|Year : 2020 | Volume
| Issue : 4 | Page : 179-186
Novel regulation of PKC-induced inflammation by Akt and protein phosphatase 2A in ovarian granulosa cells
Yen-Yu Lin1, David Sun2, Yuh-Lin Wu1
1 Department of Physiology, School of Medicine, National Yang-Ming University, Taipei, Taiwan
2 Department of Obstetrics and Gynecology, Cheng Hsin General Hospital, Taipei, Taiwan
|Date of Submission||15-Jun-2020|
|Date of Acceptance||17-Jul-2020|
|Date of Web Publication||28-Aug-2020|
Prof. Yuh-Lin Wu
Room 405, Shouren Building, National Yang-Ming University, No 155, Sec. 2, Linong Street, Taipei City 112
Source of Support: This research was supported by grants from the Ministry of Science and Technology (MOST 105-2320-B-010-026-MY3; MOST 108-2320-B-010-025), the Cheng Hsin General Hospital (CY10603; CY10802), and the Taiwan Ministry of Education Aim for the Top University Plan., Conflict of Interest: None
PKC-mediated inflammation is important in ovarian physiology. The roles of Akt and protein phosphatase 2A (PP2A) in PKC-mediated inflammation in ovarian granulosa cells (GCs) remain mostly unclear. PKC activator phorbol 12-myristate 13-acetate induced the Akt phosphorylation in rat primary GCs but reduced the Akt phosphorylation in KGN human GCs. In rat GCs, an inhibitory effect of PI3K inhibitor wortmannin and a stimulatory effect of Akt activator SC79 on PKC-induced cyclooxygenase-2 (COX-2)/PGE2production were noted; wortmannin and SC79 acted oppositely in human GCs. In rat GCs, PP2A inhibitor okadaic acid further enhanced the PKC-mediated promoter activation and elevation of mRNA and protein levels of the COX-2 gene, whereas PP2A activator sodium selenate attenuated the PKC-mediated COX-2 expression and promoter activation. PKC activation did not affect PP2A phosphorylation, but okadaic acid indeed augmented the PKC-induced NF-κB nuclear translocation. Thus, PP2A appears to act as a negative modulator in PKC-mediated cellular inflammation in rat GCs, at least in part due to its attenuating effect on the PKC-induced NF-κB activation.
Keywords: Akt, granulosa cell, phorbol 12-myristate 13-acetate, protein phosphatase 2A
|How to cite this article:|
Lin YY, Sun D, Wu YL. Novel regulation of PKC-induced inflammation by Akt and protein phosphatase 2A in ovarian granulosa cells. Chin J Physiol 2020;63:179-86
|How to cite this URL:|
Lin YY, Sun D, Wu YL. Novel regulation of PKC-induced inflammation by Akt and protein phosphatase 2A in ovarian granulosa cells. Chin J Physiol [serial online] 2020 [cited 2021 Mar 1];63:179-86. Available from: https://www.cjphysiology.org/text.asp?2020/63/4/179/293586
| Introduction|| |
Ovulation is an important stage in the ovarian cycle and it proceeds as a confined inflammation-like event responsible for multiple physiological functions in the ovary. Members of the protein kinase C (PKC) family have been widely implicated in a myriad of cellular signals, primarily initiating from the cell surface receptors-mediated liberation of phospholipids from cell membrane to result in several diverse cellular activities in the ovarian system., The significance of PKCs in inflammation has been extensively-characterized, finding that PKC activation may potentially target the IKK/NF-κB and the mitogen-activated protein kinase (MAPK) pathways and subsequently lead to the production of inflammatory mediators., In ovarian granulosa cells (GCs), blocking the PCK activity would inhibit FSH-mediated cyclooxygenase-2 (COX-2) expression, and the COX-2/PGE2 signaling has been shown as an essential cascade in ovulation., Meanwhile, the PKC-mediated interleukin-8 (IL-8) expression and the significance of IL-8 in ovulation also have been highly recognized., Therefore, the PKC-mediated production of COX-2 and IL-8 appears critical in ovarian physiology.
As the mediator between the cell surface receptors and the intracellular signaling pathways, Akt, also called protein kinase B, has been extensively characterized to interact with some other protein kinases to regulate the development of follicles and the oocyte. The transcription factor NF-κB is, in general, involved in regulating cell proliferation and inflammatory pathways, and the Akt pathway has been identified to interact with the PKC-mediated inflammation by activating the NF-κB pathway., In addition, protein phosphatase 2A (PP2A) is a well-conserved protein family with ubiquitously expressed profile, and the dysregulation of PP2A has been suggested to be associated with a panel of diseases., Altogether, these studies suggest that Akt and PP2A may play important roles in PKC-mediated inflammation in the ovarian system.
Involvement of Akt and PP2A in diverse biological activities in different peripheral tissues, including ovary has been established; Akt and PP2A have been shown to interact with the PKC signaling pathways. However the aspects on the inflammation-associated roles of the Akt and PP2A in ovary have not been well-defined yet. In this study, rat primary GCs and KGN human GCs were exposed to PKC activator in combination with various inhibitors or activators for PI3K/Akt, mitogen-activated protein kinase phosphatase 1 (MKP-1) and PP2A to clarify the functional significance of Akt and PP2A in PKC-mediated cellular inflammation in ovarian GCs.
| Materials and Methods|| |
Chemicals and reagents
Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT, USA). The PKC activator phorbol 12-myristate 13-acetate (PMA) was from Sigma Chemicals (St. Louis, MO, USA). Reverse transcriptase, SYBR green reagent and Lipofectamine™ 2000 were from ThermoFisher (ThermoFisher Scientific, Waltham, MA, USA). Antibodies were purchased from different companies: rabbit polyclonal COX-2 antibody (Cayman), mouse monoclonal IL-8 antibody (R and D), mouse monoclonal α-tubulin antibody (Sigma), rabbit polyclonal NF-κB/p65 (Rel A) antibody (Thermo Scientific), phospho-PP2A antibody (Abcam), PP2A-Aα/β antibody (Santa Cruz), rabbit monoclonal phospho-Akt antibody (Ser473) (Cell Signaling), and rabbit mono Akt antibody (Cell Signaling). Unless otherwise specified, all the other chemicals and reagents used in this project were purchased from Sigma Chemicals.
Animal ethics and isolation of primary granulosa cells
Sprague-Dawley rats were obtained from the Laboratory Animal Center (National Yang-Ming University, Taipei, Taiwan). All animals were housed under controlled humidity, temperature, and light regimen. Rats were anesthetized and sacrificed using CO2. All the animal procedures were approved by the Institutional Animal Care and Use Committee of the National Yang-Ming University (Permit Number 1070908). Primary ovarian GCs were collected from rat ovarian follicles, as reported previously. The collected follicles were punctured with a 25-gauge needle to release GCs into Dulbecco's modified Eagle media/Ham's F-12 media (DMEM/F-12) (Gibco) containing 2.438 g/L NaHCO3, 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin and 2 μg/ml insulin. Harvested cells were plated onto 3.5 cm dish coated with 1 ml containing 5 μg/μl poly-L-lysine.
Human ovarian granulosa-like tumor cell line KGN purchased from the RIKEN BioResource Center (Iberaki, Japan) was maintained in DMEM/F-12 with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. Cells were kept in 5% CO2 and 37°C atmosphere.
Determination of cellular protein expression
Total cellular proteins were collected and processed as required and their protein concentrations were determined using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). Protein samples were then subjected to regular Western blotting assay to determine the expression profiles of the various target proteins.
Measurement of PGE2 and interleukin-8 by enzyme-linked immunosorbent assay
The concentrations of PGE2 and IL-8 in the culture medium were determined using enzyme-linked immunosorbent assay kit; for PGE2 this was obtained from Assay Designs (Ann Arbor, MI, USA) and for IL-8 this was obtained from R and D systems. The ELISA was performed according to the manufacturers' instructions.
mRNA measurement by quantitative real-time polymerase chain reaction
Total cellular RNAs of treated rat GCs or KGN cells were extracted with Tri-reagent according to the manufacturer's instructions (Sigma). The RNA samples were resuspended in RNase-free diethyl pyrocarbonate-treated water and then each sample underwent quantitative RT-PCR to measure the levels of mRNAs of various genes. First, 1 μg of total RNA from each sample was used to perform the reverse transcription to generate cDNAs, and consequently, 2 μl from the reverse transcription mixture was subjected to the real-time PCR. The primer sequences used were: rat COX-2: sense: 5'-CAT GAT CTA CCC TCC CCA CG-3', antisense: 5'-CAG ACC AAA GAC TTC CTG CCC-3' to give a 67 bp product; rat actin: sense: 5'-CCC ATC TAT GAG GGT TAC GC-3', antisense: 5'-TTT AAT GTC ACG CAC GAT TTC-3' to give a 150 bp product.
Monitoring mouse cyclooxygenase-2 promoter activity
Rat GCs were plated in 24-well with 8 × 104/well overnight. The mouse COX-2 promoter construct was cotransfected with an internal control plasmid expressing β-galactosidase reporter gene. The luciferase was determined and normalized against the β-galactosidase activity within the same sample.
Rat GCs were plated onto poly-L-lysine-coated coverslips overnight and then the coverslips were incubated with rabbit polyclonal NF-κB (p65; Rel A) antibody, followed by incubation with goat anti-rabbit FITC-conjugated antibody. The nuclei were then stained with DAPI, and the cells were mounted for observation, and the images were recorded by the digital camera (CoolSNAP HQ2 CCD Camera, Photometrics, Tucson, AZ, USA) installed on a microscope (Leica DM 6000B, Leica Microsystems, Wetzlar, Germany). The positive signals of p65 in the nuclei of cells were counted and calculated as fold over the control group.
Experimental data are expressed as the mean ± the standard errors of the means (SEM). The results were analyzed using one-way analysis of variance, which was followed by the least-significant difference (LSD) test in order to compare the differences between each of the treatment groups and the control group. Differences with P < 0.05 were considered statistically significant.
| Results|| |
Protein kinase C induction of cyclooxygenase-2 expression impacted by Akt
To confirm the PKC impact from PMA on COX-2 expression, rat GCs were pretreated with PKC inhibitor BIM-1 (0.1, 1, 5 μM) for 2 h and then PMA (100 nM) was included for an additional 12 h. PMA significantly induced COX-2 expression and such induction was inhibited by PKC inhibitor [Figure 1]a. To monitor the Akt activation (phosphorylation) regulated by PKC, rat GCs were treated with PMA for 10, 30, and 60 min. At all the time points, PMA dramatically induced the Akt phosphorylation [Figure 1]b. In addition, to investigate the Akt impact on PKC-mediated COX-2 expression, rat GCs were pretreated with Akt inhibitor wortmannin (0.625, 1.25, 2.5, 5 μM) or Akt activator SC79 (1, 2, 4, 8 μg/ml) for 2 h before the inclusion of PMA for an additional 12 h. Neither wortmannin nor SC79 changed the basal level of COX-2 expression [[Figure 1]c and [Figure 1]d]. The induction of COX-2 expression by PMA was attenuated by wortmannin (5 μM), but it was further enhanced by SC79 (2, 4, 8 μg/ml) [[Figure 1]c and [Figure 1]d], suggesting a positive regulatory role of Akt in PKC-induced COX-2 production in rat GCs.
|Figure 1: The PI3K/Akt impact on the PKC-induced cyclooxygenase-2 (COX-2) expression in rat granulosa cells. Rat granulosa cells were plated overnight and the plated cells were pretreated with PKC inhibitor BIM-1 (0.1, 1, 5 μM) (a), PI3K inhibitor wortmannin (0.625, 1.25, 2.5, 5 μM) (c), or Akt activator SC79 (1, 2, 4, 8 μg/ml) (d) for 2 h and then phorbol 12-myristate 13-acetate (PMA, 100 nM) was included for 12 h, or rat granulosa cells were exposed to PMA (100 nM) for 10, 30, or 60 min (b). The COX-2 protein, Akt phosphorylation and Akt protein expression profiles were analyzed by Western blotting with α-tubulin as an internal control. The results were represented as mean ± standard error mean from 3 (a) or 4 (b, c, d) independent experiments. *, P < 0.05 compared with control group within the same time point; #P < 0.05 compared with PMA treatment group.|
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Involvement of protein phosphatase 2A in protein kinase C-mediated cyclooxygenase-2 expression in rat granulosa cells
In order to investigate the potential involvement of MKP-1 and/or PP2A in PKC-mediated COX-2 expression in rat GCs, cells were pretreated with MKP-1 inhibitor sanguinarine (0.01, 0.1, 1 μM) or PP2A inhibitor okadaic acid (10, 30 nM) for 2 h before the inclusion of PMA. The basal production of COX-2 or PGE2 was not affected by sanguinarine or okadaic acid [[Figure 2]a and [Figure 2]b]. The PKC-induced COX-2/PGE2 production was not affected by sanguinarine [Figure 2]a, but interestingly it was further promoted by okadaic acid [Figure 2]b. To understand the PP2A role in PKC-mediated COX-2 mRNA expression, rat GCs were pretreated with okadaic acid for 1 h before the inclusion of PMA for an additional 6 h. Similarly, the PMA-induced COX-2 mRNA expression was also further elevated by okadaic acid [Figure 2]c. Meanwhile, to examine the potential transcriptional regulation of the COX-2 gene by PKC and PP2A, rat GCs cells were transfected with a mouse COX-2 promoter construct, followed by the pretreatment with okadaic acid (10, 30 nM) for 2 h before the inclusion of PMA for an additional 12 h. The COX-2 promoter activity was induced by PMA, and this was further augmented by okadaic acid [Figure 2]d. On the other hand, the same rat GCs were pretreated with a PP2A activator sodium selenate (0.01, 0.1, 1 mM) before the addition of PMA. Both PKC-induced COX-2 protein expression and COX-2 promoter activation were significantly suppressed [[Figure 2]e and [Figure 2]f].
|Figure 2: PKC-mediated COX-2 expression regulated by PP2A in rat granulosa cells. Plated rat granulosa cells were pretreated with MKP-1 inhibitor sanguinarine (0.01, 0.1, 1 μM) (a), PP2A inhibitor okadaic acid (10, 30 nM) (b, c, d), or PP2A activator sodium selenate (0.01, 0.1, 1 mM) for for 2 h, and then phorbol 12-myristate 13-acetate (PMA, 100 nM) was included for an additional 6 (c) or 12 h (a, b, d, e, f). The COX-2 protein expression was determined by Western blotting with α-tubulin as an internal control (a, b, e). The PGE2concentrations in the cultured media were determined by ELISA (a, b). The COX-2 mRNA expression was determined by quantitative RT-PCR with β-actin as an internal control (c). Rat granulosa cells were cotransfected with a mouse COX-2 promoter plasmid containing a luciferase reporter gene and a reference control plasmid with pCMV-β-galctosidase reporter plasmid. The COX-2 promoter activity was determined by luciferase activity assay and normalized against the β-glactosidase activity within the same sample (d, f). The results were represented as mean ± standard error of the mean from 4 independent experiments. *, P < 0.05 compared with control group; #, P < 0.05 compared with PMA treatment group. COX-2: cyclooxygenase-2.|
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Impact of Akt in protein kinase C-mediated cyclooxygenase -2 expression in KGN human granulosa cells
We have noted the enhancement effect of Akt in PKC-mediated COX-2 in rat GCs [Figure 1], and thus we attempted to examine in human GCs. In contrast to our findings in rat GCs, PMA appeared to inhibit Akt phosphorylation at 10, 30, and 60 min [Figure 3]a. Furthermore, in contrast to the observations in rat GCs [Figure 1]c, the PMA-induced COX-2/PGE2 production was further enhanced by wortmannin [Figure 3]b; similarly, PMA-induced cellular IL-8 protein expression and its secretion were also further promoted by wortmannin [Supplementary Figure 1]. However, the PMA-induced COX-2 expression was moderately suppressed by SC79 [Figure 3]c. In addition, also different from the observations in rat GCs, sanguinarine slightly reduced PMA-mediated COX-2 expression, whereas the okadaic acid did not affect PMA-mediated COX-2 expression [Figure 3]d.
|Figure 3: The PI3K/Akt impact on the PKC-induced cyclooxygenase-2 (COX-2) expression in human granulosa cells. The overnight plated KGN human granulosa cells were exposed to phorbol 12-myristate 13-acetate (PMA, 100 nM) for 10, 30, or 60 min (a), or pretreated with PI3K inhibitor wortmannin (0.625, 1.25, 2.5, 5 μM) (b), Akt activator SC79 (1, 2, 4, 8 μg/ml) (c), MKP-1 inhibitor sanguinarine (0.01, 0.1, 1 μM) or PP2A inhibitor okadaic acid (10, 30 nM) (d) for 2 h and then PMA (100 nM) was included for 12 h. The COX-2 protein, Akt phosphorylation and Akt protein expression profiles were determined by Western blotting with α-tubulin as an internal control. The PGE2concentrations in the cultured media were determined by ELISA (b). The results were represented as mean ± standard error of the mean from 4 independent experiments. *P < 0.05 compared with control group; #P < 0.05 compared with PMA treatment group.|
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Protein phosphatase 2A impact on protein kinase C-mediated NF-κB translocation from cytosol to nucleus
Previous studies have demonstrated that NF-κB is involved in PKC-induced inflammation and PP2A may play a modulatory role in NF-κB signaling., In addition, PKC activation has been shown to regulate COX-2 expression through NF-κB signaling pathways in GCs. Thus, we went on to evaluate the role of PP2A in PMA-induced NF-κB translocation in rat GCs. Rat GCs were pretreated with okadaic acid for 30 min before the inclusion with PMA for an additional 30 min and then the immunofluorescence was used to monitor the NF-κB p65 subunit translocation from cytosol to nucleus. PMA was able to induce p65 translocation and okadaic treatment (30 nM) appeared to further increase PMA-induced NF-κB translocation [Figure 4]. The PKC impact on PP2A activation (phosphorylation) was also examined in rat GCs and was found to has no effect [Supplementary Figure 2].
|Figure 4: The PP2A impact on PMA-induced NF-κB nuclear translocation in rat granulosa cells. Rat granulosa cells were plated on slide to allow overnight attachment, and cells were then pretreated with PP2A inhibitor okadaic acid (10, 30 nM) for 30 min before the inclusion of PMA (100 nM) for additional 30 min. Nuclear localization of the NF-κB subunit p65 was visualized by immunofluorescence microscopy at a magnification of ×400 (scale bar, 50 μm). The DAPI shows nuclear staining. The mouse IgG was used as a negative control. The results represent mean ± standard error mean from 4 independent experiments. *P < 0.05 compared with control group; #P < 0.05 compared with PMA treatment group. PMA: phorbol 12-myristate 13-acetate.|
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| Discussion|| |
The current study clearly demonstrated that PKC activation dramatically induces a cellular inflammation in terms of COX-2 and PGE2 production in rat GCs, and COX-2/PGE2 as well as IL-8 production in KGN human GCs. The Akt pathway appears to act as a positive regulator in rat GCs, but as a negative regulator in human GCs. In rat GCs, the PP2A is able to attenuate PKC-mediated inflammation, possibly by targeting the NF-κB pathway.
Both COX-2 and IL-8 function significantly to regulate several ovarian physiological events.,, Meanwhile, the PKC signaling has been demonstrated to increase the expression of several pro-inflammatory mediators, including COX-2 and IL-8., Thus, we endeavored to pinpoint the potential roles of the PI3K/Akt pathway in such PKC-mediated inflammatory responses in rat and human GCs. One novel but still unsolved finding in our study is that how would PKC activator PMA perform opposite regulatory roles by increasing the Akt phosphorylation in rat GCs [Figure 1]b, but decreasing the Akt activation in KGN human GCs [Figure 3]a? Previous studies have demonstrated a PKC attenuation of the Akt signaling in mouse keratinocytes and human prostate cancer cells to modulate cell viability., It is not clear if KGN is a human tumor-derived cell line, and thus, it may act more like human prostate cancer cells. With the use of primary human GCs in the future should help solve this puzzle. In relation to inflammation, the Akt activation has been shown to suppress LPS-induced inflammation and the perturbation within this signaling cascade may cause inflammatory disorders and even malignancies., Consistently, given that an augmenting effect of PI3K inhibitor wortmannin on PKC-induced production of COX-2/PGE2 and IL-8 [[Figure 3] and Supplementary [Figure 1]] and an inhibitory effect of Akt activator SC79 on PKC-mediated COX-2/PGE2 production [Figure 3] in KGN cells, the potential inhibitory role of the Akt pathway in PKC-mediated inflammation in human GCs was noted; however, the stimulatory role of the Akt signaling in PKC-mediated COX-2/PGE2 production was substantiated in rat GCs [Figure 1]. Currently, the reason causing such an inconsistency between rat and human GCs is not clear. The KGN cells have been regarded as more close to an immature GC type in the developmental stage,, whereas the rat GCs were derived from mature follicle, and thus it may be due to the difference in stage of follicle development. Alternatively, the KGN was a tumor-derived cell line, and it may not closely mimic the primary human ovarian GCs in all aspects. Regardless, the significance of Akt in cellular inflammation appears conserved in both cell types from two species. Therefore, how the Akt would affect PKC activity in both rat and human GCs should warrant further investigation.
Besides the involvement of the Akt pathway in PKC-induced COX-2 expression, several previous studies have already demonstrated that MAPKs and NF-κB are involved in regulating COX-2 expression.,, In addition, it has been reported that MAPKs and NF-kB can be potentially regulated by MKP-1 and PP2A, respectively., In fact, PP2A is the major phosphatase in eukaryotic cells in the downregulation of the activation of a variety of protein kinase cascades, including at least Akt, PKC, and MAPKs. The PP2A is not a single enzyme molecule but formed from multiple diverse protein subunits. In fact, the B subunit protein acts as the main driver for the PP2A function. The previous study have revealed that B55α subtype indeed can potentially target to PKC and Akt in acute myeloid leukemia cells and the suppressed B55α expression was accompanied with an elevation in Akt phosphorylation; the B55α molecule was able to dephosphorylate and thereby inhibit the PKC activity.,, Whether the PP2A would directly affect the PKC activity in ovarian GCs would need further investigation. In our current study, the MKP-1 inhibitor sanguinarine only moderately suppressed the PKC-mediated COX-2 expression, but the PP2A inhibitor okadaic acid did not have any effect in human GCs [Figure 3]d. Okadaic acid further enhanced the PKC activation of promoter, mRNA, and protein expression of the COX-2 gene [[Figure 2]b, [Figure 2]c, [Figure 2]d], but the PP2A activator sodium selenate dramatically suppressed the PKC-induced COX-2 protein expression and promoter activation [[Figure 2]e and [Figure 2]f] in rat GCs. All these findings highly suggest a potential negative regulatory role of PP2A in PKC-mediated inflammation in rat GCs.
According to previous literature that PP2A may down-regulate the NF-kB activity by dephosphorylating NF-κB p65 subunit, consistently in our study, okadaic acid appears to increase the PKC-induced NF-κB translocation [Figure 4], indicating that PP2A may target to NF-κB to modulate the PKC-induced COX-2 expression in rat GCs. On the other hand, the PP2A activity may also be modulated by numerous ways, such as phosphorylation, methylation and subunit expression. In fact, a previous study has demonstrated that PP2A activity could be regulated by PKC by phosphorylation; however, such a scenario was not found in rat GCs [Supplementary Figure 2]. We have noted a stimulatory role of Akt in PKC-mediated COX-2 expression in rat GCs [Figure 1], and it has been demonstrated that Akt was able to activate NF-κB by activating the IKK system, and PP2A knockdown could result in Akt activation, suggesting an inhibitory effect of PP2A on Akt signaling., Thus, PP2A may act as a negative regulator of both Akt and NF-κB in regulating the PKC induction of COX-2/PGE2 production in rat GCs.
| Conclusion|| |
This study provides clear evidence in GCs that the PI3K/Akt pathway is involved in PKC-mediated inflammation by acting as an inhibitory player in human GCs, but as a stimulatory player in rat GCs. In addition, PP2A appears to act as an inhibitor in PKC-induced inflammation in rat GCs, and this may be due to its attenuation impact on PKC-mediated NF-κB activation.
We would like to thank Dr. Ae-Kyung Yi (University of Tennessee Health Science Center, USA) for kindly providing the mouse COX-2 promoter construct.
Financial support and sponsorship
This research was supported by grants from the Ministry of Science and Technology (MOST 105-2320-B-010-026-MY3; MOST 108-2320-B-010-025), the Cheng Hsin General Hospital (CY10603; CY10802), and the Taiwan Ministry of Education Aim for the Top University Plan.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]