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Keywords:

  • Ca2+/calmodulin-dependent protein kinase II;
  • dopamine D2 receptor promoter;
  • mitogen-activated protein kinase;
  • NB2A cell;
  • Zif268

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

To investigate regulation of D2 receptor (D2R) gene expression by protein kinases, we evaluated effects of constitutively active MAPK kinase kinase (MEKK), Ca2+/calmodulin-dependent protein kinase (CaMK) II, CaMKIV and cyclic AMP-dependent protein kinase (PKA) on D2R promoter activity using luciferase reporter gene assays. A 1.5-kbp fragment containing the rat D2R promoter was cloned upstream of the reporter and transfected into D2R-expressing NB2A cells or nonexpressing NG108-15 and C6 glioma cells. MEKK and CaMKII, but not CaMKIV and PKA, increased promoter activity 4.5- and 1.5-fold, respectively, in NB2A cells. Inhibitory effects of a MEK inhibitor and lack of effect by dominant negative (DN)-JNK1 or DN-p38MAPK revealed that ERK but not JNK and p38MAPK is involved in MEKK-induced promoter activation. Deletion and mutation of the promoter revealed that the MEKK-responsive region was Sp1 site B between nucleotides −56 and −47. Overexpression of Sp1 suppressed promoter activity without affecting MEKK-induced activation. Interestingly, overexpression of Zif268 increased promoter activity through the region between nucleotides −56 and −36. Increased activity by Zif268 was additive with CaMKII-induced activation but not with activation induced by MEKK. Co-transfection with CaMKII stimulated nuclear translocation of Zif268. These results suggest that ERK and CaMKII positively regulate the D2R promoter and that Zif268 is a potential transcription factor for the CaMKII-dependent pathway.

Abbreviations used
ATF-1

activating transcription factor-1

BDNF

brain-derived neurotrophic factor

CaMKII

Ca2+/calmodulin-dependent protein kinase II

C/EBPβ

CCAAT/enhancer-binding protein β

CREB

cAMP responsive element binding protein

D2R

dopamine D2 receptor

DMEM

Dulbecco's modified Eagle's medium

DRRF

dopamine receptor regulating factor

DTT

dithiothreitol

ERK

extracellular signal-regulated kinase

JNK

c-Jun N-terminal kinase

MAPK

mitogen-activated protein kinase

MEK

MAPK kinase

MEKK

MAPK kinase kinase

p38MAPK

p38 mitogen-activated protein kinase

PBS

phosphate-buffered saline

PKA

cyclic AMP-dependent protein kinase

PMSF

phenylmethylsulfonyl fluoride

PVDF

polyvinylidene difluoride

SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SRF

serum response factor

Dopamine pathways play important roles in mammalian brain controlling a variety of functions including locomotor activity, cognition, emotion and endocrine regulation (Missale et al. 1998). Disorders affecting dopamine pathways have been associated with several pathologies of the central nervous system, such as Parkinson's disease, schizophrenia, Tourette's syndrome and hyperprolactinoma. Dopamine receptors are coupled with trimeric GTP-binding proteins and classified into distinct subfamilies, including the D1-like receptor, which includes D1 and D5 receptors, and D2-like receptors, which include D2, D3 and D4 receptors, based on pharmacological characteristics and sequence homology (Missale et al. 1998). D1-like receptors activate adenylyl cyclase by coupling to Gs, whereas D2-like receptors inhibit adenylyl cyclase by coupling to Gi/Go subunits (Missale et al. 1998). Among these dopamine receptors, the D2 receptor (D2R) is implicated in schizophrenia since all clinically effective anti-psychotic drugs act as antagonists for D2R and, except for atypical anti-psychotic drugs, their abilities to block D2R correlate with their anti-psychotic efficacy. It was first reported that the numbers of D2R were abnormally elevated in postmortem striatum samples from schizophrenic patients (Lee et al. 1978; Owen et al. 1978). Later, several in vivo brain imaging studies for the density of D2R in never-medicated schizophrenia patients using D2R selective ligands have been reported. Those results were not consistent. Initially, elevated levels of D2R were found in the striatum of schizophrenia patients (Wong and Wagner 1986). However, most in vivo brain imaging studies revealed no change of the striatal D2R density between control and schizophrenia patients (Farde et al. 1990; Farde 1997). On the other hand, Hietala et al. 1994) found a subgroup of schizophrenia patients with relatively high striatal D2R density. Considering the effectiveness of anti-psychotic drugs for schizophrenia patients and high D2R density in some group of them, elevated signal transduction pathway through D2R may be involved in pathology of positive symptoms of schizophrenia. Therefore, understanding the molecular mechanisms underlying D2R gene expression is crucial to understand the function of dopaminergic systems in psychological disorders and their therapies.

The primary structure of the promoter region of the rat D2R gene does not contain TATA and CCAAT boxes but is GC rich and contains several putative Sp1 binding sites (Minowa et al. 1992; Valdenaire et al. 1994). It has also been reported that the D2R promoter is under strong negative control by two silencing elements (D2Neg A, from nucleotides −217 to −160, and D2Neg B, from nucleotides −116 to −76) in D2R-expressing NB41A3 cells (Minowa et al. 1992 and 1994). D2Neg B consists of an Sp1 consensus sequence (Sp1 site A) and three TGGG repeats (GT box), which also interact with Sp1 (Minowa et al. 1994), Sp3 (Yajima et al. 1998) and a zinc finger type transcription factor such as dopamine receptor regulating factor (DRRF) in the D2R promoter (Hwang et al. 2001). On the other hand, Valdenaire et al. (1994) reported that there are no silencing elements in the D2R promoter in D2R-expressing MMQ cells and that the negative regulation by the silencing elements depends on the cell type used. They and other groups suggest the existence of a strong positive element in the D2R promoter between nucleotides −75 and −30, which contains an Sp1 consensus sequence (Sp1 site B) (Minowa et al. 1992; Valdenaire et al. 1994). Transcription factors, such as Sp1, Sp3 and DRRF, bind to the proximal cis-elements in vitro and, among them, Sp3 and DRRF inhibit D2R promoter activity (Minowa et al. 1994; Chernak et al. 1997; Yajima et al. 1998; Hwang et al. 2001). Whilst increase in the D2R promoter activity by treatment with retinoic acid (RA) was reported in transfected COS-1 cells (Samad et al. 1997) and C6 glioma cells (Valdenaire et al. 1998), RA-response elements from nucleotides −68 to −55 were indicated as responsible for its activation (Samad et al. 1997; Valdenaire et al. 1998). However, to our knowledge, transcriptional regulation acting through signal transduction pathways mediated by protein kinases has not been examined for the D2R promoter.

This study focuses on the roles of three major signal transduction pathways, namely, the mitogen-activated protein kinase (MAPK), Ca2+/calmodulin-dependent protein kinase (CaMK), and cyclic AMP-dependent protein kinase (PKA) pathways on the D2R promoter. To evaluate the effects of these pathways, we expressed constitutively active mutants of MEKK, PKA, CaMKII and CaMKIV. MEKK acts as an upstream kinase of three major MAPKs: extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK) (Wood and Russo 2001). These MAPKs phosphorylate transcription factors such as Elk-1, c-Myc, c-Jun and c-Fos and regulate their transcriptional activities (Davis 1995; Fukunaga and Miyamoto 1998; Curtis and Finkbeiner 1999). CaMKIV and PKA stimulate gene expression through phosphorylation of Ser133 of the cAMP responsive element binding protein (CREB) (Enslen et al. 1994; Montminy 1997). CaMKII phosphorylates not only Ser133 but also Ser142 of CREB, thereby blocking its function (Mattews et al. 1994). CaMKII also phosphorylates CCAAT/enhancer-binding protein β (C/EBPβ) (Wegner et al. 1992; Yano et al. 1996), activating transcription factor-1 (ATF-1) (Shimomura et al. 1996) and serum response factor (SRF) (Miranti et al. 1995) and stimulates expression of atrial natriuretic factor (Ramirez et al. 1997) and brain-derived neurotrophic factor (BDNF) (Takeuchi et al. 2000). Here we isolated the rat D2 receptor promoter region from nucleotides −1518–275 and evaluated the effects of constitutively active protein kinases on promoter activity using luciferase reporter assays. We further investigated the elements and transcription factors regulated by these protein kinases for activation of the D2R promoter.

Cell culture and transfection

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

NB2A cells were grown in modified Eagle's medium (MEM) containing 10% fetal calf serum, 10 mm glutamine and 7.5% NaHCO3; NG108-15 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing additions as described by Higashida et al. (1986); and C6 glioma cells were grown in DMEM-containing 10% fetal calf serum. Cells were cultured in a humidified incubator with a 5% CO2 atmosphere at 37°C. NB2A, NG108-15 and C6 glioma cells were plated in 35-mm dishes and cultured in standard medium for 24 h. Cells were then transfected with FuGENE 6 transfection reagent and DNA at a ratio of 3 : 1 (µL : µg) in 1.5 mL of serum-free medium for 6 h. The culture medium was changed to the standard medium and cells were further cultured for 40–48 h.

RT-PCR

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

Total RNAs were prepared from the rat striatum and NB2A cells using TRIZOL LS Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Messenger RNA was reverse-transcribed using an oligo(dT) primer (Promega, Madison, WI, USA) and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Grand Island, NY, USA). PCRs were performed using a primer pair for rat D2R, consisting of a sense primer (5′-AGAGCCAACCTGAAGACACCA-3′) and an antisense primer (5′-GATGATGAACACACCGAGAAC-3′). D2R primers were positioned upstream and downstream of the variable domain of D2R. PCR products were separated by electrophoresis on 1.0% agarose gels and visualized by ethidium bromide staining. As positive controls PCR products derived from D2LR and D2SR expression vector templates (Takeuchi et al. 2002) were electrophoresed at the same time.

Construction of plasmids

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

The pCAGGSneo expression vector was kindly provided by Professor J. Miyazaki (Osaka University, Japan). cDNAs for the CaMKIIδ2 isoform were obtained and inserted into the EcoRI site of pCAGGSneo under control of the chicken β-actin promoter (Takeuchi et al. 1999). To obtain a constitutively active mutant of CaMKII (Act KII), we replaced Thr287 of CaMKIIδ2 with Asp (Hughes et al. 2001). The C-terminal of Act KII, in which Thr287 was replaced with Asp, was obtained by amplification using PCR with a sense primer (5′-TCAACGTTCTACTGTTGCCTCCATGATGCACAGGCAGGAGGATGTAGA-3′) and an antisense primer (5′-TCTCGAGTTAGAAGACCCAAATGTGAA-3′). The sense primer contains an AclI site at the 5′ end; sequences changing Thr287 to Asp are shown by underlining. The antisense primer corresponds to nucleotides 1507–1526 and contains an EcoRI site at the 5′ end. The PCR products were cut with AclI. The N-terminal of Act KII was excised with EcoRI and AclI from cDNA encoding wild-type CaMKIIδ2. Both fragments were ligated with the AclI sites to each other and then inserted into pCAGGSneo, which was verified by sequencing (termed pActKII). To obtain constitutively active CaMKIV (Act KIV), the cDNA for the entire coding region of CaMKIVα was obtained from the rat cerebellum by RT-PCR using a sense primer (5′-ATGCTCAAAGTCACGGTGCCCTCC-3′), and an antisense primer (5′-TTAGTACTCTGGCAGAATAGCATC-3′). Act KIV was obtained by amplification of the catalytic domain of CaMKIV (1–313 amino acids) using PCR with the sense primer (5′-TTCTCGAGCTATGCTCAAAGTCACGGTG-3′) and the antisense primer (5′-TTCTCGAGAAAGCTTTTTCTGAGCGGTATC-3′), both of which contain an XhoI site 5′ end of each primer. The PCR products were cut with XhoI and inserted into the XhoI site of pEYFP-Nuc expression vector (termed pKIV-Nuc). Dominant negative JNK 1 (DN-JNK1) was constructed by replacing Tyr185 and Thr183, which had to be phosphorylated for activation, with Ala and Phe, respectively. The cDNA of the entire JNK1 coding region was obtained from RNA from NB2A cells by RT-PCR using a sense primer (5′-TTCTCGAGATGAGCAGAAGCAAACGT-3′) and an antisense primer (5′-TTCTCGAGTCATTGCTGCACCTGTGCTA-3′), both of which contain 5′XhoI sites. The C-terminal of DN-JNK1 was obtained by PCR with the antisense primer used to amplify the coding region of JNK1 and a sense primer (5′-AACAAGCTTTATGATGTTTCCTGCAGTGGT-3′), which contained an HindIII site and in which Tyr185 and Thr183 codons were replaced with Ala and Phe codons, respectively. The DN-JNK1 N-terminus was obtained by PCR with the sense primer used for amplification of the coding region of JNK1 and an antisense primer (5′-ATAAAGCTTGTTCCTGCAGTCCTC-3′), which contained a 5′HindIII site. Both amplified fragments were cut with HindIII and ligated with the HindIII sites to each other. The ligated fragments were inserted into pCAGGSneo and sequenced (termed pDN-JNK1). Similarly, the cDNA encoding all of p38MAPK was obtained from NB2A cell RNA by RT-PCR using a sense primer (5′-TTCTCGAGATGTCGCAGGAAAGGCCCACGTTC-3′) and an antisense primer (5′-TTCTCGAGTCAGGACTCCATTTCTTCTT GGTC-3′), which contain 5′XhoI sites. The C-terminus of dominant negative p38MAPK (DN-p38MAPK) was obtained by PCR with the antisense primer described above and a sense primer (5′-GTGGTACCGAGCCCCAGAGATCAT-3′). The DN-p38MAPK N-terminus was obtained by PCR with the sense primer described above and an antisense primer (5′-TCGGTACCACCTGGTAGCCACTGCGCCAAACATCTC-3′), in which Tyr182 and Thr180 codons were replaced with Ala and Phe codons, respectively. Both amplified fragments were cut with KpnI and ligated with the KpnI sites to each other. The ligated fragments were inserted into pCAGGSneo and sequenced (termed pDN-p38MAPK). The following vectors were obtained from the Path Detect System (Stratagene, La Jolla, CA, USA): pFc-MEKK (encoding amino acids 380–672 of MEKK), pFc-PKA (encoding the PKA catalytic subunit), and pSRE-Luci and pCRE-Luci. The expression vector for constitutively active MEKK encodes amino acids 380–672 of MEKK, in which the regulatory N-terminal domain is deleted. The expression vector for constitutively active PKA encodes the catalytic subunit. The cDNAs for Sp1 were obtained by amplification from NB2A cell RNA by RT-PCR with the sense primer (5′-ATGAATTCATGAGCGACCAAGATCACTCAAT-3′) and an antisense primer (5′-TTGAATTCCTCAGAAACCATTGCCACTGATA-3′), both of which contain 5′-EcoRI sites. The cDNAs for Zif268 were obtained by RT-PCR amplification from NB2A cell RNA using a sense primer (5′-ATGAATTCATGAGCGACCAAGATCACTCAAT-3′) and an antisense primer (5′-TTGAATTCCTCAGAAACCATTGCCACTGATA-3′), which also contain 5′-EcoRI sites. The cDNAs of Sp1 and Zif268 were cut with EcoRI and inserted into pCAGGSneo (termed pSp1 and pZif268, respectively). To construct DRRF-HA, we first amplified the coding region of DRRF by RT-PCR from NB2A cell RNA with a sense primer (5′-GGAATTCATGTCGGCGGCCGTGGCGTGTGT-3′), which contains a 5′-EcoRI site, and an antisense primer (5′-AACATCGTATGGGTACAAGCCAGCAGGAGCTGGGCT-3′), which contains part of the sequence of the HA-tag at the 5′ end. Using the first PCR products as a template, the second amplification was carried out with the same sense primer and an antisense primer (5′-TTGAATTCTTAAGCGTAATCTGGAACATCGTATGGGTA-3′), which consists of a 5′-EcoRI site and the sequence of HA-tag. The cDNA of DRRF-HA was cut with EcoRI, inserted into pCAGGSneo, and sequenced (termed pDRRF-HA).

Construction of D2R promoter plasmids and luciferase assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

Rat genomic DNA was isolated from brain tissue using DNeasyTM Tissue Kit (Qiagen, Valencia, CA, USA). PCR amplification of the D2R promoter region (from nucleotides −1518–309: numbered with the transcription initiation site on exon 1 as 1) was undertaken using the sense primer (5′-TTCTCGAGCGGTGCATCTCAGAGAAATAAG-3′) containing a 5′XhoI site and the antisense primer (5′-GTTCTTACCTTCAAGCCATATG-3′). That fragment was subcloned into the pCR2.1 cloning vector and sequenced. The promoter region of D2R from nucleotides −1518–275, which contains a naturally occurring XhoI site in exon 1, was cut with XhoI and inserted into the XhoI site of pGL3-basic luciferase reporter vector (Promega)(termed pD2R-1518). The additional five truncated and two mutated promoter vectors were constructed by PCR with seven different sense primers, each containing a 5′XhoI site, and the common antisense primer (5′-GTTCTTACCTTCAAGCCATATG-3′) using pD2R-1518 as a template. The seven sense primers were as follows: 5′-TTCTCGAGCCCTGGGTGGGTGGGG-3′ for pD2R-117; 5′-TTCTCGAGCCCGGGCGCCTCGTG-3′ for pD2R-79; 5′-TTCTCGAGACCCCGCCCCCTCCTCCT-3′ for pD2R-56; 5′-TTCTCGAGCAGCGCTCTGATTCCG-3′ for pD2R-36; 5′-TTCTCGAGCTGTCCAGCCTC-3′ for pD2R-14; 5′-TTCTCGAGAGTGCCGGAGCTGGTCGC-3′ for pD2R-1; 5′-TTCTCGAGACCAAGCTTCCTCCTCCT-3′ for pD2R-56GCM; 5′-TTCTCGAGATGCTGCGAACTAAGGATCTCA-3′ for pD2R-56CCTM.

NB2A cells were co-transfected with the reporter and pRL-TK, which contains Renilla luciferase under control of the herpes simplex virus thymidine kinase promoter, without or with other vectors using FuGENE 6. Firefly and Renilla luciferase activities were measured by the Dual-Luciferase Reporter (DLR) Assay System (Promega) with a luminometer (TD-20/20) (Promega). The ratio of luminescence signal of firefly luciferase to that of Renilla luciferase was determined.

Immunostaining of NB2A cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

NB2A cells transfected with or without expression vectors were washed with phosphate-buffered saline (PBS) and fixed with 100% methanol at −20°C for 20 min. After fixation, the cells were rehydrated in PBS for 10 min. The cells were permeabilized with 0.05% Triton X-100 for 20 min and preincubated with 0.3% bovine serum albumin (BSA) in PBS (blocking solution) for 30 min. Cells transfected with or without pSp1 were incubated with the anti-Sp1 antibody in blocking solution at a dilution of 1 : 500. Immunoreactivities of Sp1 were detected with rhodamine-conjugated anti-goat IgG antibody. Cells transfected without or with pZif268 alone or pZif268 plus pActKII were incubated with anti-Zif268 antibody in blocking solution at a dilution of 1 : 400. Zif268 immunoreactivity was detected with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG antibody. Cells transfected with pDRRF-HA were incubated with anti-HA antibody diluted 1 : 200 in blocking solution. HA was detected with FITC-conjugated anti-rabbit IgG antibody. Immunostaining was examined under a confocal laser scanning light microscope (CLSM) (Olympus, Tokyo, Japan).

Immunoblot analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

NB2A cells transfected with pCAGGSneo alone (Mock) or pFC-MEKK were washed twice with PBS and frozen in liquid N2. Cells were then homogenized with 0.3 mL of homogenization buffer containing 50 mm HEPES, pH 7.5, 0.1% Triton X-100, 2 mm EGTA, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride (PMSF), 1 mm dithiothreitol (DTT), 1 mm leupeptin and 75 µm pepstatin A. Homogenates were treated with the sample solution of Laemmli (1970) and boiled for 3 min. Samples were separated using 9% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane using the method of Towbin et al. (1979). The membrane was incubated at room temperature for 1 h in blocking solution consisting of 4% skim milk in T-TBS (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 0.1% Tween 20) and then incubated overnight at 4°C with the Zif268 antibody diluted 1 : 1000 in blocking solution. Horseradish peroxidase-conjugated anti-goat IgG was used to visualize the primary antibody using the enhanced chemiluminescence (ECL) Plus Western Blotting Detection Reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturer's protocol.

Preparation of nuclear extracts

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

Nuclear extracts of NB2A cells were prepared by treatment with a modified mini-scale detergent as described (Schreiber et al. 1989). NB2A cells were cultured in 100-mm dish. After transfection, NB2A cells were harvested by scraping in 800 µL of Buffer-A (10 mm HEPES–KOH, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm DTT, 1 mm PMSF, 2 µg/mL pepstatin A, 1 µg/mL leupeptin, 100 nm calyculin A) at 4°C and kept for 15 min on ice, followed by addition of 50 µL of 10% Nonidet P-40 (NP-40), and the cells were homogenized for 10 s by vigorous stirring by a vortex. The homogenate was centrifuged at 3000 × g at 4°C for 10 min to prepare the nuclei, and the isolated nuclei in the pellets were resuspended in 30 µL of ice-cold buffer C (50 mm HEPES–KOH, pH 7.9, 400 mm NaCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm DTT, 1 mm PMSF, 2 µg/mL pepstatin A, 100 nm calyculin A). The nuclear proteins were extracted for 30 min by incubation the nuclei on ice, and then the supernatant containing the nuclear proteins was collected after centrifugation for 10 min at 8000 × g at 4°C. The obtained nuclear protein preparation was stored at −80°C until use.

Electrophoretic mobility shift assay (EMSA)

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

The probes corresponding to the D2R promoter regions from nucleotides −59 to −30 was synthesized with complimentary oligonucleotides, 5′-GGTGACCCCGCCCCCTCCTCCTGCGCAGCG-3′ and 5′-CGCTGCGCAGGAGGAGGGGGCGGGGGTCACC-3′. Double-stranded synthetic oligonucleotides were labeled at the 5′ end with [γ-32P]ATP with T4 polynucleotide kinase according to standard procedures. The nuclear protein/DNA-binding reaction was performed by incubation of the nuclear protein (5 µg) and 10 fmol of 32P-labeled probe (about 2 × 104 cpm) in the binding buffer containing 25 mm HEPES–KOH, pH 7.8, 50 mm KCl, 1 mm EDTA, 0.5 mm PMSF, 0.5 mm spermidine, 10 µg/mL leupeptin, 10 µg/mL pepstatin A, 10% glycerol, and 2 µg of poly(dI-dC) for 30 min at room temperature. In competition analysis, 10 pmol of the competitor oligonucleotide was mixed before the addition of nuclear extracts. Supershift assays were performed as described above with the exception that, after the incubation of the probes with nuclear extracts, 2 µg of antibodies to HA-tag was added and incubated for another 30 min at room temperature.

Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

To analyze expression of DR2, we performed RT-PCR in NB2A cells and rat striatum using primers upstream and downstream of the variable domain of D2R (Fig. 1a). In NB2A cells, two PCR products of 459 bp and 372 bp derived from D2LR and D2SR, which are splice variants of D2R (Giros et al. 1989), respectively, were detected and the intensity of the D2LR band was higher than that from D2SR. The D2R expression pattern in NB2A cells was similar to that in rat striatum. However, the amount of D2R mRNA was lower than that seen in striatum, where D2R is expressed abundantly. D2R mRNA levels in NB2A cells were comparable to those seen in cerebral cortex (data not shown). Next, we evaluated basal activities of the D2R promoter by luciferase assays in NB2A cells and two other cell lines that do not express D2R, NG108-15 (Takeuchi et al. 2002) and C6 glioma cells (Valdenaire et al. 1994) (Fig. 1b). Luciferase activities of pD2R-1518, which contains the region from nucleotides −1518–275, were increased 20-, 15- and 9.5-fold over that of pGL3-basic in NB2A, NG108-15 and C6 glioma cells, respectively. In addition, the luciferase activity of pD2R-1, which does not contain the promoter region of D2R, was similar to that of pGL3-basic (Fig. 1b).

image

Figure 1. Schematic structures of the promoter constructs and their luciferase activities in NA2A, NG108-15 and C6 glioma cells. (a) Expression of D2R mRNA was detected using RT-PCR of NB2A cells and rat striatum with specific primers based on the sequences of D2R. (b) NB2A, NG108-15 and C6 glioma cells were co-transfected with each reporter vector (0.5 µg) and pRL-TK (0.1 µg). Luciferase activities were determined as described in Materials and methods. Luciferase activities relative to pGL3-basic are expressed for each cell line. Values represent means±SE (bars) of data from four independent experiments.

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Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

To examine effects of protein kinases PKA, MEKK, CaMKII and CaMKIV on D2R promoter activity, we transfected vectors harboring individual constitutively active kinases or the pCAGGSneo alone (Mock) along with the pD2R-1518 reporter vector into NB2A, NG108-15 and C6 glioma cells. MEKK is an upstream kinase of MAPK pathways and can activate MAPKs such as ERK, JNK and p38MAPK. As shown in Fig. 2, MEKK increased D2R promoter activities 4.7-, 3.2- and 2.1-fold in NB2A, NG108-15 and C6 glioma cells, respectively, compared to Mock (Fig. 2). The most significant increase in D2R promoter activity was observed in NB2A cells (Fig. 2). CaMKII showed a 1.5-fold increase in D2R promoter activity in NB2A cells, but did not affect promoter activity in NG108-15 or C6 glioma cells (Fig. 2). CaMKIV and PKA showed no effect on D2R promoter activity in any cell line (Fig. 2). These results indicate that MAPKs and CaMKII up-regulate D2R promoter activity.

image

Figure 2. Effects of CaMKII, CaMKIV, MEKK and PKA on D2R promoter activity. NB2A, NG108-15, C6 glioma cells were co-transfected with pD2R-1518 (0.5 µg) and pRL-TK (0.1 µg) along with the empty vector (Mock) or individual constitutively active kinase vectors (0.5 µg). Luciferase activities were determined as described in Materials and methods and expressed relative to Mock-transfected cells. Values represent means±SE (bars) of data from four independent experiments. *p < 0.01 compared with the control.

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Inhibition of MEKK-induced D2R promoter activity by U0126

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

Although MEKK potentiates D2R promoter activity, MEKK acts as an upstream kinase of the three major MAPKs: ERK, JNK and p38MAPK (Wood et al. 2001). To determine which MAPK is involved in potentiation of D2R promoter activity, we examined effects of both selective MAPK inhibitors and dominant negative mutants of JNK1 and p38MAPK on MEKK-induced potentiation in NB2A cells. Treatment with U0126, which is a specific inhibitor of MEK and thereby blocks MEK-ERK pathway, inhibited MEKK-induced activation (Fig. 3a), while treatment with U0124, an inactive analog of U0126, or SB202190, a p38MAPK inhibitor, did not (Fig. 3b,c). Furthermore, co-transfection of MEKK with DN-JNK1 or DN-p38MAPK did not affect MEKK-induced potentiation of D2R promoter activity (Fig. 3d). These results suggest that MEK-ERK pathway is involved in MEKK-induced potentiation of the D2R promoter activity.

image

Figure 3. Effects of treatment with U0126, U0124 and SB202190 and of dominant negative mutants of JNK1 and p38MAPK on MEKK-induced potentiation of D2R promoter activity. (a–c) NB2A cells were co-transfected with pD2R-1518 (0.5 µg) and pRL-TK (0.1 µg) along with the empty vector (Mock) or pFc-MEKK (0.5 µg). Twenty-four hours later, the medium was changed to the standard medium or standard medium containing U0126 (10 µm), U0124 (10 µm) or SB202190 (20 µm), and the cells were incubated for 24 h. (d) NB2A cells were co-transfected with pD2R-1518 (0.5 µg), pRL-TK (0.1 µg) and the empty vector (Mock) or pFc-MEKK (0.5 µg) along with empty vector, pDN-JNK1 or pDN-p38MAPK (0.5 µg). Luciferase activities are expressed relative to Mock-transfected cells. Values represent means±SE (bars) of data from four independent experiments. *p < 0.01 compared with the control.

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Identification of MEKK-responsive regions in the D2R promoter

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

To identify MEKK responsive regions in the D2R promoter, we assessed the responsiveness to MEKK using truncated promoter constructs. Schematic representations of truncated D2R promoter constructs and their luciferase activities in NB2A cells are shown in Fig. 4(a). The luciferase activities of pD2R-117, -79, -56, -36 and -14 were gradually decreased from 75, 55, 40 and 20–10%, respectively, relative to pD2R-1518 in NB2A cells. We then co-transfected each of these luciferase constructs plus MEKK into NB2A cells, and the relative luciferase activities in MEKK-transfected cells were compared to that seen in Mock-transfected cells (Fig. 4b). pD2R-117, -79 and -56 showed essentially the same stimulatory effects of MEKK on promoter activity as the full-length promoter (pD2R-1518), whereas pD2R-36, -14 and -1 totally lost the stimulatory effects. These results clearly show that the region from −56 to − 36 is essential for MEKK responsiveness. Further studies to confirm this finding were performed using site-mutated promoter constructs, as shown in Fig. 5(a). pD2R-56GCM and -56CCTM contained mutations of the Sp1 site B and three CCT repeats, respectively. Basal luciferase activities of pD2R-56GCM and -56CCTM decreased to 27% and 65% of that of pD2R-56, respectively. The stimulatory effect of MEKK on promoter activity of pD2R-56CCTM was comparable to that of pD2R-56 but was totally eliminated in pD2R-56GCM (Fig. 5b). These results suggest that the MEKK/ERK pathway potentiates D2R promoter activity through Sp1 site B.

image

Figure 4. Analysis of MEKK-responsive regions of the D2R promoter. (a) Schematic structures of various promoter constructs and their relative luciferase activities. Sp1 consensus sequences (Sp1 A and Sp1 B) are indicated by boxes. NB2A cells were co-transfected with the promoter vectors (0.5 µg) plus pRL-TK (0.1 µg). Luciferase activities relative to pD2R-1518 are expressed. (b) NB2A cells were co-transfected with the indicated promoter vector (0.5 µg) and pRL-TK (0.1 µg) along with the empty vector (Mock) or pFc-MEKK (0.5 µg). Luciferase activities are expressed relative to Mock-transfected cells. Values represent means±SE (bars) of three from four independent experiments. *p < 0.01 compared with the control.

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image

Figure 5. Mutational analyses of MEKK-responsive regions of the D2R promoter. (a) Schematic structures of site mutated promoter constructs and their relative luciferase activities. Mutated sequences are indicated by dots under the nucleotides. NB2A cells were co-transfected with the promoter vectors (0.5 µg) plus pRL-TK (0.1 µg). Luciferase activities relative to pD2R-56 are shown. (b) NB2A cells were co-transfected with the indicated promoter vector (0.5 µg) and pRL-TK (0.1 µg) along with the empty vector (Mock) or pFc-MEKK (0.5 µg). Activity is expressed relative to Mock-transfected cells. Values represent means±SE (bars) of three from four independent experiments. *p < 0.01 compared with the control.

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Negative regulation of D2R promoter activity by Sp1 in NB2A cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

Since the MEKK-responsive region was found to be the Sp1 site B, we asked whether Sp1 is involved in MEKK-induced potentiation of D2R promoter activity by co-transfecting Sp1 into NB2A cells. Unexpectedly, overexpression of Sp1 (0.2 µg of pSp1) alone decreased up to 50% the luciferase activities of pD2R-117 and pD2R-79 constructs but did not affect the pD2R-56 construct (Fig. 6a). Transfection with higher amounts of Sp1 (0.5 µg of pSp1) further suppressed promoter activity of the pD2R-117 construct (to 20% of basal activity) (data not shown). Immunostaining of NB2A cells after transfection without or with pSp1 revealed that Sp1 was weakly expressed in nuclei of untransfected NB2A cells and strongly expressed in nuclei of transfected cells (Fig. 6b). We further examined the effect of Sp1 on MEKK-induced potentiation of D2R promoter activity using pD2R-1518 (Fig. 6ci), and pD2R-56 (Fig. 6cii), which lacks the Sp1 binding site. With pD2R-1518, overexpression of Sp1 suppressed to about 40% both basal (Mock) and MEKK-induced promoter activities (Fig. 6ci). On the other hand, the basal (Mock) and MEKK-induced activation of the pD2R-56 construct was not affected by overexpression of Sp1. These results suggest that overexpression of Sp1 suppressed D2R promoter activity through sequences upstream of the Sp1 site B and that Sp1 does not affect MEKK-induced potentiation of D2R promoter activity in NB2A cells.

image

Figure 6. Analyses of the effects of Sp1 on D2R promoter activity. (a) NB2A cells were co-transfected with the indicated promoter vector (0.5 µg) and pRL-TK (0.1 µg) along with the empty vector (Mock) or pSp1 (0.2 µg). Luciferase activities were expressed relative to Mock-transfected cells. (b) CLSM image of NB2A cells transfected without (wild type) or with Sp1. After transfection NB2A cells were immunostained with the anti-Sp1 antibody at a dilution of 1 : 500 and rhodamine-conjugated anti-goat antibody. (c) Effect of Sp1 on MEKK-induced increases in D2R promoter activity with pD2R-1518 (i) and pD2R-56 (ii). NB2A cells were co-transfected with the indicated promoter vector (0.5 µg), pRL-TK (0.1 µg) and the empty vector (Mock) or pFc-MEKK (0.5 µg) along with empty vector or pSp1 (0.2 µg). Luciferase activities are expressed relative to Mock-transfected cells. Values represent means±SE (bars) of three from four independent experiments. *p < 0.01 compared with the control.

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Effect of Zif268 on D2R promoter activity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

Sp1 site B is positively regulated by the ERK pathway but Sp1 may not function in up-regulation of D2R promoter activity by ERK in NB2A cells. To examine whether Zif268, which interacts with GC-boxes, positively regulates the D2R promoter, we performed similar assays overexpressing Zif268 (O'Donovan et al. 1999). Zif268 is known to be induced in striatal neurons by treatment with anti-psychotic drugs such as haloperidol (Nguyen et al. 1992; Simpson and Morris 1994). Overexpression of Zif268 increased D2R promoter activity in a dose-dependent manner with a maximum of 1.7-fold of that in control cells (Fig. 7a). Next, we identified which regions of the D2R promoter are responsive to Zif268 using truncated and site-mutated constructs. pD2R-117, -79 and -56 showed essentially the same stimulatory effects by Zif268 on promoter activity as seen with the pD2R-1518 construct (Fig. 7a), while pD2R-36, -14, -56GCM and -56CCTM showed no stimulatory effect by Zif268. These results indicate that the region responsive to Zif268 is between −56 and −36 and that not only Sp1 site B, but also the CCT repeats are required for Zif268 stimulation. Overexpression of Zif268 with MEKK did not show additive effects on promoter activity (Fig. 7c). Because ERK activation is known to be involved in the Zif268 expression in rat brain (Sgambato et al. 1998), we analyzed expression of Zif268 in the Mock- and MEKK-transfected NB2A cells by immunoblot analysis (Fig. 7d). The transfection efficiency in NB2A cells was estimated around 80% by transfection with pEYFP (data not shown). Levels of Zif268 protein were not changed by MEKK overexpression. This result suggests that MEKK and Zif268 may regulate the D2R promoter through different pathways and that MEKK may potentiate D2R promoter activity through a transcription factor other than Zif268.

image

Figure 7. Effect of Zif268 on D2R promoter activity. (a) NB2A cells were co-transfected with pD2R-1518 (0.5 µg) and pRL-TK along (0.1 µg) along with the indicated amount of pZif268. The total amount of DNA transfected was adjusted to 1.6 µg using the empty vector. (b) Identification of the Zif268-responsive region of the D2R promoter. NB2Acells were co-transfected with the indicated promoter vector (0.5 µg) and pRL-TK (0.1 µg) along with the empty vector (Mock) or pZif268 (0.5 µg). (c) Effects of Zif268 on MEKK-induced increases in D2R promoter activity. NB2A cells were co-transfected with the indicated promoter vector (0.5 µg), pRL-TK (0.1 µg) and the empty vector (Mock) 1.0 µg or pFc-MEKK (0.5 µg) along with empty vector or pZif268 (0.5 µg). (d) Immunoblot analysis of expression of Zif268 in NB2A cells transfected with empty vector (Mock) or pFc-MEKK. Extracts of transfected cells (10 µg) were subjected to immunoblot analysis with the anti-Zif268 antibody at a dilution of 1 : 1000. Luciferase activities shown in panels (a–c) are expressed relative to Mock-transfected cells. Values represent means±SE (bars) of three from four independent experiments. *p < 0.01 compared with the control.

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Regulation of the D2R promoter by CaMKII

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

Because CaMKII enhanced D2R promoter activity approximately 1.5-fold in NB2A cells (Fig. 2), we identified CaMKII-responsive regions in the D2R promoter using truncated promoter constructs in the same procedure described in Fig. 3. CaMKII stimulated the luciferase activities of pD2R-117, -79 and -56 but not of pD2R-36 or -14 in NB2A cells (Fig. 8a). These findings suggest that the CaMKII-responsive region is from −56 to −36, coinciding with the region responsive to Zif268. Indeed, overexpression of CaMKII with Zif268 in NB2A cells further enhanced D2R promoter activity over that seen with CaMKII or Zif268 alone (Fig. 8b). Zif268 was endogenously expressed in the cytosol of NB2A cells (Fig. 8ci). The overexpression of Zif268 showed the strong immunoreactivity of Zif268 in the cytosols of NB2A cells (Fig. 8cii). However, cotransfection with CaMKII greatly stimulated nuclear translocation of Zif268 in the most of the cell transfected (Fig. 8ciii). Act KII was localized in the cytosol but not in the nucleus (data not shown). We hypothesize that the additive effect of co-overexpression of Act KII with Zif268 on the D2R promoter is due the stimulation of nuclear translocation of Zif268 by CaMKII.

image

Figure 8. Analysis of CaMKII-responsive region of the D2R promoter. (a) NB2A cells were co-transfected with the indicated promoter vector (0.5 µg) and pRL-TK (0.1 µg) along with the empty vector (Mock) or pAct-KII (0.5 µg). (b) Additive effect of CaMKII and Zif268 on the D2R promoter. NB2A cells were co-transfected with pD2R-1518, pRL-TK (0.1 µg) and the empty vector (Mock) or pActKII (0.5 µg) along with empty vector or pZif268 (0.5 µg). (c) CLSM image of NB2A cells transfected without (i) or with pZif268 alone (ii) or pZif268 plus pActKII (iii). After transfection, cells were immunostained with the anti-Zif268 antibody at a dilution of 1 : 400. Luciferase activities shown in panels (a) and (b) are expressed relative to Mock-transfected cells. Values represent means±SE (bars) of three from six independent experiments. *p < 0.01 compared with the control.

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Effect of DRRF on the D2R promoter activity in NB2A cell

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

Hwang et al. (2001) recently identified DRRF, a zinc finger transcription factor, as a negative regulator of the D2R promoter. We examined effects of DRRF on both basal activity and MEKK or Zif268-induced potentiation of D2R promoter activity. To do so, DRRF cloned from NB2A cells and HA-tagged at the C-terminus (DRRF-HA) was overexpressed in NB2A cells (Fig. 9ai). Immunostaining with anti-HA-probe antibody showed that overexpressed DRRF-HA was clearly targeted to the nucleus (Fig. 9ai). Consistent with the previous report (Hwang et al. 2001), overexpression of DRRF-HA suppressed D2R promoter activity in a dose-dependent manner (Fig. 9aii). We examined the DRRF-HA-responsive region in the D2 promoter using truncated promoter constructs using the assay described in Fig. 3. DRRF-HA inhibited luciferase activities of pD2R-117, -79, -56 and -56CCTM but not of pD2R-36 or -56GCM in NB2A cells (Fig. 9bi). Thus, in NB2A cells the DRRF-HA-responsible region maps to the Sp1 site B, a region coincident with both the MEKK- and Zif268-responsive sites. To examine whether DRRF binds to the Sp1 site B to suppress D2R promoter activity, we preformed gel shift assay using an oligonucleotide from −59 to −30 containing the Sp1 site B in the nuclear extract from NB2A cells transfected with empty vector (Mock) and with DRRF-HA. Gel shift assay clearly showed two protein/DNA complex bands (termed C-I and C-II) in both the nuclear extracts from Mock and DRRF-HA-transfected cells (Fig. 9bii, lanes 1 and 2). The addition of 1000-fold excess of the unlabeled oligonucleotide probe thoroughly abolished the C-I bands and also eliminated the C-II bands (Fig. 9bii, lanes 3 and 4). Interestingly, the intensity of the C-I band was apparently weak in the DRRF-HA-transfected cells compared to that in the Mock-transfected cells, while the intensity of the C-II band showed no change between the Mock and DRRF-HA-transfected cells (Fig. 9bii, lanes 1 and 2). Supershift assay with the antibody to HA-tag showed no effect on these bands (Fig. 9bii, lines 5 and 6). We then examined the negative regulatory effect of DRRF-HA on MEKK- or Zif268-induced potentiation of D2R promoter activity. Overexpression of DRRF-HA suppressed both basal and MEKK- or Zif268-induced potentiation of D2R promoter activity (Fig. 9c). These results suggest that DRRF negatively regulates the D2R promoter activity by disturbing some positive regulatory factors, which may be included in the nuclear complex of C-I, to bind to the Sp1 site B and that ERK and CaMKII/Zif268 pathways can counteract DRRF-mediated repression.

image

Figure 9. Analyses of the effects of DRRF on D2R promoter activity. (ai) CLSM image of NB2A cells transfected with pDRRF-HA. After transfection, the cells were immunostained with the anti-HA probe antibody at a dilution of 1 : 200. (aii) Negative regulation of D2R promoter by overexpression of DRRR-HA. NB2A cells were co-transfected with pD2R-1518 (0.5 µg) and pRL-TK along (0.1 µg) along with the indicated amount of pDRRF-HA. The total amount of DNA transfected was adjusted to 1.1 µg using empty vector. (bi) Analysis of the DRRF-HA-responsive region on the D2R promoter. NB2A cells were co-transfected with the indicated promoter vector (0.5 µg) and pRL-TK (0.1 µg) along with the empty vector (Mock) or pDRRF-HA (0.5 µg). (bii) After transfection with 3.0 µg of empty vector (Mock) (lanes 1, 3 and 5) or pDRRF-HA (lanes 2, 4 and 6) into NB2A cells cultured in 100-mm dish, the nuclear extracts were prepared. The nuclear extracts were subjected to gel shift assay. The 32P-labeled oligonucleotide, corresponding to the D2R promoter region nucleotides from −59 to −30 was used. For competition analysis, 1000-fold excess of unlabeled oligonucleotide was mixed before the addition of nuclear extracts. Supershift assay was performed using the antibodies to HA-tag as described in Materials and methods. Arrows indicated the protein/DNA complexes, C-I and C-II. (c) Effect of DRRF-HA on MEKK (i)- and Zif268 (ii)-induced increases in D2R promoter activity. (i) NB2A cells were co-transfected with pD2R-1518 (0.5 µg), pRL-TK (0.1 µg) and the empty vector (Mock) or pFc-MEKK (0.5 µg) or along with empty vector or pDRRF-HA (0.5 µg). (ii) NB2A cells were co-transfected with pD2R-1518 (0.5 µg), pRL-TK (0.1 µg) and the empty vector (Mock) or pZif268 (0.5 µg) along with empty vector or pDRRF-HA (0.5 µg). Luciferase activities were expressed relative to Mock-transfected cells. Values represent means±SE (bars) of three from four independent experiments. *p < 0.01 compared with the control.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References

In the present study, we demonstrated that ERK and CaMKII pathways up-regulate rat D2R promoter activity through the region between nucleotides −56 and −36 and that Sp1 and Zif268, respectively, negatively and positively, regulate D2R promoter activity through the region around the Sp1 site B. Up-regulation by CaMKII is partly due to stimulation of the nuclear translocation of Zif268. The Sp1 site B also has a crucial role on promoter activity through negative regulation by DRRF. Although multiple protein kinase cascades play important roles in transcriptional regulation, up until now we knew little about regulation of D2R expression by these pathways. To our knowledge, this is the first report regarding regulation of D2R promoter by protein kinases.

NB2A cells normally express D2R but NG108-15 and C6 glioma cells do not. In NB2A cells, the basal luciferase activity of the D2R promoter was slightly higher than that seen in NG108-15 and C6 cells. However, an apparent cell type specificity of D2R promoter activity was not observed using construct pD2R-1518 (− 1518–275) in the present study. This finding is consistent with the results from Valdenaire et al. (1994) but inconsistent with those of Minowa et al. (1992). The latter group reported that D2R promoter activity is much higher in D2R-expressing NB41A3 cells than in nonexpressing cell lines such as C6, NIH3T3 and Hep G2 cells, using their promoter construct pCAT-D2-75 (−75–278) which lacks a silencing element located between − 75 and − 215. However, under the same conditions using the longest promoter construct, pCAT-D2-852 (− 852–278), D2R promoter activities were essentially the same between NB41A3, C6 glioma, and NIH3T3 cells (Minowa et al. 1992). Although the silencing element from −75 to −215 may account for such cell type specificity, the reason underlying these discrepancies is not clear.

In addition, we did not find any elements functioning as silencers in the D2R promoter. Luciferase assays using truncated promoter constructs indicate that the important region for basal D2R promoter activity was located between nucleotides −117 and −14, since pD2R-117 retained 75% of the activity of pD2R-1518 and tpD2R-14 lost most of this activity (Fig. 4a). Further truncations from the promoter construct of pD2R-117 showed gradually decreasing activity. Some positive regulators may be involved in this region. These findings were consistent with previous reports, which identify two promoters upstream and downstream of the −75 nucleotide and show that truncation of these promoters decreases activity in both D2R-expressing and -nonexpressing cell lines (Valdenaire et al. 1994). In contrast, Minowa et al. (1992) reported that activity of the construct pCAT-D2-75 was greater than that of longer constructs in NB41A3 cells and that silencing elements were located between −217 and −75 (Minowa et al. 1992). The silencing elements were defined as D2NegA (from −160 to −135) and D2NegB (from −116 to −76), and D2NegB contains the TGGG repeat and Sp1 site A (Minowa et al. 1994). Gel shift analysis of this region revealed that Sp1, Sp3 and DRRF bind to the D2NegB site (Hwang et al. 2001). However, negative regulatory effects of these regions were observed only in NB43A3 cells, and not in NB2A cells and MMQ cells in the present and the previous studies by Valdenaire et al. (1994), respectively. We hypothesize that silencing elements function primarily in NB41A3 cells, since NB41A3 cells express high levels of negative regulators, such as Sp1, Sp3 and DRRF. In the support of this idea, gel mobility shift assays with nucleotides −255 to −67 which contain the silencing elements showed very intense bands using NB41A3 nuclear extracts but relatively weak bands using striatal nuclear extracts (Minowa et al. 1994). In NB41A3 cells, these bands were supershifted with an anti-Sp1 antibody (Minowa et al. 1994). Here, overexpression of Sp1 suppressed D2R promoter activity through a region near this silencing element in NB2A cells (Fig. 6a).

In this study, we have demonstrated that MEKK activated the D2R promoter through the ERK pathway (Fig. 3) and that Sp1 site B was responsible for this activation in NB2A cells (Figs 4 and 5). Interestingly, treatment with estrogen increases the D2R promoter activity through the promoter region containing Sp1 site B using their promoter construct pCAT-D2-75 (−75–278) in NB41A3 cells (Lammers et al. 1999). Estrogen also elicits a rapid and sustained activation of the MAPK cascade including ERK1 and ERK2 in the developing CNS (Singh et al. 1999). ERK activity may underlie the mechanism of estrogen-induced D2R expression.

Sp1 has been reported to bind to the Sp1 site B in gel mobility shift assays in NB41A3 cells (Minowa et al. 1994). Sp1 is phosphorylated by ERK2, thereby stimulating its DNA binding activity (Merchant et al. 1999). However, the additive effect of cotransfection of both MEKK and Sp1 was not observed in this study (Fig. 6). Taken together, the target molecules of the ERK pathway for D2R promoter activation may be different from Sp1 in NB2A cells.

Zif268 (also known as Egr-1, NGFI-A and Krox24) was first identified as an immediate early gene induced by growth factors that encodes a protein binding to sequences similar to Sp1 sites (O'Donovan et al. 1999). Administration of anti-psychotic drugs such as haloperidol to rats induces Zif268 expression in striatal neurons (Nguyen et al. 1992; Simpson et al. 1994), and haloperidol and sulpiride treatment induce increases in D2R mRNA in the rat striatum (Bernard et al. 1991; Rogue et al. 1991). We found that overexpression of Zif268 increased D2R promoter activity in a dose-dependent manner without an additive effect of MEKK cotransfection (Fig. 7). Our findings suggest that the element around Sp1 site B is responsive to both ERK-dependent and Zif268-dependent signals without interacting with each other. Similarly, CaMKII increased promoter activity, and its responsive region from −56 to −36 does not contain consensus binding sequences for CREB, C/EBP, ATF-1 and SRE, which were reported to be regulated by CaMKII. The additive effect of Zif268 with CaMKII on promoter activity and the stimulatory effect of CaMKII on nuclear translocation of Zif268 suggest that CaMKII-induced activation of D2R promoter is partly mediated by Zif268.

We also examined the effect of DRRF, a zinc finger type transcription factor that binds to the TGGG-repeat and Sp site A in NB41A3 cells (Hwang et al. 2001). Hwang et al. also reported that overexpression of DRRF decreased the D2R promoter activity using their promoter construct pCATD2-116 in NB41A3 cells. In our present study, using mutated promoters, we showed that the suppressive effect of DRRF possibly acts on the Sp1 site B in NB2A cells. Interestingly, gel shift assay using the oligonucleotide containing the Sp1 site B revealed that overexpressed DRRF did not bind to the Sp1 site B but attenuated the binding of the nuclear factor complex of C-I. Therefore, DRRF probably acts to block some positive regulatory factors to bind to the Sp1 site B without binding itself to the region in NB2A cells.

In summary, we demonstrated positive regulation of the rat D2R promoter by ERK and CaMKII and identified regions responsive to these kinases. Zif268 also up-regulate D2R promoter activity through the same region responsive to CaMKII. CaMKII showed additive effects with Zif268 and enhanced nuclear localization of Zif268. However, transcription factors acting on Sp1 site B following ERK activation were not identified. Further investigation of transcription factors regulated by ERK should further define the mechanism underlying D2R gene expression.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell culture and transfection
  6. RT-PCR
  7. Construction of plasmids
  8. Construction of D2R promoter plasmids and luciferase assay
  9. Immunostaining of NB2A cells
  10. Immunoblot analysis
  11. Preparation of nuclear extracts
  12. Electrophoretic mobility shift assay (EMSA)
  13. Other procedures
  14. Statistical evaluation
  15. Results
  16. Basal D2R promoter activities in NB2A, NG108-15 and C6 glioma cells
  17. Effects of CaMKII, CaMKIV, MEKK and PKA on the D2R promoter activity
  18. Inhibition of MEKK-induced D2R promoter activity by U0126
  19. Identification of MEKK-responsive regions in the D2R promoter
  20. Negative regulation of D2R promoter activity by Sp1 in NB2A cells
  21. Effect of Zif268 on D2R promoter activity
  22. Regulation of the D2R promoter by CaMKII
  23. Effect of DRRF on the D2R promoter activity in NB2A cell
  24. Discussion
  25. References
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