Cell Mol Neurobiol (2015) 35:1175–1186 DOI 10.1007/s10571-015-0211-9
ORIGINAL RESEARCH
Up-Regulation of PKM2 Relates to Retinal Ganglion Cell Apoptosis After Light-Induced Retinal Damage in Adult Rats Xiaowei Yang1,3 • Hui Chen1,3 • Manhui Zhu1,3 • Rongrong Zhu1 • Bai Qin1 Hongda Fang1 • Ming Dai1,3 • Aimin Sang1,3 • Xiaojuan Liu2,3
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Received: 7 March 2015 / Accepted: 13 May 2015 / Published online: 20 May 2015 Ó Springer Science+Business Media New York 2015
Abstract Pyruvate kinase isozyme type M2 (PKM2), a key glycolytic enzyme, which is involved in ATP generation and pyruvate production, participates in tumor metabolism, growth, and other multiple cellular processes. However, one attractive biological function of PKM2 is that it translocates to the nucleus and induces cell apoptosis. Recently, increased PKM2 has been found in age-related macular degeneration (AMD), but little is known regarding its function in the AMD pathophysiology. To investigate whether PKM2 participated in retinal degeneration, we performed a lightinduced retinal damage model in adult rats. Western blot and immunohistochemistry analysis showed a significant upregulation of PKM2 in retinal ganglion cells (RGCs) layer (GCL) after light exposure. Immunofluorescent labeling indicated that PKM2 located mainly in RGCs. Co-localization of PKM2 and active caspase-3 as well as TUNEL in RGCs suggested that PKM2 might participate in RGC
Xiaowei Yang, Hui Chen, Aimin Sang, and Xiaojuan Liu have contributed equally to this work. & Aimin Sang [email protected] & Xiaojuan Liu [email protected] 1
Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, People’s Republic of China
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Department of Pathogen Biology, Medical College, Nantong University, Nantong 226001, Jiangsu, People’s Republic of China
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Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong 226001, Jiangsu, People’s Republic of China
apoptosis. In addition, the expression patterns of cyclin D1 and phosphorylated extracellular signal-regulated kinase (p-ERK) were parallel with that of PKM2. Furthermore, PKM2, cyclin D1, and active caspase-3 protein expression decreased by intravitreal injection of U0126, a highly selective inhibitor of MAPK/ERK kinase. Collectively, we hypothesized that PKM2 might participate in RGC apoptosis after light-induced retinal damage medicated by p-ERK through cycle re-entry mechanism. Keywords PKM2 Light-induced retinal damage RGCs Apoptosis Cyclin D1 ERK Rat
Introduction Age-related macular degeneration (AMD) is a complex disease caused by a combination of genetic and environmental factors. It is a common and painless eye condition as well as a leading cause of vision loss for people older than 50 years, but the molecule mechanisms of AMD are not fully known (den Hollander and de Jong 2014; Randolph 2014). Previous studies revealed that light exposure might be one of the contributing conditions in the pathogenesis of AMD (Sui et al. 2013; Fletcher et al. 2008). Light-induced retinal damage, a well-established in vivo model for retinal degeneration, covers most of the essential characteristics of human AMD and has been widely used to investigate the mechanisms of neuroretinal dysfunction (Marc et al. 2008). Photoreceptor apoptosis and retinal ganglion cell (RGC) death are common features in retinal degenerative diseases such as retinitis pigmentosa and AMD (Marc et al. 2008; Xu et al. 2014; Shu et al. 2014). Numerous studies have emphatically explored photoreceptor apoptosis following light exposure (Pasantes-Morales et al. 1981; Roca et al. 2004).
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However, the molecular mechanism of light-induced RGC damage remains unclear. Pyruvate kinase (PK) including four isoenzymes designated L, R, M1, and M2 mediates the final rate-limiting step of glycolysis by catalyzing the formation of pyruvate and ATP from phosphoenolpyruvate and ADP (Tsutsumi et al. 1988; Mukherjee et al. 2013). Other than a key role in glycolysis, PKM2 is expressed in cells with a high rate of nucleic acid synthesis such as normal proliferating cells, embryonic cells, and especially tumor cells (Tamada et al. 2012; Mazurek et al. 2005; Gupta and Bamezai 2010). The nuclear translocation of PKM2 induced by oxidative stress with H2O2 or UV light is linked with cell apoptosis (Stetak et al. 2007). Oxidative stress and damage from light are strongly involved in the pathogenesis of AMD (Cano et al. 2010). In addition, PKM2 was targeted by both dry and wet AMD patients by two-dimensional electrophoresis (Morohoshi et al. 2012). However, the expression of PKM2 and its role in AMD has not been elucidated. The cell cycle-related molecules play key roles in neuron death associated with tissue development and diseases (Greene et al. 2007). The elevation of markers for cell cycle re-entry was observed in the injured RGCs (Galan et al. 2014; Shu et al. 2014). Cyclin D1 is a key regulator of the G1/S transition as it activates CDK4, which phosphorylates Rb protein. As a result, Rb protein dissociates from E2F1, which in turn facilitates the transcription of genes needed for S phase progression. The increased expression of cyclin D1 in response to various neurotoxic agents is implicated in neuronal apoptosis (Kranenburg et al. 1996; Sumrejkanchanakij et al. 2003; Malik et al. 2008). Interestingly, PKM2 regulates G1/S transition by controlling cyclin D1 expression (Jiang et al. 2014). In this study, we firstly confirmed the expression and distribution of PKM2 in RGCs after light exposure and revealed its correlation with RGC apoptosis. Intravitreal injection of ERK inhibitor U0126 could reduce the PKM2, cyclin D1, and active caspase-3 expression. Our work may provide a useful clue to the pathogenesis of RGC apoptosis and may serve as a potential new therapeutic target for neurodegenerative diseases.
Materials and Methods Animals and the Light-Induced Retinal Damage Model Adult Sprague–Dawley male rats (Department of Animal Center, Nantong University, Nantong, China) were used in our study. Rats weighing 180–200 g were kept on a 12h:12h light–dark schedule, given food and water in a pathogen-free area. All experimental procedures were
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performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Before light exposure, rats were dark adapted for 1 day. After pupil dilation with compound tropicamide (Santen Pharmaceutical, Osaka, Japan), dark-adapted rats were placed in cages and exposed to white light (8000 lx) for 3 h, beginning at 9 AM during light exposure, room temperature (RT) was kept at 24 °C. The animals had free access to food and water. After light exposure, all rats were returned to darkness and randomly divided into six groups (6 h, 12 h, 1 day, 3 days, 5 days, 7 days). For Western blot, each group contained 18 rats and other 18 rats without light exposure belonged to the control group. In the control and light exposure 3-day 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 (350 mg/kg, i.p.) and sacrificed at different time points after light exposure. Then, the cornea, lens, and vitreous were removed; retina was extruded gently from the eye cup. Twelve retinas derived from six rats were put on one pool for further protein extraction and Western blot analysis. Three samples were studied at each time point. The extract samples (2.0 mg/ml, 10 ll) 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 (BioRad, Hercules, CA, USA). The membranes were blocked with 5 % nonfat milk at RT for 2 h, followed by incubation with primary antibodies against PKM2 (rabbit, 1:1000; Santa Cruz), active caspase-3 (mouse, 1:1000; Cell Signaling), cyclin D1 (mouse, 1:500; Santa Cruz), CDK4 (rabbit, 1:500; Santa Cruz), p-ERK1/2 (rabbit, 1:1000; Cell Signaling), ERK1/2 (rabbit, 1:1000; Cell Signaling), GAPDH (mouse, 1:1000; Santa Cruz), and b-actin (mouse, 1:1000; Sigma) at 4 °C overnight. Finally, 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 an LAS 3000 image analyzer (FUJIFILM, 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. Sections and Immunohistochemistry At defined survival time, normal and injured rats were terminally anesthetized and perfused through the ascending
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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-lm frozen sections were prepared. The superior hemisphere along the vertical meridian was chosen in this experiment. After the sections had been prepared, they were kept in an oven at 37 °C for 60 min and rinsed twice with 0.01 M PBS. The sections were blocked by confining liquid consisting of 10 % normal donkey serum, 1 % BSA, 0.3 % Triton X-100, 0.15 % Tween-20 for 2 h at RT, and incubated with PKM2 antibody (rabbit, 1:500; Santa Cruz) at RT for 2 h, followed by incubation with the primary and the secondary antibodies for 30 min at 37 °C, and then color reaction with a liquid mixture (0.02 % DAB, 3 % H2O2 and 0.1 % PBS). Finally, the sections were dehydrated, cleared, coverslipped, and examined under a light microscope (Leica, Germany). All the acquired images were used for quantification. Cell counting and assessment were performed in a double-blind manner using software Image J.
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fields were randomly chosen for every specimen. The number of TUNEL-positive cells was counted to obtain the ratio of the number of TUNEL-positive cells to the total number of cells. 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 1 min to reduce the reflux. The rats with intravitreal injection of 0.8 mM ERK inhibitor U0126 (Santa Cruz, Dallas, TX, sc-222395), which was dissolved in 5 % dimethyl sulfoxide (DMSO) with PBS, were divided into seven subgroups: control, 6 h, 12 h, 1 day, 3 days, 5 days, and 7 days. The animals were sacrificed after the start of light exposure, and the eyes were then enucleated for Western blot analysis. Statistical Analysis
Double Immunofluorescence Staining The cryosections were blocked with 10 % normal goat serum containing 3 % (w/v) BSA, 0.1 % Triton X-100, and 0.05 % Tween 20 at RT in order to avoid unspecific staining. Sections were incubated with PKM2 antibody (rabbit, 1:200; Santa Cruz). The co-incubated antibodies were antibodies for NeuN (mouse, 1:200; Cell Signaling), active caspase-3 (mouse, 1:200; Cell Signaling), and cyclin D1 (mouse, 1:500; Santa Cruz) separately for 10 h at 4 °C. On the following day, a mixture of FITC-, CY3-, and AMCA-conjugated secondary antibodies and Hoechst staining (Pierce Biotechnology, USA) were added in dark and incubated for 2 h at 4 °C. The stained sections were examined with a Leica fluorescence microscope (Germany). TUNEL Staining TUNEL staining was performed on the sections with an apoptosis detection kit (ApopTag Fluorescein in Situ Kit; Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Sections were rinsed with PBS and treated with 1 % Triton X-100 in PBS for 2 min on ice. Sections were rinsed in PBS again and incubated for 60 min at 37 °C with 50 ll of TUNEL reaction mixture and Label solution, respectively. After washing with PBS, the slides were analyzed with fluorescence microscopy (Leica, DM 5000B; Leica CTR 5000; Germany). Nine
All data were analyzed with IBM SPSS Statistics 19 statistical software. All values are expressed as mean ± SEM and were analyzed by one-way variance (ANOVA) followed by Tukey’s post hoc multiple comparison tests. P \ 0.05 was considered statistically significant. Experiments were performed at least in triplicate per condition.
Results PKM2 Expression in the Rat Retina After Light Exposure Western blot was performed to investigate the temporal patterns of PKM2 expression at different survival times. PKM2 expression was low in the normal retina. After lightinduced retinal damage, PKM2 expression gradually increased from 6 h and reached its peak at 3 days. After that, PKM2 expression tended to decline (Fig. 1a, b). These findings indicated that PKM2 might play an important role in the pathogenesis of light-induced retinal damage. Distribution of PKM2 in the Rat Retina After Light Exposure To determine the spatial distribution and the temporal profile of PKM2 in retina after light exposure, we then
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Fig. 1 PKM2 expression in the rat retina after light exposure. a Sample immunoblots probed for PKM2 and b-actin were shown above. b The bar chart demonstrated the ratio of PKM2 to b-actin at each time point. n = 3, *P \ 0.05; significantly different from the normal group
performed immunohistochemistry staining on transverse cryosections of rat retinal tissues. In the control group, PKM2-positive cells were observed in INL. At 3 days after light exposure, a prominent increase of PKM2 expression was detected in the GCL (Fig. 2b) compared with that of the normal retina based on quantitative analysis (Fig. 2e). Although PKM2-positive cells were also observed in the INL after light exposure, there is no statistical significance compared to the control group (Fig. 2a, b, d). In the negative control sections preincubated with PBS, PKM2 expression was undetectable (Fig. 2c). The data further confirmed that PKM2 expression increased after light exposure, hinting its function during light-induced retinal damage. To determine that PKM2-positive cells in the GCL were RGCs, we performed immunofluorescent double staining. In the normal retina, PKM2 was detected in the INL, which is same as the result of immunohistochemistry staining (Fig. 2f). After light exposure, increased PKM2positive cells were observed in the GCL (Fig. 2g) co-localizing with NeuN (a marker of RGCs) (Fig. 2h) in the merged image (Fig. 2j). The results suggested that PKM2 might be associated with RGC damage after light exposure. PKM2 was Relevant to RGC Apoptosis in Rat Retina After Light Exposure Previous research demonstrated that PKM2 translocated to the nucleus and participated in different apoptotic agents
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(Stetak et al. 2007). Moreover, clinical and experimental studies proved that increased PKM2 was involved in AMD and anti-PKM2 IgG served as a biomarker for diagnosis and prognosis of AMD (Morohoshi et al. 2012). Thus we hypothesize that PKM2 may be associated with retinal cell apoptosis after retinal damage. Caspase-3 activation is a prominent trait for neuronal apoptosis after light exposure. Therefore, to further detect the relationship between PKM2 and neuronal apoptosis in the retina, we performed Western blot to examine the expression profiles of active caspase-3. After light exposure, active caspase-3 was up-regulated from 12 h and culminated at 1 day (Fig. 3a, b). Active caspase-3 expression was positively correlated with PKM2 expression in a time-dependent manner (Fig. 3c). Additionally, immunofluorescent labeling showed that active caspase-3 expression was weak in the normal retina (Fig. 3d). The co-localization of active caspase-3 and PKM2 was detected in GCL at 3 days after light exposure (Fig. 3e–h). In addition, TUNEL-positive cells as well as PKM2positive cells were barely detected in the normal RGCs (Fig. 4a–d). While at 3 days after light exposure, a dramatically increased number of TUNEL-positive RGCs with PKM2 expression were observed (Fig. 4e–h), suggesting RGCs began to catabolize after light exposure. Besides, semi-quantitative analysis showed marked increase of TUNEL-positive and TUNEL–PKM2 double-positive RGCs in the retina at 3 days after light exposure (Fig. 4i, j). In a word, these results showed that PKM2 might play an important part in RGC apoptosis after light exposure. PKM2 was Co-Labeled with Cell Cycle Markers and May Be Regulated by p-ERK in Retina Besides its metabolic function, PKM2 has a non-metabolic function in the direct control of cell cycle progression by activating b-catenin and inducing the expression of cyclin D1, a downstream gene of b-catenin (Yang et al. 2011). In addition, evidences define a molecular pathway that includes cell cycle-related molecules and plays a required role in neuron death during normal development as well as in diseases and trauma (Greene et al. 2007). Previous study confirmed that cyclin D1 was correlated with apoptotic markers, such as active caspase-3, caspase-9, and poly (ADP-ribose) polymerase (PARP) (Fei et al. 2012). We assumed that PKM2 might participate in RGC apoptosis through its downstream gene cyclin D1. Therefore, we performed Western blot to examine the protein level of cyclin D1 and its partner CDK4, exploring the apoptosis status during light-induced retina damage. The results suggested that cyclin D1 expression and CDK4 expression were weak in the normal retina, while it was up-regulated parallel to the PKM2 and active caspase-3 expression after
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Fig. 2 PKM2 expression and distribution in the rat retina. a Normal retina. b 3 days after light exposure. c Negative control sections. d Bar chart illustrated the percentage of PKM2-positive cells in the INL. e Bar chart illustrated the percentage of PKM2-positive cells in GCL. *P \ 0.05, significantly different from the normal retina. Error bars represent SEM. f PKM2 expression was detected in INL in the
normal retina. g PKM2 expression was detected in both the GCL and INL at 3 days after light exposure. h NeuN was located in the GCL. i Hoechst staining in the retina. j The merged image showed the colocalization of PKM2 and NeuN in the GCL at 3 days after light exposure
light exposure (Figs. 1a, 3a, 5a). Cyclin D1 expression was positively correlated with PKM2 expression in a time-dependent manner (Fig. 5c). Additionally, the normal retina showed weak immunoreactivity for PKM2 and cyclin D1 (Fig. 5d). At 3 days after light exposure, PKM2 and cyclin D1 expression notably increased in the GCL (Fig. 5e, f). PKM2 (Fig. 5f) co-localized with cyclin D1 (Fig. 5e) in the merged image in the RGCs (Fig. 5h). Data above
manifested that PKM2 may exert its pro-apoptotic function via modulating cell cycle signaling. PKM2 can activate cell intrinsic apoptotic machinery to induce cell death through nuclear translocation (Stetak et al. 2007). Additionally, ERK-dependent phosphorylation and nuclear translocation of PKM2 are required for the autoregulation of PKM2 expression and PKM2-dependent gene expression (Yang et al. 2012). To explore whether
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Fig. 3 PKM2 involved in RGC apoptosis after light exposure. a The active caspase-3 protein level in the rat retina after light exposure. b Quantification for the staining intensity of caspase-3 at each time point. *P \ 0.05; significantly different from the normal group. c The correlation between active caspase-3 and PKM2 expression after light exposure. d–h Double labeling of PKM2 (red) and active caspase-3
(green) in the retina after light exposure. c The active caspase-3 immunoreactivity in the normal retina. d Three days after light exposure, active caspase-3 expression was significantly up-regulated. The active caspase-3 (green, e) overlapped with PKM2 (red, f) and Hoechst staining (blue, g) in the merged image (yellow, h) (Color figure online)
PKM2 was associated with p-ERK, Western blot was performed to examine the expression profile of p-ERK and t-ERK in the retina after light exposure. As shown in Fig. 6a, in the normal retina, p-ERK expression was feeble but still clearly detectable. After light exposure, p-ERK expression gradually increased (Fig. 6a, b), reached its peak at 3 days after light exposure, and then it gradually dropped. The t-ERK expression had no obvious change in both the normal and the damaged retina (Fig. 6a). In addition, p-ERK expression was positively correlated with PKM2 expression in a time-dependent manner (Fig. 6c). We supposed that the increase of p-ERK expression was associated with RGCs and performed the immunostaining of p-ERK. We found that p-ERK was weakly stained in the INL in the normal retina, but not in the GCL and ONL (Fig. 6d). After light exposure, the increase of p-ERK
expression was found in the GCL obviously (Fig. 6d). To determine whether there was an association between ERK kinase activity and cyclin D1 accumulation in the retina, ERK activation was inhibited with intravitreal injection of the highly selective ERK inhibitor U0126. We found p-ERK, cyclin D1, and PKM2 had no obvious change with U0126 intravitreal injection after light exposure (Fig. 6e). Besides that, as a comparison, we also measured PKM2, total caspase-3, and active caspase-3 expression in the normal and light-injured retina at 3 days with U0126 intravitreal injection. The light-injured retina treated with PBS showed significance high level of active caspase-3 and PKM2 compared to the light-injured retina treated with U0126 (Fig. 6f–h). The data indicated that PKM2 might induce RGC apoptosis through cell cycle pathway via phosphorylated ERK regulation.
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Fig. 4 Double staining with PKM2 and TUNEL. a–d The TUNEL and PKM2 weak immunoreactivity were detected in the RGCs in the normal retina. TUNEL (green, c) co-localized with PKM2 (red, b) and Hoechst staining (blue, a) in the merged image (yellow, d). e–h A clearly increased level of TUNEL and PKM2 immunoreactivity was
detected in the RGCs at 3 days after light exposure. TUNEL (green, g) co-localized with PKM2 (red, f) and Hoechst staining (blue, e) in the merged image (yellow, h). A semi-quantitative analysis of TUNEL (i) and PKM2/TUNEL (j) immunoreactive RGCs. *P \ 0.05, significant difference from the normal group (Color figure online)
Discussion
normal retina as well as in light-injured retina, suggesting that PKM2 may have no intimate relationship with photoreceptors. According to the previous study, a welldocumented correlation between RPE and PKM2 in the aged mouse has been identified (Morohoshi et al. 2012). In the present study, we focused on the role of PKM2 in the neural retina. However, it needs further study to reveal the molecular mechanism of PKM2 in RPE during the destruction of retinal structure after light exposure. As a key rate-limiting enzyme in the glycolytic regulating the Warburg effect, PKM2 is necessary for tumor growth (Ferguson and Rathmell 2008). Intensive studies demonstrated that PKM2 was up-regulated in a broad range of cancers (Fan et al. 2014; Wong et al. 2013). Recent studies indicated that nuclear PKM2 could activate gene transcription and cell proliferation closely related to
The present study constructed light-induced retinal damage model in adult rats and sought to evaluate whether PKM2 was involved in retinal damage. It was demonstrated that PKM2 expression was significantly up-regulated in RGCs after light-induced retinal damage. Interestingly, although PKM2 was observed in the INL in both the normal and light-injured retina, there is no statistical difference between the normal and post-damage 3-day groups. This phenomenon indicated that PKM2 may play a role in retinal normal function in the INL cells, such as bipolar nerve cells, horizontal cells, amacrines, and mu¨ller cells. The possible relevance between PKM2 and INL cells may need further investigation in the future. The ONL, photoreceptor layer, has no obvious PKM2-positive cells in the
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Fig. 5 Cyclin D1 and CDK4 were up-regulated after light exposure. a Cyclin D1 and CDK4 expression after light exposure. b The bar chart showed the ratio of cyclin D1 and CDK4 to GAPDH at each time point. n = 3, *, **P \ 0.05, significant difference from the normal group. c The correlation between cyclin D1 and PKM2
expression after light exposure. d Double labeling of PKM2 and cyclin D1 in the normal retina. After light exposure, cyclin D1 (red, e) overlapped with PKM2 (green, f) and Hoechst staining (blue, g) in the merged image (yellow, h) (Color figure online)
survival of cancer patients (Gao et al. 2012; Lee et al. 2008; Luo et al. 2011; Luo and Semenza 2011; Wang et al. 2014; Yang and Lu 2013; Zhang et al. 2013). PKM2 translocates to the nucleus to perform its non-metabolic activities, potentially functioning as a transcriptional coactivator. During tumor progression, growth signals convert PKM2 from active form to inactive form; therefore, the PK activity of PKM2 is changed and plays a ‘‘nonmetabolic’’ role. PKM2 stimulates the transcription of various genes by interacting with and phosphorylatingspecific nuclear proteins (Chen et al. 2014). Previous research identified that epidermal growth factor receptor (EGFR) activation induced b-catenin transactivation
through interacting with PKM2, which has a critical role in transcription of cyclin D1 (Yang et al. 2011). In addition to the function in tumorgenesis, PKM2 also plays a role in age-associated neurodegenerative diseases such as AMD (Morohoshi et al. 2012) and Alzheimer’s disease (AD) (Butterfield et al. 2006; Chen and Zhong 2013). But the detailed molecular mechanism of PKM2 in the neuronal apoptosis needs further exploration. The cell cycle is a delicately manipulated process essential for cell development, differentiation, proliferation, and death. Inappropriate activation of cell cycle regulators is implicated in the pathological process of a wide range of central nervous system (CNS) diseases, including acute
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Fig. 6 PKM2 may regulate RGC apoptosis by means of modulating cell cycle protein expression through phosphorylated ERK regulation. a The expression of p-ERK and t-ERK in the rat retina after light exposure. b The bar chart demonstrated the ratio of p-ERK to t-ERK at each time point. n = 3, *P \ 0.05; significantly different from the normal group. c The correlation between p-ERK and PKM2 expression after light exposure. d Double labeling of PKM2 (green) and p-ERK (red) in the normal retina and the light-damaged retina at 3 days. e After
U0126 intravitreal injection, sample immunoblots probed for p-ERK, t-ERK, cyclin D1, PKM2, and GAPDH were shown. f Sample immunoblots probed for PKM2, total caspase-3, and active caspase-3 in the rat retina with intravitreal injection of U0126 or PBS. g The bar chart demonstrated the ratio of active caspase-3 relative to GAPDH expression. h The bar chart demonstrated the ratio of PKM2 relative to GAPDH expression. n = 3, *P \ 0.05, significant difference from PBS intravitreal injection group (Color figure online)
damage and chronic neurodegenerative disorders (Hoglinger et al. 2007; Osuga et al. 2000; Di Giovanni et al. 2003). In terminally differentiated neurons, aberrant re-entry into the cell cycle triggers neuronal death instead of proliferation, which may be a common pathway shared by some acquired neurodegenerative disorders, even though multiple pathways of the cell cycle machinery are involved in distinct neuronal
demise in specific pathological circumstances (Clarke et al. 1992; Jacks et al. 1992; Lee et al. 1994). The cell cycle response is often mediated by the action of cyclin-dependent kinase complexes (Malumbres and Barbacid 2009). Cyclin D1 is a key cell cycle regulator and a direct target. Excessive cyclin D1 is associated with activity repression of E2F by initiating Rb phosphorylation and thus facilitates cell cycle
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progression (Berthet et al. 2006; Gerard and Goldbeter 2009). Cyclin D1 has been reported to appear in abnormal neurons in AD and RGCs after retinal damage (Bonda et al. 2009; Shu et al. 2014). Consistent with previous studies, the expression profiles of active caspase-3, Cyclin D1, and CDK4 were upregulated in the retina after light exposure, which was parallel with PKM2 expression. Immunofluorescence assay showed that the up-regulation of cyclin D1 was found in PKM2-positive RGCs, and was correlated with RGC apoptosis presented by active caspase-3 and TUNEL labeling. These results suggest that PKM2 may take part in RGC apoptosis via cell cycle re-entry. Nuclear PKM2 acts as a co-activator of b-catenin to induce downstream genes expression, such as c-Myc and cyclin D1. It is reported that PKM2 nuclear translocation is upon EGFR and ERK activation (Yang et al. 2011, 2012). ERK signaling pathway participates in multiple cell responses such as proliferation, migration, differentiation, and death (Cheng et al. 2013; Yang et al. 2013; Zhou et al. 2004). ERK signaling activation alongside JNK activation promoted apoptosis of cultured human CNS cells including neurons and microglia (Lannuzel et al. 1997). A further study found that caspase-3 activation is the downstream signal of ERK pathway (Pang et al. 2013). In the retina, ERK pathway played a key role in the regulation of apoptosis during retinal development (Donovan et al. 2011) and degeneration (Yang et al. 2008). These findings demonstrate a crucial role of the ERK signaling pathway in neuronal apoptosis, which may provide novel horizon in understanding of diseases such as AD and AMD, which contain abnormal neuron death. In conclusion, this study showed that up-regulation of PKM2 is associated with RGC apoptosis in light-induced retinal damage. The role of PKM2 in regulating RGC apoptosis may be executed by means of modulating cell cycle protein expression through ERK phosphorylation. These results provided a new view into the mechanism of neuronal apoptosis in the retina. Acknowledgments This work was supported in part by the National Natural Science Foundation of China (No. 81401365); a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Conflict of interest All authors declare that they have no conflict of interest.
References Berthet C, Klarmann KD, Hilton MB, Suh HC, Keller JR, Kiyokawa H, Kaldis P (2006) Combined loss of Cdk2 and Cdk4 results in embryonic lethality and Rb hypophosphorylation. Dev Cell 10(5):563–573. doi:10.1016/j.devcel.2006.03.004
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Cell Mol Neurobiol (2015) 35:1175–1186 Bonda DJ, Evans TA, Santocanale C, Llosa JC, Vina J, Bajic V, Castellani RJ, Siedlak SL, Perry G, Smith MA, Lee HG (2009) Evidence for the progression through S-phase in the ectopic cell cycle re-entry of neurons in Alzheimer disease. Aging 1(4):382–388 Butterfield DA, Poon HF, St Clair D, Keller JN, Pierce WM, Klein JB, Markesbery WR (2006) Redox proteomics identification of oxidatively modified hippocampal proteins in mild cognitive impairment: insights into the development of Alzheimer’s disease. Neurobiol Dis 22(2):223–232. doi:10.1016/j.nbd.2005. 11.002 Cano M, Thimmalappula R, Fujihara M, Nagai N, Sporn M, Wang AL, Neufeld AH, Biswal S, Handa JT (2010) Cigarette smoking, oxidative stress, the anti-oxidant response through Nrf2 signaling, and age-related macular degeneration. Vis Res 50(7): 652–664. doi:10.1016/j.visres.2009.08.018 Chen Z, Zhong C (2013) Decoding Alzheimer’s disease from perturbed cerebral glucose metabolism: implications for diagnostic and therapeutic strategies. Prog Neurobiol 108:21–43. doi:10.1016/j.pneurobio.2013.06.004 Chen L, Shi Y, Liu S, Cao Y, Wang X, Tao Y (2014) PKM2: the thread linking energy metabolism reprogramming with epigenetics in cancer. Int J Mol Sci 15(7):11435–11445. doi:10.3390/ ijms150711435 Cheng P, Alberts I, Li X (2013) The role of ERK1/2 in the regulation of proliferation and differentiation of astrocytes in developing brain. Int J Dev Neurosci 31(8):783–789. doi:10.1016/j.ijdevneu. 2013.09.008 Clarke AR, Maandag ER, van Roon M, van der Lugt NM, van der Valk M, Hooper ML, Berns A, te Riele H (1992) Requirement for a functional Rb-1 gene in murine development. Nature 359(6393):328–330. doi:10.1038/359328a0 den Hollander AI, de Jong EK (2014) Highly penetrant alleles in agerelated macular degeneration. Cold Spring Harbor Perspect Med. doi:10.1101/cshperspect.a017202 Di Giovanni S, Knoblach SM, Brandoli C, Aden SA, Hoffman EP, Faden AI (2003) Gene profiling in spinal cord injury shows role of cell cycle in neuronal death. Ann Neurol 53(4):454–468. doi:10.1002/ana.10472 Donovan M, Doonan F, Cotter TG (2011) Differential roles of ERK1/ 2 and JNK in retinal development and degeneration. J Neurochem 116(1):33–42. doi:10.1111/j.1471-4159.2010.07056.x Fan F-T, Shen C-S, Tao L, Tian C, Liu Z-G, Zhu Z-J, Liu Y-P, Pei C-S, Wu H-Y, Zhang L, Wang A-Y, Zheng S-Z, Huang S-L, Lu Y (2014) PKM2 regulates hepatocellular carcinoma cell epithelial-mesenchymal transition and migration upon EGFR activation. Asian Pac J Cancer Prev 15(5):1961–1970. doi:10.7314/ apjcp.2014.15.5.1961 Fei HR, Chen HL, Xiao T, Chen G, Wang FZ (2012) Caudatin induces cell cycle arrest and caspase-dependent apoptosis in HepG2 cell. Mol Biol Rep 39(1):131–138. doi:10.1007/s11033011-0721-6 Ferguson EC, Rathmell JC (2008) New roles for pyruvate kinase M2: working out the Warburg effect. Trends Biochem Sci 33(8):359–362. doi:10.1016/j.tibs.2008.05.006 Fletcher AE, Bentham GC, Agnew M, Young IS, Augood C, Chakravarthy U, de Jong PT, Rahu M, Seland J, Soubrane G, Tomazzoli L, Topouzis F, Vingerling JR, Vioque J (2008) Sunlight exposure, antioxidants, and age-related macular degeneration. Arch Ophthalmol 126(10):1396–1403. doi:10.1001/ archopht.126.10.1396 Galan A, Dergham P, Escoll P, de-la-Hera A, D’Onofrio PM, Magharious MM, Koeberle PD, Frade JM, Saragovi HU (2014) Neuronal injury external to the retina rapidly activates retinal glia, followed by elevation of markers for cell cycle re-entry and death in retinal ganglion cells. PLoS One 9(7):e101349. doi:10. 1371/journal.pone.0101349
Cell Mol Neurobiol (2015) 35:1175–1186 Gao X, Wang H, Yang JJ, Liu X, Liu ZR (2012) Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Mol Cell 45(5):598–609. doi:10.1016/j.molcel.2012.01.001 Gerard C, Goldbeter A (2009) Temporal self-organization of the cyclin/Cdk network driving the mammalian cell cycle. Proc Natl Acad Sci USA 106(51):21643–21648. doi:10.1073/pnas. 0903827106 Greene LA, Liu DX, Troy CM, Biswas SC (2007) Cell cycle molecules define a pathway required for neuron death in development and disease. Biochim Biophys Acta 1772(4): 392–401. doi:10.1016/j.bbadis.2006.12.003 Gupta V, Bamezai RN (2010) Human pyruvate kinase M2: a multifunctional protein. Protein Sci 19(11):2031–2044. doi:10. 1002/pro.505 Hoglinger GU, Breunig JJ, Depboylu C, Rouaux C, Michel PP, Alvarez-Fischer D, Boutillier AL, Degregori J, Oertel WH, Rakic P, Hirsch EC, Hunot S (2007) The pRb/E2F cell-cycle pathway mediates cell death in Parkinson’s disease. Proc Natl Acad Sci USA 104(9):3585–3590. doi:10.1073/pnas.0611671104 Jacks T, Fazeli A, Schmitt EM, Bronson RT, Goodell MA, Weinberg RA (1992) Effects of an Rb mutation in the mouse. Nature 359(6393):295–300. doi:10.1038/359295a0 Jiang Y, Li X, Yang W, Hawke DH, Zheng Y, Xia Y, Aldape K, Wei C, Guo F, Chen Y, Lu Z (2014) PKM2 regulates chromosome segregation and mitosis progression of tumor cells. Mol Cell 53(1):75–87. doi:10.1016/j.molcel.2013.11.001 Kranenburg O, van der Eb AJ, Zantema A (1996) Cyclin D1 is an essential mediator of apoptotic neuronal cell death. EMBO J 15(1):46–54 Lannuzel A, Barnier JV, Hery C, Huynh VT, Guibert B, Gray F, Vincent JD, Tardieu M (1997) Human immunodeficiency virus type 1 and its coat protein gp120 induce apoptosis and activate JNK and ERK mitogen-activated protein kinases in human neurons. Ann Neurol 42(6):847–856. doi:10.1002/ana.4104 20605 Lee EY, Hu N, Yuan SS, Cox LA, Bradley A, Lee WH, Herrup K (1994) Dual roles of the retinoblastoma protein in cell cycle regulation and neuron differentiation. Genes Dev 8(17): 2008–2021 Lee J, Kim HK, Han YM, Kim J (2008) Pyruvate kinase isozyme type M2 (PKM2) interacts and cooperates with Oct-4 in regulating transcription. Int J Biochem Cell Biol 40(5):1043–1054. doi:10. 1016/j.biocel.2007.11.009 Luo W, Semenza GL (2011) Pyruvate kinase M2 regulates glucose metabolism by functioning as a coactivator for hypoxiainducible factor 1 in cancer cells. Oncotarget 2(7):551–556 Luo W, Hu H, Chang R, Zhong J, Knabel M, O’Meally R, Cole RN, Pandey A, Semenza GL (2011) Pyruvate kinase M2 is a PHD3stimulated coactivator for hypoxia-inducible factor 1. Cell 145(5):732–744. doi:10.1016/j.cell.2011.03.054 Malik B, Currais A, Andres A, Towlson C, Pitsi D, Nunes A, Niblock M, Cooper J, Hortobagyi T, Soriano S (2008) Loss of neuronal cell cycle control as a mechanism of neurodegeneration in the presenilin-1 Alzheimer’s disease brain. Cell Cycle 7(5):637–646 Malumbres M, Barbacid M (2009) Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer 9(3):153–166. doi:10.1038/ nrc2602 Marc RE, Jones BW, Watt CB, Vazquez-Chona F, Vaughan DK, Organisciak DT (2008) Extreme retinal remodeling triggered by light damage: implications for age related macular degeneration. Mol Vis 14:782–806 Mazurek S, Boschek CB, Hugo F, Eigenbrodt E (2005) Pyruvate kinase type M2 and its role in tumor growth and spreading. Semin Cancer Biol 15(4):300–308. doi:10.1016/j.semcancer. 2005.04.009
1185 Morohoshi K, Ohbayashi M, Patel N, Chong V, Bird AC, Ono SJ (2012) Identification of anti-retinal antibodies in patients with age-related macular degeneration. Exp Mol Pathol 93(2):193–199. doi:10.1016/j.yexmp.2012.03.007 Mukherjee J, Phillips JJ, Zheng S, Wiencke J, Ronen SM, Pieper RO (2013) Pyruvate kinase M2 expression, but not pyruvate kinase activity, is up-regulated in a grade-specific manner in human glioma. PLoS One 8(2):e57610. doi:10.1371/journal.pone. 0057610 Osuga H, Osuga S, Wang F, Fetni R, Hogan MJ, Slack RS, Hakim AM, Ikeda JE, Park DS (2000) Cyclin-dependent kinases as a therapeutic target for stroke. Proc Natl Acad Sci USA 97(18):10254–10259. doi:10.1073/pnas.170144197 Pang W, Leng X, Lu H, Yang H, Song N, Tan L, Jiang Y, Guo C (2013) Depletion of intracellular zinc induces apoptosis of cultured hippocampal neurons through suppression of ERK signaling pathway and activation of caspase-3. Neurosci Lett 552:140–145. doi:10.1016/j.neulet.2013.07.057 Pasantes-Morales H, Ademe RM, Quesada O (1981) Protective effect of taurine on the light-induced disruption of isolated frog rod outer segments. J Neurosci Res 6(3):337–348. doi:10.1002/jnr. 490060309 Randolph SA (2014) Age-related macular degeneration. Workplace Health Saf 62(8):352. doi:10.3928/21650799-20140708-06 Roca A, Shin KJ, Liu X, Simon MI, Chen J (2004) Comparative analysis of transcriptional profiles between two apoptotic pathways of light-induced retinal degeneration. Neuroscience 129(3):779–790. doi:10.1016/j.neuroscience.2004.08.021 Shu Q, Xu Y, Zhuang H, Fan J, Sun Z, Zhang M, Xu G (2014) Ras homolog enriched in the brain is linked to retinal ganglion cell apoptosis after light injury in rats. J Mol Neurosci 54(2): 243–251. doi:10.1007/s12031-014-0281-z Stetak A, Veress R, Ovadi J, Csermely P, Keri G, Ullrich A (2007) Nuclear translocation of the tumor marker pyruvate kinase M2 induces programmed cell death. Cancer Res 67(4):1602–1608. doi:10.1158/0008-5472.CAN-06-2870 Sui GY, Liu GC, Liu GY, Gao YY, Deng Y, Wang WY, Tong SH, Wang L (2013) Is sunlight exposure a risk factor for age-related macular degeneration? A systematic review and meta-analysis. Br J Ophthalmol 97(4):389–394. doi:10.1136/bjophthalmol2012-302281 Sumrejkanchanakij P, Tamamori-Adachi M, Matsunaga Y, Eto K, Ikeda MA (2003) Role of cyclin D1 cytoplasmic sequestration in the survival of postmitotic neurons. Oncogene 22(54): 8723–8730. doi:10.1038/sj.onc.1206870 Tamada M, Suematsu M, Saya H (2012) Pyruvate kinase M2: multiple faces for conferring benefits on cancer cells. Clin Cancer Res 18(20):5554–5561. doi:10.1158/1078-0432.CCR-120859 Tsutsumi H, Tani K, Fujii H, Miwa S (1988) Expression of L- and M-type pyruvate kinase in human tissues. Genomics 2(1):86–89 Wang HJ, Hsieh YJ, Cheng WC, Lin CP, Lin YS, Yang SF, Chen CC, Izumiya Y, Yu JS, Kung HJ, Wang WC (2014) JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1alpha-mediated glucose metabolism. Proc Natl Acad Sci USA 111(1): 279–284. doi:10.1073/pnas.1311249111 Wong N, De Melo J, Tang D (2013) PKM2, a central point of regulation in cancer metabolism. Int J Cell Biol 2013:242513. doi:10.1155/2013/242513 Xu Y, Yu S, Shu Q, Yang L, Yang C, Wang J, Xu F, Ji M, Liang X (2014) Upregulation of CREM-1 relates to retinal ganglion cells apoptosis after light-induced damage in vivo. J Mol Neurosci 52(3):331–338. doi:10.1007/s12031-013-0153-y Yang W, Lu Z (2013) Nuclear PKM2 regulates the Warburg effect. Cell Cycle 12(19):3154–3158. doi:10.4161/cc.26182
123
1186 Yang LP, Wu LM, Guo XJ, Li Y, Tso MO (2008) Endoplasmic reticulum stress is activated in light-induced retinal degeneration. J Neurosci Res 86(4):910–919. doi:10.1002/jnr.21535 Yang W, Xia Y, Ji H, Zheng Y, Liang J, Huang W, Gao X, Aldape K, Lu Z (2011) Nuclear PKM2 regulates beta-catenin transactivation upon EGFR activation. Nature 480(7375):118–122. doi:10. 1038/nature10598 Yang W, Zheng Y, Xia Y, Ji H, Chen X, Guo F, Lyssiotis CA, Aldape K, Cantley LC, Lu Z (2012) ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect. Nat Cell Biol 14(12):1295–1304. doi:10.1038/ncb2629 Yang K, Cao F, Sheikh AM, Malik M, Wen G, Wei H, Ted Brown W, Li X (2013) Up-regulation of Ras/Raf/ERK1/2 signaling impairs
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Cell Mol Neurobiol (2015) 35:1175–1186 cultured neuronal cell migration, neurogenesis, synapse formation, and dendritic spine development. Brain Struct Funct 218(3):669–682. doi:10.1007/s00429-012-0420-7 Zhang X, He C, He C, Chen B, Liu Y, Kong M, Wang C, Lin L, Dong Y, Sheng H (2013) Nuclear PKM2 expression predicts poor prognosis in patients with esophageal squamous cell carcinoma. Pathol Res Pract 209(8):510–515. doi:10.1016/j.prp.2013.06.005 Zhou L, Del Villar K, Dong Z, Miller CA (2004) Neurogenesis response to hypoxia-induced cell death: map kinase signal transduction mechanisms. Brain Res 1021(1):8–19. doi:10.1016/ j.brainres.2004.05.115
ORIGINAL RESEARCH
Up-Regulation of PKM2 Relates to Retinal Ganglion Cell Apoptosis After Light-Induced Retinal Damage in Adult Rats Xiaowei Yang1,3 • Hui Chen1,3 • Manhui Zhu1,3 • Rongrong Zhu1 • Bai Qin1 Hongda Fang1 • Ming Dai1,3 • Aimin Sang1,3 • Xiaojuan Liu2,3
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Received: 7 March 2015 / Accepted: 13 May 2015 / Published online: 20 May 2015 Ó Springer Science+Business Media New York 2015
Abstract Pyruvate kinase isozyme type M2 (PKM2), a key glycolytic enzyme, which is involved in ATP generation and pyruvate production, participates in tumor metabolism, growth, and other multiple cellular processes. However, one attractive biological function of PKM2 is that it translocates to the nucleus and induces cell apoptosis. Recently, increased PKM2 has been found in age-related macular degeneration (AMD), but little is known regarding its function in the AMD pathophysiology. To investigate whether PKM2 participated in retinal degeneration, we performed a lightinduced retinal damage model in adult rats. Western blot and immunohistochemistry analysis showed a significant upregulation of PKM2 in retinal ganglion cells (RGCs) layer (GCL) after light exposure. Immunofluorescent labeling indicated that PKM2 located mainly in RGCs. Co-localization of PKM2 and active caspase-3 as well as TUNEL in RGCs suggested that PKM2 might participate in RGC
Xiaowei Yang, Hui Chen, Aimin Sang, and Xiaojuan Liu have contributed equally to this work. & Aimin Sang [email protected] & Xiaojuan Liu [email protected] 1
Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, People’s Republic of China
2
Department of Pathogen Biology, Medical College, Nantong University, Nantong 226001, Jiangsu, People’s Republic of China
3
Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong 226001, Jiangsu, People’s Republic of China
apoptosis. In addition, the expression patterns of cyclin D1 and phosphorylated extracellular signal-regulated kinase (p-ERK) were parallel with that of PKM2. Furthermore, PKM2, cyclin D1, and active caspase-3 protein expression decreased by intravitreal injection of U0126, a highly selective inhibitor of MAPK/ERK kinase. Collectively, we hypothesized that PKM2 might participate in RGC apoptosis after light-induced retinal damage medicated by p-ERK through cycle re-entry mechanism. Keywords PKM2 Light-induced retinal damage RGCs Apoptosis Cyclin D1 ERK Rat
Introduction Age-related macular degeneration (AMD) is a complex disease caused by a combination of genetic and environmental factors. It is a common and painless eye condition as well as a leading cause of vision loss for people older than 50 years, but the molecule mechanisms of AMD are not fully known (den Hollander and de Jong 2014; Randolph 2014). Previous studies revealed that light exposure might be one of the contributing conditions in the pathogenesis of AMD (Sui et al. 2013; Fletcher et al. 2008). Light-induced retinal damage, a well-established in vivo model for retinal degeneration, covers most of the essential characteristics of human AMD and has been widely used to investigate the mechanisms of neuroretinal dysfunction (Marc et al. 2008). Photoreceptor apoptosis and retinal ganglion cell (RGC) death are common features in retinal degenerative diseases such as retinitis pigmentosa and AMD (Marc et al. 2008; Xu et al. 2014; Shu et al. 2014). Numerous studies have emphatically explored photoreceptor apoptosis following light exposure (Pasantes-Morales et al. 1981; Roca et al. 2004).
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However, the molecular mechanism of light-induced RGC damage remains unclear. Pyruvate kinase (PK) including four isoenzymes designated L, R, M1, and M2 mediates the final rate-limiting step of glycolysis by catalyzing the formation of pyruvate and ATP from phosphoenolpyruvate and ADP (Tsutsumi et al. 1988; Mukherjee et al. 2013). Other than a key role in glycolysis, PKM2 is expressed in cells with a high rate of nucleic acid synthesis such as normal proliferating cells, embryonic cells, and especially tumor cells (Tamada et al. 2012; Mazurek et al. 2005; Gupta and Bamezai 2010). The nuclear translocation of PKM2 induced by oxidative stress with H2O2 or UV light is linked with cell apoptosis (Stetak et al. 2007). Oxidative stress and damage from light are strongly involved in the pathogenesis of AMD (Cano et al. 2010). In addition, PKM2 was targeted by both dry and wet AMD patients by two-dimensional electrophoresis (Morohoshi et al. 2012). However, the expression of PKM2 and its role in AMD has not been elucidated. The cell cycle-related molecules play key roles in neuron death associated with tissue development and diseases (Greene et al. 2007). The elevation of markers for cell cycle re-entry was observed in the injured RGCs (Galan et al. 2014; Shu et al. 2014). Cyclin D1 is a key regulator of the G1/S transition as it activates CDK4, which phosphorylates Rb protein. As a result, Rb protein dissociates from E2F1, which in turn facilitates the transcription of genes needed for S phase progression. The increased expression of cyclin D1 in response to various neurotoxic agents is implicated in neuronal apoptosis (Kranenburg et al. 1996; Sumrejkanchanakij et al. 2003; Malik et al. 2008). Interestingly, PKM2 regulates G1/S transition by controlling cyclin D1 expression (Jiang et al. 2014). In this study, we firstly confirmed the expression and distribution of PKM2 in RGCs after light exposure and revealed its correlation with RGC apoptosis. Intravitreal injection of ERK inhibitor U0126 could reduce the PKM2, cyclin D1, and active caspase-3 expression. Our work may provide a useful clue to the pathogenesis of RGC apoptosis and may serve as a potential new therapeutic target for neurodegenerative diseases.
Materials and Methods Animals and the Light-Induced Retinal Damage Model Adult Sprague–Dawley male rats (Department of Animal Center, Nantong University, Nantong, China) were used in our study. Rats weighing 180–200 g were kept on a 12h:12h light–dark schedule, given food and water in a pathogen-free area. All experimental procedures were
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performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Before light exposure, rats were dark adapted for 1 day. After pupil dilation with compound tropicamide (Santen Pharmaceutical, Osaka, Japan), dark-adapted rats were placed in cages and exposed to white light (8000 lx) for 3 h, beginning at 9 AM during light exposure, room temperature (RT) was kept at 24 °C. The animals had free access to food and water. After light exposure, all rats were returned to darkness and randomly divided into six groups (6 h, 12 h, 1 day, 3 days, 5 days, 7 days). For Western blot, each group contained 18 rats and other 18 rats without light exposure belonged to the control group. In the control and light exposure 3-day 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 (350 mg/kg, i.p.) and sacrificed at different time points after light exposure. Then, the cornea, lens, and vitreous were removed; retina was extruded gently from the eye cup. Twelve retinas derived from six rats were put on one pool for further protein extraction and Western blot analysis. Three samples were studied at each time point. The extract samples (2.0 mg/ml, 10 ll) 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 (BioRad, Hercules, CA, USA). The membranes were blocked with 5 % nonfat milk at RT for 2 h, followed by incubation with primary antibodies against PKM2 (rabbit, 1:1000; Santa Cruz), active caspase-3 (mouse, 1:1000; Cell Signaling), cyclin D1 (mouse, 1:500; Santa Cruz), CDK4 (rabbit, 1:500; Santa Cruz), p-ERK1/2 (rabbit, 1:1000; Cell Signaling), ERK1/2 (rabbit, 1:1000; Cell Signaling), GAPDH (mouse, 1:1000; Santa Cruz), and b-actin (mouse, 1:1000; Sigma) at 4 °C overnight. Finally, 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 an LAS 3000 image analyzer (FUJIFILM, 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. Sections and Immunohistochemistry At defined survival time, normal and injured rats were terminally anesthetized and perfused through the ascending
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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-lm frozen sections were prepared. The superior hemisphere along the vertical meridian was chosen in this experiment. After the sections had been prepared, they were kept in an oven at 37 °C for 60 min and rinsed twice with 0.01 M PBS. The sections were blocked by confining liquid consisting of 10 % normal donkey serum, 1 % BSA, 0.3 % Triton X-100, 0.15 % Tween-20 for 2 h at RT, and incubated with PKM2 antibody (rabbit, 1:500; Santa Cruz) at RT for 2 h, followed by incubation with the primary and the secondary antibodies for 30 min at 37 °C, and then color reaction with a liquid mixture (0.02 % DAB, 3 % H2O2 and 0.1 % PBS). Finally, the sections were dehydrated, cleared, coverslipped, and examined under a light microscope (Leica, Germany). All the acquired images were used for quantification. Cell counting and assessment were performed in a double-blind manner using software Image J.
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fields were randomly chosen for every specimen. The number of TUNEL-positive cells was counted to obtain the ratio of the number of TUNEL-positive cells to the total number of cells. 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 1 min to reduce the reflux. The rats with intravitreal injection of 0.8 mM ERK inhibitor U0126 (Santa Cruz, Dallas, TX, sc-222395), which was dissolved in 5 % dimethyl sulfoxide (DMSO) with PBS, were divided into seven subgroups: control, 6 h, 12 h, 1 day, 3 days, 5 days, and 7 days. The animals were sacrificed after the start of light exposure, and the eyes were then enucleated for Western blot analysis. Statistical Analysis
Double Immunofluorescence Staining The cryosections were blocked with 10 % normal goat serum containing 3 % (w/v) BSA, 0.1 % Triton X-100, and 0.05 % Tween 20 at RT in order to avoid unspecific staining. Sections were incubated with PKM2 antibody (rabbit, 1:200; Santa Cruz). The co-incubated antibodies were antibodies for NeuN (mouse, 1:200; Cell Signaling), active caspase-3 (mouse, 1:200; Cell Signaling), and cyclin D1 (mouse, 1:500; Santa Cruz) separately for 10 h at 4 °C. On the following day, a mixture of FITC-, CY3-, and AMCA-conjugated secondary antibodies and Hoechst staining (Pierce Biotechnology, USA) were added in dark and incubated for 2 h at 4 °C. The stained sections were examined with a Leica fluorescence microscope (Germany). TUNEL Staining TUNEL staining was performed on the sections with an apoptosis detection kit (ApopTag Fluorescein in Situ Kit; Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Sections were rinsed with PBS and treated with 1 % Triton X-100 in PBS for 2 min on ice. Sections were rinsed in PBS again and incubated for 60 min at 37 °C with 50 ll of TUNEL reaction mixture and Label solution, respectively. After washing with PBS, the slides were analyzed with fluorescence microscopy (Leica, DM 5000B; Leica CTR 5000; Germany). Nine
All data were analyzed with IBM SPSS Statistics 19 statistical software. All values are expressed as mean ± SEM and were analyzed by one-way variance (ANOVA) followed by Tukey’s post hoc multiple comparison tests. P \ 0.05 was considered statistically significant. Experiments were performed at least in triplicate per condition.
Results PKM2 Expression in the Rat Retina After Light Exposure Western blot was performed to investigate the temporal patterns of PKM2 expression at different survival times. PKM2 expression was low in the normal retina. After lightinduced retinal damage, PKM2 expression gradually increased from 6 h and reached its peak at 3 days. After that, PKM2 expression tended to decline (Fig. 1a, b). These findings indicated that PKM2 might play an important role in the pathogenesis of light-induced retinal damage. Distribution of PKM2 in the Rat Retina After Light Exposure To determine the spatial distribution and the temporal profile of PKM2 in retina after light exposure, we then
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Fig. 1 PKM2 expression in the rat retina after light exposure. a Sample immunoblots probed for PKM2 and b-actin were shown above. b The bar chart demonstrated the ratio of PKM2 to b-actin at each time point. n = 3, *P \ 0.05; significantly different from the normal group
performed immunohistochemistry staining on transverse cryosections of rat retinal tissues. In the control group, PKM2-positive cells were observed in INL. At 3 days after light exposure, a prominent increase of PKM2 expression was detected in the GCL (Fig. 2b) compared with that of the normal retina based on quantitative analysis (Fig. 2e). Although PKM2-positive cells were also observed in the INL after light exposure, there is no statistical significance compared to the control group (Fig. 2a, b, d). In the negative control sections preincubated with PBS, PKM2 expression was undetectable (Fig. 2c). The data further confirmed that PKM2 expression increased after light exposure, hinting its function during light-induced retinal damage. To determine that PKM2-positive cells in the GCL were RGCs, we performed immunofluorescent double staining. In the normal retina, PKM2 was detected in the INL, which is same as the result of immunohistochemistry staining (Fig. 2f). After light exposure, increased PKM2positive cells were observed in the GCL (Fig. 2g) co-localizing with NeuN (a marker of RGCs) (Fig. 2h) in the merged image (Fig. 2j). The results suggested that PKM2 might be associated with RGC damage after light exposure. PKM2 was Relevant to RGC Apoptosis in Rat Retina After Light Exposure Previous research demonstrated that PKM2 translocated to the nucleus and participated in different apoptotic agents
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(Stetak et al. 2007). Moreover, clinical and experimental studies proved that increased PKM2 was involved in AMD and anti-PKM2 IgG served as a biomarker for diagnosis and prognosis of AMD (Morohoshi et al. 2012). Thus we hypothesize that PKM2 may be associated with retinal cell apoptosis after retinal damage. Caspase-3 activation is a prominent trait for neuronal apoptosis after light exposure. Therefore, to further detect the relationship between PKM2 and neuronal apoptosis in the retina, we performed Western blot to examine the expression profiles of active caspase-3. After light exposure, active caspase-3 was up-regulated from 12 h and culminated at 1 day (Fig. 3a, b). Active caspase-3 expression was positively correlated with PKM2 expression in a time-dependent manner (Fig. 3c). Additionally, immunofluorescent labeling showed that active caspase-3 expression was weak in the normal retina (Fig. 3d). The co-localization of active caspase-3 and PKM2 was detected in GCL at 3 days after light exposure (Fig. 3e–h). In addition, TUNEL-positive cells as well as PKM2positive cells were barely detected in the normal RGCs (Fig. 4a–d). While at 3 days after light exposure, a dramatically increased number of TUNEL-positive RGCs with PKM2 expression were observed (Fig. 4e–h), suggesting RGCs began to catabolize after light exposure. Besides, semi-quantitative analysis showed marked increase of TUNEL-positive and TUNEL–PKM2 double-positive RGCs in the retina at 3 days after light exposure (Fig. 4i, j). In a word, these results showed that PKM2 might play an important part in RGC apoptosis after light exposure. PKM2 was Co-Labeled with Cell Cycle Markers and May Be Regulated by p-ERK in Retina Besides its metabolic function, PKM2 has a non-metabolic function in the direct control of cell cycle progression by activating b-catenin and inducing the expression of cyclin D1, a downstream gene of b-catenin (Yang et al. 2011). In addition, evidences define a molecular pathway that includes cell cycle-related molecules and plays a required role in neuron death during normal development as well as in diseases and trauma (Greene et al. 2007). Previous study confirmed that cyclin D1 was correlated with apoptotic markers, such as active caspase-3, caspase-9, and poly (ADP-ribose) polymerase (PARP) (Fei et al. 2012). We assumed that PKM2 might participate in RGC apoptosis through its downstream gene cyclin D1. Therefore, we performed Western blot to examine the protein level of cyclin D1 and its partner CDK4, exploring the apoptosis status during light-induced retina damage. The results suggested that cyclin D1 expression and CDK4 expression were weak in the normal retina, while it was up-regulated parallel to the PKM2 and active caspase-3 expression after
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Fig. 2 PKM2 expression and distribution in the rat retina. a Normal retina. b 3 days after light exposure. c Negative control sections. d Bar chart illustrated the percentage of PKM2-positive cells in the INL. e Bar chart illustrated the percentage of PKM2-positive cells in GCL. *P \ 0.05, significantly different from the normal retina. Error bars represent SEM. f PKM2 expression was detected in INL in the
normal retina. g PKM2 expression was detected in both the GCL and INL at 3 days after light exposure. h NeuN was located in the GCL. i Hoechst staining in the retina. j The merged image showed the colocalization of PKM2 and NeuN in the GCL at 3 days after light exposure
light exposure (Figs. 1a, 3a, 5a). Cyclin D1 expression was positively correlated with PKM2 expression in a time-dependent manner (Fig. 5c). Additionally, the normal retina showed weak immunoreactivity for PKM2 and cyclin D1 (Fig. 5d). At 3 days after light exposure, PKM2 and cyclin D1 expression notably increased in the GCL (Fig. 5e, f). PKM2 (Fig. 5f) co-localized with cyclin D1 (Fig. 5e) in the merged image in the RGCs (Fig. 5h). Data above
manifested that PKM2 may exert its pro-apoptotic function via modulating cell cycle signaling. PKM2 can activate cell intrinsic apoptotic machinery to induce cell death through nuclear translocation (Stetak et al. 2007). Additionally, ERK-dependent phosphorylation and nuclear translocation of PKM2 are required for the autoregulation of PKM2 expression and PKM2-dependent gene expression (Yang et al. 2012). To explore whether
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Fig. 3 PKM2 involved in RGC apoptosis after light exposure. a The active caspase-3 protein level in the rat retina after light exposure. b Quantification for the staining intensity of caspase-3 at each time point. *P \ 0.05; significantly different from the normal group. c The correlation between active caspase-3 and PKM2 expression after light exposure. d–h Double labeling of PKM2 (red) and active caspase-3
(green) in the retina after light exposure. c The active caspase-3 immunoreactivity in the normal retina. d Three days after light exposure, active caspase-3 expression was significantly up-regulated. The active caspase-3 (green, e) overlapped with PKM2 (red, f) and Hoechst staining (blue, g) in the merged image (yellow, h) (Color figure online)
PKM2 was associated with p-ERK, Western blot was performed to examine the expression profile of p-ERK and t-ERK in the retina after light exposure. As shown in Fig. 6a, in the normal retina, p-ERK expression was feeble but still clearly detectable. After light exposure, p-ERK expression gradually increased (Fig. 6a, b), reached its peak at 3 days after light exposure, and then it gradually dropped. The t-ERK expression had no obvious change in both the normal and the damaged retina (Fig. 6a). In addition, p-ERK expression was positively correlated with PKM2 expression in a time-dependent manner (Fig. 6c). We supposed that the increase of p-ERK expression was associated with RGCs and performed the immunostaining of p-ERK. We found that p-ERK was weakly stained in the INL in the normal retina, but not in the GCL and ONL (Fig. 6d). After light exposure, the increase of p-ERK
expression was found in the GCL obviously (Fig. 6d). To determine whether there was an association between ERK kinase activity and cyclin D1 accumulation in the retina, ERK activation was inhibited with intravitreal injection of the highly selective ERK inhibitor U0126. We found p-ERK, cyclin D1, and PKM2 had no obvious change with U0126 intravitreal injection after light exposure (Fig. 6e). Besides that, as a comparison, we also measured PKM2, total caspase-3, and active caspase-3 expression in the normal and light-injured retina at 3 days with U0126 intravitreal injection. The light-injured retina treated with PBS showed significance high level of active caspase-3 and PKM2 compared to the light-injured retina treated with U0126 (Fig. 6f–h). The data indicated that PKM2 might induce RGC apoptosis through cell cycle pathway via phosphorylated ERK regulation.
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Fig. 4 Double staining with PKM2 and TUNEL. a–d The TUNEL and PKM2 weak immunoreactivity were detected in the RGCs in the normal retina. TUNEL (green, c) co-localized with PKM2 (red, b) and Hoechst staining (blue, a) in the merged image (yellow, d). e–h A clearly increased level of TUNEL and PKM2 immunoreactivity was
detected in the RGCs at 3 days after light exposure. TUNEL (green, g) co-localized with PKM2 (red, f) and Hoechst staining (blue, e) in the merged image (yellow, h). A semi-quantitative analysis of TUNEL (i) and PKM2/TUNEL (j) immunoreactive RGCs. *P \ 0.05, significant difference from the normal group (Color figure online)
Discussion
normal retina as well as in light-injured retina, suggesting that PKM2 may have no intimate relationship with photoreceptors. According to the previous study, a welldocumented correlation between RPE and PKM2 in the aged mouse has been identified (Morohoshi et al. 2012). In the present study, we focused on the role of PKM2 in the neural retina. However, it needs further study to reveal the molecular mechanism of PKM2 in RPE during the destruction of retinal structure after light exposure. As a key rate-limiting enzyme in the glycolytic regulating the Warburg effect, PKM2 is necessary for tumor growth (Ferguson and Rathmell 2008). Intensive studies demonstrated that PKM2 was up-regulated in a broad range of cancers (Fan et al. 2014; Wong et al. 2013). Recent studies indicated that nuclear PKM2 could activate gene transcription and cell proliferation closely related to
The present study constructed light-induced retinal damage model in adult rats and sought to evaluate whether PKM2 was involved in retinal damage. It was demonstrated that PKM2 expression was significantly up-regulated in RGCs after light-induced retinal damage. Interestingly, although PKM2 was observed in the INL in both the normal and light-injured retina, there is no statistical difference between the normal and post-damage 3-day groups. This phenomenon indicated that PKM2 may play a role in retinal normal function in the INL cells, such as bipolar nerve cells, horizontal cells, amacrines, and mu¨ller cells. The possible relevance between PKM2 and INL cells may need further investigation in the future. The ONL, photoreceptor layer, has no obvious PKM2-positive cells in the
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Fig. 5 Cyclin D1 and CDK4 were up-regulated after light exposure. a Cyclin D1 and CDK4 expression after light exposure. b The bar chart showed the ratio of cyclin D1 and CDK4 to GAPDH at each time point. n = 3, *, **P \ 0.05, significant difference from the normal group. c The correlation between cyclin D1 and PKM2
expression after light exposure. d Double labeling of PKM2 and cyclin D1 in the normal retina. After light exposure, cyclin D1 (red, e) overlapped with PKM2 (green, f) and Hoechst staining (blue, g) in the merged image (yellow, h) (Color figure online)
survival of cancer patients (Gao et al. 2012; Lee et al. 2008; Luo et al. 2011; Luo and Semenza 2011; Wang et al. 2014; Yang and Lu 2013; Zhang et al. 2013). PKM2 translocates to the nucleus to perform its non-metabolic activities, potentially functioning as a transcriptional coactivator. During tumor progression, growth signals convert PKM2 from active form to inactive form; therefore, the PK activity of PKM2 is changed and plays a ‘‘nonmetabolic’’ role. PKM2 stimulates the transcription of various genes by interacting with and phosphorylatingspecific nuclear proteins (Chen et al. 2014). Previous research identified that epidermal growth factor receptor (EGFR) activation induced b-catenin transactivation
through interacting with PKM2, which has a critical role in transcription of cyclin D1 (Yang et al. 2011). In addition to the function in tumorgenesis, PKM2 also plays a role in age-associated neurodegenerative diseases such as AMD (Morohoshi et al. 2012) and Alzheimer’s disease (AD) (Butterfield et al. 2006; Chen and Zhong 2013). But the detailed molecular mechanism of PKM2 in the neuronal apoptosis needs further exploration. The cell cycle is a delicately manipulated process essential for cell development, differentiation, proliferation, and death. Inappropriate activation of cell cycle regulators is implicated in the pathological process of a wide range of central nervous system (CNS) diseases, including acute
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Fig. 6 PKM2 may regulate RGC apoptosis by means of modulating cell cycle protein expression through phosphorylated ERK regulation. a The expression of p-ERK and t-ERK in the rat retina after light exposure. b The bar chart demonstrated the ratio of p-ERK to t-ERK at each time point. n = 3, *P \ 0.05; significantly different from the normal group. c The correlation between p-ERK and PKM2 expression after light exposure. d Double labeling of PKM2 (green) and p-ERK (red) in the normal retina and the light-damaged retina at 3 days. e After
U0126 intravitreal injection, sample immunoblots probed for p-ERK, t-ERK, cyclin D1, PKM2, and GAPDH were shown. f Sample immunoblots probed for PKM2, total caspase-3, and active caspase-3 in the rat retina with intravitreal injection of U0126 or PBS. g The bar chart demonstrated the ratio of active caspase-3 relative to GAPDH expression. h The bar chart demonstrated the ratio of PKM2 relative to GAPDH expression. n = 3, *P \ 0.05, significant difference from PBS intravitreal injection group (Color figure online)
damage and chronic neurodegenerative disorders (Hoglinger et al. 2007; Osuga et al. 2000; Di Giovanni et al. 2003). In terminally differentiated neurons, aberrant re-entry into the cell cycle triggers neuronal death instead of proliferation, which may be a common pathway shared by some acquired neurodegenerative disorders, even though multiple pathways of the cell cycle machinery are involved in distinct neuronal
demise in specific pathological circumstances (Clarke et al. 1992; Jacks et al. 1992; Lee et al. 1994). The cell cycle response is often mediated by the action of cyclin-dependent kinase complexes (Malumbres and Barbacid 2009). Cyclin D1 is a key cell cycle regulator and a direct target. Excessive cyclin D1 is associated with activity repression of E2F by initiating Rb phosphorylation and thus facilitates cell cycle
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progression (Berthet et al. 2006; Gerard and Goldbeter 2009). Cyclin D1 has been reported to appear in abnormal neurons in AD and RGCs after retinal damage (Bonda et al. 2009; Shu et al. 2014). Consistent with previous studies, the expression profiles of active caspase-3, Cyclin D1, and CDK4 were upregulated in the retina after light exposure, which was parallel with PKM2 expression. Immunofluorescence assay showed that the up-regulation of cyclin D1 was found in PKM2-positive RGCs, and was correlated with RGC apoptosis presented by active caspase-3 and TUNEL labeling. These results suggest that PKM2 may take part in RGC apoptosis via cell cycle re-entry. Nuclear PKM2 acts as a co-activator of b-catenin to induce downstream genes expression, such as c-Myc and cyclin D1. It is reported that PKM2 nuclear translocation is upon EGFR and ERK activation (Yang et al. 2011, 2012). ERK signaling pathway participates in multiple cell responses such as proliferation, migration, differentiation, and death (Cheng et al. 2013; Yang et al. 2013; Zhou et al. 2004). ERK signaling activation alongside JNK activation promoted apoptosis of cultured human CNS cells including neurons and microglia (Lannuzel et al. 1997). A further study found that caspase-3 activation is the downstream signal of ERK pathway (Pang et al. 2013). In the retina, ERK pathway played a key role in the regulation of apoptosis during retinal development (Donovan et al. 2011) and degeneration (Yang et al. 2008). These findings demonstrate a crucial role of the ERK signaling pathway in neuronal apoptosis, which may provide novel horizon in understanding of diseases such as AD and AMD, which contain abnormal neuron death. In conclusion, this study showed that up-regulation of PKM2 is associated with RGC apoptosis in light-induced retinal damage. The role of PKM2 in regulating RGC apoptosis may be executed by means of modulating cell cycle protein expression through ERK phosphorylation. These results provided a new view into the mechanism of neuronal apoptosis in the retina. Acknowledgments This work was supported in part by the National Natural Science Foundation of China (No. 81401365); a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Conflict of interest All authors declare that they have no conflict of interest.
References Berthet C, Klarmann KD, Hilton MB, Suh HC, Keller JR, Kiyokawa H, Kaldis P (2006) Combined loss of Cdk2 and Cdk4 results in embryonic lethality and Rb hypophosphorylation. Dev Cell 10(5):563–573. doi:10.1016/j.devcel.2006.03.004
123
Cell Mol Neurobiol (2015) 35:1175–1186 Bonda DJ, Evans TA, Santocanale C, Llosa JC, Vina J, Bajic V, Castellani RJ, Siedlak SL, Perry G, Smith MA, Lee HG (2009) Evidence for the progression through S-phase in the ectopic cell cycle re-entry of neurons in Alzheimer disease. Aging 1(4):382–388 Butterfield DA, Poon HF, St Clair D, Keller JN, Pierce WM, Klein JB, Markesbery WR (2006) Redox proteomics identification of oxidatively modified hippocampal proteins in mild cognitive impairment: insights into the development of Alzheimer’s disease. Neurobiol Dis 22(2):223–232. doi:10.1016/j.nbd.2005. 11.002 Cano M, Thimmalappula R, Fujihara M, Nagai N, Sporn M, Wang AL, Neufeld AH, Biswal S, Handa JT (2010) Cigarette smoking, oxidative stress, the anti-oxidant response through Nrf2 signaling, and age-related macular degeneration. Vis Res 50(7): 652–664. doi:10.1016/j.visres.2009.08.018 Chen Z, Zhong C (2013) Decoding Alzheimer’s disease from perturbed cerebral glucose metabolism: implications for diagnostic and therapeutic strategies. Prog Neurobiol 108:21–43. doi:10.1016/j.pneurobio.2013.06.004 Chen L, Shi Y, Liu S, Cao Y, Wang X, Tao Y (2014) PKM2: the thread linking energy metabolism reprogramming with epigenetics in cancer. Int J Mol Sci 15(7):11435–11445. doi:10.3390/ ijms150711435 Cheng P, Alberts I, Li X (2013) The role of ERK1/2 in the regulation of proliferation and differentiation of astrocytes in developing brain. Int J Dev Neurosci 31(8):783–789. doi:10.1016/j.ijdevneu. 2013.09.008 Clarke AR, Maandag ER, van Roon M, van der Lugt NM, van der Valk M, Hooper ML, Berns A, te Riele H (1992) Requirement for a functional Rb-1 gene in murine development. Nature 359(6393):328–330. doi:10.1038/359328a0 den Hollander AI, de Jong EK (2014) Highly penetrant alleles in agerelated macular degeneration. Cold Spring Harbor Perspect Med. doi:10.1101/cshperspect.a017202 Di Giovanni S, Knoblach SM, Brandoli C, Aden SA, Hoffman EP, Faden AI (2003) Gene profiling in spinal cord injury shows role of cell cycle in neuronal death. Ann Neurol 53(4):454–468. doi:10.1002/ana.10472 Donovan M, Doonan F, Cotter TG (2011) Differential roles of ERK1/ 2 and JNK in retinal development and degeneration. J Neurochem 116(1):33–42. doi:10.1111/j.1471-4159.2010.07056.x Fan F-T, Shen C-S, Tao L, Tian C, Liu Z-G, Zhu Z-J, Liu Y-P, Pei C-S, Wu H-Y, Zhang L, Wang A-Y, Zheng S-Z, Huang S-L, Lu Y (2014) PKM2 regulates hepatocellular carcinoma cell epithelial-mesenchymal transition and migration upon EGFR activation. Asian Pac J Cancer Prev 15(5):1961–1970. doi:10.7314/ apjcp.2014.15.5.1961 Fei HR, Chen HL, Xiao T, Chen G, Wang FZ (2012) Caudatin induces cell cycle arrest and caspase-dependent apoptosis in HepG2 cell. Mol Biol Rep 39(1):131–138. doi:10.1007/s11033011-0721-6 Ferguson EC, Rathmell JC (2008) New roles for pyruvate kinase M2: working out the Warburg effect. Trends Biochem Sci 33(8):359–362. doi:10.1016/j.tibs.2008.05.006 Fletcher AE, Bentham GC, Agnew M, Young IS, Augood C, Chakravarthy U, de Jong PT, Rahu M, Seland J, Soubrane G, Tomazzoli L, Topouzis F, Vingerling JR, Vioque J (2008) Sunlight exposure, antioxidants, and age-related macular degeneration. Arch Ophthalmol 126(10):1396–1403. doi:10.1001/ archopht.126.10.1396 Galan A, Dergham P, Escoll P, de-la-Hera A, D’Onofrio PM, Magharious MM, Koeberle PD, Frade JM, Saragovi HU (2014) Neuronal injury external to the retina rapidly activates retinal glia, followed by elevation of markers for cell cycle re-entry and death in retinal ganglion cells. PLoS One 9(7):e101349. doi:10. 1371/journal.pone.0101349
Cell Mol Neurobiol (2015) 35:1175–1186 Gao X, Wang H, Yang JJ, Liu X, Liu ZR (2012) Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Mol Cell 45(5):598–609. doi:10.1016/j.molcel.2012.01.001 Gerard C, Goldbeter A (2009) Temporal self-organization of the cyclin/Cdk network driving the mammalian cell cycle. Proc Natl Acad Sci USA 106(51):21643–21648. doi:10.1073/pnas. 0903827106 Greene LA, Liu DX, Troy CM, Biswas SC (2007) Cell cycle molecules define a pathway required for neuron death in development and disease. Biochim Biophys Acta 1772(4): 392–401. doi:10.1016/j.bbadis.2006.12.003 Gupta V, Bamezai RN (2010) Human pyruvate kinase M2: a multifunctional protein. Protein Sci 19(11):2031–2044. doi:10. 1002/pro.505 Hoglinger GU, Breunig JJ, Depboylu C, Rouaux C, Michel PP, Alvarez-Fischer D, Boutillier AL, Degregori J, Oertel WH, Rakic P, Hirsch EC, Hunot S (2007) The pRb/E2F cell-cycle pathway mediates cell death in Parkinson’s disease. Proc Natl Acad Sci USA 104(9):3585–3590. doi:10.1073/pnas.0611671104 Jacks T, Fazeli A, Schmitt EM, Bronson RT, Goodell MA, Weinberg RA (1992) Effects of an Rb mutation in the mouse. Nature 359(6393):295–300. doi:10.1038/359295a0 Jiang Y, Li X, Yang W, Hawke DH, Zheng Y, Xia Y, Aldape K, Wei C, Guo F, Chen Y, Lu Z (2014) PKM2 regulates chromosome segregation and mitosis progression of tumor cells. Mol Cell 53(1):75–87. doi:10.1016/j.molcel.2013.11.001 Kranenburg O, van der Eb AJ, Zantema A (1996) Cyclin D1 is an essential mediator of apoptotic neuronal cell death. EMBO J 15(1):46–54 Lannuzel A, Barnier JV, Hery C, Huynh VT, Guibert B, Gray F, Vincent JD, Tardieu M (1997) Human immunodeficiency virus type 1 and its coat protein gp120 induce apoptosis and activate JNK and ERK mitogen-activated protein kinases in human neurons. Ann Neurol 42(6):847–856. doi:10.1002/ana.4104 20605 Lee EY, Hu N, Yuan SS, Cox LA, Bradley A, Lee WH, Herrup K (1994) Dual roles of the retinoblastoma protein in cell cycle regulation and neuron differentiation. Genes Dev 8(17): 2008–2021 Lee J, Kim HK, Han YM, Kim J (2008) Pyruvate kinase isozyme type M2 (PKM2) interacts and cooperates with Oct-4 in regulating transcription. Int J Biochem Cell Biol 40(5):1043–1054. doi:10. 1016/j.biocel.2007.11.009 Luo W, Semenza GL (2011) Pyruvate kinase M2 regulates glucose metabolism by functioning as a coactivator for hypoxiainducible factor 1 in cancer cells. Oncotarget 2(7):551–556 Luo W, Hu H, Chang R, Zhong J, Knabel M, O’Meally R, Cole RN, Pandey A, Semenza GL (2011) Pyruvate kinase M2 is a PHD3stimulated coactivator for hypoxia-inducible factor 1. Cell 145(5):732–744. doi:10.1016/j.cell.2011.03.054 Malik B, Currais A, Andres A, Towlson C, Pitsi D, Nunes A, Niblock M, Cooper J, Hortobagyi T, Soriano S (2008) Loss of neuronal cell cycle control as a mechanism of neurodegeneration in the presenilin-1 Alzheimer’s disease brain. Cell Cycle 7(5):637–646 Malumbres M, Barbacid M (2009) Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer 9(3):153–166. doi:10.1038/ nrc2602 Marc RE, Jones BW, Watt CB, Vazquez-Chona F, Vaughan DK, Organisciak DT (2008) Extreme retinal remodeling triggered by light damage: implications for age related macular degeneration. Mol Vis 14:782–806 Mazurek S, Boschek CB, Hugo F, Eigenbrodt E (2005) Pyruvate kinase type M2 and its role in tumor growth and spreading. Semin Cancer Biol 15(4):300–308. doi:10.1016/j.semcancer. 2005.04.009
1185 Morohoshi K, Ohbayashi M, Patel N, Chong V, Bird AC, Ono SJ (2012) Identification of anti-retinal antibodies in patients with age-related macular degeneration. Exp Mol Pathol 93(2):193–199. doi:10.1016/j.yexmp.2012.03.007 Mukherjee J, Phillips JJ, Zheng S, Wiencke J, Ronen SM, Pieper RO (2013) Pyruvate kinase M2 expression, but not pyruvate kinase activity, is up-regulated in a grade-specific manner in human glioma. PLoS One 8(2):e57610. doi:10.1371/journal.pone. 0057610 Osuga H, Osuga S, Wang F, Fetni R, Hogan MJ, Slack RS, Hakim AM, Ikeda JE, Park DS (2000) Cyclin-dependent kinases as a therapeutic target for stroke. Proc Natl Acad Sci USA 97(18):10254–10259. doi:10.1073/pnas.170144197 Pang W, Leng X, Lu H, Yang H, Song N, Tan L, Jiang Y, Guo C (2013) Depletion of intracellular zinc induces apoptosis of cultured hippocampal neurons through suppression of ERK signaling pathway and activation of caspase-3. Neurosci Lett 552:140–145. doi:10.1016/j.neulet.2013.07.057 Pasantes-Morales H, Ademe RM, Quesada O (1981) Protective effect of taurine on the light-induced disruption of isolated frog rod outer segments. J Neurosci Res 6(3):337–348. doi:10.1002/jnr. 490060309 Randolph SA (2014) Age-related macular degeneration. Workplace Health Saf 62(8):352. doi:10.3928/21650799-20140708-06 Roca A, Shin KJ, Liu X, Simon MI, Chen J (2004) Comparative analysis of transcriptional profiles between two apoptotic pathways of light-induced retinal degeneration. Neuroscience 129(3):779–790. doi:10.1016/j.neuroscience.2004.08.021 Shu Q, Xu Y, Zhuang H, Fan J, Sun Z, Zhang M, Xu G (2014) Ras homolog enriched in the brain is linked to retinal ganglion cell apoptosis after light injury in rats. J Mol Neurosci 54(2): 243–251. doi:10.1007/s12031-014-0281-z Stetak A, Veress R, Ovadi J, Csermely P, Keri G, Ullrich A (2007) Nuclear translocation of the tumor marker pyruvate kinase M2 induces programmed cell death. Cancer Res 67(4):1602–1608. doi:10.1158/0008-5472.CAN-06-2870 Sui GY, Liu GC, Liu GY, Gao YY, Deng Y, Wang WY, Tong SH, Wang L (2013) Is sunlight exposure a risk factor for age-related macular degeneration? A systematic review and meta-analysis. Br J Ophthalmol 97(4):389–394. doi:10.1136/bjophthalmol2012-302281 Sumrejkanchanakij P, Tamamori-Adachi M, Matsunaga Y, Eto K, Ikeda MA (2003) Role of cyclin D1 cytoplasmic sequestration in the survival of postmitotic neurons. Oncogene 22(54): 8723–8730. doi:10.1038/sj.onc.1206870 Tamada M, Suematsu M, Saya H (2012) Pyruvate kinase M2: multiple faces for conferring benefits on cancer cells. Clin Cancer Res 18(20):5554–5561. doi:10.1158/1078-0432.CCR-120859 Tsutsumi H, Tani K, Fujii H, Miwa S (1988) Expression of L- and M-type pyruvate kinase in human tissues. Genomics 2(1):86–89 Wang HJ, Hsieh YJ, Cheng WC, Lin CP, Lin YS, Yang SF, Chen CC, Izumiya Y, Yu JS, Kung HJ, Wang WC (2014) JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1alpha-mediated glucose metabolism. Proc Natl Acad Sci USA 111(1): 279–284. doi:10.1073/pnas.1311249111 Wong N, De Melo J, Tang D (2013) PKM2, a central point of regulation in cancer metabolism. Int J Cell Biol 2013:242513. doi:10.1155/2013/242513 Xu Y, Yu S, Shu Q, Yang L, Yang C, Wang J, Xu F, Ji M, Liang X (2014) Upregulation of CREM-1 relates to retinal ganglion cells apoptosis after light-induced damage in vivo. J Mol Neurosci 52(3):331–338. doi:10.1007/s12031-013-0153-y Yang W, Lu Z (2013) Nuclear PKM2 regulates the Warburg effect. Cell Cycle 12(19):3154–3158. doi:10.4161/cc.26182
123
1186 Yang LP, Wu LM, Guo XJ, Li Y, Tso MO (2008) Endoplasmic reticulum stress is activated in light-induced retinal degeneration. J Neurosci Res 86(4):910–919. doi:10.1002/jnr.21535 Yang W, Xia Y, Ji H, Zheng Y, Liang J, Huang W, Gao X, Aldape K, Lu Z (2011) Nuclear PKM2 regulates beta-catenin transactivation upon EGFR activation. Nature 480(7375):118–122. doi:10. 1038/nature10598 Yang W, Zheng Y, Xia Y, Ji H, Chen X, Guo F, Lyssiotis CA, Aldape K, Cantley LC, Lu Z (2012) ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect. Nat Cell Biol 14(12):1295–1304. doi:10.1038/ncb2629 Yang K, Cao F, Sheikh AM, Malik M, Wen G, Wei H, Ted Brown W, Li X (2013) Up-regulation of Ras/Raf/ERK1/2 signaling impairs
123
Cell Mol Neurobiol (2015) 35:1175–1186 cultured neuronal cell migration, neurogenesis, synapse formation, and dendritic spine development. Brain Struct Funct 218(3):669–682. doi:10.1007/s00429-012-0420-7 Zhang X, He C, He C, Chen B, Liu Y, Kong M, Wang C, Lin L, Dong Y, Sheng H (2013) Nuclear PKM2 expression predicts poor prognosis in patients with esophageal squamous cell carcinoma. Pathol Res Pract 209(8):510–515. doi:10.1016/j.prp.2013.06.005 Zhou L, Del Villar K, Dong Z, Miller CA (2004) Neurogenesis response to hypoxia-induced cell death: map kinase signal transduction mechanisms. Brain Res 1021(1):8–19. doi:10.1016/ j.brainres.2004.05.115
