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. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: J Neurochem. 2011 Jan 19;116(6):1077–1087. doi: 10.1111/j.1471-4159.2010.07163.x

p38 MAP kinase and PI3-kinase are involved in upregulation of mu opioid receptor transcription induced by cycloheximide

Do Kyung Kim *,†, Cheol Kyu Hwang *,§, Yadav Wagley *, Ping-Yee Law *, Li-Na Wei *, Horace H Loh *
PMCID: PMC3078638  NIHMSID: NIHMS260875  PMID: 21198637

Abstract

Despite several decades of efforts to develop safer, efficacious and non-addictive opioids for pain treatment, morphine remains the most valuable painkiller in contemporary medicine. Morphine and endogenous mu opioid peptides exert their pharmacological actions mainly through the mu opioid receptor (MOR). Analgesic effects of opioids in animals are dependent on the MOR expression levels, as demonstrated by studies of MOR-knockout mice (homo/heterozygotes) and MOR-less expressing mice. Surprisingly, in the course of our investigation to understand the mechanisms involved in the regulation of MOR gene expression, cycloheximide (CHX), a known protein synthesis inhibitor, markedly induced accumulation of MOR mRNAs in both MOR-negative and -positive cells. This induction was blocked by inhibitors of PI3-kinase and p38 MAP kinase, but not by a p42/44 MAP kinase inhibitor. In vitro, CHX was found to activate the MOR promoter and this activation was suppressed by inhibition of PI3-kinase. The transcriptional activator Sox18 was recruited to the MOR promoter in CHX-treated cells and this recruitment was also inhibited by the PI3-kinase and p38 MAP kinase inhibitors, Ly294002 and SB203580, respectively. Consistently, acetylation of histone H3 and induction of H3-K4 methylation were detected while reductions of HDAC2 binding and H3-K9 methylation were observed on the promoter. Furthermore, the MOR mRNA accumulation was almost completely inhibited in the presence of actinomycin D, indicating that this effect occurs mainly through activation of the transcriptional machinery. These observations suggest that CHX directly induces MOR gene transcription by recruiting the active transcription factor Sox18 to the MOR promoter through PI3- and/or p38 MAP kinase pathways.

Keywords: cycloheximide, morphine, opioid receptor, p38 MAP kinase, PI3-kinase, transcription

Introduction

Opioid analgesics have been widely used for severe acute pain and chronic cancer-related pain. The pharmacological and physiological effects of opioid drugs, including endogenous opioid peptides and the well-known analgesic drug morphine, are mediated through their binding to opioid receptors. Among the three major types of opioid receptors (mu, delta and kappa) several studies have suggested that the mu opioid receptor (MOR) plays a key role in mediating the major clinical effects of morphine, as well as the development of tolerance and physical dependence with chronic administration (Law et al. 2004). The analgesic effects of morphine were blocked or reduced in homozygote MOR–knockout (KO) mice (Loh et al. 1998, Sora et al. 1997) and heterozygote MOR–KO mice (Sora et al. 1997), respectively. Analgesia was also reduced in recombinant-inbred strain CXBK mice which have lower MOR expression levels (Ikeda et al. 2001), suggesting that the in vivo activities of morphine depend on the amount of the mu receptor present.

MOR activity is regulated at different levels, including epigenetic (Hwang et al. 2009), transcriptional (Law et al. 2004), posttranscriptional (Kim et al. 2008), translational (Song et al. 2007) and even at the protein level (El Kouhen et al. 2001). The MOR promoter contains many specific regulatory elements, including regions mediated by PU.1 (Hwang et al. 2004), IL-4 (Kraus et al. 2001), Sox (Hwang et al. 2003), Sp1 (Ko et al. 1998), PCBPs (Choi et al. 2008), and NRSE (Kim et al. 2006). Transcription of the MOR gene is regulated by morphine, IL-1, a bacterial endotoxin (LPS), non-opioid drugs (dopaminergic drugs, such as cocaine and haloperidol), HDAC inhibitors, and demethylating agents (Chang et al. 2001, Hwang et al. 2007). However, the exact molecular mechanisms of MOR gene regulation are still not fully understood.

In eukaryotes, it was originally shown that CHX exerts its effects by inhibiting the translocation step in protein synthesis thus blocking translational elongation. However, there is increasing evidence that the other protein synthesis inhibitors (PSI) such as anisomycin and puromycin paradoxically possess induction properties for numerous genes. This phenomenon, called superinduction, occurs through several distinct signaling pathways and has been demonstrated for immediate early-genes and various cytokine genes (Ogura et al. 2008, Lutter et al. 2000, Radulovic & Tronson 2008). CHX induces IL-6 gene expression in MDA-MB-231 and HeLa cells (Faggioli et al. 1997) and potentiates induction of IL-6 in IL-1β–treated entrocytes through activating NF-kappaB (Hershko et al. 2004). The latter study found that CHX treatment increased IL-6 mRNA and protein levels (despite partial inhibition of protein synthesis) through mRNA stabilization. CHX also suppressed IκBα resynthesis and prolonged p38 MAP kinase activation, which is associated with sustained NF-kappaB activation (Hershko et al. 2004). Similar observations of superinduction of IL-6 or other genes by CHX have been reported (Lutter et al. 2000, Newton et al. 1996). On the other hand, CHX (and its derivative Ac-CHX) blocked the TNF-α-induced activation of NF-kappaB in A549 human lung carcinoma cells by downregulating TNF receptor 1 via activation of ERK and p38 MAP kinase (Ogura et al. 2008). These findings suggest that CHX may exert its regulatory effects through distinct signaling pathways depending on the pre-treating inducer and cell type. The effect of PSI on learning and memory processes were also reported, showing impairment of auditory and contextual fear conditioning in C57BL/6N mice given CHX (Stiedl et al. 1999).

To our surprise, while investigating the mechanism of upregulation of the MOR gene, we found evidence that CHX stimulates MOR gene transcription in a dose/time-dependent manner in P19 cells. The current study was initiated to determine which of regulatory mechanisms were involved in the effect of CHX on the induction of MOR transcription.

Materials and methods

Materials

Actinomycin-D (Act-D), cycloheximide, leptomycin B (leptoB), okadaic acid (OA), PDTC, and sodium nitroprusside (SNP) were purchased from Sigma. Anisomycin, puromycin, PP2, QNZ, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), and SP600125 (SP) were purchased from EMD Biosciences. Ly294002, KT5720, PD98059, rapamycin, SB203580, and U0126 were purchased from Cell Signaling.

Cell culture and transfection

Cultures of P19 cells and the procedures to differentiate P19 cells (AP4d) have been described previously (Hwang et al. 2007). For drug treatment (Fig. 1A), P19 cells were plated to a density of 4×105 cells/well in 12-well culture plates 15 h before treatment. PDTC (100 µM), leptoB (10 ng/ml), Act-D (5 µg/ml), CHX (10 µg/ml), and SNP (100 µM) were treated to each well of P19 cells for 6 h. Cells were harvested for RNA isolation.

Fig. 1.

Fig. 1

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CHX treatment induced MOR gene expression in P19 cells. A. On left panel, the results of RT-PCR analyses of the MOR and KOR mRNA levels in P19 cells treated with different chemicals as indicated on top of gel. Significantly increased MOR band marked with asterisk. Arrowhead indicates a new isoform (named MOR-1z) produced by CHX treatment. On right panel, the effect of MOR induction by CHX was a time-dependent. Cells were treated with a 10 µg/ml concentration of CHX for 30 min to 14 hr as indicated. T½ (half time) is approximately 2 hr. B. The effect of different concentrations of CHX on the MOR and KOR genes. Cells were treated with the indicated concentrations of CHX for 6 hr. On right panel, MOR mRNA was also induced by CHX in neuroblastoma NMB cells. The MOR PCR primers were hMOR-S and hMOR-AS (Suppl. table 1) for NMB cells. The identities of the PCR products were confirmed by sequencing. Graphs under each gel indicate the averages from at least three representative experiments. Asterisk in graph indicates statistically significant findings (*: p < 0.01), relative to the untreated control. Error bars indicate the range of standard errors. β-actin was used as a control.

For CHX treatment in neuroblastoma NMB cells, cells were plated to a density of 1×106 cells/well in 6-well culture plates 15 h before CHX treatment (Fig. 1B). Transfections into NMB cells with promoter plasmids were performed as described previously (Hwang et al. 2004). MOR promoter plasmid (pL4.7k)(Ko et al. 1997) contains the full-length of MOR promoter. Cells were harvested 48 h after transfection and isolated total RNA for real-time qRT-PCR using LUC primers (Suppl. table 1). Each value was normalized by protein amount of cells.

Reverse transcription-PCR (RT-PCR) and real-time quantitative RT-PCR (qRT-PCR)

Total RNA was isolated using TRI Reagent and analyzed by RT-PCR using the MOR gene-specific primers mMOR_E3-S and mMOR_E4-AS (Hwang et al. 2007). RT-PCR was performed using a Qiagen OneStep RT-PCR kit. Similar reactions were carried out using primers for KOR (mKOR-S1 and mKOR-AS1, Suppl. table 1) and β-actin as an internal control (Hwang et al. 2007).

Real-time quantitative RT-PCR was performed as described previously (Hwang et al. 2010) using the same above MOR primer set and Quantitect SYBR Green PCR kit (Qiagen). The relative mRNA gene expression was analyzed as described previously (Pfaffl 2001, Hwang et al. 2007). The number of target molecules was normalized against that obtained for β-actin, used as an internal control. The specificity of qRT-PCR primers was determined using a melt curve after the amplification to show that only a single species of qPCR product resulted from the reaction. Single PCR products were also verified on an agarose gel. The RT-PCR and real-time qRT-PCR experiments were repeated at least three times to obtain statistical significance.

Chromatin immunoprecipitation (ChIP) assays and Western blot analyses

ChIP assays were performed as reported previously (Kim et al. 2004). The antibodies of acetylated histone H3 (AceH3; 06-599), Brm-related gene 1 (SNF2β/Brg1; 07-478), Lys4 dimethylated histone H3 (H3dmK4; 07-030), Lys9 trimethylated histone H3 (H3tmK9; 07-442) were purchased from Millipore and antibodies of histone deacetylase 2 (HDAC2; sc-9959), PCBP1 (sc-16504), Sox18 (sc-20100x), and Sp1 (sc-59x) were purchased from Santa Cruz. All ChIP assays were controlled by performing parallel experiments with no antibody, normal rabbit serum, and nonspecific Gal4 antibody (sc-577; Santa Cruz) pulldowns. Each immunoprecipitated DNA sample was analyzed by real-time qPCR using the indicated PCR primers in Fig. 5. ChIP assays were repeated at least three times. Western blot analyses were performed on protein samples prepared as described (Hwang et al. 2007). The antibodies were anti-phospho-GSK-3α/β (Ser21/9; 9331, Cell Signaling), anti-phospho-p38 MAPK (Thr180/Tyr182; 9215, Cell Signaling), anti-Sox18 (sc-20100x; Santa Cruz), and anti-β-actin (GTX109639; GeneTex).

Fig. 5.

Fig. 5

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Increased interaction of active transcription factors on the MOR promoter after CHX stimulation analyzed by chromatin immunoprecipitation (ChIP) using real-time qPCR. A. Enhanced recruitment of the active transcription factor Sox18 to the MOR promoter analyzed by ChIP real-time qPCR. P19 cells were treated with 1 or 10 µg/ml CHX for 6 hr (left graph) or with 10 µg/ml CHX for 6 hr (right graph) with either Ly294 (50 µM) or SB (25 µM). ChIP-PCR primers for distal promoter (DP, P1) were S-731 and AS-623 (Hwang et al. 2007) and primers for proximal promoter (PP, P2) were S-408 and AS-285 (Hwang et al. 2009). Asterisks indicate statistically significant findings (*: p < 0.05, relative to the no-treated control and **: p < 0.05, relative to the CHX-treated sample. Bottom gel, western blot analysis of Sox18 expression in CHX-stimulated cells with different time treatments. B. CHX also triggers epigenetic changes on the MOR promoter. P19 cells were treated with CHX for 6 hr, followed by ChIP assays. The interaction changes of various factors on both promoter regions (DP and PP) were determined by ChIP assay. Antibody Gal4 was used as a negative control. The graphs were representative of three independent experiments. Bottom panel, a proposed molecular mechanism for MOR gene stimulation by CHX through epigenetic changes and transcription factors.

Results

Induction of the MOR gene by cycloheximide

To find a specific compound that upregulated the MOR gene at the transcriptional level, MOR negative cells (P19) were treated with several chemicals (Fig. 1A). Treatment with cycloheximide (CHX) increased the MOR mRNA about 8 times (asterisk, lane 5) compared to untreated cells (lane 1). Treatment with leptoB (a specific nuclear export inhibitor), PDTC (an inhibitor of NF-κB activation), Act-D (a transcription inhibitor), and SNP (a nitric oxide donor) had slight effects on MOR transcription, (see graph below the gel image) but these were not statistically significant. Transcription of the KOR gene, another member of the opioid receptors used as a negative control, was decreased in PDTC- and Act-D-treated samples. The upper band (arrowhead) of MOR in lane 5 was identified as a new isoform (named MOR-1z) by DNA sequencing (details in Fig. 2) and this transcript encoded a different and shorter C-terminal sequence compared to full-length MOR. Fig. 1A on the right gel image shows that CHX increased MOR mRNA in a time-dependent manner, but at the longest treatment time (14 hr, lane 7) decreased MOR mRNA was observed, possibly due to cell death. The increased accumulation of MOR mRNA could be detected at 1 hr (1.9 +/− 0.3-fold) and the maximal accumulation occurred at 6 hr (8.2 +/− 0.5-fold, p < 0.05). In Fig. 1B, MOR mRNA was gradually elevated when the concentration of CHX was increased from 0.1 µg/ml to 50 µg/ml, indicating that MOR was increased by CHX in a dose-dependent manner while both KOR and β–actin were not changed.

Fig. 2.

Fig. 2

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Effects of a PI3-kinase inhibitor and serum on MOR expression. A. Effect of several inhibitors on the CHX stimulation of MOR expression. P19 cells were pretreated with 100 µM SNP, 10 nM rapamycin (rapam), 50 µM Ly294002 (Ly294), 20 µM PD98059 (PD), 5 µM PP2 for 1 hr before 4hr CHX treatment. For “serum-free” samples, cells were incubated with serum-free media for 30 min prior to CHX stimulation for 3 hr. The asterisk indicates significant inhibition or increased induction of CHX stimulation by Ly294 (*) and serum-free (**), respectively. On bottom left gel, the inhibitory effect of different concentrations of Ly294 on CHX-stimulated MOR gene analyzed by RT-PCR. Cells were pretreated with either Act-D (5 µg/ml) or the indicated concentrations of Ly294 before 4hr CHX treatment. On bottom right gel, effect of reduced serum on MOR expression. Cells were incubated for 4 hr in media containing different percentages of serum as indicated. Cells treated with CHX in serum-free media are used as a positive control. B. Western blot analysis of phospho-GSK-3α/β (Ser21/9) and phospho-p38 MAPK (Thr180/Tyr182) expression in CHX-stimulated cells with different time treatments. Anti-β-actin was used as a control. C. Identification of a novel MOR isoform induced by CHX. Numbers on the left for DNA and amino acids sequences are based on the start codon designated at +1. The gray box in bold indicates a newly found exon (exon Z) encoding a new isoform and an extra 119 bases between exons 3 and 4. Since the coding frame of the isoform contains a TGA stop codon (in box) before proceeding to the original stop codon (TAA) in exon 4, the isoform encode a shorter polypeptides as drawn in the figure underlined.

MOR transcription induction by CHX was also observed in MOR-positive cells (neuroblastoma NMB) as shown in Fig. 1B. However, in the NMB cells MOR induction began at 10 µg/ml CHX, which is 100-fold higher than observed in P19 cells (from 0.1 µg/ml CHX). Therefore, we used P19 cells for further experiments because the use of lower CHX concentrations may avoid the interference of protein synthesis inhibition.

CHX induces MOR expression via activation of PI3-kinase

To determine which members of downstream signaling pathways are essential for the biological activities of CHX, several compounds were employed as blocking agents. Inhibitory activities of the nitric oxide donor SNP, serum-free media (for abolishing the steady-state translation), the mTOR inhibitor rapamycin (translational inhibition involved in the mTOR-mediated translational pathway), the PI3-kinase inhibitor Ly294002, the p42/44 MAP kinase inhibitor PD98059, and the Src-family tyrosine kinase inhibitor PP2, were investigated using cells in the absence or presence of CHX by RT-PCR (Fig. 2A). Only the pretreatment with Ly294 could significantly block the CHX-mediated stimulation of MOR (marked *). The inhibition of translation initiation caused by serum-free media and rapamycin did not prevent MOR stimulation. Rather cell starvation by serum-free media enhanced the CHX-mediated stimulation of MOR (marked **). These results suggest that the translational control pathway mediated by mTOR and associated by serum starvation were not involved in CHX stimulation of MOR, while the PI3-kinase-associated pathway was directly associated with the stimulation. To test whether the stimulation occurs at the transcriptional level, cells were pretreated with the transcription inhibitor Act-D prior to CHX treatment (Fig. 2A bottom left gel). Act-D drastically abolished the stimulation (marked *), indicating that the CHX stimulation occurs at the transcriptional level. In addition, pretreatments with different concentrations of Ly294 (2 µM to 100 µM) were included and showed that the inhibition of CHX stimulation began at 10 µM concentration. Since serum starvation enhanced the CHX stimulation, we tested the direct effect of serum by itself using 0 to 3 % serum media (Fig. 2A bottom right gel). The results showed no change of MOR mRNA with exposure to serum-free or reduced serum media.

Glycogen synthase kinase 3 (GSK-3) is a critical downstream element of the PI3-kinase pathway, and its activity can be inhibited by phosphorylation of GSK-3α at Ser21 and GSK-3β at Ser9. Results of Fig. 2B showed that phosphorylation of GSK-3β at Ser9 increased with 15 min and 30 min CHX treatments and was decreased at both 1 and 2 hrs followed by a slight increase at 4 hr and then decreased at 6 hr. Phosphorylation of GSK-3α at Ser21 had a slight but insignificant increase. Since p38 MAPK inhibitor (SB) treatment also blocked CHX stimulation, phosphorylation of p38 MAPK was tested. The phosphorylation (p38 MAPK activation) in response to CHX treatment began at 15 min and reached the maximum at 30 min, followed by decreased phosphorylation after 30 min. These indicate that the cytosolic events (phosphorylations of GSK-3 and p38 MAPK) induced by CHX occur earlier than CHX-mediated MOR induction in nucleus.

As mentioned earlier, an upper band (arrowhead in Fig. 1A) of MOR PCR products appeared in CHX-treated samples. Since the band size was bigger than expected, the band was isolated from agarose gel and sequenced. As shown in Fig. 2C, the shaded DNA sequence (119 bp, position from 1159 to 1277 in between known exons 3 and 4) was identified as a new exon and encoded a new isoform (named MOR-1z) with shorter C-terminal sequence and may function differently compared to MOR.

p38 MAP kinase and NF-kappaB were involved in CHX stimulation

In Fig. 3A, pretreatment with the p38 MAP kinase inhibitor SB blocked the CHX stimulation of MOR, whereas the MEK1/2 kinase inhibitor U0126 did not. The p42/44 MAP kinase inhibitor PD and PKA inhibitor KT were not capable of inhibiting stimulation. NF-kappaB inhibitors (PDTC and QNZ) and protein phosphatases 1/2A inhibitor OA could block the CHX stimulation of MOR transcription. Without CHX co-treatment (bottom gel of Fig. 3A), all compounds had no effect by themselves except that the c-Jun NH(2)-terminal kinase (JNK) phosphorylation inhibitor SP had its own effect on MOR induction. These results suggested that CHX may function to positively regulate MOR transcription by activating the p38 MAP kinase and NF-kappaB pathways; however pathways involving protein phosphatases 1/2A were not involved with this upregulation.

Fig. 3.

Fig. 3

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Effects of various inhibitors on CHX stimulation. A. P19 cells were pretreated with the indicated inhibitors for 1 hr before being treated with or without CHX (10 µg/ml) for 6 hr. Treated concentrations: 50 µM Ly294, 25 µM SB203580 (SB), 20 µM PD, 10 µM U0126, 25 µM SP600125 (SP), 2 µM KT5720 (KT), 100 µM PDTC, 10 nM QNZ, and 10 nM okadaic acid (OA). Below gel shows pretreated samples without CHX as a negative control. B. Effects of other protein synthesis inhibitors on MOR expression. Cells were treated separately with 50 µg/ml puromycin (puro) and 10 µM anisomycin (aniso) as well as 10 µg/ml CHX for 6 hr in indicated % serum media. The asterisk indicates a statistically significant difference (**, p < 0.05) relative to the CHX-treated sample in 10 % serum media (10 % Se). C. Differential effects of various inhibitors on MOR stimulations by puromycin and anisomycin compared to CHX-mediated stimulation. Right gel, effects of Ly294 and SB treatments on endogenous MOR gene in MOR-positive NMB cells. D. Pretreatment with PI3-kinase inhibitor Ly294 shows the inhibition of activation (phosphorylation) of the PI3-K downstream factor GSK-3α/β in either 10% serum or serum-free media analyzed by western blot. Arrowhead indicates distinct phosphorylation of GSK-3α (upper band) by CHX in serum-free media compared to the GSK-3α in CHX treated cells in 10% serum media (left gel). Anti-β-actin was used as a control. Bottom gel, RT-PCR showed the integrity of the above western blot experiment and comparison of MOR induction by CHX in 10% serum and serum-free media.

Different protein synthesis inhibitors act similarly to induce MOR but depending on serum percentages in media

There are several protein synthesis inhibitors (PSI) for eukaryotes with different modes of action, but similar abilities to inhibit de novo protein synthesis. When these PSIs possess gene regulation function, they often do so through different signaling pathways. To determine the mode of action of MOR stimulation by these inhibitors, we used puromycin, anisomycin, and CHX in media containing either 10 % or 1 % serum (Fig. 3B). The reduced serum (1%) condition was used to give an additional stress element to cells since we observed enhanced CHX stimulation in serum-free media in Fig. 2A. Incubation of P19 cells with puromycin and anisomycin increased MOR transcription even more than cycloheximide in 10 % serum media. But the anisomycin-enhanced effect was decreased (marked *) in 1 % media compared to 10 % serum media, indicating that anisomycin-mediated stimulation requires normal serum media for its maximal effect. On the other hand, CHX-mediated stimulation requires reduced media for its maximal effect (marked **) and puromycin treatment shows a similar stimulation effect between the two serum media concentrations. Thus, each drug may function differently to stimulate MOR transcription depending on the presence of cellular stress. To further profile these differences, these three PSI-mediated stimulations were tested using several inhibitors (Fig. 3C). Stimulation of MOR transcription by the three PSIs was blocked by co-treatment with Ly294, as well as co-treatment with Act-D. In SB co-treatment, anisomycin-mediated stimulation was decreased even more than CHX treatment with SB, while SB had no effect on puromycin treatment. Co-treatment with MEK1/2 kinase inhibitor U0126 led to a decrease in the effect of anisomycin but had no effect on both puromycin and CHX treatments. In MOR-positive NMB cells, the basal MOR level was also reduced by treatments of Ly294 and SB (Fig. 3C right gel).

In Fig. 3D, a downstream regulator of PI3-K pathway GSK-3α/β was activated (phosphorylated) by CHX in 20 min treatment and the activation was blocked by Ly294. GSK-3β at Ser9 was phosphorylated by CHX treatment in 10% serum media, while both GSK-3α/β at Ser21/9 were phosphorylated by CHX in serum-free media. Thus, phosphorylation of GSK-3α at Ser21 seems to be involved in enhancement of CHX-mediated stimulation for the MOR gene in serum-free media shown in Fig. 2A (**) and Fig. 3B/D (**) compared to CHX treatment in 10 % media. The bottom gel image is RT-PCR for MOR showing the integrity of experiments.

CHX treatment in P19 cells did not induce any cellular differentiation

Since the MOR gene is highly induced by CHX treatment, we decided to determine whether the drug itself also causes neuronal or other cellular differentiation in P19 cells. mRNA (Suppl. Fig. 1B) levels of glial fibrillary acidic protein (GFAP) were not changed in the CHX-treated cells analyzed by real-time qRT-PCR, indicating no astrocyte differentiation in P19 cells treated with the drug. Other cell type-specific markers, microtubule-associated protein 2 (Mtap2; neuronal cells) and integrin-α M (Itgam; microglia) were increased 2.8- and 2.1-fold, respectively, in the treated cells. However this magnitude of increase is not significant when compared to the 40~134-fold increases seen in adult mouse brain. Induction of MOR by CHX was also confirmed by real-time qRT-PCR (Suppl. Fig. 1A). Surprisingly, we found that CHX treatment led also to enhanced Oct4 and Nanog mRNA levels and the increased Nanog was blocked by Ly294 co-treatment but Oct4 was not (Fig. 4A). This may suggest that these two embryonic stem (ES) cell markers are induced upon CHX stimulation through different signaling pathways and P19 cells maintain their embryonic status with CHX treatment. Neuronal differentiated P19 (AP4d) and mouse brain were included as negative controls for the two genes.

Fig. 4.

Fig. 4

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Effects of CHX on embryonic stem cell markers and MOR promoter analyzed by real-time quantitative RT-PCR (qRT-PCR). A. Expression of the stem cell marker genes in P19 cells with CHX analyzed by real-time qRT-PCR. CHX (10 µg/ml) was added with or without Ly294 (50 µM) to P19 cells. Neuronally differentiated P19 cells (AP4d) (Hwang et al. 2007) and mouse brain were included as controls. PCR primers for Oct4 and Nanog were described in Suppl. table 1. B. Transcriptional effect of CHX on the MOR promoter in neuroblastoma NMB cells. MOR positive NMB cells were transfected with 1 µg of a MOR promoter plasmid (pL4.7k). Cells were harvested 48 h after transfection and total RNA was isolated for real-time qRT-PCR. “no RT” indicates real-time qRT-PCR reaction without reverse transcription. CHX (10 µg/ml) was added with or without Ly294 (10 µM) in the cells. Graphs indicate the averages from at least three representative experiments. Asterisks indicate statistically significant findings (*: p < 0.05, relative to the no-treated control and **: p < 0.05, relative to the CHX-treated sample). Error bars indicate the range of standard errors.

Treatment of P19 cells with CHX results in upregulation of the MOR promoter

As shown in Fig. 2, induction of MOR expression in CHX-treated P19 cells was alleviated by treatment with the PI3-kinase inhibitor Ly294 and the transcription inhibitor Act-D. Thus, it was important to determine whether the transcriptional level of the MOR gene promoter was changed in P19 cells treated with CHX. Real-time qRT-PCR analyses revealed that the mRNA of MOR promoter (pL4.7k) was increased in the presence of CHX (Fig. 4B) and this increase was blocked by Ly294 treatment. This correlates with the MOR mRNA regulation observed in P19 cells by treatment with CHX, Ly294, and Act-D (Fig. 2). These results indicate that CHX stimulation acts through the MOR promoter at the transcriptional level.

CHX triggers epigenetic changes and led to recruit active transcription factor Sox18 to the MOR promoter

We performed ChIP assays assisted by real-time qPCR analyses using antibodies against some known active transcription factors (Sox18, Sp1, and PCBP1) for the MOR gene (Fig. 5). These three transcription factors are known to up-regulate the MOR promoter (Hwang et al. 2003, Ko et al. 1998, Choi et al. 2008). ChIP analysis of distal promoter (DP) sequences (covered by P1 primers) showed an increased interaction (about 6.2-fold in 10 µg CHX concentration) with Sox18 in CHX-treated cells in a dose dependent manner (left graph of Fig. 5A) compared to non-treated cells. This interaction was reduced by about 35 % and 43 % in Ly294/CHX and SB/CHX co-treated cells (right graph), respectively, compared to CHX-treated cells. A region (drawn Sox18 in oval, Fig. 5B) of the DP is known to be bound by Sox proteins including Sox18 (Hwang et al. 2003). However, ChIP assay using P2 primers covering the proximal MOR promoter (PP) showed no increased binding of Sox18 and rather showed less Sox18 interaction in CHX-treated cells compared to the basal binding level of non-treated cells. This indicated that Sox18 bound exclusively to the Sox-binding site of DP MOR promoter. In the bottom gel image of Fig. 5A, the amount of Sox18 protein in CHX treatment at later time points was slightly decreasing, but not significantly relative to β–actin. This suggested that the increased interaction of Sox18 by CHX was not due to changes in the amount of Sox18 protein. ChIP assay showed binding of Sp1 and PCBP1 to their binding sites on the PP were also increased (Fig. 5B) but the increases (1.5- to 3-fold ranges in 1 µg/ml and 10 µg/ml CHX) were lower than Sox18 (4- to 6.2-fold). Histone modifications were also investigated for the two promoter (DP and PP) regions by ChIP assays (Fig. 5B). These results showed that histone H3 was hyperacetylated on the DP region in a dose-dependent manner in CHX-treated cells while it was not hyperacetylated on the PP region. Trimethylation of H3 at lysine 9 (H3tmK9, as a hallmark of inactive transcription) for the DP region was decreased by 30–40 % in both doses of CHX (1 and 10 µg/ml) but the trimethylation was not changed on PP region in CHX-treated cells. For dimethylation of H3 at lysine 4 (H3dmK4, as a hallmark of active transcription), in both DP and PP regions the dimethylation was increased in a lower dose of CHX (1 µg/ml) with higher levels in the PP than in the DP. The dimethylations were decreased in cells treated with 10 µg/ml CHX but still higher than that of non-treated. Concurrently, interaction of HDAC2 (as a hallmark of inactive transcription) with the two regions was decreased in CHX-treated cells. The chromatin-remodeling factor Brg1 is known to interact with active MOR promoter in both in vivo and in vitro (Hwang et al. 2009, Hwang et al. 2010) but in CHX-treated cells the interaction of Brg1 was not changed. This suggests that CHX-mediated transcription may exclude the involvement of the chromatin-remodeling factor for MOR induction. These results (see also the schematic drawing in Fig. 5B) suggested that CHX triggered epigenetic changes (acetylated H3, H3dmK4, H3tmK9, and inhibition of HDAC binding) and recruited active transcription factors to the promoter resulting in active transcription of MOR gene via the PI3-K and p38 MAP kinase pathways.

Discussion

During our search to find any compounds that upregulate the MOR gene at the transcriptional level, treatment with an unexpected compound (CHX) was shown to increase the MOR mRNA over 8-fold compared to non-treated cells. We found that CHX increased MOR gene transcription by recruiting the active transcription factor Sox18 to the MOR promoter through the PI3- and/or p38 MAP kinase pathways. Transcription factor recruitment was enhanced by and associated with opening or modifying the chromatin structure of the MOR promoter region triggered by the CHX treatment.

Similar MOR stimulatory effects were also observed with anisomycin and puromycin, other protein synthesis inhibitors (PSI) (Fig. 3). In several studies, PSIs have been suggested to induce various genes in many biological activities, such as memory impairments (Radulovic & Tronson 2008), cytokine productions (Hershko et al. 2004), selective depletion of macrophages through apoptosis (Croons et al. 2009), superinduction of apolipoprotein II mRNA without affecting apoII mRNA stability (Sensel et al. 1994), and induction of RNA stability (Ichikawa et al. 2003). A recent study (Ravni et al. 2006) reported a microarray analysis using a hormone (PACAP) with CHX co-treatment in PC12 cells and showed most of the several categories of genes or mRNAs identified were even more induced compared to hormone treatment alone. Nevertheless, the report indicated that CHX could contribute globally to superinduction of numerous genes that are either dependent or not dependent on protein synthesis. This paradoxical increase of various genes by PSIs is a process known as superinduction. Superinduction of genes by PSIs occurs in either the presence or absence of co-stimulants, is cell-specific and the mechanisms involved are not fully understood, but are usually attributed to decreased mRNA degradation. However, there is increasing evidence that activation of signaling cascades and increased transcriptional activation may also be involved. Independent of its ability to block translation, CHX intrinsically initiates intracellular signals and immediate-early gene induction.

In many cases, CHX superinduces immediate early genes (IEGs) by increasing mRNA stability of IEGs which have a short half-life (Herdegen & Leah 1998). CHX superinduces c-jun and junB, IEG components of the AP-1 transcription factor, which degrade rapidly initiated by an AU-rich element and AUUUA motifs, respectively (Chen & Shyu 1994, Mechta et al. 1989). Moreover, one report showed that CHX and anisomycin at low concentrations that do not affect protein synthesis induced the c-fos and c-jun expression via phosphorylation of two chromatin-associated proteins (Edwards & Mahadevan 1992). Similarly at a low dose of CHX (0.01–1 µg/ml) that does not inhibit protein synthesis, the xanthine oxidoreductase (XOR) gene was induced in epithelial cells and that expression was blocked by inhibitors of p38 MAP kinase (Seymour et al. 2006). The low dose of CHX stimulated phospho-p38 MAP kinase and nuclear accumulation of the C/EBPβ transcription factor. C/EBPβ activated the XOR promoter and this activation was further induced by the low dose of CHX. In our unpublished results, MOR has longer mRNA half-life (about 9 hrs) compared to the above IEGs (usually less than 1 hour). In the 3’-UTR sequence of MOR gene, there is no obvious sequences of AU-rich and AUUUA motifs which were present in IEGs. Additionally, based on results of Act-D co-treatments (Fig. 2A) and promoter assay (Fig. 4B), MOR stimulation by CHX was not due to increasing mRNA stability rather it was increased by CHX at the transcription level.

Both Oct4 and Nanog are known to regulate pluripotency in mouse embryonic stem (ES) cells. The P19 embryonal carcinoma (EC) cells have gene expression profiles similar to ES cells (You et al. 2009) and the presences of the Oct4 and Nanog transcription factors are essential for mammalian embryogenesis and pluripotency of the P19 EC cells (Urano et al. 2006). The reduction of Nanog from CHX-mediated stimulation by a PI3-K inhibitor (Fig. 4A) is consistent with a previous report that the role of PI3-K in regulation of murine ES cell self-renewal is regulated by leukemia inhibitory factor-independent mechanisms including Nanog signaling (Paling et al. 2004). Previous studies have shown that inhibition of PI3-K activity resulted in decreased expression of Nanog mRNA and protein (Storm et al. 2007). PI3-Ks regulate Nanog expression by inhibiting GSK-3 activity. The regulatory effects of PI3-K inhibition on Nanog were reversed effectively by blocking GSK-3 activity and subsequently self-renewal was restored (Storm et al. 2007). In Fig. 4A, induction of Oct4 by CHX was not changed by the PI3-K inhibitor Ly294. Previous studies in human ES cells have shown that inhibition of PI3-K resulted in decreased expression of mRNA for Oct4 (Lee et al. 2009), suggesting that expression of Oct4 may be regulated by PI3-K differently in the presence or absence of stimulants like CHX.

As mentioned above, both CHX and anisomycin selectively depleted macrophages that play a major role in atherosclerotic plaque destabilization. Selective removal of these macrophages is a promising therapeutic approach to stabilize plaques. The selective depletion of macrophages by these drugs was blocked by the p38 MAP kinase inhibitor SB202190 without affecting smooth muscle cells viability (Croons et al. 2009). SB202190 decreased anisomycin-induced p38 MAP kinase phosphorylation, did not alter JNK phosphorylation, and increased ERK1/2 phosphorylation. These findings indicate that anisomycin, as well as CHX, selectively decreased the macrophage content in rabbit atherosclerotic plaques through p38 MAP kinase, but not ERK1/2 or JNK (Croons et al. 2009). As shown in Fig. 3, CHX-mediated MOR stimulation was blocked by the p38 MAP kinase inhibitor SB203580, much like the depletion of macrophages was blocked, although Croon's study and ours used different cell-types. CHX stimulation (Fig. 3) was not affected by a MEK1/2 inhibitor (U0126), or a JNK inhibitor (SP600125, rather it has its own stimulatory effect on MOR), indicating that the distinct mechanism of MOR stimulation by CHX is associated exclusively with p38 MAP kinase but not with MEK1/2 kinase, and JNK.

Alpha 1-adrenergic receptors were also induced by CHX (Hu & Hoffman 1993). The alpha 1-receptors, along with MOR, belong to the G protein-coupled receptor (GPCR) superfamily. Alpha 1 receptors play important roles in mediating a wide range of important cellular responses and the regulation of these receptors’ expression may have pathophysiological significance in diseases such as hypertension. Treatment of smooth muscle cells with CHX led to a marked concentration- and time-dependent accumulation of the alpha 1B receptor mRNA. This induction was due to an increase of the transcription rate and not due to a change of the alpha 1B receptor mRNA stability. Two additional inhibitors, anisomycin and emetine, had similar effects to those of CHX, while puromycin did not induce alpha 1B receptor mRNAs. The report suggested that CHX induces alpha 1B receptor gene expression through direct activation of gene transcription rather than inhibition of protein synthesis (Hu & Hoffman 1993). On the other hand, anisomycin and CHX, but not puromycin and emetine, acutely increased the mRNA of the beta 1 receptor in the pineal gland, but this increase was not transcriptionally mediated (Carter 1993). The mechanism underlying this mode of regulation was associated with control of the cellular immediate early gene c-fos. These two studies suggest that the physiological and in vitro changes in mRNA expression of alpha 1B and beta 1 receptors may provide further insight into the complexity of the molecular regulation of GPCRs. For the MOR gene, the three compounds, CHX, anisomycin, and puromycin, they have similar effects on MOR stimulation (Fig. 3). However, each showed somewhat different stimulation rates depending on the serum contents in media and different downstream elements involvement, such as p38-MAPK (SB) and MEK1/2 kinase (U0126). Therefore, the gene of each member of the GPCR family may have a distinct response to those three compounds and each may have a different mechanism of regulation.

In immune cells, PI3-K-mediated pathways were recently suggested to be involved in the induction of MOR gene transcription (Liu et al. 2010). These data showed that morphine treatment enhanced the level of phosphorylated PI3-K. The enhanced phosphorylation by morphine was abolished by the PI3-K inhibitor LY294002, implying specific involvement of PI3-K. Although we tested with a different cell-type, CHX-mediated MOR stimulation may share the PI3-K pathways with MOR gene regulation in response to morphine treatment. On the other hand, one report suggested that in neuroblastoma N2A cells the rate of MOR degradation stimulated by a MOR agonist (DAMGO) was decreased by CHX through the inhibition of protein synthesis (Afify 2002). Based on our results, it is worth to noting that CHX itself has MOR induction effect, presumably resupplying the receptor in case of receptor reduction. These contradicting phenomena have not been further confirmed yet in either study.

We demonstrate that MOR transcription was stimulated by CHX and the stimulation was blocked by inhibitors of PI3-K and p38 MAP kinase. We speculate that CHX treatment lead to chromatin modifications that enhanced the interaction of Sox18 with the MOR promoter and ultimately upregulated MOR transcription. These results may help provide new insight into how to turn on the MOR transcription switch with a specific compound; a phenomenon that is rarely observed other than with known inducers, such as chromatin modifiers.

Supplementary Material

Supp Fig S1 & Table S1

Acknowledgements

This work was supported by the National Institutes of Health [Grants DA000564, DA001583, DA011806, K05-DA070554 (HHL), DA011190, DA013926 (LW)], and by the A&F Stark Fund of the Minnesota Medical Foundation. We thank Mr. Bradley J. Stish for editorial assistance with the manuscript.

Abbreviations used

MOR

mu opioid receptor

KOR

kappa opioid receptor

DP

distal promoter

PP

proximal promoter

QNZ

6-amino-4-(4-phenoxyphenylethylamino) quinazoline

PDTC

pyrrolidine dithiocarbamate

MAP kinase

mitogen-activated protein kinase

NF-kappaB

nuclear factor-kappaB

mTOR

mammalian target of rapamycin

MEK

Mitogen-activated protein/extracellular signal-regulated kinase

PI3-K

phosphoinositide 3-kinase

CHX

cycloheximide

Ac-CHX

acetoxycycloheximide

Footnotes

The authors declare no conflict of interest.

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Supplementary Materials

Supp Fig S1 & Table S1
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