TCDD induces UbcH7 expression and synphilin-1 protein degradation in the mouse ventral midbrain

Abstract

UbcH7 is an ubiquitin-conjugating enzyme that interacts with parkin, an E3 ligase. The UbcH7–parkin complex promotes the ubiquitination and degradation of several proteins via the 26S proteasome. Cellular accumulation of the UbcH7–parkin targets alpha-synuclein and synphilin-1 has been associated with Parkinson disease. In mouse liver, 2,3,7,8-tetrachlorodibenzo-p-dioxin, an aryl hydrocarbon receptor ligand, induces UbcH7 expression. Therefore, the aim of the present study was to determine whether 2,3,7,8-tetrachlorodibenzo-p-dioxin induces Ubch7 mRNA and UbcH7 protein expression in the mouse brain, to characterize the molecular mechanism, and the effect on synphilin-1 half-life. We found that 2,3,7,8-tetrachlorodibenzo-p-dioxin promotes the aryl hydrocarbon receptor binding to Ubch7 gene promoter as well as its transactivation, resulting in an induction of UbcH7 levels in the olfactory bulb, ventral midbrain, hippocampus, striatum, cerebral cortex, brain stem, and medulla oblongata. In parallel, 2,3,7,8-tetrachlorodibenzo-p-dioxin promoted synphilin-1 degradation in an aryl hydrocarbon receptor-dependent way.

1 |. INTRODUCTION

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon and an environmental contaminant. Exposure to TCDD, the most toxic dioxin, results in several deleterious effects, including wasting syndrome, immunotoxicity, hepatotoxicity, teratogenicity, and cancer.^[1] These effects are mediated by the aryl hydrocarbon receptor (AHR) a ligand-dependent transcription factor that is a member of the bHLH-PAS (basic-helix-loop-helix-Per-ARNT-Sim) superfamily. Upon binding to TCDD, AHR translocates from the cytoplasm to the nucleus and binds to xenobiotic response elements (XREs) located in the promoter regions of its target genes. The result is an upregulation of the expression of a battery of genes encoding xenobiotic-metabolizing enzymes^[2] as well as genes involved in liver development,^[3] homeostasis of the immune system,^[4–6] neurogenesis,^[7] cholesterol and glucose metabolism,^[8,9] and cell proliferation and apoptosis.^[10]

Recently, we reported that induction of Ubch7 gene expression through the activation of the AHR results in ubiquitin proteasome system-dependent p53 and c-Fos degradation in mouse liver and Hepa-1c1c7 cells.^[11,12] UBCH7¹ is a ubiquitin-conjugating enzyme with the conserved catalytic domain UBC and is classified as an E2 class I enzyme.^[13] UbcH7 is necessary for normal mouse development, and its inactivation results in abnormal placental development, causing a lethal phenotype.^[14] UbcH7 interacts with parkin a ubiquitin E3 ligase that catalyzes the ubiquitination of several proteins including α-synuclein, Pael-R, Crel-1, and synphilin-1.^[15] The ubiquitinated target protein is usually recognized and degraded by the 26S proteasome.^[16] It was shown that the interaction between parkin and the UBCH7 is required for substrate ubiquitination.^[17] UBCH7 transfers ubiquitin (Ub) to substrates identified by parkin, and therefore plays an important role in the degradation of several parkin targets, including those whose accumulation leads to dopaminergic cell death.^[18,19] Accumulation of UBCH7–parkin substrates such as synphilin-1 in dopamine neurons has also been associated with Parkinson disease (PD).^[20,21].

Because UbcH7 is essential for parkin function, the aim of the present study was to determine whether TCDD induces UbcH7 expression in the mouse brain, particularly in the ventral midbrain where dopaminergic neurons are housed, and, if so, to determine whether this induction results in degradation of synphilin-1.

2 |. MATERIALS AND METHODS

2.1 |. Materials

TCDD was purchased from AccuStandard (New Haven, CT). Phenylmethanesulfonyl fluoride (PMSF) and dimethyl sulfoxide (DMSO) were acquired from Sigma (St. Louis, MO).

2.2 |. Animals and treatments

The development of Ahr-null mice was described previously.^[[4] Wild-type (WT) littermates on a C57BL/6J background were used as controls. The animals were housed in a pathogen-free facility and fed with autoclaved Purina rodent chow (St. Louis, MO) with water available ad libitum. All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals, as adopted and enforced by the U.S. National Institutes of Health and the Mexican Regulation of Animal Care and Maintenance (NOM-062-ZOO-1999, 2001). Mice were distributed randomly into TCDD and vehicle treatment groups (N=3/group), and treatments were carried out as described previously.^[22] TCDD was dissolved in corn oil, and a single dose of TCDD (250 μg/kg) or corn oil alone (vehicle) was administered by gavage to C57BL/6J WT and Ahr-null male mice (8–9 weeks old). Seven days later, the mice were anesthetized with pentobarbital and killed by decapitation. The brains were rapidly removed, and the olfactory bulb, ventral midbrain, hippocampus, striatum, cortex, stem, and medulla oblongata dissected out. Liver sections were also extracted. Brain and liver samples were snap frozen and stored at –70^◦C.

2.3 |. Real-time quantitative PCR

Total RNA was prepared from mouse liver and dissected brain region sections using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Camarillo, CA). RNA was quantified spectrophotometrically at an optical density of 260. RNA integrity was evaluated by electrophoresis on 1% agarose gels. cDNA was prepared for the quantitative PCR from 2 μg of total RNA using the SuperScript First-Strand Synthesis kit (Invitrogen, Camarillo, CA) and oligo dT. PCRs were performed in a StepOne Real-Time PCR System (Applied Biosystems, Branchburg, NJ) and analyzed using the comparative threshold cycle (C_T) method. The mRNAs encoding Ahr and Ubch7, with 18S ribosomal RNA (rRNA, endogenous) were amplified in a single PCR reaction to allow for normalization of the mRNA data. The PCR reaction mixture contained 2 μL of cDNA, 1xTaqMan universal PCR master mix (Applied Biosystems, Branchburg, NJ) and 0.9 and 0.25 μM primers and probes, respectively. The primer and probe sequences used for Ubch7 were as follows: 5^′-TGCCAGTCATTAGTGCTGAAAACT-3^′ (forward), 5^′-GGGTCATTCACCAGTGCTATGAG-3^′ (reverse), and probe (FAM): AAGACTGACCAAGTAATCC. The probes used for Ahr mRNA and the 18S rRNA were obtained from Applied Biosystems (Branchburg, NJ) with identification numbers Mm01291777_m1 and Mm00507222_s1, respectively.

2.4 |. Western blotting

Brain sections were homogenized in buffer containing 20 mM HEPES, 350 mM NaCl, 1 mM MgCl₂, 0.5 mM EDTA, 0.1 mM EGTA, 1% Non-idet P40, 0.5 mM DTT, 0.1% PMSF, and Mini cOmplete protease inhibitor cocktail (1 tablet/10 mL; Roche, Mannhein, Germany). The protein suspension was homogenized completely by sonication for 30 s. Protein concentrations were determined using the Bradford assay (Bio-Rad, Hercules, CA). Aliquots (40 μg) were solubilized in sample buffer (60 mM Tris–HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 20% glycerol, 2% mercaptoethanol, and 0.001% bromophenol blue) and subjected to 12% SDS polyacrylamide gel electrophoresis. Protein extracts were transferred to a nitrocellulose membrane using a mini trans-blot (Bio-Rad, Hercules, CA). The transfer was performed at a constant voltage of 80 V for 2 h in transfer buffer (48 mM Tris– HCl, 39 mM glycine, pH 8.3, and 20% methanol). Following the transfer, membranes were blocked overnight at 4^◦C in the presence of 2% nonfat dry milk and 0.5% bovine serum albumin (BSA) in blocking buffer (25 mM Tris–HCl, pH 7.5, 150 mM NaCl) and subsequently incubated at 4^◦C for 3 h with goat polyclonal anti-UbcH7 (1:2000; Abcam, Cambridge, MA), rabbit anti-synphilin-1 (1:500; Santa Cruz Biotechnology, Visalia, CA) or β-actin (1:1000; Zymed, San Francisco, CA) diluted in buffer (25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20, 0.05% nonfat dry milk, and 0.05% BSA). After washing, the membranes were incubated with the relevant horseradish peroxidase (HRP)-conjugated secondary antibodies HRP-goat anti-rabbit IgG (Zymed, San Francisco, CA), or HRP-rabbit anti-goat IgG (Pierce, Rockford, IL), for 2 h at 4^◦C. The membranes were washed and the immunoreactive protein detected using an ECL Western blotting detection kit (Amersham, Arlington Heights, IL). The integrated optical density of the bands was quantified using scanning densitometry (GS-800 calibrated densitometer; Bio-Rad, Hercules, CA).

2.5 |. Immunofluorescence

Localization of UbcH7 protein was determined by inmmunofluorescence in substantia nigra pars compacta of adult mouse brain and to identify dopaminergic neurons tyrosine hydroxylase (TH) was used. Mice were injected with an overdose of pentobarbital (80 mg/kg) and perfused intracardially with saline, subsequently the brains were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and postfixed in the same solution for 48 h, then incubated in 20% sucrose PBS. Coronal sections of 30-μm thick were cut in a cryostat and used for immunohistochemical assays. Briefly, free-floating sections were incubated at room temperature for 1 h in 0.25% Triton X-100/PBS buffer and subsequently in 5% BSA, 0.25% Triton X100/PBS buffer for 3 h. Then, sections were incubated in 3% BSA-0.25% Triton X-100/PBS solution containing the UbcH7 (1:100) and TH (1:1000) antibodies (Cell Signaling Technologies, MA). After a 48-h incubation, sections were incubated with secondary antibodies (1:400) (Alexa 488 anti-rabbit and Alexa 594 anti-mouse IgG; Thermo Fisher Scientific, Waltham, MA) diluted in PBS with 0.25% Triton X-100. Sections were counterstained with (4^′,6-Diamidino-2-Phenylindole, Dihydrochloride) DAPI to reveal the nuclei and observed under a confocal microscope. Triple-labeled images were obtained using a confocal laser-scanning microscope (Leica TCS-SPE, Wetzlar, Germany) in the XYZ (Z-stacks) mode using a 63× objective. The Z-stacks (three to four optical slices) were then converted into three-dimensional projection images using the Leica LAS AF lite software. Quantification of UbcH7 fluorescence levels in dopaminergic cells was determined as described in Burgess et al. Briefly, using Image J software, mean fluorescence measurements were obtained, along with several adjacent background readings. Fluorescence readings of eight ROIs (defined as Substantia nigra) per treatment were calculated.^[23]

2.6 |. In silico analysis

The mouse Ubch7 promoter was analyzed using the web-based bioinformatics tool TRANSFAC (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3). Score >75.

2.7 |. Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) assays were performed according to the kit protocol (Santa Cruz Biotechnology, Visalia, CA) with an anti-AHR antibody (Thermo Scientific, Rockland, IL). The PCR product corresponding to the Ubch7 proximal gene promoter was generated from an aliquot of immunoprecipitated material. Brain homogenates from WT mice treated with a single oral dose of TCDD or vehicle (corn oil) were washed with PBS buffer and cross-linked with 1% formaldehyde. After chromatin isolation, the DNA was fragmented, and immunoprecipitation was performed. The cross-linking was reversed, the DNA was purified, and PCR amplification was performed as follows: initial denaturation at 94^◦C for 3 min, 25 cycles of denaturation at 94^◦C for 30 s, annealing at 65^◦C for 30 s, and extension at 72^◦C for 3 s. A final extension cycle at 72^◦C for 10 min was added to the end of the program. The oligonucleotides used for PCR amplification were 5^′-GGCTAGAACCCCCTCACTTC-3^′ (forward) and 5´-GGCTAGAACCCCCTCACTTC-3 (reverse).

2.8 |. Cell culture and transfections

SH-SY5Y cells were obtained from American Type Culture Collection (CRL-2266; Manassas, VA). The cells were grown in 100-mm dishes with DMEM high glucose medium (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gibco, Logan, UT) and 1% antibiotic/antimycotic (Invitrogen, Carlsbad, CA) at 37^◦C in a humidified incubator with a 5% CO₂ atmosphere. The reporter plasmid pGL4/-832UbcH7 containing the Ubch7 gene promoter was described previously.^[12] SH-SY5Y cells were cultured in DMEM high glucose medium as described above. Transfection was performed using Escort IV (Sigma, San Louis, MO). Each culture (1 × 10⁶ cells/ml) was transfected with 5 μg of pGL4/-832UbcH7 and 1 μg of pRL-CMV as an internal control. The media was replaced with fresh media containing TCDD for 48 h posttransfection. After 48 h, the cells were homogenized by incubation with Passive Lysis Buffer (Promega, Madison, WI) for 15 min at room temperature. Luciferase activity was performed using the Dual-Glo Luciferase Reporter Assay System (Promega, Madison,WI) according to the manufacturer’s instructions in a Modulus luminometer (Turner Biosystems, Sunnyvale, CA). Blanks were obtained by performing the luciferase activity assays in mock-transfected cells. Firefly luciferase activity levels were normalized by comparison to the activity levels of Renilla luciferase.

2.9 |. Statistical analysis

Results are presented as the mean values ± standard deviation (SD). The statistical significance of the data was evaluated using the Student’s t-test. In all cases, the differences between groups were considered to be statistically significant when the p value was less than 0.05.

3 |. RESULTS

A previous study showed that activation of hepatic AHR results in UbcH7 gene induction.^[11] UBCH7 is the reported E2 binding partner of parkin^[17] and the combined activities of these enzymes conjugate Ub onto several substrates such as synphilin-1 for subsequent degradation by the 26S proteasome.^[15] Accumulation of UBCH7–parkin substrates was also associated with PD.^[18] Therefore, we investigated whether activation of AHR by TCDD regulates UbcH7 expression in the mouse ventral midbrain, thereby promoting synphilin-1 protein degradation.

Initially, the Ahr mRNA was measured in olfactory bulb, ventral mid-brain, hippocampus, striatum, cortex, brain stem, and medulla oblongata tissue samples, with liver serving as a positive control reference tissue (Figure 1). Ahr mRNA was observed in all brain regions analyzed, with the highest levels found in the cortex followed by the ventral midbrain, striatum, and hippocampus. Olfactory bulb, brain stem, and medulla oblongata samples had the lowest levels of Ahr mRNA expression among the brain regions analyzed. Notably, the Ahr mRNA levels in the cortex and ventral midbrain were quite high at 70% and 40% that found in liver levels, respectively.

FIGURE 1.

AHR mRNA expression in brain. Total mRNA was extracted from the liver, olfactory bulb, ventral midbrain, hippocampus, striatum, cortex, brain stem, and medulla oblongata from sacrificed mice. Ahr mRNA levels were determined by qPCR and normalized to 18S ribosomal RNA. Ahr mRNA expression levels for each brain section are reported relative to expression in the liver. Results are expressed as the mean ± SD of samples from three different mice

Liver and cortex samples exhibited a fivefold Ubch7 mRNA induction upon TCDD treatment compared to their respective controls (Figure 2). Ubch7 mRNA was increased approximately twofold after TCDD treatment in samples isolated from the other brain regions including the ventral midbrain. In contrast, TCDD treatment failed to induce Ubch7 mRNA expression in brain samples from Ahr-null mice, indicating that this effect is AHR dependent.

FIGURE 2.

AHR-dependent induction of brain Ubch7 mRNA levels. WT and Ahr-null mice were treated with a single oral dose of TCDD or corn oil (vehicle). Total mRNA was extracted from the liver, ventral midbrain, olfactory bulb, hippocampus, cortex, striatum, brain stem, and medulla oblongata. Ubch7 mRNA levels were determined by qPCR and normalized to 18S ribosomal RNA. Results are expressed as the mean ± SD of samples from three different mice. *p ≤ 0.05, treatments vs. control

Furthermore, Western blot analysis showed that the TCDD treatment increased UbcH7 protein levels in the ventral midbrain (where dopaminergic neurons are present) to more than twofold of control levels. Confocal microscopy showed that UbcH7 immunopositivity colocalized with TH (dopaminergic cell marker) immunopositivity, indicating that this ubiquitin-conjugating enzyme is indeed expressed in dopaminergic neurons. Moreover, quantification of the fluorescence intensity of the UbcH7 signal showed that TCDD treatment increased UbcH7 levels relative to control levels in WT mice (data not shown). In contrast, TCDD treatment fails to induce UbcH7 in Ahr-null dopaminergic neurons (Figure 3).

FIGURE 3.

TCDD treatment induced UbcH7 protein expression in the ventral midbrain. WT and Ahr-null mice were treated with TCDD or corn oil (vehicle). (A) Western blot of UbcH7 from ventral midbrain samples of WT mice. β-actin was used as a loading control. S1 = sample 1 and S2 = sample 2. (B) Relative expression of UbcH7 protein levels. Results are expressed as the mean±SD of samples from three different mice. *p ≤ 0.05, treatments vs. control. (C) Representative confocal microscopy images of UbcH7 expression in the ventral midbrain region. TH, UbcH7, and DAPI were visualized as green, red, and blue, respectively. Insets show in more detail the colocalization of the TH and UbcH7

In silico analysis of the mouse Ubch7 gene promoter for identification of potential transcription factor binding sites revealed common regulatory sequences, such as TATA box, Oct-1, GATA, c-ETS, as well as Pbx-1 and Cdxa binding sites between position –595 and the putative transcription start site (+1). Four putative (XREs) were also identified (data not shown). ChIP assays of brain extracts from WT mice treated with TCDD or vehicle revealed that AHR bound the Ubch7 gene promoter in the control sample and that this interaction increases notably after TCDD treatment (Figure 4). After confirming, by con-focal microscopy that SH-SY5Y cell expresses AHR (data not shown), transactivation studies were performed. Following TCDD treatment, the luciferase activity of SH-SY5Y cells that had been transfected with a pGL4 reporter vector containing the Ubch7 gene promoter upstream from the luciferase open reading frame (pGL4/-832UbcH7) was elevated in a dose response manner, reaching threefold increase, compared to untreated controls, with the 50 nM dose, indicating that the TCDD treatment trans-activated the Ubch7 gene promoter (Figure 4).

FIGURE 4.

AHR binds to and transactivates the Ubch7 promoter. (A) ChIP analysis of AHR binding to the Ubch7 gene promoter region. Immunoprecipitated material was obtained from brain samples from mice treated with vehicle (line 2) or TCDD (line 3). PCR products corresponding to the –832/–20 bp of the Ubch7 promoter region were generated. Genomic DNA was used as positive control (line 4). (B) Luciferase activity of SH-SY5Y cells (1 × 10⁶) transfected with 5 μg of the pGL4/-832UbcH7 vector containing the firefly luciferase open reading under the control of the Ubch7 promoter and then treated with TCDD or DMSO (vehicle) for 48 h. Luciferase activity was normalized to that of Renilla luciferase and expressed as the mean ± SD of three independent experiments. * p ≤ 0.05; ** p ≤ 0.01, control vs. treatments

We then examined whether the induction of UbcH7 correlates with a decrease on synphilin-1 protein levels. Western blots analysis revealed that TCDD treatment resulted in a 40% decrease in levels of the UbcH7–parkin substrate synphilin-1 in ventral midbrain of WT mice (Figure 5). In contrast, TCDD treatment failed to decrease synphilin-1 protein levels in Ahr-null mice, indicating that this effect is AHR-dependent.

FIGURE 5.

TCDD treatment decreased synphilin-1 protein levels in an AHR-dependent way. A) Western blot of synphilin-1 from ventral midbrain samples of WT and Ahr-null mice treated with TCDD or corn oil (control). β-actin was used as a loading control. B) Relative expression of synphilin-1 protein levels. Results are expressed as the mean ± S.D. of samples from three different mice. * p ≤ 0.05, treatments vs. control

4 |. DISCUSSION

In agreement with previous studies reporting the expression of Ahr mRNA in the rodent brain,^[24] in this study Ahr mRNA was detected in all brain regions analyzed. The highest Ahr mRNA expression levels were observed in the cortex and might be linked to NMDA receptor expression and activity as suggested previously by Lin et al.^[25] High levels of Ahr mRNA were also detected in the ventral midbrain. This result agrees with earlier findings, where AHR was detected in the substantia nigra in rats.^[26] The above results suggest that AHR may play an important role in brain physiology by regulating the expression of genes in several brain regions, in particular in the ventral midbrain.

Activation of AHR by TCDD resulted in an increase in Ubch7 mRNA levels in all brain regions analyzed, including the ventral midbrain, that were approximately double the control levels. This induction is likely AHR dependent because no increases were observed in the Ahr-null mice after TCDD treatment. According to the in silico study, the Ubch7 promoter contains four putative XREs. The ChIP assay suggested that, most likely, activation by TCDD causes AHRs to bind one or more of these XREs. Interestingly, AHR-Ubch7 promoter interaction was detected in controls, indicating that this transcription factor might control not only Ubch7 induction but also its basal expression. Regarding this point, several endogenous AHR ligands, derived from tryptophan metabolism, have been identified^[27] and may regulate Ubch7 basal expression through their binding to the AHR. Moreover, transfection studies established that AHR activation and binding to the Ubch7 promoter, after TCDD treatment, resulted in promoter transactivation. Taken together, these results demonstrated that the Ubch7 gene is under control of the AHR not only in the liver but also in the mouse brain.

Because UbcH7 is the partner required for proper parkin function, we focused on the ventral midbrain region to evaluate UbcH7 protein levels. TCDD treatment resulted in a twofold increase in ventral midbrain UbcH7 protein levels. Additionally, confocal microscopy indicated that AHR activation induces UbcH7 expression only in dopamine neurons from WT mice. Although previous reports have shown expression of UBCH7 and the rat ortholog UbcR7 in the substantia nigra,^[28,29] the present study demonstrated expression of UbcH7 specifically in dopaminergic neurons.

The increase in AHR activation observed in response to TCDD treatment might augment ubiquitination and degradation of UbcH7– parkin substrates. In agreement with this possibility, TCDD treatment decreased synphilin-1 protein levels in the ventral midbrain in an AHR-dependent way.

To our knowledge, there is no information regarding UBCH7 gene variations (such as polymorphisms) that have been associated with any neuropathology, such as PD. However, some UBCH7 gene variant haplotypes that result in increased levels of this E2 enzyme were associated with autoimmune diseases, such as lupus erythematosus and Crohn’s disease.^[28,30] Therefore, UBCH7 gene polymorphisms or exposure to chemical agents such as TCDD that modify UBCH7 levels might promote the development of certain pathologies.

In conclusion, these results identify the AHR as an indirect modulator of the degradation of UbcH7–parkin substrates in the mouse brain. Future studies are needed to characterize the consequences of AHR-mediated induction of UbcH7 and to explore both the toxicological implications and the therapeutic potential of this pathway.