoxycholate, 5 mM EDTA, Phosphatases inhibitor, and Protease Inhibitor) and centrifugated at 12,000×g, 4 °C for 15 min. After determination of the supernatant concentration with the Bradford assay (Bio-Rad), the samples were subjected to SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene diflouride (PVDF) filter membrane. The membranes were blocked with 5 % non-fat milk and incubated with primary antibody overnight. After washing with Tris-buffered saline containing 0.1 % Triton x-100 (TBST), the blots were incubated with secondary antibodies for 1.5 h at room temperature (RT) and detected using an enhanced chemiluminescence (ECL) kit (Pierce) or with the Odyssey infrared imaging system (LI-COR Bioscience). All results shown are representative of at least three independent experiments.

Immunohistochemistry and Immunofluorescence

Experimental group that was 12 h after ICH or the sham group (6 rats per group) were anesthetized and perfused with 500 mL of 0.9 % saline, followed by 4 % paraformaldehyde. After perfusion, the brains were removed and post-fixed in the same fixative for 24 h, then transferred to 20 % sucrose for 2–3 days and to 30 % sucrose for another 2–3 days. The brains were then cut at 5 µm with a cryostat, and the sections were stored at −20 °C until use.

For immunohistochemistry, brain sections were washed in phosphate-buffered saline (PBS), and treated with 3 % H2O2 for 10 min at RT to reduce endogenous peroxidase activity. The sections were blocked with confining liquid consisting of 10 % donkey serum, 1 % bovine serum albumin (BSA), 0.3 % Triton X-100, and 0.15 % Tween-20 for 2 h at RT, then incubated with anti-c-Fos antibodies (rabbit; 1:50; Santa Cruz) for 2 h. After washed in PBS, the sections were incubated in secondary antibodies at 37 °C for 30 min and color-reacted with the liquid mixture (0.02 % daminobenzidine tetrahydrochloride (DAB), 0.1 % PBS, and 3 % H2O2). At the end, the sections were air-dried, dehydrated, covered with coverslips, and examined using Leica microscope (Germany).

For double immunofluorescent labeling, sections were prepared as previously. After air-dried for 2 h, sections were first blocked with normal serum blocking solution (10 % donkey serum, 1 % BSA, 0.3 % Triton X-100, and 0.15 % Tween-20) for 2 h at RT. Then, the sections were incubated with primary antibodies against c-Fos (rabbit; 1:50; Santa Cruz), NeuN (mouse; 1:300; Chemicon), CNPase (mouse; 1:200; Sigma), GFAP (mouse; 1:100; Sigma), and active caspase-3 (mouse or rabbit; 1:200; Santa Cruz). Briefly, sections were incubated with two kinds of primary antibodies overnight at 4 °C, followed by a mixture of FITC- and TRITC-conjugated secondary antibodies (Jackson ImmunoResearch) for 2 h at 4 °C. Images were captured using a Leica fluorescence microscope.

Quantitative Analysis

For cell quantification in immunohistochemistry, the number of c-Fos-positive cells in the basal ganglia 2 mm from the hematoma was counted at 400× magnification using a Leica microscope. To avoid counting the same cell in more than one section, we counted every fifth section. For each section, the number of c-Fos-positive cells of three separate regions (per square millimeter) in basal ganglia was calculated. The final statistical values of per square millimeter were the representative of six sections.

Electrophoretic Mobility Shift Assay (EMSA)

EMSA assays were performed using the Gel Shift Assay System (Promega) according to the manufacturer’s protocol. The double-stranded oligonucleotides containing the putative AP-1 binding sites (underlined) were as follows: 5′-CGCTTGATGAGTCAGCCGGAA-3′. In the binding reactions, γ32P-labeled DNA probes were incubated with 10 μg of nuclear extract. Binding reactions were performed at RT for 20 min, and the DNA–protein complexes were resolved by electrophoresis on 5 % non-denaturing Tris–Borate–EDTA polyacrylamide gels and visualized by autoradiography. Signals were quantified using PhosphorImager and ImageQuant™ software. For supershift experiments, the nuclear extracts were pre-incubated with 200 ng antibodies against c-Fos for 20 min.

Primary Cortical Neurons Cultures and Transfection

Primary cortical neurons were dissociated from Sprague–Dawley at embryonic day 18 according to a previously described procedure with some modifications (Banker and Cowan 1977). Briefly, after the cortex was digested with trypsin, the cell suspensions were re-suspended with Dulbecco’s Modified Eagle Medium (DMEM) containing 10 % fetal bovine serum to stop the reaction, followed by filtered through 40-µm sieve. Isolated cells were seeded into plates pre-coated with 100 mg/l poly-l-lysine at a density of 2 × 106 cells/well in 6-well plates or 2 × 104 cells/well in 96-well plates. After incubated at 37 °C with 5 % CO2 for 4 h, the cells were cultured with NEUROBASAL™ medium (Gibco) supplemented with 2 % B27, 0.5 mM glutamine, and 100 µg/ml penicillin/streptomycin for 7 days. The isolated cells were exposed to 75 μM hemin for indicated time points and then analyzed for CCK-8 assays or Western blot experiments.

For transfection, after 2 days culture, the isolated cells were transfected with c-Fos siRNA or negative control siRNA (Santa Cruz) using Lipofectamine 2000 (Invitrogen). 48 h later, cells were treated with hemin for 12 h, and then used for the subsequent experiments.

CCK-8 Assay

CCK-8 assay was performed using the commercial CCK-8 kit in accordance with the manufacturer’s protocol (Dojindo). For measurement of the effects of hemin on neuronal viability, primary cortical neurons were treated with different concentrations of hemin or with fixed amounts of hemin for different time points. After that, CCK-8 solution was added to each well at a final concentration of 10 % (v/v) and incubated for 2.5 h at 37 °C. The absorbance was measured at 490/630 nm by the microplate reader (Bio-Rad). The experiments were repeated at least 6 replicates of each treatment.

Statistical Analysis

The data were presented as the mean ± standard deviation (SD) of at least three independent experiments. Student’s t test was used for pairwise comparisons. One-way ANOVA, with Dunnet’s t test, was used for multiple comparisons. p < 0.05 was considered as statistically significant.

Results

Changes in the Expression and Distribution of c-Fos Following ICH

Rats in the sham and ICH groups were subjected to forelimb placing and corner turn test at different survival times, respectively. Rats of ICH with significant neurological function deficits were chosen for subsequent experiments. From Fig. 1a, b, we showed that rat in ICH group was obviously impaired compared with the sham group in the first 5 days, whereas by 7 days and thereafter, all rats had recovered neurological test scores.

Fig. 1.

Fig. 1

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Variations of c-Fos protein levels following ICH. Forelimb placing scores (a) and corner turn testing (b) were displayed at various time points following ICH (*p < 0.05). c Western blot was employed to detect the protein levels of c-Fos surrounding the hematoma at different survival times. The expression of c-Fos was relatively low in the sham-operated group, then remarkably up-regulated at 3 h after ICH, peaked at 12 h, and declined soon afterward. d The relative protein content of c-Fos was calculated by densitometric analysis, and data were normalized to that of the corresponding β-actin. Values are presented as mean ± SEM (n = 3, *p < 0.05). *p < 0.05, indicated significantly difference from the sham-operated group

To detect the c-Fos expression after ICH, Western blot was performed to investigate the temporal levels of c-Fos in rat basal ganglia around the hematoma at various time points after ICH. Figure 1c, d shows that c-Fos protein level was relatively low in the sham group, then remarkably increased at 3 h after ICH and peaked at 12 h. To further verify the up-regulation of c-Fos after ICH, we detected the distribution of c-Fos at 12 h following ICH by immunohistochemistry. The sham-operated group showed faint c-Fos-positive staining (Fig. 2a, b), which was consisted with the result from Western blot. By contrast, the number of c-Fos-positive cells in the ICH group was increased, and the signaling of c-Fos-positive was enhanced surrounding the hematoma compared with the sham-operated group (Fig. 2c, d). As the antibody control, there was no IgG staining in the negative control sections (Fig. 2e). Additionally, we also assessed the expression of c-Fos in the sham-operated group and the ICH group by cell counting, and statistics results demonstrated that there was significant increase of c-Fos-expressing cells after ICH (Fig. 2f). From the data above, we can see that c-Fos protein profiles had a temporal change after ICH, suggesting its relevant biological function following ICH.

Fig. 2.

Fig. 2

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Immunohistochemistry of c-Fos surrounding the hematoma. Faint signals of c-Fos immunoactivity were found in the sham-operated group (a, b). However, the ICH group (12 h after ICH) showed enhanced c-Fos signals (c, d). e No positive signals were found in the IgG control. f The number of c-Fos cells was largely increased comparing the ICH group with the sham-operated group. *p < 0.05 indicated that the ICH group was different from the sham-operated group. Asterisk indicates the hematoma. Scale bar, left column, 400 μm; right column, 50 μm

Cell Type of c-Fos Expression

To further identify which kind of cell type may be relevant to the increased expression of c-Fos after ICH, we used double immunofluorescent microscopy studying on transverse cryosections of brain tissues by co-labeling c-Fos with different cell-specific markers, such as NeuN, CNPase, and GFAP, which represent neurons, oligodendrocytes, and astrocytes, respectively. Following ICH, c-Fos staining was observed in NeuN-positive cells (Fig. 3a–c), while not in CNPase (Fig. 3d–f), or GFAP-positive cells (Fig. 3g–i), indicating that neurons specifically contain c-Fos. No IgG-positive cells were detected in the negative control (Fig. 3j, k).

Fig. 3.

Fig. 3

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Immunofluorescence for c-Fos with different phenotype-specific markers surrounding the hematoma. c-Fos (red, a, d, g) was co-labeled with neuronal marker NeuN (green, b), oligodendrocyte marker CNPase (green, e), and astrocyte marker GFAP (green, h). Arrows in the merged images (c) represented the co-localization of c-Fos with NeuN. No detectable signal was found in the IgG control (red, j, green, k). Scale bars, 50 μm (j, k) (Color figure online)

c-Fos Relevant to Neuronal Apoptosis After ICH

To investigate whether c-Fos is related to neuronal apoptosis after rat ICH, we detected the protein levels of active caspase-3. As expected, the expression profiles of active caspase-3 changed after ICH, and peaked at day 1 and 2 (Fig. 4a, b), which kept the temporal consistency with that of the c-Fos. In addition, double immunofluorescent staining displayed that the active caspase-3 not only located in neurons, but also co-located with c-Fos at 12 h after ICH around the hematoma (Fig. 4c). These data indicated that c-Fos up-regulation in neurons might be connected with neuronal apoptosis after ICH.

Fig. 4.

Fig. 4

Fig. 4

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Correlations of c-Fos with neuronal apoptosis following ICH. a Western blot analysis showed the expression of active caspase-3 was increased with the peak at day 1 and 2 following ICH. b Values were expressed as the mean ± SEM (n = 3, *p < 0.05). c Double immunofluorescent staining showed the co-localization of active caspase-3 with NeuN as well as with c-Fos in brain basal ganglia around the hematoma at 12 h after ICH. Scale bars, 50 μm. d Electromobility shift assays (EMSA) were performed using nuclear extracts from sham-operated and ipsilateral groups of ICH, and then a labeled oligonucleotide encoding the AP-1 binding consensus sequence was added to the extracts that were pre-treated with or without anti-c-Fos antibodies. AP-1 binding activity remarkably increased following ICH compared with the sham-operated groups, and peaked at 12 h. e Values were the AP-1 activities (%) of sham group in extracts untreated with anti-c-Fos antibodies, and presented as the mean ± SEM (n = 3, *p < 0.05). f Western blot analysis showed the protein levels of FasL, Fas, and active caspase-8 were increased from 3 h after ICH, and peaked at day 1 and 2. g The bar graph indicated the relative protein levels of FasL/Fas/active caspase-8 versus β-actin at each time point. Data were presented as mean ± SEM (n = 3, *,#,^ p < 0.05). p < 0.05, indicated significantly difference from the sham-operated group (Color figure online)

It has been demonstrated c-Fos, a member of AP-1 complex, can exert its pro-apoptotic activity via AP-1, which binds to AP-1 binding site in FasL promoter region and initiates its expression (Lauricella et al. 2006; Matsumoto et al. 2007). Coincidently, FasL/Fas-mediated extrinsic apoptotic pathway has been reported in central nervous system diseases, such as traumatic brain injury (Beer et al. 2000), cerebral ischemia (Sairanen et al. 2006), and ICH (Delgado et al. 2008), although the molecular mechanism of FasL up-regulation in ICH has not been yet defined. Therefore, it promotes us to investigate the DNA-binding activities of AP-1 and FasL expression level after ICH. We labeled AP-1 binding consensus sequence 5’-CGCTTGATGAGTCAGCCGGAA-3’DNA fragment with [γ-32p] ATP and used it as a probe in EMSA. Nuclear extracts from either sham- or ICH-operated groups were incubated with the probe. Figure 4d, e shows that ICH-operated groups exhibited increased AP-1 binding activity with the highest at 12 h when compared with the sham-operated groups, which had the similar variation trend with that of c-Fos expression. In addition, we incubated nuclear extracts with anti-c-Fos antibodies, which caused a supershift of the antibody-AP-1 complex, indicating the presence of c-Fos in the complex (Fig. 4d). Concomitant with the activation of AP-1, we detected a parallel increase of FasL protein expression (Fig. 4f, g). Moreover, the temporal profiles of Fas receptor and active caspase-8 were parallel with that of c-Fos and active caspase-3, and peaked at day 1 and 2, respectively (Fig. 4f, g). These results together indicated that c-Fos might implicate in neuronal apoptosis following ICH via inducing FasL/Fas extrinsic apoptotic pathway.

Up-Regulation of c-Fos and Cellular Apoptosis in Vitro ICH

Our above data showed that c-Fos was relevant with neuronal apoptosis in vivo rat ICH. To further investigate the role of c-Fos on neuronal apoptosis after ICH, we exposed primary cortex neurons to hemin to imitate ICH in vitro, since hemin is released from hemoglobin accumulating in blood outside vessel, and plays an important role in brain damage associated with ICH (Lin et al. 2012; Wang and Dore 2008). We initially incubated primary cortex neurons with 25, 50, 75, 100, and 150 μM of hemin at indicative time points, and CCK-8 assays showed that neuronal viability was significantly reduced after exposure to 75 μM hemin for 9 h compared to untreated neuronal cells (Fig. 5a). Moreover, at this concentration, cells’ viability decreased in a time-dependent manner (Fig. 5b). Also, the protein levels of apoptotic marker, active caspase-3, increased concomitantly with cells’ viability loss (Fig. 5c). These data indicated that the in vitro ICH model was successfully established. Based on the in vitro ICH model, c-Fos expression and AP-1 binding activities were increased in parallel and reached the summit at 12 h (Fig. 5c–f). Simultaneously, Fas, FasL, active caspase-8, and active caspase-9 were up-regulated, suggesting that FasL/Fas system was activated and its downstream caspase cascade was initiated in vitro ICH model (Fig. 5g, h).

Fig. 5.

Fig. 5

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Up-regulation of c-Fos in primary cortical neurons after hemin exposure. a CCK-8 assays showed that hemin treatment reduced neuronal viability in a dose-dependent manner at the indicated time points (3, 9, and 18 h) compared to untreated cells (control) (*p < 0.05). b Hemin (75 μM) treatment reduced neuronal viability in a time-dependent manner compared to untreated cells assayed by CCK-8 (*p < 0.05). c The protein levels of active caspase-3 and c-Fos were increased in cultured primary cortical neurons after hemin exposure. d The relative protein levels of active caspase-3 and c-Fos were calculated by densitometric analysis, and data were normalized to that of the corresponding NeuN (*p < 0.05). e EMSA was performed on nuclear extracts from primary cortical neurons treated with hemin (75 μM) for different times, and then they were treated or untreated with anti-c-Fos antibodies, followed by incubated with a labeled oligonucleotide encoding the AP-1 binding consensus sequence. f Data were presented as a percentage of the corresponding control cells in nuclear extracts without c-Fos antibodies, and the values were expressed as the mean ± SEM (n = 3, *p < 0.05). g Upon hemin stimulation, FasL, Fas, active caspase-8, and active caspase-9 were up-regulated concomitantly and peaked at 18 h. h The bar chart indicated the density of four proteins in (g) versus NeuN (*,#,^,& p < 0.05). p < 0.05, indicated significantly difference from the control cells

c-Fos Regulation of Neuronal Apoptosis Via FasL/Fas Apoptotic Pathway

To further confirm the function of c-Fos on neuronal apoptosis following ICH, c-Fos-specific RNAi was used to knock down the expression of c-Fos in cultured primary cortex neurons. Western blot results revealed that c-Fos-specific RNAi largely reduced c-Fos protein level in primary cortex neurons compared with non-specific RNAi after 12 h hemin treatment (Fig. 6a, b). Meanwhile, down-regulation of c-Fos expression not only inhibited AP-1 binding activities (Fig. 6c, d), but also suppressed the protein levels of FasL and active caspase-3 in the presence of hemin (Fig. 6e, f). Taken together, these results demonstrate that c-Fos might exert its pro-apoptosis effect on neurons via FasL/Fas apoptotic signaling pathway.

Fig. 6.

Fig. 6

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c-Fos regulation of neuronal apoptosis via Fas/FasL apoptotic pathway. a Western blot analysis showed siRNA silenced c-Fos in cultured primary cortical neurons after 12 h hemin exposure. c Knockdown of c-Fos decreased AP-1 binding activities in primary cortical neurons treated with hemin. d Data were expressed as a percentage of the corresponding control cells in nuclear extracts without c-Fos antibodies, and were presented as the mean ± SEM (n = 3, *p < 0.05). e Knockdown of c-Fos suppressed levels of FasL and active caspase-3 in hemin-treated primary cortical neurons. Data showed in b and f were represented the relative protein contents of c-Fos in (a) and FasL and active caspase-3 in (e), respectively, which were calculated by densitometric analysis and normalized to that of the corresponding NeuN. Values were presented as mean ± SEM (n = 3, *p < 0.05). p < 0.05, indicated significantly difference

Discussion

ICH is one of the most detrimental sub-types of stroke and accounts for 10–15 % of all strokes (Kuramatsu et al. 2013). Up to now, all randomized controlled trials investigating treatment approaches in ICH have failed to document improvements on clinical endpoints (Chuang et al. 2009; Sansing et al. 2009; Thiex and Tsirka 2007). Therefore, getting a better understanding about the underlying molecular and cellular mechanisms of damage following ICH is urgent for individuals and societies. Based on the amount of bleeding and the predilection site in clinical scenarios, the present study established an ICH rat model to simulate clinical ICH. In this in vivo model, c-Fos protein level was up-regulated around the hematoma, and the temporal changes were striking in apoptotic neurons, but not in astrocytes or oligodendrocytes. Since c-Fos is an important member of the activating protein 1 (AP-1) transcription complex, we examined AP-1 activation and discovered that AP-1 exhibited high DNA-binding activities in the course of ICH. It reminded us that the c-Fos transcription factor might function as an immediate early gene to initiate apoptosis-related protein expression after ICH. As expected, the expression levels of FasL, active caspase-8 and active caspase-3 were elevated in vivo and in vitro studies. Moreover, silencing of c-Fos by siRNA in primary cortex neurons can attenuate hemin-induced expression of FasL and active caspase-3. From the results above, we speculate that c-Fos might be involved in neuronal apoptosis following ICH via FasL/Fas apoptotic signaling pathway.

Rupture of blood vessels within the brain parenchyma can lead to primary and secondary injuries, which both contribute to the injury after the onset of ICH. Excluding the primary damage caused by mass effect, the resulting hematoma can trigger a series of un-favorable events which lead to secondary insults and finally severe neurological deficits or death (Aronowski and Zhao 2011). Animal models of ICH have demonstrated that apoptosis is a main contributor to brain damage, and neuronal apoptosis is found both around the hematoma and in the surrounding tissue (Gong et al. 2001). Apoptosis, also known as programed cell death, can be triggered by either intrinsic or extrinsic signaling pathway. In these two pathways, the intrinsic pathway is induced by cytotoxic stimuli and leads to eventual activation of downstream caspases, whereas the extrinsic pathway is triggered by ligation of death receptors and directly initiates the activation of upstream caspases (Choi and Benveniste 2004). However, ultimate caspase-3 activation is the overlaps between the two pathways and causes cell apoptosis. FasL/Fas-mediated apoptosis pathway belongs to the latter, and Fas is a death receptor located on the cell surface that belongs to the tumor necrosis factor receptor family (Nagata and Golstein 1995). The interaction between Fas and its ligand, FasL, can transfer apoptotic signaling and trigger cell apoptosis. Both of them are normally expressed in the central nervous system and can be up-regulated in various pathological conditions. Also, FasL/Fas-mediated apoptosis has been described in central nervous system diseases, such as traumatic brain injury (Beer et al. 2000), cerebral ischemia (Sairanen et al. 2006), and ICH (Delgado et al. 2008). Consistent with previous studies, our data showed that FasL/Fas system was activated in vivo and in vitro ICH model, and neuronal apoptosis concomitantly appeared.

c-Fos, an important member of the activating protein 1 (AP-1) transcription factor, represents a prototypical “immediate early” gene, because its expression is rapidly and dramatically induced by different extracellular stimuli (Ferrero et al. 2012; Niu et al. 2012). c-Fos cannot be homodimerized, but can be heterodimerized with Jun family proteins to form stable complex (also called AP-1), thereby enhancing its DNA-binding activity. Once activated, AP-1 transcription complex binds to sites locating in the regulatory region of target genes to modulate the late-response expression of critical factors for a wide range of biologic processes, such as transformation, proliferation, differentiation, and apoptosis (Bossis et al. 2005; Caputto et al. 2014; Chinenov and Kerppola 2001; Hu et al. 2002). In some situation, the c-Fos gene is required during all phases of the cell cycle in exponentially growing cells, and is a potent inducer of cell proliferation (Pai and Bird 1994; Shaulian and Karin 2001). In immortalized human hepatocyte, c-Fos plays a positive role in cell cycle regulation, and overexpression of c-Fos could contribute to hepatocarcinogenesis through stabilization of Cyclin D1 within the nucleus (Guller et al. 2008). However, there are emerging studies suggesting that abnormal expression of c-Fos poorly contributes to proliferation, or even leads to cell apoptosis (Mikula et al. 2003; Niu et al. 2012). Mohammad Asim disclosed that phospho(p)-c-Fos/c-Jun heterodimer binds to the c-Myc promoter in macrophages and thus causes inducing the expression of c-Myc and ornithine decarboxylase (ODC) and final cellular apoptosis (Asim et al. 2010). Soultana Markopoulou indicated that vanadium-mediated apoptosis is promoted by c-Fos, leading to alterations in clusterin (CLU) isoform processing and up-regulation of the pro-death nuclear CLU protein (Markopoulou et al. 2009). Moreover, several papers suggest that AP-1 might mediate apoptosis through transcriptional regulation of the expression of the FasL gene to start the extrinsic apoptotic pathway (Kasibhatla et al. 1998; Kolbus et al. 2000; Le-Niculescu et al. 1999). Similar to the latter reports, in this study, our result showed that concomitant with the expression elevation of c-Fos, the DNA-binding activities of AP-1 and the protein levels of FasL, Fas, and active caspase-3 were increased in vivo and in vitro ICH model. Besides, c-Fos always co-localized well with active caspase-3 in neurons after rat ICH. Moreover, knockdown of c-Fos by its specific RNAi effectively suppressed AP-1 activities and the expression levels of FasL and active caspase-3, and also protected neurons from hemin-induced apoptosis. Therefore, these data indicate that c-Fos may be related to neuronal apoptosis after ICH via regulating FasL expression.

However, in some cases, FasL expression can be transcriptionally regulated by different compositions of AP-1 transcription complex. For example, Wen-Hsin Liu demonstrated that arachidonic acid induces FasL up-regulation in human leukemia U937 cells via activation of ATF-2/c-Jun heterodimer (AP-1) and suppression of c-Fos pathway (Liu and Chang 2009). Andrea Kolbus suggested that ATF-2/c-Jun rather than c-Fos/c-Jun is responsible for transcriptional activation of FasL in response to alkylating agents (Kolbus et al. 2000). On the contrary, in the human T Lymphocytes, it is c-Fos/c-Jun and NF-kB transcription complex that are required for FasL expression induced by genotoxic agents (Kasibhatla et al. 1998). These optional discordances of AP-1 composition for FasL regulation might be relevant with cell-stimulating factors or cell type-specific effect.

Taken together, our data suggest that c-Fos promotes neuronal apoptosis after ICH via FasL/Fas-mediated extrinsic apoptotic pathway. However, the intrinsic molecular mechanism that c-Fos regulates neuronal apoptosis following ICH needs to be further confirmed in vivo. In future, more studies should be executed to inquire the therapeutic potentials of c-Fos for the purpose of achieving better prognosis following ICH.

Acknowledgments

We thank Dr. Aiguo Shen of Nantong University for his advice and great help on this paper. This work was supported by the National Key Basic Research Program of China (2103CB531603) to Keke Huo.

Conflict of interest

The authors declare no conflict of interest.

References

  1. Aronowski J, Zhao X (2011) Molecular pathophysiology of cerebral hemorrhage: secondary brain injury. Stroke 42(6):1781–1786 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Asim M, Chaturvedi R, Hoge S, Lewis ND, Singh K, Barry DP, Algood HS, de Sablet T, Gobert AP, Wilson KT (2010) Helicobacter pylori induces ERK-dependent formation of a phospho-c-Fos c-Jun activator protein-1 complex that causes apoptosis in macrophages. J Biol Chem 285(26):20343–20357 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bacigaluppi S, Retta SF, Pileggi S, Fontanella M, Goitre L, Tassi L, La Camera A, Citterio A, Patrosso MC, Tredici G, Penco S (2013) Genetic and cellular basis of cerebral cavernous malformations: implications for clinical management. Clin Genet 83(1):7–14 [DOI] [PubMed] [Google Scholar]
  4. Banker GA, Cowan WM (1977) Rat hippocampal neurons in dispersed cell culture. Brain Res 126(3):397–1342 [DOI] [PubMed] [Google Scholar]
  5. Beer R, Franz G, Schopf M, Reindl M, Zelger B, Schmutzhard E, Poewe W, Kampfl A (2000) Expression of Fas and Fas ligand after experimental traumatic brain injury in the rat. J Cereb Blood Flow Metab 20(4):669–677 [DOI] [PubMed] [Google Scholar]
  6. Bossis G, Malnou CE, Farras R, Andermarcher E, Hipskind R, Rodriguez M, Schmidt D, Muller S, Jariel-Encontre I, Piechaczyk M (2005) Down-regulation of c-Fos/c-Jun AP-1 dimer activity by sumoylation. Mol Cell Biol 25(16):6964–6979 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brusselbach S, Mohle-Steinlein U, Wang ZQ, Schreiber M, Lucibello FC, Muller R, Wagner EF (1995) Cell proliferation and cell cycle progression are not impaired in fibroblasts and ES cells lacking c-Fos. Oncogene 10(1):79–86 [PubMed] [Google Scholar]
  8. Caputto BL, Cardozo Gizzi AM, Gil GA (2014) c-Fos: an AP-1 transcription factor with an additional cytoplasmic, non-genomic lipid synthesis activation capacity. Biochim Biophys Acta 1841(9):1241–1246 [DOI] [PubMed] [Google Scholar]
  9. Chinenov Y, Kerppola TK (2001) Close encounters of many kinds: Fos-Jun interactions that mediate transcription regulatory specificity. Oncogene 20(19):2438–2452 [DOI] [PubMed] [Google Scholar]
  10. Choi C, Benveniste EN (2004) Fas ligand/Fas system in the brain: regulator of immune and apoptotic responses. Brain Res Brain Res Rev 44(1):65–81 [DOI] [PubMed] [Google Scholar]
  11. Chuang YC, Chen YM, Peng SK, Peng SY (2009) Risk stratification for predicting 30-day mortality of intracerebral hemorrhage. Int J Qual Health Care 21(6):441–447 [DOI] [PubMed] [Google Scholar]
  12. Creutzfeldt CJ, Weinstein JR, Longstreth WT Jr, Becker KJ, McPharlin TO, Tirschwell DL (2009) Prior antiplatelet therapy, platelet infusion therapy, and outcome after intracerebral hemorrhage. J Stroke Cerebrovasc Dis 18(3):221–228 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. da Silva JC, Scorza FA, Nejm MB, Cavalheiro EA, Cukiert A (2014) c-FOS expression after hippocampal deep brain stimulation in normal rats. Neuromodulation 17(3):213–217 discussion 216–217 [DOI] [PubMed] [Google Scholar]
  14. Delgado P, Cuadrado E, Rosell A, Alvarez-Sabin J, Ortega-Aznar A, Hernandez-Guillamon M, Penalba A, Molina CA, Montaner J (2008) Fas system activation in perihematomal areas after spontaneous intracerebral hemorrhage. Stroke 39(6):1730–1734 [DOI] [PubMed] [Google Scholar]
  15. Ferrero GO, Velazquez FN, Caputto BL (2012) The kinase c-Src and the phosphatase TC45 coordinately regulate c-Fos tyrosine phosphorylation and c-Fos phospholipid synthesis activation capacity. Oncogene 31(28):3381–3391 [DOI] [PubMed] [Google Scholar]
  16. Gomes JA, Manno E (2013) New developments in the treatment of intracerebral hemorrhage. Neurol Clin 31(3):721–735 [DOI] [PubMed] [Google Scholar]
  17. Gong C, Boulis N, Qian J, Turner DE, Hoff JT, Keep RF (2001) Intracerebral hemorrhage-induced neuronal death. Neurosurgery 48(4):875–882 discussion 882–873 [DOI] [PubMed] [Google Scholar]
  18. Guller M, Toualbi-Abed K, Legrand A, Michel L, Mauviel A, Bernuau D, Daniel F (2008) c-Fos overexpression increases the proliferation of human hepatocytes by stabilizing nuclear Cyclin D1. World J Gastroenterol 14(41):6339–6346 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Habib GM (2010) Arsenite causes down-regulation of Akt and c-Fos, cell cycle dysfunction and apoptosis in glutathione-deficient cells. J Cell Biochem 110(2):363–371 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hata R, Maeda K, Hermann D, Mies G, Hossmann KA (2000) Dynamics of regional brain metabolism and gene expression after middle cerebral artery occlusion in mice. J Cereb Blood Flow Metab 20(2):306–315 [DOI] [PubMed] [Google Scholar]
  21. Herrera DG, Robertson HA (1996) Activation of c-fos in the brain. Prog Neurobiol 50(2–3):83–107 [DOI] [PubMed] [Google Scholar]
  22. Hu Y, Jin X, Snow ET (2002) Effect of arsenic on transcription factor AP-1 and NF-kappaB DNA binding activity and related gene expression. Toxicol Lett 133(1):33–45 [DOI] [PubMed] [Google Scholar]
  23. Hua Y, Schallert T, Keep RF, Wu J, Hoff JT, Xi G (2002) Behavioral tests after intracerebral hemorrhage in the rat. Stroke 33(10):2478–2484 [DOI] [PubMed] [Google Scholar]
  24. Hwang BY, Appelboom G, Ayer A, Kellner CP, Kotchetkov IS, Gigante PR, Haque R, Kellner M, Connolly ES (2011) Advances in neuroprotective strategies: potential therapies for intracerebral hemorrhage. Cerebrovasc Dis 31(3):211–222 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Karabiyikoglu M, Hua Y, Keep RF, Ennis SR, Xi G (2004) Intracerebral hirudin injection attenuates ischemic damage and neurologic deficits without altering local cerebral blood flow. J Cereb Blood Flow Metab 24(2):159–166 [DOI] [PubMed] [Google Scholar]
  26. Kasibhatla S, Brunner T, Genestier L, Echeverri F, Mahboubi A, Green DR (1998) DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-kappa B and AP-1. Mol Cell 1(4):543–551 [DOI] [PubMed] [Google Scholar]
  27. Kolbus A, Herr I, Schreiber M, Debatin KM, Wagner EF, Angel P (2000) c-Jun-dependent CD95-L expression is a rate-limiting step in the induction of apoptosis by alkylating agents. Mol Cell Biol 20(2):575–582 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kovary K, Bravo R (1991) The jun and fos protein families are both required for cell cycle progression in fibroblasts. Mol Cell Biol 11(9):4466–4472 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kuramatsu JB, Huttner HB, Schwab S (2013) Advances in the management of intracerebral hemorrhage. J Neural Transm 120(Suppl 1):S35–S41 [DOI] [PubMed] [Google Scholar]
  30. Laderoute KR (2005) The interaction between HIF-1 and AP-1 transcription factors in response to low oxygen. Semin Cell Dev Biol 16(4–5):502–513 [DOI] [PubMed] [Google Scholar]
  31. Lauricella M, Emanuele S, D’Anneo A, Calvaruso G, Vassallo B, Carlisi D, Portanova P, Vento R, Tesoriere G (2006) JNK and AP-1 mediate apoptosis induced by bortezomib in HepG2 cells via FasL/caspase-8 and mitochondria-dependent pathways. Apoptosis 11(4):607–625 [DOI] [PubMed] [Google Scholar]
  32. Le-Niculescu H, Bonfoco E, Kasuya Y, Claret FX, Green DR, Karin M (1999) Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to Fas ligand induction and cell death. Mol Cell Biol 19(1):751–763 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Li L, Ke K, Tan X, Xu W, Shen J, Zhai T, Xu L, Rui Y, Zheng H, Zhai P, Zhao J, Cao M (2013) Up-regulation of NFATc4 involves in neuronal apoptosis following intracerebral hemorrhage. Cell Mol Neurobiol 33(7):893–905 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lin S, Yin Q, Zhong Q, Lv FL, Zhou Y, Li JQ, Wang JZ, Su BY, Yang QW (2012) Heme activates TLR4-mediated inflammatory injury via MyD88/TRIF signaling pathway in intracerebral hemorrhage. J Neuroinflamm 9:46 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Liu WH, Chang LS (2009) Arachidonic acid induces Fas and FasL upregulation in human leukemia U937 cells via Ca2+/ROS-mediated suppression of ERK/c-Fos pathway and activation of p38 MAPK/ATF-2 pathway. Toxicol Lett 191(2–3):140–148 [DOI] [PubMed] [Google Scholar]
  36. Markopoulou S, Kontargiris E, Batsi C, Tzavaras T, Trougakos I, Boothman DA, Gonos ES, Kolettas E (2009) Vanadium-induced apoptosis of HaCaT cells is mediated by c-fos and involves nuclear accumulation of clusterin. FEBS J 276(14):3784–3799 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Matsumoto N, Imamura R, Suda T (2007) Caspase-8- and JNK-dependent AP-1 activation is required for Fas ligand-induced IL-8 production. FEBS J 274(9):2376–2384 [DOI] [PubMed] [Google Scholar]
  38. Mikula M, Gotzmann J, Fischer AN, Wolschek MF, Thallinger C, Schulte-Hermann R, Beug H, Mikulits W (2003) The proto-oncoprotein c-Fos negatively regulates hepatocellular tumorigenesis. Oncogene 22(43):6725–6738 [DOI] [PubMed] [Google Scholar]
  39. Nagata S, Golstein P (1995) The Fas death factor. Science 267(5203):1449–1456 [DOI] [PubMed] [Google Scholar]
  40. Nagy Z, Simon L, Bori Z (2002) Regulatory mechanisms in focal cerebral ischemia. New possibilities in neuroprotective therapy. Ideggyogy Sz 55(3–4):73–85 [PubMed] [Google Scholar]
  41. Niu Q, Liu H, Guan Z, Zeng Q, Guo S, He P, Guo L, Gao P, Xu B, Xu Z, Xia T, Wang A (2012) The effect of c-Fos demethylation on sodium fluoride-induced apoptosis in L-02 cells. Biol Trace Elem Res 149(1):102–109 [DOI] [PubMed] [Google Scholar]
  42. Pai SR, Bird RC (1994) c-fos expression is required during all phases of the cell cycle during exponential cell proliferation. Anticancer Res 14(3A):985–994 [PubMed] [Google Scholar]
  43. Sairanen T, Karjalainen-Lindsberg ML, Paetau A, Ijas P, Lindsberg PJ (2006) Apoptosis dominant in the periinfarct area of human ischaemic stroke: a possible target of antiapoptotic treatments. Brain 129(Pt 1):189–199 [DOI] [PubMed] [Google Scholar]
  44. Sansing LH, Messe SR, Cucchiara BL, Cohen SN, Lyden PD, Kasner SE (2009) Prior antiplatelet use does not affect hemorrhage growth or outcome after ICH. Neurology 72(16):1397–1402 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sharma HS, Westman J, Gordh T, Nyberg F (2000) Spinal cord injury induced c-fos expression is reduced by p-CPA, a serotonin synthesis inhibitor. An experimental study using immunohistochemistry in the rat. Acta Neurochir Suppl 76:297–301 [DOI] [PubMed] [Google Scholar]
  46. Shaulian E, Karin M (2001) AP-1 in cell proliferation and survival. Oncogene 20(19):2390–2400 [DOI] [PubMed] [Google Scholar]
  47. Smeyne RJ, Vendrell M, Hayward M, Baker SJ, Miao GG, Schilling K, Robertson LM, Curran T, Morgan JI (1993) Continuous c-fos expression precedes programmed cell death in vivo. Nature 363(6425):166–169 [DOI] [PubMed] [Google Scholar]
  48. Thiex R, Tsirka SE (2007) Brain edema after intracerebral hemorrhage: mechanisms, treatment options, management strategies, and operative indications. Neurosurg Focus 22(5):E6 [DOI] [PubMed] [Google Scholar]
  49. Tsai YW, Yang YR, Wang PS, Wang RY (2011) Intermittent hypoxia after transient focal ischemia induces hippocampal neurogenesis and c-Fos expression and reverses spatial memory deficits in rats. PLoS One 6(8):e24001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wang J, Dore S (2008) Heme oxygenase 2 deficiency increases brain swelling and inflammation after intracerebral hemorrhage. Neuroscience 155(4):1133–1141 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yamamura Y, Hua X, Bergelson S, Lodish HF (2000) Critical role of Smads and AP-1 complex in transforming growth factor-beta -dependent apoptosis. J Biol Chem 275(46):36295–36302 [DOI] [PubMed] [Google Scholar]

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