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Retinoic acid downregulates the expression of ciliary neurotrophic factor in rat Schwann cells
Neuroscience Letters 339 (2003) 13–16 www.elsevier.com/locate/neulet
Retinoic acid downregulates the expression of ciliary neurotrophic factor in rat Schwann cells Verena Johann, Nina Jeliaznik, Kirsten Schrage, Jo¨rg Mey* Institut fu¨r Biologie II, RWTH-Aachen, Kopernikusstrasse 16, 52074 Aachen, Germany Received 18 October 2002; received in revised form 5 December 2002; accepted 5 December 2002
Abstract Neuropoietic cytokines, which serve as mediators in neuroglial interactions, are differentially regulated after peripheral nerve injury. In Schwann cells, the expression of ciliary neurotrophic factor (CNTF) decreases. Pursuing the hypothesis that retinoic acid (RA) serves as a regulator of lesion-induced cytokine signaling we found that all RA receptors and retinoid X receptors are expressed in Schwann cell primary cultures. Using quantitative reverse transcription-polymerase chain reaction, we have investigated the effect of RA on the expression of CNTF in these cells. After treatment with 10 nM all-trans RA for 22 h the concentration of CNTF mRNA was reduced to 63% of the control level, reminiscent of the regulation after nerve injury in vivo. In addition to CNTF, the mRNAs of leukemia inhibitory factor, interleukin-6, ciliary neurotrophic factor receptor component a and gp130 were detected in the Schwann cells. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cytokines; Ciliary neurotrophic factor; Schwann cells; Retinoic acid; Rat; Retinoic acid Receptors; Retinoid X-receptors
An injury in the adult peripheral nervous system causes physiological interactions of macrophages, Schwann cells and neurons that can lead to axonal growth and functional recovery. The communication between Schwann cells and neurons plays a major role for this process of regeneration. An important pathway of intercellular signal transduction after nerve injury is provided by neuropoietic cytokines, including ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). CNTF is highly concentrated in the cytoplasm of Schwann cells. Although after a nerve lesion CNTF-expression is reduced in Schwann cells, this cytokine is released from the damaged cells [14] and exerts neurotrophic effects on motor neurons [5,6,13]. In the context of therapeutic strategies after nerve injury we wish to understand the mechanisms of traumatic gene regulation of neuropoietic cytokines. One possible regulator is retinoic acid (RA) because RA-dependent gene expression of cytokines has been described in various cell cultures [10]. To study the role of RA as a transcriptional regulator in peripheral nerve regeneration, we are examining its effects on signal * Corresponding author. Tel.: þ 49-241-8024852; fax: þ 49-2418022133. E-mail address: [email protected] (J. Mey).
transduction in Schwann cells, which provide a growthpermissive environment for regenerating axons in the PNS. Primary cultures of Schwann cells were prepared from new-born Sprague– Dawley rats. In one preparation 20 sciatic nerves were dissected, freed from blood vessels and fatty tissue, cut up in small pieces and digested for 1 h at 37 8C in 10 ml Dulbecco’s Modified Eagle’s Medium (DMEM) containing 0.06% collagenase and 0.25% trypsin. To obtain single cells the nerve pieces were gently triturated (0.7 mm, then 0.4 mm gauge cannulae). The cell suspension was plated out in uncoated culture flasks (25 cm2) with 5 ml DMEM containing 10% fetal calf serum (FCS). Fibroblast growth was reduced by the addition of 10 mM cytosine arabinoside (Ara C) to the medium for 4 days. To eliminate remaining fibroblasts, the cells were incubated 30 min with Thy 1.1 antibody (Sigma) at 37 8C followed by treatment with baby rabbit complement (Linaris) for another 30 min. Schwann cells were then plated out in a poly-L -lysine coated culture flask (25 cm2) with 5 ml DMEM containing 10% FCS, 2 mM forskolin (ICN) and 100 mg/ml bovine pituitary extract (Life Technologies). After the cells had reached confluence, complement lysis was repeated and cells were cultured in poly-L -lysine coated flasks (75 cm2) with 10 ml DMEM containing 10% FCS and 2 mM forskolin. The identity of cultured Schwann cells was confirmed with S-
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 0 4 - 3 9 4 0 ( 0 2 ) 0 1 4 2 7 - 1
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V. Johann et al. / Neuroscience Letters 339 (2003) 13–16
100 immunocytochemistry (Fig. 1a). For RA-treatment, cells were cultivated in medium without forskolin for at least 24 h before 10 nM all-trans RA was added and cells were kept in the dark for another 22 h at 37 8C/5% CO2. Control cells received an equal amount of dimethyl sulfoxide, used as a solvent of RA. The absence of RA activity in medium and serum was checked with high pressure liquid chromatography and with a RA sensitive reporter cell line that detects all-trans RA at levels as low as 0.01 nM [11,16]. Total RNA was isolated using Trizol based on the method of Chomczynski and Sacchi [4]. Two culture flasks were pooled to generate one sample. Samples of 500 ng total RNA were treated with DNase and reverse transcribed with oligo dT-adaptor primers and M-MLV reverse transcriptase (Life Technologies) according to the manufacturer’s instructions. PCR primers, synthesized at MWG-Biotech (Ebersberg, Germany), and experimental conditions for reverse transcription-polymerase chain reaction (RT-PCR) were used as published previously [12]. After 35 cycles the PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide to verify their expected sizes: CNTF (214 bp), LIF (247 bp), IL-6 (503 bp), glyceraldehyde phosphate dehydrogenase (GAPDH) (246 bp), gp130 (657 bp) and CNTFRa (848 bp). To quantify the amounts of CNTF and LIF-mRNA after
RA-treatment, PCRs were performed by means of the Roche Light Cycler System using the DNA Master SYBR Green I kit [12]. With every PCR amplification of CNTF or LIF mRNA the expression of GAPDH was used as a nonregulated control. For all primer combinations various concentrations of the extracts were run to calculate efficiency of the PCR. Six independent experiments with pairs of RA-treated and control cells were performed and statistically analyzed with Jmp software (SAS institute). For this, logarithms of the mRNA ratios of RA-treated to control samples were calculated, and corrected for differences in the extract concentrations based on the GAPDH analysis. After confirming the normal distribution of these data (Shapiro – Wilk W-test) we tested the difference between the mean and zero with a two-tailed t-test. The identity of PCR products was confirmed with melting curve analysis and agarose gel electrophoresis. In addition, the amplified cDNA fragments of CNTF and LIF were cloned (TOPO-TA cloning, Invitrogen) and sequenced. For immunoblotting, Schwann cells and rat sciatic nerves were homogenized in hypotonic buffer with 0.5% Triton X – 100, and protease inhibitors 1 mM PMSF, 1 mM leupeptin, 1% aprotinin. After 15 min centrifugation at 12.000 g, 4 8C, soluble protein extracts were separated with discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred by semidry blotting onto a nitrocellulose
Fig. 1. Expression of the RAR and RXR in Schwann cell primary cultures from rat sciatic nerve. (a) Phase contrast and S-100 immunofluorescence micrographs of Schwann cell primary cultures from P0 rat sciatic nerves. (b) Detection of receptor mRNAs in Schwann cells. RT-PCR products were separated in an agarose gel stained with ethidium bromide. bp: position of 100 bp molecular weight markers, Aa: RARa, Xa RXRa etc. (c) Western blot analysis of the RARb and RXRb in Schwann cells (Ab, Xa) and rat sciatic nerves (ScN; left lane RAR, right lane RXR), 10 mg protein per lane. Bars indicate positions of molecular weight markers (kDa).
V. Johann et al. / Neuroscience Letters 339 (2003) 13–16
membrane. Rabbit antisera binding specifically to RAreceptors (RAR)b or retinoid X-receptors (RXR)a, purchased from Santa Cruz (sc-552, sc-553), were used in 1/1000 dilution. Detection of immunoreactivity was based on peroxidase-conjugated secondary antibodies (Sigma) and the enhanced chemoluminescence method (Amersham). Since the presence of its nuclear receptors is a condition for RA to act as a transcriptional regulator we determined their expression in rat sciatic nerve and in Schwann cell primary cultures from the sciatic nerve. RT-PCR amplification with primers for all six retinoid receptors detected the transcripts of the RAR (RAR-a, -b, -g) and of RXR (RXRa, -b and -g) in sciatic nerves as well as in Schwann cells. Fig. 1b shows the amplified fragments of the RAR- and RXR-genes in Schwann cells. In addition to the mRNA expression, protein immunoreactivity was determined by Western blot analysis. Polyclonal sera that react either with RARb or with RXRa types bound to immunoreactive proteins in Schwann cells and sciatic nerves and (Fig. 1c). With RT-PCR we successfully amplified transcripts of the cytokines CNTF, IL-6, LIF and the cytokine receptors CNTFRa and gp130 in rat Schwann cells. All genes were expressed in RA-treated and untreated primary cultures. Fig. 2a shows all the amplified fragments separated in an agarose gel stained with ethidium bromide. In vivo the expression of CNTF decreases after sciatic nerve injury, but it is still not clear how this downregulation is controlled. To investigate the role of RA as a possible regulator of transcription, we quantified the amount of LIFand CNTF-mRNA after 22 h 10 nM RA-treatment. For this we performed quantitative PCRs with the Light Cycler System (Roche). Using the fluorescent dye SYBR Green I, which is activated after binding to double stranded DNA, the fluorescence is recorded at the end of each elongation phase, and increasing amounts of PCR product can be monitored as the reaction proceeds. For relative quantification of the initial concentration of the target sequence, in each reaction the PCR cycle was determined when the amplification curve increased above background fluorescence (crossing point analysis; Light Cycler Software Version 3, Roche). When comparing RA-treated with control samples, we also obtained the relative concentrations of PCR products with GAPDH primers in each case. These results were used to correct for different cDNA concentrations in the samples. Fig. 2b shows quantitative PCR results for GAPDH- and CNTF-PCRs with cDNAs of RA-treated and untreated cells. After RA-treatment the mRNA expression of CNTF was down-regulated to 63% of the control level (n ¼ 6, SEM ¼ 4%). This effect was highly significant (P , 0:0005) and suggests a possible role of RA in mechanisms for traumatic changes in gene expression observed in vivo. In case of LIF-mRNA, we found no significant effect of RA on the concentration of its transcript. Neuropoietic cytokines like CNTF are important signals during the neuroglial interactions in the injured nervous
15
Fig. 2. Expression of neuropoietic cytokines and their receptors in Schwann cells. (a) Amplified fragments of LIF, CNTF, IL-6, GAPDH, gp130 and CNTFRa separated in an ethidium bromide stained agarose gel. M: position of 100 bp molecular weight markers, results with two different primer combinations shown for IL-6. RA: Amplification of mRNAs from cells treated with all-trans RA. (b) Online-PCR with primers for GAPDH (grey curves, left) and CNTF (black curves, right). Amplifications of cDNA from Schwann cells treated 22 h with 20 nM all-trans RA (dotted lines) and from cells treated with vehicle (continuous lines). The horizontal line at 0.3 indicates the fluorescence level used to determine crossing points in the semilogarithmic graphs.
system. CNTF and LIF both have neurotrophic effects on motor neurons and are retrogradely transported in their axons after peripheral nerve damage [5 – 7,13]. In adult rats CNTF is expressed in Schwann cells, the protein itself is highly concentrated in the cytoplasm of these cells. A crush lesion causes the release of CNTF, presumably from damaged Schwann cells, while its mRNA decreases after the injury [14]. In contrast, LIF-expression is upregulated in Schwann cells [8], as is the expression of IL-6 [2], and after sciatic nerve transection the amount of phosphorylated STAT-3 (signal transducer and activator of transcription) rises, possibly activating downstream targets of cytokine signaling [15]. From a therapeutic point of view the regulation of these intercellular signals is of great interest. Because of its role in neuronal and glial differentiation RA has been suggested to regulate gene expression also in regenerating nerves [10]. As shown in the present report Schwann cells express nuclear receptors that make them putative targets of RA signaling. To test RA as a potential regulator in nerve regeneration we addressed the question
16
V. Johann et al. / Neuroscience Letters 339 (2003) 13–16
whether injury induced changes in gene expression can be reproduced with RA treatment. This was shown for primary cultures of Schwann cells from rat sciatic nerves. Future experiments based on interference with RA signaling in vivo will be required to further test the relevance of this effect in peripheral nervous system (PNS) regeneration. It is unlikely that the downregulation of CNTF was a direct effect of RA via RA responsive elements (RARE) upstream of the cytokine gene, because there is no evidence for the existence of such RAREs in the CNTF promoter so far and the known RAREs generally cause an upregulation of the associated genes when activated by retinoid receptordimers [9]. Cases of gene inhibition by RA have been reported, however: A negative regulation by 9-cis RA was shown for the mouse gonadotropin releasing hormone (GnRH) gene in GT1-1 cells. In addition to the RARE in the promoter region of the GnRH gene, which exerts an enhancing effect on the GnRH-expression, Cho et al. found a specific RA-responsive sequence that leads to a decreased gene expression [3]. It is more likely that RA receptors upregulate expression of other proteins which directly act as transcription inhibitors. The molecular interactions between RA and neuropoietic cytokine genes have yet to be addressed. Abe and coworkers provide a possible explanation for the downregulation of CNTF in Schwann cells. Their findings suggest that an inactive state of extracellularsignal-regulated kinase (ERK) is crucial for the CNTF expression in Schwann cells, and that activation of ERK following nerve injury critically influences the expression of CNTF [1]. Thus, RA might indirectly affect CNTFexpression via interactions with the ERK-pathway, which is activated by neurotrophins. RA-dependent upregulation of neurotrophin receptors has already been shown in the developing nervous system [10,17].
Acknowledgements The authors thank Dr Frank Bosse for very helpful suggestion regarding the Schwann cell primary cultures and Marion Reisdorf for technical assistance with Western blotting/ECL. This work was supported by the Deutsche Forschungsgemeinschaft, SFB 542 Teilprojekt A6. NJ receives a scholarship from the Graduiertenfo¨rderung Nordrhein Westfalen, Germany.
References [1] K. Abe, K. Namikawa, M. Honma, T. Iwata, I. Matsuoka, K. Watabe, H. Kiyama, Inhibition of Ras extracellular-signal-regulated kinase (RK) mediated signaling promotes ciliary neurotrophic factor (CNTF) expression in Schwann cells, J. Neurochem. (2001) 700– 703. [2] L.M. Bolin, A.N. Verity, J.E. Silver, E.M. Shooter, J.S. Abrams, Interleukin-6 production by Schwann cells and induction in sciatic nerve injury, J. Neurochem. 64 (1995) 850 –858. [3] S. Cho, J. Chung, J. Han, B.J. Lee, D.H. Kim, K. Rhee, K. Kim, 9-cisRetinoic acid represses transcription of the gonadotropin-releasing hormone (GnRH) gene via proximal promoter region that is distinct from all-trans-retinoic acid response element, Mol. Brain Res. 87 (2001) 214 –222. [4] P. Chomszynski, N. Sacchi, Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162 (1987) 156–159. [5] R. Curtis, K. Adryan, Y. Zhu, P. Harkness, R. Lindsay, P. DiStephano, Retrograde axonal transport of ciliary neurotrophic factor is increased by peripheral nerve injury, Nature 365 (1993) 253–255. [6] R. Curtis, S.S. Scherer, R. Somogyi, Retrograde axonal transport of LIF is increased by peripheral nerve injury: correlation with increased LIF in distal nerve, Neuron 12 (1994) 191 –204. [7] S. Gianola, F. Rossi, Evolution of the Purkinje cell response to injury and regenerative potential during postnatal development of the rat cerebellum, J. Comp. Neurol. 430 (2001) 101–117. [8] Y. Ito, M. Yamamoto, M. Li, M. Doyu, F. Tanaka, T. Mutch, T. Mitsuma, G. Sobue, Differential temporal expression of mRNAs for ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and their receptors (CNTFRa, LIFRb, IL-6Ra and gp130) in injured peripheral nerves, Brain Res. 793 (1998) 321 –327. [9] D.J. Mangelsdorf, R.M. Evans, Retinoid receptors as transcription factors, Transcriptional regulation, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1992, pp. 1137–1167. [10] J. Mey, Retinoic acid as a regulator of cytokine signaling after nerve injury, Z. Naturforsch. C 56c (2001) 163–176. [11] J. Mey, S. Hammelmann, OLN-93 oligodendrocytes synthesize alltrans retinoic acid in vitro, Cell Tissue Res. 302 (2000) 49–58. [12] J. Mey, L. Henkes, Retinoic acid enhances leukemia inhibitory factor expression in OLN-93 oligodendrocytes, Cell Tissue Res. 310 (2002) 155 –161. [13] M. Sendtner, G.W. Kreutzberg, H. Thoenen, Ciliary neurotrophic factor prevents the degeneration of motor neurons after axotomy, Nature 345 (1990) 440 –441. [14] M. Sendtner, K.A. Sto¨ckli, H. Thoenen, Synthesis and localization of ciliary neurotrophic factor in the sciatic nerve of the adult rat after lesion and during regeneration, J. Cell Biol. 118 (1992) 139–148. [15] J.Y. Sheu, D.J. Kulhanek, F.P. Eckenstein, Differential patterns of ERK and STAT3 phosphorylation after sciatic nerve transection in the rat, Exp. Neurol. 166 (2000) 392 –402. [16] M. Wagner, B. Han, T.M. Jessell, Regional differences in retinoid release from embryonic neuronal tissue detected by an in vitro reporter assay, Development 116 (1992) 55– 66. [17] S. Wyatt, R. Andres, H. Rohrer, A.M. Davies, Regulation of neurotrophin receptor expression by retinoic acid in mouse sympathetic neuroblasts, J. Neurosci. 19 (1999) 1062–1071.
Retinoic acid downregulates the expression of ciliary neurotrophic factor in rat Schwann cells Verena Johann, Nina Jeliaznik, Kirsten Schrage, Jo¨rg Mey* Institut fu¨r Biologie II, RWTH-Aachen, Kopernikusstrasse 16, 52074 Aachen, Germany Received 18 October 2002; received in revised form 5 December 2002; accepted 5 December 2002
Abstract Neuropoietic cytokines, which serve as mediators in neuroglial interactions, are differentially regulated after peripheral nerve injury. In Schwann cells, the expression of ciliary neurotrophic factor (CNTF) decreases. Pursuing the hypothesis that retinoic acid (RA) serves as a regulator of lesion-induced cytokine signaling we found that all RA receptors and retinoid X receptors are expressed in Schwann cell primary cultures. Using quantitative reverse transcription-polymerase chain reaction, we have investigated the effect of RA on the expression of CNTF in these cells. After treatment with 10 nM all-trans RA for 22 h the concentration of CNTF mRNA was reduced to 63% of the control level, reminiscent of the regulation after nerve injury in vivo. In addition to CNTF, the mRNAs of leukemia inhibitory factor, interleukin-6, ciliary neurotrophic factor receptor component a and gp130 were detected in the Schwann cells. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cytokines; Ciliary neurotrophic factor; Schwann cells; Retinoic acid; Rat; Retinoic acid Receptors; Retinoid X-receptors
An injury in the adult peripheral nervous system causes physiological interactions of macrophages, Schwann cells and neurons that can lead to axonal growth and functional recovery. The communication between Schwann cells and neurons plays a major role for this process of regeneration. An important pathway of intercellular signal transduction after nerve injury is provided by neuropoietic cytokines, including ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). CNTF is highly concentrated in the cytoplasm of Schwann cells. Although after a nerve lesion CNTF-expression is reduced in Schwann cells, this cytokine is released from the damaged cells [14] and exerts neurotrophic effects on motor neurons [5,6,13]. In the context of therapeutic strategies after nerve injury we wish to understand the mechanisms of traumatic gene regulation of neuropoietic cytokines. One possible regulator is retinoic acid (RA) because RA-dependent gene expression of cytokines has been described in various cell cultures [10]. To study the role of RA as a transcriptional regulator in peripheral nerve regeneration, we are examining its effects on signal * Corresponding author. Tel.: þ 49-241-8024852; fax: þ 49-2418022133. E-mail address: [email protected] (J. Mey).
transduction in Schwann cells, which provide a growthpermissive environment for regenerating axons in the PNS. Primary cultures of Schwann cells were prepared from new-born Sprague– Dawley rats. In one preparation 20 sciatic nerves were dissected, freed from blood vessels and fatty tissue, cut up in small pieces and digested for 1 h at 37 8C in 10 ml Dulbecco’s Modified Eagle’s Medium (DMEM) containing 0.06% collagenase and 0.25% trypsin. To obtain single cells the nerve pieces were gently triturated (0.7 mm, then 0.4 mm gauge cannulae). The cell suspension was plated out in uncoated culture flasks (25 cm2) with 5 ml DMEM containing 10% fetal calf serum (FCS). Fibroblast growth was reduced by the addition of 10 mM cytosine arabinoside (Ara C) to the medium for 4 days. To eliminate remaining fibroblasts, the cells were incubated 30 min with Thy 1.1 antibody (Sigma) at 37 8C followed by treatment with baby rabbit complement (Linaris) for another 30 min. Schwann cells were then plated out in a poly-L -lysine coated culture flask (25 cm2) with 5 ml DMEM containing 10% FCS, 2 mM forskolin (ICN) and 100 mg/ml bovine pituitary extract (Life Technologies). After the cells had reached confluence, complement lysis was repeated and cells were cultured in poly-L -lysine coated flasks (75 cm2) with 10 ml DMEM containing 10% FCS and 2 mM forskolin. The identity of cultured Schwann cells was confirmed with S-
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 0 4 - 3 9 4 0 ( 0 2 ) 0 1 4 2 7 - 1
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V. Johann et al. / Neuroscience Letters 339 (2003) 13–16
100 immunocytochemistry (Fig. 1a). For RA-treatment, cells were cultivated in medium without forskolin for at least 24 h before 10 nM all-trans RA was added and cells were kept in the dark for another 22 h at 37 8C/5% CO2. Control cells received an equal amount of dimethyl sulfoxide, used as a solvent of RA. The absence of RA activity in medium and serum was checked with high pressure liquid chromatography and with a RA sensitive reporter cell line that detects all-trans RA at levels as low as 0.01 nM [11,16]. Total RNA was isolated using Trizol based on the method of Chomczynski and Sacchi [4]. Two culture flasks were pooled to generate one sample. Samples of 500 ng total RNA were treated with DNase and reverse transcribed with oligo dT-adaptor primers and M-MLV reverse transcriptase (Life Technologies) according to the manufacturer’s instructions. PCR primers, synthesized at MWG-Biotech (Ebersberg, Germany), and experimental conditions for reverse transcription-polymerase chain reaction (RT-PCR) were used as published previously [12]. After 35 cycles the PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide to verify their expected sizes: CNTF (214 bp), LIF (247 bp), IL-6 (503 bp), glyceraldehyde phosphate dehydrogenase (GAPDH) (246 bp), gp130 (657 bp) and CNTFRa (848 bp). To quantify the amounts of CNTF and LIF-mRNA after
RA-treatment, PCRs were performed by means of the Roche Light Cycler System using the DNA Master SYBR Green I kit [12]. With every PCR amplification of CNTF or LIF mRNA the expression of GAPDH was used as a nonregulated control. For all primer combinations various concentrations of the extracts were run to calculate efficiency of the PCR. Six independent experiments with pairs of RA-treated and control cells were performed and statistically analyzed with Jmp software (SAS institute). For this, logarithms of the mRNA ratios of RA-treated to control samples were calculated, and corrected for differences in the extract concentrations based on the GAPDH analysis. After confirming the normal distribution of these data (Shapiro – Wilk W-test) we tested the difference between the mean and zero with a two-tailed t-test. The identity of PCR products was confirmed with melting curve analysis and agarose gel electrophoresis. In addition, the amplified cDNA fragments of CNTF and LIF were cloned (TOPO-TA cloning, Invitrogen) and sequenced. For immunoblotting, Schwann cells and rat sciatic nerves were homogenized in hypotonic buffer with 0.5% Triton X – 100, and protease inhibitors 1 mM PMSF, 1 mM leupeptin, 1% aprotinin. After 15 min centrifugation at 12.000 g, 4 8C, soluble protein extracts were separated with discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred by semidry blotting onto a nitrocellulose
Fig. 1. Expression of the RAR and RXR in Schwann cell primary cultures from rat sciatic nerve. (a) Phase contrast and S-100 immunofluorescence micrographs of Schwann cell primary cultures from P0 rat sciatic nerves. (b) Detection of receptor mRNAs in Schwann cells. RT-PCR products were separated in an agarose gel stained with ethidium bromide. bp: position of 100 bp molecular weight markers, Aa: RARa, Xa RXRa etc. (c) Western blot analysis of the RARb and RXRb in Schwann cells (Ab, Xa) and rat sciatic nerves (ScN; left lane RAR, right lane RXR), 10 mg protein per lane. Bars indicate positions of molecular weight markers (kDa).
V. Johann et al. / Neuroscience Letters 339 (2003) 13–16
membrane. Rabbit antisera binding specifically to RAreceptors (RAR)b or retinoid X-receptors (RXR)a, purchased from Santa Cruz (sc-552, sc-553), were used in 1/1000 dilution. Detection of immunoreactivity was based on peroxidase-conjugated secondary antibodies (Sigma) and the enhanced chemoluminescence method (Amersham). Since the presence of its nuclear receptors is a condition for RA to act as a transcriptional regulator we determined their expression in rat sciatic nerve and in Schwann cell primary cultures from the sciatic nerve. RT-PCR amplification with primers for all six retinoid receptors detected the transcripts of the RAR (RAR-a, -b, -g) and of RXR (RXRa, -b and -g) in sciatic nerves as well as in Schwann cells. Fig. 1b shows the amplified fragments of the RAR- and RXR-genes in Schwann cells. In addition to the mRNA expression, protein immunoreactivity was determined by Western blot analysis. Polyclonal sera that react either with RARb or with RXRa types bound to immunoreactive proteins in Schwann cells and sciatic nerves and (Fig. 1c). With RT-PCR we successfully amplified transcripts of the cytokines CNTF, IL-6, LIF and the cytokine receptors CNTFRa and gp130 in rat Schwann cells. All genes were expressed in RA-treated and untreated primary cultures. Fig. 2a shows all the amplified fragments separated in an agarose gel stained with ethidium bromide. In vivo the expression of CNTF decreases after sciatic nerve injury, but it is still not clear how this downregulation is controlled. To investigate the role of RA as a possible regulator of transcription, we quantified the amount of LIFand CNTF-mRNA after 22 h 10 nM RA-treatment. For this we performed quantitative PCRs with the Light Cycler System (Roche). Using the fluorescent dye SYBR Green I, which is activated after binding to double stranded DNA, the fluorescence is recorded at the end of each elongation phase, and increasing amounts of PCR product can be monitored as the reaction proceeds. For relative quantification of the initial concentration of the target sequence, in each reaction the PCR cycle was determined when the amplification curve increased above background fluorescence (crossing point analysis; Light Cycler Software Version 3, Roche). When comparing RA-treated with control samples, we also obtained the relative concentrations of PCR products with GAPDH primers in each case. These results were used to correct for different cDNA concentrations in the samples. Fig. 2b shows quantitative PCR results for GAPDH- and CNTF-PCRs with cDNAs of RA-treated and untreated cells. After RA-treatment the mRNA expression of CNTF was down-regulated to 63% of the control level (n ¼ 6, SEM ¼ 4%). This effect was highly significant (P , 0:0005) and suggests a possible role of RA in mechanisms for traumatic changes in gene expression observed in vivo. In case of LIF-mRNA, we found no significant effect of RA on the concentration of its transcript. Neuropoietic cytokines like CNTF are important signals during the neuroglial interactions in the injured nervous
15
Fig. 2. Expression of neuropoietic cytokines and their receptors in Schwann cells. (a) Amplified fragments of LIF, CNTF, IL-6, GAPDH, gp130 and CNTFRa separated in an ethidium bromide stained agarose gel. M: position of 100 bp molecular weight markers, results with two different primer combinations shown for IL-6. RA: Amplification of mRNAs from cells treated with all-trans RA. (b) Online-PCR with primers for GAPDH (grey curves, left) and CNTF (black curves, right). Amplifications of cDNA from Schwann cells treated 22 h with 20 nM all-trans RA (dotted lines) and from cells treated with vehicle (continuous lines). The horizontal line at 0.3 indicates the fluorescence level used to determine crossing points in the semilogarithmic graphs.
system. CNTF and LIF both have neurotrophic effects on motor neurons and are retrogradely transported in their axons after peripheral nerve damage [5 – 7,13]. In adult rats CNTF is expressed in Schwann cells, the protein itself is highly concentrated in the cytoplasm of these cells. A crush lesion causes the release of CNTF, presumably from damaged Schwann cells, while its mRNA decreases after the injury [14]. In contrast, LIF-expression is upregulated in Schwann cells [8], as is the expression of IL-6 [2], and after sciatic nerve transection the amount of phosphorylated STAT-3 (signal transducer and activator of transcription) rises, possibly activating downstream targets of cytokine signaling [15]. From a therapeutic point of view the regulation of these intercellular signals is of great interest. Because of its role in neuronal and glial differentiation RA has been suggested to regulate gene expression also in regenerating nerves [10]. As shown in the present report Schwann cells express nuclear receptors that make them putative targets of RA signaling. To test RA as a potential regulator in nerve regeneration we addressed the question
16
V. Johann et al. / Neuroscience Letters 339 (2003) 13–16
whether injury induced changes in gene expression can be reproduced with RA treatment. This was shown for primary cultures of Schwann cells from rat sciatic nerves. Future experiments based on interference with RA signaling in vivo will be required to further test the relevance of this effect in peripheral nervous system (PNS) regeneration. It is unlikely that the downregulation of CNTF was a direct effect of RA via RA responsive elements (RARE) upstream of the cytokine gene, because there is no evidence for the existence of such RAREs in the CNTF promoter so far and the known RAREs generally cause an upregulation of the associated genes when activated by retinoid receptordimers [9]. Cases of gene inhibition by RA have been reported, however: A negative regulation by 9-cis RA was shown for the mouse gonadotropin releasing hormone (GnRH) gene in GT1-1 cells. In addition to the RARE in the promoter region of the GnRH gene, which exerts an enhancing effect on the GnRH-expression, Cho et al. found a specific RA-responsive sequence that leads to a decreased gene expression [3]. It is more likely that RA receptors upregulate expression of other proteins which directly act as transcription inhibitors. The molecular interactions between RA and neuropoietic cytokine genes have yet to be addressed. Abe and coworkers provide a possible explanation for the downregulation of CNTF in Schwann cells. Their findings suggest that an inactive state of extracellularsignal-regulated kinase (ERK) is crucial for the CNTF expression in Schwann cells, and that activation of ERK following nerve injury critically influences the expression of CNTF [1]. Thus, RA might indirectly affect CNTFexpression via interactions with the ERK-pathway, which is activated by neurotrophins. RA-dependent upregulation of neurotrophin receptors has already been shown in the developing nervous system [10,17].
Acknowledgements The authors thank Dr Frank Bosse for very helpful suggestion regarding the Schwann cell primary cultures and Marion Reisdorf for technical assistance with Western blotting/ECL. This work was supported by the Deutsche Forschungsgemeinschaft, SFB 542 Teilprojekt A6. NJ receives a scholarship from the Graduiertenfo¨rderung Nordrhein Westfalen, Germany.
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