Retinoic acid enhances Erk phosphorylation in the chick retina

Neuroscience Letters 426 (2007) 18–22

Retinoic acid enhances Erk phosphorylation in the chick retina Eric Kampmann, J¨org Mey ∗ Institut f¨ur Biologie II, RWTH-Aachen, Kopernikusstrasse 16, D-52074 Aachen, Germany Received 24 May 2007; received in revised form 13 July 2007; accepted 17 July 2007

Abstract The transcriptional activator retinoic acid (RA) is a regulator of neural development and regeneration. Synergistic effects with brain-derived neurotrophic factor suggested that RA influences neurotrophin signaling. To test this hypothesis RA was administered systemically to E17 chick embryos, and retinas were prepared 12 h and 24 h later to measure mRNA or protein expression. While there was no significant influence on activation of Akt, CREB and STAT-3, RA-treatment caused elevated levels of Erk-phosphorylation, a kinase involved in Trk signaling. A small but significant increase in the expression of TrkB mRNA and protein was observed but no significant change in TrkA, TrkC and p75 expression. © 2007 Published by Elsevier Ireland Ltd. Keywords: Regeneration; Transcription factor; Neurotrophin; Retinoids

The transcriptional activator retinoic acid (RA) has recently been investigated as a potential therapeutic tool to induce axonal regeneration [5,17]. After synthesis from blood-derived vitamin A by local aldehyde dehydrogenases, RA is secreted and diffuses through cell membranes to exert its activity by binding to specific nuclear receptors, which act as ligand-activated transcription factors [3]. In addition, nongenomic modes of actions are known for RA, e.g., direct inhibition of NF␬B or phosphorylation of Erk [4,5]. Positive effects of RA on neuronal survival and axonal regeneration were shown in vitro and in vivo [5,17,21,25].

Abbreviations: BCA, bicinchoninic acid; BDNF, brain derived neurotrophic factor; CNS, central nervous system; CREB, cAMP response element binding protein; DMSO, dimethyl sulfoxide; DRG, dorsal root ganglion; E, embryonic day; Erk, extracellular signal regulated kinase; Erk-P, phosphorylated Erk; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAP kinase, mitogen activated protein kinase; NF␬B, nuclear factor kappa B; NGF, nerve growth factor; NT, neurotrophin; PNS, peripheral nervous system; RA, retinoic acid; RAR, retinoic acid receptor; RARE, retinoic acid response element; RGC, retinal ganglion cell(s); RT-PCR, reverse transcription polymerase chain reaction; RXR, retinoid X receptor; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; S.E.M., standard error of the mean; STAT, signal transducer and activator of transcription; Trk, tropomyosin related kinase (or tyrosine kinase) ∗ Corresponding author at: Institut f¨ ur Biologie II, RWTH-Aachen, Kopernikusstrasse 16, D-52056 Aachen, Germany. Tel.: +49 241 8024852; fax: +49 241 8022133. E-mail address: [email protected] (J. Mey). 0304-3940/$ – see front matter © 2007 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2007.07.039

Experimental lesions of retinal ganglion cell axons have provided important insights into the molecular mechanisms required for axonal growth in the CNS [12]. The retinotectal pathway of the chick is a suitable model to investigate the neurotrophic potential of RA because RA is an important morphogenetic factor in development of the visual system and because it has been shown to enhance neurotrophin-dependent fiber growth from chick retina explants [18]. In adult birds, as in mammals, retinal ganglion cells do not regenerate within the optic nerve. Since eye development in precocial bird species, including the chicken, is completed at the time of hatching, the chick retina shortly before hatching can be used for the investigation of factors that allow or inhibit regeneration. The neurotrophins are intercellular messengers implied in neuronal differentiation and survival that can also support regeneration [16]. They activate the receptor tyrosine kinases TrkA, TrkB, and TrkC, which are localized on the cell surface and display overlapping affinities to specific neurotrophins: NGF activates TrkA, BDNF and NT-4/5 activate TrkB, and NT-3 binds to TrkC. Another neurotrophin receptor, which belongs to the tumor necrosis factor receptor family, is p75. It binds all neurotrophins with low affinity and is often coexpressed with Trks. It can facilitate neurotrophic effects of Trk signaling but is also a trigger of apoptosis [15]. For retinal ganglion cells BDNF is the neurotrophin with best-characterized effects on cell survival and axonal growth [19,24]. Although RA treatment alone did not increase axonal regeneration from retinal ganglion cells, a preceding study revealed a

E. Kampmann, J. Mey / Neuroscience Letters 426 (2007) 18–22

synergistic effect of RA on BDNF-dependent neurite outgrowth in cultivated retinal explants [18]. It was conjectured that this may have been caused by an effect of RA on BDNF/TrkB signaling. Since systemic RA treatment is used in oncology and dermatology its pharmacological properties are well described. As a small, fat-soluble molecule RA passes the blood brain barrier. The present study was undertaken to test whether systemic application of all-trans RA affects the expression of neurotrophin receptors and/or has an influence on the phosphorylation of downstream signals. Experiments were performed with White Leghorn chick embryos (Gallus gallus domesticus), supplied by a local poultry farm (Van den Boom, Kelpen, NL). Eggs were opened at embryonic day (E)3 and embryos were treated in ovo at E17 as described previously [18]. A single dose of 10 ␮l 1 mM all-trans RA (Sigma R2625; aliquots diluted in DMSO) was dropped once onto the chorio-allantoic membrane. As a control procedure 10 ␮l DMSO were given. After 12 h and 24 h the retinas were prepared for RNA (right eye) and protein (left eye) extraction. Isolation of total retinal RNA was performed with Trizol (Invitrogen). RNA-extracts were treated with deoxyribonuclease I and reverse transcribed to cDNA using Omniscript Reverse Transcription (Qiagen), oligo(dT)12–18 primer and RNase out (all Invitrogen) with calculated concentrations of 50 ng/␮l total RNA. Primers (IBA-NAPS, G¨ottingen, Germany) with the following sequences were used at 0.5 ␮M; TrkA, sense: GTG GGA TCA GAG CCA TCT GT, anti-sense: CAG AGA GGA GCA GGA TCA CC, product size: 190 bp; TrkB, CCC AAA CTG CGA CTT ACC AT, ACA GTG AAT GGA ATG CAC CA, 347 bp; p75, AAG AAG CCA ACG AAG AAG CA, GGT TAT CCA CCT TCA AGG CA, 243 bp; TrkC, TCC TCT GGG AGA TCT TCA CCT A, GGA TGT CCA GGT AGA TTG GTG T, 237 bp; GAPDH, CCT CTC TGGC AAA GTC CAA G, CAT CTG CCC ATT TGA TGT TG, 209 bp; RAR␤, CGT AGC ATC AGT GCA AAA GG, TGC ACC ATA GGG GAT TGA CT, 212 bp. Samples were diluted 1/10 after reverse transcription, and 2 ␮l were used for amplification with the Light Cycler (Roche). Reactions were carried out in 10 ␮l volume/capillary with the QuantiTect kit (Qiagen) containing 5 ␮l Qiagen PCR master mix, 1 ␮l primers, 2 ␮l nuclease-free water, and the cDNA sample. The experimental protocol consisted of 900 s enzyme activation at 95 ◦ C, followed by 45 cycles of 15 s denaturation 94 ◦ C, 20 s annealing 50 ◦ C, 20 s amplification 72 ◦ C, and 5 s fluorescence measurement at 80 ◦ C, followed by melting curve analysis and agarose gel electrophoresis. RT-PCR amplification of GAPDH was used to normalize expression of target genes. Relative mRNA concentrations were determined using crossing point analysis of log/linear plots of fluorescence/cycle number (crossing line at 0.02). For every primer pair we calibrated PCR efficiency with concentration curves of retina extracts. For protein extracts, the retinas were briefly sonicated in lysis buffer (20 mM HEPES, 2% Triton X-100, 1 mM PMSF, 1 ␮M Leupeptin, 1% Aprotinin, pH 7.4) and centrifuged to remove insoluble material (15,000 × g for 15 min). Protein extracts were separated with SDS-PAGE (20 ␮g per lane, determined with BCA protein assay) and transferred to nitrocellulose mem-

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branes. The primary antibody against TrkB (rabbit polyclonal serum, dilution 1/5000) was kindly provided by Dr. Lefcort, Montana State University. Erk, Erk-P, STAT-P and CREB-P were detected with rabbit polyclonal antisera from Cell Signaling Technology (#9102, #9101, #9131, all 1/1000) and Upstate (#06-519, 1/1000). Akt-P was stained with a rabbit monoclonal antibody (Cell Signaling Technology #4058, 1/1000) and p75 with a mouse monoclonal antibody (Chemicon, MAB365, 1/500). Peroxidase-coupled secondary antibodies were purchased from Sigma (anti-rabbit IgG: A6154; anti-mouse IgG: A3682) and detected with the enhanced chemiluminescence method (rapid protocol, Amersham Biosciences). Films were exposed for 30 s, 1, 2, 5, 10 and 20 min, and exposures in the linear range of intensity, i.e., before photochemical saturation, were selected for densitometric evaluation with a digital image analysis system. To check the protein content of each sample after blotting, all membranes were stained with Ponceau S. For normalization of immunoreactive signals Western blots were stripped and probed with a rabbit actin antiserum (Sigma A-2066, 1/500). In accordance with previous analyses of gene expression [14] we detected immunoreactivity of the retinoic acid receptors RAR␣/␤ and RXR␣/␤/␥ in the chick retina (data not shown). Transcriptional or cytosolic effects of retinoic acid are therefore possible. To determine whether RA affected the intracellular pathway of neurotrophin signaling we determined the Erk-P/Erk ratio. Erk-1 and Erk-2 belong to the family of mitogen-activated protein (MAP) kinases, which are phosphorylated after Trk activation. Their activity is necessary for the neuritogenic effects of BDNF [2,16]. Chickens only express the homologue of mammalian Erk-2 [23]. Western blots from retina extracts were immunostained with antibodies against Erk-P and Erk (Fig. 1a). After 12 h of RA incubation the Erk-P/Erk ratio was elevated by a factor of 1.43 ± 0.13 and significantly after 24 h by a factor of 1.74 ± 0.20 (mean ± S.E.M., n = 7; t-test, p < 0.05). In addition, we assessed the concentration of phosphorylated Akt, CREB and STAT-3. Intracellular signals of PI3K (Akt), and cAMP (CREB) were not affected by RA in our experiments. Phosphorylation of the transcription factor STAT-3, which is induced by IL-6 type cytokines, was found in peripheral nerve regeneration and after noxious stimulation in the retina but not after RA treatment [27]. We also detected no differences following RA treatment of the embryos (Fig. 1). For the induction of retinal gene expression, E17 chick embryos were treated 12 or 24 h with 10 nmol all-trans RA in 10 ␮l DMSO. Conventional RT-PCR amplification resulted in single bands of PCR products for each of the four neurotrophin receptors TrkA, TrkB, TrkC, p75 and for GAPDH. Effects of RA treatment on mRNA concentrations of these genes were quantified with Light Cycler RT-PCR. Fig. 2a–d shows amplification plots from experiments using the retinas of one RA treated and one control embryo. The left shift of the corresponding PCR curve demonstrates that the expression of TrkB was increased after 24 h RA treatment, while transcript concentrations of TrkA, TrkC, p75 and GAPDH were the same as under the control condition. Statistical analysis of all experi-

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Fig. 1. Effect of RA treatment on phosphorylation of intracellular signals of neurotrophins and cytokines in the chick retina. (a) Specific antibodies against Erk and phosphorylated Erk (Erk-P) demonstrated that the Erk-P/Erk ratio was elevated as a consequence of RA treatment. Phosphorylated STAT-3, Akt and CREB levels did not change; Ponceau-S staining of corresponding molecular weight proteins below indicates sample loading. (b) Quantification of immunoblots, white columns show results from control experiments and grey columns from RA-treated embryos. Error bars indicate S.E.M., *p < 0.05, n = 5–7.

ments (Fig. 2e) revealed that RA treatment almost doubled gene expression of TrkB to 1.90 ± 0.19 (mean ± S.E.M.), which was significant (n = 7; p < 0.05). The apparent mRNA reduction of p75 (0.62 ± 0.14; n = 8) and TrkC (0.68 ± 0.10; n = 6) after RA treatment was not significant, and changes were also not statistically significant after 12 h treatment. The retinoic acid receptor RAR␤, a known target of RA, was significantly upregulated by the treatment (1.21 ± 0.04; n = 6; p < 0.05). Melting curves showed single, characteristic peaks for all PCR products, and all PCR fragments obtained with the Light cycler had the expected sizes. To confirm the effect of RA, protein concentrations were measured with SDS-PAGE and Western blotting. As there was a downward tendency in p75 mRNA expression after RA treatment, the p75 immunoreactivity was assessed together with TrkB. Embryos were treated as described for PCR analysis. The antibodies against p75 detected two bands with relative molecular weights of about 37 and 45 kDa. RA treatment caused no concentration differences of either isoform (Fig. 3). Changes of the signal were 1.00 ± 0.10 and 0.97 ± 0.08 for the upper and lower band, respectively (n = 6, n.s.). We used a wellcharacterized antibody against the extracellular domain of chick TrkB [20]. This antibody showed three immunoreactive bands with calculated molecular weights between 73 and 125 kDa (Fig. 3). For chick TrkB several splice forms are known, some of which are truncated proteins that lack the intracellular tyrosine kinase domain [http://www.pir.uniprot.org/index.shtml; primary accession no.: Q91987; annotated 2006-10-31 (entry version 67)]. The protein concentrated in the high molecular weight band was considered to be the full-length receptor, and

the proteins of the two remaining bands were assumed to be shorter splice variants. Following RA administration the concentration level of the full-length receptor was elevated by a factor of 1.41 ± 0.19 after 12 h and by 1.16 ± 0.04 after 24 h (n = 5, p < 0.05). Among the lower TrkB immunoreactive bands a protein with 73 kDa also showed an RA-dependent increase of 1.45-fold after 24 h (S.E.M. = 0.07, n = 5, p < 0.05) but no significant change after 12 h. The other splice variant was not affected by the treatment. The positive effect of RA on Erk phosphorylation is in accordance with a synergistic effect of RA on BDNF-induced regeneration of retinal ganglion cells published before, because BDNF/TrkB signaling also acts via activation of this kinase. Whether the small RA-induced increase of TrkB levels can account for this observation remains open, especially since a direct effect of RA on Erk phosphorylation was demonstrated in neuronal cultures from embryonic rat cortex [4]. In addition, a number of other intracellular signal transduction pathways converge on the phosphorylation of MAP kinases. Several publications report interactions between the neurotrophin and retinoic acid signaling pathways. In dorsal root ganglia NGF appears to be upstream of the RA synthesizing retinaldehyde dehydrogenase 2 [6], whereas in developing sympathetic neurons RA regulates the expression of neurotrophin receptors [13]. NGF in return may induce the expression of RAR␤ [7]. Neurotrophins and their receptors are involved in cell proliferation, survival and differentiation and are expressed in the retina of all vertebrates examined so far. The response of nerve cells to neurotrophins may change during ontogenesis, e.g., chick RGC develop a sensitivity for BDNF during

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Fig. 2. Effect of RA treatment on mRNA expression of neurotrophin receptors. (a–d) Examples of quantitative RT-PCR curves of TrkA (a), TrkB (b), TrC (c) and p75 (d); Sybr-Green fluorescence, plotted against cycle number increases as a function of the increasing concentration of PCR products. Dotted lines represent results from RA-treated tissue, continuous lines the control condition. In (b) a control PCR amplification without reverse transcription is shown. Arrow heads indicate crossing points. (d) Statistical analysis of five to eight independent experiments show a significant, positive effect of RA on the expression of TrkB. Grey columns show data from non-treated and black columns from RA-treated samples. Error bars indicate S.E.M., *p < 0.05.

the time when retinofugal axons innervate central targets [22]. In the visual system of ferrets the ratio of full-length to truncated TrkB splice variants lacking the tyrosine kinase domain is high at early developmental stages, declines during the period of ganglion cell death, and adult animals have truncated variants as the predominant receptor in the retina [1]. In the E18 chick we found at least three different isoforms of TrkB with similar levels of immunoreactivity. The function of the shorter TrkB proteins can only be guessed, because different studies about truncated TrkB forms suppose inhibitory [11] or activating functions with respect to BDNF-signaling or neurite outgrowth [26]. Treatment with RA raised expression of the non-truncated form, which mediates classic neurotrophin signaling. The antibody against the low-affinity neurotrophin receptor p75 detected two protein bands of about 37 and 45 kDa, which were not affected by RA treatment. According to its amino acid sequence the chick p75 protein is expected to have a size of 19 kDa [http://www.ensembl.org/Gallus gallus; peptide id: ENSGALP00000020402 (release 42, December 2006)], however this is without glycosylation. Dechant et al. [9] described

crosslinked NT3–p75 complexes from chicken to migrate at 60 and 80 kDa, which is in accordance with our results of p75 immunoreactivity not linked to the neurotrophin. Of two known splice variants of this receptor the shorter form differs from the full-length protein by the absence of three extracellular cysteinerich domains and has no affinity to neurotrophins. Neurotrophins were thought of as retrograde signaling molecules, which are taken up in axonal target zones and transported retrogradely to the cell body, but they may also act in an autocrine or paracrine fashion and can be anterogradely transported as well [28]. Both, the Erk and the PI3-kinase pathways are typically activated by neurotrophins. The PI3-kinase pathway with its downstream kinase Akt has been implicated in neurite outgrowth. Other studies postulate that the MAP kinase activity is required or that both pathways are necessary. By blocking these pathways in sensory neurons of mice Markus et al. [16] showed that Erk is mainly involved in neurite outgrowth whereas Akt is more important for terminal branching and axon calibre growth. Akt posphorylation was not influenced by RA treatment in our experiments. In accordance with this concept

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Fig. 3. Effect of RA treatment on protein concentrations of p75 and TrkB. Western blot analysis of retinal extracts after the indicated time of RA exposure in vivo. Plots show quantification of the immunoreactive signals of full-length proteins. The TrkB immunoreactive bands corresponding to the full-length receptor (fl) and one truncated form (*) were increased after RA treatment. Error bars indicate S.E.M., *p < 0.05, n = 5–7.

there is further evidence that Erk phosphorylation is important for neurite outgrowth in chicken retinal ganglion cells [10]. Our results imply that RA signaling may contribute to this process by inducing TrkB expression and enhancing Erk phosphorylation. In addition to retinal ganglion cells, other retinal cell types that express RARs [14] may be involved. Our data also show that systemically administered RA, which was applied to extra-embryonic blood vessels, influences the CNS, confirming a number of previous studies with mice and rats, where intraperitoneal injections of RA elicited effects on brain physiology and behavior [8]. Acknowledgements We thank Frances Lefcort, Montana State University for the TrkB antiserum. Nanette Rombach established the quantitative RT-PCR method in the laboratory. References [1] K.L. Allendoerfer, R.J. Cabelli, E. Escandon, D.R. Kaplan, K. Nikolics, C.J. Shatz, Regulation of neurotrophin receptors during the maturation of the mammalian visual system, J. Neurosci. 14 (1994) 1795–1811. [2] J.K. Atwal, B. Massie, F.D. Miller, D.R. Kaplan, The TrkB-Shc site signals neuronal survival and local axon growth via MEK and PI3-kinase, Neuron 27 (2000) 265–277. [3] J. Bastien, C. Rochette-Egly, Nuclear retinoid receptors and the transcription of retinoid-target genes, Gene 328 (2004) 1–16. [4] E. Ca˜no´ n, J.M. Cosgaya, S. Scsucova, A. Aranda, Rapid effects of retinoic acid on CREB and ERK phosphorylation in neuronal cells, Mol. Biol. Cell 15 (2004) 5583–5592. [5] M. Clagett-Dame, E.M. McNeill, P.D. Muley, The role of all-trans retinoic acid in neurite outgrowth and axonal elongation, J. Neurobiol. 66 (2006) 739–756. [6] J. Corcoran, M. Maden, Nerve growth factor acts via retinoic acid synthesis to stimulate neurite outgrowth, Nat. Neurosci. 2 (1999) 307–308.

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