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MEK–ERK signaling in adult newt retinal pigment epithelium cells is strengthened immediately after surgical induction of retinal regeneration
Neuroscience Letters 523 (2012) 39–44
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
MEK–ERK signaling in adult newt retinal pigment epithelium cells is strengthened immediately after surgical induction of retinal regeneration Aki Mizuno a,1 , Hirofumi Yasumuro a,1 , Taro Yoshikawa a , Wataru Inami a , Chikafumi Chiba b,∗ a b
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan Faculty of Life and Environmental Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan
h i g h l i g h t s ERK-mediated signaling activity in RPE cells is elevated quickly (in 30 min) upon retinectomy. MEK–ERK signaling is intensified by itself through up-regulation of the expression of constituent molecules in the pathway. Blockade of initial MEK–ERK signaling inhibits cell-cycle re-entry of RPE cells.
a r t i c l e
i n f o
Article history: Received 21 May 2012 Accepted 13 June 2012 Keywords: Retina Regeneration Newt MEK ERK Retinal pigment epithelium
a b s t r a c t Adult newt retinal pigment epithelium (RPE) cells are mitotically quiescent in the physiological condition, but upon a traumatic injury of the neural retina (NR) they re-enter the cell-cycle and eventually regenerate the missing NR. Here, to understand the mechanism underlying the cell-cycle re-entry of RPE cells following NR injury, we first investigated changes in MEK–ERK signaling activity in RPE cells upon removal of the NR (retinectomy) from the eye of living animals, and found that ERK-mediated signaling activity is elevated quickly (in 30 min) upon retinectomy. In addition, we found, in in vitro analyses, that immediate early activation of MEK–ERK signaling may occur in RPE cells upon NR injury, intensifying the MEK–ERK signaling itself through up-regulation of the expression of constituent molecules in the pathway, and that 1-h blockade of such early MEK–ERK signaling interferes with the cell-cycle re-entry, which occurs 5–10 days later. Together, these results provide us with insight that elevation of MEK–ERK signaling activity upon NR injury may be a key process for mitotically quiescent RPE cells to re-enter the cell-cycle, leading to retinal regeneration. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The retinal pigment epithelium (RPE) is a highly specialized monolayer with pigmented microvilli that is located between the neural retina (NR) and the choriocapillaris in the eye, being indispensable for physiological functions of the NR or vision [15]. The mature RPE cells are, as a rule, mitotically quiescent in physiological conditions, but upon a traumatic injury of the NR these cells change their phenotype and start to proliferate. In humans, such changes to RPE cells are responsible for retinal diseases such as proliferative vitreoretinopathy (PVR) leading to vision loss [6,10,13]. In contrast, certain urodele amphibians such as newts can regenerate, even in adulthood, their entire retinas through proliferation and transdifferentiation of the RPE cells [2,3,5,8,12,18,19]. Therefore, the study of retinal regeneration in the adult newt may provide
∗ Corresponding author. Tel.: +81 29 853 4667; fax: +81 29 853 6614. E-mail address: [email protected] (C. Chiba). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.06.037
clues for the treatment of PVR, and even for retinal regeneration in patients’ eyes. The mechanism underlying the cell-cycle re-entry of RPE cells upon NR injury is not fully understood in either humans or newts, although it can be a clinical target for manipulating both retinal diseases and regeneration. In the last study using a ‘retina-less eye-cup (RLEC)’ culture system of adult newt, we provided evidence suggesting that activation of the MEK–ERK cascade, a part of the intracellular signaling pathways [11,17], may be a requisite for the first cell-cycle entry of RPE cells upon NR injury [21]. Since MEK–ERK signalling activity is elevated during preparation of the RLEC (within 1 h), immediate early activation of MEK–ERK signaling might be essential for this important event in retinal regeneration. However, it remains to be studied whether such early activation of MEK–ERK signaling really occurs upon NR injury in vivo. Moreover, there is no evidence to show if early activation of MEK–ERK signaling affects the first cell-cycle entry of RPE cells that occurs 5–10 days later [21]. Therefore, in the current study, to clarify these points we investigated changes in the expression and activity of MEK–ERK in RPE cells immediately after retinectomy (surgical removal of the
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NR from the eye of a living animal) by means of immunoblotting (IB) and immunohistochemistry (IHC). In addition, using the RLEC culture system we examined the effects of a blockade of the early activation (in 1 h) of MEK–ERK signaling on the first cell-cycle entry of RPE cells in 10 days.
2. Materials and methods
To inhibit MEK–ERK signaling in the period of RLEC preparation, the eye-cup was soaked in PBS containing 5 M U0126 (MEK1/2 specific inhibitor; V1121, Promega, Madison, WI, USA) and its solvent 0.25% DMSO (D2650, Sigma–Aldrich) for 1 h at RT. For the mock control, only solvent was administered. In the current study, two eyeballs from one animal were used for the test and the control respectively. The ratio of BrdU + RPE cells in the RLEC at 10 days in culture was estimated as in [21].
2.1. Animals, retinectomy and RLEC culture
2.2. IHC and IB
Adult newts Cynops pyrrhogaster (total body-length: 9–12 cm) were housed in laboratory [1]. Retinectomy was carried out by removing the NR together with the lens from the left eye of anesthetized animal [2]. The operated animals were laid on a plastic tray the left side up at room temperature (RT: ∼22 ◦ C) until use within 2 h. RLECs were prepared and cultured as in [21]. Briefly, the eyeball was cut along the equator, its anterior half containing the lens was carefully removed, the posterior half (i.e., the eye-cup) was soaked in PBS for 1 h at RT, and finally the NR was carefully removed to make a RLEC (empirically, for in vitro retinectomy, this 1-h incubation of eye-cups in PBS is necessary to weaken the attachment of the NR to the RPE, allowing removal of the NR from the eye-cup without loss/damage of RPE cells). The RLEC was transferred into a small chamber filled with 200 l of 80% L-15 culture medium (Invitrogen, Carlsbad, CA, USA) containing 2% penicillin–streptomycin (Liquid; 15140-122, Life Technologies) and 5 g/ml BrdU (Sigma–Aldrich, St. Louis, MO, USA), and incubated at 25 ◦ C.
The primary antibodies used: rabbit anti-MEK1/2 polyclonal (1:400; 9122, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-MEK1/2 monoclonal (1:500; 9154S, Cell Signaling Technology), rabbit anti-ERK1/2 polyclonal (1:300; p44/42 MAP Kinase antibody, 9102, Cell Signaling Technology), rabbit antiphospho-ERK1/2 polyclonal (1:500; Phospho-p44/42 MAP Kinase antibody, 9101S, Cell Signaling Technology), mouse anti--actin monoclonal (1:3000; ab6276, Abcam, Cambridge, UK) and mouse anti-RPE65 monoclonal (1:500; MAB5428, Millipore, MA, USA). The secondary antibodies used: biotinylated goat anti-rabbit IgG (1:300; BA-1000, Vector), biotinylated goat anti-mouse IgG (1:300; BA-9200, Vector) and FITC conjugated goat anti-mouse IgG (1:150; 115-035-003, Jackson Immuno Research, West Grove, USA). For IHC, tissue sections were prepared from normal eyeballs immediately (0 min) and at 30, 60 and 120 min after retinectomy [16]. To localize MEK and ERK proteins in these tissues, immunoperoxidase labeling was carried out as in [2] with some modifications: in this study, HRP-labeled streptavidin (#426062, Nichirei
Fig. 1. Expression and activity of MEK/ERK proteins in the intact RPE cells. (A) Immunoblotting of proteins extracted from either retina-less eye-cups (RLECs) or RPE-choroid tissues isolated from RLECs. In both samples, MEK1, MEK2, ERK1 and ERK2, and their phosphorylated forms (p-MEK1, p-MEK2, p-ERK1 and p-ERK2) were detected, although in the RPE-choroid tissues the amount of MEK1 and MEK2 was lower than that in the RLECs. Control: negative control with no primary antibody. (B) RPE in a section of the normal eyeball. Top: the choroid side; bottom: the neural retina side. (C–G) Immunolabeling of sections of the normal eyeball with either MEK1/2- (C), p-MEK1/2- (D), ERK1/2- (E) or p-ERK1/2-antibody (F), and a negative control with no primary antibody (G). Melanin pigments in these sections were bleached. Arrows in each panel indicate the nuclei of RPE cells. The nucleus indicated by the arrow* is enlarged in the right-hand panel. Scale bar: (G) (for B–G) 50 M.
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Fig. 2. Change in the expression and activity of MEK/ERK proteins in RPE cells after retinectomy. (A) Immunohistochemistry with p-ERK1/2-antibody. Sections were prepared from eyes right after retinectomy (0 min) and from those at 30 and 60 min after retinectomy. Orientation of the tissues was the same as that in Fig. 1. Horizontal bars indicate the thickness of the RPE layer. Immunoreactivity of RPE cell nuclei increased obviously at 30 and 60 min after retinectomy (arrows). Melanin pigments in these tissues were bleached. Control: negative control with no primary antibody. Scale bar: 100 M. For immunoreactivity to either MEK1/2-, p-MEK1/2- or ERK1/2-antibody, see Supplementary Fig. S1. (B) Immunoblotting with MEK1/2-, p-MEK1/2-, ERK1/2- and ERK1/2-antibody. Proteins were extracted from eye-cups right after retinectomy (0 min) and those at 30, 60 and 120 min after retinectomy. RPE-specific protein RPE65 (∼63 kDa) was used as the internal control. Under this condition, the amount of MEK1 and p-MEK1 was too low to be detected.
Bioscience, Tokyo, Japan) was applied to minimize background staining. RPE cells were always identified by simultaneous FITCimmunofluorescence labeling of RPE65. After labeling, melanin pigments in the tissues were bleached [2]. For IB, protein samples were collected as in [21] from (i) RLECs prepared from eye-cups which had been incubated in either PBS or U0126-containing PBS for 1 h at RT, (ii) RPE-choroid tissues isolated from the RLECs in PBS at RT, and (iii) posterior halves of retinectomized eyes which were collected from animals immediately (0 min) and at 30, 60 and 120 min after surgery (the retinectomized eye was cut into posterior and anterior halves in chilled PBS in 3 min). SDS-PAGE and IB were carried out [16]. Protein bands were quantified using a function of ImageJ 1.44p software
(Wayne Rasband, National Institutes of Health, USA; http://imagej.nih.gov/ij). RPE65 was used as the internal control. 2.3. Data analysis Images of tissues were acquired using a colour CCD camera (C4742-95 ORCA-ER system, Hamamatsu Photonics, Japan) connected to a personal computer. Figures were prepared using Photoshop Extended CS5. Image, brightness, contrast, and sharpness were adjusted. Statistical data were presented as the mean ± SEM. Non-parametric tests were carried out to evaluate the statistical significance of the data, using Ekuseru-Toukei 2008 software (Social Survey Research Information, Tokyo, Japan).
Fig. 3. Semi-quantitative analysis of the changes in the expression and activity of ERK1 (A–C) and ERK2 (D–F) proteins in RPE cells after retinectomy. Immunoblotting with ERK1/2- and p-ERK1/2-antibody was carried out as done in Fig. 2B, and data sets from 6 independent rounds were analyzed. The values were normalized against those at 0 min. Statistical differences according to the Mann–Whitney’s U-test are indicated by *P < 0.05 and **P < 0.01.
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Fig. 4. Effects of U0126 on the expression and activity of MEK2 (A–C), ERK1 (D–F) and ERK2 (G–I) proteins in RPE cells in 1 h after preparation of the eye-cup. After eye-cups were prepared from normal eyeballs and incubated in PBS containing either 0.25% DMSO only (Mock) or 5 M U0126 + 0.25% DMSO for 1 h, RLECs were prepared by removing the neural retina from the eye-cup and used for immunoblotting. Data sets from more than 5 independent rounds (>4 RLECs/round) were analyzed as done in Fig. 3. The values were normalized against those of Mock. n: number of rounds. Statistical differences are indicated by *P < 0.05; for the Sheffe’s test following the Friedman test, black asterisks; for the Jonckheere–Terpstra test, white asterisks. Note that the statistical difference in (F) was P = 0.058 according to the Jonckheere–Terpstra test.
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The original research reported herein was performed under the guidelines established by the University of Tsukuba Animal Use and Care Committee. 3. Results 3.1. Expression and activity of MEK/ERK proteins in the intact RPE cells IB was carried out with protein extracts from either RLECs or RPE-choroid tissues that were prepared from normal eyeballs; in these samples, both MEK and phosphorylated MEK (p-MEK) antibodies labelled two identical bands (∼40 and ∼38 kDa) corresponding to MEK1 and MEK2, and both ERK and phosphorylated ERK (p-ERK) antibodies also labelled two bands (∼39 and ∼37 kDa) corresponding to ERK1 and ERK2, respectively (Fig. 1A). To localize these proteins in the posterior eye, IHC was carried out with sections of normal eyeballs; every antibody lightly labelled both the cytoplasm and nuclei of almost all RPE cells (Fig. 1B–G), whereas positive signals were not obvious in other tissues in the posterior eye except for the NR (data not shown). These results confirm the presence of a MEK–ERK signaling cascade in intact RPE cells. 3.2. Change in the expression and activity of MEK/ERK proteins in RPE cells after retinectomy IHC was carried out with eye section at 0, 30 and 60 min after retinectomy; change in immunoreactivity along the RPE was not clear in either MEK-, p-MEK- or ERK-antibody, but p-ERK immunoreactivity in the nuclei of RPE cells obviously increased in 30 min (Fig. 2A and Supplementary Fig. S1). Such nuclear localization of p-ERK suggests activation of ERK-mediated signaling [4]. It must be noted that during this period, no apparent changes in morphology occurred in most tissues including the RPE in the posterior eye, except for the choroid in which, like the inflammatory response, the capillaries started to expand, so a large amount of blood was supplied to the choroid, and that some cells, possibly of leukocytes, which were labeled with every antibody appeared in the blood (data not shown). IB was carried out with protein extracts from eye-cups prepared from eyes at 0, 30, 60 and 120 min after retinectomy. Fig. 2B shows a particular data set. In this case, both MEK2 and its active form (pMEK2) seemed to increase in 30 min after retinectomy, and then the level of MEK2 was almost sustained until 120 min, whereas the level of p-MEK2 gradually decreased. ERK1 and ERK2 also increased in 30 min, but both decreased thereafter. Correlated with those changes, their active forms (p-ERK1 and p-ERK2) also increased in 30 min and then decreased. Semi-quantitative analysis was carried out with data sets of IB from more than 3 independent rounds (>4 eye-cups/round); unexpectedly, significant changes were not observed in either the amount of MEK2 or its activity (calculated by p-MEK2/MEK2) in this period (data not shown). On the other hand, the amount of both ERK1 and ERK2 were, and their active forms (p-ERK1 and p-ERK2) were also, increased significantly in 30 min after retinectomy and then decreased (Fig. 3). However, interestingly, their activities (pERK1/ERK1 and p-ERK2/ERK2) did not change in this period (Fig. 3C and F). Thus, immediately after retinectomy, ERK-mediated signaling in RPE cells was likely to be reinforced through up-regulation of ERK protein expression. On the other hand, regulation of MEK (upstream kinase of ERK) after retinectomy was obscure. Since the amount of MEK proteins in the RPE is assumed to be much smaller than that of ERK proteins (Fig. 1A), the quantification of MEK proteins in the RPE by IB might have been seriously affected by the inflow of
Fig. 5. Effect of 1-h treatment of eye-cups by U0126 on the cell-cycle entry of RPE cells in the following 10-day RLEC culture. RLECs prepared from eye-cups, which had been incubated in PBS containing either 0.25% DMSO only (Mock) or 5 M U0126 + 0.25% DMSO for 1 h, were cultured in the presence of BrdU for 10 days. BrdU-positive (BrdU+) nuclei of RPE cells in the RLEC were counted at 10 days, and the ratio of BrdU + RPE cells was calculated against the total cell number in the whole RPE sheet. n: number of RLECs. Statistical difference according to the Sheffe’s test following the Friedman test is indicated by *P < 0.05.
MEK/ERK-positive blood cells from outside the eye. Therefore, to avoid this possible noise due to blood inflow, we used an in vitro system in the following experiments. 3.3. Effects of a MEK1/2-specific inhibitor U0126 on initial changes in the expression and activity of MEK/ERK proteins in RPE cells IB was carried out with a protein extract from RLECs prepared from eye-cups which had been incubated in PBS containing 5 M U0126 and its solvent 0.25% DMSO for 1 h, and the expression and activity of MEK/ERK proteins were compared with those in the RLECs prepared from eye-cups treated only with solvent (Fig. 4); the amount of MEK2, ERK1 and ERK2, and their active forms (p-MEK2, p-ERK1 and p-ERK2) after 1-h incubation decreased significantly in the presence of U0126; the activity of ERK1 (p-ERK1/ERK1) tended to be and that of ERK2 (p-ERK2/ERK2) was significantly suppressed in the presence of U0126, whereas that of MEK2 (p-MEK2/MEK2) was not affected. These results suggest that MEK-mediated activation of ERK occurs in RPE cells during 1-h incubation of the eye-cup in PBS, and that this signal activity up-regulates the expression of constituent proteins of the MEK–ERK cascade, reinforcing the signal activity itself. 3.4. Effect of 1-h inhibition of initial MEK–ERK signaling on the cell-cycle entry of RPE cells RLECs prepared after 1-h incubation of eye-cups in the presence of U0126 were cultured in 80% L-15 medium containing 5 g/ml BrdU for 10 days, and then BrdU-positive (BrdU+) nuclei of RPE cells were counted; the ratio of BrdU+ RPE cells was significantly low compared with that of a mock control in which the eye-cups were treated with the solvent only (Fig. 5), suggesting that 1-h inhibition of MEK–ERK signaling seriously affected cell-cycle entry of RPE cells in the following 10 days. 4. Discussion The current in vitro study suggests that immediate early activation of the MEK–ERK signaling pathway may occur in RPE cells following NR injury, intensifying the MEK–ERK signaling itself through up-regulation of the expression of constituent molecules in the pathway (i.e., via a positive feedback). Such a positive feedback in MEK–ERK signaling has been reported in other systems
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[14,20]. The current in vivo study demonstrated that, as suggested in vitro, ERK-mediated signaling in RPE cells is strengthened quickly (within 30 min) after retinectomy, although changes in the expression and activity of MEK in RPE cells were not able to be determined. In IB analysis, the amount of ERK and p-ERK proteins appeared to decrease gradually after 30 min (Figs. 2 and 3). Consistently, in the IHC of eye sections at 6 h after retinectomy, p-ERK-immunoreactivity of RPE cells was almost the same as that of the 0-min sample (data not shown). Taken together, MEK–ERK signaling in RPE cells is likely to be strengthened temporarily correlating with the onset of retinal regeneration. This event may be a key process for mitotically quiescent RPE cells to re-enter the cell-cycle upon NR injury. In mammals, such immediately early activation of MEK–ERK signaling in RPE cells is known to occur upon NR injury such as in retinal detachment [4]. In humans, MEK–ERK signaling is involved in proliferation and metaplastic transformation of RPE cells, sometimes leading to pathogenesis such as PVR [7,9,13]. Therefore, it is interesting to address whether the same signalling pathway involved in PVR works in adult newts as a constituent of the trigger signal for retinal regeneration. We have just initiated experiments aimed at trying to regulate MEK–ERK activity in RPE cells in vivo by intraocular drug administration and transgenesis. Acknowledgements This work was supported by MEXT (23124502) and JSPS (20650060; 24650229; 21300150). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.neulet.2012.06.037. References [1] M.N. Casco-Robles, S. Yamada, T. Miura, K. Nakamura, T. Haynes, N. Maki, K. Del Rio-Tsonis, P.A. Tsonis, C. Chiba, Expressing exogenous genes in newts by transgenesis, Nature Protocols 6 (2011) 600–608. [2] C. Chiba, A. Hoshino, K. Nakamura, K. Susaki, Y. Yamano, Y. Kaneko, O. Kuwata, F. Maruo, T. Saito, Visual cycle protein RPE65 persists in new retinal cells during retinal regeneration of adult newt, The Journal of Comparative Neurology 495 (2006) 391–407.
[3] C. Chiba, V.I. Mitashov, Cellular and molecular events in the adult newt retinal regeneration, in: C. Chiba (Ed.), The Strategies for Retinal Tissue Repair and Regeneration in Vertebrates: from Fish to Human 2007, Research Signpost, Kerala, India, 2007, pp. 15–33. [4] S.F. Geller, G.P. Lewis, S.K. Fisher, FGFR1, signaling, and AP-1 expression after retinal detachment: reactive Müller and RPE cells, Investigative Ophthalmology & Visual Science 42 (2001) 1363–1369. [5] T. Haynes, K. Del Rio-Tsonis, Retina repair, stem cells and beyond, Current Neurovascular Research 1 (2004) 231–239. [6] L.K. Kim, J.G. Arroyo, Mechanisms in proliferative vitreoretinopathy, Ophthalmology Clinics of North America 15 (2002) 81–86. [7] R.E. King, K.D. Kent, J.A. Bomser, Resveratrol reduces oxidation and proliferation of human retinal pigment epithelial cells via extracellular signal-regulated kinase inhibition, Chemico-biological Interactions 151 (2005) 143–149. [8] V.I. Mitashov, Mechanisms of retina regeneration in urodeles, The International Journal of Developmental Biology 40 (1996) 833–844. [9] R.L. Pacheco-Domínguez, I.P. Palma-Nicolas, E. López, A.M. López-Colomé, The activation of MEK–ERK1/2 by glutamate receptor-stimulation is involved in the regulation of RPE proliferation and morphologic transformation, Experimental Eye Research 86 (2008) 207–219. [10] J.C. Pastor, Proliferative vitreoretinopathy: an overview, Survey of Ophthalmology 43 (1998) 3–18. [11] G. Pearson, F. Robinson, T. Beers Gibson, B.E. Xu, M. Karandikar, K. Berman, M.H. Cobb, Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions, Endocrine Reviews 22 (2001) 153–183. [12] T.A. Reh, C. Pittack, Transdifferentiation and retinal regeneration, Seminars in Cell Biology 6 (1995) 137–142. [13] E. Salero, T.A. Blenkinsop, B. Corneo, A. Harris, D. Rabin, J.H. Stern, S. Temple, Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives, Cell Stem Cell 10 (2012) 88–95. [14] S.Y. Shin, O. Rath, S.M. Choo, F. Fee, B. McFerran, W. Kolch, K.H. Cho, Positiveand negative-feedback regulations coordinate the dynamic behaviour of the Ras–Raf–MEK–ERK signal transduction pathway, Journal of Cell Science 122 (2009) 425–435. [15] O. Strauss, The retinal pigment epithelium in visual function, Physiological Reviews 85 (2005) 845–881. [16] K. Susaki, C. Chiba, MEK mediates in vitro neural transdifferentiation of the adult newt retinal pigment epithelium cells: is FGF2 an induction factor, Pigment Cell Research 20 (2007) 364–379. [17] G.M. Thomas, R.L. Huganir, MAPK cascade signaling and synaptic plasticity, Nature Reviews. Neuroscience 5 (2004) 173–183. [18] P.A. Tsonis, Regeneration in vertebrates, Developmental Biology 221 (2000) 273–284. [19] P.A. Tsonis, K. Del Rio-Tsonis, Lens and retina regeneration: transdifferentiation, stem cells and clinical applications, Experimental Eye Research 78 (2004) 161–172. [20] J. Wang, A. Yen, A MAPK-positive feedback mechanism for BLR1 signaling propels retinoic acid-triggered differentiation and cell cycle arrest, The Journal of Biological Chemistry 283 (2008) 4375–4386. [21] T. Yoshikawa, A. Mizuno, H. Yasumuro, W. Inami, M.N. Vergara, K. Del RioTsonis, C. Chiba, MEK–ERK and heparin-susceptible signaling pathways are involved in cell-cycle entry of the wound edge retinal pigment epithelium cells in the adult newt, Pigment Cell Melanoma Research 25 (2012) 66–82.
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
MEK–ERK signaling in adult newt retinal pigment epithelium cells is strengthened immediately after surgical induction of retinal regeneration Aki Mizuno a,1 , Hirofumi Yasumuro a,1 , Taro Yoshikawa a , Wataru Inami a , Chikafumi Chiba b,∗ a b
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan Faculty of Life and Environmental Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan
h i g h l i g h t s ERK-mediated signaling activity in RPE cells is elevated quickly (in 30 min) upon retinectomy. MEK–ERK signaling is intensified by itself through up-regulation of the expression of constituent molecules in the pathway. Blockade of initial MEK–ERK signaling inhibits cell-cycle re-entry of RPE cells.
a r t i c l e
i n f o
Article history: Received 21 May 2012 Accepted 13 June 2012 Keywords: Retina Regeneration Newt MEK ERK Retinal pigment epithelium
a b s t r a c t Adult newt retinal pigment epithelium (RPE) cells are mitotically quiescent in the physiological condition, but upon a traumatic injury of the neural retina (NR) they re-enter the cell-cycle and eventually regenerate the missing NR. Here, to understand the mechanism underlying the cell-cycle re-entry of RPE cells following NR injury, we first investigated changes in MEK–ERK signaling activity in RPE cells upon removal of the NR (retinectomy) from the eye of living animals, and found that ERK-mediated signaling activity is elevated quickly (in 30 min) upon retinectomy. In addition, we found, in in vitro analyses, that immediate early activation of MEK–ERK signaling may occur in RPE cells upon NR injury, intensifying the MEK–ERK signaling itself through up-regulation of the expression of constituent molecules in the pathway, and that 1-h blockade of such early MEK–ERK signaling interferes with the cell-cycle re-entry, which occurs 5–10 days later. Together, these results provide us with insight that elevation of MEK–ERK signaling activity upon NR injury may be a key process for mitotically quiescent RPE cells to re-enter the cell-cycle, leading to retinal regeneration. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The retinal pigment epithelium (RPE) is a highly specialized monolayer with pigmented microvilli that is located between the neural retina (NR) and the choriocapillaris in the eye, being indispensable for physiological functions of the NR or vision [15]. The mature RPE cells are, as a rule, mitotically quiescent in physiological conditions, but upon a traumatic injury of the NR these cells change their phenotype and start to proliferate. In humans, such changes to RPE cells are responsible for retinal diseases such as proliferative vitreoretinopathy (PVR) leading to vision loss [6,10,13]. In contrast, certain urodele amphibians such as newts can regenerate, even in adulthood, their entire retinas through proliferation and transdifferentiation of the RPE cells [2,3,5,8,12,18,19]. Therefore, the study of retinal regeneration in the adult newt may provide
∗ Corresponding author. Tel.: +81 29 853 4667; fax: +81 29 853 6614. E-mail address: [email protected] (C. Chiba). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.06.037
clues for the treatment of PVR, and even for retinal regeneration in patients’ eyes. The mechanism underlying the cell-cycle re-entry of RPE cells upon NR injury is not fully understood in either humans or newts, although it can be a clinical target for manipulating both retinal diseases and regeneration. In the last study using a ‘retina-less eye-cup (RLEC)’ culture system of adult newt, we provided evidence suggesting that activation of the MEK–ERK cascade, a part of the intracellular signaling pathways [11,17], may be a requisite for the first cell-cycle entry of RPE cells upon NR injury [21]. Since MEK–ERK signalling activity is elevated during preparation of the RLEC (within 1 h), immediate early activation of MEK–ERK signaling might be essential for this important event in retinal regeneration. However, it remains to be studied whether such early activation of MEK–ERK signaling really occurs upon NR injury in vivo. Moreover, there is no evidence to show if early activation of MEK–ERK signaling affects the first cell-cycle entry of RPE cells that occurs 5–10 days later [21]. Therefore, in the current study, to clarify these points we investigated changes in the expression and activity of MEK–ERK in RPE cells immediately after retinectomy (surgical removal of the
40
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NR from the eye of a living animal) by means of immunoblotting (IB) and immunohistochemistry (IHC). In addition, using the RLEC culture system we examined the effects of a blockade of the early activation (in 1 h) of MEK–ERK signaling on the first cell-cycle entry of RPE cells in 10 days.
2. Materials and methods
To inhibit MEK–ERK signaling in the period of RLEC preparation, the eye-cup was soaked in PBS containing 5 M U0126 (MEK1/2 specific inhibitor; V1121, Promega, Madison, WI, USA) and its solvent 0.25% DMSO (D2650, Sigma–Aldrich) for 1 h at RT. For the mock control, only solvent was administered. In the current study, two eyeballs from one animal were used for the test and the control respectively. The ratio of BrdU + RPE cells in the RLEC at 10 days in culture was estimated as in [21].
2.1. Animals, retinectomy and RLEC culture
2.2. IHC and IB
Adult newts Cynops pyrrhogaster (total body-length: 9–12 cm) were housed in laboratory [1]. Retinectomy was carried out by removing the NR together with the lens from the left eye of anesthetized animal [2]. The operated animals were laid on a plastic tray the left side up at room temperature (RT: ∼22 ◦ C) until use within 2 h. RLECs were prepared and cultured as in [21]. Briefly, the eyeball was cut along the equator, its anterior half containing the lens was carefully removed, the posterior half (i.e., the eye-cup) was soaked in PBS for 1 h at RT, and finally the NR was carefully removed to make a RLEC (empirically, for in vitro retinectomy, this 1-h incubation of eye-cups in PBS is necessary to weaken the attachment of the NR to the RPE, allowing removal of the NR from the eye-cup without loss/damage of RPE cells). The RLEC was transferred into a small chamber filled with 200 l of 80% L-15 culture medium (Invitrogen, Carlsbad, CA, USA) containing 2% penicillin–streptomycin (Liquid; 15140-122, Life Technologies) and 5 g/ml BrdU (Sigma–Aldrich, St. Louis, MO, USA), and incubated at 25 ◦ C.
The primary antibodies used: rabbit anti-MEK1/2 polyclonal (1:400; 9122, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-MEK1/2 monoclonal (1:500; 9154S, Cell Signaling Technology), rabbit anti-ERK1/2 polyclonal (1:300; p44/42 MAP Kinase antibody, 9102, Cell Signaling Technology), rabbit antiphospho-ERK1/2 polyclonal (1:500; Phospho-p44/42 MAP Kinase antibody, 9101S, Cell Signaling Technology), mouse anti--actin monoclonal (1:3000; ab6276, Abcam, Cambridge, UK) and mouse anti-RPE65 monoclonal (1:500; MAB5428, Millipore, MA, USA). The secondary antibodies used: biotinylated goat anti-rabbit IgG (1:300; BA-1000, Vector), biotinylated goat anti-mouse IgG (1:300; BA-9200, Vector) and FITC conjugated goat anti-mouse IgG (1:150; 115-035-003, Jackson Immuno Research, West Grove, USA). For IHC, tissue sections were prepared from normal eyeballs immediately (0 min) and at 30, 60 and 120 min after retinectomy [16]. To localize MEK and ERK proteins in these tissues, immunoperoxidase labeling was carried out as in [2] with some modifications: in this study, HRP-labeled streptavidin (#426062, Nichirei
Fig. 1. Expression and activity of MEK/ERK proteins in the intact RPE cells. (A) Immunoblotting of proteins extracted from either retina-less eye-cups (RLECs) or RPE-choroid tissues isolated from RLECs. In both samples, MEK1, MEK2, ERK1 and ERK2, and their phosphorylated forms (p-MEK1, p-MEK2, p-ERK1 and p-ERK2) were detected, although in the RPE-choroid tissues the amount of MEK1 and MEK2 was lower than that in the RLECs. Control: negative control with no primary antibody. (B) RPE in a section of the normal eyeball. Top: the choroid side; bottom: the neural retina side. (C–G) Immunolabeling of sections of the normal eyeball with either MEK1/2- (C), p-MEK1/2- (D), ERK1/2- (E) or p-ERK1/2-antibody (F), and a negative control with no primary antibody (G). Melanin pigments in these sections were bleached. Arrows in each panel indicate the nuclei of RPE cells. The nucleus indicated by the arrow* is enlarged in the right-hand panel. Scale bar: (G) (for B–G) 50 M.
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Fig. 2. Change in the expression and activity of MEK/ERK proteins in RPE cells after retinectomy. (A) Immunohistochemistry with p-ERK1/2-antibody. Sections were prepared from eyes right after retinectomy (0 min) and from those at 30 and 60 min after retinectomy. Orientation of the tissues was the same as that in Fig. 1. Horizontal bars indicate the thickness of the RPE layer. Immunoreactivity of RPE cell nuclei increased obviously at 30 and 60 min after retinectomy (arrows). Melanin pigments in these tissues were bleached. Control: negative control with no primary antibody. Scale bar: 100 M. For immunoreactivity to either MEK1/2-, p-MEK1/2- or ERK1/2-antibody, see Supplementary Fig. S1. (B) Immunoblotting with MEK1/2-, p-MEK1/2-, ERK1/2- and ERK1/2-antibody. Proteins were extracted from eye-cups right after retinectomy (0 min) and those at 30, 60 and 120 min after retinectomy. RPE-specific protein RPE65 (∼63 kDa) was used as the internal control. Under this condition, the amount of MEK1 and p-MEK1 was too low to be detected.
Bioscience, Tokyo, Japan) was applied to minimize background staining. RPE cells were always identified by simultaneous FITCimmunofluorescence labeling of RPE65. After labeling, melanin pigments in the tissues were bleached [2]. For IB, protein samples were collected as in [21] from (i) RLECs prepared from eye-cups which had been incubated in either PBS or U0126-containing PBS for 1 h at RT, (ii) RPE-choroid tissues isolated from the RLECs in PBS at RT, and (iii) posterior halves of retinectomized eyes which were collected from animals immediately (0 min) and at 30, 60 and 120 min after surgery (the retinectomized eye was cut into posterior and anterior halves in chilled PBS in 3 min). SDS-PAGE and IB were carried out [16]. Protein bands were quantified using a function of ImageJ 1.44p software
(Wayne Rasband, National Institutes of Health, USA; http://imagej.nih.gov/ij). RPE65 was used as the internal control. 2.3. Data analysis Images of tissues were acquired using a colour CCD camera (C4742-95 ORCA-ER system, Hamamatsu Photonics, Japan) connected to a personal computer. Figures were prepared using Photoshop Extended CS5. Image, brightness, contrast, and sharpness were adjusted. Statistical data were presented as the mean ± SEM. Non-parametric tests were carried out to evaluate the statistical significance of the data, using Ekuseru-Toukei 2008 software (Social Survey Research Information, Tokyo, Japan).
Fig. 3. Semi-quantitative analysis of the changes in the expression and activity of ERK1 (A–C) and ERK2 (D–F) proteins in RPE cells after retinectomy. Immunoblotting with ERK1/2- and p-ERK1/2-antibody was carried out as done in Fig. 2B, and data sets from 6 independent rounds were analyzed. The values were normalized against those at 0 min. Statistical differences according to the Mann–Whitney’s U-test are indicated by *P < 0.05 and **P < 0.01.
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Fig. 4. Effects of U0126 on the expression and activity of MEK2 (A–C), ERK1 (D–F) and ERK2 (G–I) proteins in RPE cells in 1 h after preparation of the eye-cup. After eye-cups were prepared from normal eyeballs and incubated in PBS containing either 0.25% DMSO only (Mock) or 5 M U0126 + 0.25% DMSO for 1 h, RLECs were prepared by removing the neural retina from the eye-cup and used for immunoblotting. Data sets from more than 5 independent rounds (>4 RLECs/round) were analyzed as done in Fig. 3. The values were normalized against those of Mock. n: number of rounds. Statistical differences are indicated by *P < 0.05; for the Sheffe’s test following the Friedman test, black asterisks; for the Jonckheere–Terpstra test, white asterisks. Note that the statistical difference in (F) was P = 0.058 according to the Jonckheere–Terpstra test.
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The original research reported herein was performed under the guidelines established by the University of Tsukuba Animal Use and Care Committee. 3. Results 3.1. Expression and activity of MEK/ERK proteins in the intact RPE cells IB was carried out with protein extracts from either RLECs or RPE-choroid tissues that were prepared from normal eyeballs; in these samples, both MEK and phosphorylated MEK (p-MEK) antibodies labelled two identical bands (∼40 and ∼38 kDa) corresponding to MEK1 and MEK2, and both ERK and phosphorylated ERK (p-ERK) antibodies also labelled two bands (∼39 and ∼37 kDa) corresponding to ERK1 and ERK2, respectively (Fig. 1A). To localize these proteins in the posterior eye, IHC was carried out with sections of normal eyeballs; every antibody lightly labelled both the cytoplasm and nuclei of almost all RPE cells (Fig. 1B–G), whereas positive signals were not obvious in other tissues in the posterior eye except for the NR (data not shown). These results confirm the presence of a MEK–ERK signaling cascade in intact RPE cells. 3.2. Change in the expression and activity of MEK/ERK proteins in RPE cells after retinectomy IHC was carried out with eye section at 0, 30 and 60 min after retinectomy; change in immunoreactivity along the RPE was not clear in either MEK-, p-MEK- or ERK-antibody, but p-ERK immunoreactivity in the nuclei of RPE cells obviously increased in 30 min (Fig. 2A and Supplementary Fig. S1). Such nuclear localization of p-ERK suggests activation of ERK-mediated signaling [4]. It must be noted that during this period, no apparent changes in morphology occurred in most tissues including the RPE in the posterior eye, except for the choroid in which, like the inflammatory response, the capillaries started to expand, so a large amount of blood was supplied to the choroid, and that some cells, possibly of leukocytes, which were labeled with every antibody appeared in the blood (data not shown). IB was carried out with protein extracts from eye-cups prepared from eyes at 0, 30, 60 and 120 min after retinectomy. Fig. 2B shows a particular data set. In this case, both MEK2 and its active form (pMEK2) seemed to increase in 30 min after retinectomy, and then the level of MEK2 was almost sustained until 120 min, whereas the level of p-MEK2 gradually decreased. ERK1 and ERK2 also increased in 30 min, but both decreased thereafter. Correlated with those changes, their active forms (p-ERK1 and p-ERK2) also increased in 30 min and then decreased. Semi-quantitative analysis was carried out with data sets of IB from more than 3 independent rounds (>4 eye-cups/round); unexpectedly, significant changes were not observed in either the amount of MEK2 or its activity (calculated by p-MEK2/MEK2) in this period (data not shown). On the other hand, the amount of both ERK1 and ERK2 were, and their active forms (p-ERK1 and p-ERK2) were also, increased significantly in 30 min after retinectomy and then decreased (Fig. 3). However, interestingly, their activities (pERK1/ERK1 and p-ERK2/ERK2) did not change in this period (Fig. 3C and F). Thus, immediately after retinectomy, ERK-mediated signaling in RPE cells was likely to be reinforced through up-regulation of ERK protein expression. On the other hand, regulation of MEK (upstream kinase of ERK) after retinectomy was obscure. Since the amount of MEK proteins in the RPE is assumed to be much smaller than that of ERK proteins (Fig. 1A), the quantification of MEK proteins in the RPE by IB might have been seriously affected by the inflow of
Fig. 5. Effect of 1-h treatment of eye-cups by U0126 on the cell-cycle entry of RPE cells in the following 10-day RLEC culture. RLECs prepared from eye-cups, which had been incubated in PBS containing either 0.25% DMSO only (Mock) or 5 M U0126 + 0.25% DMSO for 1 h, were cultured in the presence of BrdU for 10 days. BrdU-positive (BrdU+) nuclei of RPE cells in the RLEC were counted at 10 days, and the ratio of BrdU + RPE cells was calculated against the total cell number in the whole RPE sheet. n: number of RLECs. Statistical difference according to the Sheffe’s test following the Friedman test is indicated by *P < 0.05.
MEK/ERK-positive blood cells from outside the eye. Therefore, to avoid this possible noise due to blood inflow, we used an in vitro system in the following experiments. 3.3. Effects of a MEK1/2-specific inhibitor U0126 on initial changes in the expression and activity of MEK/ERK proteins in RPE cells IB was carried out with a protein extract from RLECs prepared from eye-cups which had been incubated in PBS containing 5 M U0126 and its solvent 0.25% DMSO for 1 h, and the expression and activity of MEK/ERK proteins were compared with those in the RLECs prepared from eye-cups treated only with solvent (Fig. 4); the amount of MEK2, ERK1 and ERK2, and their active forms (p-MEK2, p-ERK1 and p-ERK2) after 1-h incubation decreased significantly in the presence of U0126; the activity of ERK1 (p-ERK1/ERK1) tended to be and that of ERK2 (p-ERK2/ERK2) was significantly suppressed in the presence of U0126, whereas that of MEK2 (p-MEK2/MEK2) was not affected. These results suggest that MEK-mediated activation of ERK occurs in RPE cells during 1-h incubation of the eye-cup in PBS, and that this signal activity up-regulates the expression of constituent proteins of the MEK–ERK cascade, reinforcing the signal activity itself. 3.4. Effect of 1-h inhibition of initial MEK–ERK signaling on the cell-cycle entry of RPE cells RLECs prepared after 1-h incubation of eye-cups in the presence of U0126 were cultured in 80% L-15 medium containing 5 g/ml BrdU for 10 days, and then BrdU-positive (BrdU+) nuclei of RPE cells were counted; the ratio of BrdU+ RPE cells was significantly low compared with that of a mock control in which the eye-cups were treated with the solvent only (Fig. 5), suggesting that 1-h inhibition of MEK–ERK signaling seriously affected cell-cycle entry of RPE cells in the following 10 days. 4. Discussion The current in vitro study suggests that immediate early activation of the MEK–ERK signaling pathway may occur in RPE cells following NR injury, intensifying the MEK–ERK signaling itself through up-regulation of the expression of constituent molecules in the pathway (i.e., via a positive feedback). Such a positive feedback in MEK–ERK signaling has been reported in other systems
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[14,20]. The current in vivo study demonstrated that, as suggested in vitro, ERK-mediated signaling in RPE cells is strengthened quickly (within 30 min) after retinectomy, although changes in the expression and activity of MEK in RPE cells were not able to be determined. In IB analysis, the amount of ERK and p-ERK proteins appeared to decrease gradually after 30 min (Figs. 2 and 3). Consistently, in the IHC of eye sections at 6 h after retinectomy, p-ERK-immunoreactivity of RPE cells was almost the same as that of the 0-min sample (data not shown). Taken together, MEK–ERK signaling in RPE cells is likely to be strengthened temporarily correlating with the onset of retinal regeneration. This event may be a key process for mitotically quiescent RPE cells to re-enter the cell-cycle upon NR injury. In mammals, such immediately early activation of MEK–ERK signaling in RPE cells is known to occur upon NR injury such as in retinal detachment [4]. In humans, MEK–ERK signaling is involved in proliferation and metaplastic transformation of RPE cells, sometimes leading to pathogenesis such as PVR [7,9,13]. Therefore, it is interesting to address whether the same signalling pathway involved in PVR works in adult newts as a constituent of the trigger signal for retinal regeneration. We have just initiated experiments aimed at trying to regulate MEK–ERK activity in RPE cells in vivo by intraocular drug administration and transgenesis. Acknowledgements This work was supported by MEXT (23124502) and JSPS (20650060; 24650229; 21300150). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.neulet.2012.06.037. References [1] M.N. Casco-Robles, S. Yamada, T. Miura, K. Nakamura, T. Haynes, N. Maki, K. Del Rio-Tsonis, P.A. Tsonis, C. Chiba, Expressing exogenous genes in newts by transgenesis, Nature Protocols 6 (2011) 600–608. [2] C. Chiba, A. Hoshino, K. Nakamura, K. Susaki, Y. Yamano, Y. Kaneko, O. Kuwata, F. Maruo, T. Saito, Visual cycle protein RPE65 persists in new retinal cells during retinal regeneration of adult newt, The Journal of Comparative Neurology 495 (2006) 391–407.
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