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Differential and coordinated regulation of expression of norepinephrine transporter in catecholaminergic cells in culture
Brain Research 776 Ž1997. 181–188
Research report
Differential and coordinated regulation of expression of norepinephrine transporter in catecholaminergic cells in culture Ichiro Matsuoka ) , Masashi Kumagai, Kenzo Kurihara Faculty of Pharmaceutical Sciences, Hokkaido UniÕersity, Sapporo 060, Japan Accepted 12 August 1997
Abstract The norepinephrine transporter ŽNET. terminates noradrenergic neurotransmission at synapse by high-affinity sodium-dependent reuptake into presynaptic terminals, and thus serves as a marker of differentiation of noradrenergic neurons. In the present study, we studied the regulatory mechanism of the expression of NET-mRNA in cultured neurons from newborn rat superior cervical ganglia ŽSCG. and in clonal rat pheochromocytoma cells ŽPC12.. SCG neurons in culture expressed a high level of NET-mRNA, which was further increased 2.5–5 fold from day 1 to day 13. Treatment of SCG neurons with the cholinergic differentiation factor ŽCDF.rleukemia inhibitory factor ŽLIF. and ciliary neurotrophic factor ŽCNTF., neurokines known to induce the switch from adrenergic to cholinergic phenotype in SCG neurons, led to the suppression of the level of NET-mRNA in a concentration dependent manner, concomitantly with the suppression of mRNA for tyrosine hydroxylase ŽTH., an adrenergic marker enzyme in cultured SCG neurons. On the other hand, retinoic acid, a compound which is also known to increase the expression of choline acetyltransferase, a cholinergic marker enzyme, and suppress the expression of TH in the cultured SCG neurons and PC12 cells, rather increased the level of NET-mRNA in these two cell populations. Alterations of the Naq-dependent norepinephrine transport activity which paralleled the changes in the NET-mRNA levels were confirmed by the w 3 Hxnorepinephrine uptake assay. These results indicate that cell extrinsic factors regulate the expressions of NET and TH genes by a common as well as by distinct mechanisms. q 1997 Elsevier Science B.V. Keywords: Norepinephrine transporter; Tyrosine hydroxylase; Leukemia inhibitory factor; Ciliary neurotrophic factor; Retinoic acid; Superior cervical ganglia; PC12 cell
1. Introduction Neurotransmitter phenotype is an important characteristic of neuronal cells and is determined at certain stages during neuronal differentiation. Transmitter phenotype associates with the neuronal ability to synthesize, store, release, degrade and re-utilize particular transmitter. Thus, transmitter phenotype is established through expressions of group of genes concerning synthesis and degradation of transmitter and transport of transmitter through plasma membrane and intracellular vesicles, etc. To elucidate the mechanism of establishment of transmitter phenotype and its plasticity, it is important to analyze the mechanism regulating the expression of transmitter related genes in neuronal cells. In the past, the expression of transmitter synthesizing enzyme has been used as the most popular marker of transmitter phenotype and its plasticity ) Corresponding author. Fax: q81 Ž11. 706-4991; E-mail: [email protected]
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 0 1 6 - 0
w14,22,23,26,33x. The regulation of expressions of transmitter synthesizing enzymes, in the most part, has been analyzed using in vitro culture systems. For example, transcriptional mechanisms of tyrosine hydroxylase ŽTH., key enzyme in catecholamine synthesis and choline acetyltransferase ŽChAT., ACh synthesizing enzyme have been extensively studied using cultured neural cells such as PC12 and sympathetic neurons from superior cervical ganglia ŽSCG. w14,22,23,26,27,33x. The transmitter phenotype of the newborn rat SCG neurons changes from adrenergic to cholinergic upon stimulation with various external signals. Both leukemia inhibitory factor ŽLIF. and ciliary neurotrophic factor ŽCNTF. suppress the expression of TH gene, but increase the expression of ChAT gene w30,34x. We have also shown that treatment of SCG neurons with all-trans-retinoic acid Žretinoic acid. induced the expression of ChAT gene and suppressed the TH gene w13x. Retinoic acid exerted a similar cholinergic differentiation effect on PC12 cells w22x. On the other hand, the sodium-dependent neurotrans-
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mitter transporter on the plasma membrane is an another type of marker of the transmitter phenotype of neuronal cells w1,2,18x. For example, the expression pattern of sodium-dependent norepinephrine transporter ŽNET. in both the CNS and PNS generally corresponds to the distribution of noradrenergic neurons w18,25x. Thus, the expression of sodium-dependent transmitter transporter is expected to be linked to that of transmitter synthesizing enzymes w6,7,18,28x. The regulatory mechanism of the transporter expression, however, has remained largely unknown. Recent identification of gene family of transporter enabled us to use produce gene probes to analyze in detail the distribution and mechanism of expression of the transporters in CNS and PNS w1,2,17,31x. In the present paper we characterized the mode of NET gene regulation in adrenergic sympathetic neurons of rat superior cervical ganglia ŽSCG. and clonal pheochromocytoma cells ŽPC12. in culture. The results indicate that expressions of NET and TH in these cell populations were regulated coordinately by LIF and CNTF, but were regulated differentially by retinoic acid.
2. Materials and methods 2.1. Cell culture SCG were dissected from 2-day-old Wistar rats and digested sequentially with 3 mgrml collagenase ŽSigma. for 30 min and 2 mgrml trypsin ŽDifco. for 30 min at 378C. Dissociated cells were plated onto 35 mm culture dishes precoated with poly-D-lysine ŽSigma. and laminin ŽCollaborative Research. at a density of approximately 5 ganglia per dish w14,21x. Cells were fed with Ham’s F12 medium ŽSigma. containing 40 ngrml NGF ŽPromega., 0.1 mgrml BSA, 50 Urml penicillin, 0.1 mgrml streptomycin and N2 supplement w5x. On the day following plating, the culture medium was replenished and 10 mM Ara-C ŽSigma. was added for 3 days to prevent proliferation of nonneuronal cells. All-trans retinoic acid ŽSigma., leukemia inhibitory factor ŽLIF, AMRAD. or ciliary neurotrophic factor ŽCNTF, Genzyme. were added into the medium from one day after plating of the cells. PC12 cells Žsubclone h w9x. were grown in Dulbecco’s modified Eagle’s medium ŽSigma. containing 5% fetal calf serum, 5% horse serum, 50 Urml penicillin and 0.1 mgrml streptomycin w22x. The culture dishes were coated with rat tail collagen. 2.2. Cloning of rat NET cDNA A pair of degenerate oligonucleotide primers, corresponding to the regions of high sequence identity among the human NET and other neurotransmitter transporters, located between the putative transmembrane domains 1–4
of the human NET sequence w25x, were synthesized with the following sequence: sense, TTŽTrC.CCITAŽTrC.ITITGŽTrC.TAIŽArC.AŽArG.AAŽTrC.GGIGG; antisense, ACŽTrC.TTICCIŽGrC.ŽArT.IGTŽTrC.TTIACICCŽTrC.TTCCA. Polymerase chain reaction ŽPCR. was performed with above oligonucleotide primers and the cDNA prepared from rat PC12 cells according to the following scheme: 948C Ž1 min., 508C Ž2 min. and 728C Ž3 min.; total number of PCR cycle, 45 cycle. PCR products were cloned into the plasmid vector pCR II ŽInvitrogen. and sequenced by the dideoxy method. 2.3. RNA preparation and quantitation of NET-mRNA leÕels by Northern blot analysis Total RNA was prepared from the cells according to the method of Chomczynski and Sacchi with slight modifications as described previously w13,21x. Total RNA Ž4 mg for PC12 cells or 2 mg for SCG neurons. was subjected to electrophoresis through 1.5% agarose gel and transferred to a nylon membrane ŽHybond-N, Amersham.. The membrane was hybridized with 32 P-labeled cRNA probe at 658C in 50% formamide. The membrane initially hybridized with rat NET-cRNA probe was strip-washed and rehybridized with rat TH and mouse b-actin cRNA probes. Radioactivity bound to NET-, TH- and b-actin-mRNA was quantified with BAS2000 Bio-Image-Analyzer ŽFUJI.. NET-and TH-mRNA levels were normalized for the bactin-mRNA expression to correct the slight variance of RNA amount applied, since b-actin-mRNA levels were not altered significantly by retinoic acid Žsee Fig. 1.. 2.4. [ 3 H]Norepinephrine uptake The uptake of w 3 Hxnorepinephrine was measured according to the method of Lingen et al. w16x. The culture medium was replaced by 1 ml of uptake buffer of the following composition: 125 mM NaCl, 2.4 mM K 2 SO4 , 1.2 mM KH 2 PO4 , 1.2 mM MgSO4 , 25 mM HEPES, 5.6 mM glucose, 1 mM ascorbic acid, 0.1 mM pargyline; pH of the solution was adjusted to 7.4 with Tris. The cells were preincubated for 15 min at 378C in the absence or presence of 1 mM desipramine and then incubated for 1 min in the presence of 50 nM w 3 Hxnorepinephrine ŽNEN.. After the incubation, cells were rinsed three times with the ice-cold uptake buffer and then dissolved with 0.5 ml 1 N NaOH and neutralized with 0.5 ml 1 N HCl. Radioactivity of the solubilized cells was determined by scintillation counting. Specific uptake was defined as the difference between total uptake and the uptake in the presence of desipramine. In case of SCG neurons, the uptake data was expressed as per culture dish basis. In case of PC12 cells,
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Fig. 1. The partial amino acid sequence of the rat norepinephrine transporter predicted by RT-PCR using mRNA from cultured sympathetic neurons of 2-days old rat SCG. A cDNA corresponding to base pairs 334–816 of the human NET gene was cloned and sequenced as described in Section 2 and its amino acid sequence was deduced assuming the same reading frame as for the human NET cDNA. Only the residues different in human NET cDNA were indicated below the rat sequence. Underlined positions indicate the putative transmembrane domains ŽTM2–TM4..
amount of the solubilized cells was measured and the uptake data was expressed as per mg cell protein basis.
3. Results 3.1. Cloning and expression of of rat norepinephrine transporter Using degenerate PCR primers based on the conserved regions of published human and bovine NET cDNA and total RNA prepared from newborn rat superior cervical ganglia ŽSCG., we isolated a 542 bp cDNA. Sequence analysis of this cDNA fragment Žrat NET cDNA. revealed 88% nucleotide identity, and 94% amino acid identity to the human NET Žamino acid 92–252.. Using antisense cRNA probe transcribed from the partial rat NET- and TH-cDNAs, we performed Northern blot analysis on total RNA prepared from PC12 and newborn rat SCG neurons. Both the SCG neurons and PC12 cells possess adrenergic properties as indicated by high levels of TH-mRNA expression ŽFig. 2.. NET-mRNA was also detected at high levels in both the SCG neurons and PC12 cells. The Northern blot analysis indicated both SCG neurons and PC12 cells expressed NET-mRNA approximately at 5.8 kb ŽFig. 2, see also Ref. w34x.. As shown in Fig. 3B, TH-mRNA in cultured SCG neurons increased 4-fold from day 1 to day 13 indicating the development and maturation of adrenergic neurons. NET-mRNA levels in the cultured SCG neurons increased in a similar manner during the course of culture; i.e. it increased about 3-fold between day 1 and day 13 ŽFig. 3A..
and continuously observed during the whole culture period Ž13 days.. As shown in Fig. 3C,D, both LIF and CNTF suppressed the NET- and TH-mRNA levels in a dose-dependent manner. LIF at concentrations above 1 ngrml suppressed NET-mRNA and TH-mRNA to less than 10% and 25% of the original levels, respectively. Half-maximal concentrations of LIF to suppress the NET- and TH-mRNA levels were between 0.01 and 0.1 ngrml. CNTF at 100 ngrml suppressed NET- and TH-mRNA to less than 30% of the original levels. Half-maximal concentrations of
3.2. Suppression of NET-mRNA by LIF and CNTF Treatment of SCG neurons with 1 ngrml LIF or 10 ngrml CNTF for 6 days caused a suppression of the expression of NET-mRNA with concomitant suppression of TH-mRNA. As shown in Fig. 3A,B, the suppressions of NET- and TH-mRNA levels by LIF and CNTF were evident at least from 3 days after addition of these factors
Fig. 2. Northern blot analysis of NET- TH- and b-actin-mRNA expressions in rat pheochromocytoma cells ŽPC12 cells. and SCG Neurons. PC12 cells were cultured in the absence or presence of 10 mM retinoic acid ŽRA. for 2 days. SCG neurons were cultured in the absence or presence of 10 nM retinoic acid ŽRA., 1 ngrml LIF or 10 ngrml CNTF for 6 days. Northern blot analysis was performed as described in Section 2. Each lane contained 2 mg ŽSCG neurons. or 4 mg ŽPC12 cells. total RNA.
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CNTF to suppress the NET- and TH-mRNA levels were between 5 and 20 ngrml. Both LIF Žup to 10 ngrml. and CNTF Žup to 100 ngrml. were not toxic to SCG neurons
Fig. 4. Effects of retinoids on the NET-mRNA levels in the cultured SCG neurons. ŽA. SCG neurons were cultured in the absence or presence of 10 nM retinoic acid ŽRA., 10 nM retinol or 10 nM retinal for 6 days. Then, total RNA was prepared and subjected to the quantitative Northern blot analysis. ŽB. Retinoic acid Ž10y8 M. was added to SCG neurons on the next day after plating. Then, the neurons were cultured for the indicated period Žclosed circle.. The mRNA levels in the control culture without retinoic acid are shown with open circles. Bars represent the mean and range of duplicate determinations.
and did not alter the development of extensive network of neurites, although LIF above 10 ngrml was slightly toxic to SCG neurons as indicated by deterioration of neurites. To confirm whether the suppression of the norepinephrine transporter mRNA levels by LIF and CNTF leads to alteration of norepinephrine transport activity of SCG neurons, we performed w 3 Hxnorepinephrine uptake assay. In the control culture of SCG neurons, non-specific w 3 Hxnorepinephrine uptake, which was not inhibited by desipramine, a specific inhibitor of norepinephrine transporter, was less than 20% of the total uptake Ždata not
Fig. 3. Effects of LIF and CNTF on the NET ŽA,C.- and TH ŽB,D.-mRNA levels in the cultured SCG neurons. ŽA,B. LIF Ž1 ngrml, closed circle. or CNTF Ž10 ngrml, closed square. was added to SCG neurons on the next day after plating. Then, neurons were cultured for the indicated periods. The mRNA levels in the control culture without additive are shown with open circles. ŽC,D. SCG neurons were cultured for 6 days in the presence of indicated concentrations of LIF Žclosed circle. or CNTF Žclosed square.. Then, total RNA was prepared and subjected to Northern blot analysis. NET- and TH-mRNA levels were quantitated as described in Section 2. Points represent the mean and range of two independent cultures.
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shown.. As shown in Fig. 7A, the treatment of the SCG neurons with 1 ngrml LIF and 10 ngrml CNTF for 6 days reduced the specific uptake of w 3 Hxnorepinephrine to less than 10% and to 30% of the original level. 3.3. Up-regulation of NET-mRNA by retinoic acid We have previously shown that retinoic acid suppresses TH-mRNA levels and TH activities in both SCG neurons and PC12 cells while it increases mRNA level and enzymatic activity of ChAT. In the present study, we examined whether retinoic acid alters expression of norepinephrine transporter in SCG neurons and PC12 cells. Among three retinoids examined at 10y8 M, retinoic acid specifically increased the NET-mRNA levels in SCG neurons Ž2.5-fold increase with 6-days treatment., while other two retinoids had no effect ŽFig. 4A.. As shown in Fig. 4B, the NETmRNA-inducing effect of retinoic acid on SCG neurons was detectable at 7 days in culture and continued at least for 14 days. The increase of NET expression was confirmed also by NET activity. As shown in Fig. 7A, w 3 Hxnorepinephrine uptake in SCG neurons was increased
Fig. 6. Effects of retinoic acid on the NET-mRNA levels in PC12 cells. ŽA. PC 12 cells were cultured in the presence of 10y5 M retinoic acid for the indicated period. ŽB. PC12 cells were cultured for 2 days in the presence of indicated concentrations of retinoic acid. NET-mRNA levels were measured by the quantitative Northern blot analysis. Points represent the mean and range of two independent cultures.
Fig. 5. Effects of retinoic acid on the NET- and TH-mRNA levels in the cultured SCG neurons. SCG neurons were cultured for 9 days in the presence of the indicated concentrations of retinoic acid. Then, total RNA was prepared and subjected to the quantitative Northern blot analysis for the NET ŽA.- and TH ŽB.-mRNAs. Points represent the mean and range of two independent cultures.
1.4-fold of original level by 10y8 M retinoic acid added for 6 days. The analysis of concentration dependence revealed that the regulation of NET-mRNA levels was more sensitive to retinoic acid than that of TH-mRNA levels. Thus, the NET-mRNA-inducing effect on SCG neurons was detectable from concentrations of retinoic acid as low as 10y1 0 M ŽFig. 5A., while the TH-suppression effect was detectable with retinoic acid above 10y8 M ŽFig. 5B.. These results suggest that retinoic acid regulates the expressions of NET and TH genes by distinct molecular mechanisms. The NET-mRNA inducing effect of retinoic acid was also observed in PC12 cells. The NET-mRNA-inducing effect on PC12 cells was rather quick; i.e. maximal Ž9-fold. increase was observed at 2 days of treatment with retinoic acid and the NET-mRNA levels decreased slowly thereafter. Corresponding to this observation, w 3 Hxnorepinephrine uptake to PC12 cells was also increased 2-fold of the original level within 2 days of treatment with 10y5 M retinoic acid ŽFig. 7B.. On the other hand, the induction of NET-mRNA in PC12 cells required much higher concen-
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similarity with regard to adrenergic functions and also dissimilarity with regard to different cell context; i.e. SCG neurons are primary cultured yet developing neuronal cells, while PC12 cells are clonal cell line of extrachromaffin cells. Two cholinergic differentiation factors, LIF and CNTF suppressed the NET expression only in SCG neurons, while retinoic acid increased the NET expression in both SCG neurons and PC12 cells. Effects of these factors are discussed separately below. 4.1. Effects of LIF and CNTF
Fig. 7. Effects of retinoic acid, LIF and CNTF on the uptake of w 3 Hxnorepinephrine in the cultured SCG neurons and PC12 cells. ŽA. SCG neurons were cultured for 6 days in the presence of 10y5 M retinoic acid ŽRA., 1 ngrml LIF or 10 ngrml CNTF. Data were corrected for nonspecific transport defined as accumulation of w 3 Hxnorepinephrine in the presence of 1 mM desipramine. Bars represent means"S.D. of duplicate determination of three independent cultures. ŽB. Time course of the effects of 10y5 M retinoic acid on the uptake of w 3 Hxnorepinephrine in PC12 cells. Points represent the mean and range of two independent cultures, with values corrected as described above.
trations of retinoic acid as compared to cultured SCG neurons ŽFig. 6B, see also Fig. 5A..
4. Discussion Since administration of blockers of neurotransmitter reuptake results in powerful physiologic effects, elucidation of the regulatory mechanism of the function of the neurotransmitter transporter has been under keen focus of the pharmacologists for the development of potentially useful therapeutic drugs. However, mechanisms regulating the expression of neurotransmitter transporters under developmental, cell-environmental and chronic contexts have remained largely unknown in spite of their potential importance. In the present study, we have shown that the expression of NET in two adrenergic cell populations, namely, SCG neurons and PC12 cell line is regulated by various extrinsic factors. These two cell populations have
Both LIF and CNTF are the potent survival factor for the cholinergic spinal motor neurons w3,15,20,32x. CNTF and LIF act on neuronal cells via shared signaling pathways that involve the IL-6 signal transducing receptor component gp130 w10x. LIF and CNTF was shown to down-regulate the TH activity and TH-mRNA levels, while they up-regulate the ChAT activity and ChAT-mRNA levels in SCG neurons in culture w11,30,34x. More recently, it was shown that LIF and CNTF have ability to up-regulate also vesicular ACh transporter ŽVAChT. in cultured SCG neurons w4,24x. The VAChT coding sequence was shown to be contained within the first intron of the ChAT gene w8x. The unusual organization of the VAChT and ChAT genes strongly suggests that they may share some transcriptional mechanisms to facilitate the process of determination of transmitter phenotype. Our observation that LIF and CNTF have ability to suppress NET and TH expressions with a similar concentration dependence in adrenergic neurons suggest that a similar mechanism operates also in the case of NET and TH gene regulations, although linkage of the organization of NET gene to TH is not likely to occur w29x. The coordinated up-regulation of mRNAs for ChAT and VAChT as well as coordinate down-regulation of mRNAs for NET and TH by LIF and CNTF suggests that common signaling pathways control genes for transmitter synthesizing enzymes and neurotransmitter transporters in a mutually exclusive manner to choose one type of transmitter phenotype. These common-signaling pathways may well be functioning during development when neuronal cells choose their transmitter phenotype. 4.2. Effects of retinoic acid Retinoic acid has been shown to affect pattern formation and cell differentiation in vertebrate embryogenesis including development of nervous system w19x. We have previously shown that retinoic acid has an ability to induce ChAT genes and suppress TH genes in both the SCG neurons and PC12 cells, although its physiological significance has remained obscure w12,22x. More recently, it was reported that retinoic acid has ability to increase mRNA levels for VAChT gene w4,24x. These observations sug-
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gested that retinoic acid acts as a cholinergic inducing factor on a terminally differentiated neurons to specify their transmitter phenotype. However, our observation that retinoic acid increased the NET-mRNA levels in SCG neurons and PC12 cells at the same time it suppressed TH-mRNA levels contradict the above notion at least for cells which had previously acquired adrenergic properties such as the expression of TH and NET genes. Therefore, it is very important to examine whether retinoic acid induces the expression of NET-mRNA also in other types of neuronal cells and neuronal progenitors whose transmitter phenotype is not yet determined. In this context, it is also interesting to examine whether LIFrCNTF and retinoic acid have ability to up-regulate mRNA levels for choline transporter, another marker gene for cholinergic neurons, which is not cloned so far. On the other hand, differential in vivo regulations of NET and TH genes in pharmacological context are recently reported for adrenergic cell populations in both CNS and periphery w7x. Reserpine, an inhibitor of vesicular monoamine transporters, administered to rat decreased the expression of NET-mRNA in adrenal medulla and locus ceruleus, while the expression of TH genes in these two regions was increased by the administration of reserpine w7x. These observations were interpreted as the homeostatic response of adrenergic cells to meet with increased demand for catecholamine secretion by elevating catecholamine synthesis and suppressing reuptake of norepinephrine. The differential regulation of NET and TH genes by retinoic acid presents ‘pharmacological’ similarity to the reserpine action and may be described as a ‘opposite’ homeostatic response of adrenergic cells to the changing demand for catecholamine storage within the cells, although primary target of retinoic acid in this situation is not known. However, differential sensitivity of NET and TH genes to retinoic acid in SCG neurons rather suggests that retinoic acid regulates these two genes by distinct mechanisms. In any case, it remains to be clarified in which physiological context retinoic acid exerts its ability to alter these gene expressions in adrenergic cells. Clarification of the molecular mechanisms of the retinoic acid action should await further studies. In conclusion, we demonstrated that NET and TH genes were regulated by LIFrCNTF and retinoic acid in coordinated and differential manner. Cultured SCG neurons and PC12 cells, thus, provide suitable models to further clarify the molecular mechanisms that regulate transcription of these genes.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas Ž04268103. from the Ministry of Education, Science and Culture, Japan.
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Research report
Differential and coordinated regulation of expression of norepinephrine transporter in catecholaminergic cells in culture Ichiro Matsuoka ) , Masashi Kumagai, Kenzo Kurihara Faculty of Pharmaceutical Sciences, Hokkaido UniÕersity, Sapporo 060, Japan Accepted 12 August 1997
Abstract The norepinephrine transporter ŽNET. terminates noradrenergic neurotransmission at synapse by high-affinity sodium-dependent reuptake into presynaptic terminals, and thus serves as a marker of differentiation of noradrenergic neurons. In the present study, we studied the regulatory mechanism of the expression of NET-mRNA in cultured neurons from newborn rat superior cervical ganglia ŽSCG. and in clonal rat pheochromocytoma cells ŽPC12.. SCG neurons in culture expressed a high level of NET-mRNA, which was further increased 2.5–5 fold from day 1 to day 13. Treatment of SCG neurons with the cholinergic differentiation factor ŽCDF.rleukemia inhibitory factor ŽLIF. and ciliary neurotrophic factor ŽCNTF., neurokines known to induce the switch from adrenergic to cholinergic phenotype in SCG neurons, led to the suppression of the level of NET-mRNA in a concentration dependent manner, concomitantly with the suppression of mRNA for tyrosine hydroxylase ŽTH., an adrenergic marker enzyme in cultured SCG neurons. On the other hand, retinoic acid, a compound which is also known to increase the expression of choline acetyltransferase, a cholinergic marker enzyme, and suppress the expression of TH in the cultured SCG neurons and PC12 cells, rather increased the level of NET-mRNA in these two cell populations. Alterations of the Naq-dependent norepinephrine transport activity which paralleled the changes in the NET-mRNA levels were confirmed by the w 3 Hxnorepinephrine uptake assay. These results indicate that cell extrinsic factors regulate the expressions of NET and TH genes by a common as well as by distinct mechanisms. q 1997 Elsevier Science B.V. Keywords: Norepinephrine transporter; Tyrosine hydroxylase; Leukemia inhibitory factor; Ciliary neurotrophic factor; Retinoic acid; Superior cervical ganglia; PC12 cell
1. Introduction Neurotransmitter phenotype is an important characteristic of neuronal cells and is determined at certain stages during neuronal differentiation. Transmitter phenotype associates with the neuronal ability to synthesize, store, release, degrade and re-utilize particular transmitter. Thus, transmitter phenotype is established through expressions of group of genes concerning synthesis and degradation of transmitter and transport of transmitter through plasma membrane and intracellular vesicles, etc. To elucidate the mechanism of establishment of transmitter phenotype and its plasticity, it is important to analyze the mechanism regulating the expression of transmitter related genes in neuronal cells. In the past, the expression of transmitter synthesizing enzyme has been used as the most popular marker of transmitter phenotype and its plasticity ) Corresponding author. Fax: q81 Ž11. 706-4991; E-mail: [email protected]
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 0 1 6 - 0
w14,22,23,26,33x. The regulation of expressions of transmitter synthesizing enzymes, in the most part, has been analyzed using in vitro culture systems. For example, transcriptional mechanisms of tyrosine hydroxylase ŽTH., key enzyme in catecholamine synthesis and choline acetyltransferase ŽChAT., ACh synthesizing enzyme have been extensively studied using cultured neural cells such as PC12 and sympathetic neurons from superior cervical ganglia ŽSCG. w14,22,23,26,27,33x. The transmitter phenotype of the newborn rat SCG neurons changes from adrenergic to cholinergic upon stimulation with various external signals. Both leukemia inhibitory factor ŽLIF. and ciliary neurotrophic factor ŽCNTF. suppress the expression of TH gene, but increase the expression of ChAT gene w30,34x. We have also shown that treatment of SCG neurons with all-trans-retinoic acid Žretinoic acid. induced the expression of ChAT gene and suppressed the TH gene w13x. Retinoic acid exerted a similar cholinergic differentiation effect on PC12 cells w22x. On the other hand, the sodium-dependent neurotrans-
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mitter transporter on the plasma membrane is an another type of marker of the transmitter phenotype of neuronal cells w1,2,18x. For example, the expression pattern of sodium-dependent norepinephrine transporter ŽNET. in both the CNS and PNS generally corresponds to the distribution of noradrenergic neurons w18,25x. Thus, the expression of sodium-dependent transmitter transporter is expected to be linked to that of transmitter synthesizing enzymes w6,7,18,28x. The regulatory mechanism of the transporter expression, however, has remained largely unknown. Recent identification of gene family of transporter enabled us to use produce gene probes to analyze in detail the distribution and mechanism of expression of the transporters in CNS and PNS w1,2,17,31x. In the present paper we characterized the mode of NET gene regulation in adrenergic sympathetic neurons of rat superior cervical ganglia ŽSCG. and clonal pheochromocytoma cells ŽPC12. in culture. The results indicate that expressions of NET and TH in these cell populations were regulated coordinately by LIF and CNTF, but were regulated differentially by retinoic acid.
2. Materials and methods 2.1. Cell culture SCG were dissected from 2-day-old Wistar rats and digested sequentially with 3 mgrml collagenase ŽSigma. for 30 min and 2 mgrml trypsin ŽDifco. for 30 min at 378C. Dissociated cells were plated onto 35 mm culture dishes precoated with poly-D-lysine ŽSigma. and laminin ŽCollaborative Research. at a density of approximately 5 ganglia per dish w14,21x. Cells were fed with Ham’s F12 medium ŽSigma. containing 40 ngrml NGF ŽPromega., 0.1 mgrml BSA, 50 Urml penicillin, 0.1 mgrml streptomycin and N2 supplement w5x. On the day following plating, the culture medium was replenished and 10 mM Ara-C ŽSigma. was added for 3 days to prevent proliferation of nonneuronal cells. All-trans retinoic acid ŽSigma., leukemia inhibitory factor ŽLIF, AMRAD. or ciliary neurotrophic factor ŽCNTF, Genzyme. were added into the medium from one day after plating of the cells. PC12 cells Žsubclone h w9x. were grown in Dulbecco’s modified Eagle’s medium ŽSigma. containing 5% fetal calf serum, 5% horse serum, 50 Urml penicillin and 0.1 mgrml streptomycin w22x. The culture dishes were coated with rat tail collagen. 2.2. Cloning of rat NET cDNA A pair of degenerate oligonucleotide primers, corresponding to the regions of high sequence identity among the human NET and other neurotransmitter transporters, located between the putative transmembrane domains 1–4
of the human NET sequence w25x, were synthesized with the following sequence: sense, TTŽTrC.CCITAŽTrC.ITITGŽTrC.TAIŽArC.AŽArG.AAŽTrC.GGIGG; antisense, ACŽTrC.TTICCIŽGrC.ŽArT.IGTŽTrC.TTIACICCŽTrC.TTCCA. Polymerase chain reaction ŽPCR. was performed with above oligonucleotide primers and the cDNA prepared from rat PC12 cells according to the following scheme: 948C Ž1 min., 508C Ž2 min. and 728C Ž3 min.; total number of PCR cycle, 45 cycle. PCR products were cloned into the plasmid vector pCR II ŽInvitrogen. and sequenced by the dideoxy method. 2.3. RNA preparation and quantitation of NET-mRNA leÕels by Northern blot analysis Total RNA was prepared from the cells according to the method of Chomczynski and Sacchi with slight modifications as described previously w13,21x. Total RNA Ž4 mg for PC12 cells or 2 mg for SCG neurons. was subjected to electrophoresis through 1.5% agarose gel and transferred to a nylon membrane ŽHybond-N, Amersham.. The membrane was hybridized with 32 P-labeled cRNA probe at 658C in 50% formamide. The membrane initially hybridized with rat NET-cRNA probe was strip-washed and rehybridized with rat TH and mouse b-actin cRNA probes. Radioactivity bound to NET-, TH- and b-actin-mRNA was quantified with BAS2000 Bio-Image-Analyzer ŽFUJI.. NET-and TH-mRNA levels were normalized for the bactin-mRNA expression to correct the slight variance of RNA amount applied, since b-actin-mRNA levels were not altered significantly by retinoic acid Žsee Fig. 1.. 2.4. [ 3 H]Norepinephrine uptake The uptake of w 3 Hxnorepinephrine was measured according to the method of Lingen et al. w16x. The culture medium was replaced by 1 ml of uptake buffer of the following composition: 125 mM NaCl, 2.4 mM K 2 SO4 , 1.2 mM KH 2 PO4 , 1.2 mM MgSO4 , 25 mM HEPES, 5.6 mM glucose, 1 mM ascorbic acid, 0.1 mM pargyline; pH of the solution was adjusted to 7.4 with Tris. The cells were preincubated for 15 min at 378C in the absence or presence of 1 mM desipramine and then incubated for 1 min in the presence of 50 nM w 3 Hxnorepinephrine ŽNEN.. After the incubation, cells were rinsed three times with the ice-cold uptake buffer and then dissolved with 0.5 ml 1 N NaOH and neutralized with 0.5 ml 1 N HCl. Radioactivity of the solubilized cells was determined by scintillation counting. Specific uptake was defined as the difference between total uptake and the uptake in the presence of desipramine. In case of SCG neurons, the uptake data was expressed as per culture dish basis. In case of PC12 cells,
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Fig. 1. The partial amino acid sequence of the rat norepinephrine transporter predicted by RT-PCR using mRNA from cultured sympathetic neurons of 2-days old rat SCG. A cDNA corresponding to base pairs 334–816 of the human NET gene was cloned and sequenced as described in Section 2 and its amino acid sequence was deduced assuming the same reading frame as for the human NET cDNA. Only the residues different in human NET cDNA were indicated below the rat sequence. Underlined positions indicate the putative transmembrane domains ŽTM2–TM4..
amount of the solubilized cells was measured and the uptake data was expressed as per mg cell protein basis.
3. Results 3.1. Cloning and expression of of rat norepinephrine transporter Using degenerate PCR primers based on the conserved regions of published human and bovine NET cDNA and total RNA prepared from newborn rat superior cervical ganglia ŽSCG., we isolated a 542 bp cDNA. Sequence analysis of this cDNA fragment Žrat NET cDNA. revealed 88% nucleotide identity, and 94% amino acid identity to the human NET Žamino acid 92–252.. Using antisense cRNA probe transcribed from the partial rat NET- and TH-cDNAs, we performed Northern blot analysis on total RNA prepared from PC12 and newborn rat SCG neurons. Both the SCG neurons and PC12 cells possess adrenergic properties as indicated by high levels of TH-mRNA expression ŽFig. 2.. NET-mRNA was also detected at high levels in both the SCG neurons and PC12 cells. The Northern blot analysis indicated both SCG neurons and PC12 cells expressed NET-mRNA approximately at 5.8 kb ŽFig. 2, see also Ref. w34x.. As shown in Fig. 3B, TH-mRNA in cultured SCG neurons increased 4-fold from day 1 to day 13 indicating the development and maturation of adrenergic neurons. NET-mRNA levels in the cultured SCG neurons increased in a similar manner during the course of culture; i.e. it increased about 3-fold between day 1 and day 13 ŽFig. 3A..
and continuously observed during the whole culture period Ž13 days.. As shown in Fig. 3C,D, both LIF and CNTF suppressed the NET- and TH-mRNA levels in a dose-dependent manner. LIF at concentrations above 1 ngrml suppressed NET-mRNA and TH-mRNA to less than 10% and 25% of the original levels, respectively. Half-maximal concentrations of LIF to suppress the NET- and TH-mRNA levels were between 0.01 and 0.1 ngrml. CNTF at 100 ngrml suppressed NET- and TH-mRNA to less than 30% of the original levels. Half-maximal concentrations of
3.2. Suppression of NET-mRNA by LIF and CNTF Treatment of SCG neurons with 1 ngrml LIF or 10 ngrml CNTF for 6 days caused a suppression of the expression of NET-mRNA with concomitant suppression of TH-mRNA. As shown in Fig. 3A,B, the suppressions of NET- and TH-mRNA levels by LIF and CNTF were evident at least from 3 days after addition of these factors
Fig. 2. Northern blot analysis of NET- TH- and b-actin-mRNA expressions in rat pheochromocytoma cells ŽPC12 cells. and SCG Neurons. PC12 cells were cultured in the absence or presence of 10 mM retinoic acid ŽRA. for 2 days. SCG neurons were cultured in the absence or presence of 10 nM retinoic acid ŽRA., 1 ngrml LIF or 10 ngrml CNTF for 6 days. Northern blot analysis was performed as described in Section 2. Each lane contained 2 mg ŽSCG neurons. or 4 mg ŽPC12 cells. total RNA.
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CNTF to suppress the NET- and TH-mRNA levels were between 5 and 20 ngrml. Both LIF Žup to 10 ngrml. and CNTF Žup to 100 ngrml. were not toxic to SCG neurons
Fig. 4. Effects of retinoids on the NET-mRNA levels in the cultured SCG neurons. ŽA. SCG neurons were cultured in the absence or presence of 10 nM retinoic acid ŽRA., 10 nM retinol or 10 nM retinal for 6 days. Then, total RNA was prepared and subjected to the quantitative Northern blot analysis. ŽB. Retinoic acid Ž10y8 M. was added to SCG neurons on the next day after plating. Then, the neurons were cultured for the indicated period Žclosed circle.. The mRNA levels in the control culture without retinoic acid are shown with open circles. Bars represent the mean and range of duplicate determinations.
and did not alter the development of extensive network of neurites, although LIF above 10 ngrml was slightly toxic to SCG neurons as indicated by deterioration of neurites. To confirm whether the suppression of the norepinephrine transporter mRNA levels by LIF and CNTF leads to alteration of norepinephrine transport activity of SCG neurons, we performed w 3 Hxnorepinephrine uptake assay. In the control culture of SCG neurons, non-specific w 3 Hxnorepinephrine uptake, which was not inhibited by desipramine, a specific inhibitor of norepinephrine transporter, was less than 20% of the total uptake Ždata not
Fig. 3. Effects of LIF and CNTF on the NET ŽA,C.- and TH ŽB,D.-mRNA levels in the cultured SCG neurons. ŽA,B. LIF Ž1 ngrml, closed circle. or CNTF Ž10 ngrml, closed square. was added to SCG neurons on the next day after plating. Then, neurons were cultured for the indicated periods. The mRNA levels in the control culture without additive are shown with open circles. ŽC,D. SCG neurons were cultured for 6 days in the presence of indicated concentrations of LIF Žclosed circle. or CNTF Žclosed square.. Then, total RNA was prepared and subjected to Northern blot analysis. NET- and TH-mRNA levels were quantitated as described in Section 2. Points represent the mean and range of two independent cultures.
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shown.. As shown in Fig. 7A, the treatment of the SCG neurons with 1 ngrml LIF and 10 ngrml CNTF for 6 days reduced the specific uptake of w 3 Hxnorepinephrine to less than 10% and to 30% of the original level. 3.3. Up-regulation of NET-mRNA by retinoic acid We have previously shown that retinoic acid suppresses TH-mRNA levels and TH activities in both SCG neurons and PC12 cells while it increases mRNA level and enzymatic activity of ChAT. In the present study, we examined whether retinoic acid alters expression of norepinephrine transporter in SCG neurons and PC12 cells. Among three retinoids examined at 10y8 M, retinoic acid specifically increased the NET-mRNA levels in SCG neurons Ž2.5-fold increase with 6-days treatment., while other two retinoids had no effect ŽFig. 4A.. As shown in Fig. 4B, the NETmRNA-inducing effect of retinoic acid on SCG neurons was detectable at 7 days in culture and continued at least for 14 days. The increase of NET expression was confirmed also by NET activity. As shown in Fig. 7A, w 3 Hxnorepinephrine uptake in SCG neurons was increased
Fig. 6. Effects of retinoic acid on the NET-mRNA levels in PC12 cells. ŽA. PC 12 cells were cultured in the presence of 10y5 M retinoic acid for the indicated period. ŽB. PC12 cells were cultured for 2 days in the presence of indicated concentrations of retinoic acid. NET-mRNA levels were measured by the quantitative Northern blot analysis. Points represent the mean and range of two independent cultures.
Fig. 5. Effects of retinoic acid on the NET- and TH-mRNA levels in the cultured SCG neurons. SCG neurons were cultured for 9 days in the presence of the indicated concentrations of retinoic acid. Then, total RNA was prepared and subjected to the quantitative Northern blot analysis for the NET ŽA.- and TH ŽB.-mRNAs. Points represent the mean and range of two independent cultures.
1.4-fold of original level by 10y8 M retinoic acid added for 6 days. The analysis of concentration dependence revealed that the regulation of NET-mRNA levels was more sensitive to retinoic acid than that of TH-mRNA levels. Thus, the NET-mRNA-inducing effect on SCG neurons was detectable from concentrations of retinoic acid as low as 10y1 0 M ŽFig. 5A., while the TH-suppression effect was detectable with retinoic acid above 10y8 M ŽFig. 5B.. These results suggest that retinoic acid regulates the expressions of NET and TH genes by distinct molecular mechanisms. The NET-mRNA inducing effect of retinoic acid was also observed in PC12 cells. The NET-mRNA-inducing effect on PC12 cells was rather quick; i.e. maximal Ž9-fold. increase was observed at 2 days of treatment with retinoic acid and the NET-mRNA levels decreased slowly thereafter. Corresponding to this observation, w 3 Hxnorepinephrine uptake to PC12 cells was also increased 2-fold of the original level within 2 days of treatment with 10y5 M retinoic acid ŽFig. 7B.. On the other hand, the induction of NET-mRNA in PC12 cells required much higher concen-
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similarity with regard to adrenergic functions and also dissimilarity with regard to different cell context; i.e. SCG neurons are primary cultured yet developing neuronal cells, while PC12 cells are clonal cell line of extrachromaffin cells. Two cholinergic differentiation factors, LIF and CNTF suppressed the NET expression only in SCG neurons, while retinoic acid increased the NET expression in both SCG neurons and PC12 cells. Effects of these factors are discussed separately below. 4.1. Effects of LIF and CNTF
Fig. 7. Effects of retinoic acid, LIF and CNTF on the uptake of w 3 Hxnorepinephrine in the cultured SCG neurons and PC12 cells. ŽA. SCG neurons were cultured for 6 days in the presence of 10y5 M retinoic acid ŽRA., 1 ngrml LIF or 10 ngrml CNTF. Data were corrected for nonspecific transport defined as accumulation of w 3 Hxnorepinephrine in the presence of 1 mM desipramine. Bars represent means"S.D. of duplicate determination of three independent cultures. ŽB. Time course of the effects of 10y5 M retinoic acid on the uptake of w 3 Hxnorepinephrine in PC12 cells. Points represent the mean and range of two independent cultures, with values corrected as described above.
trations of retinoic acid as compared to cultured SCG neurons ŽFig. 6B, see also Fig. 5A..
4. Discussion Since administration of blockers of neurotransmitter reuptake results in powerful physiologic effects, elucidation of the regulatory mechanism of the function of the neurotransmitter transporter has been under keen focus of the pharmacologists for the development of potentially useful therapeutic drugs. However, mechanisms regulating the expression of neurotransmitter transporters under developmental, cell-environmental and chronic contexts have remained largely unknown in spite of their potential importance. In the present study, we have shown that the expression of NET in two adrenergic cell populations, namely, SCG neurons and PC12 cell line is regulated by various extrinsic factors. These two cell populations have
Both LIF and CNTF are the potent survival factor for the cholinergic spinal motor neurons w3,15,20,32x. CNTF and LIF act on neuronal cells via shared signaling pathways that involve the IL-6 signal transducing receptor component gp130 w10x. LIF and CNTF was shown to down-regulate the TH activity and TH-mRNA levels, while they up-regulate the ChAT activity and ChAT-mRNA levels in SCG neurons in culture w11,30,34x. More recently, it was shown that LIF and CNTF have ability to up-regulate also vesicular ACh transporter ŽVAChT. in cultured SCG neurons w4,24x. The VAChT coding sequence was shown to be contained within the first intron of the ChAT gene w8x. The unusual organization of the VAChT and ChAT genes strongly suggests that they may share some transcriptional mechanisms to facilitate the process of determination of transmitter phenotype. Our observation that LIF and CNTF have ability to suppress NET and TH expressions with a similar concentration dependence in adrenergic neurons suggest that a similar mechanism operates also in the case of NET and TH gene regulations, although linkage of the organization of NET gene to TH is not likely to occur w29x. The coordinated up-regulation of mRNAs for ChAT and VAChT as well as coordinate down-regulation of mRNAs for NET and TH by LIF and CNTF suggests that common signaling pathways control genes for transmitter synthesizing enzymes and neurotransmitter transporters in a mutually exclusive manner to choose one type of transmitter phenotype. These common-signaling pathways may well be functioning during development when neuronal cells choose their transmitter phenotype. 4.2. Effects of retinoic acid Retinoic acid has been shown to affect pattern formation and cell differentiation in vertebrate embryogenesis including development of nervous system w19x. We have previously shown that retinoic acid has an ability to induce ChAT genes and suppress TH genes in both the SCG neurons and PC12 cells, although its physiological significance has remained obscure w12,22x. More recently, it was reported that retinoic acid has ability to increase mRNA levels for VAChT gene w4,24x. These observations sug-
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gested that retinoic acid acts as a cholinergic inducing factor on a terminally differentiated neurons to specify their transmitter phenotype. However, our observation that retinoic acid increased the NET-mRNA levels in SCG neurons and PC12 cells at the same time it suppressed TH-mRNA levels contradict the above notion at least for cells which had previously acquired adrenergic properties such as the expression of TH and NET genes. Therefore, it is very important to examine whether retinoic acid induces the expression of NET-mRNA also in other types of neuronal cells and neuronal progenitors whose transmitter phenotype is not yet determined. In this context, it is also interesting to examine whether LIFrCNTF and retinoic acid have ability to up-regulate mRNA levels for choline transporter, another marker gene for cholinergic neurons, which is not cloned so far. On the other hand, differential in vivo regulations of NET and TH genes in pharmacological context are recently reported for adrenergic cell populations in both CNS and periphery w7x. Reserpine, an inhibitor of vesicular monoamine transporters, administered to rat decreased the expression of NET-mRNA in adrenal medulla and locus ceruleus, while the expression of TH genes in these two regions was increased by the administration of reserpine w7x. These observations were interpreted as the homeostatic response of adrenergic cells to meet with increased demand for catecholamine secretion by elevating catecholamine synthesis and suppressing reuptake of norepinephrine. The differential regulation of NET and TH genes by retinoic acid presents ‘pharmacological’ similarity to the reserpine action and may be described as a ‘opposite’ homeostatic response of adrenergic cells to the changing demand for catecholamine storage within the cells, although primary target of retinoic acid in this situation is not known. However, differential sensitivity of NET and TH genes to retinoic acid in SCG neurons rather suggests that retinoic acid regulates these two genes by distinct mechanisms. In any case, it remains to be clarified in which physiological context retinoic acid exerts its ability to alter these gene expressions in adrenergic cells. Clarification of the molecular mechanisms of the retinoic acid action should await further studies. In conclusion, we demonstrated that NET and TH genes were regulated by LIFrCNTF and retinoic acid in coordinated and differential manner. Cultured SCG neurons and PC12 cells, thus, provide suitable models to further clarify the molecular mechanisms that regulate transcription of these genes.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas Ž04268103. from the Ministry of Education, Science and Culture, Japan.
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