Gene Expression In PC12 Cells Research Articles

Phoenix dactylifera L. Pollen and Fluvoxamine Maleate Protect PC12 Cells Against H2O2-Induced Oxidative Stress by Involvement of Nrf2 and SIGMAR1 Gene Expression

Background: The association between oxidative stress and the pathogenesis of neurodegenerative diseases has been documented. Besides, there is evidence that antidepressant agents, such as fluvoxamine maleate (Flv), can ameliorate neurotoxicity. The date palm (Phoenix dactylifera L.) pollen (DPP) contains various compounds with antioxidant capacity; however, its underlying mechanism of function has not been fully understood. Objectives: The present study aimed to compare the neuroprotective effects of DPP and Flv on H2O2-induced oxidative damage and their effects on Nrf2, SIGMAR1, and Bcl2 gene expression in PC12 cells. Methods: First, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay examined the toxicities of DPP, Flv, and H2O2 at various concentrations on the PC12 cells. Real-time polymerase chain reaction (PCR) measured the expression of Nrf2, SIGMAR1, and Bcl2 in PC12 cells in the presence or absence of DPP or Flv after 4 h of treatment with 100 μM H2O2. Results: Based on the MTT results, DPP at concentrations of 200 - 1000 µg/mL had no toxic effect on PC12 cells. The IC50 was evaluated at 57.26 μM and 109.5 μM under treatment with Flv and H2O2. Real-time PCR analysis showed a decrease in the expression of Nrf2 and SIGMAR1 in PC12 cells treated with 100 μM H2O2, an indicator of oxidative stress recruitment in PC12 cells. Pretreatment with DPP and Flv (500 µg/mL and 10 µM, respectively) for 24 h resulted in the upregulation of Nrf2 relative to the vehicle control. In addition, pretreatment with DPP increased the level of SIGMAR1 in PC12 cells compared with H2O2-exposed cells. Considering the role of SIGMAR1 in endoplasmic reticulum oxidative stress, the SIGMAR1 level should be evaluated at the translational level. Compared to the untreated cells, the expression of Bcl2 decreased in all the experimental cases, and the difference in Bcl2 expression was not significant between the co-treatment experimental cases. Conclusions: Taken together, DPP and Flv have neuroprotective effects against oxidative damage in PC12 cells. Exposure of PC12 cells to 500 μg/mL DPP and 10 μM Flv for 24 h protected the morphology and function of PC12 cells under H2O-induced oxidative stress via regulating oxidative stress-involved genes.

NGF activates NFAT via the MEK1/2 pathway in PC12 cells

Nerve growth factor (NGF) plays a key role in controlling growth, maintenance and survival of neurons as well as nociceptor development. Many of its critical actions are believed to be mediated by activation of corresponding transcription factors. Here I attempt to reveal signaling cascades that relay NGF stimulation to NFAT, a family of Ca2+/calcineurin-dependent transcription factors that are implicated in playing a pivotal role in neuronal development and survival, and pain sensation. With a luciferase gene reporter assay, it was confirmed that NGF can trigger NFAT-dependent gene expression in PC12 cells. Subsequent research with pharmacological tools uncovers that this effect was mediated by the TrkA-MEK1/2 pathway. Furthermore, it was determined that NFATc3 is the predominant isoform by calculating an absolute copy number of each NFAT isoform with quantitative PCR. Taken together, it is proposed that elucidating cellular connections linking NGF and NFAT may help reveal novel therapeutic targets, which can be exploited to devise innovative remedies for the treatment of NGF and NFAT-associated clinical disorders such as anomalous neuronal development and hyperalgesia.

Hypothermia Regulates Endoplasmic Reticulum (ER) Stress through the X-box Binding Protein-1 (XBP1) Gene Expression in PC12 Cells

Hypothermia Regulates Endoplasmic Reticulum (ER) Stress through the X-box Binding Protein-1 (XBP1) Gene Expression in PC12 Cells

Phenylethanolamine N-methyltransferase gene expression in PC12 cells exposed to intermittent hypoxia

Epidemiological studies show a strong correlation between Obstructive Sleep Apnea (OSA) and cardiovascular disorders. OSA patients experience intermittent hypoxia (IH), characterized by brief, but recurring episodes of cessation in breathing. These patients have higher levels of circulating catecholamines and an increased incidence of hypertension; however the mechanisms defining this association are not clearly established. Genetic linkage studies have associated the phenylethanolamine N-methyltransferase (PNMT) gene to the development of hypertension. PNMT, the terminal enzyme in the catecholamine biosynthetic pathway, directly responsible for adrenaline synthesis, is elevated in hypertensive animals. Recent studies utilizing PC12 cells show an increase in the expression of PNMT and its regulatory transcription factors when exposed to continuous hypoxia. The current study examined the regulation of PNMT under conditions of IH. The mRNA of PNMT was analyzed to assess if the regulation of PNMT expression entails alternative splicing. The mRNA and protein of transcription factors HIF1α, Egr-1, GR, and Sp1, were analyzed to assess the cellular pathways involved in regulating PNMT expression. A PNMT promoter-driven luciferase assay was performed to evaluate promoter activity under IH. Preliminary results lay an antecedent for the regulation of PNMT by IH conceivably via an altered regulation of its transcription factors and establish a possible role for PNMT in IH mediated hypertension in OSA patients.

MAP Kinase Phosphatase 3 (MKP3) Preserves Norepinephrine Transporter Activity by Modulating ERK1/2 Kinase-Mediated Gene Expression.

The norepinephrine transporter (NET) mediates the clearance of norepinephrine (NE) from the extracellular space and is a target of therapeutic antidepressants and psychostimulants. Previously we identified a MAP kinase phosphatase 3 (MKP3), as an important modulator of protein kinase C (PKC) mediated internalization of the related dopamine transporter (DAT). Here we show that MKP3 decreases PKC-mediated down regulation of NET expressed in PC12 cells. We demonstrate that this process involves a PKC-stimulated decrease of NET surface expression that is dependent on dynamin. Surprisingly, MAP kinase inhibitors have no effect on the PKC-mediated regulation of NET activity, suggesting that, like PKC-mediated regulation of the DAT, the acute activation of MAP kinases is not likely to be involved. To elucidate potential mechanisms we used a substrate trap-based assay to identify extracellular-signal-regulated kinase (ERK)1/2 as the predominant substrate of MKP3. Furthermore we also established that brief chemical stabilization of a modified destabilized MKP3 does not alter PKC-mediated down regulation of NET. Finally, the expression of a dominant negative version of H-Ras, an upstream activator of ERK1/2, abolishes phorbol 12-myristate 13-acetate (PMA)-mediated down regulation of NET in a manner similar to MKP3. Taken together we propose that chronic MKP3 expression regulates surface NET through the sustained inhibition of ERK1/2 MAP kinase signaling that alters gene expression in PC12 cells. This is supported by gene expression data from naïve and MKP3-expressing PC12 cells that reveal robust decreases in gene expression of several genes in the MKP3-tranfected cells. Interestingly, caveolin-1, a protein with a critical role in membrane protein trafficking is down regulated by MKP3 expression. We further show that selective silencing of the caveolin-1 gene in naïve PC12 cells attenuates PKC-mediated downregulation of NET activity, consistent with a potential role for caveolins in regulating NET surface expression. In summary, these results suggest that chronic MKP3 expression alters the expression of genes in PC12 cells that are involved in the regulation of NET surface expression.

Hypothermia Regulates Insulin-like Growth Factor 1 Gene Expression in PC12 Cells

In this study, we evaluated the effects of various hypothermic conditions (32℃), including lithium chloride treatment, on insulin-like growth factor 1 (IGF-1) gene expression in PC12 cells. The results show that short-term hypothermic treatment ( 1 day). Repeated switching between normal temperature and hypothermia every 2 h increased IGF-1 gene expression approximately 3-4-fold. These findings indicate that hypothermia dynamically regulates IGF-1 gene expression. This study could be helpful for the development of treatment and diagnostic strategies for ischemia.

Glucose deprivation regulates the progranulin-sortilin axis in PC12 cells.

Progranulin (PGRN) is a growth factor implicated in several neurodegenerative diseases, such as frontotemporal lobar degeneration. Despite its important role in the central nervous system (CNS), the mechanisms controlling PGRN expression in the CNS are largely unknown. Recent evidence, however, suggested that several stressors, such as hypoxia, acidosis, or oxidative stress, induce PGRN expression. The present study was mainly aimed at determining whether and, if so, how glucose deprivation affects PGRN expression in PC12 cells. Initially, it was found that glucose deprivation gradually induced PGRN gene expression in PC12 cells. To elucidate the underlying molecular mechanisms, several intracellular signalings that were modified in response to glucose deprivation were examined. Both adenosine monophosphate kinase (AMPK) activation and changes in osmotic pressure, which are modified by extracellular glucose concentration, had no effect on PGRN gene expression; on the other hand, p38 activation in response to glucose deprivation played an important role in inducing PGRN gene expression. It was also found that expression of sortilin, a PGRN receptor implicated in PGRN endocytosis, was dramatically reduced by glucose deprivation. In contrast to glucose‐dependent regulation of PGRN gene expression, AMPK activation played a central role in reducing sortilin expression. Overall, the present study suggests that the PGRN–sortilin axis is modulated by glucose deprivation via two distinct mechanisms. As PGRN is neuroprotective, this system may represent a new neuroprotective mechanism activated by glucose deprivation in the CNS.

Poly(Dimethylsiloxane) (PDMS) Affects Gene Expression in PC12 Cells Differentiating into Neuronal-Like Cells

IntroductionMicrofluidics systems usually consist of materials like PMMA - poly(methyl methacrylate) and PDMS - poly(dimethylsiloxane) and not polystyrene (PS), which is usually used for cell culture. Cellular and molecular responses in cells grown on PS are well characterized due to decades of accumulated research. In contrast, the experience base is limited for materials used in microfludics chip fabrication.MethodsThe effect of different materials (PS, PMMA and perforated PMMA with a piece of PDMS underneath) on the growth and differentiation of PC12 (adrenal phaeochromocytoma) cells into neuronal-like cells was investigated using cell viability, cell cycle distribution, morphology, and gene expression analysis.Results/ConclusionsAfter differentiation, the morphology, viability and cell cycle distribution of PC12 cells grown on PS, PMMA with and without PDMS underneath was the same. By contrast, 41 genes showed different expression for PC12 cells differentiating on PMMA as compared to on PS. In contrast, 677 genes showed different expression on PMMA with PDMS underneath as compared with PC12 cells on PS. The differentially expressed genes are involved in neuronal cell development and function. However, there were also many markers for neuronal cell development and functions that were expressed similarly in cells differentiating on PS, PMMA and PMMA with PDMS underneath. In conclusion, it was shown that PMMA has a minor impact and PDMS a major impact on gene expression in PC12 cells.

Extracellular ATP activates NFAT-dependent gene expression in neuronal PC12 cells via P2X receptors

BackgroundTreatment of neuronal PC12 cells with ATP induces depolarisation and increases intracellular calcium levels via purinergic receptors. In many cell types, sustained elevation of intracellular calcium levels cause changes in gene expression via activation of the transcription factor NFAT (nuclear factor of activated T cells). We have therefore characterised the signalling pathway by which ATP regulates NFAT-dependent gene expression in PC12 cells.ResultsThe activation of NFAT transcriptional activity by extracellular ATP was characterised with the help of reporter gene assays. Treatment of PC12 cells with ATP elicited a dose-dependent increase in luciferase activity (EC50 = 78 μM). UTP, 4-benzoylbenzoyl ATP and α,β-methylene ATP did not mimic the effect of ATP, which was abolished by treatment with the P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS). This pharmacological characterisation provides evidence for a critical role of ionotropic P2X receptors. Blockade of L-type voltage-dependent calcium channels by nifedipine reduced the response of NFAT to ATP, indicating that a depolarisation-mediated calcium influx was required for maximal NFAT activation. Inhibition of store-operated calcium entry by the pyrazole derivative BTP2 also diminished ATP-dependent NFAT activation. Furthermore, ATP-induced NFAT activation was associated with the activation of the mitogen-activated protein kinases ERK1/2. Finally, treatment with ATP increased the levels of the NFAT target transcripts, RCAN1-4 (regulator of calcineurin) and BDNF (brain derived neurotrophic factor).ConclusionThe present data show that ATP induces NFAT-dependent changes in gene expression in PC12 cells by acting on P2X receptors. Maximal NFAT activation depends on both depolarisation-induced calcium influx and store-operated calcium entry and requires the activity of the protein phosphatase calcineurin and the mitogen-activated protein kinase cascade.

Rab22 controls NGF signaling and neurite outgrowth in PC12 cells

Rab22 is a small GTPase that is localized on early endosomes and regulates early endosomal sorting. This study reports that Rab22 promotes nerve growth factor (NGF) signaling-dependent neurite outgrowth and gene expression in PC12 cells by sorting NGF and the activated/phosphorylated receptor (pTrkA) into signaling endosomes to sustain signal transduction in the cell. NGF binding induces the endocytosis of pTrkA into Rab22-containing endosomes. Knockdown of Rab22 via small hairpin RNA (shRNA) blocks NGF-induced pTrkA endocytosis into the endosomes and gene expression (VGF) and neurite outgrowth. Overexpression of human Rab22 can rescue the inhibitory effects of the Rab22 shRNA, suggesting a specific Rab22 function in NGF signal transduction, rather than off-target effects. Furthermore, the Rab22 effector, Rabex-5, is necessary for NGF-induced neurite outgrowth and gene expression, as evidenced by the inhibitory effect of shRNA-mediated knockdown of Rabex-5. Disruption of the Rab22-Rabex-5 interaction via overexpression of the Rab22-binding domain of Rabex-5 in the cell also blocks NGF-induced neurite outgrowth, suggesting a critical role of Rab22-Rabex-5 interaction in the biogenesis of NGF-signaling endosomes to sustain the signal for neurite outgrowth. These data provide the first evidence for an early endosomal Rab GTPase as a positive regulator of NGF signal transduction and cell differentiation.

Visible Light-Regulated Gene Expression and Neurite Outgrowth of Nerve Cells

The neurite outgrowth and gene expression of PC12 cells on collagen-coated glass plates under light-emitting diode (LED) irradiation and mixed light irradiation were investigated at several wavelengths (470, 525, 600, and 630 nm). We found that visible light regulated the neurite outgrowth of PC12 cells. Therefore, we investigated the expression level of p75NTR and TrkA in PC12 cells in order to evaluate whether gene expression in PC12 cells changed according to the wavelength of light used to irradiate these cells. When the PC12 cells were irradiated with mixed light or LED light for 10 min or 30 h in the presence of nerve growth factor (NGF), p75NTR expression was downregulated compared to that in PC12 cells cultured in the dark with NGF in the culture medium. Light irradiation influenced the expression of TrkA in the PC12 cells cultured with or without NGF. Light irradiation of PC12 cells regulated the expression of specific genes as well as neurite outgrowth.

Quercetin exerts a neuroprotective effect through inhibition of the iNOS/NO system and pro-inflammation gene expression in PC12 cells and in zebrafish

Flavonoids have been reported to be potent antioxidants and beneficial in the treatment of oxidative stress-related diseases. Quercetin, a major flavonoid naturally occurring in plants, deserves attention because of its beneficial effects observed in various in vitro and in vivo neural damage models; however, the actions of quercetin are paradoxical. In an effort to confirm the neuroprotective effect of quercetin and to elucidate its mechanism of action, the neuroprotective effects of quercetin in PC12 cells and in zebrafish models were investigated. In this study, the selective dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA), was used to induce neural damage in PC12 cells and zebrafish models. Pretreatment with quercetin offered neuroprotection against 6-OHDA-induced PC12 cell death. Moreover, quercetin could prevent 6-OHDA-induced PC12 cell apoptosis and 6-OHDA-stimulated dopaminergic neuron loss in zebrafish. Interestingly, quercetin was able to protect, but not rescue the dopaminergic neuron damage when zebrafish were treated with quercetin at different maturation stages of the blood brain barrier. A mechanistic study showed that quercetin could inhibit NO over-production and iNOS over-expression in PC12 cells and could down-regulate the over-expression of pro-inflammatory genes (e.g. IL-1ß, TNF-α and COX-2) in zebrafish, suggesting that these genes play a role in the neuroprotective effect of quercetin. The objective of this study was to provide a scientific rationale for the clinical use of quercetin, leading to its development as an effective therapeutic agent for the treatment of Parkinson's disease.

Molecular mechanisms associated with peptidergic control of adrenergic function

Phenylethanolamine N‐methyltransferase (PNMT), which produces epinephrine, is a marker of adrenergic function. While pituitary adenylate cyclase activating polypeptide (PACAP), independently and cooperatively with NGF, activates PNMT promoter‐driven and endogenous PNMT gene expression in PC12 cells, molecular mechanisms remain unknown. Transfection assays in PC12 cells and PKA‐deficient and PLCγ1‐deficient PC12 cells using PNMT promoter‐luciferase gene constructs and signaling inhibitors showed that PLCγl and cAMP‐dependent PKA signaling are critical for PACAP activation and PI3K, PKC, ERK1/2 MAPK, p38 MAPK downstream. The inhibitors also abrogated PACAPergic induction of endogenous PNMT. Western analysis of nuclear protein from PACAP‐treated PC12 cells showed that Egr‐1 and AP‐2 underlie PACAP responses. Results with nested deletion or site‐directed mutant constructs support this possibility. Exposure of PACAP‐treated transfected and untransfected PC12 cells to histone deacetylase (HDAC) inhibitors, Na butyrate or Trichostatine A, incrementally activated the PNMT promoter and endogenous PNMT. Findings indicate that PACAP transcriptionally activates the PNMT gene with HDAC being limiting. Presence of long and short forms of PNMT mRNA further suggest post‐transcriptional regulation by PACAP. Support: Spunk Fund, Inc., Sobel‐Keller Fund and McLean Hospital.

Rat mesenchymal stem cells increase tyrosine hydroxylase expression and dopamine content in ventral mesencephalic cells in vitro

Mesenchymal stem cells (MSCs) are pluripotent adult stem cells. It has been shown that MSCs secrete neurotrophic factors involving nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Also, these neurotrophic factors can upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells and neural stem cells. Here, we investigated the effect of co-culturing rat E13.5 ventral mesencephalic cells (VMCs) with MSCs from rat bone marrow on TH expression and dopamine (DA) content. The study consisted of 3 groups: MSC, VMC and a combined MSC+VMC group. All groups were cultured in serum-free neuro-basal medium for 3 days. Thereafter, each group was analyzed by RT-PCR, western blotting, and HPLC. The co-culture group showed a higher expression at TH and DA than the VMC group. However, TH and DA were not present in the MSC group. These observations suggest that MSCs could be an alternative source for treating neurodegenerative diseases such as Parkinson's disease (PD).

Selenoprotein T is a PACAP‐regulated gene involved in intracellular Ca2+mobilization and neuroendocrine secretion

Selenoproteins contain the essential trace element selenium, the deficiency of which is associated with cancer or accelerated aging. Although selenoproteins are thought to be instrumental for the effects of selenium, the biological function of many of these proteins remains unknown. Here, we studied the role of selenoprotein T (SelT), a selenocysteine (Sec) -containing protein with no known function, which we have identified as a novel target gene of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) during PC12 cell differentiation. SelT was found to be ubiquitously expressed throughout embryonic development and in adulthood in rat. Immunocytochemical analysis revealed that SelT is mainly localized to the endoplasmic reticulum through a hydrophobic domain. PACAP and cAMP induced a rapid and long-lasting increase in SelT gene expression in PC12 cells, in a Ca(2+)-dependent manner. These results suggested a possible role of SelT in PACAP signaling during PC12 cell differentiation. Indeed, overexpression of SelT in PC12 cells provoked an increase in the concentration of intracellular Ca(2+) ([Ca(2+)](i)) that was dependent on the Sec residue. Conversely, SelT gene knockdown inhibited the PACAP-induced increase in [Ca(2+)](i) and reduced hormone secretion. These findings demonstrate the implication of a selenoprotein in the regulation of Ca(2+) homeostasis and neuroendocrine secretion in response to a cAMP-stimulating trophic factor.

Effects of tri butyl tin acetate on dopamine biosynthesis and l-dopa-lnduced cytotoxicity in pc12 cells

The effects of tributyltin acetate (TBTA) on dopamine biosynthesis and L-3,4-dihydroxyphenylalanine (L-DOPA)-induced cytotoxicity in PC12 cells were examined. TBTA at concentrations of 0.1-0.2 microM inhibited dopamine biosynthesis by reducing tyrosine hydroxylase (TH) activity and TH gene expression in PC12 cells. TBTA at 0.1-0.4 microM also reduced L-DOPA (20-50 microM)-induced increases in dopamine content for 24 h in PC12 cells. TBTA at concentrations up to 0.3 microM did not affect cell viability. However, TBTA at concentrations higher than 0.4 microM caused apoptotic cytotoxicity. Exposure of PC12 cells to non-cytotoxic (0.1 and 0.2 microM) or cytotoxic (0.4 microM) concentrations of TBTA with L-DOPA (20, 50 and 100 microM) significantly increased the cell loss and the percentage of apoptotic cells after 24 or 48 h compared with TBTA or L-DOPA alone. These data suggest that TBTA inhibits dopamine biosynthesis and enhances L-DOPA-induced cytotoxicity in PC12 cells.

Enhanced tyrosine hydroxylase expression in PC12 cells co-cultured with feline mesenchymal stem cells

Mesenchymal stem cells (MSCs) secrete a variety of neuroregulatory molecules, such as nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor, which upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells. Enhancing TH gene expression is a critical step for treatment of Parkinson's disease (PD). The objective of this study was to assess the effects of co-culturing PC12 cells with MSCs from feline bone marrow on TH protein expression. We divided the study into three groups: an MSC group, a PC12 cell group, and the combined MSC + PC12 cell group (the co-culture group). All cells were cultured in DMEM-HG medium supplemented with 10% fetal bovine serum for three days. Thereafter, the cells were examined using western blot analysis and immunocytochemistry. In western blots, the co-culture group demonstrated a stronger signal at 60 kDa than the PC12 cell group (p<0.001). TH was not expressed in the MSC group, either in western blot or immunocytochemistry. Thus, the MSCs of feline bone marrow can up-regulate TH expression in PC12 cells. This implies a new role for MSCs in the neurodegenerative disease process.

Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells

Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2α, c-myc, and the peroxisome proliferator-activated receptor-γ were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions.

Opioid-induced regulation of gene expression in PC12 cells stably transfected with mu-opioid receptor

It has been postulated that opiates induce addictive behaviour via changes in gene expression. PC12 cells were stably transfected with the recombinant human mu-opioid receptor (MOR) to study opioid-induced gene expression. Expression was verified by binding assay, immunocytochemistry, and immunblotting experiments. Forskolin-induced cAMP formation was inhibited by [D-Ala 2, N-Me-Phe 4, Gly 5-ol]-enkephalin (DAMGO 1 μM), a specific MOR agonist. This effect was completely antagonized by naloxone. By using cDNA arrays, including approximately 1200 well-defined genes normally expressed in neural tissue, we monitored semi-quantitative changes in gene expression after 3 h short-term exposure to DAMGO. Incubation with DAMGO increased mRNA levels for 13 genes and down-regulated 13 other genes. Annexin V, RGS4 and CREB genes showed pronounced increase in expression after stimulation with DAMGO. Quantitative RT-PCR confirmed that DAMGO increased mRNA levels of Annexin V, an apoptosis-induced gene. We suggest that the PC12 cell transfected with the recombinant human MOR is a useful tool for identification of opioid-induced genes that may provide information on opiate effects of relevance for dependence.

NIBP, a Novel NIK and IKKβ-binding Protein That Enhances NF-κB Activation

The transcription factor NF-kappaB plays an important role in both physiological and pathological events in the central nervous system. Nevertheless, the mechanisms of NF-kappaB-mediated regulation of gene expression, and the signaling molecules participating in the NF-kappaB pathway in the central nervous system are, to date, poorly understood. To identify such molecules, we conducted a yeast two-hybrid screen of a human brain cDNA library using NIK as bait. As a result, we identified a novel NIK and IKK(beta) binding protein designated NIBP that is mainly expressed in brain, muscle, heart, and kidney. Interestingly, low levels of expression were detected in immune tissues such as spleen, thymus, and peripheral blood leukocytes, where NF-kappaB is known to modulate immune function. We demonstrated by immunohistochemistry that NIBP expression in the brain is localized to neurons. NIBP physically interacts with NIK, IKK(beta), but not IKK(alpha) or IKK(gamma). NIBP overexpression potentiates tumor necrosis factor-alpha-induced NF-kappaB activation through increased phosphorylation of the IKK complex and its downstream I(kappa)B(alpha) and p65 substrates. Finally, knockdown of NIBP expression by small interfering RNA reduces tumor necrosis factor-alpha-induced NF-kappaB activation, prevents nerve growth factor-induced neuronal differentiation, and decreases Bcl-xL gene expression in PC12 cells. Our data demonstrate that NIBP, by interacting with NIK and IKK(beta), is a new enhancer of the cytokine-induced NF-(kappa)B signaling pathway. Because of its neuronal expression, we propose that NIBP may be a potential target for modulating the NF-(kappa)B signaling cascade in neuronal pathologies dependent upon abnormal activation of this pathway.