Protein kinase A regulates cholinergic gene expression in PC12 cells: REST4 silences the silencing activity of neuron-restrictive silencer factor/REST

Abstract

The role of protein kinase A in regulating transcription of the cholinergic gene locus, which contains both the vesicular acetylcholine transporter gene and the choline acetyltransferase gene, was investigated in PC12 cells and a protein kinase A-deficient PC12 mutant, A126.1B2, in which transcription of the gene is reduced. The site of action of protein kinase A was localized to a neuron-restrictive silencer element/repressor element 1 (NRSE/RE-1) sequence within the cholinergic gene. Neuron-restrictive silencer factor (NRSF)/RE-1-silencing transcription factor (REST), the transcription factor which binds to NRSE/RE-1, was expressed at similar levels in both PC12 and A126.1B2 cells. Although nuclear extracts containing NRSF/REST from A126.1B2 exhibited binding to NRSE/RE-1, nuclear extracts from PC12 cells did not. The NRSF/REST isoform REST4 was expressed in PC12 cells but not in A126.1B2. REST4 inhibited binding of NRSF/REST to NRSE/RE-1 as determined by gel mobility shift assays. Coimmunoprecipitation was used to demonstrate interaction between NRSF/REST and REST4. Expression of recombinant REST4 in A126.1B2 was sufficient to transcriptionally activate the cholinergic gene locus. Thus, in PC12 cells, protein kinase A promotes the production of REST4, which inhibits repression of the cholinergic gene locus by NRSF/REST.

FIG. 1

Expression of the PKA catalytic subunit in the cytosolic and nuclear fractions of PC12 cell lines. Nuclear and cytosolic extracts (10 μg) from the indicated cell lines were subjected to SDS-PAGE followed by Western blot analysis with a rabbit anti-PKA catalytic subunit antibody and then detection by enhanced chemiluminescence.

FIG. 2

PKA activity and ChAT activity in control cells and cells transfected with the PKA catalytic β subunit. A126.1B2 cells stably transfected with either the plasmid pcDNA3-Catβ, containing the catalytic subunit of PKA (A126.1B2^Catβ), or the plasmid pcDNA3-CatβM, containing an inactive catalytic subunit of PKA (A126.1B2^CatβM), were grown in DMEM containing G418. The medium was changed every 3 days, and cells were subcultured every 6 days. After 3 weeks, cells were harvested and lysates were prepared. Also shown are results obtained with untreated wild-type PC12 cells and wild-type PC12 cells treated for 3 days with 10 μM H89. ChAT and PKA activity were assayed as described in Materials and Methods. Data are means ± standard errors of the means (n = 6). ∗∗, P < 0.01 versus the respective control value (Student’s t test).

FIG. 3

Transient-transfection assays using the 5′ noncoding region of the human ChAT gene driving the luciferase reporter gene. A schematic showing the 5′ noncoding region of the human cholinergic gene and the constructs generated from this region is shown on the bottom. The position of the VAChT gene is indicated, while R represents the first exon of the cholinergic gene. The positions of restriction sites for EcoRI, NheI, and XhoI are shown. Constructs were named on the basis of the restriction fragment inserted into the pXP2 vector, with N corresponding to the NheI site, X corresponding to the XhoI site, and E corresponding to the EcoRI site. PXP2EX-m contains a mutated inactive NRSE. H89 is a PKA inhibitor. Constructs were transiently transfected into the indicated cell lines as described in Materials and Methods. After 48 h, cells were harvested, and extracts prepared from them were subsequently assayed for luciferase activity. Luciferase activity was normalized to β-galactosidase activity.

FIG. 4

NRSE binding activity in PC12 and A126.1B2 cell lines. (A) Electrophoretic mobility shift assay. Nuclear cell extracts prepared from the indicated cell lines were analyzed for binding to the cholinergic NRSE/RE-1 by electrophoretic mobility shift assays. (B) Western blot analysis of NRSF. Nuclear extracts (100 μg) from each cell line were subjected to SDS-PAGE followed by Western blotting with an anti-NRSF MAb and detection with the ECL+Plus detection system. PC12 cells were treated with 10 μM H89 as for Fig. 2. (C) Supershifting of NRSE/RE-1 gel shift band. Electrophoretic mobility shift assays were conducted as described for panel B except that the nuclear extract was preincubated with 2 μg of anti-NRSF MAb or 2 μg of a control MAb prepared against mouse ChAT where indicated.

FIG. 5

Analysis of NRSF/REST and REST4 mRNA expression in PC12 and A126.1B2 cell lines and the effect of forskolin. (A) NRSF/REST and REST4 mRNA levels in PC12 cell lines. PCR was performed with primer pairs specific for NRSF/REST, REST4, and actin as described in Materials and Methods. The bottom pattern is a control in which the primer pairs for REST4 were used without RT of RNA. PC12 cells were treated with H89 as described in the legend to Fig. 2. (B) Effect of forskolin on NRSF/REST, REST4, and ChAT mRNAs. PC12 cells were untreated (−) or were treated with 10 μM forskolin (+) for the time periods indicated. PCR was performed as described in Materials and Methods.

FIG. 6

REST4 inhibits binding of NRSF/REST to the NRSE element of the cholinergic gene. (A) Electrophoretic mobility shift assays performed with nuclear extracts from HEK293 cells expressing NRSF/REST (lane 1), REST4 (lane 2), and a 1:1 mixture of the two extracts (lane 3). (B) Western blot analysis of HEK293 cells transfected with NRSF/REST or REST4. The positions of molecular mass markers are shown on the right. (C) Electrophoretic mobility shift assays performed with 10 μg of a nuclear extract from HEK293 cells expressing NRSF and increasing amounts (0 to 10 μg) of a HEK293 nuclear extract expressing REST4 (+REST4) or control plasmid (−REST4). The arrow indicates the gel shift band produced by the binding of NRSF/REST to the NRSE from the cholinergic gene. The faster-moving gel shift band is a nonspecific band seen in all experiments.

FIG. 7

REST4 forms a hetero-oligomer with NRSF/REST. Nuclear extracts were prepared from HEK293 cells expressing either FLAG-REST4 or myc-NRSF/REST. The individual extracts or a mixture of the two were subjected to immunoprecipitation (IP) with either an immobilized goat IgG to the FLAG epitope (FLAG), an immobilized goat IgG to the myc epitope (Myc), or immobilized nonimmune goat IgG (NI). The resulting samples were subjected to SDS-PAGE and probed with MAb 12C11-1, which is directed against the N-terminal region of NRSF/REST and thus recognizes both NRSF/REST and REST4. The bands corresponding to REST4 and NRSF/REST are indicated. The positions of molecular mass markers are shown on the left.

FIG. 8

ChAT activity and ChAT and VAChT mRNAs in A126.1B2 cells transfected with REST4. (A) A126.1B2 was transfected with pCS2+MT-REST4 (+REST4) or vector alone (−REST4), using SuperFect transfection reagent. After 72 h, cell extracts were prepared and assayed for REST4 expression by Western blot analysis (top) or for ChAT activity (bottom) as described in Materials and Methods. (B) PCR analysis for ChAT mRNA (30 cycles) and VAChT mRNA (35 cycles) of A126.1B2 treated as described above.