Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10423

RESEARCH ARTICLE

Neurite outgrowth resistance to rho kinase inhibitors in PC12 Adh cell Hua Yin1, Xiaolin Hou1, Tingrui Tao1, Xiaoman Lv1, Luyong Zhang2 and Weigang Duan1,3* 1 Key Laboratory of Molecular Biology for Sinomedicine, Yunnan University of Traditional Chinese Medicine, 1076, Yuhua Road, University City of Chenggong, Kunming 650500, China 2 Jiangsu Center for Drug Screening, China Pharmaceutical University, 24, Tongjiaxiang, Nanjing 210009, China 3 Initiative team of Microenvironment, Yunnan Key Laboratory for Enthnomedicine, 1076, Yuhua Rd, Chenggong District, Kunming 650500, China

Abstract Rho kinase (ROCK) inhibitor is a promising agent for neural injury disorders, which mechanism is associated with neurite outgrowth. However, neurite outgrowth resistance occurred when PC12 Adh cell was treated with ROCK inhibitors for a longer time. PC12 Adh cells were treated with ROCK inhibitor Y27632 or NGF for different durations. Neurite outgrowth resistance occurred when PC12 Adh cell exposed to Y27632 (33 mM) for 3 or more days, but not happen when exposed to nerve growth factor (NGF, 100 ng/mL). The gene expression in the PC12 Adh cells treated with Y27632 (33 mM) or NGF (100 ng/mL) for 2 or 4 days was assayed by gene microarray, and the reliability of the results were confirmed by real-time RT-PCR. Cluster analysis proved that the gene expression profile of PC12 Adh cell treated with Y27632 for 4 days was different from that treated with Y27632 for 2 days and those treated with NGF for 2 and 4 days, respectively. Pathway analysis hinted that the neurite outgrowth resistance could be associated with up-regulation of inflammatory pathways, especially rno04610 (complement and coagulation cascades), and down-regulation of cell cycle pathways, especially rno04110. Keywords: cell differentiation; neurite outgrowth resistance; PC12 Adh cell; rho kinase inhibitor; Y27632

Introduction Rho kinase (ROCK), also known as rho-associated coiledcoil forming protein serine/threonine kinase, is one of the central regulatory biomacromolecules for cytoskeleton movement (Leung et al., 1996) and neurite outgrowth (Hirose et al., 1998). Two isoforms encoded by two different genes of ROCK have been described: ROCK I (ROK b or p160 ROCK) and ROCK II (ROK a) (Duan et al., 2006; Darenfed et al., 2007). The enzyme is activated when bound to rho-GTP (especially rho A-GTP), and the active ROCK will phosphorylate myosin light chain (MLC) and other proteins, which results in cytoskeleton regulation(Amano et al., 1996). ROCK II is preferentially expressed in nervous system, while ROCK I shows higher expression levels in non-neuronal tissues, including heart, lung, and skeletal muscles (Mueller et al., 2005). ROCK II expression in bovine brain was observed mainly in the pyramidal neurons of the hippocampus and cerebral cortex, and in the Purkinje cells



of the cerebellum (Mueller et al., 2005). In PC12-related cell (PC12 Adh cell and PC12 cell) (Yang et al., 2010; Que et al., 2011) and Neuro-2a cell (Wylie and Chantler, 2003), ROCK II expression was also observed. It is believed that neurite outgrowth is the physical foundation for recovery from neural injury and neural disorders, which are associated with neuron death and/or glial scar formation (Mueller et al., 2005; Chan et al., 2007; Kubo and Yamashita, 2007). ROCK over-activation or upregulation was reported to be correlated with neural injury and most neural disorders (Mueller et al., 2005; Kubo and Yamashita, 2007). Otherwise, ROCK inhibition by Y27632 or fasudil and ROCK down-regulation by small interfering RNA (siRNA) were both able to promote neural fiber regeneration and functional recovery (Fournier et al., 2003; Hiraga et al., 2006; Impellizzeri et al., 2012). Even further, cell models demonstrated that ROCK over-activation caused neural growth cone collapse (Wahl et al., 2000), while ROCK inhibition caused neurite outgrowth (Mueller et al., 2005).

Corresponding author: e-mail: [email protected]

Cell Biol Int 39 (2015) 563–576 © 2015 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

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Thus, the relationship between ROCK inhibition and neural recovery was well established. In cell models, the morphological pattern of neurite outgrowth caused by ROCK inhibitors was very similar to that caused by nerve growth factor (NGF) in early observation (less than 3 days) (Fujita et al., 2001; Nusser et al., 2002; Minase et al., 2010). Because NGF was able to down-regulate ROCK expression, and the neurite outgrowth effect was almost reversed by ROCK activators, a rough conclusion was drawn that it is via ROCK inhibition that NGF induced neurite outgrowth (Fujita et al., 2001; Nusser et al., 2002; Minase et al., 2010). According to the above conclusion, it can be deduced that, both the morphological profile of neurite outgrowth in early observation (less than 3 days) and later observation (3 days and more) should also be similar. The profile of neural function including neurotransmitter synthesis, release, and some functional protein expression such as synapsin I should also be similar. However, experiments did not support the speculations. Actually, when PC12-related cell was treated with ROCK inhibitor of Y27632 for 6 days, expression of tyrosine hydroxylase and synapsin I was down-regulated, followed with norepinephrine synthesis and release decreased, which were different from those in PC12-related cell treated with NGF (Fukuda et al., 2005; Duan et al., 2009). Our previous study in PC12 Adh cell (Que et al., 2011; Duan et al., 2012) further suggested that, different from NGF, ROCK inhibitor could cause neurite outgrowth resistance, since the neurite outgrowth would cease or collapse when the cell was exposed to ROCK inhibitors for more than 3 days. The differences could be a result of the different gene expression profiles induced by ROCK inhibitor at different times. PC12 cell is a pheochromocytoma cell line from Rattus norvegicus, a common tool cell line used for studying neural differentiation and neural function. About 5 years ago, there was only one cell line related to PC12 (PC12 cell) deposited in American Type Culture Collection (ATCC). However, there are two recently: one is PC12 cell and the other is PC12 Adh cell. The description of them by ATCC is different from each other in morphology and culture method, though they derived from the same ancestral cell. Our previous study found that PC12 Adh cell is more sensitive to ROCK inhibitors in neurite outgrowth than PC12 cell (Que et al., 2011). Our previous study on PC12 Adh cell even found that the neurite outgrowth resistance was associated with cyclooxygenase-2 (COX-2) pathway up-regulation (Duan et al., 2012). However, COX-2 pathway suppression only partly improved the resistance (Duan et al., 2012). The results strongly suggested that there could be more important pathways or genes involved. Since total gene expression analysis is a good approach to study gene expression and pathway regulation, the present work applied 564

this approach to attempt to find the mechanism of the resistance in PC12 Adh cell caused by ROCK inhibitor Y27632. Materials and methods

Materials PC12 cell and PC12 Adh cell were purchased from ATCC, and were identified based on the ATCC’s description. ROCK inhibitor Y27632 ([(R)-(þ)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide  2HCl], purity  95%) was purchased from Alexis Corporation (Lausen, Switzerland). NGF-7S (130 kDa) was purchased from Sigma-Aldrich (St. Louis, MO). Ham’s F12K medium, Roswell Park Memorial Institute-1640 (RPMI-1640) Medium, horse serum, fetal bovine serum, Trizol reagent, and 1 TRIzol Plus RNA Purification kit were produced by Invitrogen (Carlsbad, CA).

Cell culture PC12 cells were incubated in RPMI-1640 medium with 10% heat-inactivated horse serum and 5% fetal bovine serum in an atmosphere containing 5% carbon dioxide (CO2) and 95% air at 37 C (http://www.atcc.org/Products/All/CRL1721.aspx#culturemethod). PC12 Adh cells were incubated in Ham’s F12K medium with 15% horse serum and 2.5% fetal bovine serum in an atmosphere containing 5% CO2 and 95% air at 37 C (http://www.atcc.org/Products/All/CRL1721.1.aspx#culturemethod).

Positive cell observation When cells were about to occupy 80% of the flask, they were dispersed and seeded in 96-well plates coated with 0.01% polylysine. After 4 h, cells were exposed to Y27632 (33 mM) or NGF (300 ng/mL). The medium was renewed every 2 days, and the concentration of Y27632 or NGF in the medium was maintained. The morphological observation was carried out every 24 h. The treated PC12 cells were categorized into three groups based on their morphology: neurite-positive cells, round cells, and other cells. The cells with longer neurite(s) than their soma were defined as neurite-positive cells (Duan et al., 2009). The other cells had various features including microspikes, ruffles, and a flattened appearance. More than 100 cells in one well were randomly observed. If fewer than 100, all of them were observed. The area occupied by cells was less than 50% throughout the observation. Neurite-positive cell rate was calculated by Formula 1 (Duan et al., 2009).

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Neurite-positive cell rateð%Þ neurite-positive cells  100% ¼ total cells observed

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ðFormula1Þ

NimbleGen rat gene expression microarrays Cell culture for gene expression microarray When PC12 Adh cells were about to occupy 80% of the flask, they were dispersed and equally seeded in five new flasks (25 cm2). Four hours later, cells in two flasks were exposed to Y27632 (33 mM) for 48 h (Y276322D) or 96 h (Y276324D), cells in the other two flasks were exposed to NGF (100 ng/ mL) for 48 h (NGF2D) or 96 h (NGF4D), and the cells in the last flask were exposed to phosphate-buffered saline (PBS) of the same volume for 72 h (PC12RNA). The medium was renewed every 2 days, and the concentration of Y27632 or NGF in the medium was maintained. At the end of 2 days, the culture medium was gently removed and the flasks were kept at 4 C. Then, 2 mL cold Trizol reagent was drawn into the flasks, and cells were gently piped to lysate. The lysate was kept at 80 C, and total RNA in the lysate was extracted and purified by TRIzol Plus RNA Purification kit. RNA sample quality control RNA quantity and quality were measured by NanoDrop ND-1000 spectrophotometer (PeqLab, Erlangen, Germany). RNA integrity was assessed by standard denaturing agarose gel electrophoresis. RNA labeling and array hybridization This part of experiment followed MIAME standards for microarrays (Brazma et al., 2001). Double-strand cDNA (dscDNA) was synthesized from total RNA using an Invitrogen SuperScript ds-cDNA synthesis kit in the presence of 100 pM oligo dT primers. ds-cDNA was cleaned and labeled in accordance with the NimbleGen Gene Expression Analysis protocol (NimbleGen Systems, Inc., Madison, WI, USA). Briefly, ds-cDNA was incubated with 4 mg RNase A at 37 C for 10 min and cleaned using phenol, chloroform, isoamyl alcohol, followed by ice-cold absolute ethanol precipitation. The purified cDNA was quantified using a NanoDrop ND-1000. For Cy3 labeling of cDNA, the NimbleGen One-Color DNA labeling kit was used according to the manufacturer’s guidelines detailed in the Gene Expression Analysis protocol (NimbleGen Systems, Inc.). ds-cDNA (1 mg) was incubated for 10 min at 98 C with 1 OD of Cy3–9mer primer. Later 100 pM of deoxynucleoside triphosphates and 100 U of the Klenow fragment (New England Biolabs, Ipswich, MA, USA) were added and the mixture was incubated at 37 C for 2 h. The reaction was

stopped by adding 0.1 volume of 0.5 M ethylenediaminetetraacetic acid, and the labeled ds-cDNA was purified by isopropanol/ethanol precipitation. Microarrays were hybridized at 42 C for 16 to 20 h with 4 mg of Cy3-labeled dscDNA in NimbleGen hybridization buffer/hybridization component A in a hybridization chamber (Hybridization System; NimbleGen Systems, Inc.). Following hybridization, washing was done using the NimbleGen Wash Buffer kit (NimbleGen Systems, Inc.). After being washed in an ozonefree environment, the slides were scanned using the Axon GenePix 4000B microarray scanner (Molecular Devices Corporation, CA, USA). Data collection and treatment Slides were scanned at 5 mm pixel resolution using an Axon GenePix 4000B microarray scanner piloted by GenePix Pro 6.0 software (Axon). Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. The Probe level (*_norm_RMA.pair) files and Gene level (*_RMA.calls) files were generated after normalization. All gene level files were imported into Agilent GeneSpring GX software (version 11.5.1) for further analysis. Differentially expressed genes were identified through Fold Change filtering. Hierarchical clustering was performed using the Agilent GeneSpring GX software (version 11.5.1). Gene ontology (GO) analysis and pathway analysis were performed using the standard enrichment computation method. Data analysis The box plot is a traditional method for visualizing the distribution of a dataset (Battke et al., 2010). They are most useful for comparing the distributions of several datasets. Here, a box plot view was used to observe and compare the distributions of expression values for the samples in an experiment after normalization. Scatter plot is a visualization method used for assessing the variation in gene expression (or reproducibility) between two compared arrays (Meng et al., 2008; Battke et al., 2010). The values of X and Y axes in the scatter plot are the normalized signal values of each sample (log2 scaled). The green lines are fold change lines; and the default fold change value given was 2.0. Genes above the top green line and below the bottom green line indicated more than 2.0-fold change of genes between two compared arrays. Hierarchical clustering is one of the simplest and widely used clustering techniques for analysis of gene expression data (Battke et al., 2010; Yu and Peng, 2013). Cluster analysis arranges samples into groups based on their expression levels, which allow us to hypothesize about the relationships

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among samples. The dendrogram shows the relationships among the expression levels of samples. Pathway analysis Based on the latest Kyoto Encyclopedia of Genes and Genomes (KEGG) database, pathway analysis by differentially expressed genes was provided. This analysis allowed to determine the biological pathway and identified a significant enrichment of differentially expressed genes. The P-value denoted the significance of the pathway (Subramanian et al., 2005). The lower the P-value, the more significant the pathway (the cutoff P-value is 0.05.). Enrichment Score is calculated by Formula 2. The original Enrichment Score is a positive value, and a minus is placed before the value if the pathway is down-regulated.

EnrichmentScore ¼ log 10 ðP-valueÞ

ðFormula2Þ

microarray (see RNA labeling and array hybridization). Primers of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, as an internal control) and the selected genes were designed online http://www.ncbi.nlm.nih.gov/tools/ primer-blast/ (Table 1), and synthesized by Shanghai KangChen Bio-Tech (Shanghai, China). Real-time PCR was performed in ABI PRISM 7900 system (Applied Biosystems, NY, USA). The results of real time RT-PCR of the selected genes were scaled by Formula 3. The scaled results were analyzed with one-way analysis of variance, and the means comparison of the scaled results between samples was performed using Student-Newman-Keuls method (if equal variances were assumed) or Tamhane’s T2 method (if equal variances were not assumed) of post hoc test. Statistically significant differences were accepted at P < 0.05.

ScaledResult ¼ log 2 ð Real-time reverse transcription-polymerase chain reaction (RT-PCR) Twelve genes related to neurite outgrowth and/or to neural differentiation were selected for real-time RT-PCR based on the results of gene expression microarray. The total cDNA was measured from an aliquot for gene expression

Geneselected Þ GAPDH

ðFormula3Þ

Bivariate correlation between the scaled results from realtime RT-PCR and those of gene expression microarray of the same gene was performed using Pearson’s method, and statistically significant differences were accepted at P < 0.05 (two tailed).

Table 1 Information on primers of the selected genes

Gene

Seq. ID

Function

Primers

GAPDH

XM_576394.3

Atp1a2

NM_012505

Atp2b2

NM_012508

As an internal control in the present study. GO:0043005(neuron projection); GO:0043197(dendritic spine) GO:0030425 (dendrite)

Syp

NM_212523

GO:0043005 (neuron projection)

Map2

NM_013066

GO:0016358(dendrite development)

Slc1a2

NM_031967

development-related protein

Vgf

NM_031783

GO:0043005 (neuron projection)

Nefl

NM_053865

GO:0030182 (neuron differentiation)

Ndrg4

NM_012618

GO:0043005 (neuron projection)

Stmn2

NM_017215

GO:0043197 (dendritic spine)

Rtn1

NM_053440

GO:0030182 (neuron differentiation)

Kif5a

NM_012664

S100a4

NM_030997

GO:0048168 (regulation of neuronal synaptic plasticity) GO:0030182 (neuron differentiation)

GGAAAGCTGTGGCGTGAT AAGGTGGAAGAATGGGAGTT TACACCCTGACCAGCAACATC AGCCGCTTCGTATGCTAATG TGCCTGCTATTTCCGTTCA TCTCTCCTCTTCTCCCTTGTCTA ACAGCCGTGTTCGCTTTCA GGGTCCCTCAGTTCCTTGC CTTGCCTATGTCTTGCCTTGA CCATCGTTCCGCTAGTGTTG GGAAGAAGAACGACGAGGTGT GAGATGACTGCCTTGGTTGTATT CGTCCTCTTCTGCTTCCTTCTAC TCCTCAGCTACCTGCCCATT AGAAGAAGGTGGTGAGGGTGA CCTGGGATAGTTGGGAATGG AGCGTATGTGCTCGCCAAGT GGCAGCCCAATCAATCCAG ACTTGGAGACTGTGAGCTGGTT TTGCCCTACACTGGGAATGA ATGGCTTATTGAGTTCTGATTCTG ACAGGAGATGGCTGGTTAGGT CCCAGATTGCTAAGCCTGTGAG CTGTGGCGTTTCCATTGTCC ACTTGGACAGCAACAGGGAC ATGGCAATGCAGGACAGGA

566

Anneal ( C)

Product (bp)

60

308

60

150

60

128

60

152

60

110

60

169

60

114

60

129

60

106

60

102

60

259

60

243

60

70

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Results

Y27632 induced neurite outgrowth in PC12 Adh cell rather than in PC12 cell Normal PC12 cell is morphologically similar to PC12 Adh cell. NGF (300 ng/mL) was able to induce neurite outgrowth in both cells in 48 h, while ROCK inhibitors Y27632 and fasudil of 33 mM were not able to do so in PC12 cell (Figure 1) (the results of fasudil shown in Supporting Information Figure S1). The results were consistent with our previous findings (Que et al., 2011).

Y27632 induced neurite outgrowth in PC12 Adh cell but caused resistance NGF (300 ng/mL) was able to induce neurite outgrowth in PC12 Adh cell in a time-dependent manner (Figure 2A and 2B). The effect of Y27632 (33 mM) and fasudil (33 mM, shown in Figure S2) in inducing neurite outgrowth was similar to that of NGF in 48 h, but the effect became weaker later (Figures 2A and 2B). The results confirmed our previous findings (Duan et al., 2012).

Results of gene expression analysis The integrity of RNA was assessed by electrophoresis on a denaturing agarose gel. Intact total RNA run on a denaturing gel had sharp 28S and 18S rRNA bands (eukaryotic samples).

Neurite outgrowth resistance to rho kinase inhibitors

The 28S rRNA band was approximately twice as intense as the 18S rRNA band. This 2:1 intensity ratio indicates that the RNA is intact. OD260/OD280 ratio of the total RNA samples was higher than 1.8, later detected by the NanoDrop ND1000. An Axon GenePix 4000B microarray scanner scanned the microarray, and signals of 26,419 genes in every sample were collected. After normalization, the distributions of log2 ratios among the samples were nearly the same (Figure 3A). To compare distribution between every two samples, scatter plots were also performed (see Figure S3). Hierarchical clustering was performed based on “All Targets Value”. The result of hierarchical clustering showed distinguishable gene expression profiling among the five samples (Figure 3B). The results showed that, there was a discontinuous change of gene expression profile in PC12 Adh from Y276322D to Y276324D, interrupted by PC12RNA (the control), and there was a continuous change of gene expression profile from NGF2D to NGF4D.

Pathway analysis Based on the latest (Nov 26, 2013) KEGG database, pathway analysis by differentially expressed genes was provided. Compared with control sample (PC12RNA), top 10 downor up-regulated pathways of NGF-treated samples (Figure 4) and Y27632-treated samples (Figure 5) were listed. There were 34–46 significant down- or up-regulated pathways in NGF- or Y27632-treated samples.

Figure 1 Rather than PC12 cell, PC12 Adh cell was sensitive to ROCK inhibitor Y27632 (33 mM) in neurite outgrowth, although both cells were able to grow neurites when exposed to NGF (300 ng/mL) for 48 h. bar ¼ 50 mm.

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Figure 2 Resistance occurred when PC12 Adh cell was exposed to ROCK inhibitor Y27632 for 3 or more days. Both Y27632 (33 mM) and NGF (300 ng/mL) were able to similarly induce neurite outgrowth in PC12 Adh cell within 2 days. However, if exposed to Y27632 for 3 or more days, the neurites did not grow as before, and the positive rate decreased; but neurites induced by NGF were growing all the way. Another ROCK inhibitor fasudil (33 mM) had similar effect in neurite outgrowth as Y27632. (A) The morphological data; (B) Statistical data calculated from morphological data (mean  SD, n ¼ 3), *P < 0.05, compared with NGF of the same day. bar ¼ 50 mm.

In NGF-treated PC12 Adh cell (Figures 4A and 4B), seven pathways were down-regulated among the top 10 downregulated pathways both in 2 days and 4 days; and three pathways were up-regulated both in 2 days and 4 days. However, in Y27632-treated PC12 Adh cell (Figures 5A and 5B), only four pathways were down-regulated among the top 10 down-regulated pathways both in 2 days and 4 days; and only two pathways were up-regulated both in 2 days and 4 568

days. But the extent of the down- or up-regulated pathways in samples treated with Y27632 was much smaller than those in samples treated with NGF. Comparing Figure 4A (treated with NGF for 2 days) with Figure 5A (treated with Y27632 for 2 days), we found there were seven pathways (GABAergic synapse, Insulin secretion, Dopaminergic synapse, Morphine addiction, Retrograde endocannabinioid signaling, Glutamatergic

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Figure 3 Distribution of gene expression of five samples (A): the distribution of five samples was almost the same; Heat map and hierarchical clustering for gene expression profile in PC12 Adh cell. Hierarchical clustering for “All Targets Value” (B): “Red” indicates high relative expression and “green” indicates low relative expression. NGF4D, NGF2D, and Y276322D stood together, while Y276324D stood at the opposite side of the map separated by PC12RNA. NGF2D: PC12 Adh cell treated with NGF (100 ng/mL) for 2 days; NGF4D: treated with NGF (100 ng/mL) for 4 days; Y276322D: treated with Y27632 (33 mM) for 2 days; Y276324D: treated with Y27632 (33 mM) for 4 days; PC12RNA: treated normally as control.

synapse, and Nicotine addiction) down-regulated in both, and there were three pathways (ECM-receptor interaction, Focal adhesion, and PI3K-Akt signaling pathway) upregulated in both groups. When comparing Figure 4B (treated with NGF for 4 days) with Figure 5B (treated with Y27632 for 4 days), we found there were four pathways (GABAergic synapse, Nicotine addiction, Synaptic vesicle cycle, and Glutamatergic synapse) down-regulated in both groups, and seven pathways (Staphylococcus aureus infection, Complement and coagulation cascades, Pertussis, Osteoclast differentiation, Hematopoietic cell lineage, Leishmaniasis, and Malaria) up-regulated in both groups. The both down-regulated and both up-regulated pathways could contribute to neurite outgrowth, and the other pathways in Y27632-treated samples could contribute to neurite outgrowth resistance. Although most pathways shown in Figures 4 and 5 might be related to neurite outgrowth, they must provide different contribution from time to time. In order to find the different pathways that could cause resistance effect in inducing neurite outgrowth by Y27632, the Enrichment Scores of Y276324D and NGF4D were

calculated by comparing with Y276322D and NGF2D, respectively. Enrichment Score above 3.0 is displayed in Figure 5. The regulation of pathway varied drastically from the 2nd to 4th days in Y27632-treated PC12 Adh cell; but varied within a narrow range in NGF-treated PC12 Adh cell (Figure 5). There was only one pathway with Enrichment Score above 4.0 in NGF-treated group (Figure 5D), but there were nine in Y27632-treated group (Figure 5C). Pathways with Enrichment Score above 4.0 and their physiological function are listed in Table 2.

Result of real-time RT-PCR Compared with the scaled result of the 2nd day (NGF2D), four of the selected genes were down-regulated (P < 0.05) and two up-regulated (P < 0.05) on the 4th day (NGF4D); however, five genes were down-regulated (P < 0.05) and only one was up-regulated (P < 0.05) (Figure 6). The expression pattern of the selected genes from the 2nd day to the 4th day caused by NGF (NGF4D vs. NGF2D) was obviously different from that caused by Y27632 (Y276324D vs. Y276322D) (Figure 6).

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Figure 4 Ten of the most up- or down-regulated pathways in NGF (100 ng/mL)-treated PC12 Adh cells for 2 days (A) and for 4 days (B). Pathways down-regulated both on the 2nd day and on the 4th day; Pathways up-regulated both on the 2nd day and on the 4th day; *Pathway may be related to neurite. n ¼ 3.

Correlation between scaled results from real-time RTPCR and those from gene expression microarray To evaluate reliability of the results from gene expression microarray, correlation between the results of the selected genes from real-time RT-PCR and those from gene expression microarray was performed (Figure 7). The scatter plot (Figure 7) showed that there were a good correlation between them (P < 0.01, r ¼ 0.525, two tailed). Discussion PC12 cell was widely used as a classical tool to study neurite outgrowth, neural differentiation and related functions (Chung et al., 2014). However, in ATCC, two cell lines associated with “PC12” with different culture methods were found (http://www.atcc.org/en/Search_Results.aspx? dsNav¼Ntk:PrimarySearch%7cpc12%7c3%7c,Ny:True, Ro:0,N:1000552&searchTerms ¼ pc12&redir ¼ 1). One was “PC12 cell”, and the other was “PC12 Adh cell”. Our previous and present study found PC12 Adh cell was more sensitive to ROCK inhibitors (Y27632 and fasudil; the data of fasudil shown in Figure S1) in neurite outgrowth. Therefore, PC12 Adh cell was used in our subsequent study. Neurite outgrowth resistance caused by ROCK inhibitors (Y27632 and fasudil, the morphological data of fasudil shown in Figure S2) was confirmed in the present study. The resistance occurred when PC12 Adh cell was exposed to Y27632 for 3 or more days. These results were consistent 570

with our previous study (Duan et al., 2012). Based on the findings, samples of PC12 Adh cells treated with Y27632 or NGF for 2 and 4 days were collected for gene expression microarray, The reliability of the results from gene expression microarray was verified by real-time RT-PCR (Figure 7). Gene expression profiles of the five samples confirmed the results of Figure 2, for NGF4D, NGF2D, and Y276322D were clustered together, while Y276324D was separated by PC12RNA (the control) (Figure 3B). To find which pathways contribute to the resistance, the enrichment analysis was applied. Part of pathways down- or up-regulated in Y276322D, Y276324D, NGF2D, and NGF4D was similar, and some of them were different from each other. Since neurite outgrowth resistance occurred when PC12 Adh cell was treated with Y27632 for 3 or more days, pathway analysis between the 2nd and 4th day was performed (Figure 5 and Table 2). There are three tendencies of a living PC12 cell: one is at stable state, others are proliferation and differentiation; and there could be more than one direction for differentiation (Hughes et al., 2000). Based on the data in Table 2, pathways contributing to the resistance should come from the nine pathways. Among them, there were three up-regulated pathways (rno05150, rno04610, and rno04670) associated with inflammation, and four down-regulated pathways (rno04110, rno03460, rno00240, and rno03008) possibly related to cell cycle (cell urvival or proliferation). By

Cell Biol Int 39 (2015) 563–576 © 2015 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology

H. Yin et al.

Neurite outgrowth resistance to rho kinase inhibitors

Figure 5 Pathway analysis Ten of the most down- or up-regulated pathways in PC12 Adh cells treated with Y27632 (33 mM) for 2 days (A) and for 4 days (B). Pathways down-regulated both on the 2nd day and on the 4th day; Pathways up-regulated both on the 2nd day and on the 4th day. The down-regulated, or up-regulated pathways with Enrichment Scores above 3.0 in PC12 Adh cell treated with Y27632 (33 mM) or NGF (100 ng/mL) for 4 days were listed, respectively compared with those for 2 days: (Y276324D vs. Y276322D) (C), and (NGF4D vs. NGF2D) (D). A minus placed before Enrichment Score indicated down-regulation. Names of pathway in bold appeared both in (C) and (D). * Pathway may be related to neurite outgrowth. n ¼ 3.

comparing the pathway analysis of NGF4D vs NGF2D, pathway of rno04727 (associated with GABAergic synapse) should be excluded as a contributor for neurite outgrowth resistance (Table 2).

As for neurite outgrowth, neural growth cone formation is the first step. It was widely reported that, during inflammatory process, ROCK pathway was up-regulated and activated, and inflammatory reaction would cause

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Table 2 Pathways (P.) with Enrichment Score (E. Score) above 4.0 and their physiological function P. ID

Definition

E. Score

rno04110

*Cell cycle

8.218838

rno04514

*Cell adhesion molecules (CAMs)

7.210357

rno05150

Staphylococcus aureus infection

5.783308

rno04610

Complement and coagulation cascades

4.915016

rno04727

*GABAergic synapse

4.83888

rno04670

*Leukocyte transendothelial migration

4.523616

rno03460

Fanconi anemia pathway

-4.476784

rno00240

*Pyrimidine metabolism

4.43126

rno03008

*Ribosome biogenesis in eukaryotes

-4.215352

rno04727

*GABAergic synapse

4.278259

Involved Genes (A) Y276324D vs. Y276322D ANAPC1/ANAPC10/ANAPC7/BUB1/BUB3/CCNA2/ CCND1/CDC16/CDC23/CDC25C/CDC45L/CDC6/ CDC7/CDK1/CDK2/CDK4/CDK7/CHEK2/CUL1/DBF4/ ESPL1/MAD2L1/ /MCM4/MCM5/ORC1L/ORC2L/ ORC6L/SFN/ SKP2/STAG1/TTK/WEE1 CD34/CD4/CD40/CD99/CDH4/CDH5/CLDN10/ CLDN11/CLDN19/CLDN23/CLDN5/CLDN6/ CNTN1/ CNTN2/ESAM/ITGAL/ITGAM/ITGB2/ JAM2/LRRC4/ LRRC4C/MAG/MPZ/NCAM2/ NEGR1/NFASC/NLGN2/ NLGN3/NRCAM/NRXN1/ NRXN2/OCLN/RT1-CE3/ SDC3/SDC4/SELPLG C1QA/C1QB/C1QC/C3/C4B/CFD/CFH/FCGR1A/ FCGR2B/FCGR3A/ITGAL/ITGAM/ITGB2/MASP1/ PTAFR/SELPLG

A2M/BDKRB1/C1QA/C1QB/C1QC/C3/C4B/ C7/C8G/ CD59/CFD/CFH/CR2/MASP1/ SERPINF2/SERPING1/ THBD/VWF ABAT/ADCY2/ADCY4/ADCY5/ADCY6/ CACNA1A/ CACNA1C/GABBR1/GABRA1/ GABRA2/GABRB1/ GLUL/GNAI1/GNAO1/ GNG3/GNGT2/PRKCB/ SLC38A3/SLC6A1/SLC6A11/SLC6A13 CD99/CDH5/CLDN10/CLDN11/CLDN19/ CLDN23/ CLDN5/CLDN6/CXCR4/CYBB/ ESAM/GNAI1/ITGAL/ ITGAM/ITGB2/ JAM2/MMP2/NCF1/NCF4/OCLN/ PRKCB/ RAC2/TXK/VAV1 ATRIP/BRCA1/BRIP1/EME1/FANCA/ FANCD2/ MTMR15/PALB2/PMS2/RAD51C/ REV3L/UBE2T/USP1/ WDR48 DTYMK/NME2/NT5C1B/NT5C3/NT5C3L/ NT5E/ PNPT1/POLD2/POLD3/POLE/POLE2/ POLR1A/POLR1E/ POLR3H/PRIM2/ RGD1565904/RRM1/RRM2B/ TWISTNB/ TXNRD1/UCK2/UMPS CIRH1A/EFTUD1/FBL/GTPBP4/HEATR1/ LSG1/ MPHOSPH10/NOB1/NOP58/POP1/POP4/ POP5/ RPP38/RPP40/SPATA5/TAF9/ UTP14A/UTP18/WDR3 (B) NGF4D vs. NGF2D ADCY2/GABRD/GABRG2/GLUL/GNAO1/ GNG7/NSF/ PRKCA/SLC12A5/SLC32A1/ SLC38A1/SLC6A1/ SLC6A11

Function(s)

Cell cycle

Cell adhesion

Prevention of membrane attack complex formation; Suppression of chemotaxis and phagocyte activation; Suppression of neutrophil transmigration; inflammation Inflammation, prostaglandin biosynthesis, nitric oxide biosynthesis, ect. Neural hyperpolarization, decrease excitability Early endosome degradation Transendothelial migration

Nuclear foci formation; Mismatch repair; Homologous recombination; cell survival and proliferation pyrimidine metabolism; cell survival and proliferation Ribosome biogenesis in eukaryotes; cell survival and proliferation Neural hyperpolarization, decrease excitability; Early endosome degration

A minus placed before Enrichment Score labeled the down-regulation. Pathways in bold appeared both in (A) and (B). * Pathway may be related to neurite.

neural growth cone collapse, neurite retraction, and even neuron death (Wahl et al., 2000; Mueller et al., 2005; Chan et al., 2007). Thus, inflammatory factors from inflammatory pathway up-regulation or activation were negative for neurite outgrowth (Alamdary et al., 2013). The up-regulated pathways of rno05150 and 04670 (Table 2) could not be 572

directly associated with neurite outgrowth resistance in the PC12 Adh cell model, because the two pathway upregulation needed other inflammatory cells (such as leukocyte and endothilia) for participation; but they could take action in vivo. The up-regulated pathways of rno04610 in Table 2 would cause biosynthesis of prostaglandin, and

Cell Biol Int 39 (2015) 563–576 © 2015 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology

H. Yin et al.

Neurite outgrowth resistance to rho kinase inhibitors

Figure 6 Results of real time RT-PCR (Mean  SD, n ¼ 3). The selected genes were sorted from small to large by the sum of continued changes. The bold line was the trendline of the continued fold change caused by NGF, and the dotted line was the trendline of that caused by Y27632. The bold and dotted lines indicated that the expression patterns of the selected genes were different from each other. Continued fold change ¼ (Scaled Result) 4D(Scaled Result) 2D. * P < 0.05, the scaled result of 4th day compared with that of 2nd day. NGF2D: PC12 Adh cell treated with NGF (100 ng/mL) for 2 days; NGF4D: treated with NGF (100 ng/mL) for 4 days; Y276322D: treated with Y27632 (33 mM) for 2 days; Y276324D: treated with Y27632 (33 mM) for 4 days.

according to common understanding, though some factors (like MAP1B 1–126 and militarinone A) were able to induce neurite outgrowth and cell death / apoptosis in PC12 cell or neuronal cell almost simultaneously (Küenzi et al., 2008; Gomi and Uchida, 2012). A favorable factor should not only induce neurite outgrowth but also promote neural cell

3 2 1

RT-PCR

many prostaglandins such as prostaglandin E2 (Tamiji and Crawford, 2010), or related products such as lysophosphatidic acid (PLA) (Matsui et al., 1996) were able to suppress neurite outgrowth, and even cause neurite retraction. Otherwise, the suppression of some of them by COX-2 inhibitor such as NS398 was able to improve the resistance to some extent (Duan et al., 2012). Inflammatory pathway up-regulation induced by ROCK inhibitor may be associated with p300 acetyltransferase which takes a pivotal role in gene transcription by acetylating histone (Ling and Lobie, 2004; Tanaka et al., 2006), since the activity of the enzyme is mainly regulated by nuclear ROCK. ROCK inhibitor may pass through nuclear membrane, inhibit the enzyme, and then cause inflammatory pathway up-regulation. However, this is only a hypothesis and needs further investigation. The pathway analysis found that pathways of rno04110, rno 03460, rno00240, and rno03008 which associated with cell cycle were down-regulated (Table 2). These pathways were able to promote cell survival or proliferation; if suppressed, would cause cell proliferation retarded, even cell death or apoptosis. Beside neurite outgrowth resistance, PC12 Adh cell treated with Y27632 for 4 days became fibrinoid which suggested cell survival retardation or apotosis occurred; but this phenomenon did not appear in those treated with NGF (Figure 2). Cell survival retardation and apotosis is not a favorable factor for neurite outgrowth

0 -1 -2 -3 -4 -5 -6

-4

-2

0

2

4

6

Microarray

Figure 7 Correlation between the scaled results of the selected genes from real time RT-PCR and those from gene expression microarray. RT-PCR: scaled results of the selected genes from real time RT-PCR; Microarray: scaled results from gene expression microarray. P < 0.01 (two tailed), r ¼ 0.525.

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ROCK inhibitor -

Inhibition

ROCK II Cell cycle

Staphylococcus aureus infection

Pyrimidine metabolism

Complement and coagulation cascades

Ribosome biogenesis in eukaryotes

Down-regulate

Up-regulate

...

Leukocyte transendothelial migration …

Neurite Outgrowth Cell cycle retardation

Neurite Retraction

?

Figure 8 The supposed mechanism of resistance to neurite outgrowth caused ROCK inhibitor in PC12 Adh cell. ROCK inhibitor induces PC12 Adh cell growing neurite at first. At the same time and then, ROCK inhibitor could down-regulate some pathways that suppress cell proliferation (or inducing cell apoptosis), and could up-regulate some pathways associated with inflammation that induce neurite retraction; which could be the cause of resistance. Arrow with dotted line means suppression. ? means possible but insufficient evidence.

survival at least not to cause cell death / apoptosis; for NGF was both able to do so in PC12 cell (Fuenzalida et al., 2007). As a result from negative feedback, up-regulation and down-regulation are common in bio-functional adaptation. The well-known example is the effect of beta-adrenergic blockers on their receptors. Chronic administration of betaadrenergic blocker could cause its receptors up-regulation and its effect would become weaker and weaker and vice versa (Bohm et al., 1997; Ferguson, 2001; Marchese et al., 2008). It is expected that the neurite outgrowth resistance caused by Y27632 could be explained as a result from a negative feedback also at large, though the detailed mechanism could be different from that of beta-adrenergic blockers. The hypothesis was summarized in Figure 8. Taken together, the profile of neurite outgrowth caused by ROCK inhibitor was different from that caused by NGF. Neurite outgrowth resistance became manifested when PC12 Adh cell was exposed to ROCK inhibitor for 3 or more days, and the mechanism could be associated with upregulation of inflammatory pathways (especially rno04610) and down-regulation of cell cycle pathways (especially rno04110). Acknowledgement and funding This study was financially supported by the National Natural Science Foundation of China (NSFC), No. 81060109.

Conflicts of interest None.

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Yu T, Peng H (2013) Hierarchical clustering of high-throughput expression data based on general dependences. IEEE/ACM Trans Comput Biol Bioinform 10: 1080–5. Received 26 May 2014; accepted 26 December 2014. Final version published online 2 February 2015.

Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site. Figure S1: ROCK inhibitors (Y27632 and fasudil) of 33 mM almost could not induce neurite outgrowth in PC12 cell, but NGF was able to do so in the cell. B was statistical analysis of A (morphological data). (ANOVA, *P < 0.05, vs. 0 day, n ¼ 3) bar ¼ 100 mm. Figure S2: ROCK inhibitors (Y27632 and fasudil) of 33 mM were able to induce neurite outgrowth in PC12 Adh cell. The effect was similar to that of NGF within 2 days, but a resistance became manifest in the 3rd day and later. B was

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statistical analysis of A (morphological data). (ANOVA, *P < 0.05, vs. 0 day, n ¼ 3) bar ¼ 100 mm. Figure S3: The scatterplot is a visualization that is useful for assessing the variation (or reproducibility) between chips. The Scatter-Plot is a visualization method used for assessing the gene expression variation (or reproducibility) between the two compared arrays. The values of X and Y axes in the Scatter-Plot are the normalized signal values of each sample (log2 scaled). The green lines are Fold Change Lines (The default fold change value given is 2.0). Genes above the top green line and below the bottom green line indicated more than 2.0 fold change of genes between two compared arrays. The firs row from left to right is Scatter-Plot for N2_vs_C, Scatter-Plot for N4_vs_C, ScatterPlot for Y2_vs_C, and Scatter-Plot for Y4_vs_C; the second row is Scatter-Plot for N4_vs_N2, Scatter-Plot for Y4_vs_Y2, Scatter-Plot for Y2_vs_N2, and Scatter-Plot for Y4_vs_N4. N2, PC12 Adh cell treated with NGF for 2 days; N4, PC12 Adh cell treated with NGF for 4 days; C, PC12 Adh cell cultured normally as control; Y2, PC12 Adh cell treated with Y27632 for 2 days; Y4, PC12 Adh cell treated with Y27632 for 4 days.

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Neurite outgrowth resistance to rho kinase inhibitors in PC12 Adh cell.

Rho kinase (ROCK) inhibitor is a promising agent for neural injury disorders, which mechanism is associated with neurite outgrowth. However, neurite o...
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