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. 2016 Mar 22;37(1):83–92. doi: 10.1007/s10571-016-0346-3

Involvement of Fra-1 in Retinal Ganglion Cell Apoptosis in Rat Light-Induced Retina Damage Model

Xiaojuan Liu 1,4,#, Xiaowei Yang 2,4,#, Rongrong Zhu 2,4, Ming Dai 2,4, Manhui Zhu 2,4, Yuntian Shen 3, Hongda Fang 2,4, Aimin Sang 2,4,✉,#, Hui Chen 2,4,✉,#
PMCID: PMC11482155  PMID: 27002720

Abstract

Cell cycle re-entry, in which Fra-1 (transcription factor FOS-related antigen 1) plays an important role, is a key process in neuronal apoptosis. However, the expression and function of Fra-1 in retinal ganglion cell (RGC) apoptosis are unknown. To investigate whether Fra-1 was involved in RGC apoptosis, we performed a light-induced retinal damage model in adult rats. Western blot revealed that up-regulation of Fra-1 expression appeared in retina after light exposure (LE). Immunostaining indicated that increased Fra-1 was mainly expressed in RGCs in retinal ganglion cell layer (GCL) after LE. Co-localization of Fra-1 with active caspase-3 or TUNEL-positive cells in GCL after LE was also detected. In addition, Fra-1 expression increased in parallel with cyclin D1 and phosphorylated mitogen-activated protein kinase p38 (p-p38) expression in retina after LE. Furthermore, Fra-1, cyclin D1, and active caspase-3 protein expression decreased by intravitreal injection of SB203580, a highly selective inhibitor of p38 MAP kinase (p38 MAPK). All these results suggested that Fra-1 may be associated with RGC apoptosis after LE regulated by p38 MAPK through cell cycle re-entry mechanism.

Keywords: Fra-1, Light-induced retinal damage, Retinal ganglion cell, Cell cycle re-entry, Apoptosis, P38 MAPK

Introduction

Retina ganglion cells (RGCs) are the ultimate neurons in retina that respond to light and transfer light into electrochemical signals in the brain. Loss of RGCs occurs in multiple conditions such as glaucomatous optic neuropathy, anterior ischemic optic neuropathy, arterial and venous vessel occlusions, traumatic optic neuropathy, and diabetic retinopathy (Chidlow et al. 2014). Light impinging on the retina in situ is potentially toxic to retinal cells and leads to retinal degeneration, especially in cells with compromised functions such as glaucoma, diabetic retinopathy, and ischemia (Li et al. 2011a). Although apoptotic RGCs have already been demonstrated in light-induced retinal damage model, the precise pathologic mechanisms underlying RGC apoptosis are still unclear.

A pathway including cell cycle-related molecules plays a required role in neuronal death during normal development as well as in disease and trauma (Greene et al. 2007). Neuronal apoptosis and cell cycle activation share common pathways. Neuronal cell cycle re-entry is an apoptotic route in major neurological disorders (Folch et al. 2012). In the retina, extra-retinal injury to RGCs initiates signaling cascades which are associated with molecular events leading to neuronal cell cycle re-entry, DNA hyperploidy, and RGC death (Galan et al. 2014; Wu et al. 2014).

AP-1 transcription factors which are leucine zipper proteins belonging to members of the Jun family (c-Jun, JunB, and JunD) and Fos family (c-Fos, FosB, Fra-1, and Fra-2), control rapid responses of mammalian cells to stimuli that impact proliferation, differentiation, transformation, and apoptosis (Shaulian and Karin 2002). In normal cells, AP-1 is involved in cell proliferation and, depending on the cellular context, exerts both proapoptotic and antiapoptotic functions in response to a wide range of environmental stimuli such as mitogens, stress-inducing agents, and inflammatory cytokines. Recently, important proof was provided on the role of Fra-1 in the regulation of cell cycle (Burch et al. 2004). Thus, we hypothesize that Fra-1 may be greatly critical for RGC apoptosis after LE.

In this study, we aimed to determine Fra-1 expression and function in adult rat light-induced retinal damage model. Intravitreal injection of p38 MAPK inhibitor SB203580 reduced the light-induced Fra-1, cyclin D1, and active caspase-3 expression. Present study may gain a better insight into the possible roles of Fra-1 in light-induced RGC apoptosis.

Materials and Methods

Animals and the Light-Induced Retinal Damage Model

Adult male Sprague–Dawley rats aged 10 weeks and weighing 220–270 g (Laboratory Animal Center, Nantong University) were used in our study. All rats were housed in a 12-h light–dark cycle with access to food and water ad libitum. All experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

As previously described (Sang et al. 2013), before LE, rats were dark adapted for 2 weeks. After pupil dilation with compound tropicamide (Santen Pharmaceutical, Osaka, Japan), dark-adapted rats were placed in cages and exposed to white light (the light wavelength ranges from 400 to 760 nm, 8000 lx) for 3 h, beginning at 9 a.m. During LE, the room temperature was kept at 24 °C. The rats had free access to food and water. After light exposure (LE), all rats were returned to darkness and randomly divided into six groups (6 h, 12 h, 1, 3, 5, 7 days). For Western blot, each group contained 18 rats (the 18 rats were pooled into three groups of 6 rats each group) and other 18 rats without LE belonged to the normal group. In the normal and LE 3 days groups, another three rats (six eyeballs) each were used for immunohistochemistry, immunofluorescence, and TUNEL assay.

Western Blot

To obtain samples for Western blot, all rats were given an overdose of chloral hydrate (400 mg/kg, i.p.) and sacrificed at the designated time points. Then, the cornea, lens, and vitreous were removed; retina was extruded gently from the eye cup. Retinas were put into one pool for further protein extraction and analysis. The extract samples (2.0 mg/ml, 10 μl) were loaded, subjected to 10 % SDS-PAGE, and electro-blotted onto PVDF membranes using the Mini-PROTEAN 3 Electrophoresis System and the Mini Trans-Blot Electrophoretic Transfer System (Bio-Rad, Hercules, CA). The membranes were blocked with 5 % nonfat milk for 2 h, followed by incubation with primary antibodies against Fra-1 (rabbit, 1:500; Santa Cruz Biotechnology Inc., Santa Cruz, USA), active caspase-3 (rabbit, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 (mouse, 1:500; Santa Cruz), p-p38 (mouse, 1:2000; Santa Cruz), p38 (rabbit, 1:3000; Santa Cruz), and GAPDH (mouse, 1:3000; Santa Cruz) at 4 °C overnight. Next, immunoreactive bands were detected by chemiluminescence using corresponding HRP-conjugated secondary antibodies (1:2000; GE Healthcare, Piscataway, NJ, USA), enhanced chemiluminescence detection reagents (GE Healthcare), and LAS 3000 image analyzer (Fujifilm, Tokyo, Japan). Quantitative changes in band intensities were evaluated with Image Quant 5.2 software (GE Healthcare). Values are responsible for at least three independent experiments.

Quantitative Real-Time PCR

Quantitative real-time PCR (qPCR) was used to determine Fra-1 mRNA level at different times after LE. Total RNA was isolated from retina of rats using RISO reagent (Biomics, Nantong, Jiangsu, China) and treated with DNase I. Complementary DNA (cDNA) was synthesized by reverse transcriptase from total RNA with oligo-d (T) primers. The qPCR analysis was performed with the Bio-Rad IQ5 real-time PCR detection system (Bio-Rad, Hercules, CA) using the SYBR Master mixture (Biomics, Nantong, Jiangsu, China). The PCR reactions were performed in triplicate on each cDNA template along with triplicate reactions of a house-keeping gene, GAPDH. We used the following primers for Fra-1, forward (5-AAGTTGGAGGATGAGAAA-TCGG-3) and reverse (5-CCTTCTTGTCTTCTTCTGGGATT-3); for GAPDH, forward (5-GAAGGTGAAGGTCGGAGTC-3) and reverse (5-GAAGATGGTGATG-GGATTTC-3). The specific amplification was verified by melting curve analysis. The data were normalized against GAPDH. The expression levels were determined using the ΔΔCT method (IQ5 software version 2.0, Bio-Rad) and presented as fold changes.

Sections and Immunohistochemistry

In the normal and LE 3 d groups, rats (n = 3 per group) were terminally anesthetized and perfused through the ascending aorta with saline, followed by 4 % paraformaldehyde. After perfusion, the superior conjunctiva was sutured with 8.0 vicryl. The eyes were fixed in 4 % paraformaldehyde solution, followed by immersion in sucrose solution for cryoprotection. Then, the tissues were embedded in OCT compound and 7-μm frozen sections were prepared. The superior hemisphere along the vertical meridian was chosen in the study. For immunohistochemistry, sections were blocked with 10 % normal serum blocking solution and incubated overnight at 4 °C with Fra-1 antibody (rabbit, 1:200; Santa Cruz) in blocking solution and then reacted with the biotinylated goat anti-rabbit secondary antibody (1:200; Vector Laboratories, Burlingame, CA) for 2 h. Sections were processed in the complex avidin–biotin-peroxidase (ABC Kit, Vector Laboratories) for 40 min at 37 °C. Slides were examined by a Leica light microscope (Leica Microsystems, Wetzlar, Germany).

Double Immunostaining

The cryosections were blocked and then incubated with Fra-1 antibody (rabbit, 1:200; Santa Cruz). The antibodies against Brn3B (mouse, 1:200; Santa Cruz), active caspase-3 (mouse, 1:200; Cell Signaling), or cyclin D1 (Rabbit, 1:500; Santa Cruz) were co-incubated for 10 h at 4 °C. On the following day, a mixture of FITC-, CY3-, and AMCA-conjugated secondary antibodies and Hoechst (Pierce Biotechnology, Radford, IL, USA) were added in dark and incubated for 2 h at 4 °C. The stained sections were examined with a Leica fluorescence microscope (Leica Microsystems).

TUNEL Staining

Terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (TUNEL) staining was employed using the in situ cell death detection kit, fluorescein (Roche Applied Science, Mannheim, Germany). The cryosections were rinsed with PBS and treated with 1 % Triton X-100 in PBS for 2 min on ice. Then, the slides were rinsed in PBS and incubated with 50 μl of TUNEL reaction mixture for 60 min at 37 °C. After washing with PBS three times, the slides were analyzed using a Leica fluorescence microscope (Leica Microsystems).

Intravitreal Injection

After the rats were anesthetized, their corneas were anesthetized with a drop of 0.5 % proparacaine hydrochloride (Alcaine; Alcon-Couvreur, Puurs, Belgium), pupils were dilated with 1 % tropicamide, and then the eyes were gently protruded with a rubber sleeve. Intravitreal injection of different remedies was performed 1 mm behind the limbus with a 33-gage blunt-tip needle (Hamilton, Reno, NV, USA) and leaving the needle for 2 min to reduce the reflux. The rats with intravitreal injection of 10 nmol glycine, with SB203580 (Calbiochem, San Diego, CA), which was dissolved in 5 % dimethyl sulfoxide (DMSO) or with PBS, were divided into two subgroups: normal and 3 d. The animals were sacrificed after LE. The eyes were then enucleated for Western blot analysis. Any rat with visible lens damage or vitreous hemorrhage was excluded.

Quantitative Analysis

The cells were counted using the software Image-Pro plus 6.0. To avoid counting the same cell in more than one section, we counted the number of positive cells in the retina every fifth Sect. (50 μm apart) at ×40 magnification. For each section, three separate regions were examined. The cell counts in the three sections were then used to determine the total number of positive cells and whole retinal cells per square millimeter.

Statistical Analysis

All data were analyzed with IBM SPSS Statistics 19 software. All values were expressed as mean ± SEM and were analyzed by one-way variance (ANOVA) followed by Tukey’s post hoc multiple comparison test. P < 0.05 was considered statistically significant. Experiments were performed at least in triplicate per condition.

Results

The Fra-1 Expression and Distribution Following LE

First, Western blot was used to identify Fra-1 expression in retina after LE, showing that Fra-1 expression was low in the normal retina and maintained low level at 6 and 12 h after LE. However, Fra-1 expression reached a peak at 3 days and then dropped from 5 days, returned to normal level at 7 days (Fig. 1a, b). Fra-1 mRNA level determined by qPCR, increased and reached its peak at 3 days after LE, parallel to its protein expression (Fig. 1c). To further determine the temporal profile and spatial distribution of Fra-1 in retina, we chose 3 days after LE at which Fra-1 protein expression reached a maximum as the time point for morphological examination. Immunohistochemistry assay showed that only weak expression of Fra-1 was observed in the INL of normal retina (Fig. 1d). After LE, Fra-1 expression increased in the GCL, INL, and ONL (Fig. 1e–j) compared with normal retina. To further identify the cell location of Fra-1, immunofluorescent double staining was performed with Brn3B (a RGC marker) in the injured retina. Fra-1 was mainly expressed in Brn3B-positive cells after LE (Fig. 1h–l). The LE retinas showed an obviously progressive photoreceptor loss and thinner width in ONL compared with normal retina, which was the evidence of retinal degeneration after LE (Fig. 1g–j). These data were consistent with Western blot results, showing the increased Fra-1 expression induced by LE. Besides, Fra-1 expression was primarily confined to the RGCs.

Fig. 1.

Fig. 1

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The Fra-1 expression and distribution in rat retina following LE. a Fra-1 protein expression after LE by Western blot. b Quantification graph showed relative optical intensity of Fra-1 to GAPDH. The data are mean ± SEM (n = 3; *P < 0.05, significantly different from the normal group). c Fra-1 mRNA level was detected by qPCR. The data are mean ± SEM (n = 3; *P < 0.05, significantly different from the normal group). d–j Cross sections of the retina were immunostained with anti-Fra-1 antibody. k–o. Double labeling of Fra-1 (red) and Brn3B (green) in retina. Fra-1 (red, l) was co-localized with Brn3b (green, m) in the merged image (yellow, o) after LE, nuclear Hoechst staining (blue, n) (Color figure online)

Fra-1 was Associated with RGC Apoptosis

To study RGC apoptosis after LE, Western blot was performed to detect active caspase-3 (an apoptosis marker) protein expression. Active caspase-3 expression was low in normal retina, then gradually increased after LE and reached a peak at 3 days (Fig. 2a, b), positively correlated with Fra-1 expression in a time-dependent manner (Fig. 2c). Double immunofluorescent staining revealed that co-localization of active caspase-3 and Fra-1 was observed at 3 days in GCL after LE (Fig. 2d–h). TUNEL staining was applied at 3 days after LE to further determine whether Fra-1 was involved in RGC apoptosis. The number of TUNEL-positive cells was relatively low in normal retina (Fig. 3a). At 3 days after LE, TUNEL-positive cells increased (Fig. 3b), co-localizing with Fra-1 (Fig. 3b–e). Semi-quantitative analysis showed that a markedly increased number of TUNEL and Fra-1/TUNEL-positive cells in the GCL at 3 days after LE (Fig. 4f–g). These results indicated that Fra-1 might be associated with RGC apoptosis after LE.

Fig. 2.

Fig. 2

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The expression and location of active caspase-3 in rat retina after LE. a Active caspase-3 protein expression was examined in retina after LE by Western blot. b The bar chart showed the ratio of active caspase-3 to GAPDH; the data are mean ± SEM (n = 3, *P < 0.05, significantly different from the normal group). c The correlation between active caspase-3 and Fra-1 expression in rat retina. d–h. Double immunofluorescence staining was performed for Fra-1 (red) and active caspase-3 (green) in retina. Active caspase-3 (green, e) was co-localized with Fra-1 (red, f) in the merged image (yellow, h) after LE, nuclear Hoechst staining (blue, g) (Color figure online)

Fig. 3.

Fig. 3

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Fra-1 was expressed in TUNEL-positive cells after LE. a TUNEL staining showing the expression of TUNEL-positive cells (green) in normal retina. b–e The number of TUNEL-positive cells was increased at 3 d after LE and was co-localized with Fra-1 in GCL. TUNEL-positive cells (green, b); Fra-1 (red, c); nuclear Hoechst staining (blue, d). e The yellow color visualized in the merged images represented the co-localization of Fra-1 with TUNEL-positive cells. Scale bar 60 μm. f, g Semi-quantitative analysis indicated an obvious increase in the number of TUNEL and FRA-1/TUNEL-positive cells in GCL at 3 d after LE. The data are mean ± SEM (n = 3; *P < 0.05, significantly different from the normal group) (Color figure online)

Fig. 4.

Fig. 4

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The cyclin D1 expression and distribution after LE. a Cyclin D1 protein expression after LE was detected by Western blot. b Quantification graph showed the intensity of cyclin D1 to GAPDH. The data are mean ± SEM (n = 3; *P < 0.05, significantly different from the normal group). c The correlation between cyclin D1 and active caspase-3 expression. d, e Dramatically up-regulated cyclin D1 expression was observed in injured retina at 3 d after LE by immunofluorescence staining (e), compared with the normal retina (d). e–h Cyclin D1 expression (red, e) was overlapped with active caspase-3 (green, f) expression and Hoechst (blue, g) in merged image (yellow, h) (Color figure online)

Increased Expression of Cyclin D1 in RGCs After LE

Western blot was used to analyze cyclin D1 expression after LE. Cyclin D1 expression was weak in normal retina, increased after LE (Fig. 4a, b), positively correlated with active caspase-3 expression in a time-dependent manner (Fig. 4c). Furthermore, immunofluorescence assay showed that cyclin D1 was hardly detectable in normal retina (Fig. 4d), but it was rapidly up-regulated and overlapped with active caspase-3 after LE (Fig. 4e–h). These results suggested that Fra-1 may exert apoptotic function via cell cycle re-entry signaling.

P38 MAPK Regulates Fra-1 Expression After LE

Extracellular signal regulated p38 MAPK plays a central role in regulating AP-1 family member expression in response to toxic stimuli in a wide variety of cell types (Whitmarsh and Davis 1996; Wang et al. 2015). Besides, toxic-inducible Fra-1 expression was attenuated by p38 MAPK inhibitor (Zhang et al. 2005). Thus, we sought to identify whether p38 MAPK was involved in regulating Fra-1 expression after LE. In normal retina, p-p38 expression was feeble. After LE, p-p38 expression gradually increased (Fig. 5a, b), reached its peak at 3 days after LE, and then gradually dropped, positively correlated with Fra-1 expression in a time-dependent manner (Fig. 5c). The p38 expression had no obviously change in both normal and injured retinas (Fig. 5a).

Fig. 5.

Fig. 5

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Fra-1 may regulate RGC apoptosis by modulating cell cycle protein expression through regulating p38 MAPK phosphorylation. a The expression of p-p38 and p38 in rat retina after light exposure. b The bar chart demonstrated the ratio of p-p38 to p-p38. n = 3, *P < 0.05; significantly different from the normal group. c The correlation between p-p38 and Fra-1 expression after LE. d Double labeling of Fra-1 (green) and p-p38 (red) in normal and injured retina at 3 d. e After SB203580 intravitreal injection, sample immunoblots probed for p-p38 and p38 in normal and injured retina. f Sample immunoblots probed for Fra-1, cyclin D1, and active caspase-3 in injured retina with intravitreal injection of SB203580 or PBS. g The bar chart demonstrated the ratio of Fra-1, cyclin D1, and active caspase-3 relative to GAPDH. n = 3, *P < 0.05, significant difference from PBS intravitreal injection group (Color figure online)

To determine whether the increase of p-p38 expression was associated with RGCs, we performed the immunostaining of p-p38, finding that p-p38 was weakly stained in the GCL of normal retina. After LE, the obvious increase of p-p38 expression was observed in the GCL at 3 days after LE, co-localizing with Fra-1. These results suggest that LE potently activates p38 MAPK pathway in RGCs.

To determine whether p38 MAPK pathway regulates LE induced Fra-1 expression, we used SB203580, a p38 MAPK inhibitor, for intravitreal injection. Fra-1 induction was markedly suppressed by intravitreal injection of SB203580 (Fig. 5e). Moreover, we also measured Fra-1, active caspase-3, and cyclin D1 expression in the LE group at 3 days with SB203580 or PBS intravitreal injection. The injured retina treated with SB203580 showed markedly low level of active caspase-3, Fra-1, and cyclin D1 compared to PBS injection group (Fig. 5f, g). The data indicated that Fra-1 might induce RGC apoptosis through cell cycle re-entry via p38 MAPK pathway regulation.

Discussion

Light-induced retinal damage is used as a model to study retinal degenerative diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), which have an important feature—apoptotic photoreceptor cell death (Rezaie et al. 2012; Marc et al. 2008). RGC death is also a common feature of retinal degenerative diseases (Chen et al. 2013; Garcia-Ayuso et al. 2011). Previous study demonstrated that RGC death was caused by RGC axonal compression, which is followed retinal pigment epithelial (RPE) migration, formation of RPE-vascular complexes, and vascular displacement (Marco-Gomariz et al. 2006). Recently, visible light has been reported to induce RGC line. Visible light might also directly trigger the death pathway by affecting the nucleus, which is an important center for DNA transcription and duplication (Li et al. 2011b). Furthermore, accumulating evidences show that light impinging on the retina in situ has the capacity to induce RGC apoptosis (Liu et al. 2012; Li et al. 2011b). In the studies of vision restoration in blind individuals using bionic devices, researchers found that retinal stimulation could restore limited visual perception to patients with RP. However, loss of RGCs precludes this approach (Lewis et al. 2015). Thus, it is important to explore molecular and cellular events involved in RGC apoptosis. To date, the mechanisms or trigger factors responsible for the onset of light-induced retinal damage are unknown.

The present study mimicked light-induced retinal damage and explored the cellular mechanism after LE in adult rats. Our data revealed that Fra-1 was gradually up-regulated in the retina after LE. Immunohistochemistry and immunofluorescent double staining suggested that Fra-1 was mainly localized in RGCs. The co-localization of Fra-1 with active caspase-3 or TUNEL was detected in RGCs. Moreover, active caspase-3 expression was in parallel with Fra-1 expression. Based on these data, Fra-1 might be associated with RGC apoptosis after LE.

Aberrant neuronal re-entry into the cell cycle is emerging as a potential pathological mechanism in Alzheimer disease (AD) (Bonda et al. 2009). Delayed intravenous administration of honokiol, a cell cycle inhibitor, could effectively improve functional recovery and attenuate neuronal cell death (Wang et al. 2014). The results indicate that cell cycle re-entry has a causative role in neurodegeneration (Marathe et al. 2015; Bonda et al. 2010). Besides the pivotal role in central nervous system, cell cycle re-entry may also participate in RGC apoptosis. In the retina, Ski-interacting protein (SKIP) plays an important part in cell cycle re-entry and relates to RGC apoptosis after optic nerve crush (Wu et al. 2014). Besides, RGC axon injury rapidly conveys a signal that activates RGC cycle re-entry, DNA hyperploidy, and neuronal apoptosis (Galan et al. 2014). Cell cycle re-entry includes complex physiological and biochemical mechanisms. Once cell cycle re-entry is initiated, increased expression of cell cycle regulators induces neuronal apoptosis (Camins et al. 2007; De Falco et al. 2006; Verdaguer et al. 2007). One of the vital mechanisms of cell cycle re-entry is G1/S transition regulation (Sancar et al. 2004). Furthermore, cell cycle activation is coordinated by cyclin D1 which is rate limiting and essential for the progression through G1 phase of cell cycle (Galan et al. 2014). Cyclin D1 is up-regulated in rat traumatic brain injury and ischemic reperfused retina. Cyclin-dependent kinase inhibitors can protect neurons against apoptosis (Lu et al. 2014; Sakamoto et al. 2011). It is illuminated that cyclin D1 is a key regulator in cell cycle re-entry inducing apoptosis of neuronal cells including RGCs.

Fra-1 is a regulator of mitotic progression and frequently overexpressed in cancers and implicated in invasiveness (Casalino et al. 2007). Researchers have delineated a dynamic program of chromatin trafficking of c-Fos and Fra-1 required for cyclin D1 expression during cell cycle re-entry by using cell cycle arrest in response to oxidative stress (Burch et al. 2004). Inhibiting entry of Fra-1 into the nucleus can block cyclin D1 expression (Burch et al. 2004). Blocking induction of Fra-1 can repress the expression of cyclin D1 which can induce terminal cell cycle arrest, associated with myogenesis (Rajabi et al. 2014). Interestingly, as our data show, Fra-1 expression was increased in parallel with cyclin D1 expression after LE. Additionally, cyclin D1 expression was increased in parallel with active caspase-3 expression in RGCs. We hypothesize that Fra-1 might be associated with RGC apoptosis after light-induced retinal damage via cell cycle re-entry.

p38 MAPK plays an important role in cellular processes such as differentiation and apoptosis as well as pathological conditions such as neurodegenerative disorders, diabetes, and inflammatory disease (O’Callaghan et al. 2014). Specially, it is an important mediator of signal transduction that responds to oxidative stress (McCubrey et al. 2006). In retina, p38 MAPK was involved in signal transduction after multiple toxic stimulations such as oxidative stress, ischemia/reperfusion, and hypoxia. Inhibiting p38 MAPK pathway through SB203580 decreased the number of ganglion cell loss and partially protected retinal damage (Jiang et al. 2012; Zhang et al. 2015; Hong et al. 2007). In our study, we demonstrate that inhibiting p38 MAPK phosphorylation can decrease the Fra-1, cyclin D1, and active caspase-3 expression, showing that p38 MAPK signaling pathway might regulate cell cycle re-entry to affect RGC apoptosis.

In conclusion, this study showed that up-regulation of Fra-1 is associated with RGC apoptosis in light-induced retinal damage. The role of Fra-1 in regulating RGC apoptosis may be executed by modulating cell cycle protein expression through p38 MAPK phosphorylation. These results provided a new view into the molecular mechanism of RGC apoptosis in the retina.

Acknowledgments

This work was supported in part by the National Natural Science Foundation of China (No. 81401365) and Nantong Science and Technology Innovation Project (Nos. MS12015056, HS2013014); a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Compliance with Ethical Standards

Conflicts of Interest

The authors claim no conflicts of interest.

Footnotes

Xiaojuan Liu, Xiaowei Yang, Aimin Sang and Hui Chen have contributed equally to this work.

Contributor Information

Aimin Sang, Email: aiminsang@163.com.

Hui Chen, Email: Chenhuieye@126.com.

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