Retinoic acid up-regulates ciliary neurotrophic factor receptors in cultured chick neurons and cardiomyocytes

Neuroscience Letters 240 (1998) 9–12

Retinoic acid up-regulates ciliary neurotrophic factor receptors in cultured chick neurons and cardiomyocytes Xin Wang, Stanley W. Halvorsen* Department of Biochemical Pharmacology, 447 Hochstetter Hall, Box 601200, SUNY at Buffalo, Buffalo, NY 14260-1200, USA Received 26 August 1997; received in revised form 13 November 1997; accepted 21 November 1997

Abstract Retinoic acid is an important developmental factor in the heart and nervous system and regulates the expression of trophic factor receptors in neural cell lines. Here we show the effects of retinoic acid on cytokine responsiveness in embryonic chick neurons and myocytes. Treatment of cultured cardiomyocytes and retinal and ciliary ganglion neurons with retinoic acid resulted in increased expression of receptors for the neuropoietic cytokine, CNTF. All-trans-retinoic acid induced as much as a 3-fold increase in CNTF receptor a subunit mRNA in a time and concentration dependent manner and resulted in an enhanced CNTFinduced tyrosine phosphorylation of the transcription factor, STAT3. These results indicate that neurons and myocytes expressing CNTF receptors are responsive to retinoic acid and suggest that retinoids may regulate cell sensitivity to cytokines during development.  1998 Elsevier Science Ireland Ltd.

Keywords: Retinoic acid; STAT; Ciliary neurotrophic factor; mRNA; Retina; Ciliary ganglia; Cardiomyocyte; Cytokines

Ciliary neurotrophic factor (CNTF) is a member of the neuropoietic cytokine family. It was originally described as a potential neuronal survival factor for developing chick ciliary ganglion neurons but has since been implicated in survival and differentiation of a variety of avian and mammalian peripheral and central neurons and glia, and its receptor is also found in both skeletal and cardiac myocytes [7,20,25,30]. The expression of CNTF, CNTF receptors and cell sensitivity to CNTF each show evidence of developmental regulation [5,7,13,15]. The expression of CNTF receptors is critical to normal embryonic development. Null mutants of CNTF receptors show dramatic defects in mice and are non-viable at birth [2]. The known receptor for CNTF is a tripartite complex composed of this peripheral ligand-binding a subunit and two additional signal-transducing components, leukemia inhibitory factor (LIF) receptor b (gp190) and gp130 [29]. The gp130 and LIF receptor b subunits are shared with several other members of the neuropoietic cytokine family, but the a subunit is unique to the CNTF receptor [7]. The signal transducing subunits associ* Corresponding author. Tel.: +1 716 6453936; fax: +1 716 6453850; e-mail: [email protected]

ate with members of the cytosolic tyrosine kinases, JAK/ TYK, resulting in activation of downstream effectors including the signal transducers and activators of transcription, STAT, family of transcription factors [28]. We have been examining mechanisms for regulating CNTF receptors in neuronal model systems and found that the expression of CNTF receptors is regulated during differentiation of neural crest-derived SH-SY5Y human neuroblastoma cells. Induction of an adrenergic phenotype in SH-SY5Y cells by growth in phorbol esters results in a decreased level of CNTF receptor via a post translational mechanism [18]. Induction of a cholinergic phenotype by treatment with retinoic acid produces an increase in CNTF receptor levels through an increase in gene expression of the CNTF receptor a subunit [18]. Retinoic acid is implicated as an important regulator of neuronal development, especially of neural crest-derived cells, in the chick [4,11,23]. Retinoic acid up-regulates nerve growth factor receptor expression in chick sympathetic neurons [23] and down regulates alpha7-containing neural nicotinic acetylcholine receptors in neuroblastoma cells [6]. Here we report that CNTF receptors on chick primary neurons and cardiomyocytes are up-regulated by

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(97) 00927- 0

10

X. Wang, S.W. Halvorsen / Neuroscience Letters 240 (1998) 9–12

retinoic acid and further that the increase in receptor expression results in an increase in sensitivity of the cells to CNTF-mediated phosphorylation of STAT3. Cells were dissociated from 8- to 9-day-old chick embryos (White Leghorn, Gawlak Farms, Lawton, NY, USA). Ciliary ganglion neurons were cultured on a substratum of poly-d-lysine and mouse laminin as previously described [12]. The medium contained 3% chick eye extract for neuronal survival [12,21]. Retinal neurons were processed similarly except that cells were plated on poly-dlornithine (10 mg/cm2) coated tissue culture dishes at a final density of 5 × 105 cells/cm2 in media without eye extract. Atria were separated, minced and dissociated by sequential trypsin treatments, preplated on uncoated plastic dishes to remove fibroblasts, and cultured in gelatin-coated 35 mm plastic tissue culture dishes at a density of ~106 cells per dish as previously described [9,17]. Total RNA (20 mg/lane) isolated from cultured cells by a single step method using guanidinium thiocyanate-phenolchloroform extraction [1] was resolved on formaldehyde/ agarose gels and transferred onto nylon membranes by standard capillary blotting techniques. A chick CNTF receptor a subunit gene (also referred to as chick growth promoting activity receptor a) [7] was used to generate a 571 nt fragment (591–1161) as a specific probe for CNTF receptor a mRNA. The fragment was cut from the amplified plasmid, gel purified and random-labeled with [a-32P]dATP using the Klenow fragment. After hybridization and high-stringency washes the blots were exposed to X-ray film. Hybridization with a partial cDNA fragment of the chick ribosomal L-27

Fig. 1. Retinoic acid regulates CNTF receptor a subunit mRNA in cultured chick neurons and cardiac myocytes. Ciliary ganglion neurons (A), retinal neurons (B) and atrial cardiomyocytes (C) were grown for 2–3 days before treatment with retinoic acid (10 mM) for the number of hours indicated. Total RNA was analyzed for the CNTF receptor a subunit (CNTFR; upper panels) followed by analysis of ribosomal protein L-27 mRNA (lower panels).

Fig. 2. Concentration dependence of retinoic acid regulation of CNTF receptor in ciliary ganglia. Ciliary ganglion neurons were plated as in Fig. 1 and treated for the final 18 h with retinoic acid. Total RNA was analyzed by Northern blotting for both CNTF receptor a (CNTFRa) and L-27 mRNA. Data are the mean ± SEM (n = 2–4) of the ratio of CNTFRa to L-27 signal relative to that in untreated cells.

gene was used as a loading control [14,16]. Autoradiographs exposed in the linear range of the film were analyzed by scanning densitometry and relative density determined by peak area [12]. Neurons or myocytes were grown in culture for the indicated time, rinsed and incubated in serum-free, unsupplemented media for 3 h then treated as indicated with human CNTF, washed in Eagle’s minimal essential medium (MEM) containing 1 mM Na3VO4 and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis as previously described [12]. Immunoblots were probed first with a polyclonal antibody to tyrosine phosphorylated (Y705) STAT3 (New England Biolabs, Beverly, MA, USA) and analyzed. They were then stripped and reprobed with a monoclonal antibody to STAT3 for normalization (Transduction Laboratories, Lexington, KY, USA) as previously described [12,18,31]. Bound antibody was visualized after the appropriate secondary antibody by enhanced chemiluminescence and quantified by scanning densitometry. To determine if CNTF receptors in primary neurons were regulated by retinoic acid we tested its effects on peripheral and central nervous system (CNS) neurons of embryonic chick. Treatment of cultured ciliary ganglion neurons with all-trans-retinoic acid induced a 2.3-fold (±0.4 SEM, n = 7) increase in mRNA for CNTF receptor a within 6 h (Fig. 1a). Neurons treated with retinoic acid showed an average 3.0fold (±0.6, n = 4) increase in CNTF receptor mRNA levels compared with control cells after 24 h and the levels remained elevated for up to 46 h. Retinoic acid induced similar increases in CNTF receptor mRNA in cultured retinal neurons where after 24 h of continuous treatment levels were 2.4 (±0.5, n = 3) times that of the control (Fig. 1b). Embryonic chick atria also express functional CNTF receptors [30]. Cultured chick atrial cardiomyocytes treated with retinoic acid showed an increase of CNTF receptor mRNA of 1.6-fold (±0.1, n = 3) over untreated controls (Fig. 1c). Ciliary ganglion neurons treated for 18 h with retinoic

X. Wang, S.W. Halvorsen / Neuroscience Letters 240 (1998) 9–12

Fig. 3. Retinoic acid enhances the CNTF response of neurons. Ciliary ganglion neurons were plated as described in Fig. 1 and either untreated (X) or treated with retinoic acid (10 mM, B) during the final 24 h. Neurons were exposed to the indicated concentration of CNTF for 15 min before culture lysates were analyzed by Western blotting. Data points are the mean ± SEM (n = 3) of the ratios of phosphotyrosine-STAT3 to STAT3 signal in CNTF-stimulated cells normalized to that in unstimulated cells.

acid showed initial sensitivity between 10 nM and 1 mM, an estimated EC50 of 100 nM and a maximal response at 10 mM (Fig. 2). The effects on CNTF receptor expression were selective for retinoic acid. Treatment of ciliary ganglia neurons with either the developmental factor, basic fibroblast growth factor, or the nerve growth factor, NGF (each at 50 ng/ml for 24 h) failed to increase CNTF receptor mRNA levels (99 ± 4%, n = 2 and 88 ± 5% n = 3, respectively, vs. control). Thus, CNTF receptors in both central and peripheral neurons as well as in cardiac cells were up-regulated after retinoic acid treatment. We tested if the increase in CNTF receptor a subunit resulted in a change in response of the cells to CNTF. The effect of retinoic acid on sensitivity of cells to CNTFinduced tyrosine phosphorylation of STAT3 was determined in ciliary ganglion neurons. Neurons treated for 24 h with retinoic acid responded to CNTF at concentrations up to 10 times lower then untreated cells (Fig. 3). Thus, retinoic acid treatment increased the sensitivity of neurons to CNTF. We have shown here that both cardiomyocytes and primary neurons from the peripheral parasympathetic nervous system and CNS respond to retinoic acid by up-regulating CNTF receptor expression. Retinoids are metabolites of vitamin A that regulate gene expression in embryos and adults by binding to nuclear receptors of the steroid hormone receptor superfamily of ligand-activated transcription factors. The two major classes of nuclear retinoid-receptor genes found in vertebrates are the retinoic acid receptors (RAR-a, -b and -g) and the retinoid receptors (RXR-a, -b and -g) [10,24]. These genes are developmentally regulated and differentially expressed in tissues, including neurons and heart [27]. Concentrations of all-trans-retinoic acid of 0.1–10 mM are active on chick RAR-a and -b receptors [8,22] and mediate differentiation of heart [3] and peripheral and central neurons [4,11,23,26]. The identity of the specific retinoid receptor mediating the regulation of CNTF recep-

11

tors is not yet known. All-trans-retinoic acid binds to cloned and expressed RAR-a receptors with an EC50 of 0.2 mM and binds maximally at 10 mM [8] and tissue levels of all-transretinoic acid have been estimated in young embryos to be substantially higher than 0.5 mM [19]. Thus, our finding that all-trans-retinoic acid up regulates CNTF receptors with an EC50 of 0.1 mM is consistent with an interaction at RAR-a receptors. Further, a potential RAR response element is located in the untranslated region of the human CNTF receptor gene [8]. However, all-trans-retinoic acid may contain minor contaminating isoforms or be metabolized by the cells to 9-, 11- or 13-cis-retinoic acid, so it is possible that one of these is the active agent [8,11]. These results suggest mechanisms for the regulation of CNTF responsiveness of cells during development. They demonstrate that CNTF receptors on primary chick neurons and myocytes are regulated by retinoic acid. Levels of retinoids in tissues and/or retinoic acid receptors in cells may serve as a switch for cells to enhance their sensitivity to CNTF by increasing synthesis of new receptor protein. The increased CNTF response in developing neurons could result in, for example, enhanced cholinergic differentiation or cell survival. Since retinoic acid and receptors for retinoic acid are present in developing neural crest and cardiac cells we suggest that these mechanisms regulate CNTF and related cytokine responsiveness and are likely important during in vivo development. Supported by grants from NIH-NINDS, NSF and AHANYS to S.W.H. and by a grant from the Mark Diamond Fund of SUNY-Buffalo to X.W. We thank Regeneron Pharmaceuticals for recombinant human CNTF. [1] Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156–159. [2] DeChiara, T., Vejsada, R., Poueymirou, W., Acheson, A., Suri, C., Conover, J., Friedman, B., McClain, J., Pan, L., Stahl, N., Ip, N., Kato, A. and Yancopoulos, G., Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth, Cell, 83 (1995) 313–322. [3] Dickman, E.D. and Smith, S.M., Selective regulation of cardiomyocyte gene expression and cardiac morphogenesis by retinoic acid, Dev. Dyn., 206 (1996) 39–48. [4] Dupin, E. and Le Douarin, N.M., Retinoic acid promotes the differentiation of adrenergic cells and melanocytes in quail neural crest cultures, Dev. Biol., 168 (1995) 529–548. [5] Finn, T. and Nishi, R., Expression of ciliary neurotrophic factor in targets of ciliary ganglion neurons during and after the cell death phase, J. Comp. Neurol., 366 (1996) 559–571. [6] Halvorsen, S.W., Jiang, N. and Malek, R., Regulation of nicotinic acetylcholine receptors on human neuroblastoma cells during differentiation, Biochem. Pharmacol., 50 (1995) 1655– 1661. [7] Heller, S., Finn, T.P., Huber, J., Nishi, R., Geiben, M., Pu¨schel, A. and Rohrer, H., Analysis of function and expression of the chick GPA receptor (GPARa) suggests multiple roles in neuronal development, Development, 121 (1995) 2681–2693. [8] Heyman, R.A., Mangelsdorf, D.J., Dyck, J.A., Stein, R.B., Eichele, G., Evans, R.M. and Thaller, C., 9-Cis retinoic acid is

12

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

X. Wang, S.W. Halvorsen / Neuroscience Letters 240 (1998) 9–12 a high affinity ligand for the retinoid X receptor, Cell, 68 (1992) 397–406. Hunter, D.D. and Nathanson, N.M., Biochemical and physical analyses of newly synthesized muscarinic acetylcholine receptors in cultured embryonic chicken cardiac cells, J. Neurosci., 6 (1986) 3739–3748. Kastner, P., Mark, M. and Chambon, P., Nonsteroid nuclear receptors: what are genetic studies telling us about their role in real life?, Cell, 83 (1995) 859–869. Kelley, M.W., Turner, J.K. and Reh, T.A., Retinoic acid promotes differentiation of photoreceptors in vitro, Development, 120 (1994) 2091–2102. Koshlukova, S., Finn, T.P., Nishi, R. and Halvorsen, S.W., Identification of functional receptors for ciliary neurotrophic factor on chick ciliary ganglion neurons, Neuroscience, 72 (1996) 821– 832. Kotzbauer, P., Lampe, P., Estus, S., Milbrandt, J. and Johnson, E., Postnatal development of survival responsiveness in rat sympathetic neurons to leukemia inhibitory factor and ciliary neurotrophic factor, Neuron, 12 (1994) 763–773. Lebeau, M.-C., Alvarez-Bolado, G., Braissant, O., Wahli, W. and Catsicas, S., Ribosomal protein L27 is identical in chick and rat, Nucleic Acids Res., 19 (1991) 1337. Leung, D.W., Parent, A.S., Cachianes, G., Esch, F., Coulombe, J.N., Nikolich, K., Eckenstein, F.P. and Nishi, R., Cloning, expression during development, and evidence for release of a trophic factor for ciliary ganglion neurons, Neuron, 8 (1992) 1045–1053. Lu, C. and Halvorsen, S.W., Channel activators regulate ATPsensitive potassium channel (Kir6.1) expression in chick cardiomyocytes, FEBS Lett., 412 (1997) 121–125. Lu, C., Hegde, P. and Halvorsen, S.W., Regulation of glibenclamide receptors in cultured chick cardiomyocytes, Pharmacol. Res., 32 (1995) 149–153. Malek, R.L. and Halvorsen, S.W., Opposing regulation of ciliary neurotrophic factor receptors on neuroblastoma cells by distinct differentiating agents, J. Neurobiol., 32 (1997) 81–94. McCaffery, P., Posch, K.C., Napoli, J.L., Gudas, L. and Drager, U.C., Changing patterns of the retinoic acid system in the developing retina, Dev. Biol., 158 (1993) 390–399.

[20] Nishi, R., Target-derived molecules that influence the development of neurons in the avian ciliary ganglion, J. Neurobiol., 25 (1994) 612–619. [21] Nishi, R. and Berg, D.K., Two components from eye tissue that differentially stimulate growth and development of ciliary ganglion neurons in culture, J. Neurosci., 1 (1981) 505–513. [22] Repa, J.J., Plum, L.A., Tadikonda, P.K. and Clagett-Dame, M., All-trans 3,4-didehydroretinoic acid equals all-trans retinoic acid in support of chick neuronal development, FASEB J., 10 (1996) 1078–1084. [23] Rodriguez-Tebar, A. and Rohrer, H., Retinoic acid induces NGF-dependent survival response and high affinity NGF receptors in immature chick sympathetic neurons, Development, 112 (1991) 813–820. [24] Ross, A.C., Overview of retinoid metabolism, J. Nutrition, 123 (1993) 346–350. [25] Sendtner, M., Carroll, P., Holtmann, B., Hughes, R.A. and Thoenen, H., Ciliary neurotrophic factor, J. Neurobiol., 25 (1994) 1436–1453. [26] Shiga, T., Gaur, V.P., Yamaguchi, K. and Oppenheim, R.W., The development of interneurons in the chick embryo spinal cord following in vivo treatment with retinoic acid, J. Comp. Neurol., 360 (1995) 463–474. [27] Smith, S.M. and Eichele, G., Temporal and regional differences in the expression pattern of distinct retinoic acid receptor-b transcripts in the chick embryo, Development, 111 (1991) 245–252. [28] Stahl, N. and Yancopoulos, G., The alphas, betas, and kinases of cytokine receptor complexes, Cell, 74 (1993) 587–590. [29] Stahl, N. and Yancopoulos, G.D., The tripartite CNTF receptor complex – activation and signaling involves components shared with other cytokines, J. Neurobiol., 25 (1994) 1454– 1466. [30] Wang, X. and Halvorsen, S.W., Developmental regulation of ciliary neurotrophic factor receptor in embryonic chick heart, Soc. Neurosci. Abstr., 22 (Part 2 ) (1996) 1477. [31] Wishingrad, M.A., Koshlukova, S. and Halvorsen, S.W., Ciliary neurotrophic factor stimulates the phosphorylation of two forms of STAT3 in chick ciliary ganglion neurons, J. Biol. Chem., 272 (1997) 19752–19757.