Nerve Growth Factor-Induced Differentiation of PC12 Cells Is Accompanied by Elevated Adenylyl Cyclase Activity

Rat pheochromocytoma (PC12) cells characteristically undergo differentiation when cultured with nerve growth factor (NGF). Here we show that NGF dramatically increased the adenylyl cyclase-activating property of forskolin in PC12 cells. This effect of NGF was well maintained even when NGF was removed after 4 days, even though the morphological features of neuronal differentiation were rapidly lost on removal of NGF. The enhanced cAMP production in response to forskolin could be due to a synergistic interaction between forskolin and endogenously released agonists acting on G s -coupled receptors. However, responses to forskolin were not attenuated by antagonists of adenosine A2 receptors or pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, suggesting that adenosine and PACAP were not involved. Adenylyl cyclases 3, 6 and 9 were the predominant isoforms expressed in PC12 cells, but we found no evidence for NGF-induced changes in expression levels of any of the 9 adenylyl cyclase isoforms, nor in the expression of G s . These findings highlight that NGF has a subtle influReceived: December 14, 2009 Accepted after revision: February 4, 2010 Published online: April 14, 2010 Dr. H. Wise School of Biomedical Sciences, Faculty of Medicine The Chinese University of Hong Kong Shatin, NT, Hong Kong, SAR (China) Tel. +852 2609 6849, Fax +852 2603 5139, E-Mail helenwise @ cuhk.edu.hk © 2010 S. Karger AG, Basel 1424–862X/10/0181–0032$26.00/0 Accessible online at: www.karger.com/nsg D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /6 /2 01 7 4: 10 :5 9 A M Effect of NGF on cAMP in PC12 Cells Neurosignals 2010;18:32–42 33 The cyclooxygenase-1 enzyme responsible for prostaglandin production behaves as a delayed response gene in PC12 cells exposed to NGF [6, 10] . During studies to assess the role of prostaglandins during PC12 cell differentiation [11] , we noted a marked increase in forskolin-stimulated adenylyl cyclase (AC) activity over a 6-day treatment period with NGF. Forskolin directly activates AC and can amplify cAMP-dependent signaling activated by G s -coupled receptors [12] . Thus, G s -promoted enhancement of AC activity in response to forskolin occurs not only when cells are incubated with exogenously administered agonists for G s -coupled receptors but also by agonists that can be endogenously released by cells. Many cells are capable of releasing AMP which can undergo extracellular degradation to adenosine via ectonucleotidases [13] . Indeed, endogenously released adenosine has been shown to act synergistically with forskolin to enhance cAMP production through A2aR in PC12 cells [14] . Furthermore, NGF can increase PACAP production by PC12 cells [15] which could stimulate PACAP-specific receptors (PAC1R) to generate cAMP and inositol phosphates by coupling to G s and G q proteins, respectively [16] . Therefore, if NGF caused the increased expression of components of G s -dependent cell signaling pathways (e.g., G s -coupled receptors and/or their endogenous ligands), then forskolin could synergize with these factors and generate the responses we observed. Cyclic AMP-dependent protein kinases have an essential role in neuronal differentiation and synaptic plasticity [17–19] , and modulation of cAMP-dependent signaling may be crucial in developing methodologies to generate pure populations of neurons from human neural stem cells [20] . Therefore, the aim of this study was to identify which factor(s) were responsible for the enhanced responses to forskolin observed in our PC12 cell line following incubation with NGF and to determine the relationship between increased AC activity and the process of neuronal differentiation. Having previously shown that NGF increased A2aR and PACAP mRNA expression in PC12 cells [6] , we initially focused on these factors in addition to other components of cAMP-dependent cell signaling pathways in PC12 cells. Materials and Methods Materials Mouse nerve growth factor (mNGF 2.5s) was purchased from Alomone Labs (Jerusalem, Israel). [ 3 H]adenine was purchased from GE Healthcare (Hong Kong). TRIzol, DNase I (Amp Grade), oligo-d(T) 20 primer, dNTPs and M-MVL were from Invitrogen (Carlsbad, Calif., USA), and FastStart TaqDNA Polymerase was from Roche Diagnostics (Mannheim, Germany). GelRed TM was purchased from Biotium (Hayward, Calif., USA). PACAP-(1–38) and PACAP-(6–38) were purchased from Phoenix Pharmaceuticals (Beijing, China), and ZM241385 was purchased from Tocris Bioscience (Bristol, UK). All other compounds were supplied by Invitrogen or Sigma Chemical Co. (St. Louis, Mo., USA). Cell Culture PC12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with heat-inactivated horse serum (HIHS: 10%, v/v), fetal bovine serum (5%, v/v), penicillin (100 U/ ml) and streptomycin (100  g/ml). Cells were routinely grown on 100-mm tissue culture dishes at 37 ° C in a humidified atmosphere of 5% CO 2 /95% air, and medium was changed every 3 days. Undifferentiated cells were plated in collagen-coated (0.2 mg/ml) tissue culture plates for assay. To study the effect of NGF, cells were cultured for 1 day in collagen-coated assay plates, then washed and incubated in low serum medium (DMEM containing 1% HIHS). NGF (50 ng/ml) or an equivalent volume of DMEM was then added (taken as day 0). Medium 8 NGF was replaced every 2 days. cAMP Assay AC activity was assayed in Hepes-buffered saline containing 1 m M 3-isobutyl-1-methylxanthine (IBMX) to inhibit cyclic nucleotide phosphodiesterase activity, as previously described [21] . Briefly, PC12 cells (3 ! 10 5 cells/well) were cultured in 0.5 ml medium in 24-well plates, and incubated with [ 3 H]adenine (2 Ci/ml; 1 Ci/well) for 24 h prior to assay. The production of [ 3 H]cAMP from cellular [ 3 H]ATP was estimated as the ratio of radiolabeled cAMP to total AXP (i.e. cAMP, ADP and ATP), and is expressed as [cAMP]/[total AXP] ! 100 (i.e. % conversion). All assays were performed in duplicate. Cells were preincubated with antagonists/inhibitors for 30 min as appropriate and then incubated with forskolin (1 M ) or receptor agonists for 30 min. Morphometric Analysis PC12 cells (5 ! 10 4 cells/well) were cultured in 2 ml medium in 6-well plates. At specific time points, phase-contrast images were captured (DS Camera Control Unit DS-L1, Nikon) under 100 ! magnification from at least 5 views per well. The images were quantified using Scion Image with the observer unaware of the treatment group. One hundred cells per well were scored for differentiation defined as (i) the proportion of cells with neurite/s equal to or greater than the diameter of the cell body, (ii) the number of neurites per cell, and (iii) the length of longest neurite. RT-PCR Total RNA was extracted with TRIzol and treated with DNase I to eliminate any contaminating genomic DNA. First-strand complementary DNA was synthesized using the M-MLV reverse transcriptase system followed by FastStart TaqDNA Polymerase for PCR amplification. The primers for AC1–9 and GAPDH were taken from published sequences [22] . Primer sequences for A2aR, A2bR, G s were from Castillo et al. [23] and those for PACAP and PAC1R from Hashimoto et al. [15] and Braas and May [24] , respectively. PCR was initiated by a 5-min denaturation at 95 ° C, followed by 34 cycles (for AC3, AC6, AC7, AC9, A2bR, PACAP, PAC1R and G s and GAPDH) or 40 cycles (for AC1, AC2, AC4, D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /6 /2 01 7 4: 10 :5 9 A M Yung /Lai /Chow /Ip /Tsim /Wong /Wu / Wise Neurosignals 2010;18:32–42 34 AC5, AC8 and A2bR) of 30 s at 54 ° C and 50 s at 72 ° C, and terminated by a final extension for 7 min at 72 ° C. The PCR products were resolved using a 1.5% agarose gel containing GelRed TM , and the DNA bands were visualized by UV illumination and were quantified by Quantity One software (Bio-Rad Laboratories, Hercules, Calif., USA). The relationship between the amount of PCR products and the number of amplification cycles (27–36 for A2aR and G s ; 27–39 for PACAP; 30–39 for AC1, 6, 7, 9, GAPDH and PAC1R; 36–42 for AC1, 2, 4, 5, 8 and A2bR) was linear (data not shown). Data Analysis Values reported are means 8 SEM. Comparisons between groups were made using ANOVA with Bonferroni’s post-tests. All analyses were performed using GraphPad Prism software version 5.1 (GraphPad Software Inc., San Diego, Calif., USA). Statistical significance was taken as p ! 0.05.