Expression of arachidonic acid-metabolizing cytochrome P450s in human megakaryocytic Dami cells

Cytochrome P450s (P450s) are involved in the metabolism of arachidonic acid (ARA), and ARA metabolites are associated with various cellular signaling pathways, such as blood hemostasis and inflammation. The present study demonstrates the expression of ARA-metabolizing P450s in the human megakaryocytic Dami cells using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunublotting analysis followed by activity assays using ARA as a substrate. In addition to the previously identified CYP5A1, both protein and mRNAs of CYP1A1, 2U1, and 2J2 bands were detected. Ethoxyresorufin-O-deethylase (EROD) activity was observed in Dami cells, and its activity was significantly decreased after treatment with the P450 inhibitor SKF-525A when compared to the control groups (60% reduction, P < 0.001). CYP1A1 protein expression in Dami cells was induced by 3-methylenecholantheren. This increase in CYP1A1 protein level was correlated with enhanced EROD activity (fourfold increase vs. the control), as well as with increased metabolites, such as 20-hydroxyeicosatrienoic acid (20-HETE), 14, 15-EET (14-,15-epoxyeicosatrienoic acid), and 14, 15-dihydroxyeicosatrienoic acid (14, 15-DHET). The expression of soluble epoxide hydrolase, an enzyme responsible for the synthesis of DHETs from EETs, was confirmed by RT-PCR. Furthermore, 15 ARA metabolites, including 8,9-EET, 14,15-EET, and 20-HETE, were detected by LC-MS/MS in ARA-treated Dami cells, and their levels were decreased with the treatment of the SKF-525A. The present data suggest the possibility that the P450s play a role in the metabolism of ARA and other CYP-related substrates in human megakaryocytes and that P450 expression in megakaryocytic cell lines may predict their existences in platelets with functional activities.


Introduction
The cytochromes P450 (CYPs) constitute a superfamily of hemoprotein enzymes (Zhu and Silverman 2008). They are well recognized for their major roles in the synthesis, activation, and metabolism of many endogenous and xenobiotic compounds, such as hormones, fatty acids, drugs, and carcinogens (Spriet et al. 2009). It has been repeatedly reported that multiple P450s metabolize arachidonic acid (ARA) (Devos et al. 2010;Mitra et al. 2011;Theken et al. 2011;Xu et al. 2011). ARA is a fatty acid found in the human cell membrane, which is the precursor of eicosanoids that play important biological roles in the regulation of blood homeostasis and inflammation process (Imig 2012). ARA metabolites have also been reported to be involved in bone marrow haematopoiesis, and its abnormal regulation has been associated with the case of cardiovascular diseases (Lutton et al. 1989;Abraham et al. 1991;Pfister et al. 2010). In humans, ARA is epoxidized to epoxyeicosatrienoic acids (EETs) by CYP2C and CYP2J subfamilies, while CYP4A, 4F, and 1A subfamilies and CYP2U1 enzyme hydroxylate ARA to 20-HETE (Lasker et al. 2000;Pearson et al. 2009;Devos et al. 2010). The expression of ARA-matabolizing P450s varies among different tissues. For example, CYP2J2 is expressed in cardiac tissues more predominantly than in other tissues (Wu et al. 1996). Furthermore, CYP2C subfamily is found in significantly higher quantities in the liver and gastrointestinal tract than in bone marrow (Bieche et al. 2007). These differences in ARA-metabolizing P450 expression levels may lead to different levels of ARA metabolites, thus affecting tissues' biological regulation.
Megakaryocytes are the bone marrow cells responsible for the production of blood platelets. In humans, megakaryocytes normally account for approximately 0.05−0.1% of all nucleated bone marrow cells. Each megakaryocyte produces 1,000-3,000 enucleate platelets through a thrombopoietin-mediated process (Deutsch and Tomer 2006). Although thromboxane synthase (CYP5A1) has been found to be highly expressed in megakaryocytes and platelets (Nakahata 2008), other P450 expressions in megakaryocytes and their daughter platelets are, as yet, poorly understood. Interestingly, ARA and some of its metabolites have been identified in platelets (Sheppard et al. 1992), suggesting the possible presence of ARA-metabolizing P450s in megakaryocytes. Since platelets, being cell fragments derived from the precursor megakaryocytes, cannot proliferate in the cell culture system due to a lack of nucleus, there are no cell lines available for the molecular study of platelets. The megakaryocytic cell line (Dami cells) has frequently been used for the study of platelets (Khetawat et al. 2000;Shi et al. 2010;Lev et al. 2011).The goal of the present study was to determine which P450s are expressed in Dami cells with the functional activity for the metabolism of ARA.
Cell culture. The human megakaryocytic Dami cell line was obtained from the American Type Culture Collections (Rockville, MD). Dami cells were cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cultures were maintained in a humidified atmosphere of 5% CO 2 at 37°C as previously described (Khetawat et al. 2000). Dami cells were treated with 10 μM of 3-MC for 72 h in order to test the induction of CYP1A1 and determine the effect of CYP1A1 induction on ARA metabolism. For these experiments, 3-MC was dissolved in dimethyl sulfoxide (DMSO), and the final DMSO concentration in the culture medium a Gene-specific primers were designed in the present study was 0.1% (v/v). The control culture received the same volume of DMSO without 3-MC. After 3-MC treatment, the cells were incubated with 100 μM ARA for 12 h (Rehfeldt et al. 1993).
Reverse transcriptase-polymerase chain reaction. RNAs from Dami cells, and human liver were extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Liver tissue samples were obtained from the Inje Pharmacogenomics Research Center Biobank (Inje University College of Medicine, Busan, Korea). The research protocol for the usage of human liver tissues was approved by the Institutional Review Board of Busan Paik Hospital (Inje University, Busan, Korea). Three micrograms of total isolated RNA was added to a reaction mixture containing 100 pmol oligo(dT), 2.5 mM dNTP, 0.1M DTT, 5× first strand buffer, 200 U of M-MLV reverse transcriptase and RNAase-free water for synthesis of cDNA. The reaction mixtures were incubated at 42°C for 50 min. Next, conventional PCR was performed by adding 330 ng of cDNA to a mixture containing 10 mM dNTPs, 25 mM MgCl 2 , 2× D-Taq polymerase buffer, 10 pmoles from each of the forward and reverse primers (Table 1), and 1.5 U of D-Taq DNA polymerase. The PCR products were separated on a 2% agarose gel and visualized using ethidium bromide staining.
Immunoblot analysis. Dami cell cultures were centrifuged at 1,000 rpm for 3 min. Then, lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 0.25% Nadeoxycholate, and protease inhibitor cocktail] was added to the Dami cell pellet. The mixture was vortexed vigorously, sonicated twice for 10 s, and the resulting lysate was incubated on ice for 50 min. The lysates were stored at −80°C until use. Then, 35 μg of protein was boiled at 95°C for 5 min with 4× loading buffer containing 0.1 M Tris-HCl (pH 6.8), 4% sodium dodecyl sulfate (SDS), 1.5% bromophenol blue, 20% glycerol, and 5% βmercaptoethanol. The denatured proteins were separated with 12% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane in a buffer containing 25 mM Tris-HCl, 192 mM glycine, and 20% (v/v) methanol. The membrane was blocked by 5% skimmed milk in Tris-Buffered Saline supplemented with 0.1% Tween 20 solution. The membrane was probed with polyclonal goat anti-CYP1A1 IgG and anti-CYP2U1 IgG, and monoclonal mouse anti-CYP2J2 IgG, separately, and reproved with  The standard curve of known concentrations of resorufin was between 50 and 500 pmoles (Kennedy et al. 1993). After measurement, the cells were lysed, and the protein concentration was determined using the Bradford dye method (Martin et al. 1995). The resurfin formation was expressed as nanomoles per minute per milligram of protein.
Statistical analysis. All values represent the mean±standard deviation of triplicate reactions. Statistical significance was analyzed using a two-tailed Student's t test. All statistical analyses were performed using the SAS program (version 9.1.3; SAS Institute, Cary, NC). Statistically significant differences as compared with the control groups are represented as *P<0.05, **P<0.01, and ***P<0.001.

Results
Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we screened the mRNA expression of 15 major P450s which are reported to metabolize ARA (Devos et al. 2010;Mitra et al. 2011;Theken et al. 2011;Xu et al. 2011). The screened ARA-P450s were as follows: CYP1A1, 1A2, 1B1, 2C8, 2C9, 2C19, 2D6, 2J2, 2U1, 4A11, 4F2, 4F3, 3A4, and 3A5 in addition to CYP5A1 which is known to be expressed in platelets. We detected mRNAs of CYP1A1, 2U1, and 2J2 in Dami cells, as well as the mRNA of CYP5A1 (Fig. 1). Specific amplification was confirmed as their predicted sizes as shown in Table 1. There were no detectable bands in the PCR reactions performed with RNA samples that were not treated with reverse transcriptase; these reactions served as a control to monitor genomic DNA contamination. Immunobloting analysis showed the detection of CYP1A1, 2U1, and 2J2 proteins, with bands size of 50-60 kDa, in Dami cells (Fig. 1B). The detected bands were in agreement with the theoretical size of 50-60 kDa for P450s and with the liver microsome sample used as a reference standard for the detection of P450s. EROD assays of CYP1A1 were performed with Dami cells. The activity of resorufin formation was 112± 10 nmol/min/mg protein. To confirm the activity of P450, 40 μM of the P450 inhibitor SKF-525A was added to the reactions and compared with the non-SKF-525A-treated samples. Figure 2 shows that EROD activity decreased significantly with the addition of SKF-525A to Dami cells (60%, P< 0.001) when compared to the control groups. Because CYP1A1 (this study) and the nuclear Aryl hydrocarbon receptor (AhR) (Lindsey and Papoutsakis 2011) are both expressed in megakaryocytes, we investigated the inducibility of CYP1A1 in Dami cells using 3-MC which is an AhR agonist, after confirming the expression of AhR in Dami cells (Fig. 3A). Immunoblot analysis showed that the CYP1A1 protein level increased in Dami cells after 3-MC treatment (Fig. 3B), and that this increased protein level correlated with the increase in EROD activity (fourfold, P<0.001) as compared with the DMSO-treated groups (Fig. 3C).

Discussion
Although ARA and its metabolites are important signal molecules in blood hemostasis, ARA metabolism by

P450s in megakaryocytes and megakaryocytic Dami cells remains unclear. Megakaryocytic Dami cells have been used
to study the biological function of megakaryocytes and platelets, because circulating platelets have no nucleus (Khetawat et al. 2000;Lev et al. 2011;Lee et al. 2012). In the current study, we investigated ARA-metabolizing P450s in Dami cells. In addition to CYP5A1, we found that CYP1A1, 2U1, and 2J2 were also expressed in Dami cells. CYP1A1, 2U1, and 2J2 have been reported to metabolize ARA and its derivatives (Devos et al. 2010;Gaedigk et al. 2006). Therefore, it can be suggested that these P450s may play a role in the metabolism of ARA and its related eicosanoid compounds in the megakaryocytes and the platelets. The literature reports several instances of similarities in the P450 expression profiles of Dami cells and bone marrow. For example, in human bone marrows, CYP2U1 and 1A1 are expressed at high levels, but CYP3A and 2C are not present (Bieche et al. 2007). Similarly, in this study CYP1A1 and 2U1 were strongly expressed in Dami cells, while CYP3A4, 3A5, 2C8, 2C9, and 2C19 were not detectable. The similar expression profiles of P450s in bone marrow tissues and Dami cells may indicate that CYP2U1 and 1A1 in bone marrow could be derived, at least in part, from megakaryocytes; however, it cannot rule out the possibility that other bone marrow cell types can also express CYP1A1 and 2U1.
CYP1A1 expression was detected, and its expression was induced by 3-MC in Dami cells. This increase in protein expression correlated with the increase in EROD activity. These results suggest that there could be variations in CYP1A1 expression levels in megakaryocytes induced by environmental stimuli, such as cigarette smoking and other aromatic hydrocarbons with the potential to alter the metabolism of ARA as well as other CYP1A1 substrates. CYP1A1 metabolizes some drugs, such as theophylline and caffeine (Yang and Lee 2008;Amin et al. 2011). It is expressed more in cancer tissues and activates procarcinogenic compounds, such as polycyclic aromatic hydrocarbons (Levova et al. 2011). We found that EROD activity was inhibited by 40 μM SKF-525A, confirming that the reaction was mediated by P450s in megakaryocytic Dami cells. Because EROD activity is mediated by the CYP1A subfamily and CYP1B1 (Smith et al. 2011), EROD activity in Dami cells appears to be a result of CYP1A1. Our results showed that 14,15-EET and 14,15-DHET were increased in Dami cells after treatment with 3-MC. It is known that 14,15-EET is metabolized to 14, 15-DHET by epoxide hydrolases (Seidegard et al. 1984). This finding is consistent with a previous report that 14,15-EET synthesis increases after induction of the CYP1A subfamily by different AhR nuclear receptor agonists in human HepG2 cells (Diani-Moore et al. 2006). In the present study, the expression of soluble epoxide hydrolase was evidenced in Dami cells, and the experiment using the inhibitor of epoxide hydrolase enzyme would add additional information for further translation of the metabolic fate for EETs and DHETs. CYP4A and 4F subfamilies have been identified as the main P450s for 20-HETE production (Lasker et al. 2000;Pearson et al. 2009), although multiple studies showed that CYP1A1 hydroxylases ARA to 20-HETE The HPLC chromatogram shows the 15 identified ARA metabolites in Dami cells. The cells were incubated with 100 μM ARA for 12 h. ARA metabolites were then extracted by ethyl acetate (pH 3−4). Ethyl acetate was evaporated, and the residue was dissolved in 100 μl ethanol, followed by LC-MS/ MS screening for ARA metabolites. Further details are included in the "Materials and Methods" section. 20-HETE-6 (100 μg/ml) was used as internal standard. (Aboutabl et al. 2009;Arnold et al. 2010). Furthermore, the formation of 20-HETE is inhibited by 7-ER in bone marrow (Abraham et al. 1991). These results potentially indicate an essential role for CYP1A1 in the formation of 20-HETE in bone marrow. Our results showed that induction of CYP1A1 by 3-MC in Dami cells increased the metabolism of ARA to 20-HETE. These results indicate that altered levels of the metabolite may cause variation in megakaryocyte and platelet functions. The relatively high expression of CYP1A1 in megakaryocytes could increase our understanding of hematologic diseases and hematotoxicity. Benzenes cause bone marrow toxicity and hematotoxicity (Hirabayashi and Inoue 2010), and the aryl hydrocarbon receptor (AhR) was found to mediate benzene-induced toxicity (Hayashibara et al. 2003). The potent carcinogen 3-MC is an alkylated derivative of benzo(a)anthracene. In this study, we confirmed the expression of AhR in Dami cells by RT-PCR and observed that 3-MC induced CYP1A1 expression, which is predicted due to the activation of AhR by 3-MC. Polyaromatic hydrocarbons identified in cigarette smoke revealed to activate AhR and induce CYP1A1 (Roth et al. 2001). Moreover, smoking showed to increase platelet aggregation (Li et al. 2011). Therefore, it is suggested to investigate the effect of CYP1A1 induction on platelet aggregation variations. The results of the present study indicate that Dami cells may be a good cell line for studying CYP1A1 induction and metabolism for human megakaryocytes and human platelets, at least in part. Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) have been reported to be involved in the regulation of CYP2J2 (Siest et al. 2008;Ellfolk et al. 2009). However, CAR and PXR were not detected by RT-PCR in our hand (data not shown), prompting us to study the induction of CYP1A1 in the present study. CYP2U1 is an extrahepatic P450. It is expressed more in the thyroid, brain, heart, and bone marrow than in the liver (Karlgren et al. 2005;Dutheil et al. 2009). We found that CYP2U1 was also expressed in Dami cells. Information on the molecular regulation of CYP2U1 is very limited, and there is still no specific inhibitor and inducer of CYP2U1. However, CYP2U1 metabolized ARA to 20-HETE (Devos et al. 2010), suggesting that 20-HETE, which was detected in Dami cells, could be produced, at least in part by CYP2U1. CYP2J2 is highly expressed in the cardiovascular system (Wu et al. 1996). It affects blood hemostasis via the metabolism of ARA to EETs, which are known to decrease blood pressure and to inhibit platelet aggregation (Spiecker and Liao 2006). Western blot analysis of CYP2J2 revealed one major band with a molecular weight of 50-60 kDa in Dami cells. However, we noticed that CYP2J2 in liver tissue was revealed by two distinct bands. Wu et al. (1996) also showed two bands in the liver and major one band of CYP2J2 in other tissues in immunoblot analysis. These results may indicate that CYP2J2 in human heart, kidney, and the platelet precursor cells megakaryocytes share similar immunochemical reactions. CYP2J2 is suggested to contribute to the formation of epoxidation of ARA (Wu et al. 1996). Expression of CYP2J2 in megakaryocytes might play a role in the formation of ARA metabolites, and they may be involved in the biological function of megakaryocytes and platelets.
In summary, we identified ARA-metabolizing P450s and confirmed their expressions with functional activities in Dami cells. These P450s may play important roles in the metabolism of ARA as well as other xenobiotic compounds in megakaryocytes. Particularly, because CYP1A1 expression levels are largely induced by environmental polycyclic aromatic hydrocarbons, altered levels of CYP1A1 expression may cause variations in CYP1A1-mediated metabolism in megakaryocytes. The present information on P450 expressions in megakaryocytic Dami cells would further extend our knowledge on the roles of P450s in megakaryocytes as well as platelets.