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Glial cell line–derived neurotrophic factor (GDNF) is a potent growth factor essential to the development, survival, and function of dopaminergic neurons (Airaksinen and Saarma 2002). The molecular mechanisms underlying GDNF expression remain elusive; thus, we set out to identify a signaling pathway that governs GDNF levels. We found that treatment of both differentiated dopaminergic-like SH-SY5Y cells and rat midbrain slices with the dopamine D2 receptor (D2R) agonist, quinpirole, triggered an increase in the expression of GDNF that was temporally preceded by an increase in the levels of zinc-finger protein 268 (Zif268), a DNA-binding transcription factor encoded by an immediate-early gene. Moreover, the D2R inhibitor raclopride blocked the increase of both GDNF and Zif268 expression following potassium-evoked dopamine release in SH-SY5Y cells. We used adenoviral delivery of small hairpin RNA (shRNA) targeting Zif268 to down-regulate its expression and found that Zif268 is specifically required for the D2R-mediated up-regulation of GDNF. Furthermore, the D2R-mediated induction of GDNF and Zif268 expression was dependent on Gβγ-mediated signaling and activation of extracellular signal–regulated kinase 1/2. Importantly, using chromatin immunoprecipitation assay, we identified a direct association of Zif268 with the GDNF promoter. These results suggest that D2R activation induces a Gβγ- and extracellular signal–regulated kinase 1/2-dependent increase in the level of Zif268, which functions to directly up-regulate the expression of GDNF.
Glial cell line–derived neurotrophic factor (GDNF), a secreted growth factor belonging to the transforming growth factor-β (TGF-β) superfamily, was first isolated from a rat glioma cell line (Lin et al. 1993). The expression of GDNF is widespread throughout the CNS during development (Schaar et al. 1993; Stromberg et al. 1993; Choi-Lundberg and Bohn 1995), but becomes largely relegated to discrete regions in the adult brain, such as the striatum, hippocampus, cortex, and thalamus (Trupp et al. 1997; Golden et al. 1998, 1999; Barroso-Chinea et al. 2005). In addition, neurons, as opposed to glia, appear to be the main source of GDNF expression in the adult brain (Pochon et al. 1997). GDNF plays an important role in the function and survival of dopaminergic neurons in the midbrain (Airaksinen and Saarma 2002), where its receptor, the Ret receptor tyrosine kinase, and its coreceptor, GDNF family receptor-α 1 (GFRα1), are expressed (Trupp et al. 1997; Glazner et al. 1998). GDNF's interaction with GFRα1 promotes the recruitment, dimerization, and autophosphorylation of the Ret receptor (Airaksinen and Saarma 2002). This leads to the subsequent activation of the extracellular signal–regulated kinase 1/2 (Erk1/2), phosphoinositide 3-kinase (PI3K), phospholipase C-γ (PLCγ), and other signaling pathways (Airaksinen and Saarma 2002). The potent neuroprotective and neuroregenerative effects of GDNF on midbrain dopaminergic neurons present great therapeutic potential for the treatment of Parkinson's disease, a condition characterized by the specific loss of dopaminergic neurons in the substantia nigra (SN) region of the midbrain (Rangasamy et al. 2010). GDNF plays an important role in the regulation of dopaminergic neuron firing rates (Yang et al. 2001; Wang et al. 2010), dopamine release (Wang et al. 2010; Barak et al. 2011b), and in the maintenance of learning processes throughout senescence (Miyazaki et al. 2003). Interestingly, low levels of GDNF are associated with alcohol addiction in humans (Heberlein et al. 2010), and infusion of GDNF reduces the intake of alcohol in rats (Carnicella et al. 2008, 2009b; Carnicella and Ron 2009; Barak et al. 2011a). Finally, GDNF-mediated signaling in the CNS was shown to prevent depression- and anxiety-like behaviors in mice subjected to chronic stress (Uchida et al. 2011).
Constitutive, non-activity-dependent (i.e., passive) release comprises a major mode of GDNF secretion (Oh-hashi et al. 2009; Lonka-Nevalaita et al. 2010), indicating that the transcription and translation of GDNF is an important regulatory step for the function of this growth factor. However, little is known about the control of GDNF expression; thus, we set out to identify a possible signaling mechanism that regulates the expression of this growth factor. Previously, in vitro treatment of midbrain neuronal (Guo et al. 2002) or astrocytic (Ohta et al. 2000) cultures with apomorphine, which activates both dopamine D1 and D2 receptors, results in the up-regulation of GDNF mRNA. In addition, we previously demonstrated that the dopamine D1/D2 receptor agonist, cabergoline, increases the expression of GDNF in the human dopaminergic-like SH-SY5Y cell line, as well as in the midbrain of rats and mice following in vivo systemic administration of this compound (Carnicella et al. 2009a). Interestingly, endogenous GDNF expression levels are partially reduced in both the midbrain and striatum of mice lacking the dopamine D2 Gαi protein-coupled receptor (D2R) (Bozzi and Borrelli 1999; Saavedra et al. 2008). Together, these studies suggest that D2R-mediated signaling may contribute the expression of GDNF. Therefore, we tested whether, and if so, how, specific activation of D2Rs results in the induction of GDNF expression.
TRIzol reagent and pre-cast sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels were purchased from Invitrogen (Carlsbad, CA, USA). Phosphatase inhibitor cocktails II and III, deoxyribonuclease (DNase), and ethidium bromide were purchased from Sigma (St. Louis, MO, USA). The protease inhibitor mini-tablets were purchased from Roche (Indianapolis, IN, USA). The reverse transcription system and 2X PCR master mix were purchased from Promega (Madison, WI, USA). Quinpirole, Raclopride, U0126, PD98059, and gallein were purchased from Tocris Bioscience (Minneapolis, MN, USA). The BCA Protein Assay Kit was purchased from Pierce Biotechnology (Rockford, IL, USA). The enhanced chemiluminescence detection reagents were purchased from Fisher Scientific (Pittsburgh, PA, USA). The pRNAT-H1.1/Shuttle vector was obtained from the GenScript Corporation (Piscataway, NJ, USA). The Adeno-X vector, Expression System, and Purification and Rapid Titer kits were purchased from Clontech (Mountain View, CA, USA). The Chromatin Immunoprecipitation (ChIP) Assay Kit was obtained from Millipore (Billerica, MA, USA).
Anti-GDNF antibody (AF-212-NA) was obtained from R&D systems (Minneapolis, MN, USA). Anti-zinc-finger protein 268 (Zif268) antibody (#4153) was purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase–conjugated secondary antibodies, anti-Erk2 (sc-1647), anti-phosphoErk1/2 (pErk1/2; sc-7976), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sc-25778) polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dilutions of the antibodies were as follows: anti-GDNF, 1 : 250; anti-GAPDH, 1 : 5000; anti-Erk2, 1 : 2000; anti-pErk1/2, 1 : 2000; and anti-Zif268, 1 : 500.
The human dopaminergic-like SH-SY5Y cells were plated at a density of 2 × 105 cells/ml in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS, 10%), penicillin/streptomycin, and non-essential amino acids. Cells were differentiated into a neuronal-like phenotype by supplementing a 1% FBS DMEM solution containing 10 μM retinoic acid (RA) for 3 days, at which point the medium was changed again, to 1% FBS DMEM without RA for an additional 18–24 h. This was done to washout the RA, which can itself activate intracellular signaling pathways, such as Erk1/2 (Miloso et al. 2004). Cells were then treated with quinpirole (50 μM) in saline vehicle. For indicated pre-treatments, U0126, PD98059, or gallein were added to the cell medium 10 min before the addition of quinpirole. All inhibitors were prepared in dimethylsulfoxide, and remained in the cellular medium throughout the quinpirole treatment. Controls were treated with dimethylsulfoxide at a final concentration of 0.1%. For KCl-evoked dopamine release, the cellular medium was supplemented with 100 mM KCl. At the end of treatments, cells were briefly washed in phospho-buffered saline, then lysed and collected in either TRIzol reagent, for RT-PCR analysis, or radio-immunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 120 mM NaCl, 1% NP-40, 0.1% deoxycholate, and 0.5% sodium dodecyl sulfate), for western blot analysis. Alternatively, for ChIP experiments, cells were treated with 1% paraformaldehyde for 10 min at 37°C to cross-link DNA and proteins.
Acute slice treatments
Male Sprague–Dawley rats (P23–26) were purchased from Harlan Inc., Indianapolis, IN, USA. All experimental protocols involving rats were approved by the Ernest Gallo Research Center Institutional Animal Care and Use Committee (IACUC), and were conducted in agreement with the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. Rats were deeply anesthetized with isoflurane, then killed by decapitation. The brains were quickly removed and immediately placed in ice-cold artificial cerebral spinal fluid (aCSF; 126 mM NaCl, 1.2 mM KCl, 1.2 mM NaH2PO4, 1 mM MgCl2, 2.4 mM CaCl2, 18 mM NaHCO3, 11 mM glucose) saturated with oxygen (95% O2 and 5% CO2 mixture). Horizontal slices (150 μm) containing the midbrain were prepared using a Leica vibratome (Leica Biosystems, Buffalo Grove, IL, USA) in ice-cold aCSF. The ventral midbrain, containing the ventral tegmental area and the substantia nigra (SN), were dissected from the slices, and allowed to rest in oxygen-saturated aCSF at 22°C for 45 min prior to treatment. Slices were treated with 50 μM QP for an additional 30 or 240 min in oxygen-saturated, 22°C aCSF. At the end of the treatment, the slices were mechanically homogenized in TRIzol in preparation for RT-PCR analysis.
Reverse Transcription Polymerase Chain Reaction
Total RNA was isolated from cells lysed in TRIzol reagent according to the manufacturer's recommended protocol. Following DNase treatment, messenger RNA (mRNA) was selectively reverse transcribed using the Reverse Transcription System and oligo(dT) primers. The resulting cDNA was used in PCR reactions for GDNF, Zif268, and the housekeeping gene GAPDH using the following primers: human GDNF upstream: 5′- TGC CAG AGG ATT ATC CTG ATC AGT TCG ATG -3′; human GDNF downstream: 5′- GAT ACA TCC ACA CCT TTT AGC GGA ATG CTT -3′; human Zif268 upstream: 5′- TGA CCG CAG AGT CTT TTC CT -3′; human Zif268 downstream: 5′- TGG GTT GGT CAT GCT CAC TA -3′; human GAPDH upstream: 5′- TGA AGG TCG GTG TCA ACG GAT TTG GC -3′; human GAPDH downstream: 5′- CAT GTA GGC CAT GAG GTC CAC CAC -3′. For GDNF, 33–35 amplification cycles were used, while 27 cycles were used for Zif268 and GAPDH. Equal volumes of the PCR products were resolved on 1.8% agarose gels containing 0.05% ethidium bromide for visualization under UV light. Images were captured using the Eagle Eye 2 software. Relative band intensities were quantified using NIH ImageJ software (Bethesda, MD, USA), and normalized to GAPDH.
Cells were lysed and collected in radio-immunoprecipitation assay buffer containing protease and phosphatase inhibitors. Lysates were briefly sonicated, and then placed on ice for 30 min. Protein concentrations were determined using the BCA Protein Assay kit. Equal amounts of protein (15–25 μg) were resolved on NuPAGE 10% (for Zif268 and Erks 1/2) or 4–12% gradient (for GDNF) Bis-Tris gels, and transferred onto nitrocellulose membranes overnight. For pErk1/2 and Erk2 detection, membranes were first probed for pErk1/2, then incubated in stripping buffer (25 mM glycine-HCl, 1% sodium dodecyl sulfate, pH 3, for 30 min at 22°C) and reprobed for Erk2. Membranes were incubated with primary antibody for 4 h at 22°C, followed by a 2-h 22°C incubation with the appropriate Horseradish peroxidase-conjugated secondary antibodies to detect immunoreactivity via an enhanced chemiluminescence reaction. Images were developed on Kodak film (Fisher Scientific, Pittsburgh, PA, USA), and digitally scanned for relative densitometric quantification using NIH ImageJ software.
Following the paraformaldehyde-mediated cross-linking of DNA and proteins, cells were washed twice, briefly, with phospho-buffered saline and collected. ChIP was conducted as described in the manufacturer's protocol. Shearing of DNA was accomplished by using three rounds of 30-pulse, low-power sonication. Zif268-containing complex was IP-ed overnight at 4°C with 10 μg of anti-Zif268 (rabbit polyclonal IgG). Control IP with normal rabbit IgG was conducted in parallel. Putative Zif268-binding sites were identified by manually scanning the 1000-base-pair region immediately upstream of the transcription start site of the human GDNF gene (NCBI Ref Seq: NG_011675.2) for the Zif268 consensus binding sequence. Primer sequences for the PCR reactions were as follows: putative Zif268 binding site #1 upstream: 5′- CTC GGA CCT CGG CTT CTG -3′; putative Zif268 binding site #1 downstream: 5′- AAC AGG TCA GGG GCA CGC GT -3′; putative Zif268 binding site #2 upstream: 5′- AGC TCC TTT TCT GCC ACT -3′; putative Zif268 binding site #2 downstream: 5′- GGG TGG ATC AAA AAT CGA GA -3′; negative control upstream: 5′- CTC GCT GCT CTC CTC TCC T -3′; negative control downstream: 5′- AGT CCC GTG AAG ACA TGA GG -3′. PCR products were amplified using 42 amplification cycles, and were resolved on a 1.8% agarose gel. Bands were visualized as described above for RT-PCR.
Adenoviral-mediated shRNA down-regulation of Zif268
A double-stranded oligonucleotide encoding a 21-base-pair short hairpin RNA (shRNA) sequence against base pairs 1236–1257 of the coding region of Zif268 mRNA was subcloned into the BamHI and HindIII sites of the pRNAT-H1.1/Shuttle vector containing a green fluorescent protein (GFP) marker. This sequence was previously shown to silence the expression of human Zif268 (Ma et al. 2009). The resulting shZif268 expression cassette was then subcloned into the Adeno-X viral genome. Adenoviral particles were packaged and amplified in HEK293 cells as described in the Adeno-X Maxi Purification kit protocol. The adenoviral titer was determined using the Adeno-X Rapid Titer kit. Preparation of a control virus expressing a non-related control sequence was performed as previously described (Jeanblanc et al. 2009). For adenoviral delivery to SH-SY5Y cells, 2 × 106 infectious units (ifu)/mL of either virus was added to the cell medium after differentiation. All experiments were conducted 48 h later.
Data are expressed as the mean ± SEM. One- or two-way analysis of variance (anova) were used to determine the statistical significance in experiments comparing more than two groups. Significant main effects or interactions of the anovas were further investigated with post hoc Bonferroni or Student–Newman–Keul's tests, as indicated.
Activation of D2Rs up-regulates GDNF expression
Differentiated human SH-SY5Y cells exhibit a number of features characteristic of dopaminergic neurons. For example, once differentiated, these cells cease dividing and assume neuron-like morphology (Pahlman et al. 1984) and express tyrosine hydroxylase, dopamine transporter, and dopamine β-hydroxylase, proteins necessary for the biosynthesis, uptake, and metabolism of dopamine (Ault and Werling 2000). In addition, these cells form synaptic vesicles and synapses (Sarkanen et al. 2007), propagate action potentials (Tosetti et al. 1998), and are capable of releasing neurotransmitter following potassium-mediated depolarization (Murphy et al. 1991; Ault and Werling 2000; Gibb et al. 2011). Thus, we first tested whether activation of D2Rs results in increased GDNF expression in this widely used dopaminergic cell culture model. To do so, cells were treated with the D2R agonist quinpirole (50 μM), and the mRNA and protein levels of the growth factor were assessed. As shown in Fig. 1, quinpirole treatment caused a significant up-regulation of GDNF mRNA (Fig. 1a), which corresponded to an increase of the level of GDNF protein (Fig. 1b). Next, we tested whether D2R activation would enhance GDNF expression in the midbrain, where approximately 75% of neurons are dopaminergic (Fields et al. 2007) and endogenously express the D2R (Chen et al. 1991). To do so, we treated slices prepared from the rat ventral midbrain for 240 min with 50 μM quinpirole and found that activation of D2Rs caused an up-regulation of GDNF expression in midbrain neurons (Fig. 1c). Together, these results suggest that the expression of this growth factor in dopaminergic neurons is increased following D2R activation.
Activation of D2Rs up-regulates Zif268 expression
Next, we set out to determine the molecular mechanism of the D2R-mediated up-regulation of GDNF expression. Zif268 (also known as early growth response protein-1 [Egr-1], Krox24, and nerve growth factor–induced gene A [NGF-IA]) belongs to the immediate-early gene family of transcription factors (Knapska and Kaczmarek 2004). Interestingly, the up-regulation of Zif268 expression in response to fibroblast growth factor-2 (FGF2) and fibroblast growth factor-1 (FGF1) treatment enhanced the expression of GDNF in cultured rat astrocytes and the rat PC12 cell line, respectively (Lin et al. 2009; Shin et al. 2009). If Zif268 mediates the increase in GDNF expression in response to D2R activation, then the transcription factor expression should be induced first in response to D2R agonist treatment. Cells were therefore treated with quinpirole and the mRNA and protein levels of the transcription factor were assessed. We found a significant, and transient, increase in the levels of Zif268 mRNA (Fig. 2a) and protein (Fig. 2b) at 30 and 60 min following quinpirole treatment, respectively. Thus, D2R activation leads to a time-dependent increase in Zif268 mRNA, which consequently translates to an increase in Zif268 protein levels. Importantly, a 30-min quinpirole treatment of rat ventral midbrain slices likewise resulted in an increase in the mRNA levels of the transcription factor (Fig. 2c). Together, these findings suggest that activation of D2Rs results in an increase in the levels of this transcription factor.
Evoked dopamine release results in the sequential up-regulation of Zif268 and GDNF in a D2R-dependent mechanism
As previously mentioned, the dopaminergic SH-SY5Y cells rapidly release the neurotransmitter dopamine following potassium-induced depolarization (Ault and Werling 2000; Gibb et al. 2011). We therefore tested whether evoked dopamine release, and the subsequent activation of D2Rs causes an up-regulation of Zif268 and/or GDNF. To do so, cells were treated with 100 mM KCl in the presence and absence of the D2R inhibitor, raclopride. We observed an up-regulation of Zif268 30 min following KCl treatment (Fig. 3a and b), which was followed by an increase in the level of GDNF mRNA at 240 min (Fig. 3a and c). Importantly, raclopride attenuated both of these effects (Fig. 3a–c). These findings suggest that the KCl-induced dopamine release leads to the up-regulation of these transcription and growth factors via D2R-mediated signaling.
D2R activation up-regulates GDNF via Zif268
As the rise in Zif268 levels in response to D2R activation is followed at a later time point by the up-regulation of GDNF, we hypothesized that D2R-mediated GDNF expression involves the Zif268 transcription factor. To address this possibility, we constructed an adenovirus expressing GFP and shRNA targeting the coding region of Zif268 (adenovirus [AdV] shZif268). Cells were first infected with either the AdV-shZif268, or adenovirus expressing a non-related control sequence (AdV-Ctrl) (Jeanblanc et al. 2009), and the level of Zif268 protein was assessed 48 h later in the presence or absence of quinpirole. Visualization of GFP confirmed a high level of expression of the shZif268 construct (Fig. 4a), and AdV-shZif268 significantly reduced basal Zif268 protein levels compared with AdV-Ctrl (Fig. 4b). Importantly, treatment of cells expressing the shZif268 construct with quinpirole (50 μM for 60 min) blocked the D2R-mediated induction of Zif268, as compared with the quinpirole-treated AdV-Ctrl cells (Fig. 4b).
As shown in Fig. 4c and d, quinpirole treatment caused an increase in the mRNA and protein levels of GDNF in the AdV-Ctrl-infected cells. However, infection of cells with AdV-shZif268 blocked the D2R-mediated up-regulation of GDNF (Fig. 4c–d). Together, these results indicate that Zif268 is required for the increase in GDNF expression resulting from D2R activation.
D2R activation increases Zif268 and GDNF expression via Gβγ and Erk1/2
Next, we set out to determine which signaling pathway underlies the D2R-mediated increase of Zif268 expression. Activation of D2R, a Gαi-protein-coupled receptor, has been shown to stimulate the Erk1/2 signaling pathway through a mechanism involving the Gβγ subunit complex (Faure et al. 1994; Choi et al. 1999; Ghahremani et al. 2000; Beaulieu and Gainetdinov 2011). In addition, increases in the level of Zif268 in vitro in response to cholinergic agonists (Greenwood and Dragunow 2002), calcium influx (Rusanescu et al. 1995), or growth factor treatments (Kumahara et al. 1999; Shin et al. 2009) are dependent on the Erk1/2 signaling pathway (Knapska and Kaczmarek 2004). We therefore tested whether the D2R-mediated up-regulation of Zif268 is a consequence of a Gβγ-mediated activation of Erk1/2. First, we established that Erk1/2 is activated in response to quinpirole treatment in our model system. To do so, we measured levels of phosphorylated, and thus activated, Erk1/2 (pErk1/2) in the presence and absence of quinpirole. As shown in Fig. 5a, quinpirole treatment caused a significant increase in the levels of pErk1/2 within 15 min. To test whether the D2R-mediated activation of Erk1/2 requires Gβγ, we pre-treated the cells with gallein, a small molecule that binds to a domain on Gβγ that is essential for its association with downstream targets (Bonacci et al. 2006). As shown in Fig. 5b, we found that pre-treatment of cells with gallein (20 μM) blocked the increase in Erk1/2 phosphorylation in response to quinpirole. Together, these results show that D2R activation leads to a Gβγ-dependent activation of the Erk1/2 signaling pathway. To determine whether the D2R-mediated up-regulation of Zif268 expression and the subsequent increase in GDNF are the consequence of the Gβγ-mediated activation of the Erk1/2 signaling pathway, cells were pre-treated for 10 min with either gallein (20 μM), or the mitogen-activated protein kinase kinase (MEK, the kinase immediately upstream of Erk1/2) inhibitors U0126 (10 μM), or PD98059 (10 μM) prior to a 30- or 240-min treatment with 50 μM quinpirole, and the levels of Zif268 and GDNF mRNA, respectively, were assessed. As shown in Fig. 6a, the Gβγ inhibitor attenuated the D2R-mediated increase of Zif268, as compared with the vehicle-pre-treated cells. In addition, both MEK inhibitors lowered the basal level of Zif268 expression and blocked the D2R-mediated up-regulation of Zif268 mRNA, as compared with the vehicle-pre-treated controls (Fig. 6a). Moreover, the expression levels of GDNF after D2R activation were also attenuated in the presence of either the MEK or the Gβγ inhibitors (Fig. 6b). Taken together, our findings suggest that quinpirole-mediated activation of the D2R leads to a Gβγ-dependent activation of the Erk1/2 signaling pathway, which is necessary for both Zif268 and GDNF expression.
D2R activation induces a direct interaction of Zif268 with the human GDNF promoter region
Finally, we set out to determine whether Zif268 directly associates with the GDNF promoter after D2R activation. Zif268 binds DNA at its consensus sequence (5′-GCG[G/T]GGGCG-3′), thus directly influencing gene expression (Christy and Nathans 1989). By searching for this consensus sequence in the 1000-bp region upstream of the GDNF transcription start site, we identified two putative Zif268-binding sites (Fig. 7a). The ChIP technique was then used to determine whether Zif268 directly binds the GDNF promoter at either of these sites in the presence of quinpirole. Following a 60-min quinpirole treatment, Zif268-containing DNA–protein complexes were IP-ed from cross-linked cellular lysates, and PCR was conducted to assess whether Zif268 was associated with either putative Zif268-binding site #1 and/or #2 (PCRs 1 and 2; Fig. 7a) in the presence or absence of quinpirole. As a negative control, we conducted a PCR to amplify an intermediate region of the GDNF promoter that was not expected to associate with Zif268 (PCR 3; Fig. 7a). We found that activation of the D2R caused an association of Zif268 with putative binding site #2, but not with putative binding site #1 (Fig. 7b). As expected, Zif268 did not associate with the intermediate region, either in the presence or absence of quinpirole (Fig. 7b). These findings indicate that D2R-mediated signaling promotes the association of Zif268 with a novel binding site at the GDNF promoter.
Here, we show that dopamine, via the activation of the D2R, causes a Gβγ-dependent phosphorylation, and thus activation, of Erk1/2 that leads to an increase in the level of the transcription factor, Zif268. Zif268 then directly interacts with a specific site at the GDNF promoter and up-regulates the levels of the growth factor (model, Fig. 8). Together, our findings illustrate a novel mechanism by which the release of dopamine and the consequent activation of the D2R increases the expression of GDNF.
D2R signaling induces a Zif268-mediated up-regulation of GDNF
We show that potassium-evoked dopamine release in differentiated SH-SY5Y cells and the subsequent activation of D2Rs cause a sequential increase in the expression levels of first the transcription factor, Zif268, and then GDNF. Importantly, we demonstrate that the up-regulation of both Zif268 and GDNF via D2Rs occurs in the rat ventral midbrain, a dopaminergic region that endogenously expresses this receptor (Chen et al. 1991). Finally, we found that D2R-mediated up-regulation of GDNF expression occurs via the induction of expression of the transcription factor, Zif268. Together, our findings suggest that following physiological activation of D2Rs, the levels of GDNF may be increased in a Zif268-dependent manner. Dopamine, which is released in response to dopaminergic neuron excitation, binds to D2Rs to regulate both dopamine synthesis and release via a negative feedback mechanism, in addition to activating various molecular signaling pathways that contribute to the expression of dopamine-driven functions, such as locomotion and reward-seeking behaviors, including drug addiction (Picetti et al. 1997; Usiello et al. 2000). Thus, it is highly plausible that neuronal activity that causes the release of dopamine may, under certain circumstances, induce the expression of GDNF via a D2R-mediated increase in Zif268 expression.
Erk1/2-mediated induction of Zif268 expression up-regulates GDNF
We show that D2R-mediated activation of the Erk1/2 pathway contributes to an increase in the level of Zif268, and is required for the subsequent expression of GDNF. Erk1/2 activation by several different types of stimuli can result in increased Zif268 levels, consequently altering gene expression (Rusanescu et al. 1995; Kumahara et al. 1999; Greenwood and Dragunow 2002; Shin et al. 2009). Thus, activation of Erk1/2 signaling through pathways other than that mediated by the D2R, as we describe, may cause an up-regulation of GDNF via Zif268. For example, growth factors, such as GDNF, are known to cause Erk1/2 pathway activation (Airaksinen and Saarma 2002). Interestingly, we previously showed that GDNF-activated Erk1/2 contributes to an up-regulation of this growth factor's own expression, both in SH-SY5Y cells (He and Ron 2006) and in the ventral tegmental area of rats in vivo (Barak et al. 2011a). In light of our present findings, it is possible that the positive autoregulation of GDNF mRNA by GDNF-activated Erk1/2 occurs via Zif268; however, this possibility is yet to be determined. Interestingly, inhibition of Erk1/2 activation resulted in a decrease in the basal level of Zif268 in SH-SY5Y cells. This is likely because of the reduction of constitutive basal Erk1/2 activity in immortalized cell lines by MEK inhibitors (Kress et al. 2010). This finding puts forward the possibility that a basal level of Zif268 is maintained by baseline Erk1/2 pathway activity. This prospect, as well as whether this is a specific characteristic of dopaminergic cell types, remains to be tested.
Direct association of Zif268 with the human GDNF promoter
We found that following D2R activation, Zif268 is directly associated with a binding sequence approximately 700 base pairs upstream of the human GDNF transcription start site, which we describe as putative Zif268 binding site #2. A previous study reported a direct association of Zif268 with the rat GDNF promoter (Shin et al. 2009) following FGF2 treatment of cultured rat astrocytes. We note that the Zif268-binding motif reported by Shin and colleagues bears a close positional homology to what we describe in our study as putative Zif268 binding site #1. However, we found no association of Zif268 with the human putative Zif268 binding site #1 in either the presence or absence of quinpriole. It is possible that the site-specific binding of Zif268 to the GDNF promoter may depend on whether growth factor or neurotransmitter receptors are activated. It is also possible that Zif268 binds to and enhances the activity of the GDNF promoter in a species- or cell-type-specific manner. In addition, previous sequence analyses of the human GDNF proximal promoter region have revealed putative binding sites for several transcription factors including specificity protein-1 (Sp1), activating protein-2 (AP2), cAMP response element binding, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and metal regulatory element-binding protein (MRE-BP) (Woodbury et al. 1998; Baecker et al. 1999). Thus, whether D2R-induced Zif268 acts alone or in concert with one or more of these transcription factors to up-regulate the expression of GDNF remains to be determined.
Increased dopamine release in the reward circuitry of the brain is a hallmark of exposure to all drugs of abuse (Nestler 2001). Intriguingly, the expression of Zif268 may be altered in response to cocaine and alcohol in brain regions that are targeted by drugs of abuse (Koob et al. 1998; Bertran-Gonzalez et al. 2008; Vilpoux et al. 2009). In addition, Zif268 has been found to play a critical role in the reconsolidation of both heroin- and cocaine-associated memories, which ultimately affects drug-seeking behaviors (Lee et al. 2005; Hellemans et al. 2006). Separately, the expression of GDNF, a potent negative regulator of the rewarding properties of drugs of abuse (Carnicella and Ron 2009; Ghitza et al. 2010), in the CNS is subject to alterations in response to cocaine (Green-Sadan et al. 2003), heroin (Airavaara et al. 2011), phencyclidine (Semba et al. 2004), and alcohol (Ahmadiantehrani et al. 2013). Together with our present findings, it is possible that drug-induced dopamine release can, via a D2R-mediated increase in the levels of Zif268, influence the expression of the growth factor to mediate the physiological effects of drugs of abuse.
The D2R-Gβγ-Zif268-GDNF pathway may also be implicated in hippocampal learning and memory. An age-related decline in the hippocampal expression of endogenous GDNF has been implicated in senescence-related deficits in learning and memory (Miyazaki et al. 2003), while increased levels of GDNF in the hippocampus may function to repair learning and memory impairments caused by chronic stress (Bian et al. 2012). Notably, Zif268 has long been known to play a role in long-term potentiation (Knapska and Kaczmarek 2004; James et al. 2005), as well as in various models of sensory-based learning (Mello et al. 1992; Okuno and Miyashita 1996; Da Costa et al. 1997; Mello 2002) and long-term memory (Jones et al. 2001). More recently, long-term potentiation, synaptic plasticity, and memory formation were found to be dependent on D2R and Gβγ signaling (Saab et al. 2009). Although GDNF, Zif268, and D2R-activated Gβγ have been separately shown to contribute to the learning and memory functions of the hippocampus, whether they act in concert, or in the sequential pathway that we have identified in this study, remains to be tested.
In summary, we found that dopamine-mediated D2R activation leads to a Gβγ- and Erk1/2-mediated increase in the levels of Zif268. Importantly, this elevation in Zif268 levels is specifically required for the D2R-mediated up-regulation of GDNF (model, Fig. 8). Our results are an important contribution to the understanding of the regulation of this critical growth factor. Moreover, these findings illustrate a novel pathway by which dopaminergic signaling, via the D2R, alters GDNF expression, with possible ramifications on learning and memory mechanisms, as well as for the treatment of Parkinson's disease and drug addiction.
This research was supported by funds provided by the National Institutes of Health–National Institute on Alcohol Abuse and Alcoholism (NIH–NIAAA) RO1 AA014366 (D.R.), the State of California for medical research on alcohol and substance abuse through the University of California San Francisco (D.R.), NIH–NIAAA F31 AA017801 (S.A.). The authors have no conflict of interest to declare.