p38 Mitogen-activated protein kinase and PI3-kinase are involved in up-regulation of mu opioid receptor transcription induced by cycloheximide

p38 Mitogen-activated protein kinase and PI3-kinase are involved in up-regulation of mu opioid receptor transcription induced by cycloheximide
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  , , *  Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN, USA   Department of Oral Physiology, Chosun University School of Dentistry, Gwangju, South Korea Opioid analgesics have been widely used for severe acute pain and chronic cancer-related pain. The pharmacologicaland physiological effects of opioid drugs, including endog-enous opioid peptides and the well-known analgesic drugmorphine, are mediated through their binding to opioidreceptors. Among the three major types of opioid receptors(mu, delta, and kappa) several studies have suggested that themu opioid receptor (MOR) plays a key role in mediating themajor clinical effects of morphine, as well as the development of tolerance and physical dependence with chronic adminis-tration (Law  et al.  2004). The analgesic effects of morphinewere blocked or reduced in homozygote MOR–knockout (KO) mice (Sora  et al.  1997; Loh  et al.  1998) and heterozy-gote MOR–KO mice (Sora  et al.  1997), respectively. Anal-gesia was also reduced in recombinant-inbred strain CXBK  Received July 27, 2010; revised manuscript received November 10,2010; accepted December 17, 2010.§Address correspondence and reprint requests to Dr Cheol KyuHwang, Department of Pharmacology, University of Minnesota, 6-120Jackson Hall, 321 Church St. S.E., Minneapolis, MN 55455, USA.E-mail:  Abbreviations used  : Act-D, actinomycin-D; Brg1, Brm-related gene 1;ChIP, chromatin immunoprecipitation; CHX, cycloheximide; DP, distal promoter; ERK, extracellular-regulated kinase; ES, embryonic stem;GPCR, G protein-coupled receptor; GSK-3, glycogen synthase kinase 3;HDAC 2, histone deacetylase 2; IEGs, immediate early genes; JNK, c-Jun NH(2)-terminal kinase; KO, knockout; KOR, kappa opioid receptor;MEK, mitogen-activated protein/extracellular signal-regulated kinase;MOR, mu opioid receptor; mTOR, mammalian target of rapamycin; NF- j B, nuclear factor-kappaB; OA, okadaic acid; PDTC, pyrrolidinedithiocarbamate; PI3-K, phosphoinositide 3-kinase; PP, proximal pro-moter; PSI, protein synthesis inhibitors; qRT-PCR, quantitative RT-PCR;SNP, sodium nitroprusside. Abstract Despite several decades of efforts to develop safer, effica-cious, and non-addictive opioids for pain treatment, morphineremains the most valuable painkiller in contemporary medi-cine. Morphine and endogenous mu opioid peptides exerttheir pharmacological actions mainly through the mu opioidreceptor (MOR). Analgesic effects of opioids in animals aredependent on the MOR expression levels, as demonstratedby studies of MOR-knockout mice (homo/heterozygotes) andMOR-less expressing mice. Surprisingly, in the course of ourinvestigation to understand the mechanisms involved in theregulation of MOR gene expression, cycloheximide (CHX), aknown protein synthesis inhibitor, markedly induced accu-mulation of MOR mRNAs in both MOR-negative and -positivecells. This induction was blocked by inhibitors of phospho-inositide 3-kinase (PI3-K) and p38 MAPK, but not by a p42/44MAPK inhibitor.  In vitro  , CHX was found to activate the MORpromoter and this activation was suppressed by inhibition ofPI3-K. The transcriptional activator Sox18 was recruited to theMOR promoter in CHX-treated cells and this recruitment wasalso inhibited by the PI3-K and p38 MAPK inhibitors,Ly294002 and SB203580, respectively. Consistently, acety-lation of histone H3 and induction of H3-K4 methylation weredetected while reductions of histone deacetylase 2 bindingand H3-K9 methylation were observed on the promoter. Fur-thermore, the MOR mRNA accumulation was almost com-pletely inhibited in the presence of actinomycin-D, indicatingthat this effect occurs mainly through activation of the tran-scriptional machinery. These observations suggest that CHXdirectly induces MOR gene transcription by recruiting the ac-tive transcription factor Sox18 to the MOR promoter throughPI3- and/or p38 MAPK pathways. Keywords:  cycloheximide, morphine, opioid receptor, p38mitogen-activated protein kinase, phosphoinositide 3-kinase,transcription. J. Neurochem  . (2011)  116 , 1077–1087. JOURNAL OF NEUROCHEMISTRY   | 2011 | 116 | 1077–1087 doi: 10.1111/j.1471-4159.2010.07163.x   2011 The AuthorsJournal of Neurochemistry    2011 International Society for Neurochemistry,  J. Neurochem.  (2011)  116 , 1077–1087  1077  mice which have lower MOR expression levels (Ikeda  et al. 2001), suggesting that the  in vivo  activities of morphinedepend on the amount of the mu receptor present.MOR activity is regulated at different levels, includingepigenetic (Hwang  et al.  2009), transcriptional (Law  et al. 2004), post-transcriptional (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 specificregulatory 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 neuron-restrictive silencer element (NRSE) (Kim  et al. 2006). Transcription of the MOR gene is regulated bymorphine, IL-1, a bacterial endotoxin (lipopolysaccharide),non-opioid drugs (dopaminergic drugs, such as cocaine andhaloperidol), histone deacetylase (HDAC) inhibitors, anddemethylating 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 srcinally shown that cycloheximide(CHX) exerts its effects by inhibiting the translocation step in protein synthesis thus blocking translational elongation.However, there is increasing evidence that the other proteinsynthesisinhibitors(PSI), suchasanisomycinandpuromycin, paradoxically possess induction properties for numerousgenes. This phenomenon, called superinduction, occursthrough several distinct signaling pathways and has beendemonstrated for immediate early genes (IEGs) and variouscytokine genes (Lutter   et al.  2000; Ogura  et al.  2008;Radulovic and Tronson 2008). CHX induces IL-6 geneexpression in MDA-MB-231 and HeLa cells (Faggioli  et al. 1997) and potentiates induction of IL-6 in IL-1 b -treatedentrocytes through activating nuclear factor-kappaB (NF- j B)(Hershko  et al.  2004). The latter study found that CHXtreatment increased IL-6 mRNA and protein levels (despite partial inhibition of protein synthesis) through mRNA stabil-ization.CHXalsosuppressedI j B a resynthesis andprolonged p38 MAPKactivation, which is associated with sustained NF- j B activation (Hershko  et al.  2004). Similar observations of superinduction of IL-6 or other genes by CHX have beenreported(Newton  et al. 1996;Lutter  et al. 2000). Ontheother hand, CHX (and its derivative acetoxycycloheximide) blocked the tumor necrosis factor (TNF)- a -induced activationof NF- j B in A549 human lung carcinoma cells by down-regulating TNF receptor 1 via activation of extracellular-regulated kinase (ERK) and p38 MAPK (Ogura  et al.  2008).These findings suggest that CHX may exert its regulatoryeffects through distinct signaling pathways depending on the pre-treating inducer and cell type. The effects of PSI onlearning and memory processes were also reported, showingimpairment of auditory and contextual fear conditioning inC57BL/6N mice given CHX (Stiedl  et al.  1999).To our surprise, while investigating the mechanism of up-regulation of the MOR gene, we found evidence that CHXstimulates MOR gene transcription in a dose/time-dependent manner in P19 cells. This study was initiated todetermine which regulatory mechanisms were involved inthe effect of CHX on the induction of MOR transcription. Materials and methods Materials Actinomycin-D (Act-D), CHX, leptomycin B (leptoB), okadaic acid(OA), pyrrolidine dithiocarbamate (PDTC), and sodium nitroprus-side (SNP) were purchased from Sigma (St Louis, MO, USA).Anisomycin, puromycin, 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ), PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)yrazolo[3,4-d]pyrimidine), and SP600125 (SP) were purchased fromEMD Biosciences (Gibbstown, NJ, USA). Ly294002, KT5720,PD98059, rapamycin, SB203580, and U0126 were purchased fromCell Signaling Technology (Beverly, MA, USA). 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  ·  10 5 cells/well in 12-well culture plates 15 h before treatment.PDTC (100  l M ), leptoB (10 ng/mL), Act-D (5  l g/mL), CHX(10  l g/mL), and SNP (100  l M ) were treated to each well of P19cells for 6 h. Cells were harvested for RNA isolation.For CHX treatment in neuroblastoma NMB cells, cells were plated to a density of 1  ·  10 6 cells/well in six-well culture plates15 h before CHX treatment (Fig. 1b). Transfections into NMB cellswith 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 wereharvested 48 h after transfection and isolated total RNA for real-time qRT-PCR using LUC primers (Table S1). Each value wasnormalized by protein amount of cells. RT-PCR and real-time quantitative RT-PCR (qRT-PCR) Total RNA was isolated using TRI Reagent (Molecular ResearchCenter, Cincinnati, OH, USA) and analyzed by RT-PCR using theMOR gene-specific primers mMOR_E3-S and mMOR_E4-AS(Hwang  et al.  2007). RT-PCR was performed using a QiagenOneStep RT-PCR kit (Valencia, CA, USA). Similar reactions werecarried out using primers for kappa opioid receptor (KOR; mKOR-S1 and mKOR-AS1; Table S1) and  b -actin as an internal control(Hwang  et al.  2007).Real-time qRT-PCR was performed as described previously(Hwang  et al.  2010) using the same above MOR primer set andQuantitect SYBR Green PCR kit (Qiagen). The relative mRNA geneexpression was analyzed as described previously (Pfaffl 2001;Hwang  et al.  2007). The number of target molecules was normal-ized against that obtained for   b -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 werealso verified on an agarose gel. The RT-PCR and real-time qRT-PCR experiments were repeated at least three times to obtain statisticalsignificance. Journal of Neurochemistry    2011 International Society for Neurochemistry,  J. Neurochem.  (2011)  116 , 1077–1087   2011 The Authors 1078 |  D. K. Kim  et al.  Chromatin immunoprecipitation (ChIP) assays and western blotanalyses Chromatin immunoprecipitation assays were performed as reported previously (Kim  et al.  2004). The antibodies of acetylated histoneH3 (06-599), Brm-related gene 1 (SNF2 b /Brg1; 07-478), Lys4dimethylated histone H3 (H3dmK4; 07-030), and Lys9 trimethylat-ed histone H3 (H3tmK9; 07-442) were purchased from MilliporeCorporation (Bedford, MA, USA) and antibodies of HDAC2 (sc-9959), PCBP1 [poly(C)-binding protein 1; sc-16504], Sox18 (sc-20100x), and Sp1 (sc-59x) were purchased from Santa CruzBiotechnology (Santa Cruz, CA, USA). All ChIP assays werecontrolled by performing parallel experiments with no antibody,normal rabbit serum, and non-specific Gal4 antibody (sc-577; SantaCruz) pulldowns. Each immunoprecipitated DNA sample wasanalyzed by real-time qPCR using the indicated PCR primers inFig. 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-3 a /  b (Ser21/9; 9331; Cell Signaling), anti-phospho-p38 MAPK (Thr180/ Tyr182; 9215; Cell Signaling), anti-Sox18 (sc-20100x; Santa Cruz),and anti- b -actin (GTX109639; GeneTex, Irvine, CA, USA). Results Induction of the MOR gene by CHX To finda specific compound that up-regulate the MORgene at the transcriptional level, MOR-negative cells (P19) weretreated with several chemicals (Fig. 1a). Treatment with CHXincreased the MOR mRNA about 8 times (asterisk, lane 5)compared with untreated cells (lane 1). Treatment with leptoB(aspecificnuclearexportinhibitor),PDTC(aninhibitorofNF- j B activation), Act-D (a transcription inhibitor), and SNP (anitric oxide donor) had slight effects on MOR transcription(seegraphbelowthegelimage),butthesewerenotstatisticallysignificant.TranscriptionoftheKORgene,anothermemberof the opioid receptors used as a negative control, was decreasedin PDTC- and Act-D-treated samples. The upper band(arrowhead) of MOR in lane 5 was identified as anew isoform(named MOR-1z) by DNA sequencing (details in Fig. 2) andthis transcript encoded a different and shorter C-terminalsequence compared to full-length MOR. Figure 1(a) on theright gel image shows that CHX increased MOR mRNA in atime-dependent manner, but at the longest treatment time(14 h, lane 7) decreased MOR mRNAwas observed, possibly because of cell death. The increased accumulation of MOR mRNA could be detected at 1 h (1.9 ± 0.3-fold) and themaximal accumulation occurred at 6 h (8.2 ± 0.5-fold,  p  < 0.05). In Fig. 1(b), MOR mRNA was gradually elevatedwhen the concentration of CHX was increased from 0.1  l g/ mL to 50  l g/mL,indicating that MOR was increased byCHXinadose-dependentmannerwhilebothKORand b -actinwerenot changed.MOR transcription induction by CHX was also observedin MOR-positive cells (neuroblastoma NMB) as shown inFig. 1(b). However, in the NMB cells MOR induction beganat 10  l g/mL CHX, which is 100-fold higher than observed inP19 cells (from 0.1  l g/mL CHX). Therefore, we used P19cells for further experiments because the use of lower CHXconcentrations may avoid the interference of protein synthe-sis inhibition. CHX induces MOR expression via activation of phosphoinositide 3-kinase (PI3-K) 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- MOR (a)(b)   A  c   t  -   D  C   H   X  S   N   P –    P   D   T  C    L  e  p   t  o   B KOR   -actin 1 2 3 4 5 6 MOR CHX : 0.01, 0.1, 1, 10, 50   (µg)1 2 3 4 5 6 KOR   -actin – 0246800.010.111050 CHX (µg) MORKOR * (h) T1/2 = ~2 h CHX (10 µg)0 0.5 1 2 4 6 141 2 3 4 5 6 7 MORKOR   -actin 0246800.5124614 MORKOR (h) *******    R  e   l  a   t   i  v  e   b  a  n   d   i  n   t  e  n  s   i   t  y   t  o         β   -  a  c   t   i  n MOR CHX (µg)0 1 10 501 2 3 4 -actin NMB cells –    R  e   l  a   t   i  v  e   b  a  n   d   i  n   t  e  n  s   i   t  y   t  o         β   -  a  c   t   i  n   A  c   t  -   D  C   H   X  S   N   P   P   D   T  C    L  e  p   t  o   B * 40268 MORKOR CHX (µg) ** MOR β -actin0 1 10 5005. Fig. 1  CHX treatment induced MOR gene expression in P19 cells.(a) On left panel, the results of RT-PCR analyses of the MOR andKOR mRNA levels in P19 cells treated with different chemicals asindicated on top of gel. Significantly increased MOR band marked withasterisk. Arrowhead indicates a new isoform (named MOR-1z) pro-duced by CHX treatment. On right panel, the effect of MOR inductionby CHX was a time-dependent. Cells were treated with a 10  l g/mLconcentration of CHX for 30 min to 14 h as indicated. T½ (half time) isapproximately 2 h. (b) The effect of different concentrations of CHX onthe MOR and KOR genes. Cells were treated with the indicated con-centrations of CHX for 6 h. On right panel, MOR mRNA was alsoinduced by CHX in neuroblastoma NMB cells. The MOR PCR primerswere hMOR-S and hMOR-AS (Table S1) for NMB cells. The identitiesof the PCR products were confirmed by sequencing. Graphs undereach gel indicate the averages from at least three representativeexperiments. Asterisk in graph indicates statistically significant find-ings (* p   < 0.01), relative to the untreated control. Error bars indicatethe range of standard errors.  b -Actin was used as a control.   2011 The AuthorsJournal of Neurochemistry    2011 International Society for Neurochemistry,  J. Neurochem.  (2011)  116 , 1077–1087 Control of MOR transcription by PI3- and p38 MAP kinases  | 1079  free media (for abolishing the steady-state translation), themammalian target of rapamycin (mTOR) inhibitor rapamycin(translational inhibition involved in the mTOR-mediatedtranslational pathway), the PI3-K inhibitor Ly294002, the p42/44 MAPK inhibitor PD98059, and the Src-familytyrosine kinase inhibitor PP2, were investigated using cellsin the absence or presence of CHX by RT-PCR (Fig. 2a).Only the pre-treatment with Ly294 could significantly block the CHX-mediated stimulation of MOR (marked *). Theinhibition of translation initiation caused by serum-freemedia and rapamycin did not prevent MOR stimulation.Rather cell starvation by serum-free media enhanced theCHX-mediated stimulation of MOR (marked **). Theseresults suggest that the translational control pathway med-iated by mTOR and associated by serum starvation were not involved in CHX stimulation of MOR, while the PI3-K-associated pathway was directly associated with the stimu-lation. To test whether the stimulation occurs at thetranscriptional level, cells were pre-treated with the tran-scription 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 (c)  903 CATCAAAGCA CTGATCACGA TTCCAGAAAC CACTTTCCAG ACTGTTTCCT 953 GGCACTTCTG CATTGCCTTG GGTTACACAA ACAGCTGCCT GAACCCAGTT1003 CTTTATGCGT TCCTGGATGA AAACTTCAAA CGATGTTTTA GAGAGTTCTG1053 CATCCCAACT TCCTCCACAA TCGAACAGCA AAACTCTGCT CGAATCCGTC1103 AAAACACTAG GGAACACCCC TCCACGGCTA ATACAGTGGA TCGAACTAAC1153 CACCAG  AAAA TCACCTGACA TTAAAAGCAG GAGTAACCGG GCGTGGTGGC1203 GCACACTTTT AATCCCAGCA CTCGGGAGGC AGAGACTGGC GGATTTCTGA 1253 GTTCGAGGCC AGCCTGGTCT ACAGA  CTAGA AAATCTGGAA GCATAAACTG1303 CTCCATTGCC C TAA  Encoded peptides  mMOR_E3-S mMOR_E3-ASexon 3 end exon 4 begin   exon Z PTSSTIEQQNSARIRQNTREHPSTANTVDRTNHQKITPTSSTIEQQNSARIRQNTREHPSTANTVDRTNHQLENLEAETAPLP  MOR isoform  357357 ********************************** Novel isoform ( in above (a)) CHX (10 µg)    S   N   P   S  e  r  u  m  -  f  r  e  e   R  a  p  a  m   L  y   2   9  4   P   D   P   P   2 * MOR (a)(b)   -actin CHX *** **   C   H   X   A  c   t  -   D –Ly294 (µM) MOR   -actin 2 5 10 25 50 100 p-GSK-3 α (Ser21)     -actin CHX (h)   1   5   m   i  n – 0.5 1 2 4 6 Western blot p-GSK-3 β (Ser9)p-p38 MAPK    R  e   l  a   t   i  v  e   b  a  n   d   i  n   t  e  n  s   i   t  y   t  o         β   -  a  c   t   i  n CHX (h)   1   5   m   i  n – 0.5 1 2 4 6    B  a  n   d   i  n   t  e  n  s   i   t  y 01324 p-GSK-3 α   (Ser21)     -actinp-GSK-3   (Ser9)p-p38 MAPK CHX 10 – 0.5 1 3 – Serum (%) 04812 * ***** +–+–+–+–+–+–+– +–++++++++–+–––––+–+–+–+–+–+– Fig. 2  Effects of a PI3-kinase inhibitor and serum on MOR expres-sion. (a) Effect of several inhibitors on the CHX stimulation of MORexpression. P19 cells were pre-treated with 100  l M  SNP, 10 n M rapamycin (rapam), 50  l M  Ly294002 (Ly294), 20  l M  PD98059 (PD),5  l M  PP2 for 1 h before 4 h CHX treatment. For ‘serum-free’ samples,cells were incubated with serum-free media for 30 min prior to CHXstimulation for 3 h. The asterisk indicates significant inhibition or in-creased induction of CHX stimulation by Ly294 (*) and serum-free (**),respectively. On bottom left gel, the inhibitory effect of differentconcentrations of Ly294 on CHX-stimulated MOR gene analyzed byRT-PCR. Cells were pre-treated with either Act-D (5  l g/mL) or theindicated concentrations of Ly294 before 4 h CHX treatment. Onbottom right gel, effect of reduced serum on MOR expression. Cellswere incubated for 4 h in media containing different percentages ofserum as indicated. Cells treated with CHX in serum-free media areused as a positive control. (b) Western blot analysis of phospho-GSK-3 a  /  b  (Ser21/9) and phospho-p38 MAPK (Thr180/Tyr182) expressionin CHX-stimulated cells with different time treatments. Anti- b -actin wasused as a control. (c) Identification of a novel MOR isoform induced byCHX. Numbers on the left for DNA and amino acids sequences arebased on the start codon designated at +1. The gray box in boldindicates a newly found exon (exon Z) encoding a new isoform and anextra 119 bases between exons 3 and 4. Since the coding frame of theisoform contains a TGA stop codon (in box) before proceeding to thesrcinal stop codon (TAA) in exon 4, the isoform encode a shorterpolypeptides as drawn in the figure underlined. Journal of Neurochemistry    2011 International Society for Neurochemistry,  J. Neurochem.  (2011)  116 , 1077–1087   2011 The Authors 1080 |  D. K. Kim  et al.  transcriptional level. In addition, pre-treatments with differ-ent concentrations of Ly294 (2–100  l M ) were included andshowed that the inhibition of CHX stimulation began at 10  l M  concentration. Since serum starvation enhanced theCHX stimulation, we tested the direct effect of serum byitself using 0–3% serum media (Fig. 2a, bottom right gel).The results showed no change of MOR mRNAwith exposureto serum-free or reduced serum media.Glycogen synthase kinase 3 (GSK-3) is a critical down-stream element of the PI3-K pathway, and its activity can beinhibited by phosphorylation of GSK-3 a  at Ser21 and GSK-3 b  at Ser9. Results of Fig. 2(b) showed that phosphorylationof GSK-3 b  at Ser9 increased with 15 min and 30 min CHXtreatments and was decreased at both 1 and 2 h followed by aslight increase at 4 h and then decreased at 6 h. Phosphor-ylation of GSK-3 a  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) inresponse to CHX treatment began at 15 min and reached themaximum at 30 min, followed by decreased phosphorylationafter 30 min. These indicate that the cytosolic events(phosphorylations of GSK-3 and p38 MAPK) induced byCHX occur earlier than CHX-mediated MOR induction innucleus.As mentioned earlier, an upper band (arrowhead inFig. 1a) of MOR PCR products appeared in CHX-treatedsamples. Since the band size was bigger than expected, the band was isolated from agarose gel and sequenced. Asshown in Fig. 2(c), 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   C   H   X  MOR   -actin –    P  u  r  o   A  n   i  s  o  C   H   X  –    P  u  r  o   A  n   i  s  o 10% serum1% serum –CHXPuroAniso 10% Se1% Se 05101520 *** NMB cells    L  y  2  9  4   S   B – + CHX MOR (a)(b)(c)(d)   -actin   C   H   X  –    L  y  2  9  4   S   B   P   D   U  0  1  2  6   S   P   K   T   P   D   T  C   Q   N   Z  O  A MOR   -actin w/o CHX    L  y  2  9  4   S   B   P   D   U  0  1  2  6   S   P   K   T   P   D   T  C   Q   N   Z  O  A – MOR   -actin    L  y  2  9  4   S   B ––   A  c   t  -   D    U  0  1  2  6 – + + + + + Puro MOR   -actin– + + + + + Aniso MOR   -actin    R  e   l  a   t   i  v  e   b  a  n   d   i  n   t  e  n  s   i   t  y   t  o         β   -  a  c   t   i  n p-GSK-3 α  /    (Ser21/9)   -actin CHX    L  y  2  9  4   S   B – – + + + – Western blot 10% serum    L  y  2  9  4   S   B – – + + + – Serum-freeMOR   -actin    L  y  2  9  4   S   B ––    L  y  2  9  4   S   B ––CHX – + + +10% serum– + + +Serum-free RT-PCR p-3 α p-3   ***** MOR   -actin– + + + + + CHX Fig. 3  Effects of various inhibitors on CHX stimulation. (a) P19 cellswere pre-treated with the indicated inhibitors for 1 h before beingtreated with or without CHX (10  l g/mL) for 6 h. Treated concentra-tions: 50  l M  Ly294, 25  l M  SB203580 (SB), 20  l M  PD, 10  l M  U0126,25  l M  SP600125 (SP), 2  l M  KT5720 (KT), 100  l M  PDTC, 10 nM6-amino-4-(4-phenoxyphenylethylamino) quinazoline (QNZ), and10 nM OA. Below gel shows pre-treated samples without CHX as anegative control. (b) Effects of other protein synthesis inhibitors onMOR expression. Cells were treated separately with 50  l g/mLpuromycin (puro) and 10  l M  anisomycin (aniso) as well as 10  l g/mLCHX for 6 h in indicated % serum media. The asterisk indicates astatistically significant difference (** p   < 0.05) relative to the CHX-treated sample in 10% serum media (10% Se). (c) Differential effectsof various inhibitors on MOR stimulations by puromycin and aniso-mycin compared with CHX-mediated stimulation. Right gel, effects ofLy294 and SB treatments on endogenous MOR gene in MOR-po-sitive NMB cells. (d) Pre-treatment with PI3-kinase inhibitor Ly294shows the inhibition of activation (phosphorylation) of the PI3-Kdownstream factor GSK-3 a  /  b  in either 10% serum or serum-freemedia analyzed by western blot. Arrowhead indicates distinct phos-phorylation of GSK-3 a  (upper band) by CHX in serum-free mediacompared with the GSK-3 a  in CHX-treated cells in 10% serummedia (left gel). Anti- b -actin was used as a control. Bottom gel,RT-PCR showed the integrity of the above western blot experimentand comparison of MOR induction by CHX in 10% serum andserum-free media.   2011 The AuthorsJournal of Neurochemistry    2011 International Society for Neurochemistry,  J. Neurochem.  (2011)  116 , 1077–1087 Control of MOR transcription by PI3- and p38 MAP kinases  | 1081
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