14,15‐epoxyeicosatrienoic acid produced by cytochrome P450s enhances neurite outgrowth of PC12 and rat hippocampal neuronal cells

Abstract Polyunsaturated fatty acids, such as arachidonic acid, are accumulated in brain and induce neuronal differentiation. Arachidonic acid is metabolized to epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) by cytochrome P450s. In this study, we found that 14,15‐EET and 20‐HETE‐enhanced NGF‐induced rat pheochromocytoma PC12 cell neurite outgrowth even at the concentration of 100 nmol L−1. LC‐MS analysis revealed that 14,15‐EET was effectively produced from arachidonic acid by rat CYP2C11, 2C13, and 2C23, and these P450s were expressed in PC12 cells. An inhibitor of these P450s, ketoconazole, inhibited neurite outgrowth, whereas inhibition of soluble epoxide hydrolase, which hydrolyzes EETs to their corresponding diols enhanced neurite outgrowth. To determine the mechanism of neurite formation enhancement by arachidonic acid metabolites, we focused on transient receptor potential (TRP) channels expressed in PC12 cells. The TRPV4 inhibitor HC067047, but not the TRPV1 inhibitor capsazepine, inhibited the effects of 14,15‐EET on neurite outgrowth of PC12. Furthermore, 14,15‐EET increased the cytosolic calcium ion concentration and this increase was inhibited by HC067047. 14,15‐EET also enhanced neurite outgrowth of primary cultured neuron from rat hippocampus. This study suggests that arachidonic acid metabolites produced by P450 contribute to neurite outgrowth through calcium influx.

There are many HETE isomers produced by P450s, including 5,8,9,11,12,15,16,17,18,19, and 20-HETEs. 6 19-HETE is a stimulator of renal Na + -K + -ATPase 7 and 20-HETE is a potent vasoconstrictor in isolated rat aortas. 8 EETs were initially identified as endotheliumderived hyperpolarizing factors (EDHFs) 9 and attracted much interest. However, EETs are now suggested to be more than just vasodilators. EETs were also found to influence many biological processes, including ion channel regulation, mitogenesis, and inflammation. 10 There are four EET isomers, 5,6-, 8,11,and 14,EET were demonstrated to be powerful mitogens, using kidney-and endothelium-derived culture cells. 11 11,12-EET is also involved in angiogenesis under hypoxic conditions. 12 Although specific receptors for EETs and HETEs have not been identified, EETs activate some receptors such as Ca 2+ -activated K + channels and transient receptor potential (TRP) channels. 13 In the brain, HETEs and EETs are released from neurons, astrocytes, and cerebral blood vessels, and contribute to cerebral blood flow and angiogenesis, 14 but their effects on neural functions are not well understood. The neurite outgrowth of neurons is very important for the function of the central nervous system. Rat phenochromocytoma PC12 cells are used as a model system for neurodegenerative diseases and as an in vitro model system to study cell differentiation. Nerve growth factor (NGF) induces neural differentiation of PC12 cells, leading to neurite outgrowth. NGF also induces expression of phospholipase A 2 in PC12 cells. 15 We also used primary cultured neuron isolated from rat fetal hippocampus. In this study, we investigate biological functions of metabolites of arachidonic acid by P450s in neurite outgrowth of these cells. 2C23, 2E1, 4A2, sEH, NADPH-cytochrome P450 reductase, and β-actin were prepared as described previously. [16][17][18] β-NADPH was purchased from Oriental Yeast Co. LTD (Tokyo, Japan), horse serum was from Equitech-Bio (Kerrville, TX), and nerve growth factor (NGF) was form Alomone Labs (Jerusalem, Israel). HC067047 and Fura-2 AM were from Abcam plc (Cambridge, UK). Fluo-4AM was from AAT Bioquest (Sunnyvale, CA).

| Cell culture and PC12 cell neurite extension
Rat pheochromocytoma (PC12) cells were obtained from RIKEN cell bank (Tsukuba, Japan). Cells were cultured in DMEM containing 10% horse serum, 5% fetal calf serum(FCS), penicillin (100 units/mL), and streptomycin (100 μg/mL), and maintained at 37°C in 5% CO 2 and 95% air. For the neurite outgrowth assay, cells were seeded at 1 × 10 4 cells per well in 24-well plates. After 1 day, culture medium was replaced with DMEM containing 0.25% HS, 0.13% FCS, and NGF (50 ng/mL) with EETs, DHETs or HETEs at the indicated concentrations. Differentiated cells with neurites were defined as those with neurite length greater than the cell body of individual cell, and ratio of differentiated cells to total number of cells was determined.
Neurite length of differentiated cells was measured using Image J.

| Isolation of rat hippocampal neuronal calls
Neuron-rich cultures were prepared from hippocampus of Wistar rats (Clea Japan, Inc., Tokyo) at embryonic day 18 as described previously. 19,20 Cells were plated at 1 × 10 5 cells per cloning ring on the poly-ethylene-imine coated dish with Neurobasal Medium (Thermo Fisher Scientific, MA) containing 2% B27 Supplement (Thermo Fisher Scientific), penicillin (100 units/mL) (Thermo Fisher Scientific), streptomycin (100 μg/mL) (Thermo Fisher Scientific), 5 μg/mL Insulin (Sigma Chemical), and 0.5 mmol L −1 L-Glutamine. 21,22 After 24 hours, the cloning ring was removed, and 14,15-EET (100 nmol L −1 ), 14,15-DHET (100 nmol L −1 ), or TRPV4 agonist RN-1747 (10 μmol L −1 ) were added to the cell medium. After 24 hours, the length of neurite diffused into the free space from cells was measured. For immunofluorescence analysis, the cells exposed to 14,15-EET for 48 hours were fixed with 4% PFA, following BSA blocking for 1 hour, and incubated with anti-neurofilament antibody (Cell Signaling), and Dylight594-conjugated secondary antibody (Vector Laboratories, CA, USA) in Immuno-enhancer solution (Wako, Osaka, Japan). Image was obtained by confocal microscopy (Nikon A1, Nikon, Tokyo, Japan). All experiments were conducted in accordance with guidelines on the welfare of experimental animals and with the approval of the Ethics Committee on the use of animals of Kwansei Gakuin University.

| Arachidonic acid metabolism by P450s
Purification of rat P450 (CYP1A1, 1A2, 2A1, 2B1, 2C11, 2C13, 2C23, 2D1, 2E1, 2J3, 4A2, and 4F1), NADPH-cytochrome P450 reductase, and cytochrome b 5 was performed as described previously. 23,24 The reaction of arachidonic acid with P450 was carried out by the method described previously with a brief modification. 6,23,25 As some of the arachidonic acid purchased from Sigma was converted to HETEs or EETs by autoxidation, the mixture was separated by HPLC and purified arachidonic acid was used for the substrate of P450. P450 (50 pmol) with cytochrome b 5 (50 pmol), NADPH-cytochrome P450 reductase Mobile phase A (20% methanol and 0.1% acetic acid) and mobile phase B (80% acetonitrile, 20% methanol, and 0.1% acetic acid) were used, and chromatography was done at a flow rate of 0.2 mL/minute by gradient elution as follows: a linear gradient from 100% A to 60% B at 0-18 minute, 60% B at 18-23 minute a linear gradient from 60% B to 100% B at 23-25 minute, and 100% B at 25-26 minute. Mass spectrometry was carried out using a Nanofrontier LD mass spectrometer (Hitachi, Tokyo, Japan) and then ionized by electrospray ionization (ESI). ESI was accomplished in the negative ion mode with a spray potential of 3200 V. The analytes were detected by a tandem TOF monitored for total ions at m/z 319.2 for HETEs or EETs. The amount of produced HETEs and EETs was determined by a calibration curve prepared with authentic metabolites.

| Statistical analysis
The differential significance of the results obtained was determined by One-way ANOVA followed by a Bonferroni/Dunn post hoc test, and P < 0.05 was considered statistically significant.  EET was most efficiently produced by CYP2C23. Dihydroxyeicosatrienoic acids (DHETs) were not produced in these conditions.

PC12 cells
We found that the most effective arachidonic acid metabolites to enhance neurite outgrowth of PC12 cells were 14,15-EET which mainly produced by CYP2C and 2E1, and 20-HETE produced by CYP4A (Figures 1 and 2). Next, we investigated protein levels of P450s which produce 14,15-EET or 20-HETE in PC12 cells ( Figure 3A). CYP2C11, 2C13, and 2C23 were clearly detected in PC12 cells. However, CYP4A2, which produces 20-HETE, was not detected. NADPH-cytochrome P450 reductase and sEH proteins were detected in PC12 cells.

P450 inhibitors
To investigate the contribution of 14,15-EET-producing P450 to PC12 cell neurite outgrowth, cells were treated with P450 inhibitors. First, we OGURO ET AL.

| Inhibition of calcium influx and neurite outgrowth in PC12 cells by TRP inhibitors
Although the receptors for EETs remain unknown, EETs has been recently shown to activate TRPV1 and TRPV4. 13,29 These TRP proteins were expressed in PC12 cells. 30 Figures 5A and B). In addition, 14,15-EET increased neurofilament-positive neurons which are thought to be mature axons 33 (Figure 5C). In rat hippocampus, and cultured hippocampal neurons, expression of TRPV4 has been shown. 34 We found that a TRPV4 agonist, RN-1747 also enhanced neurite outgrowth of cells (Figure 5B)  EET concentration was estimated to be 120 ng/g wet tissue in rat. 37 14,15-EET was mainly produced by rat CYP2C11, 2C13, and 2C23 from arachidonic acid. CYP2C13 reveals relatively low activity toward drugs and testosterone. 38,39 We found that CYP2C13 efficiently produced 16-HETE and 14,15-EET from arachidonic acid.

| Enhancement of neurite outgrowth of rat primary cultured neuron by 14,15-EET
These results are consistent with those of El-Sheberi and El-Kadi 40 using the microsomes of cells expressing recombinant rat CYP2C13.
Although human CYP2J was isolated as an arachidonic acid epoxygenase, 41 rat CYP2J3 exhibited little epoxidase activity toward arachidonic acid in this study.
In this study, we detected 14,15-EET-producing P450s (CYP2C11, 2C13, and 2C23) in PC12 cells, and their inhibitor, ketoconazole, abrogated NGF-enhanced neurite outgrowth, suggesting the contribution of these P450s to neurogenesis. Ketoconazole also inhibit EET-induced relaxation in monkey cerebral artery. 42 Expression of CYP2C in rat brain has been shown in several reports. 23,43 CYP2C11 is expressed in perivascular nerves in rat brain, 44   Our study suggests that arachidonic acid metabolites, 14,15-EETs, produced by cytochrome P450 contribute to neurite outgrowth through activation of TRPV4. Regulation of EET levels using sEH inhibitors, which hydrolyze EETs in the brain, has been explored as therapy for cerebral vascular diseases, such as stroke, because EETs will improve cerebral blood flow as vasodilators. 14 Therefore, the increase in EET levels by suppression of sEH activity in neuronal cells may also be effective in enhancing neurogenesis.