Involvement of the MEK-ERK/p38-CREB/c-fos signaling pathway in Kir channel inhibition-induced rat retinal Müller cell gliosis

Our previous studies have demonstrated that activation of group I metabotropic glutamate receptors downregulated Kir channels in chronic ocular hypertension (COH) rats, thus contributing to Müller cell gliosis, characterized by upregulated expression of glial fibrillary acidic protein (GFAP). In the present study, we explored possible signaling pathways linking Kir channel inhibition and GFAP upregulation. In normal retinas, intravitreal injection of BaCl2 significantly increased GFAP expression in Müller cells, which was eliminated by co-injecting mitogen-activated protein kinase (MAPK) inhibitor U0126. The protein levels of phosphorylated extracellular signal-regulated protein kinase1/2 (p-ERK1/2) and its upstream regulator, p-MEK, were significantly increased, while the levels of phosphorylated c-Jun N-terminal kinase (p-JNK) and p38 kinase (p-p38) remained unchanged. Furthermore, the protein levels of phosphorylated cAMP response element binding protein (p-CREB) and c-fos were also increased, which were blocked by co-injecting ERK inhibitor FR180204. In purified cultured rat Müller cells, BaCl2 treatment induced similar changes in these protein levels apart from p-p38 levels and the p-p38:p38 ratio showing significant upregulation. Moreover, intravitreal injection of U0126 eliminated the upregulated GFAP expression in COH retinas. Together, these results suggest that Kir channel inhibition-induced Müller cell gliosis is mediated by the MEK-ERK/p38-CREB/c-fos signaling pathway.

Previous studies have shown that some intracellular signaling pathways may be activated in Müller cells under retinal pathological conditions. For instance, expression of phosphorylated extracellular signal-regulated protein kinase (p-ERK) in Müller cells was increased in a rat model of retinal ischemia-reperfusion 29 . In glaucomatous human eyes, both the immunostaining intensity of mitogen-activated protein kinases (MAPKs) and the number of MAPK-positive cells were greater than that in control eyes, and elevated expression of p-ERK was localized to glial cells 30 . However, it is not yet absolutely definite whether or which of these signal molecules are involved in Müller cell gliosis following glaucoma onset. In this study, we explored the underlying mechanisms that link Kir channel inhibition and upregulation of GFAP expression in rat retinal Müller cells.

Involvement of the MAPK/ERK signaling pathway in Müller cell gliosis due to inhibition of Kir channels.
We first confirmed that inhibition of Kir channels by BaCl 2 indeed induced upregulation of GFAP expression in normal retinas 11 . BaCl 2 was injected intravitreally and retinas were collected 7 d after the injection for immunohistochemistry and Western blot analysis. As shown in Fig. 1A, GFAP expression was strictly localized to the endfeet of Müller cells in the ganglion cell layer (GCL) of the retinal section obtained from saline-injected eye (control) (a1 and a3). A significant increase in GFAP expression was observed in the section obtained from BaCl 2 -injected retina (Fig. 1B,b1 and b3). We then examined the possible involvement of MAPK signaling in BaCl 2 -induced upregulation of GFAP expression. The upregulation of GFAP expression was significantly reduced by co-injecting U0126, a MAPK inhibitor (Fig. 1C). Co-injection of U0124, an inactive analog of U0126, did not affect the BaCl 2 effect on expression of GFAP (Fig. 1D). Consistently, Western blotting revealed that total GFAP protein levels extracted from BaCl 2 -injected retinas were profoundly increased, with an average protein density of 159.9 ± 10.3% that of controls (n = 6, P = 0.001), which was rescued by U0126 (102.5 ± 2.5% of control, n = 6, P = 0.647), but not U0124 (156.7 ± 9.0% of control, n = 6, P < 0.001 vs. control and P = 0.849 vs. BaCl 2 -injected retinas) (Fig. 1E,F). These results suggest that the MAPK signaling pathway is involved in Kir channel inhibition-induced upregulation of GFAP expression in Müller cells.

Inhibition of Kir channels increases p-CREB and c-fos levels in BaCl 2 -injected retinas. p-ERK
can translocalize into the nucleus and activate its downstream targets, including cAMP response element binding protein (CREB) and c-fos, thus influencing transcription, translation, and protein synthesis. We first examined changes in expression of p-CREB in BaCl 2 -injected retinas. As shown in Fig. 4, BaCl 2 injection did not affect total CREB levels, but profoundly increased p-CREB levels to 284.2 ± 74.3% of control (n = 6, P = 0.023) as well as the p-CREB:CREB ratio to 197.3 ± 29.2% of control (n = 6, P = 0.048). In addition, FR180204, a selective ERK inhibitor, which was co-injected with BaCl 2 , rescued the enhanced p-CREB levels and p-CREB:CREB ratio to control levels ( Fig. 4C,D). Similarly, c-fos protein levels were increased by BaCl 2 -injection (149.3 ± 12.6% of control, n = 6, P = 0.008) and were reversed by FR180204 (Fig. 4E,F).

Inhibition of the MAPK/ERK signaling pathway reduces GFAP expression in COH reti-
nas. Finally, we tested whether inhibition of MAPK/ERK signaling affected Müller cell gliosis in COH retinas. The rat COH model was used, as our previous reports 11,32,33 . The average IOPs of operated eyes from day 1 to 1 week (G1d to G1w) ranged from 24.8 ± 0.3 to 25.5 ± 1.6 mmHg (n = 12), which was significantly higher than that for unoperated eyes (19.0 ± 0.3 mmHg, n = 12) and for sham-operated eyes (19.0 ± 0.6 mmHg, n = 6) (P all <0.001). Fig. 6A shows that GFAP expression was significantly increased in retinal vertical section obtained from COH rat at G1w (a2), as compared to that of sham-operated rat (control) (a1), which is consistent with our previous study 11 . Intravitreal injection of U0126 3d prior to the COH operation eliminated the increase of GFAP expression in COH retina (a3). Consistently, the retinal GFAP level, assessed by Western blotting, was increased to 131.6 ± 7.4% of control (n = 6, P = 0.002) in operated left eyes (Fig. 6B,C). Intravitreal injection of U0126 completely eliminated the IOP elevation-induced upregulation of GFAP levels (109.0 ± 6.2% of control, n = 6, P = 0.146) (Fig. 6B,C). These results strongly suggest that the MAPK/ERK signaling pathway is involved in Müller cell gliosis.

Discussion
Increasing evidence has demonstrated that downregulation of Kir channels is a key step for Müller cell gliosis in experimental glaucoma models and in patients with glaucoma 6,11,18,22 . It is commonly believed that inhibition of Kir channels leads to Müller cell depolarization 6,23,27,28 , which results in a loss of the strongly hyperpolarized resting potential that is important for the physiological functions of these cells. In this study, we found that intravitreal injection of BaCl 2 induced Müller cell gliosis in rat retinas, similar to our previous report 11 . Since we have demonstrated that the BaCl 2 -induced GFAP upregulation was due to inhibition of Kir channels, and not by its nonspecific effects 11 , it is reasonable to say that intravitreal injection of BaCl 2 resulting in Kir channel inhibition is an effective method for the induction of Müller cell gliosis. It should be noted that Ba 2+ is not a selective Kir channel blocker. Previous study has shown that Ba 2+ could also inhibit hyperpolarization-activated cation current (I h ) channels in a low affinity manner in rat hippocampal CA1 pyramidal neurons 34 . Modulation of I h channels may affect the neuronal excitability in retina 35 . However, there is no evidence showing that I h channels are expressed in retinal Müller cells. Therefore, Ba 2+ -induced Müller cell gliosis was indeed by inhibiting Kir channels in Müller cells.
Our work provides direct evidence showing that the MEK-ERK-CREB/c-fos signaling pathway mediated Kir channel inhibition-induced upregulation of GFAP expression in rat Müller cells. This is supported by the following facts. First, the MEK inhibitor U0126 completely inhibited BaCl 2 injection-induced GFAP upregulation in retinal Müller cells, as shown using immunohistochemistry and Western blotting (Fig. 1). Next, the protein levels of p-ERK1/2 and p-MEK, and the ratios of p-ERK1/2:ERK1/2 and p-MEK:MEK were significantly increased in BaCl 2 -injected retinas (Figs 2-4). U0126, but not U0124, reversed the effects of BaCl 2 on protein levels of p-ERK1/2 and the p-ERK1/2:ERK1/2 ratio, suggesting that the activity of the MEK/ERK pathway was enhanced. In addition, it should be noted that total and phosphorylated protein levels of JNK and p38, another two types of MAPKs, did not show significant changes in BaCl 2 injected retinas. Indeed, a previous study has shown that in retinas of patients with glaucoma, p-ERK, but not p-JNK and p-p38, was detected predominately in Müller cells although the intensity of immunostaining for the MAPKs and the number of MAPK-positive cells were greater than those of control eyes 30 . In a rat retinal ischemia-reperfusion model induced by acutely elevating IOP to 110 mmHg for 45-60 min, expression of p-ERK in Müller cells increased significantly 29 . Furthermore, intravitreal injection of BaCl 2 induced the upregulation of p-CREB and c-fos protein levels as well as the p-CREB:CREB ratio. The BaCl 2 -induced effects on p-CREB and c-fos expression were reversed by FR180204, a specific ERK inhibitor, strongly suggesting that the elevation of p-CREB and c-fos expression was triggered by p-ERK. It has been shown that ERK plays an important role in regulating neuronal functions 36 . CREB can be phosphorylated not only by protein kinase A (PKA), but also by other kinases, such as ERK 37 . ERK can be activated by numerous stimuli (in this study it was stimulated by inhibition of Kir channels by BaCl 2 ); in turn, p-ERK may activate its downstream targets including CREB and c-fos. Activating the p-ERK/p-CREB signaling pathway affects translation and new protein synthesis 38,39 . Usually, the basal level of c-fos expression in neurons is low, but it can be elevated following stimulation through second messenger systems 40 . Activated c-fos can bind to DNA and regulate the transcription of various target genes, such as GFAP. Therefore, it is reasonable to deduce that p-ERK increased the activities of p-CREB and c-fos, thus increasing GFAP protein synthesis.
In addition to Müller cells, Kir channels are also expressed in retinal neurons, such as RGCs 35,41 . To exclude the possible involvement of these retinal neurons in BaCl 2 -induced upregulation of GFAP expression, purified cultured rat Müller cells were treated with BaCl 2 and changes in GFAP and MAPK protein levels were examined by Western blotting. Our results clearly showed that BaCl 2 treatment of cultured Müller cells induced an increase in GFAP expression, suggestive of Müller cell gliosis. Significant upregulation of p-ERK1/2, p-MEK, p-CREB, and c-fos protein levels, as well as p-ERK1/2:ERK1/2 ratios, was detected in BaCl 2 -treated cells, confirming the involvement of the MEK-ERK-CREB/c-fos signaling pathway by Kir channel inhibition. However, it should be noted that in BaCl 2 -treated Müller cells, the p-p38 protein levels and the p-p38:p38 ratio were also significantly increased, which was inconsistent with the observations from BaCl 2 -injected retinas. Two factors may contribute to this inconsistency. First, among the three major types of MAPKs, ERK is expressed mainly in glial cells, while p38 and JNK are localized in RGCs and amacrine cells 29,30 . Under pathological conditions, elevated levels of p-ERK were detected in Müller cells, and p-JNK and p-p38 were associated with nonglial cells 29,30 . In addition, some faint p-p38 was also detectable in scattered glial cells or their processes 29 . In BaCl 2 -injected retinas, changes in p-p38 may occur only in Müller cells. Therefore, in whole retinal homogenates obtained from BaCl 2 -injected rats, p-p38 protein levels may be too low to be detected. Second, in BaCl 2 -injected retinas, Ba 2+ may inhibit Kir channels in retinal neurons, and subsequently influence p-p38 protein levels in these neurons, resulting in unchanged total p-p38 levels and p-p38:p38 ratio in whole retinal extracts. The elevated level of p-p38 appeared when retinal neurons were absent in purified Müller cell cultures. Nevertheless, upregulation of GFAP expression in Müller cells induced by Kir channel inhibition may be mediated by increased p-ERK and p-p38, with p-ERK being the predominant mediator. Furthermore, in BaCl 2 -treated Müller cells, total MEK protein levels were increased in addition to upregulated p-MEK. This resulted in an unchanged p-MEK:MEK ratio, even though p-MEK was significantly increased. We speculate that this may be due to the continuous strong depolarization induced by BaCl 2 treatment. This issue should be investigated in our future studies. In addition, a remained question is how to link Müller cell depolarization to MEK/ERK signaling activation. It was reported that MEK/ERK signaling pathway could be activated by elevating intracellular Ca 2+ in neurons 42 . Voltage-gated Ca 2+ channels, such as T-and L-type Ca 2+ channels, were functionally expressed in retinal Müller cells 43 . Inhibition of Kir channels by Ba 2+ could result in membrane depolarization of Müller cells, in turn activate voltage-gated Ca 2+ channels and increase Ca 2+ influx, thus activating Ca 2+ -dependent MEK/ERK signaling.
It is noteworthy that in the retina, different types of MAPKs may have distinct functions depending on their differential expression in different cells. The ERK signaling pathway is generally activated by extracellular stimuli and other factors, such as mitogens. ERK signaling is involved in modulation of transcriptional activity, thus influencing cell growth and differentiation 44 . In this work, we found that inhibition of Kir channels activates ERK signaling, as evidenced by increased p-ERK levels. p-ERK further activates CREB and c-fos, thus increasing GFAP protein synthesis. On the other hand, the p38 and JNK pathways are strongly activated by cytokines, such as tumor necrosis factor (TNF)-α 45 , which have been implicated in neuronal death [46][47][48][49][50] . For instance, p38 was involved in axotomized RGC death in chick embryos 47 , and in RGC apoptosis mediated by glutamate neurotoxicity in rats 50,51 . Blocking the apoptosis signal-regulating kinase 1 (ASK1)-p38 pathway could prevent RGC death following optic nerve injury 52 . In addition, previous studies have demonstrated that JNK may play a major role in various forms of neuronal death. In the rat retinal ischemia-reperfusion model, inhibition of JNK activation significantly decreased cell apoptosis in the GCL, the inner nuclear layer (INL), and the photoreceptor layer 53 . In mice, JNK signaling was activated after axonal injury of RGCs, which may be the major early pathway triggering RGC death after axonal injury, and may directly link axonal injury to the transcriptional activity that controls RGC death 54 . In BaCl 2 -injected retinas, p38 and JNK signaling pathways remained unchanged, suggesting that inhibition of Kir channels in retinal neurons was not sufficient to activate these two pathways, even though it was sufficient to strongly activate ERK in Müller cells. Moreover, inhibition of ERK signaling pathway by intravitreal injection of U0126 eliminated GFAP expression upregulation in COH retinas, further demonstrating the involvement of ERK signaling pathway in Müller cell gliosis.
In conclusion, we here demonstrate that the MEK/ERK-p38/CREB-c-fos signaling pathway mediates the Kir channel inhibition-induced upregulation of GFAP in rat Müller cells. Previous studies have demonstrated that activated Müller cells may release cytotoxic factors, such as TNF-α and nitric oxide (NO), which acted on RGCs and resulted in RGC damage 6 . Our present results suggest that appropriate reduction of MEK/ERK signaling pathway could alleviate Müller cell gliosis, which may be an effective way for preventing the loss of RGCs in glaucoma.

Methods
Animals. All experimental procedures described were approved by the Laboratory Animal Care and Use Committee at Fudan University (Shanghai, China) and are in accordance with the National Institutes of Health (NIH) guidelines. Male Sprague-Dawley rats, purchased from SLAC Laboratory Animal Co., Ltd (Shanghai, China), were maintained on a 12-h light/dark cycle.