2024 Volume 47 Issue 6 Pages 1196-1203
Microglia, resident immune cells in the central nervous system (CNS), play a critical role in maintaining CNS homeostasis. However, microglia activated in response to brain injury produce various inflammatory mediators, including nitric oxide (NO) and proinflammatory cytokines, leading to considerable neuronal damage. NO generated by inducible NO synthase (iNOS) rapidly reacts with superoxide to form a highly toxic product, peroxynitrite. Therefore, iNOS is considered to be a putative therapeutic target for cerebral ischemia. Here, we examined the effects of panobinostat (Pano), a histone deacetylase inhibitor, on lipopolysaccharide (LPS)-induced iNOS expression using rat immortalized microglia HAPI cells. Pano inhibited LPS-induced expression of iNOS mRNA and NO production in a dose-dependent manner; however, it had little effect on the LPS-induced activation of c-Jun N-terminal kinase (JNK) and p38 or nuclear translocation of nuclear factor-κB (NF-κB). The interferon-β (IFN-β)/signal transducer and activator of transcription (STAT) pathway is essential for LPS-induced iNOS expression in macrophages/microglia. We also examined the effects of Pano on LPS-induced IFN-β signaling. Pano markedly inhibited LPS-induced IFN-β expression and subsequent tyrosine phosphorylation of STAT1. However, the addition of IFN-β restored the decreased STAT1 phosphorylation but not the decreased iNOS expression. In addition, Pano inhibited the LPS-increased expression of octamer binding protein-2 and interferon regulatory factor 9 responsible for iNOS expression, but IFN-β addition also failed to restore the decreased expression of these factors. Thus, we conclude that the inhibitory effects of Pano are due not only to the inhibition of the IFN-β/STAT axis but also to the downregulation of other factors not involved in this axis.
Accumulating evidence suggests that inflammation is closely related to neuronal injury observed in several central nervous system (CNS) disorders, such as cerebral ischemia and Alzheimer’s disease.1,2) Microglia, resident immune cells in the CNS, play a critical role in maintaining CNS homeostasis. However, the overactivation of microglia following brain injury produces various inflammatory mediators, including nitric oxide (NO), superoxide, and proinflammatory cytokines, and exacerbates neuronal damage.1) NO generated by inducible NO synthase (iNOS) in activated microglia rapidly reacts with superoxide to form a highly toxic product, peroxynitrite. This product has been reported to promote ischemia-induced brain injury.3) Indeed, ischemic damage and neurological deficits following focal cerebral ischemia have been shown to be alleviated in iNOS-deficient mice.4) Therefore, iNOS is thought to be a promising therapeutic target for cerebral ischemia.
In microglia and macrophages the expression of iNOS is regulated by toll-like receptor 4 (TLR4).5) The stimulation of TLR4 by lipopolysaccharide (LPS), an exogenous TLR4 ligand, activates several signaling pathways including nuclear factor-κB (NF-κB) and the mitogen-activated protein kinase (MAPK) pathways. These pathways play key roles in LPS-induced iNOS expression.6) In addition, interferon-β (IFN-β), which is rapidly produced in response to LPS, serves as an autocrine mediator in LPS-induced iNOS expression.7) Several studies have shown that TLR4 is involved in the exacerbation of ischemic brain injury. For example, brain damage following cerebral ischemia is ameliorated in TLR4-knockout mice.8) Damage-associated molecular patterns (DAMPs), such as high mobility group box 1, are molecules that are released from damaged brain cells and promote neuroinflammation via the activation of TLR4.9)
There is evidence to suggest that histone deacetylase (HDAC) inhibitors are effective at treating cerebral ischemia. Treatment with HDAC inhibitors, such as valproic acid (VPA), reduces infarct volumes and improve neurological deficits.10) As described above, inflammatory processes are closely associated with neuronal injury following cerebral ischemia. VPA suppresses microglial activation observed in the ischemic boundary zone.11) Other HDAC inhibitors, such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA), prevent the production of inflammatory mediators such as iNOS and proinflammatory cytokines.12,13) These findings suggest that the anti-inflammatory effects of HDAC inhibitors are involved in the protection against ischemic injury. However, the mechanism by which HDAC inhibitors elicit anti-inflammatory effects is not fully understood. Panobinostat (Pano) is a potent oral pan-HDAC inhibitor approvable for the therapy of multiple myeloma.14) In this study, we examined the effects of Pano on LPS-induced iNOS expression using rat immortalized microglia HAPI cells and explored the underlying mechanisms.
Pano was purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.). Anti-NF-κB p65 antibody (sc-8008) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Anti-phospho-JNK (#9255), anti-JNK (#9252), anti-phospho-p38 MAPK (#9215), anti-p38 MAPK (#9212), anti-phospho-STAT1 (#7649), anti-Stat1 (#14994), and anti-histone H3 (#4499) antibodies were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.). Anti-β-actin (MAB1501) and anti-acetyl-Histone H3 (06-599) antibodies were purchased from Merck (Burlington, MA, U.S.A.). LPS (Escherichia coli 0111: B4) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Rat IFN-β was purchased from U-CyTech biosciences (Utrecht, The Netherlands).
Cell CultureRat immortalized microglia HAPI cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal calf serum (FCS), 4 mM L-glutamine, 100 units/mL penicillin G, and 0.1 mg/mL streptomycin in a humidified 5% CO2/95% air incubator at 37 °C. HAPI cells were seeded in a 3.5 cm-diameter dish at a density of 6.0 × 105 cells/dish and a 6 cm-diameter dish at a density of 2.0 × 106 cells/dish in DMEM containing 1% heat-inactivated FCS (1% FCS DMEM). The next day, cells were treated with LPS. Pano was added to the cultures 30 min prior to treatment with LPS.
RNA Isolation and Real-Time RT-PCRTotal RNA was extracted from the treated cells with TRI Reagent (Molecular Research Center, Inc, Cincinnati, OH, U.S.A.). First-strand cDNA was synthesized from 1 µg of total RNA using a ReverTra Ace kit (Toyobo, Osaka, Japan). Real-time quantitative PCR (qPCR) was performed using Thunderbird SYBR qPCR Mix (Toyobo). Aliquots of the cDNA solution were amplified using the following specific primers: rat iNOS (forward, 5′-TTGCTTCTGTGCTAATGCGG-3′; reverse, 5′-CAGAACTGAGGGTACATGCT-3′), IFN-β (forward, 5′-ATCGACTACAAGCAGCTCCA-3′; reverse, 5′-ACCTTTGTACCCTCCAGTAA-3′), interferon regulatory factor 1 (IRF1) (forward, 5′-CAACAAGGATGCCTGTCTGT-3′; reverse, 5′-GCTGTGTAACTGCTGTGGTC-3′), Oct-2 (forward, 5′-GCTCTATGGCAACGACTTCA-3′; reverse, 5′-TGTGCAGCTGCTCTGCAATC-3′), IRF9 (forward, 5′-AGCAATGTCTGTGGTGGTGGCA-3′; reverse, 5′-TCTAGGCTGTGCACCTGGATCT-3′), and 18S ribosomal RNA (rRNA) (forward, 5′-CGGCTACCACATCCAAGGAA-3′; reverse, 5′-GCTGGAATTACCGCGGCTT-3′). qPCR was carried out as follows: for iNOS, IRF1, Oct-2, and IRF9, 95 °C for 1 min, followed by 40 cycles of 95 °C for 10 s, 58 °C for 30 s, and 72 °C for 1 min; for IFN-β, 95 °C for 1 min, followed by 40 cycles of 95 °C for 10 s, 54 °C for 30 s, and 72 °C for 1 min; for 18S rRNA, 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. The mRNA levels were normalized relative to 18S rRNA levels in each sample.
Measurement of NitriteHAPI cells were seeded in a 24-well plate at a density of 2.0 × 105 cells in 1% FCS DMEM. The next day, the cells were treated with 100 ng/mL LPS in the presence or absence of Pano for 24 h. Nitrite concentrations in the medium were measured using the Griess method.15)
Western BlottingHAPI cells were washed twice with ice-cold phosphate-buffered saline (PBS); then, whole cell and nuclear extracts were prepared as in our previous report.15) Whole cell (20 µg) or nuclear (5 µg) extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with each primary antibody (1 : 3000) and sequentially with horseradish peroxidase (HRP)-conjugated second antibody (1 : 5000). Proteins were detected using SuperSignal West Pico PLUS (Thermo Fisher Scientific, Waltham, MA, U.S.A.) or ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and visualized using the ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, U.S.A.).
Plasmid ConstructionA DNA fragment of the promoter region (−101 to + 39) of the IFN-β gene was created by PCR using specific primers (forward primer, 5′-GCCTCGAGTTAGAAACTACTAAAATGTA-3′; reverse primer, 5′-CAAGATCTAAAGGTTGCAGTTAGAATGT-3′) from human genomic DNA. The fragment was digested with Bgl II and Xho I, and the digested fragment was subcloned into the Bgl II/Xho I site of the pGL4.12 reporter vector (Promega, Madison, WI, U.S.A.). This plasmid was designated as IFN-β pGL4.
Reporter AssayHAPI cells were seeded in a 24-well microplate at a density of 1.8 × 105 cells/well. Cells were transfected with 400 ng of the IFN-β pGL4 and 10 ng of the internal reference pRL-TK (Promega) using TransIT-LT1 Transfection Reagent (Mirus Bio, Madison, WI, U.S.A.) according to the manufacturer’s protocol. After treatment, the cells were washed twice with PBS and lysed. The luciferase activity in the cell lysates was then measured using the dual-luciferase reporter assay system (Promega). Renilla luciferase (pRL-TK) activity was used to normalize the transfection efficiency. The experiments were carried out in triplicate.
Statistical AnalysisData were analyzed using ANOVA followed by Bonferroni’s or Holm’s post hoc test. A p-value less than 0.05 was considered significant.
First, we examined the effects of Pano on LPS-induced NO production. HAPI cells were treated with LPS (100 ng/mL) for 24 h in the presence of various concentrations of Pano; then, nitrite levels in the conditioned medium were measured. LPS markedly stimulated NO production in HAPI cells (Fig. 1A). Pano inhibited LPS-induced NO production in a dose-dependent manner (Fig. 1A). In general, NO production elicited by LPS is regulated by the expression of iNOS. Indeed, the treatment of HAPI cells with LPS increased the expression levels of iNOS mRNA (Fig. 1B). Pano reduced LPS-induced iNOS mRNA expression in a dose-dependent manner (Fig. 1B).
(A) Effects of Pano on LPS-induced NO production. HAPI cells were treated with LPS (100 ng/mL) for 24 h in the presence or absence of Pano (10, 30, or 100 nM). After treatment, nitrite was measured using the Griess assay. Values (mean ± standard deviation (S.D.), n = 3) represent the concentrations of nitrite in the conditioned medium. ** p < 0.01 (vs. LPS alone). (B) Effects of Pano on LPS-induced iNOS expression. HAPI cells were treated with LPS (100 ng/mL) for 6 h in the presence or absence of Pano (10, 30, or 100 nM). After treatment, total RNA was extracted and then RT-qPCR was performed. Values (mean ± standard error (S.E.), n = 3) are expressed relative to mRNA levels in LPS-treated cells. ** p < 0.01 (vs. LPS alone). (C) Effects of Pano on histone acetylation in HAPI cells. HAPI cells were treated with Pano (100 nM), TSA (500 nM), or VPA (2 mM) for 1 h. After treatment, whole cell lysates were prepared from the treated cells, and the lysates were subjected to Western blot analysis. A representative blot from three independent experiments is shown. (D) Effects of HDAC inhibitors on LPS-induced iNOS expression. HAPI cells were treated with LPS (100 ng/mL) for 6 h in the presence of Pano (100 nM), TSA (500 nM), or VPA (2 mM). HDAC inhibitors were present 30 min prior to and during LPS treatment. After treatment, total RNA was extracted and then RT-qPCR was performed. Values (mean ± S.E., n = 3) are expressed relative to mRNA levels in LPS-treated cells. ** p < 0.01 (vs. LPS alone).
Pano is an HDAC inhibitor used in therapy for multiple myeloma. To determine whether Pano can inhibit HDAC activity in HAPI cells at the concentration used in this study, we examined the effects of Pano on histone acetylation. HAPI cells were treated with Pano (100 nM) for 1 h; we then detected acetylated histone using Western blotting. Pano, as well as trichostatin A (TSA) and VPA, other HDAC inhibitors, increased the acetylation levels of histone H3 (Fig. 1C). In addition, we examined the effects of TSA and VPA on LPS-induced iNOS expression. Consistent with the results of previous studies, the HDAC inhibitors suppressed the induction of iNOS mRNA (Fig. 1D).
Effects of Pano on LPS-Induced Activation of Signaling PathwaysSome HDAC inhibitors are reported to influence various signaling pathways.16,17) LPS-induced expression of iNOS has been demonstrated to be regulated by the MAPK and NF-κB pathways. TLR4 stimulation by LPS promotes the activation of MAPK, especially JNK and p38, and the translocation of NF-κB to the nucleus. We examined the effects of Pano on the LPS-induced activation of these pathways. LPS caused phosphorylation of JNK and p38 in HAPI cells (Fig. 2A) and nuclear accumulation of NF-κB (Fig. 2B). However, Pano had little effect on these processes (Figs. 2A, B). Some studies have demonstrated that activation of the transcription factor STAT1 is indispensable for LPS-induced iNOS expression in macrophages.18) We also investigated whether Pano affects LPS-induced STAT1 activation. Pano inhibited the increased tyrosine phosphorylation of STAT1 by LPS (Fig. 2C).
(A) Effects of Pano on LPS-induced activation of JNK and p38. HAPI cells were treated with LPS (100 ng/mL) for 1 h in the presence or absence of Pano (100 nM). After treatment, whole cell extracts were prepared from the treated cells, and the extracts were subjected to Western blot analysis. (B) Effects of Pano on LPS-induced NF-κB nuclear translocation. HAPI cells were treated with LPS (100 ng/mL) for 1 h in the presence or absence of Pano (100 nM). After treatment, nuclear extracts were prepared from the treated cells, and the extracts were subjected to Western blot analysis. (C) Effects of Pano on LPS-induced SATA1 activation. HAPI cells were treated with LPS (100 ng/mL) for 3 h in the presence or absence of Pano (100 nM). After treatment, whole cell extracts were prepared from the treated cells, and the extracts were subjected to Western blot analysis. A representative blot from three independent experiments is shown. Values (mean ± S.E., n = 3 or 4) are expressed as fold-changes relative to untreated cells (Ctrl). * p < 0.05, ** p < 0.01 (vs. Ctrl); ## p < 0.01 (vs. LPS alone).
Previous reports have shown that the induction of IFN-β expression and subsequent STAT1 activation are essential for LPS-induced iNOS expression in macrophages and microglia.7,18) Blockade of the IFN-β/STAT pathway attenuates LPS-induced iNOS expression.18) We examined the effects of Pano on LPS-induced IFN-β signaling. LPS stimulated the induction of IFN-β mRNA in HAPI cells, and Pano markedly inhibited this induction (Fig. 3A). Next, to evaluate the inhibitory effects of Pano on LPS-induced IFN-β gene expression, we performed a reporter assay using the reporter plasmid IFN-β pGL4. Pano reduced the increased IFN-β promoter activity induced by LPS, indicating that Pano suppresses LPS-induced IFN-β expression at the transcriptional level (Fig. 3B).
(A) Effects of Pano on LPS-induced IFN-β expression. HAPI cells were treated with LPS (100 ng/mL) for 1 h in the presence or absence of Pano (100 nM). After treatment, RT-qPCR was performed. Values (mean ± S.E., n = 3) are expressed relative to mRNA levels in LPS-treated cells. ** p < 0.01 (vs. LPS alone). (B) Effects of Pano on IFN-β promoter activity. Upper panel: Schematic diagram of the IFN-β reporter plasmid. Lower panel: HAPI cells were transiently transfected with IFN-β pGL4 (400 ng) and pRL-TK (10 ng). The next day, the culture medium was replaced with 1% FCS DMEM. Two-four hours later, the cells were treated with LPS (100 ng/mL) for 3 h in the presence or absence of Pano (100 nM). After treatment, the cells were lysed and the luciferase activity in the lysate was measured using a luminometer. Values (mean ± S.D., n = 3) are expressed as promoter activity (Firefly luciferase [IFN-β pGL4]/Renilla luciferase [pRL-TK]). ** p < 0.01 (vs. LPS alone).
Since IFN-β activates STAT signaling, the suppression of LPS-induced STAT1 phosphorylation by Pano is likely attributable to its inhibitory effect on the induced expression of IFN-β (Fig. 2C). To determine whether the addition of IFN-β restores the decrease in STAT1 phosphorylation caused by Pano, after HAPI cells had been treated with LPS and Pano for 2 h, IFN-β was added to the culture. Its addition abolished the inhibitory effect of Pano on LPS-induced STAT1 phosphorylation (Fig. 4A). Next, we examined whether STAT1 activation by adding IFN-β is able to restore the reduction by Pano of LPS-induced iNOS expression. After treatment of HAPI cells with LPS and Pano for 2 h, IFN-β was added to the culture, and the cells were incubated for another 4 h. However, the decreased iNOS mRNA expression only slightly, but significantly, recovered by the addition of IFN-β (Fig. 4B).
(A) Effects of IFN-β addition on the inhibition of STAT1 phosphorylation by Pano. HAPI cells were treated with LPS (100 ng/mL) for 3 h in the presence or absence of Pano (100 nM). Two hours after the addition of LPS, IFN-β (5000 units/mL) was added to the culture. After treatment, whole cell extracts were prepared from the treated cells, and the extracts were subjected to Western blot analysis. A representative blot from three independent experiments is shown. Values (mean ± S.E., n = 3) are expressed as fold-changes relative to untreated cells (Ctrl). * p < 0.05, ** p < 0.01 (vs. Ctrl); # p < 0.05. (B) Effects of IFN-β addition on the decreased iNOS expression by Pano. HAPI cells were treated with LPS (100 ng/mL) for 6 h in the presence or absence of Pano (100 nM). Two hours after the addition of LPS, IFN-β (5000 units/mL) was added to the culture. After treatment, RT-qPCR was performed. Values (mean ± S.E., n = 3) are expressed relative to mRNA levels in LPS-treated cells. ** p < 0.01 (vs. LPS alone); ## p < 0.01 (vs. LPS plus Pano).
To determine why STAT1 activation by adding IFN-β did not fully recover the inhibition by Pano of LPS-induced iNOS expression, we investigated the contribution of other factors involved in LPS-induced iNOS expression. IRF1 is a target gene of STAT1 and participates in LPS-induced iNOS expression.18–20) LPS promoted IRF1 mRNA expression, and Pano suppressed LPS-induced IRF1 mRNA expression (Fig. 5A). The addition of IFN-β abolished the reduction in IRF1 mRNA expression caused by Pano (Fig. 5A), indicating that the inhibition of IFN-β/STAT signaling by Pano is responsible for this phenomenon. In addition to IRF1, octamer binding protein-2 (Oct-2) and Interferon regulatory factor 9 (IRF9) have also been shown to play important roles in LPS-induced iNOS expression.21–23) Therefore, we examined the involvement of Oct-2 and IRF9 in the inhibitory effects of Pano. LPS induced Oct-2 and IRF9 mRNAs in HAPI cells, and Pano prevented their induction. However, the addition of IFN-β failed to restore the decreased levels of Oct-2 and IRF9 mRNAs (Figs. 5B, C).
HAPI cells were treated with LPS (100 ng/mL) for 6 h in the presence or absence of Pano (100 nM). Two hours after the addition of LPS, IFN-β (5000 units/mL) was added to the culture. After treatment, RT-qPCR was performed. Values (mean ± S.E., n = 3) are expressed as fold changes relative to Ctrl. * p < 0.05, ** p < 0.01 (vs. Ctrl); # p < 0.05, ## p < 0.01 (vs. LPS alone); †† p < 0.01. NS, not significant.
It has previously been demonstrated that HDAC inhibitors protect against neuronal cell death associated with cerebral ischemia.24) For example, SAHA, a pan-HDAC inhibitor, has been shown to suppress ischemia/reperfusion brain injury by inhibiting microglia activation.25) In addition, the administration of butylate, another HDAC inhibitor, decreases the production of inflammatory cytokines caused by middle cerebral artery occlusion, leading to a reduction in the infarct volume.26) These findings suggest that the protective effects of HDAC inhibitors against brain injury following cerebral ischemia are related to their anti-inflammatory properties. Al Shoyaib et al. reported that delayed treatment with Pano does not facilitate motor recovery after ischemic stroke.27) The reason remains unclear. Since microglia are activated rapidly after ischemia and elicit inflammatory responses,28) the timing of Pano administration may need to be considered to exert its protective effects. However, how HDAC inhibitors elicit anti-ischemic effects is not fully understood. In this study, we found that Pano markedly suppressed LPS-induced iNOS expression in microglial HAPI cells; we then explored the underlying mechanism.
LPS-induced iNOS expression is regulated via various signaling pathways including NF-κB and MAPK, especially JNK and p38. HDAC inhibitors such as TSA and KBH-A42 have been shown to prevent LPS-induced iNOS expression.22,29) TSA suppresses LPS-induced JNK phosphorylation, although it does not affect the nuclear translocation of NF-κB. KBH-A42 has little or no effect on the activation of NF-κB and JNK, but it reduces p38 phosphorylation. Here, we demonstrated that Pano does not affect the LPS-induced activation of these pathways, indicating that the NF-κB and MAPK signaling pathways are unlikely to be involved in the inhibitory effects of Pano. On the other hand, Pano prevented LPS-induced STAT1 phosphorylation in HAPI cells. It has been shown that LPS cannot elicit iNOS induction in STAT1-deficient macrophages.18) Therefore, the IFN-β/STAT1 axis is considered to play a key role in LPS-induced iNOS expression. In the present study, we found that Pano suppressed LPS-induced IFN-β expression. The addition of IFN-β recovered the reduced STAT-1 phosphorylation by Pano. Therefore, the inhibition by Pano of LPS-induced IFN-β expression is likely involved in preventing the activation of STAT1.
HDACs have been shown to be closely associated with inflammatory responses.12,13) Chen et al.30) reported that the defect of HDAC3 in macrophages impairs the IFN-β/STAT1 axis and suppresses LPS-induced expression of IFN-β-dependent genes. In addition, the transcriptional activation of the IFN-β gene elicited by viral infection or LPS is suppressed by HDAC inhibitors, including TSA.31) However, the mechanism underlying this is not fully understood. LPS regulates IFN-β gene expression via the activation of IRF3.32) Scriptaid, an HDAC inhibitor, suppresses virus-induced IFN-β expression by blocking the phosphorylation and nuclear translocation of IRF3.33) In addition, the acetylation of IRF3 prevents its recruitment to the IFN-β promoter and reduces IFN-β expression induced by vesicular stomatitis virus.34) We found that Pano suppressed LPS-induced activation of the IFN-β promoter. As the IFN-β promoter region used in the current study included an IRF binding element, the decrease in IFN-β expression caused by Pano may be due to Pano-elicited functional alterations of IRF3 via phosphorylation or acetylation. Further studies are needed to elucidate the mechanism by which Pano suppresses IFN-β expression.
The IFN-β/STAT signaling pathway plays an important role in LPS-induced iNOS expression. Indeed, we found that Pano suppressed LPS-induced IFN-β expression and STAT1 phosphorylation in HAPI cells. However, STAT1 activation by adding IFN-β only slightly recovered the decreased iNOS expression by Pano. These results suggest that the IFN-β/STAT signaling pathway on its own may be insufficient for full expression of iNOS by LPS. Therefore, we examined whether other factors involved in iNOS induction contribute to the inhibitory effects of Pano. Oct-2 binds to the octamer binding site in the iNOS gene and directly regulates its expression. Oct-2 knockdown has been reported to prevent LPS-induced-iNOS expression.35) These findings indicate that Oct-2 is a key factor for LPS-induced iNOS expression. Lu et al.22) demonstrated that the HDAC inhibitor TSA decreases LPS-induced iNOS expression through the downregulation of Oct-2 in macrophages. Consistent with these observations, we found that Pano suppressed LPS-induced Oct-2 mRNA expression in HAPI cells. In addition to Oct-2, interferon-stimulated gene factors (ISGFs) have been shown to bind to an IFN-stimulated responsive element in the regulatory region of the iNOS gene and cooperate with NF-κB to regulate iNOS expression.21) IRF9 is associated with phosphorylated STAT1/STAT2 and forms a heterotrimer, termed ISGF3.36) Bacterial pathogens fail to cause iNOS induction in IRF9-deficient macrophages.21) These observations indicate that IRF9 plays a key role in IFN-β-dependent iNOS expression. In the present study, we found that Pano also decreased the expression of IRF9 mRNA. However, the addition of IFN-β failed to recover the decreased expression of Oct-2 and IRF9 mRNA. The expression of the Oct-2 and IRF9 genes is likely regulated independently of the IFN-β/STAT1 signaling pathway. The reason why the addition of IFN-β only slightly recovered the suppression of the iNOS gene may be due in part to the downregulation of Oct-2 and IRF9 by Pano. In addition, TSA increases histone H4 acetylation at the iNOS promoter and leads to reduction in interleukin (IL)-1β-induced iNOS expression.37) Therefore, Pano may also change the acetylation status of histone H4 in the promoter region of the iNOS gene, resulting in the decreased iNOS expression.
In conclusion, we have demonstrated that Pano suppresses the induction of iNOS in activated microglia. This effect of Pano is likely attributable to the inhibition of LPS-induced activation of IFN-β/STAT signaling and the decreased expression of Oct-2 and IRF9. It was recently reported that Pano penetrates the blood-brain barrier.38) Therefore, our findings support the possibility that Pano could be a potential therapeutic drug for neuronal injury following cerebral brain ischemia.
The authors declare no conflict of interest.
