A Novel Protein Containing cdc10/SWI6 Motifs Regulates Expression of mRNA Encoding Catecholamine Biosynthesizing Enzymes*

Catecholaminergic (dopaminergic, noradrenergic, and adrenergic) transmitter phenotypes require the cooperative actions of four biosynthetic enzymes: tyrosine hydroxylase, aromaticl-amino acid decarboxylase, dopamine β-hydroxylase, and phenylethanolamine N-methyltransferase. Mechanisms that control expression of these enzymes in a transmitter phenotype-specific manner, however, are poorly understood. Here, we provide evidence that overexpression of a novel cdc10/SWI6 motif-containing protein, V-1, elicits the coordinate up-regulation of tyrosine hydroxylase, aromaticl-amino acid decarboxylase, and dopamine β-hydroxylase mRNAs in the neuronal cell line PC12D, and as a result, catecholamine levels are increased. Furthermore, V-1 is strongly expressed in the cytoplasm of rat chromaffin cells of adrenal medulla. Thus, V-1 may act as a cytoplasmic protein/protein adapter and be involved in control of the catecholaminergic phenotype expression via an intracellular pathway signaling to the nucleus.

V-1 is a novel soluble protein consisting of 117 amino acids that contains 2.5 tandem repeats of the cdc10/SWI6 motif, also known as the ankyrin repeat (see Fig. 1A) (20,21). This motif has been demonstrated to be crucial for protein-protein interactions (22,23). Our recent studies have revealed a characteristic temporal profile for the expression of the V-1 gene in developing murine brain. During embryonic stages, expression of the V-1 gene is detectable but weak. After birth, expression of V-1 mRNA gradually increases to reach a maximal level during the first to second postnatal weeks, declining thereafter to adult levels by postnatal day 28. 2 However, strong expression of the V-1 gene persists in regions of synaptic plasticity even after the second postnatal week (24). We have established stable transfectants that overexpress V-1 using the catecholamine-producing neuronal cell line PC12D to examine functional roles of V-1 in neuronal cells and analyzed the neuronal phenotypes of these cells using techniques of molecular biology, neurochemistry, biochemistry, and electrophysiology. In this study, we also demonstrate that the V-1 protein is intensely co-expressed with TH protein in catecholamine-producing tissues in situ and provide evidence that V-1 functions in the control of the catecholaminergic transmitter phenotype.
Stable Cell Transfections-The PC12D subclone of rat pheochromocytoma cells (PC12) was cultured as described previously (27). For stable transfection experiments, 3 ϫ 10 6 cells/100-mm dish were incubated overnight in 10 ml of the culture medium. The following day, cells were transfected with 10 -12 g/dish pEFSAneo (28,29) human elongation factor 1␣ gene promoter-driven expression vector carrying the rat cDNA (21) inserted into the XbaI/EcoRV site or the vector alone, using mammalian cell transfection kit (Stratagene). Selection was performed in the culture medium containing 560 g/ml G418 (Life Technologies, Inc.). Eighty G418-resistant clones transfected with the DNA construct directing V-1 overproduction were screened by Western blot analysis with anti-V-1 antibody as described below, and several V-1overexpressing clones were thereby isolated. For the subsequent experiments, two clones highly expressing V-1, V1-46, and V1-69 were used. Similarly, two stable transfectants with the vector alone (C-7 and C-9) were isolated and used as vector control clones.
Western Blot Analyses-For Western blot analysis, 1 ϫ 10 6 cells were plated on 60-mm tissue culture dishes and cultured for 48 h. Cells were lysed in SDS-PAGE sample buffer (2% SDS, 10% glycerol, 62.5 mM Tris-HCl (pH 6.8)) including protease inhibitors (24). The lysates were analyzed by immunoblotting with anti-V-1 antibody (24, 25), a monoclonal antibody to TH (26), anti-14 -3-3 ␤ protein antibody (sc-628, Santa Cruz Biotechnology), and anti-erk 2 monoclonal antibody (Upstate Biotechnology, Inc.), and signals were visualized with enhanced * This work was supported partially by Research Grant 5B-4 for Nervous and Mental Disorders from the Japanese National Ministry of Health and Welfare. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

RNA Preparation and Northern Blot Analyses-For
Northern analysis, 6 ϫ 10 6 cells were plated on 100-mm tissue culture dishes and harvested 48 h later. RNA preparation and Northern analysis were performed as described previously (21). Human TH cDNA (30), rat AADC cDNA (31), human DBH cDNA (32), mouse PNMT cDNA (33), and mouse Phox2 cDNA (34) were used as specific probes. Following autoradiography, hybridization signals were quantified using a densitometer.
TH Enzymatic Activity and Catecholamine Content Assays-For assays of TH enzymatic activity and catecholamines, 1 ϫ 10 6 cells were plated on 35-mm tissue culture dishes and harvested 48 h later. Cells were homogenized in 0.5 ml of 0.32 M sucrose to measure TH activity as described previously (35). Quantification of dopamine and norepinephrine was performed by high performance liquid chromatography-electrochemical detection (EICOM) following extraction of the cells with 0.4 ml of 0.4 N perchloric acid. Dopamine and norepinephrine were separated on a Eicompak MA-5ODS column (4.6 ϫ 150 mm), with mobile phase consisting of 0.05 M citrate-phosphate buffer (pH 3.5) including 100 mg/1 EDTA and l mM sodium heptanesulfonate, and of 0.1 M sodium phosphate buffer (pH 3.2) containing 100 mg/1 EDTA, respectively, at a flow rate of 0.7 ml/min. A graphite electrode maintained at a potential of ϩ 0.6 V versus Ag/AgCl was used as a detection system.

RESULTS AND DISCUSSION
Our extensive immunohistochemical analyses indicated that the V-1 protein is expressed in a wide variety of different central neurons (24) as well as in rat ovary (25) and other rat tissues including thymus, spleen, and kidney. 2 Interestingly, strong expression of the V-1 protein coincided with TH expression in the cytoplasm of catecholaminergic cells, including adrenergic chromaffin cells of adrenal gland (Fig. 1B) and noradrenergic neurons of the sympathetic ganglia and locus coeruleus. 2 To examine the potential role of the V-1 protein in the regulation of the catecholaminergic phenotype, we generated stable transfectants that overexpress the V-1 protein using PC12D cells. Among stable transfectants harboring V-1expression constructs, two clones V1-46 and V1-69 were chosen for phenotype analyses. V-1 protein levels of the V-1overexpressing clones were 5-6-fold higher than that of the parent cell, which also endogenously expresses the V-1 protein.
Increases in the V-1 protein result from an increase in V-1 mRNA level as demonstrated by Northern analysis (Fig. 2). On the other hand, there were no significant changes in the V-1 protein levels in control clones stably transfected with the vector alone (C-7 and C-9) compared with the parent cell (Fig.  2, A and B).
We initially examined the functional role of V-1 in the regulation of expression of genes encoding TH (36), AADC (37), DBH (38), and PNMT (39,40), which catalyze the four synthetic successive steps from tyrosine to dopa, dopamine, norepinephrine, and epinephrine. As shown in Fig. 3 (A and B), mRNA expression of the first enzyme, TH, was increased in both V-1-overexpressing clones from 2.4-to 8.1-fold compared with those in the two control clones. Two sizes of mRNA species of the second enzyme AADC could be detected (Fig. 3, A and B). Levels of the larger AADC mRNA species were increased 20.2-68.2-fold in V-1-overexpressing clones compared with those of the control clones, where AADC mRNA expression appeared to be down-regulated in comparison with that of parent cell (Fig.  3, A and B). 3 Three mRNA species of the third enzyme DBH were detected in all transfectants. The expression of two DBH mRNA species of higher molecular weight was differentially up-regulated 6.2-19.3-fold in the V-1-overexpressing clones compared with those in the control clones (Fig. 3, A and B). In contrast, no mRNA expression of the fourth enzyme PNMT was 3 T. Yamakuni, unpublished observations.

FIG. 2. Analyses of the V-1 protein (A and B) and its mRNA levels (C) in V-1-overexpressing clones (V1-46 and V1-69), two control clones (C-7 and C-9), and parental cells (D). A, proteins of cell lysates prepared from the transfectants or parent cells (45 g/lane)
were separated on 18% SDS-PAGE and assayed by Western blot analysis with anti-V-1 antibody (upper panel), followed by reprobing with anti-14-3-3 ␤ protein antibody (as an internal control; lower panel). B, the histogram indicates the results of three independent Western blot experiments. Data represent the means Ϯ S.E. Differences in V-1 protein levels among two V-1-overexpressing clones and parent and two control clones are significant at p Ͻ 0.05 (paired t-test). C, total cellular RNAs (10 g) isolated from transfectant clones and parent cells were analyzed by Northern blotting.

V-1 Protein Overexpression in PC12 Cells
detectable in the parent cell by Northern analysis, 3 consistent with previous data showing that there is no detectable level of PNMT enzymatic activity in PC12 cells (41). PNMT mRNA was also not detected in the V-1-overexpressing clones. 3 These results suggest that the V-1 protein positively controls the coordinate expression of TH, AADC, and DBH mRNA in PC12D cells.
To obtain additional evidence at the enzymatic and functional levels, we assayed protein levels and enzymatic activities of TH and the catecholamine contents of cells. Western blot analysis using an antibody specific for TH showed that the levels of TH protein were 1.8 -5.3 times higher in the V-1overexpressing clones compared with control clones (Fig. 3, C  and D). TH enzymatic activities in homogenates prepared from the V-1-overexpressing clones were also 1.8 -3.8-fold higher than those prepared from control clones (Fig. 4A), indicating that increases in TH enzymatic activities parallel the changes in the protein and mRNA levels of TH. Dopamine levels in V-1-overexpressing clones were 6 -36-fold higher, and norepinephrine levels were 10 -100-fold higher compared with those in the control cells (Fig. 4B). The results of our biochemical and neurochemical experiments suggest that up-regulation of the expression of TH, AADC and DBH mRNAs observed in the V-1-overexpressing clones is indeed responsible for the increases in dopamine and norepinephrine production.
In addition, among the V-1-overexpressing clones and the control clones, there were no discernible changes in the magnitudes of Na ϩ and Ca 2ϩ channel currents, as determined by the whole cell voltage-clamp recording method 7 days after treatment with nerve growth factor. 4 We therefore conclude that the remarkable increases in catecholamine production observed in both V1-46 and V1-69 clones are the result of a change in phenotype induced by V-1 overproduction.

FIG. 3. Up-regulation of TH, AADC, and DBH mRNAs (A and B), and TH protein levels (C and D) in the V-1overexpressing clones.
Each photograph is representative of three and five independent Northern and Western analyses, respectively. The histograms (B and D) indicate results compiled from these experiments. Data represent the means Ϯ S.E. A, two autoradiographic images for DBH mRNA were obtained at different exposure times. Total cellular RNAs (10 g) of transfectants were subjected to Northern blotting. Staining gels with ethidium bromide (bottom panel) and reprobing blots with mouse Phox2 cDNA were performed to confirm equivalent loading. B, differences in TH, AADC, and DBH mRNA levels among the V-1-overexpressing clones and control clones are significant at p Ͻ 0.05. C, cell lysates (1.2 g of protein) of these clones were subjected to 10% SDS-PAGE, blotted onto polyvinylidene difluoride membrane, and probed with anti-TH antibody and successively with anti-erk 2 (as an internal control) antibody. Differences in TH protein levels among the V-1-overexpressing clones and control clones are significant at p Ͻ 0.05 (paired t test). the involvement of V-1 in the control of a neuronal function, i.e. regulation of catecholamine biosynthesis. The fact that the V-1 protein is expressed in various rat tissues as described above raises the possibility that V-1 may have additional functions. There are neither putative DNA binding domains nor other functional domains that seem to be involved in gene expression regulation within the V-1 protein (20,21). The V-1 protein also could not be detected in nuclear extracts prepared from the V-1-overexpressing clones, as assayed by Western blot analysis, 3 which suggests that the V-1 protein may function in the cytoplasm. Nevertheless, overexpression of V-1 resulted in an increase in mRNA expression for three enzymes that determine the catecholaminergic phenotype. An unusual characteristic of the V-1 protein is that about 73% of the entire molecule is composed of 2.5 contiguous repeats of the cdc10/SWI6 motif (Fig. 1A). It is now widely recognized that the cdc10/SWI6 motif serves as an interface for protein-protein interactions between cdc10/SWI6 motif-containing transcription regulators and transcription factors (22,23). Therefore, it is quite possible that up-regulation of the expression of TH, AADC, and DBH mRNAs observed in the two independently isolated V-1-overexpressing clones results from the co-interactive action of V-1 with an as-yet-unidentified partner protein. Furthermore, our results of Northern blot analysis of DBH mRNA raise the possibility that V-1 might be involved in regulation of RNA processing after transcription of the DBH gene. Control of gene expression occurs by transcriptional and post-transcriptional regulation. The steady state level of mRNA is known to be regulated post-transcriptionally (3,42). For example, it has been shown that stimulation of cytolasmic Ca 2ϩ /phospholipiddependent protein kinase enhances the stability of TH mRNA, and resultantly the steady state level of TH mRNA is increased (42). Therefore, we cannot exclude the possibility that V-1 stabilizes TH, AADC, and DBH mRNAs. It seems likely that V-1 acts as a protein/protein adapter in the cytoplasm of cells and indirectly influences mechanisms that control the coordinate regulation of TH, AADC, and DBH genes through an intracellular signaling pathway to the nucleus. This model is supported by the observations that strong expression of V-1 occurs in the cytoplasm of norepinephrine-2 and epinephrineproducing cells (Fig. 1B).
Our present data suggest an important role for V-1 in catecholamine biosynthesis control, whereas the precise mechanism by which V-1 regulates gene expression of TH, AADC, and DBH remains to be revealed. The elucidation of an intracellular signaling pathway linked with the V-1-mediated phenotype regulation may offer new approaches to therapy for neurological and cardiovascular diseases.