- Email: [email protected]
Expression and physiological regulation of BDNF receptors in the neuroendocrine melanotrope cell of Xenopus laevis
General and Comparative Endocrinology 153 (2007) 176–181 www.elsevier.com/locate/ygcen
Expression and physiological regulation of BDNF receptors in the neuroendocrine melanotrope cell of Xenopus laevis Adhanet H. Kidane, Sander H.J. van Dooren, Eric W. Roubos, Bruce G. Jenks
*
Department of Cellular Animal Physiology, Integrative Physiology, EURON European Graduate School of Neuroscience, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands Received 29 September 2006; revised 28 March 2007; accepted 1 April 2007 Available online 11 April 2007
Abstract Brain-derived neurotrophic factor (BDNF) and a-melanophore-stimulating hormone (a-MSH) are co-sequestered in secretory granules in melanotrope cells of the pituitary pars intermedia of the amphibian Xenopus laevis. a-MSH is responsible for pigment dispersion in dermal melanophores during the process of black-background adaptation. BDNF-production in melanotrope cells is increased by placing animals on a black background, and BDNF acts as an autocrine stimulatory factor on the melanotrope cells. However, the repertoire of possible neurotrophin receptors of the melanotrope is unknown. In this study we have established the expression of full length TrkB (TrkB.FL), truncated TrkB (TrkB.T) and p75NTR receptors in the Xenopus neurointermediate lobe by RT-PCR. In situ hybridization reveals the presence of TrkB.FL mRNA and p75NTR mRNA in melanotrope cells. Quantitative RT-PCR shows that in animals on a black background the amounts of TrkB.T and p75NTR mRNA are about three times higher than in white backgroundadapted animals. We suggest that the amount of p75NTR sets the sensitivity of the melanotrope cells for the stimulatory action of BDNF during physiological adaptation to background light intensity. 2007 Elsevier Inc. All rights reserved. Keywords: BDNF; TrkB; p75NTR; Melanotrope; a-MSH; Pars intermedia
1. Introduction The actions of neurotrophins can be very diverse because these growth factors have multiple receptors, each receptor having its own unique signaling properties. The best known neurotrophin receptors belong to the tropomyosin receptor kinase (Trk) family of receptors, which signal through their intrinsic tyrosine kinase activity to promote growth (Chao, 2003). Signaling by Trk receptors involves dimerization of receptor molecules leading to intramolecular phosphorylation, a prerequisite for the activation of intracellular signaling cascades (Ullrich and Schlessinger, 1990; Jing et al., 1992). Truncated Trk receptors, lacking the intracellular kinase domain, have been thought to act as negative effectors of full-length receptors (Luikart
*
Corresponding author. Fax: +31 243652714. E-mail address: [email protected] (B.G. Jenks).
0016-6480/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2007.04.001
et al., 2003), although there is recent evidence that such truncated receptors also may have signaling properties themselves (Ohira et al., 2005). A third important neurotrophin receptor is the p75 neurotrophin receptor (p75NTR). While this receptor has low affinity for the neurotrophins, it can form a complex with Trk receptors to form high-affinity binding sites for neurotrophins, enabling the receptor to participate in the stimulation of growth processes (Esposito et al., 2001). The signaling pathway of p75NTR is very versatile, depending on the type of ligand and on the nature of the receptor binding partners. The receptor displays high affinity binding with precursor forms of neurotrophins (pro-neurotrophins) and can induce apoptosis by interacting with sortillin (Lee et al., 2001; Nykjaer et al., 2004). In contrast, p75NTR inhibits growth by interacting with the Nogo receptor (Nykjaer et al., 2005). It is unclear how p75NTR selects between its partners and through which intracellular signaling cascade it exerts these opposite effects.
A.H. Kidane et al. / General and Comparative Endocrinology 153 (2007) 176–181
A suitable, well-defined cell type to study the modes of neurotrophin signaling is the neuroendocrine melanotrope cell in the intermediate lobe of the pituitary gland of the amphibian Xenopus laevis. This cell produces a-melanophore-stimulating hormone (a-MSH), a peptide that controls pigment dispersion in dermal melanophores during the process of adaptation to changed background light intensity (Weatherhead et al., 1971; Jenks et al., 2003; Roubos et al., 2005). The melanotrope cell produces not only a-MSH but also brain-derived neurotrophic factor (BDNF) (Kramer et al., 2002). The cell displays marked hypertrophy in animals adapting to a black background and its cell volume increase is accompanied by enhanced production of a-MSH (Martens et al., 1987) and BDNF (Kramer et al., 2002). BDNF and a-MSH are co-sequestered in and presumably co-released from melanotrope secretory granules (Wang et al., 2004). BDNF stimulates melanotrope cell production of the precursor protein of a-MSH, pro-opiomelanocortin (Kramer et al., 2002). Therefore, endogenous BDNF likely has an autocrine/paracrine stimulatory action on melanotrope cells (Kramer et al., 2002). This action proceeds presumably through one or more BDNF-specific receptors, but the repertoire of neurotrophin receptor expression by the melanotrope cell is unknown. The aim of the present study was to determine which of the receptors known to be involved in BDNF signaling, full length TrkB (TrkB.FL), truncated TrkB (TrkB.T) and p75NTR, are expressed by the Xenopus melanotrope cell and, furthermore, to test if BDNF receptor expression is physiologically regulated, by the background light condition. We show that Xenopus melanotropes produce various neurotrophin receptors and, moreover, that the expression of some of these receptors is differentially regulated during the process of background adaptation. 2. Materials and methods 2.1. Animals Young-adults of the South African clawed toad X. laevis, aged 6 months, were reared in our laboratory under standard conditions, kept in tap water at 22 C, and fed beef heart and trout pellets (Touvit, Trouw, Putten, The Netherlands). Full background skin adaptation was achieved by keeping the animals on a white or black background with continuous light, for 3 weeks. Animal treatment was in agreement with the Declaration of Helsinki and the Dutch law concerning animal welfare, as verified by the committee for animal experimentation of Radboud University Nijmegen.
2.2. RNA extraction and cDNA synthesis After decapitation, freshly dissected neurointermediate lobes (NILs) were individually collected in 500 ll ice-cold Trizol (Life Technologies, Paisley, UK) and homogenized by sonification. After chloroform extraction and isopropyl alcohol precipitation, RNA was dissolved in 25 ll RNAse-free H2O. Total RNA was measured with an Eppendorf Biophotometer (Vaudaux-Eppendorf AG, Basel, Switzerland). First strand cDNA synthesis was performed with 1 lg RNA and 5 mU/ll random primers (Roche, Mannheim, Germany), at 70 C for 10 min, followed by
177
double strand synthesis in strand buffer (Life Technologies) with 10 mM DTT, 20 U Rnasin (Promega, Madison, WI, USA), 0.5 mM dNTPs (Roche) and 100 U Superscript II reverse transcriptase (Life Technologies), at 37 C for 75 min and at 95 C for 10 min.
2.3. Reverse transcriptase polymerase chain reaction (RT-PCR) PCR was performed in a total volume of 25 ll in buffer containing 5 ll of template cDNA, 3 mM MgCl2, 0.625 U FastStart Taq DNA polymerase (Roche), 0.25 mM dNTPs (Roche) and 0.3 mM of each primer. Primers were designed for TrkB.FL (Accession No. S69713), TrkB.T (Accession No. BC044959), p75NTR (Accession No. AF246462) and GAPDH (Accession No. U41753), on the basis of the published X. laevis mRNA sequences. The following primer pairs were used: TrkB.FL, forward 5 0 -ACCTCTACCGCGAGCAAGAC-3 0 and reverse 5 0 -GAGT AACTCTGCTTCCCG ATGAA-3 0 (specific for the intracellular domain; product size 101 bp); TrkB.T, forward 5 0 -CAGCATTAGTTTGTACTGG CC-3 0 and reverse 5 0 -CTACCCATTCAGAGGAACCG-3 0 (product size 116 bp). The reverse primer for TrkB.T is specific for the unique short intracellular domain; p75NTR, forward 5 0 -CACTATCTGTGAGGACGG TG-3 0 and reverse 5 0 -GGGAAAGTCTGAGCTTGCTG-3 0 (product size 118 bp); and GAPDH, forward 5 0 -GCTCCTCTCGCAAAGGTCAT-3 0 and reverse 5 0 -GGGCCATCCACTGTCTTCTG-3 0 (product size: 118 bp). The optimum temperature cycling protocol was determined to be 95 C for 30 s, 58 C for 30 s and 72 C for 2 min, using a programmable thermal cycler (Eppendorf, Mastercycler gradient, Hamburg, Germany). After PCR, the reaction products were run on a 2% agarose gel and visualized with ethidium bromide to check the length of the amplified cDNA.
2.4. Quantitative RT-PCR Quantitative RT-PCR was performed in a total volume of 25 ll in a buffer solution containing 5 ll of template cDNA, 1· SYBR Green buffer (Applied Biosystems, Foster City, CA, USA), 3 mM MgCl2, 0.625 U AmpliTaq Gold and 0.2 mM dNTPs (Applied Biosystems), and 0.6 lM of each primer (the same primer sets as in RT-PCR were used). The optimum temperature cycling was 95 C for 10 min followed by 35 reaction cycles of 95 C for 15 s and 60 C for 1 min, using a 5700 GeneAmp PCR system (Applied Biosystems). For each reaction, the cycle threshold (Ct) was determined, i.e., the cycle number at which fluorescence was detected above an arbitrary threshold (0.8). At this threshold Ct-values are within the exponential phase of the amplification. To compare the relative amounts of the mRNA of interest in NILs from black- versus white-adapted animals, Ct-values were normalized to those for GAPDH, by subtracting the Ct-values for the RNA of interest from the Ct-values for GAPDH.
2.5. Preparation of DIG-labeled TrkB.FL and p75NTR riboprobes To synthesize a Xenopus riboprobe for in situ hybridization, PCR was used to amplify 297 and 281 bp PCR fragments of TrkB.FL (specific for the intracellular tyrosine kinase domain) and p75NTR, respectively. The forward and reverse primers for TrkB.FL were 5 0 -GAACCCGCAGTA CTTTGGAA-3 0 and 5 0 -GGGTCACCTTCCACACAAAC-3 0 , respectively, and for p75NTR 5 0 -AAGAGCGAACAGTGCAAACT-3 0 and 5 0 -GACG TCCCTGTGTAGGGAAA-3 0 , respectively. PCR products were checked for size in 2% agarose gel, ligated into pGEM-T plasmid (Promega) and sequenced to confirm identity. After linearization of the plasmid with speI or NcoI (Roche), 11-UTP digoxigenin (DIG)-labeled antisense and sense probes were prepared as run-off transcripts using T7 and SP6 RNA polymerase (Roche).
2.6. In situ hybridization Three black- and white-adapted animals were anesthetized by immersion in a solution of 0.1% tricaine methane sulfonate (MS22; Novartis, Basel, Switzerland) in tap water, and transcardially perfused with an
178
A.H. Kidane et al. / General and Comparative Endocrinology 153 (2007) 176–181
ice-cold 0.6% NaCl solution, for 5 min, followed by 250 ml Bouin’s fixative, for 15 min. After decapitation, the brain and the pituitary gland were dissected and postfixed by immersion in the same fixative, for 16 h at 4 C, rinsed in 70% ethanol to eliminate excess of picric acid, for 24 h, dehydrated in a graded ethanol series, and embedded in paraffin. Coronal sections (7 lm) were mounted on poly-L-lysine-coated slides and allowed to air-dry, for 16 h at 45 C, deparaffinized and rehydrated. Then sections were treated with 0.1% pepsin in 0.2 N HCl, for 15 min at 37 C, postfixed in 4% formaldehyde in sodium phosphate-buffered saline (PBS), for 5 min, incubated in 1% hydroxyl ammonium chloride, for 15 min, dehydrated in ethanol, and air-dried. Hybridization took place for 16 h at 62 C in hybridization buffer consisting of 10% sodium dextran sulfate, 50% formamide, four times concentrated standard saline citrate buffer (4· SSC; pH 7.0), 1· Denhardt’s and 200 lg/ml yeast t-RNA, with 500 ng/ml mRNA probe. Then sections were treated with 10 lg/ml ribonuclease A (Roche), for 30 min at 37 C, followed by stringency washes in 2· SSC, 1· SSC, 0.5· SSC, for 30 min at 20 C, and 0.1· SSC, for 30 min at 37 C. The alkaline phosphatase (AP) method with nitroblue tetrazolium chloride/ 5-bromo-4-chloro-3-indolyl phosphate, toluidine salt (NBT/BCIP) as substrate, was used to visualize DIG label. Sections were rinsed for 10 min in Tris-buffered saline (TBS), blocked in 1% bovine serum albumin (Sigma Chemical, St. Louis, MO) and 2% normal goat serum in TBS, for 30 min, and incubated in anti-DIG-AP (1:500, Roche) in blocking solution, for 16 h at 4 C. After 3 rinses of 10 min in TBS, they were pre-incubated for 10 min in buffer containing 100 mM Tris, 100 mM NaCl, 50 mM MgCl2; pH 9.5 (AP-buffer). Then they were stained by NBT/BCIP (Roche) in AP-buffer until sufficient color development. Specificity was checked by hybridization with the sense mRNA probes.
2.7. Statistics Quantitative data were analyzed by Student’s t-test (a = 5%), using Microsoft Excel software.
3. Results 3.1. BDNF receptors in Xenopus melanotrope cells The presence of TrkB.FL, TrkB.T and p75NTR mRNA was demonstrated in NILs by RT-PCR, using primers designed to amplify 101, 116 and 118 bp products, respectively (Fig. 1). With in situ hybridization strong signals of TrkB.FL and p75NTR transcripts were detected throughout the pars intermedia of the pituitary gland in both blackadapted and white-adapted animals. A representative result for a black-adapted animal is shown in Fig. 2. Some parts in the distal pituitary lobe were also positively stained, whereas the neural lobe was completely devoid of staining. With the sense probes no positive staining was visible, indicating the staining with the antisense probe was specific. 3.2. Quantitative RT-PCR of TrkB.FL, TrkB.T and p75NTR Quantitative RT-PCR revealed that there was no statistically significant difference in the expression levels of TrkB.FL in NILs of black- and white-adapted animals (Table 1). There was, however, a significantly higher level of mRNA for TrkB.T and p75NTR in NILs of blackadapted animals (Table 1). A decrease in the DCt value by 1 represents an increase in mRNA level by a factor of 2. Therefore, the difference in DCt values between black-
Fig. 1. Agarose gel electrophoresis of reaction product of RT-PCR on total mRNA from NILs of black-adapted Xenopus laevis, using primers for TrkB.FL, TrkB.T and p75NTR. Molecular weight markers (M) are indicated on the left.
adapted and white-adapted animals for reflects a 3.2-fold increase in mRNA adapted animals (i.e., 21.69); for p75NTR was also a 3.2-fold increase in mRNA adapted animals (i.e., 21.67).
TrkB.T (1.69) levels in blackexpression there levels in black-
4. Discussion Our in situ hybridization analysis demonstrates the presence of both TrkB.FL and p75NTR mRNAs in virtually every cell in the intermediate lobe of the pituitary gland of X. laevis. In Xenopus the pars intermedia is a relatively homogeneous tissue with over 95% of the cell content being melanotrope cells. Therefore, we conclude that the melanotropes are expressing the TrkB.FL and p75NTR receptors, a conclusion consistent with our earlier observation that the melanotropes are target cells for the action of BDNF (Kramer et al., 2002). Since the specific intracellular domain of TrkB.T is very short (11 amino acids), this receptor cannot reliably be identified in NILs by in situ hybridization. However, since the neural lobe largely consists of axons and axon terminals and contains only few mRNA-producing cells (pituicytes), the TrkB.T PCR-signal found in the NIL most likely represents TrkB.T mRNA in melanotropes in the pituitary intermediate lobe. It should be noted that it is technically not possible to separate the intermediate lobe of the Xenopus pituitary gland from the neural lobe, as the lobes are very intimately connected. TrkB.FL receptors are usually associated with stimulation of growth processes and we therefore suggest that they are involved in the hypertrophy of melanotrope cells in black-adapted animals. In fact, this hypertrophy is a marked phenomenon of Xenopus melanotropes as they double in size during prolonged black background adaptation (de Rijk et al., 1990). TrkB.FL receptors interact with p75NTR to form high-affinity receptor complexes for BDNF (Huang and Reichardt, 2003). In view of the fact that p75NTR mRNA is more highly expressed in melanotropes in black animals than in white ones, we propose that under the dark environmental condition melanotropes insert a high amount of high-affinity TrkB.FL–p75NTR
A.H. Kidane et al. / General and Comparative Endocrinology 153 (2007) 176–181
179
Fig. 2. In situ hybridization analysis of TrkB.FL and p75NTR mRNA in the pituitary of black adapted Xenopus. Sections were hybridized with an antisense Dig-labeled p75NTR riboprobe (A and B), or TrkB.FL riboprobe (C and D). B and D are details of A and C, respectively. Arrows indicate nucleus and arrowhead indicates cytoplasm. pd, pars distalis; pi, pars intermedia; pn, pars nervosa; scale bar A and C, 50 lm; B and D, 20 lm.
Table 1 Real-time RT-PCR analysis of the expression of TrkB.FL, TrkB.T and P75NTR DCt
Mean SEM P
TrkB.FL
P75NTR
TrkB.T
BA
WA
BA
WA
BA
WA
5.35 5.95 4.97
5.94 5.52 6.14
4.8 5.82 5.24
6.37 7.06 7.52
5.34 4.46 5.13 4.26
5.53 6.49 7.00 6.81
5.42 0.29 n.s.
5.87 0.18
5.29 0.3 0.019
6.98 0.33
4.79 0.26 0.007
6.46 0.33
Each PCR determination included an analysis of the expression of the housekeeping gene GAPDH. Each Ct-value for receptor expression was subtracted from the corresponding Ct-value for GAPDH to give the DCt value. The DCt values between black-adapted (BA) and white-adapted (WA) animals were tested for statistical significance using Student’s t-test (n.s., not significant).
receptor complexes into their plasma membrane. Since we find no difference in TrkB.FL expression between white and black animals, TrkB.FL expression in Xenopus melanotrope cells may be constitutive. Presumably, the sensitivity of the melanotrope cell for BDNF is controlled through the regulation of p75NTR.
In contrast to TrkB.FL mRNA, the expression of TrkB.T mRNA in the Xenopus melanotrope appears to be strongly regulated, as it is higher in melanotropes of black animals than of white ones. Such a differential regulation of TrkB.FL and TrkB.T is in line with the situation in mouse embryonic cortical neurons, where these receptors are differentially regulated in a calcium-dependent way (Kingsbury et al., 2003). TrkB.T, which is devoid of an intracellular tyrosine kinase domain, has been proposed to function as a negative regulator of TrkB.FL (Biffo et al., 1995; Eide et al., 1996; Ninkinal et al., 1997). The high sequence homology between the short intracellular domain of the Xenopus TrkB.T with TrkB.T of mammals (Fig. 3), has led us to designate this receptor as Xenopus TrkB.T (xTrkB.T). Among mammals there is a 100% sequence conservation in this intracellular domain (Klein et al., 1990; Middlemas et al., 1991; Shelton et al., 1995), whereas xTrkB.T exhibits 75% sequence identity with mammalian TrkB.T (Fig. 3). This suggests that this part of the TrkB.T has a particular, evolutionary well-conserved significance. It has been suggested that TrkB.T has its own signaling cascade, independent of TrkB.FL. Such signaling seems to include RhoA signaling (Ohira et al., 2005, 2006), the mobilization of intracellular Ca2+ (Rose et al., 2003) and a neu-
180
A.H. Kidane et al. / General and Comparative Endocrinology 153 (2007) 176–181 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
hTrkB.T
k l a r h s k f g m k g f v l f h k I p l d g
mTrkB.T
k l a r h s k f g m k g f v l f h k I p l d g
rTrkB.T
k l a r h s k f g m k g f v l f h k I p l d g
xTrkB.T
k f g r h s k f g l k g f v l f h s v p l n g
Fig. 3. The intracellular domain of TrkB.T of Xenopus and mammals. The first 12 amino acids are identical with full length TrkB (TrkB.FL), and the remaining 11 residues (bold) are specific for truncated TrkB (TrkB.T). The intracellular domain of TrkB.T has a serine residue (italics), which is conserved. Shading indicates homology. xTrkB.T: Xenopus laevis truncated TrkB, Accession No. AAH44959; mTrkB.T: Mus musculus truncated TrkB, Accession No. AAA40482; rTrkB.T: Rattus norvegicus truncated TrkB, Accession No. AAP21832; hTrkB.T: Homo sapiens truncated TrkB, Accession No. AAM77876.
rotrophin-independent interaction with p75NTR (Hartmann et al., 2004). Although the intracellular domain of TrkB.T lacks tyrosine and threonine, it does possess a conserved serine residue that might be phosphorylated as a component in a signaling pathway. Our observation that TrkB.T mRNA expression depends on the state of background light intensity, suggests that TrkB.T plays a role in regulating the neuroendocrine background adaptation reflex. In conclusion, Xenopus melanotrope cells, besides producing BDNF, express mRNAs for the major receptors involved in BDNF signaling, namely TrkB.FL, TrkB.T and p75NTR. The upregulation of TrkB.T and p75NTR in melanotropes of animals adapted to a dark background indicates that these receptors play a regulatory role during adaptation. Possibly, the upregulation of p75NTR and the complexing of this receptor with constitutively expressed TrkB.FL receptors to form a high-affinity site for BDNF, sets the sensitivity of the melanotrope cell to actions of autocrine/paracrine BDNF. Acknowledgements The authors are grateful to Mrs. Ron J.C. Engels and P.M.J.M. Cruijsen for technical assistance. This work was supported by a grant from The Netherlands Organization for Scientific Research, NWO (#813.07.001). References Biffo, S., Offenhauser, N., Carter, B.D., Barde, Y.A., 1995. Selective binding and internalisation by truncated receptors restrict the availability of BDNF during development. Development 121, 2461–2470. Chao, M., 2003. Neurotrophins and their receptors: a convergence point for many signalling pathways. Nature Rev. Neurosci. 4, 299–309. De Rijk, E.P.C.T., Jenks, B.G., Wendelaar Bonga, S.E., 1990. Morphology of the pars intermedia and the melanophore-stimulating cells in Xenopus laevis in relation to background adaptation. Gen. Comp. Endocrinol. 79, 74–82. Eide, F.F., Vining, E.R., Eide, B.L., Zang, K., Wang, X.-Y., Reichardt, L.F., 1996. Naturally occurring truncated TrkB receptors have dominant inhibitory effects on Brain-Derived Neurotrophic Factor signaling. J. Neurosci. 16, 3123–3129. Esposito, D., Patel, P., Stephens, R.M., Perez, P., Chao, M.V., Kaplan, D.R., Hempstead, B.L., 2001. The cytoplasmic and transmembrane domains of the p75 and Trk A receptors regulate high affinity binding to nerve growth factor. J. Biol. Chem. 276, 32687–32695.
Hartmann, M., Brigadski, T., Erdmann, K.S., Holtmann, B., Sendtner, M., Narz, F., Lessmann, V., 2004. Truncated TrkB receptor-induced outgrowth of dendritic filopodia involves the p75 neurotrophin receptor. J. Cell Sci. 117, 5803–5814. Huang, E.J., Reichardt, L.F., 2003. Trk receptors: roles in neuronal transduction. Annu. Rev. Biochem. 72, 609–642. Jenks, B.G., Roubos, E.W., Scheenen, W.J.J.M., 2003. Ca2+ oscillations in melanotropes of Xenopus laevis: their generation, propagation, and function. Gen. Comp. Endocrinol. 131, 209–219. Jing, S., Tapley, P., Barbacid, M., 1992. Nerve growth factor mediates signal transduction through trk homodimer receptors. Neuron 9, 1067–1069. Kingsbury, T.J., Murray, P.D., Bambrick, L.L., Krueger, B.K., 2003. Ca(2+)-dependent regulation of TrkB expression in neurons. J. Biol. Chem. 17, 407–448. Klein, R., Conway, D., Parada, L.F., Barbacid, M., 1990. The TrkB tyrosine protein kinase gene codes for a second neurogenic receptor that lacks the catalytic kinase domain. Cell 65, 189–197. Kramer, B.M.R., Cruijsen, P.M.J.M., Ouwens, D.T.W.M., Coolen, M.W., Martens, G.J.M., Roubos, E.W., Jenks, B.G., 2002. Evidence that brain-derived neurotrophic factor acts as an autocrine factor on pituitary melanotrope cells of Xenopus laevis. Endocrinology 143, 1337–1345. Lee, R., Kermani, P., Teng, K.K., Hempstead, B.L., 2001. Regulation of cell survival by secreted proneurotrophins. Science 294, 1945–1948. Luikart, B.W., Nef, S., Shipman, T., Parada, L.F., 2003. In vivo role of truncated trkb receptors during sensory ganglion neurogenesis. Neuroscience 117, 847–858. Martens, G.J.M., Weterings, K.A.P., van Zoest, I.D., Jenks, B.G., 1987. Physiologically-induced changes in proopiomelanocortin mRNA levels in the pituitary gland of the amphibian Xenopus laevis. Biochem. Biophys. Res. Comm. 143, 678–684. Middlemas, D.S., Lindberg, R.A., Hunter, T., 1991. TrkB, a neural receptor protein–tyrosine kinase: evidence for a full-length and two truncated receptors. Mol. Cell. Biol. 11, 143–153. Ninkinal, N., Grashchuck, M., Buchman, V.L., Davies, A.M., 1997. TrkB variants with deletions in the leucine-rich motifs of the extracellular domain. J. Biol. Chem. 272, 13019–13025. Nykjaer, A., Lee, R., Teng, K.K., Jansen, P., Madesen, P., Nielsen, M.S., Jacobsen, C., Kliemannel, M., Schwartz, E., Willnow, T.E., Hempstead, B.L., Petersen, C.M., 2004. Sortilin is essential for proNGFinduced neuronal cell death. Nature 427, 843–848. Nykjaer, A., Willnow, T.E., Peterson, M.C., 2005. P75NTR—live or let die. Curr. Opin. Neurobiol. 15, 49–57. Ohira, K., Homma, K.J., Hirai, H., Nakamura, S., Hayashi, M., 2006. TrkB-T1 regulates the RhoA signaling and actin cytoskeleton in glioma cells. Biochem. Biophys. Res. Commun. 342, 867–874. Ohira, K., Kumanogoh, H., Sahara, Y., Homma, K.J., Hirai, H., Nakamura, S., Hayashi, M., 2005. A truncated tropo-myosinerelated kinase B receptor, T1, regulates glial cell morphology via Rho GDP dissociation inhibitor 1. J. Neurosci. 25, 1343– 1353.
A.H. Kidane et al. / General and Comparative Endocrinology 153 (2007) 176–181 Rose, C.R., Blum, R., Pichler, B., Lepier, A., Kafitz, K.W., Konnerth, A., 2003. Truncated TrkB-T1 mediates neurotrophin-evoked calcium signaling in glia cells. Nature 426, 74–78. Roubos, E.W., Scheenen, W.J.J.M., Jenks, B.G., 2005. Neuronal, nonneuronal, and autocrine control of Xenopus melanotrope cell activity. Ann. NY Acad. Sci. 1040, 172–183. Shelton, D.L., Sutherland, J., Gripp, J., Camerato, T., Armanini, M.P., Philips, H.S., Carroll, K., Spencer, S.D., Levinson, A.D., 1995. Human trks: molecular cloning, tissue distribution, and expression of extracellular domain immunoadhesins. J. Neurosci. 15, 477–491.
181
Ullrich, A., Schlessinger, J., 1990. Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203–212. Wang, L.C., Meijer, H.K., Humbel, B.M., Jenks, B.G., Roubos, E.W., 2004. Activity-dependent dynamics of coexisting brain derived neurotrophic factor, pro-opiomelanocortin and a-melanophore-stimulating hormone in melanotrope cells of Xenopus laevis. J. Neuroendocrinol. 16, 19–25. Weatherhead, B., Thornton, V.F., Whur, P., 1971. Effects of change of background colour on the ultrastructure of the’MSH cells’ of the pars intermedia of Xenopus laevis. J. Endocrinol. 49, 25–26.
Expression and physiological regulation of BDNF receptors in the neuroendocrine melanotrope cell of Xenopus laevis Adhanet H. Kidane, Sander H.J. van Dooren, Eric W. Roubos, Bruce G. Jenks
*
Department of Cellular Animal Physiology, Integrative Physiology, EURON European Graduate School of Neuroscience, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands Received 29 September 2006; revised 28 March 2007; accepted 1 April 2007 Available online 11 April 2007
Abstract Brain-derived neurotrophic factor (BDNF) and a-melanophore-stimulating hormone (a-MSH) are co-sequestered in secretory granules in melanotrope cells of the pituitary pars intermedia of the amphibian Xenopus laevis. a-MSH is responsible for pigment dispersion in dermal melanophores during the process of black-background adaptation. BDNF-production in melanotrope cells is increased by placing animals on a black background, and BDNF acts as an autocrine stimulatory factor on the melanotrope cells. However, the repertoire of possible neurotrophin receptors of the melanotrope is unknown. In this study we have established the expression of full length TrkB (TrkB.FL), truncated TrkB (TrkB.T) and p75NTR receptors in the Xenopus neurointermediate lobe by RT-PCR. In situ hybridization reveals the presence of TrkB.FL mRNA and p75NTR mRNA in melanotrope cells. Quantitative RT-PCR shows that in animals on a black background the amounts of TrkB.T and p75NTR mRNA are about three times higher than in white backgroundadapted animals. We suggest that the amount of p75NTR sets the sensitivity of the melanotrope cells for the stimulatory action of BDNF during physiological adaptation to background light intensity. 2007 Elsevier Inc. All rights reserved. Keywords: BDNF; TrkB; p75NTR; Melanotrope; a-MSH; Pars intermedia
1. Introduction The actions of neurotrophins can be very diverse because these growth factors have multiple receptors, each receptor having its own unique signaling properties. The best known neurotrophin receptors belong to the tropomyosin receptor kinase (Trk) family of receptors, which signal through their intrinsic tyrosine kinase activity to promote growth (Chao, 2003). Signaling by Trk receptors involves dimerization of receptor molecules leading to intramolecular phosphorylation, a prerequisite for the activation of intracellular signaling cascades (Ullrich and Schlessinger, 1990; Jing et al., 1992). Truncated Trk receptors, lacking the intracellular kinase domain, have been thought to act as negative effectors of full-length receptors (Luikart
*
Corresponding author. Fax: +31 243652714. E-mail address: [email protected] (B.G. Jenks).
0016-6480/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2007.04.001
et al., 2003), although there is recent evidence that such truncated receptors also may have signaling properties themselves (Ohira et al., 2005). A third important neurotrophin receptor is the p75 neurotrophin receptor (p75NTR). While this receptor has low affinity for the neurotrophins, it can form a complex with Trk receptors to form high-affinity binding sites for neurotrophins, enabling the receptor to participate in the stimulation of growth processes (Esposito et al., 2001). The signaling pathway of p75NTR is very versatile, depending on the type of ligand and on the nature of the receptor binding partners. The receptor displays high affinity binding with precursor forms of neurotrophins (pro-neurotrophins) and can induce apoptosis by interacting with sortillin (Lee et al., 2001; Nykjaer et al., 2004). In contrast, p75NTR inhibits growth by interacting with the Nogo receptor (Nykjaer et al., 2005). It is unclear how p75NTR selects between its partners and through which intracellular signaling cascade it exerts these opposite effects.
A.H. Kidane et al. / General and Comparative Endocrinology 153 (2007) 176–181
A suitable, well-defined cell type to study the modes of neurotrophin signaling is the neuroendocrine melanotrope cell in the intermediate lobe of the pituitary gland of the amphibian Xenopus laevis. This cell produces a-melanophore-stimulating hormone (a-MSH), a peptide that controls pigment dispersion in dermal melanophores during the process of adaptation to changed background light intensity (Weatherhead et al., 1971; Jenks et al., 2003; Roubos et al., 2005). The melanotrope cell produces not only a-MSH but also brain-derived neurotrophic factor (BDNF) (Kramer et al., 2002). The cell displays marked hypertrophy in animals adapting to a black background and its cell volume increase is accompanied by enhanced production of a-MSH (Martens et al., 1987) and BDNF (Kramer et al., 2002). BDNF and a-MSH are co-sequestered in and presumably co-released from melanotrope secretory granules (Wang et al., 2004). BDNF stimulates melanotrope cell production of the precursor protein of a-MSH, pro-opiomelanocortin (Kramer et al., 2002). Therefore, endogenous BDNF likely has an autocrine/paracrine stimulatory action on melanotrope cells (Kramer et al., 2002). This action proceeds presumably through one or more BDNF-specific receptors, but the repertoire of neurotrophin receptor expression by the melanotrope cell is unknown. The aim of the present study was to determine which of the receptors known to be involved in BDNF signaling, full length TrkB (TrkB.FL), truncated TrkB (TrkB.T) and p75NTR, are expressed by the Xenopus melanotrope cell and, furthermore, to test if BDNF receptor expression is physiologically regulated, by the background light condition. We show that Xenopus melanotropes produce various neurotrophin receptors and, moreover, that the expression of some of these receptors is differentially regulated during the process of background adaptation. 2. Materials and methods 2.1. Animals Young-adults of the South African clawed toad X. laevis, aged 6 months, were reared in our laboratory under standard conditions, kept in tap water at 22 C, and fed beef heart and trout pellets (Touvit, Trouw, Putten, The Netherlands). Full background skin adaptation was achieved by keeping the animals on a white or black background with continuous light, for 3 weeks. Animal treatment was in agreement with the Declaration of Helsinki and the Dutch law concerning animal welfare, as verified by the committee for animal experimentation of Radboud University Nijmegen.
2.2. RNA extraction and cDNA synthesis After decapitation, freshly dissected neurointermediate lobes (NILs) were individually collected in 500 ll ice-cold Trizol (Life Technologies, Paisley, UK) and homogenized by sonification. After chloroform extraction and isopropyl alcohol precipitation, RNA was dissolved in 25 ll RNAse-free H2O. Total RNA was measured with an Eppendorf Biophotometer (Vaudaux-Eppendorf AG, Basel, Switzerland). First strand cDNA synthesis was performed with 1 lg RNA and 5 mU/ll random primers (Roche, Mannheim, Germany), at 70 C for 10 min, followed by
177
double strand synthesis in strand buffer (Life Technologies) with 10 mM DTT, 20 U Rnasin (Promega, Madison, WI, USA), 0.5 mM dNTPs (Roche) and 100 U Superscript II reverse transcriptase (Life Technologies), at 37 C for 75 min and at 95 C for 10 min.
2.3. Reverse transcriptase polymerase chain reaction (RT-PCR) PCR was performed in a total volume of 25 ll in buffer containing 5 ll of template cDNA, 3 mM MgCl2, 0.625 U FastStart Taq DNA polymerase (Roche), 0.25 mM dNTPs (Roche) and 0.3 mM of each primer. Primers were designed for TrkB.FL (Accession No. S69713), TrkB.T (Accession No. BC044959), p75NTR (Accession No. AF246462) and GAPDH (Accession No. U41753), on the basis of the published X. laevis mRNA sequences. The following primer pairs were used: TrkB.FL, forward 5 0 -ACCTCTACCGCGAGCAAGAC-3 0 and reverse 5 0 -GAGT AACTCTGCTTCCCG ATGAA-3 0 (specific for the intracellular domain; product size 101 bp); TrkB.T, forward 5 0 -CAGCATTAGTTTGTACTGG CC-3 0 and reverse 5 0 -CTACCCATTCAGAGGAACCG-3 0 (product size 116 bp). The reverse primer for TrkB.T is specific for the unique short intracellular domain; p75NTR, forward 5 0 -CACTATCTGTGAGGACGG TG-3 0 and reverse 5 0 -GGGAAAGTCTGAGCTTGCTG-3 0 (product size 118 bp); and GAPDH, forward 5 0 -GCTCCTCTCGCAAAGGTCAT-3 0 and reverse 5 0 -GGGCCATCCACTGTCTTCTG-3 0 (product size: 118 bp). The optimum temperature cycling protocol was determined to be 95 C for 30 s, 58 C for 30 s and 72 C for 2 min, using a programmable thermal cycler (Eppendorf, Mastercycler gradient, Hamburg, Germany). After PCR, the reaction products were run on a 2% agarose gel and visualized with ethidium bromide to check the length of the amplified cDNA.
2.4. Quantitative RT-PCR Quantitative RT-PCR was performed in a total volume of 25 ll in a buffer solution containing 5 ll of template cDNA, 1· SYBR Green buffer (Applied Biosystems, Foster City, CA, USA), 3 mM MgCl2, 0.625 U AmpliTaq Gold and 0.2 mM dNTPs (Applied Biosystems), and 0.6 lM of each primer (the same primer sets as in RT-PCR were used). The optimum temperature cycling was 95 C for 10 min followed by 35 reaction cycles of 95 C for 15 s and 60 C for 1 min, using a 5700 GeneAmp PCR system (Applied Biosystems). For each reaction, the cycle threshold (Ct) was determined, i.e., the cycle number at which fluorescence was detected above an arbitrary threshold (0.8). At this threshold Ct-values are within the exponential phase of the amplification. To compare the relative amounts of the mRNA of interest in NILs from black- versus white-adapted animals, Ct-values were normalized to those for GAPDH, by subtracting the Ct-values for the RNA of interest from the Ct-values for GAPDH.
2.5. Preparation of DIG-labeled TrkB.FL and p75NTR riboprobes To synthesize a Xenopus riboprobe for in situ hybridization, PCR was used to amplify 297 and 281 bp PCR fragments of TrkB.FL (specific for the intracellular tyrosine kinase domain) and p75NTR, respectively. The forward and reverse primers for TrkB.FL were 5 0 -GAACCCGCAGTA CTTTGGAA-3 0 and 5 0 -GGGTCACCTTCCACACAAAC-3 0 , respectively, and for p75NTR 5 0 -AAGAGCGAACAGTGCAAACT-3 0 and 5 0 -GACG TCCCTGTGTAGGGAAA-3 0 , respectively. PCR products were checked for size in 2% agarose gel, ligated into pGEM-T plasmid (Promega) and sequenced to confirm identity. After linearization of the plasmid with speI or NcoI (Roche), 11-UTP digoxigenin (DIG)-labeled antisense and sense probes were prepared as run-off transcripts using T7 and SP6 RNA polymerase (Roche).
2.6. In situ hybridization Three black- and white-adapted animals were anesthetized by immersion in a solution of 0.1% tricaine methane sulfonate (MS22; Novartis, Basel, Switzerland) in tap water, and transcardially perfused with an
178
A.H. Kidane et al. / General and Comparative Endocrinology 153 (2007) 176–181
ice-cold 0.6% NaCl solution, for 5 min, followed by 250 ml Bouin’s fixative, for 15 min. After decapitation, the brain and the pituitary gland were dissected and postfixed by immersion in the same fixative, for 16 h at 4 C, rinsed in 70% ethanol to eliminate excess of picric acid, for 24 h, dehydrated in a graded ethanol series, and embedded in paraffin. Coronal sections (7 lm) were mounted on poly-L-lysine-coated slides and allowed to air-dry, for 16 h at 45 C, deparaffinized and rehydrated. Then sections were treated with 0.1% pepsin in 0.2 N HCl, for 15 min at 37 C, postfixed in 4% formaldehyde in sodium phosphate-buffered saline (PBS), for 5 min, incubated in 1% hydroxyl ammonium chloride, for 15 min, dehydrated in ethanol, and air-dried. Hybridization took place for 16 h at 62 C in hybridization buffer consisting of 10% sodium dextran sulfate, 50% formamide, four times concentrated standard saline citrate buffer (4· SSC; pH 7.0), 1· Denhardt’s and 200 lg/ml yeast t-RNA, with 500 ng/ml mRNA probe. Then sections were treated with 10 lg/ml ribonuclease A (Roche), for 30 min at 37 C, followed by stringency washes in 2· SSC, 1· SSC, 0.5· SSC, for 30 min at 20 C, and 0.1· SSC, for 30 min at 37 C. The alkaline phosphatase (AP) method with nitroblue tetrazolium chloride/ 5-bromo-4-chloro-3-indolyl phosphate, toluidine salt (NBT/BCIP) as substrate, was used to visualize DIG label. Sections were rinsed for 10 min in Tris-buffered saline (TBS), blocked in 1% bovine serum albumin (Sigma Chemical, St. Louis, MO) and 2% normal goat serum in TBS, for 30 min, and incubated in anti-DIG-AP (1:500, Roche) in blocking solution, for 16 h at 4 C. After 3 rinses of 10 min in TBS, they were pre-incubated for 10 min in buffer containing 100 mM Tris, 100 mM NaCl, 50 mM MgCl2; pH 9.5 (AP-buffer). Then they were stained by NBT/BCIP (Roche) in AP-buffer until sufficient color development. Specificity was checked by hybridization with the sense mRNA probes.
2.7. Statistics Quantitative data were analyzed by Student’s t-test (a = 5%), using Microsoft Excel software.
3. Results 3.1. BDNF receptors in Xenopus melanotrope cells The presence of TrkB.FL, TrkB.T and p75NTR mRNA was demonstrated in NILs by RT-PCR, using primers designed to amplify 101, 116 and 118 bp products, respectively (Fig. 1). With in situ hybridization strong signals of TrkB.FL and p75NTR transcripts were detected throughout the pars intermedia of the pituitary gland in both blackadapted and white-adapted animals. A representative result for a black-adapted animal is shown in Fig. 2. Some parts in the distal pituitary lobe were also positively stained, whereas the neural lobe was completely devoid of staining. With the sense probes no positive staining was visible, indicating the staining with the antisense probe was specific. 3.2. Quantitative RT-PCR of TrkB.FL, TrkB.T and p75NTR Quantitative RT-PCR revealed that there was no statistically significant difference in the expression levels of TrkB.FL in NILs of black- and white-adapted animals (Table 1). There was, however, a significantly higher level of mRNA for TrkB.T and p75NTR in NILs of blackadapted animals (Table 1). A decrease in the DCt value by 1 represents an increase in mRNA level by a factor of 2. Therefore, the difference in DCt values between black-
Fig. 1. Agarose gel electrophoresis of reaction product of RT-PCR on total mRNA from NILs of black-adapted Xenopus laevis, using primers for TrkB.FL, TrkB.T and p75NTR. Molecular weight markers (M) are indicated on the left.
adapted and white-adapted animals for reflects a 3.2-fold increase in mRNA adapted animals (i.e., 21.69); for p75NTR was also a 3.2-fold increase in mRNA adapted animals (i.e., 21.67).
TrkB.T (1.69) levels in blackexpression there levels in black-
4. Discussion Our in situ hybridization analysis demonstrates the presence of both TrkB.FL and p75NTR mRNAs in virtually every cell in the intermediate lobe of the pituitary gland of X. laevis. In Xenopus the pars intermedia is a relatively homogeneous tissue with over 95% of the cell content being melanotrope cells. Therefore, we conclude that the melanotropes are expressing the TrkB.FL and p75NTR receptors, a conclusion consistent with our earlier observation that the melanotropes are target cells for the action of BDNF (Kramer et al., 2002). Since the specific intracellular domain of TrkB.T is very short (11 amino acids), this receptor cannot reliably be identified in NILs by in situ hybridization. However, since the neural lobe largely consists of axons and axon terminals and contains only few mRNA-producing cells (pituicytes), the TrkB.T PCR-signal found in the NIL most likely represents TrkB.T mRNA in melanotropes in the pituitary intermediate lobe. It should be noted that it is technically not possible to separate the intermediate lobe of the Xenopus pituitary gland from the neural lobe, as the lobes are very intimately connected. TrkB.FL receptors are usually associated with stimulation of growth processes and we therefore suggest that they are involved in the hypertrophy of melanotrope cells in black-adapted animals. In fact, this hypertrophy is a marked phenomenon of Xenopus melanotropes as they double in size during prolonged black background adaptation (de Rijk et al., 1990). TrkB.FL receptors interact with p75NTR to form high-affinity receptor complexes for BDNF (Huang and Reichardt, 2003). In view of the fact that p75NTR mRNA is more highly expressed in melanotropes in black animals than in white ones, we propose that under the dark environmental condition melanotropes insert a high amount of high-affinity TrkB.FL–p75NTR
A.H. Kidane et al. / General and Comparative Endocrinology 153 (2007) 176–181
179
Fig. 2. In situ hybridization analysis of TrkB.FL and p75NTR mRNA in the pituitary of black adapted Xenopus. Sections were hybridized with an antisense Dig-labeled p75NTR riboprobe (A and B), or TrkB.FL riboprobe (C and D). B and D are details of A and C, respectively. Arrows indicate nucleus and arrowhead indicates cytoplasm. pd, pars distalis; pi, pars intermedia; pn, pars nervosa; scale bar A and C, 50 lm; B and D, 20 lm.
Table 1 Real-time RT-PCR analysis of the expression of TrkB.FL, TrkB.T and P75NTR DCt
Mean SEM P
TrkB.FL
P75NTR
TrkB.T
BA
WA
BA
WA
BA
WA
5.35 5.95 4.97
5.94 5.52 6.14
4.8 5.82 5.24
6.37 7.06 7.52
5.34 4.46 5.13 4.26
5.53 6.49 7.00 6.81
5.42 0.29 n.s.
5.87 0.18
5.29 0.3 0.019
6.98 0.33
4.79 0.26 0.007
6.46 0.33
Each PCR determination included an analysis of the expression of the housekeeping gene GAPDH. Each Ct-value for receptor expression was subtracted from the corresponding Ct-value for GAPDH to give the DCt value. The DCt values between black-adapted (BA) and white-adapted (WA) animals were tested for statistical significance using Student’s t-test (n.s., not significant).
receptor complexes into their plasma membrane. Since we find no difference in TrkB.FL expression between white and black animals, TrkB.FL expression in Xenopus melanotrope cells may be constitutive. Presumably, the sensitivity of the melanotrope cell for BDNF is controlled through the regulation of p75NTR.
In contrast to TrkB.FL mRNA, the expression of TrkB.T mRNA in the Xenopus melanotrope appears to be strongly regulated, as it is higher in melanotropes of black animals than of white ones. Such a differential regulation of TrkB.FL and TrkB.T is in line with the situation in mouse embryonic cortical neurons, where these receptors are differentially regulated in a calcium-dependent way (Kingsbury et al., 2003). TrkB.T, which is devoid of an intracellular tyrosine kinase domain, has been proposed to function as a negative regulator of TrkB.FL (Biffo et al., 1995; Eide et al., 1996; Ninkinal et al., 1997). The high sequence homology between the short intracellular domain of the Xenopus TrkB.T with TrkB.T of mammals (Fig. 3), has led us to designate this receptor as Xenopus TrkB.T (xTrkB.T). Among mammals there is a 100% sequence conservation in this intracellular domain (Klein et al., 1990; Middlemas et al., 1991; Shelton et al., 1995), whereas xTrkB.T exhibits 75% sequence identity with mammalian TrkB.T (Fig. 3). This suggests that this part of the TrkB.T has a particular, evolutionary well-conserved significance. It has been suggested that TrkB.T has its own signaling cascade, independent of TrkB.FL. Such signaling seems to include RhoA signaling (Ohira et al., 2005, 2006), the mobilization of intracellular Ca2+ (Rose et al., 2003) and a neu-
180
A.H. Kidane et al. / General and Comparative Endocrinology 153 (2007) 176–181 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
hTrkB.T
k l a r h s k f g m k g f v l f h k I p l d g
mTrkB.T
k l a r h s k f g m k g f v l f h k I p l d g
rTrkB.T
k l a r h s k f g m k g f v l f h k I p l d g
xTrkB.T
k f g r h s k f g l k g f v l f h s v p l n g
Fig. 3. The intracellular domain of TrkB.T of Xenopus and mammals. The first 12 amino acids are identical with full length TrkB (TrkB.FL), and the remaining 11 residues (bold) are specific for truncated TrkB (TrkB.T). The intracellular domain of TrkB.T has a serine residue (italics), which is conserved. Shading indicates homology. xTrkB.T: Xenopus laevis truncated TrkB, Accession No. AAH44959; mTrkB.T: Mus musculus truncated TrkB, Accession No. AAA40482; rTrkB.T: Rattus norvegicus truncated TrkB, Accession No. AAP21832; hTrkB.T: Homo sapiens truncated TrkB, Accession No. AAM77876.
rotrophin-independent interaction with p75NTR (Hartmann et al., 2004). Although the intracellular domain of TrkB.T lacks tyrosine and threonine, it does possess a conserved serine residue that might be phosphorylated as a component in a signaling pathway. Our observation that TrkB.T mRNA expression depends on the state of background light intensity, suggests that TrkB.T plays a role in regulating the neuroendocrine background adaptation reflex. In conclusion, Xenopus melanotrope cells, besides producing BDNF, express mRNAs for the major receptors involved in BDNF signaling, namely TrkB.FL, TrkB.T and p75NTR. The upregulation of TrkB.T and p75NTR in melanotropes of animals adapted to a dark background indicates that these receptors play a regulatory role during adaptation. Possibly, the upregulation of p75NTR and the complexing of this receptor with constitutively expressed TrkB.FL receptors to form a high-affinity site for BDNF, sets the sensitivity of the melanotrope cell to actions of autocrine/paracrine BDNF. Acknowledgements The authors are grateful to Mrs. Ron J.C. Engels and P.M.J.M. Cruijsen for technical assistance. This work was supported by a grant from The Netherlands Organization for Scientific Research, NWO (#813.07.001). References Biffo, S., Offenhauser, N., Carter, B.D., Barde, Y.A., 1995. Selective binding and internalisation by truncated receptors restrict the availability of BDNF during development. Development 121, 2461–2470. Chao, M., 2003. Neurotrophins and their receptors: a convergence point for many signalling pathways. Nature Rev. Neurosci. 4, 299–309. De Rijk, E.P.C.T., Jenks, B.G., Wendelaar Bonga, S.E., 1990. Morphology of the pars intermedia and the melanophore-stimulating cells in Xenopus laevis in relation to background adaptation. Gen. Comp. Endocrinol. 79, 74–82. Eide, F.F., Vining, E.R., Eide, B.L., Zang, K., Wang, X.-Y., Reichardt, L.F., 1996. Naturally occurring truncated TrkB receptors have dominant inhibitory effects on Brain-Derived Neurotrophic Factor signaling. J. Neurosci. 16, 3123–3129. Esposito, D., Patel, P., Stephens, R.M., Perez, P., Chao, M.V., Kaplan, D.R., Hempstead, B.L., 2001. The cytoplasmic and transmembrane domains of the p75 and Trk A receptors regulate high affinity binding to nerve growth factor. J. Biol. Chem. 276, 32687–32695.
Hartmann, M., Brigadski, T., Erdmann, K.S., Holtmann, B., Sendtner, M., Narz, F., Lessmann, V., 2004. Truncated TrkB receptor-induced outgrowth of dendritic filopodia involves the p75 neurotrophin receptor. J. Cell Sci. 117, 5803–5814. Huang, E.J., Reichardt, L.F., 2003. Trk receptors: roles in neuronal transduction. Annu. Rev. Biochem. 72, 609–642. Jenks, B.G., Roubos, E.W., Scheenen, W.J.J.M., 2003. Ca2+ oscillations in melanotropes of Xenopus laevis: their generation, propagation, and function. Gen. Comp. Endocrinol. 131, 209–219. Jing, S., Tapley, P., Barbacid, M., 1992. Nerve growth factor mediates signal transduction through trk homodimer receptors. Neuron 9, 1067–1069. Kingsbury, T.J., Murray, P.D., Bambrick, L.L., Krueger, B.K., 2003. Ca(2+)-dependent regulation of TrkB expression in neurons. J. Biol. Chem. 17, 407–448. Klein, R., Conway, D., Parada, L.F., Barbacid, M., 1990. The TrkB tyrosine protein kinase gene codes for a second neurogenic receptor that lacks the catalytic kinase domain. Cell 65, 189–197. Kramer, B.M.R., Cruijsen, P.M.J.M., Ouwens, D.T.W.M., Coolen, M.W., Martens, G.J.M., Roubos, E.W., Jenks, B.G., 2002. Evidence that brain-derived neurotrophic factor acts as an autocrine factor on pituitary melanotrope cells of Xenopus laevis. Endocrinology 143, 1337–1345. Lee, R., Kermani, P., Teng, K.K., Hempstead, B.L., 2001. Regulation of cell survival by secreted proneurotrophins. Science 294, 1945–1948. Luikart, B.W., Nef, S., Shipman, T., Parada, L.F., 2003. In vivo role of truncated trkb receptors during sensory ganglion neurogenesis. Neuroscience 117, 847–858. Martens, G.J.M., Weterings, K.A.P., van Zoest, I.D., Jenks, B.G., 1987. Physiologically-induced changes in proopiomelanocortin mRNA levels in the pituitary gland of the amphibian Xenopus laevis. Biochem. Biophys. Res. Comm. 143, 678–684. Middlemas, D.S., Lindberg, R.A., Hunter, T., 1991. TrkB, a neural receptor protein–tyrosine kinase: evidence for a full-length and two truncated receptors. Mol. Cell. Biol. 11, 143–153. Ninkinal, N., Grashchuck, M., Buchman, V.L., Davies, A.M., 1997. TrkB variants with deletions in the leucine-rich motifs of the extracellular domain. J. Biol. Chem. 272, 13019–13025. Nykjaer, A., Lee, R., Teng, K.K., Jansen, P., Madesen, P., Nielsen, M.S., Jacobsen, C., Kliemannel, M., Schwartz, E., Willnow, T.E., Hempstead, B.L., Petersen, C.M., 2004. Sortilin is essential for proNGFinduced neuronal cell death. Nature 427, 843–848. Nykjaer, A., Willnow, T.E., Peterson, M.C., 2005. P75NTR—live or let die. Curr. Opin. Neurobiol. 15, 49–57. Ohira, K., Homma, K.J., Hirai, H., Nakamura, S., Hayashi, M., 2006. TrkB-T1 regulates the RhoA signaling and actin cytoskeleton in glioma cells. Biochem. Biophys. Res. Commun. 342, 867–874. Ohira, K., Kumanogoh, H., Sahara, Y., Homma, K.J., Hirai, H., Nakamura, S., Hayashi, M., 2005. A truncated tropo-myosinerelated kinase B receptor, T1, regulates glial cell morphology via Rho GDP dissociation inhibitor 1. J. Neurosci. 25, 1343– 1353.
A.H. Kidane et al. / General and Comparative Endocrinology 153 (2007) 176–181 Rose, C.R., Blum, R., Pichler, B., Lepier, A., Kafitz, K.W., Konnerth, A., 2003. Truncated TrkB-T1 mediates neurotrophin-evoked calcium signaling in glia cells. Nature 426, 74–78. Roubos, E.W., Scheenen, W.J.J.M., Jenks, B.G., 2005. Neuronal, nonneuronal, and autocrine control of Xenopus melanotrope cell activity. Ann. NY Acad. Sci. 1040, 172–183. Shelton, D.L., Sutherland, J., Gripp, J., Camerato, T., Armanini, M.P., Philips, H.S., Carroll, K., Spencer, S.D., Levinson, A.D., 1995. Human trks: molecular cloning, tissue distribution, and expression of extracellular domain immunoadhesins. J. Neurosci. 15, 477–491.
181
Ullrich, A., Schlessinger, J., 1990. Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203–212. Wang, L.C., Meijer, H.K., Humbel, B.M., Jenks, B.G., Roubos, E.W., 2004. Activity-dependent dynamics of coexisting brain derived neurotrophic factor, pro-opiomelanocortin and a-melanophore-stimulating hormone in melanotrope cells of Xenopus laevis. J. Neuroendocrinol. 16, 19–25. Weatherhead, B., Thornton, V.F., Whur, P., 1971. Effects of change of background colour on the ultrastructure of the’MSH cells’ of the pars intermedia of Xenopus laevis. J. Endocrinol. 49, 25–26.