Epinephrine Increases Phosphorylation of MAP-2c in Rat Pheochromocytoma Cells (PC12 Cells) via a Protein Kinase C- and Mitogen Activated Protein Kinase-Dependent Mechanism Lu Tie,† Jian-Zhao Zhang,† Yan-Hua Lin,† Tian-Hao Su,† Yu-Hua Li,† Hong-Li Wu,† You-Yi Zhang,‡ He-Ming Yu,§ and Xue-Jun Li*,† Department of Pharmacology, School of Basic Medical Sciences and State Key Laboratory of Natural & Biomimetic Drugs, Peking University, Beijing 100083, China, Institute of Vascular Medicine, Peking University Third Hospital, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing 100083, P.R. China, and National Research Institute for Family Planning, Beijing 100081, P.R. China Received November 6, 2007
Adrenoceptors mediate effects of endogenous catecholamines and have been shown to affect the neuronal development. Microtubule-associated protein-2 (MAP-2) is an important cytoskeleton protein whose phosphorylation in response to extracellular signal is involved in the regulation of neurite outgrowth and neuronal plasticity. The present study was designed to determine the effect of activation of adrenoceptor by epinephrine on MAP-2 phosphorylation in differentiation PC12 cells and, if so, to explore the mediating mechanism. We found that epinephrine could significantly increase the phosphorylation of MAP-2c at ser136 in a dose- and time-dependent manner in differentiated PC12 cells as well as microtubule arrays. Differentiated PC12 cells express R2A-adrenoceptor, whose antagonists could block these mentioned effects of epinephrine, and clonidine which is the agonist of R2-adrenoceptor could mimic the effect of epinephrine. Moreover phosphorylation of ERK and PKC was induced by epinephrine, and ERK and PKC specific inhibitors concentration-dependently prevented epinephrine-induced phosphorylation of MAP-2c at ser136. In addition, pretreatment of PC12 cells with epinephrine partly inhibited 30 µM nocodazole induced neurites retraction. These findings suggest that epinephrine induces phosphorylation of MAP-2c at ser136 through a R2-adrenoceptor mediated, ERK/PKC-dependent signaling pathway, which may contribute to the stabilization of neurites. Keywords: microtubule-associated protein 2c • phosphorylation • R2-adrenoceptor • ERK • PKC • PC12 cells • AP18
Introduction Endogenous catecholamines mediate a variety of functions in the central nervous system and peripheral nervous system. Epinephrine as an adrenergic neurotransmitter could transmit its biological signals to control these functions via R-adrenoceptors (R1 and R2) and β-adrenoceptors (β1, β2, and β3).1 Recently, intensive studies focusing on R2-adrenoceptors (R2ARs) showed that R2-ARs exert neuroprotective effect both in vivo and in vitro.2–4 The R2-ARs belong to the GPCR superfamily, including three subtypes R2A, R2B, and R2C. The three R2-ARs subtypes differ in the pharmacological properties and tissue distribution, and display different expression profile during the early brain development.5–9 In subsequent cell cultures, noradrenaline pretreatment of amphibian embryos affects neuronal differentiation, which is blocked by R-AR * Corresponding author: Xue-Jun Li, Professor, Department of Pharmacology, School of Basic Medical Science, Peking University, 38 Xueyuan Road, Beijing, 100083, P.R. China. Tel: (8610) 82802863. Fax: (8610) 82802863. E-mail:
[email protected]. † Peking University. ‡ Peking University Third Hospital. § National Research Institute for Family Planning.
1704 Journal of Proteome Research 2008, 7, 1704–1711 Published on Web 02/22/2008
antagonists, suggesting R2-ARs may be involved in the neuron differentiation.10 Microtubule (MT) and microtubule-associated proteins (MAPs) are the main components of neurite and are organized into parallel arrays with increasing stability during the neuron differentiation.11 Microtubule-associated protein-2 (MAP-2) is one of the most abundant MAPs within the mammalian brain, consisting of multiple isoforms generated by alternative splicing of a single gene. MAP-2 contains the high molecular weight MAP-2a, MA-2b, and MAP-2e (with apparent molecular masses of 270–280 kDa), and the low molecular weight MAP-2c and MAP-2d isoforms (with apparent molecular masses of 70–75 kDa).12,13 All MAP-2 isoforms are developmentally regulated in the nervous system. MAP-2b and MAP-2c are expressed during early fetal development, while MAP-2a and MAP-2d appear at later stages of development.14,15 In neurons, MAP-2 interacted with MT regulates the assembly and stability of cytoskeletal dynamics, as well as the spacing between MT, dendritic elongation, the formation of the dendrite scaffold, and oligodendrocyte process outgrowth. MAP-2 has multiple phosphorylation sites which can be phosphorylated or dephosphorylated by several protein kinases, 10.1021/pr700711s CCC: $40.75
2008 American Chemical Society
Epinephrine Increases Phosphorylation of MAP-2c in PC12 Cells
research articles
16,17
such as PKA, PKC, ERK, and phosphases. Alteration in the phosphorylation of MAP-2 affects the dendrites growth and plasticity in neurons, and phosphorylation at distinct sites may differentially affect the function of MAP-2.18–20 The purpose of this study was to test whether triggering of adrenergic receptor evoked the variation of phosphorylation of MAP-2. In the present study, PC12 cells, a rat pheochromocytoma cell line served as an established model of neuronal differentiation,21 were explored to investigate whether the activation of adrenergic receptor by epinephrine could affect the phosphorylation and protein expression of MAP-2, and we explored the mediating mechanism and uncovered its initial functional significance.
Experimental Procedures Reagents and Antibodies. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco (Grand land, NY). Rat PC12 cells were kindly provided by Dr. Jianhui Liang (National Institute on Drug Dependence, Peking University, Beijing, China). Murine 2.5S Nerve Growth Factor was purchased from Promega Corp. (Madison, WI). Monoclonal mouse antiphosphorylated form of MAP-2a,b,c (Clone AP18) came from Laboratory Vision Corp. (Fremont, CA), and monoclonal mouse anti-R tubulin antibody was purchased from Zymed Laboratories (South San Francisco, CA). FITC-conjugated goat anti-mouse IgG was purchased from Santa Cruz (CA). Monoclonal mouse anti-phospho-ERK1/2 (Thr202/Tyr204), polyclonal rabbit anti-ERK1/2 and polyclonal rabbit anti-phosphoPKC (pan) antibodies were purchased from Cell Signal Technology, Inc. (Beverly, MA). Monoclonal mouse anti-PKC R (H-7) antibody was purchased from Santa Cruz (CA). H-89 and PD98059 were obtained from Calbiochem (San Diego, CA). The 10-mm glass bottom dishes were purchased from MatTek Corporation (Ashland, MA). Epinephrine, prazosin hydrochloride, propranolol hydrochloride, yohimbine hydrochloride, clonidine hydrochloride, nocodazole, and chelerythrine chloride were purchased from Sigma-Aldrich (St. Louis, MO). Other chemicals were purchased from the Chinese Chemical Co. Cell Culture. Rat PC12 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% horse serum, 5% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin, and maintained in a humidified atmosphere of 5% CO2 at 37 °C. To acquire neuronal differentiation, PC12 cells were subcultured at a density of 5 × 104 cells/mL onto collagen-coated 10-mm glass bottom dishes for immunofluorescence and confocal microscopy analysis or onto collagen-coated 100-mm Petri plates for Western blot analysis. One day after seeding, cells were rinsed once with serum-free DMEM, and then exposed to 50 ng/mL mouse Nerve Growth Factor 2.5S (DMEM supplemented with 1% horse serum, 50 U/mL penicillin, and 50 U/mL streptomycin) for 8 days. The medium, including NGF, was replaced every other day during the experiment. Immunoblot Analysis. Cells were washed three times with ice-cold PBS and resuspended in high salt lysis buffer (1% Triton-X100, 50 mM Tris-HCl (pH 8.0), 0.25 M NaCl, 5 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride). The supernatant was collected following centrifugation at 12 000g for 10 min and stored at -70 °C prior to use. Protein was quantified by the Bradford method, and equal amounts of protein were analyzed by electrophoresis on a 7.5% sodium dodecyl sulfatepolyacrylamide gel and electrophoretically transferred onto polyvinylidene difluoride (PVDF) immobilion-P membranes of
Figure 1. Effect of epinephrine on MAP-2c phosphorylation at Ser136 in differentiated PC12 cells. Differentiated PC12 cells were incubated with 10 nM, 100 nM, and 1 µM epinephrine for 24 h (A) or with epinephrine for 0, 6, 12, 24, and 36 h (B), and then total proteins were extracted for examination of MAP-2c phosphorylation at Ser136 by Western blotting. The staining intensity of the bands was determined by laser scanning followed by quantitative densitometric analysis. Results are plotted in a graph. Values are mean ( SEM and are shown as a percentage of the control. (n ) 3 for each time period. *P < 0.05 compared with controls.)
0.45-µm pore size. Membranes were blocked in blocking buffer (phosphate-buffered saline plus 0.05% Tween-20 and 5% nonfat dry milk) for 1 h or more, and then incubated overnight at 4 °C with the following antibodies: mouse anti-phospho-MAP-2 AP18 (1/1000), mouse anti-MAP-2 (1/1000), mouse anti-Rtubulin (1/1000), mouse anti-phospho ERK1/2 (Thr202/Tyr204) (1/5000), rabbit anti-ERK1/2 (1/5000), rabbit anti-phospho-PKC (pan) (1/1000), mouse anti-PKC R (H-7) (1/1000). After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody, the blots were developed using an enhanced chemiluminescence (ECL) detection kit. The staining intensity of the bands was determined by densitometric image analysis with Quantity One software version 4.4.0. Immunofluorescence and Confocal Imaging. Cells plated on glass bottom dishes were washed with PBS, pH 7.4, fixed Journal of Proteome Research • Vol. 7, No. 4, 2008 1705
research articles
Tie et al.
Figure 2. Confocal image of the levels of MAP-2c phosphorylation at Ser136 and R-tubulin in differentiated PC12 cells. Pictures A and B represent the levels of MAP-2c phosphorylation at Ser136 in PC12 cells incubated with or without 1 µM epinephrine for 24 h. Pictures C and D represent the expression of R-tubulin in PC12 cells incubated with or without 1 µM epinephrine for 24 h. Arrows show microtubule arrays in differentiated PC12 cells. (E) The negative control was set without the presence of the primary antibody. (F) Phase-contrast shows the morphology of PC12 cells.
with 4% paraformaldehyde for 15 min, and pretreated with 0.5% Triton X-100 for 10 min at room temperature. Nonspecific binding sites in the cells were blocked with 5% normal goat serum for 30 min and incubated overnight at 4 °C with the anti-phospho-MAP-2 antibody (1/500) or anti-rat tubulin antibody (1/500). The following day, cells were washed with PBS and incubated for 1 h with the TRITC-conjugated goat antirabbit IgG (1/100) for 30 min in the dark at 37 °C. The immunofluorescent signal was observed using a Leica TCS SP2 confocal microscope. RNA Extraction and RT-PCR of r2-Adrenergic Receptors. Total RNA was isolated from 8 days differentiated cell cultures and rat brain using TRIzol, according to manufacturer’s 1706
Journal of Proteome Research • Vol. 7, No. 4, 2008
instructions. For RT-PCR analysis of R2A-, R2B-, and R2Cadrenoceptor, 2 µg of total RNA was reverse-transcribed to cDNA. PCR reactions were then performed by using the following primers: R2A-adrenoceptor (sense primer, 5′-GTTCACCGTGTTTGGCAACG-3′; antisense primer, 5′-TGGCGATCTGGTAGATACGC-3′; 540 bp), R2B-adrenoceptor (sense primer, 5′-TCACTGGCAGCAGCCGACAT-3′; antisense primer, 5′-GCGTTGGTCGCCCTTGTA-3′; 308 bp), and R2C-adrenoceptor (sense primer, 5′-CACCGTGCTCGTGGTGATC-3′; antisense primer, 5′-GATGACAGCCGAGATGAGCC-3′; 343 bp). The reactions were cycled 35 times from 94 °C for 40 s to 60 °C for 45 s and then 72 °C for 1 min. Negative control PCRs with a subsitution of dissection solution or total RNA without RT
Epinephrine Increases Phosphorylation of MAP-2c in PC12 Cells
research articles
Figure 3. (A) Effects of adrenoceptor antagonists on MAP-2c phosphorylation at Ser136 in differentiated PC12 cells. Differentiated PC12 cells were preincubated with prazosin (10 µM), yohimbine (10 µM), and propranolol (10 µM) for 30 min before being stimulated with 1 µM epinephrine for 24 h, and then total proteins were extracted for examination of MAP-2c phosphorylation at Ser136 by Western blotting. Values are mean ( SEM and are shown as a percentage of the control. (n ) 3 for each time period. *P < 0.05 compared with controls; #P < 0.05 compared with the cells incubated with 1 µM epinephrine alone.) (B) Effects of clonidine on MAP-2c phosphorylation at Ser136 in differentiated PC12 cells. Differentiated PC12 cells were incubated with 10 nM, 100 nM, and 1 µM clonidine, and then total proteins were extracted for examination of MAP-2c phosphorylation at Ser136 by Western blotting. Values are mean ( SEM and are shown as a percentage of the control. (n ) 3 for each time period. *P < 0.05 compared with controls.) (C) Expression of R2-adrenoceptor subtype mRNA in differentiated PC12 cells. RNA was reverse-transcribed and amplified by PCR with R2A-specific primers (lane 1), R2B-specific primers (lane 2), and R2C-specific primers (lane 3). The templates used for the PCR amplifications are indicated by A (total RNA reverse-transcribed into the first strand of cDNA), B (rat brain cDNA; positive control), C (total RNA; control for DNA contamination), and D (no template added; negative control). The figure displays an ethidium bromide-stained agarose gel with PCR products and DNA molecular weight marker (M).
reaction were performed in parallel. PCR products were separated by a 2% agarose gel and stained with ethidium bromide. Neurite Outgrowth Assay. Brightfield images were obtained using an Olympus IX70 with a CCD camera. To quantitate neurite outgrowth, the number of neurites per cell and the relative length of the neurite, in designated microscopic fields, were measured. The differentiation score was defined as follows: 0 ) no change (round or polygonal cells); 1 ) any extension of one or more neurites, each less than one body length; 2 ) extension of one or more neurite between one and two cell body length; 3 ) at least one neurite of more than two body length in size.22,23 Statistical Analysis. Data were expressed as means ( SEM. The significance of differences was evaluated with one-way ANOVA followed by Student–Newman–Keuls test. A probability level of P < 0.05 was considered statistically significant.
Results Epinephrine Increases Phosphorylation of MAP-2c at Ser136 in Differentiated PC12 Cells. Among all the phosphorylation sites, phosphorylation of MAP-2 at Ser136 located adjacent to the R[GRAPHIC]binding region at the N-terminal domain of MAP-2 has shown to be regulated
during dendrite growth and plasticity.16 The monoclonal antibody AP18 is available to react with this epitope. To assess the effects of epinephrine on the phosphorylation of this site, PC12 cells treated with NGF for 8 days were used. Treatment of PC12 cells with epinephrine enhanced the phosphorylation of MAP-2c at Ser136 in a time- and concentration-dependent manner (Figure 1). The increase in the phosphorylation of MAP-2c at Ser136 in differentiated PC12 cells reached its maximum after incubation with epinephrine (1 µM) for 24 h. Epinephrine Induces the Increase of the Microtubule Arrays. To characterize the microtubule properties of the neurites induced by exposure to epinephrine, immunofluorescence studies were carried out using MAP-2c and R-tubulin antibodies. Because MAP-2 plays an important role in the modulation of microtubule polymerization, we examined whether epinephrine-induced MAP-2 phosphorylation affects microtubule arrays in differentiated PC12 cells using immunofluorescence. As shown in Figure 2, the fluorescence intensity was remarkably increased from differentiated PC12 cells treated with 1 µM epinephrine for 24 h when antibody against MAP2c was used, which is in agreement with increased content shown in Figure 1. Epinephrine did not affect the content of Journal of Proteome Research • Vol. 7, No. 4, 2008 1707
research articles
Tie et al.
Figure 5. Effects of epinephrine on the phosphorylation of ERK1/2 (A) and PKC (B) in differentiated PC12 cells. Differentiated PC12 cells were incubated with 1 µM epinephrine for various time intervals, and then, total proteins were extracted for examination of phosphorylation of ERK1/2 and PKC and ERK1/2 and PKC protein levels by Western blotting.
Figure 4. Effects of epinephrine on the nocodazole induced a prominent retraction of neurite outgrowth in differentiated PC12 cells. Differentiated PC12 cells were treated with 30 µM nocodazole for 45 min after 24 h incubation with epinephrine. (A) Phase contrast micrographs illustrating cell morphologies of the indicated cultures. (B) Differentiation scores were calculated as described in Experimental Procedures. Values are mean ( SEM and are shown as a percentage of the control. (n ) 3 for each time period. *P < 0.05 compared with controls; #P < 0.05 compared with the cells incubated with 1 µM epinephrine alone; ## P < 0.01 compared with the cells incubated with 1 µM epinephrine alone.)
tubulin in PC12 cells, but noticeably increased the fluorescence intensity of the microtubule networks. r2-Adrenoceptor Antagonist Yohimbine Blocks EpinephrineInduced Enhanced Phosphorylation of MAP-2c at Ser136 in Differentiated PC12 Cells. To further test whether the epinphine-induced stimulation of phosphorylation of MAP-2c at Ser136 was dependent on the activation of adrenoceptor, the respective R1, R2, and β-adrenoceptor antagonists prazosin, yohimbine, and propranolol were used. We preincubated PC12 cells with 10 µM prazosin, yohimbine,and propranolol for 30 min. The results showed that addition of yohimbine abolished the phosphorylation of MAP-2c at Ser136 induced by epinephrine, but there was discernable effect from prazosin and propranolol treatment (Figure 3A). Treatment with clonidine (0.1–1.0 µM) significantly increased MAP-2c at Ser136 in a concentration-dependent manner (Figure 3B). RT-PCR was explored to investigate the expression of R2A-, R2B-, and R2C-adrenoceptor mRNA in differentiated PC12 cells. As shown in Figure 3C, only R2A-adrenoceptor mRNA was 1708
Journal of Proteome Research • Vol. 7, No. 4, 2008
detected with a predicted size of 540 bp in differentiated PC12 cells (lanes A1); the size of PCR product of R2A-adrenoceptor was the same as that detected in rat brain tissues (lanes B1). No RT-PCR product of R2B- and R2C-adrenoceptor was detected in differentiated PC12 cells (lane A2 and A3). Epinephrine Induces Partial Protection against Retraction Evoked by Nocodazole. To examine the effect from epinephrine on microtubule, differentiated PC12 cells were treated with a microtubule depolymerizing reagent, nocodazole, after 24 h incubation with epinephrine. With treatment of 30 µM nocodazole for 45 min, neurite of some cells appeared to be reduced in control cells. However, an increase in the differentiation score was observed in cells preincubated with epinephrine (Figure 4B). ERK1/2 and PKC Activities Are Required for EpinephrineInduced Phophorylation of MAP-2c at Ser136 in Differentiated PC12 Cells. Previous studies demonstrated that several protein kinases such as ERK, PKC, and GSK are responsible for the phosphorylation of MAP-2c at N-terminal region. Thus, we examined whether the activation of ERK1/2, PKC, and GSK are involved in the MAP-2c Ser136 phosphorylation induced by epinephrine. Treatment of differentiated PC12 cells with epinephrine resulted in phosphorylation of ERK1/2 at 30 min (Figure 5A), whereas the ERK inhibitor PD98059 abolished the stimulatory effects of epinephrine on phosphorylation of MAP-2c (P < 0.01) (Figures 6 and 7). In addition, phosphorylation of PKC increased at 60 min after epinephrine treatment, and the elevated level was maintained up to 24 h (Figure 5B). As shown in Figure 6, pretreatment of cells with the PKC inhibitor chelerythrine prevented the phosphorylation of MAP-2c at Ser136 induced by epinephrine. However, no phosphorylation at MAP-2c Ser136 was observed when the PC12 cells were preincubated with PKA inhibitor H-89 or GSK inhibitor LiCl (Figure 6B).
Discussion The major new findings in the present study are (1) epinephrine evoked a time- and concentration-dependent increase of MAP2c phosphorylation at Ser136 in differentiated PC12 cells via R2adrenoceptor, resulting in the elevated microtubule networks; (2) epinephrine partially protected the neurites in differentiated PC12 cells against retraction evoked by nocodazole; (3) in vitro inhibi-
Epinephrine Increases Phosphorylation of MAP-2c in PC12 Cells
research articles
Figure 7. Schematic illustration of possible mechanisms on epinephrine regulation of MAP-2c phosphorylation at Ser136 in differentiated PC12 cells. Epinephrine induces phosphorylation of MAP-2c at Ser136 through an R2-adrenoceptor mediated, ERK/ PKC-dependent signaling pathway.
Figure 6. Effects of various inhibitors on MAP-2c phosphorylation at Ser136 in differentiated PC12 cells. Differentiated PC12 cells were preincubated with PD98059, chelerythrine (A), H-89 and LiCl (B) before being stimulated with 1 µM epinephrine for 24 h, and then, total proteins were extracted for examination of MAP-2c phosphorylation at Ser136 by Western blotting. The staining intensity of the bands was determined by laser scanning followed by quantitative densitometric analysis. Results are plotted in a graph. Values are mean ( SEM and are shown as a percentage of the control. (n ) 3 for each time period. *P < 0.05 compared with controls; #P < 0.05 compared with the cells incubated with 1 µM epinephrine alone; ##P < 0.01 compared with the cells incubated with 1 µM epinephrine alone.)
tion of ERK1/2 and PKC prevented the increase of MAP-2c phosphorylation at Ser136 induced by epinephrine. MAP-2c as the low-molecular weight isoform of MAP-2 has the identical N- and C-terminal regions to the high molecular weight MAP-2 and lacks the central domain (CD).24,25 MAP-2c, as well as its mRNA, is expressed at early development stages, and it is present in developing central nervous systems only during neurite outgrowth and not during process stabilization, implying that MAP-2c might be important for CNS developing.26–28 In the present study, the epinephrine-induced increase of phosphory-
lation of MAP-2c was only found in differentiated PC12 cells, suggesting phosphorylation of MAP-2c at Ser136 is related to the neurites’ development in PC12 cells. MAP-2 promotes the assembly and stability of microtubules and is involved in the formations of crossbridge structures between microtubules and other cytoskeletal components.29,30 This function is regulated by the phosphorylation state of MAP-2 which holds a number of phosphorylation sites. Phosphorylation at distinct sites on the MAP-2 molecule may differentially influence its function.18–20 It is known that phosphorylation at the proline-rich region and tubulin-binding domain leads to the detachment of MAP-2 from microtubules and causes their destabilization.31,32 Whereas phosphorylation of Ser136 which is located at the N-terminus and close to the PKA RΠ-binding domain may not be involved in modulating binding of MAP-2 to tubulin, nor in the formation of crossbridge structures with the outermost part of the C-terminus.33 In support of this view, the mice with the first 158 amino acids of MAP-2 deleted have shown major developmental abnormalities in hippocampal architecture.34 In addition, MAP-2 is phosphorylated at Ser136 throughout postnatal development of visual cortex and cerebellum.35 Our study showed epinephrine induced the increase of phosphorylation of MAP-2c at Ser136, with the increase of microtubule network. These results suggest the phosphorylation of MAP-2 at Ser136 may serve as a different role compared with other sites. Adrenoceptors, as inhibitory autoreceptors, regulate a number of neurotransmitters in the central and peripheral nervous system. R2-Adrenoceptors are widely distributed throughout the adult CNS and play an important role in the development and final expression of the CNS functions as they subserve. During development, mRNA for the 2A subtype of R2-adrenoceptor is expressed prenatally in many brain regions while the 2B and 2C subtypes are expressed only after birth, indicating different roles for the subtypes during development.6–8 Moreover, adrenoceptor agonists show neuroprotective effects, which are mostly via the R2A-adrenoceptor.36,37 The findings of the present study are that epinephrine induced phosphorylation of MAP-2c at Ser136 in differentiated PC12 cells, mediated by the activation of R2adrenoceptors; in addition, the R2A-adrenoceptor was found to be expressed in NGF-induced differentiated PC12 cells. In support of this possibility, Taraviras and Olli-Lahdesmaki38 demonstrated that epinephrine induced differentiation of PC12R2 cells, with flattening of the cell bodies and extension of cellular outgrowths, Journal of Proteome Research • Vol. 7, No. 4, 2008 1709
research articles similar to the morphological changes induced by NGF, while this effect was not observed in the PC12R2B and PC12R2C cells. In another study, it is found that R2A-adrenoceptor activation leads to the reduction of high-molecular weight MAP-2 phosphorylation on both serine and threonine and increase of dendrite growth in primary neuronal cultures.39 This may be due to R2-adrenoceptorinduced phosphorylation of different isoform and sites of MAP-2. Several signal molecules, such as ERK, p38 MAPK, JNK, and PKC, have been shown to be involved in the neurogenesis of PC12 cells. Among all these signal molecules, PKC, ERK, and GSK could phosphorylate the MAP-2c at the N-terminus. In the present study, epinephrine resulted in the activation of ERK1/2 and PKC after 30 min and 24 h of incubation, respectively. The blockade of ERK and PKC prevented the epinephrine-induced phosphorylation of MAP-2c at Ser136. PKA and GSK were shown not to be involved in this aspect. These results suggest that phosphorylation of MAP-2c at Ser136 may not be directly caused by ERK, because the phosphorylation of MAP-2c reached its maximum late after the activation of ERK induced by epinephrine. During the NGF-induced neurite outgrowth, PKC actives ERK via Ras activation; in our studies, ERK1/2 is activated before PKC, and showed a transient response, suggesting a different manner of stimulation of epinephrine compared with NGF. In conclusion, epinephrine enhanced phosphorylation of MAP-2c at Ser136 via R2-adrenoceptor-ERK/PKC-dependent mechanism in differentiated PC12 cells with the increase of microtubule network. This study provides new insight into the effects of epinephrine on PC12 cells.
Acknowledgment. This work was supported by the National Natural Science Foundation of China (No. 30171090, 30270528, 30572202, 30772571), 973 Program of the Ministry of Science and Technology in China (No. 2004CB518902), and research fund from Ministry of Education of China (111 Projects No.B0700 and 985 Project). References (1) Hein, L. Adrenoceptors and signal transduction in neurons. Cell Tissue Res. 2006, 326 (2), 541–551. (2) Hoffman, W. E.; Kochs, E.; Werner, C.; Thomas, C.; Albrecht, R. F. Dexmedetomidine improves neurologic outcome from incomplete ischemia in the rat. Reversal by the alpha 2-adrenergic antagonist atipamezole. Anesthesiology 1991, 75 (2), 328–332. (3) Halonen, T.; Kotti, T.; Tuunanen, J.; Toppinen, A.; Miettinen, R.; Riekkinen, P. J. Alpha 2-adrenoceptor agonist, dexmedetomidine, protects against kainic acid-induced convulsions and neuronal damage. Brain Res. 1995, 693 (1–2), 217–224. (4) Laudenbach, V.; Mantz, J.; Lagercrantz, H.; Desmonts, J. M.; Evrard, P.; Gressens, P. Effects of alpha(2)-adrenoceptor agonists on perinatal excitotoxic brain injury: comparison of clonidine and dexmedetomidine. Anesthesiology 2002, 96 (1), 134–141. (5) Handy, D. E.; Flordellis, C. S.; Bogdanova, N. N.; Bresnahan, M. R.; Gavras, H. Diverse tissue expression of rat alpha 2-adrenergic receptor genes. Hypertension 1993, 21 (6 Pt. 1), 861–865. (6) Winzer-Serhan, U. H.; Leslie, F. M. Alpha2B adrenoceptor mRNA expression during rat brain development. Brain Res. Dev. Brain Res. 1997, 100 (1), 90–100. (7) Winzer-Serhan, U. H.; Raymon, H. K.; Broide, R. S.; Chen, Y.; Leslie, F. M. Expression of alpha 2 adrenoceptors during rat brain developmentsII. Alpha 2C messenger RNA expression and [3H]rauwolscine binding. Neuroscience 1997, 76 (1), 261–272. (8) Winzer-Serhan, U. H.; Raymon, H. K.; Broide, R. S.; Chen, Y.; Leslie, F. M. Expression of alpha 2 adrenoceptors during rat brain developmentsI. Alpha 2A messenger RNA expression. Neuroscience 1997, 76 (1), 241–260. (9) Civantos Calzada, B.; Aleixandre de Artinano, A. Alpha-adrenoceptor subtypes. Pharmacol. Res. 2001, 44 (3), 195–208. (10) Rowe, S. J.; Messenger, N. J.; Warner, A. E. The role of noradrenaline in the differentiation of amphibian embryonic neurons. Development 1993, 119 (4), 1343–1357.
1710
Journal of Proteome Research • Vol. 7, No. 4, 2008
Tie et al. (11) Yamada, K. M.; Spooner, B. S.; Wessells, N. K. Axon growth: roles of microfilaments and microtubules. Proc. Natl. Acad. Sci. U.S.A. 1970, 66 (4), 1206–1212. (12) Kalcheva, N.; Albala, J.; O’Guin, K.; Rubino, H.; Garner, C.; ShafitZagardo, B. Genomic structure of human microtubule-associated protein 2 (MAP-2) and characterization of additional MAP-2 isoforms. Proc. Natl. Acad. Sci. U.S.A. 1995, 92 (24), 10894–10898. (13) Kalcheva, N.; Shafit-Zagardo, B. Three unique 5′ untranslated regions are spliced to common coding exons of high- and lowmolecular-weight microtubule-associated protein-2. J. Neurochem. 1995, 65 (4), 1472–1480. (14) Binder, L. I.; Frankfurter, A.; Kim, H.; Caceres, A.; Payne, M. R.; Rebhun, L. I. Heterogeneity of microtubule-associated protein 2 during rat brain development. Proc. Natl. Acad. Sci. U.S.A. 1984, 81 (17), 5613–5617. (15) Riederer, B.; Matus, A. Differential expression of distinct microtubule-associated proteins during brain development. Proc. Natl. Acad. Sci. U.S.A. 1985, 82 (17), 6006–6009. (16) Sanchez, C.; Diaz-Nido, J.; Avila, J. Phosphorylation of microtubuleassociated protein 2 (MAP2) and its relevance for the regulation of the neuronal cytoskeleton function. Prog. Neurobiol. 2000, 61 (2), 133–168. (17) Brugg, B.; Matus, A. Phosphorylation determines the binding of microtubule-associated protein 2 (MAP2) to microtubules in living cells. J. Cell Biol. 1991, 114 (4), 735–743. (18) Audesirk, G.; Cabell, L.; Kern, M. Modulation of neurite branching by protein phosphorylation in cultured rat hippocampal neurons. Brain Res Dev Brain Res 1997, 102 (2), 247–60. (19) Johnson, G. V.; Jope, R. S. The role of microtubule-associated protein 2 (MAP-2) in neuronal growth, plasticity, and degeneration. J. Neurosci. Res. 1992, 33 (4), 505–512. (20) Sanchez Martin, C.; Diaz-Nido, J.; Avila, J. Regulation of a sitespecific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons. Neuroscience 1998, 87 (4), 861–870. (21) Greene, L. A.; Tischler, A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. U.S.A. 1976, 73 (7), 2424–2428. (22) Davidkova, G.; Zhang, S. P.; Nichols, R. A.; Weiss, B. Reduced level of calmodulin in PC12 cells induced by stable expression of calmodulin antisense RNA inhibits cell proliferation and induces neurite outgrowth. Neuroscience 1996, 75 (4), 1003–1019. (23) Acosta, R.; Montanez, C.; Fuentes-Mera, L.; Gonzalez, E.; Gomez, P.; Quintero-Mora, L.; Mornet, D.; Alvarez-Salas, L. M.; Cisneros, B. Dystrophin Dp71 is required for neurite outgrowth in PC12 cells. Exp. Cell Res. 2004, 296 (2), 265–275. (24) Kindler, S.; Schulz, B.; Goedert, M.; Garner, C. C. Molecular structure of microtubule-associated protein 2b and 2c from rat brain. J. Biol. Chem. 1990, 265 (32), 19679–19684. (25) Garner, C. C.; Matus, A. Different forms of microtubule-associated protein 2 are encoded by separate mRNA transcripts. J. Cell Biol. 1988, 106 (3), 779–783. (26) Matus, A. Microtubule-associated proteins: their potential role in determining neuronal morphology. Annu. Rev. Neurosci. 1988, 11, 29–44. (27) Nunez, J. Differential expression of microtubule components during brain development. Dev. Neurosci. 1986, 8 (3), 125–141. (28) Nunez, J. Immature and mature variants of MAP2 and tau proteins and neuronal plasticity. Trends Neurosci. 1988, 11 (11), 477–479. (29) Hirokawa, N. Microtubule organization and dynamics dependent on microtubule-associated proteins. Curr. Opin. Cell Biol. 1994, 6 (1), 74–81. (30) Mandelkow, E.; Mandelkow, E. M. Microtubules and microtubuleassociated proteins. Curr. Opin. Cell Biol. 1995, 7 (1), 72–81. (31) Ebneth, A.; Drewes, G.; Mandelkow, E. M.; Mandelkow, E. Phosphorylation of MAP2c and MAP4 by MARK kinases leads to the destabilization of microtubules in cells. Cell Motil. Cytoskeleton 1999, 44 (3), 209–224. (32) Murthy, A. S.; Flavin, M. Microtubule assembly using the microtubule-associated protein MAP-2 prepared in defined states of phosphorylation with protein kinase and phosphatase. Eur. J. Biochem. 1983, 137 (1–2), 37–46. (33) Riederer, B. M.; Draberova, E.; Viklicky, V.; Draber, P. Changes of MAP2 phosphorylation during brain development. J. Histochem. Cytochem. 1995, 43 (12), 1269–1284. (34) Khuchua, Z.; Wozniak, D. F.; Bardgett, M. E.; Yue, Z.; McDonald, M.; Boero, J.; Hartman, R. E.; Sims, H.; Strauss, A. W. Deletion of the N-terminus of murine map2 by gene targeting disrupts hippocampal ca1 neuron architecture and alters contextual memory. Neuroscience 2003, 119 (1), 101–111.
research articles
Epinephrine Increases Phosphorylation of MAP-2c in PC12 Cells (35) Philpot, B. D.; Lim, J. H.; Halpain, S.; Brunjes, P. C. Experiencedependent modifications in MAP2 phosphorylation in rat olfactory bulb. J. Neurosci. 1997, 17 (24), 9596–9604. (36) Ma, D.; Rajakumaraswamy, N.; Maze, M. alpha2-Adrenoceptor agonists: shedding light on neuroprotection. Br. Med. Bull. 2004, 71, 77–92. (37) Wheeler, L.; WoldeMussie, E.; Lai, R. Role of alpha-2 agonists in neuroprotection. Surv. Ophthalmol. 2003, 48 (Suppl. 1), S47–51. (38) Taraviras, S.; Olli-Lahdesmaki, T.; Lymperopoulos, A.; Charitonidou, D.; Mavroidis, M.; Kallio, J.; Scheinin, M.; Flordellis, C.
Subtype-specific neuronal differentiation of PC12 cells transfected with alpha2-adrenergic receptors. Eur. J. Cell Biol. 2002, 81 (6), 363–374. (39) Song, Z. M.; Abou-Zeid, O.; Fang, Y. Y. Alpha2a adrenoceptors regulate phosphorylation of microtubule-associated protein-2 in cultured cortical neurons. Neuroscience 2004, 123 (2), 405–418.
PR700711S
Journal of Proteome Research • Vol. 7, No. 4, 2008 1711
