Identification and characterization of the promoter region of the Nav1.7 voltage-gated sodium channel gene (SCN9A)

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 37 (2008) 537 – 547

Identification and characterization of the promoter region of the Nav1.7 voltage-gated sodium channel gene (SCN9A) James K.J. Diss,a,⁎ Mattia Calissano,a Duncan Gascoyne,b Mustafa B.A. Djamgoz,c and David S. Latchman a,d a

Medical Molecular Biology Unit, Institute of Child Health, University College London, Guilford Street, London WC1N 1EH, UK Molecular Haematology and Cancer Biology Unit, Institute of Child Health, University College London, Guilford Street, London WC1N 1EH, UK c Neuroscience Solutions to Cancer Research Group, Biological Sciences, Imperial College, South Kensington, London SW7 2AZ, UK d Birkbeck College, University of London, Malet Street, London WC1E 7HX, UK b

Received 30 May 2007; revised 15 November 2007; accepted 6 December 2007 Available online 15 December 2007

The Nav1.7 sodium channel plays an important role in pain and is also upregulated in prostate cancer. To investigate the mechanisms regulating physiological and pathophysiological Nav1.7 expression we identified the core promoter of this gene (SCN9A) in the human genome. In silico genomic analysis revealed a putative SCN9A 5′ non-coding exon ~ 64,000 nucleotides from the translation start site, expression of which commenced at three very closely-positioned transcription initiation sites (TISs), as determined by 5′ RACE experiments. The genomic region around these TISs possesses numerous core elements of a TATA-less promoter within a well-defined CpG island. Importantly, it acted as a promoter when inserted upstream of luciferase in a fusion construct. Moreover, the activity of the promoter-luciferase construct ostensibly paralleled endogenous Nav1.7 mRNA levels in vitro, with both increased in a quantitatively and qualitatively similar manner by numerous factors (including NGF, phorbol esters, retinoic acid, and Brn-3a transcription factor over-expression). © 2007 Elsevier Inc. All rights reserved.

Introduction Altered voltage-gated sodium channel (VGSC) transcription is increasingly recognised as an important mechanism underlying numerous disorders of excitable tissues including pain, multiple sclerosis and certain epilepsies (Waxman, 2001). There are nine different pore-forming VGSC α-subunit (VGSCα) genes in humans and rodents, as well as the related but functionally distinct non-

⁎ Corresponding author. ICH, 30 Guilford Street, London WC1N 1EH, UK. Fax: +44 20 7905 2301. E-mail address: [email protected] (J.K.J. Diss). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2007.12.002

voltage-gated sodium-sensing Nax channel (Goldin et al., 2000). Each can be functionally fine-tuned by numerous pre- and posttranslational mechanisms, such as alternative splicing and association with activity-modulating β-subunits (Diss et al., 2004; Catterall et al., 2005). The SCN9A gene gives rise to Nav1.7, preferentially expressed in nociceptive dorsal root ganglion (DRG) neurons and sympathetic ganglia (Klugbauer et al., 1995; Sangameswaran et al., 1997). This VGSCα subtype amplifies small depolarisations to generate ‘threshold currents’ close to resting potential (Cummins et al., 1998). A number of studies have indicated that Nav1.7, via mutation or altered transcription, plays a key role in pain. Levels of Nav1.7 mRNA and protein have been shown to be increased in rat DRG neurons in both acute and chronic models of inflammatory pain (Black et al., 2004; Gould et al., 2004; Strickland et al., 2007), and a marked increase of Nav1.7-immunoreactive nerve fibres has been found in the mucosal, sub-mucosal and muscle layers of patients with idiopathic rectal hypersensitivity and faecal urgency (Yiangou et al., 2007). Consistent with this, mice with the SCN9A gene specifically deleted in nociceptive sensory neurons exhibited reduced (and in some instances abolished) responses to inflammatory, mechanical and thermal pain (Nassar et al., 2004). Numerous mutations of SCN9A that cause excessive channel activity have been revealed to underlie the inherited pain sydromes erythromelalgia (Cummins et al., 2004; Yang et al., 2004a; Han et al., 2006) and paroxysmal extreme pain disorder (Fertleman et al., 2006). Perhaps most strikingly, however, three recent studies have demonstrated that individuals with a congenital inability to experience pain have mutations in SCN9A causing total loss of Nav1.7 function (Cox et al., 2006; Ahmad et al., 2007; Goldberg et al., 2007). Remarkably, effective knockout of Nav1.7 in these individuals appeared to abolish sensation of all types of pain without otherwise impairing sympathetic neuron function or altering nonnociceptive sensory functions like touch, despite total SCN9A

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Fig. 1. In silico and experimental identification of exon 1 of human Nav1.7. (A) Full-length mRNA transcripts of VGSCα subtypes possess one or multiple 5′ exons (gray boxes) typically located many thousand nucleotides (kb) upstream of the ATG translation start site-containing exon (black) (from Schade and Brown, 2000; Kasai et al., 2001; Wallace et al., 2001; Yang et al., 2004b; Drews et al., 2005; Shang and Dudley, 2005). (B) RT-PCR reactions using the PRB forward primer located in human SCN9A exon 1 in conjunction with the reverse primer in exon 2 yielded an amplicon (black box) from SH-SY5Y human neuroblastoma and PC-3 metastatic prostate cancer cell line cDNAs that spanned a 63,890 nucleotide intron. Forward primers located 1619, 994, 326, 240, 182, 165, 145, 128, 118 and 77 (‘PRA’) nucleotides upstream of PRB (Supplementary Table 1) did not yield amplicons. (C) Alignment of the Nav1.7 exon 1 region of mouse chromosome 2 with human chromosome 2 identified a homologous region 63,890 nucleotides from the translation ATG start site. The amplicon (shaded) produced from RT-PCRs using the forward PRB primer and reverse primer (indicated) contained 216 nucleotides of exon 1 of human SCN9A. (D) Results of 5′ RACE experiments performed on cDNA derived from PC-3 cells using four different reverse primers (A–D) located in SCN9A exon 2. As shown, RT-PCRs generated amplicons of ∼ 350–450 nucleotides when visualized on agarose gels. Differences in product lengths corresponded to the relative positions of the reverse primers in exon 2. Below, sequenced 5′ RACE clones possessed three different 5′ ends (denoted with asterisks) located 32, 43 and 65 nucleotides upstream of primer ‘PRB’, respectively.

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deletion in mice resulting in perinatal death (Nassar et al., 2004). Species differences in the expression profile of Nav1.7 have been suggested to account for the non-lethality of SCN9A functional knockout in humans (Ahmad et al., 2007). Taken together, these findings strongly imply that pathophysiological upregulation of Nav1.7 expression/activity plays an important role in increased pain sensation and that therapies based on blocking Nav1.7 activity or knocking out its expression may provide effective pain relief (Waxman, 2006). We have recently found that Nav1.7 expression is pathophysiologically upregulated in prostate cancer. Nav1.7 expression is switched on specifically in metastatic prostate cancer cell lines (Diss et al., 2001) and Nav1.7 mRNA levels are significantly upregulated in the prostatic tissue of prostate cancer patients compared to age-matched controls (Diss et al., 2005). Furthermore, Nav1.7 activity potentiates numerous cell behaviours integral to the metastatic cascade in vitro (e.g. Grimes et al., 1995; Laniado et al., 1997; Djamgoz et al., 2001; Fraser et al., 2003). Despite the potential therapeutic importance of Nav1.7, as yet the major factors which regulate the expression of SCN9A (and that might also trigger dysregulated expression in pain or cancer) have not been identified. Here, we identify the core promoter of SCN9A in the human genome and begin to investigate the mechanisms regulating physiological and pathophysiological Nav1.7 expression.

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Results In silico and experimental identification of human SCN9A (5′UTR) exon 1 In order to begin to identify the location and sequence of the core human SCN9A promoter, we attempted to determine the position of the transcription initiation site(s) (TIS), i.e. the start of the first SCN9A exon. Analyses of previously identified VGSCα promoters revealed that full-length VGSCα mRNA transcripts possess at least one 5′ exon upstream of the ATG translation start site-containing exon (‘exon 2’) (Fig. 1A). Although this was not present in human Nav1.7 mRNA GenBank entries (X82835, NM002977, DQ857292), a putative mouse Nav1.7 mRNA sequence was found (GenBank XM287124, determined by in silico analyses and not experimentally), containing a 497 nucleotide long 5′UTR-containing exon situated 66,165 nucleotides upstream of exon 2 (Supplementary Fig. 1). This putative mouse SCN9A exon 1 displayed high sequence similarity to a region ~ 64,000 nucleotides from the SCN9A translation start site on human chromosome 2 (Fig. 1B). RT-PCRs performed on human SH-SY5Y neuroblastoma (an in vitro model of sensory neurons) and PC-3 metastatic prostate cancer cell line cDNAs utilising primers spanning the putative human 5′ UTR intron yielded one amplicon of 291 nucleotides when the forward primer ‘PRB’ was used with the common reverse primer (outlined in Fig. 1B

Fig. 2. The putative human SCN9A promoter. Sequence alignment of the putative human SCN9A promoter with the corresponding region of the mouse genome. The major transcription initiation site (TIS) is indicated by ⁎. The CpG island is indicated with a black overline. Putative Inr, BRE, TATA and Sp1/GC boxes, egr1, NRSF/RE1 and Brn-3 binding sites identified by MatInspector are boxed. Nucleotide numbers relative to the position of the major TIS are shown.

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and Supplementary Table 1). Sequencing confirmed that this amplicon contained 216 nucleotides of human SCN9A exon 1 (and 75 nucleotides of exon 2; Fig. 1C). Reactions controlling for genomic DNA contamination did not yield this amplicon. Attempts to obtain more of the human exon 1 sequence by performing RT-PCRs with primers located 1619, 994, 326, 240, 182, 165, 145, 128, 118 and 77 nucleotides further upstream (Supplementary Table 1) were unsuccessful (Fig. 1B). Based on these data, intron 1 of human SCN9A is 63,890 nucleotides long, slightly (~3.5%) shorter than in the mouse (66,165 nucleotides), with repetitive elements and simple sequences recognised by the RepeatMasker program constituting 35% of its size. Since we were only able to roughly locate the SCN9A TIS to a 77 nucleotide region (between primers ‘PRA’ and ‘PRB’ in Fig. 1B) using RT-PCR, we performed 5′ rapid amplification of cDNA ends (5′ RACE) experiments on total RNA from PC-3 cells to more precisely determine TIS location. Four different reverse primers located in exon 2 of SCN9A were used (Supplementary Table 1) and all four generated amplicons of ~ 350–450 nucleotides, with differences in product lengths corresponding to the relative positions of the reverse primers in exon 2 (Fig. 1D). In total, 19 clones derived from reactions using these four different primers were sequenced, 10 of which were bona fide SCN9A-derived products. All represented products spanning the previously identified intron 1, and possessed the same 3′ end of exon 1. However, three different 5′ ends of exon 1 were found corresponding to TISs located 32, 43 and 65 nucleotides upstream of primer ‘PRB’, respectively (Fig. 1D). Since 50% of clones possessed 5′ ends starting 32 nucleotides upstream of primer ‘PRB’ we designated this as the major TIS, denoted ‘+1’ (Figs. 1D and 2). Analysis of the core human SCN9A promoter sequence A region of genomic DNA (from ~ −1300 to ~+ 200) around the three TISs was analysed with the Softbury-FPROM promoter prediction software (http://www.softberry.com/berry.phtml?topic= fprom&group=programs&subgroup=promoter) and found to be highly likely (score: + 9.695) to contain a functional mammalian promoter on the sense strand at +3 (Fig. 2). Importantly, this is in a 345 nucleotide long CpG island (extending from −266 to + 80; EMBOSS CpG Plot, http://www.ebi.ac.uk/emboss/cpgplot/). This highly GC-rich (74% from −150 to + 150) putative promoter region has no TATA box, similar to other VGSCα gene promoters (e.g. SCN5A and SCN8A; Yang et al., 2004b, Drews et al., 2005). However, it does possess two characteristic core elements of TATA-less promoters: a PyPyAN(T/A)PyPy initiator (Inr), at + 50, and the upstream TFIIB-binding motif, BREu (Thomas and Chiang, 2006), at − 23 (Fig. 2). Further analysis of the SCN9A promoter using MatInspector (http://genomatix.de/cgi-bin/matinspector) revealed that it contains the following: (i) four upstream Sp1/GC box-like sequences (consensus, GGGCGG), within 160 nucleotides at − 40/− 45, − 102/ − 107, − 145/− 150 and − 155/−160; (ii) seven overlapping GAGA boxes between −109 and − 136; (iii) three putative egr1 binding sites at − 2/− 9, − 42/− 49 and − 173/− 185; and (iv) a putative NRSF/RE-1 site at −308/− 326. Most of these cis-regulatory elements (especially the Inr and GC-boxes) are structurally homologous in human and mouse (Fig. 2). A second, more marginal, putative sense strand promoter was also found at −630 (score: + 2.456). This is not within a CpG island (EMBOSS CpG Plot, http://www.ebi.ac.uk/emboss/cpgplot/; Softbury-CpGFinder,

http://www.softberry.com/berry.phtml?topic=cpgfinder&group= programs&subgroup=promoter) and is not highly GC-rich (46% from − 150 to + 150), but it includes a series of putative overlapping TATA boxes (from − 657 to − 676) on both sense and antisense strands and two downstream Inr sequences, at −574 and − 562. These regulatory elements are generally not conserved between human and mouse (Fig. 2). The region around the start site of human SCN9A exon 1 possesses promoter activity 1440 nucleotides of human genomic DNA around the designated Nav1.7 transcriptional start site (from − 1280 to + 159), including all of the CpG island (−266 to + 80) were cloned upstream of the luciferase gene in the pGL3-Basic vector, generating the SCN9A–D0 fusion construct. When transfected into SH-SY5Y, PC-3 and LNCaP cells, average basal SCN9A–D0 activity was more than 10× greater than the promoter-less vector. Interestingly, basal activity of this fusion construct was qualitatively consistent (logarithmically) with the level of endogenous Nav1.7 expression in these cells (Fig. 3). Thus, compared with LNCaP cells, mean SCN9A–D0 activity was 19.6-fold higher in SH-SY5Y cells and 8.5-fold higher in PC-3 cells; correspondingly, Nav1.7 mRNA levels were greatest in SH-SY5Y cells and lowest in LNCaP cells. Quantitative differences between promoter activity and Nav1.7 expression in these cells could result from other mechanisms (including the regulation of mRNA stability and microRNAs) playing a significant role in determining Nav1.7 mRNA levels, and from regulatory elements outside of the genomic region present in the SCN9A–D0 promoter construct determining overall endogenous SCN9A promoter activity. A series of SCN9A–luciferase deletion constructs were made to localize the main functional promoter regions (Fig. 4). Promoter activity was completely reduced to background levels with total deletion of the region that included the CpG island (Fig. 4A). This

Fig. 3. The SCN9A–D0 luciferase promoter construct acts as a promoter with activity reflecting relative endogenous Nav1.7 mRNA levels. Plot showing activity of the largest construct, SCN9A–D0, containing all 1440 nucleotides of the putative SCN9A promoter compared to relative Nav1.7 mRNA expression levels (expressed as loge) in SH-SY5Y, PC-3 and LNCaP cell lines. ⁎ denotes statistical significance compared to LNCaP activity/ mRNA levels ( p b 0.05). Error bars represent standard errors. Inset, electrophoresis gel showing Nav1.7 amplicons derived from SH-SY5Y (S), PC-3 (P) and LNCaP (L) and ‘blank’ (B) RT-PCRs, after 35 cycles.

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would support our prediction that the conserved TATA-less promoter at + 3 rather than the non-conserved TATA-containing putative promoter at − 630 is the functional SCN9A core promoter. Incremental deletions of sequences upstream of the core promoter region did result in incremental reductions in promoter activity however, indicating that these upstream regions do contain regulatory elements that potentiate the activity of the SCN9A promoter (Fig. 4B). Additionally, as expected of a TATA-less promoter, basal promoter activity could not be located to a single region of the core promoter. Indeed, as shown in Fig. 4C, both (i) the area around the putative TIS and Inr sequence (−81 to + 159; construct SCN9A–D7), and (ii) the central part of the CpG region (− 205 to − 57; construct SCN9A–DG) possessed significant, and similar, promoter activities. The robust activity of the SCN9A–DG construct suggested that the seven overlapping GAGA boxes in the central part of the CpG region might (in addition to the core

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promoter around the major TIS) play a significant role in Nav1.7 transcriptional initiation. The activity of SCN9A promoter constructs parallels endogenous mRNA expression following application of Nav1.7 transcription-regulating agents:(i) Differentiating stimuli Significantly greater activity of the SCN9A promoter-fusion constructs in cells expressing high (SH-SY5Y and PC-3) compared to low (LNCaP) levels of Nav1.7 indicated that activity of these constructs paralleled endogenous Nav1.7 mRNA expression in vitro. To test this further, we treated SH-SY5Y and PC-3 cells with differentiating stimuli (nerve growth factor, NGF; phorbol 12myristate 13-acetate, PMA; retinoic acid, RA) and compared the effects of each on promoter construct activity and Nav1.7 mRNA expression (measured using real-time RT-PCR).

Fig. 4. Identifying the core SCN9A promoter elements. (A–C) Left: A series of deletion constructs were made and transfected into SH-SY5Y and PC-3 cells (left). Primers used to generate the constructs are shown with their nucleotide position relative to the major TIS. CpG island, black box; putative Inr sequence near TIS, gray box; GAGA boxes, shaded box; putative TATA box-containing upstream promoter, white box. Right: Activity of the SCN9A deletion constructs compared to background levels of the promoter-less vector (pGL3-Basic) in SH-SY5Y cells (comparable PC-3 data not shown). ⁎ denotes statistical significance compared to the pGL3-Basic empty vector ( p b 0.05). Error bars represent standard errors.

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In SH-SY5Y sensory neurons, treatment for 24 h with 300 nM PMA and 10 μM RA (but not 100 ng/ml NGF) caused gene expression changes associated with differentiation, including upregulation of egr1 mRNA levels (Supplementary Fig. 2A). This is consistent with previous studies showing that these cells are responsive to PMA and RA but not NGF (Pahlman et al., 1984; Schulte et al., 2005). Concomitantly, Nav1.7 mRNA levels and activity of the SCN9A–D0 promoter construct were both increased ~2-fold following treatment with PMA or RA (but not NGF; Fig. 5A). In PC-3 cells (as in SH-SY5Y cells), 24 h treatment with 100 ng/ml NGF and 300 nM PMA also significantly altered egr1 levels (Supplementary Fig. 2B) and significantly increased both Nav1.7 mRNA levels and SCN9A–D0 promoter construct activity by ~2-fold (Fig. 5B). In contrast, egr1 and Nav1.7 expression, and SCN9A–D0 promoter activity were not significantly altered by 10 μM RA (Supplementary Fig. 2B and Fig. 5B), consistent with PC-3 cells being relatively insensitive to RA (Campbell et al., 1998; Murthy et al., 2003). In summary, in both SH-SY5Y and PC-3 cells we found that treatments with stimuli to which the cells were responsive resulted in (qualitatively and quantitatively) comparable effects on endogenous Nav1.7 mRNA expression and SCN9A promoter construct activity. The activity of SCN9A promoter constructs parallels endogenous mRNA expression following application of Nav1.7 transcription-regulating agents: (ii) Transcription factors We and others have shown previously that expression of the Brn3a and Brn-3b POU family transcription factors changes dramatically with sensory neuron differentiation (Turner et al., 1994; McEvilly et al., 1996; Smith and Latchman, 1996; Smith et al., 1997). Furthermore, over-expression of the short isoform of Brn-3a (Brn-3a[s]) significantly upregulates Nav1.7 mRNA expression in PC-3 cells (Diss et al., 2006). In the present study, we tested whether over-expression of Brn-3a proteins in PC-3 and SH-SY5Y cells resulted in ostensibly similar changes in Nav1.7 mRNA levels and SCN9A promoter construct activity 24 h after transfection. Indeed this appeared to be the case: in SH-SY5Y cells, both SCN9A

promoter construct activity and Nav1.7 mRNA levels were significantly increased by both Brn-3a isoforms (2- to 2.5-fold by Brn-3a[l], and ~ 1.8-fold by Brn-3a[s], Fig. 6A); in PC-3 cells, both SCN9A promoter activity and Nav1.7 mRNA levels increased significantly and consistently by 1.3-fold and 1.5-fold, respectively, with Brn-3a[s] (Fig. 6B). The effect of Brn-3a[l] was much more variable than that of Brn-3a[s], as previously documented (Diss et al., 2006). Importantly, this variability was evident in the results of experiments using both the SCN9A promoter construct and in those measuring Nav1.7 mRNA levels by real-time RT-PCR, providing further evidence that the activity of our SCN9A construct paralleled endogenous Nav1.7 mRNA expression in vitro. Discussion In the present study, we initially identified the human SCN9A promoter by in silico analyses and subsequently confirmed its location experimentally using RT-PCR and 5′ RACE methods. Like the core promoters of other VGSCα genes examined to date (e.g. Schade and Brown, 2000; Kasai et al., 2001; Wallace et al., 2001; Yang et al., 2004b; Drews et al., 2005; Shang and Dudley, 2005), the SCN9A promoter is located far (~ 64,000 nucleotides) upstream of the ATG translation start site and has the following characteristics: (i) A high degree of sequence conservation between human and mouse; (ii) an extensive CpG island around the TIS(s); and (iii) numerous GC boxes that could serve as binding sites for the Sp1 transcription factor. Consistent with other VGSCαs, the promoter lacks a functional TATA box. Transcription initiation is likely to be directed instead by an initiator (Inr) sequence, a motif observed in 60–85% of human gene promoters very close to the TIS (Suzuki et al., 2001; Gershenzon and Ioshikhes, 2005). TATA-less promoters frequently drive the expression of multiple mRNA transcripts with 5′ ends originating from alternative TISs clustered within tens to hundreds of nucleotides, as in the SCN5A and SCN8A core promoters (Yang et al., 2004b; Drews et al., 2005). Using 5′ RACE we found three SCN9A TISs clustered within a 33 nucleotide region. Expression of some VGSCα genes, including SCN5A, may be driven by a second promoter located b 1000 nucleotides from the

Fig. 5. Testing whether SCN9A promoter construct activity ostensibly parallels endogenous Nav1.7 mRNA expression in SH-SY5Y neuroblastoma and PC-3 prostate cancer cells in vitro: response to differentiating stimuli. Plots showing the effect of 100 ng/ml NGF, 300 nM PMA and 10 μM RA treatments for 24 h (control, 100%) on Nav1.7 mRNA levels (white bars) and SCN9A-promoter construct activity (black bars) in SH-SY5Y (A) and PC-3 cells (B). ⁎ denotes statistical significance compared to control treatments ( p b 0.05). Error bars represent standard errors. All real-time data shown were normalized with NADHcytochrome b5 reductase. Similar results were obtained with β2 microglobulin normalizations.

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Fig. 6. Testing whether SCN9A promoter construct activity parallels endogenous Nav1.7 mRNA expression in vitro: response to transcription factors. Plots showing the effect on Nav1.7 mRNA levels (white bars) and SCN9A-promoter activity (black bars), compared to empty vector (pLTR-poly) controls, 24 h after over-expression of long and short Brn-3a isoforms in SH-SY5Y (A) and PC-3 (B) cells. ⁎ denotes statistical significance compared to pLTR-poly controls ( p b 0.05). Error bars represent standard errors. All real-time data shown were normalized with NADH-cytochrome b5 reductase. Similar results were obtained with β2 microglobulin normalizations.

ATG start site (e.g. Schade and Brown, 2000). We found no evidence for the use of a second SCN9A promoter positioned close to the translation start site in (i) 5′ RACE experiments performed on PC-3 cells or (ii) in silico analyses of human chromosome 2. However, in silico analyses did reveal a second putative promoter possessing a number of TATA boxes ~ 600 nucleotides upstream of the SCN9A TISs (~ 65,000 nucleotides from the ATG start site). Experimental data from luciferase assays indicated, however, that this second promoter was not functional. Furthermore, the poor conservation of this region between human and mouse genomes would lead us to suggest that it does not drive Nav1.7 expression. In contrast, the putative TATA-less SCN9A promoter cloned upstream of the firefly luciferase gene did show clear promoter function. Activity was mainly localized to the 345 nucleotide long CpG island region but, like numerous TATA-less promoters, was not derived from one clearly delineated promoter location within this region. Rather, the CpG region could be divided into at least two parts with similar promoter activities (represented by constructs SCN9A–DG and SCN9A–D7). One of these possessed a series of seven overlapping GAGA boxes. GAGA factor, which recognises these motifs, is a key component of the transcription initiation complex of several promoters, generating an open chromatin structure and recruiting TFIID (Wyse et al., 2000; Leibovitch et al., 2002). Therefore, GAGA factor acting through these GAGA elements may play a role in determining the levels of RNA polymerase II associated with the SCN9A promoter, particularly in response to growth factors and steroid hormones (Wyse et al., 2000).

Regulation of SCN9A by VGSCα transcription factors Transcription factors are fundamental determinants of gene expression yet only two, REST/NRSF and Brn-3a, have been reported to regulate VGSCα expression (Chong et al., 1995; Diss et al., 2004, 2006). We have previously shown that Nav1.7 expression is upregulated by Brn-3a[s] in prostate cancer cells (Diss et al., 2006). Consistent with this, Nav1.7 levels are significantly reduced in the

sensory neurons of Brn-3a −/− mice (Eng et al., 2004). In the present study we used Brn-3a regulation of Nav1.7 expression in prostate cancer and neuroblastoma cells to test how well SCN9A promoter construct activity would reflect expression of the endogenous gene. The effect of Brn-3a over-expression on the activity of the SCN9A promoter constructs was consistent with concomitant changes in Nav1.7 mRNA levels (and was isoform-specific). Whether REST/ NRSF regulates the expression Nav1.7 was not tested. However, it is notable (i) that a conserved potential RE-1/NRSE site exists in the human and mouse SCN9A promoters (Fig. 2), (ii) that REST/NRSF is expressed at high levels in both SH-SY5Y and PC-3 cells (Di Toro et al., 2005; J.K.J. Diss, unpublished), and (iii) REST/NRSF levels, like Nav1.7, are altered in SH-SY5Y cells in response to neuronal differentiation (e.g. following exposure to IGF-1; Di Toro et al., 2005).

The effect of differentiating stimuli on Nav1.7 mRNA expression Numerous growth factors, hormones and signalling cascades have been reported to regulate VGSCα mRNA levels, and Nav1.7 in particular (reviewed in Wada, 2006). Such effectors are thought to act primarily at the level of transcription, although some regulation may occur post-transcriptionally utilising mechanisms including mRNA splicing, editing and micoRNA expression. We used three factors (NGF, PMA and RA) to test whether the activity of the proposed SCN9A promoter would ostensibly parallel Nav1.7 mRNA expression. Egr1 was used as a positive control for the three treatments since (i) it is rapidly induced by a wide range of proliferative/differentiation stimuli (Adamson and Mercola, 2002; Adamson et al., 2003) and (ii) egr1 expression is high in SH-SY5Y and PC-3 cells (Wernersson et al., 1998; Mora et al., 2005). Egr1 mRNA levels were altered in line with the cells’ ability to respond to these treatments. Thus, egr1 levels were not altered by NGF in SH-SY5Y cells which lack the trkA receptor (Schulte et al., 2005) or by RA in PC-3 cells which have very low RARβ expression (Campbell et al., 1998; Murthy et al., 2003). Importantly, numerous putative egr1 sites were identified in the SCN9A

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promoter suggesting that this transcription factor may be a key regulator of Nav1.7 expression, including by the treatments administered here. Egr1 may also provide a further mechanism through which Brn-3 transcription factors regulate SCN9A transcription (since Brn-3 proteins strongly activate the egr1 promoter; Smith et al., 1999). It is notable that Brn-3a, egr1 and Nav1.7 levels are all significantly increased in prostate cancer patient biopsies (Adamson and Mercola, 2002; Adamson et al., 2003; Diss et al., 2005, 2006).

5′ primer (Invitrogen, Paisley, U.K.) in touchdown RT-PCRs on the synthesized cDNA. Reactions were performed with Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Paisley, U.K.) as follows: 3 min of denaturing at 95 °C followed by 5 cycles of 30 s at 94 °C, 1 min at 72 °C, 5 cycles of 30 s at 94 °C, 1 min at 70 °C, and 35 cycles of 30 s at 94 °C, 30 s at 65 °C, 1 min at 68 °C. In order to permit subsequent efficient TA-cloning products were then subjected to a further PCR using GoTaq (Promega UK, Southampton, U.K.) comprising 5 cycles of 30 s at 94 °C, 30 s at 65 °C, 1 min at 72 °C, with a final extension step at 72 °C for 10 min. All products were analysed by gel electrophoresis, cloned into the pGEM-T Easy vector (Promega UK, Southampton, U.K.) and sequenced.

Concluding remarks Expression constructs

In conclusion, we have identified the core SCN9A gene promoter, the functional unit controlling Nav1.7 transcription. This is likely to assist in the determination of key factors responsible for physiological and pathophysiological Nav1.7 expression. Given the proposed importance of Nav1.7 activity to pain sensation and prostate cancer progression, identification of the SCN9A promoter could be of clinical use, especially if the generation of specific Nav1.7 protein-blocking drugs proves to be difficult. Furthermore, since variations in the sequence of the cardiac VGSCα (SCN5A) gene promoter have recently been shown to give rise to individual variability in cardiac conduction velocity, response to cardiac VGSC-blocking drugs and predispostion to cardiac arrhythmia (Bezzina et al., 2006), knowledge of the SCN9A promoter sequence could reveal similar individual variations underlying conditions associated with Nav1.7. Experimental methods

Human genomic DNA, extracted from PC-3 cells using Trizol solution (Invitrogen, Paisley, U.K.), was used as a template to amplify 11 different fragments from a region spanning 1440 nucleotides of the SCN9A promoter (from −1280 to +159). PCRs were performed with forward primers containing the SacI recognition sequence and reverse primers containing the HindIII recognition sequence near their respective 5′ ends (shown in Supplementary Table 3), as described above. Products were cloned into the pGEM-T Easy vector, digested with SacI and HindIII, and the insert subcloned into the SacI and HindIII sites of the pGL3-Basic luciferase vector (Promega UK, Southampton, U.K.). Resultant SCN9A-pGL3-Basic clones were sequenced to verify identity and sequence integrity. PCRs were also performed to amplify the same 1440 nucleotide genomic region from SH-SY5Y and LNCaP cells. These products were sequenced and found to be identical to the corresponding PC-3 product (and to the published GenBank human chromosome 2 sequence). For Brn-3a over-expression experiments, two different pLTRpoly eukaryotic expression vectors containing Brn-3a[l] and Brn-3a[s] transcription factors under the control of the MoMuLV promoter were used, as previously described (Diss et al., 2006).

Cell culture SH-SY5Y neuroblastoma cells were cultured in F12/MEM medium supplemented with 15% foetal bovine serum (FBS), 2 mM L-glutamine, 1× non-essential amino acids (all Gibco Invitrogen, Paisley, U.K.) at 37 °C in a humidified incubator containing 5% CO2. Both PC-3 and LNCaP cell lines were cultured in RPMI 1640 medium (Gibco Invitrogen, Paisley, U.K.) with 10% FBS, as described previously (Diss et al., 2001). Nav1.7 exon 1 RT-PCRs RNA was harvested from cells and single-stranded cDNA synthesised as described previously (Diss et al., 2005). RT-PCR reactions targeting the 5′ end of the human Nav1.7 transcript(s) expressed in SH-SY5Y and PC-3 cells were performed using a reverse primer located just after the ATG translation start codon of Nav1.7 (in exon 2) in conjunction with one of 11 forward primers. These forward primers targeted a 1619 nucleotide region of human chromosome 2 located ~64,000 nucleotides upstream of the reverse primer. The sequences of the primers used are shown in Supplementary Table 1. PCRs were performed for 40 cycles, as described previously (Diss et al., 2001). For each PCR, two control reactions (a ‘minus reverse transcriptase’ and a ‘blank’) were also performed to control for possible genomic/cross-contamination from other sources. All products were analysed by gel electrophoresis, cloned into the pGEM-T Easy vector (Promega UK, Southampton, U.K.) and sequenced. 5′ RACE 5′ RACE was carried out using the GeneRacer Kit (Invitrogen, Paisley, U.K.) with 5 μg of total RNA Trizol-extracted from PC-3 cells, according to the manufacturer’s instructions. Reverse transcription was primed with random hexamers. Four reverse primers located in SCN9A exon 2 (Race A–D, Supplementary Table 2) were used in conjunction with the GeneRacer

Dual luciferase gene promoter reporter assays These were carried out on cell extracts using the Dual Luciferase Reporter Assay System (Promega UK, Southampton, U.K.). The SCN9ApGL3-Basic luciferase reporter constructs were transfected into SH-SY5Y and PC-3 cells in 6-well plates (1 × 105 cells/well) with either 4 μl Lipofectamine 2000 (Invitrogen, Paisley, U.K.) for SH-SY5Y cells, or 4 μl Lipofectamine reagent (Invitrogen, Paisley, U.K.) for PC-3 and LNCaP cells, and 0.25 μg of the SCN9A-pGL3-Basic fusion construct per well. In each experiment, 25 ng pRL-TK vector (Promega) expressing Renilla luciferase was co-transfected to control for any inter-sample variability due to differing transfection efficiency or cell viability. Cells were harvested 24 h after lipofectamine/DNA application and luminescence measured as described previously (Diss et al., 2006). In each experiment, luciferase values (normalized to sample-matched Renilla luciferase readings) from three wells were averaged. Experiments were performed at least in triplicate and mean relative promoter activity calculated from the average of each experiment. The activity of the promoter-less pGL3-Basic vector was tested in each experiment to serve as a baseline. Subsequent experiments to determine the effect of differentiating stimuli and Brn-3 transcription factors on the activity of SCN9A-pGL3Basic luciferase reporter constructs were performed as above with the following modifications: (i) Experiments were performed on four wells, with the cells of one well harvested using 750 μl Trizol solution (Invitrogen, Paisley, U.K.) for use in subsequent real-time RT-PCR experiments. (ii) In experiments performed with the differentiating stimuli, NGF, PMA or RA were applied to cells (in the dark) 5 h after transfection (upon re-feeding with normal growth medium) to a final concentration of 100 ng/ml NGF, 300 nM PMA or 10 μM RA. (iii) When determining the effect of Brn-3a over-expression on SCN9A-pGL3-Basic luciferase reporter construct activity, 0.25 μg of one of the SCN9A-pGL3-Basic luciferase reporter constructs and 0.25 μg of either empty pLTRpoly vector or a pLTRpoly

J.K.J. Diss et al. / Mol. Cell. Neurosci. 37 (2008) 537–547 vector expressing one of the Brn-3a isoforms was added to cells during transfections. Real-time RT-PCRs Total RNA was harvested from SH-SY5Y and PC-3 cells in 6-well plates and single-stranded cDNA was synthesised as described above. Realtime RT-PCR was performed using Platinum SYBR Green (Invitrogen, Paisley, U.K.) on the DNA Engine Opticon system (MJ Research, Waltham, MA), as described before (Diss et al., 2005, 2006). Targets for which relative expression levels in SH-SY5Y and PC-3 cells were determined and the primer pairs used are shown in Supplementary Table 4. A second Nav1.7-specific PCR was performed and results subsequently compared to those derived from the first Nav1.7 PCR (‘Nav1.7 PCR1’; Supplementary Table 4). Duplicate reactions for each amplicon were carried out simultaneously for each sample cDNA. Relative expression levels were normalized to the levels of β2 microglobulin (B2M) and NADHcytochrome b5 reductase (Cytb5R) sample-matched normalizing genes (Supplementary Table 4). All PCRs worked with similar efficiencies, as determined by reactions performed on serial dilutions of cDNA derived from PC-3 cells (data not shown). In order to verify product composition, 5 μl of each reaction was electrophoresed on agarose gels and a final melt curve was also carried out from 65 °C to 95 °C with 0.3 °C steps. Data analysis Normalized target expression levels were calculated using the 2− ΔΔCt method (Livak and Schmittgen, 2001) and expressed as means ± standard errors. To determine statistical significance, real-time RT-PCR and dual luciferase data were analysed by one-way ANOVA followed by Bonferroni post hoc tests, and p values b0.05 were considered significant.

Acknowledgments We would like to thank John Estridge, Arthur Bannister, Sean Barry, Dr Dan Berwick, Dr Sam Bowen and Dr David Faulkes for their expert technical assistance, and Emma Sala-Soriano and Maruschka Malacos for useful discussions. For Ken and Edna Wallis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mcn.2007.12.002. References Adamson, E.D., Mercola, D., 2002. Egr1 transcription factor: multiple roles in prostate tumor cell growth and survival. Tumour Biol. 23, 93–102. Adamson, E., de Belle, I., Mittal, S., Wang, Y., Hayakawa, J., Korkmaz, K., O’Hagan, D., McClelland, M., Mercola, D., 2003. Egr1 signaling in prostate cancer. Cancer Biol. Ther. 2, 617–622. Ahmad, S., Dahllund, L., Eriksson, A.B., Hellgren, D., Karlsson, U., Lund, P.E., Meijer, I.A., Meury, L., Mills, T., Moody, A., Morinville, A., Morten, J., O’Donnell, D., Raynoschek, C., Salter, H., Rouleau, G.A., Krupp, J.J., 2007. A stop codon mutation in SCN9A causes a lack of pain sensation. Hum. Mol. Genet. 16, 2114–2121. Bezzina, C.R., Shimizu, W., Yang, P., Koopmann, T.T., Tanck, M.W., Miyamoto, Y., Kamakura, S., Roden, D.M., Wilde, A.A., 2006. Common sodium channel promoter haplotype in Asian subjects underlies variability in cardiac conduction. Circulation 113, 338–344. Black, J.A., Liu, S., Tanaka, M., Cummins, T.R., Waxman, S.G., 2004. Changes in the expression of tetrodotoxin-sensitive sodium channels

545

within dorsal root ganglia neurons in inflammatory pain. Pain 108, 237–247. Campbell, M.J., Park, S., Uskokovic, M.R., Dawson, M.I., Koeffler, H.P., 1998. Expression of retinoic acid receptor-beta sensitizes prostate cancer cells to growth inhibition mediated by combinations of retinoids and a 19-nor hexafluoride vitamin D3 analog. Endocrinology 139, 1972–1980. Catterall, W.A., Goldin, A.L., Waxman, S.G., 2005. International Union of Pharmacology. XLVII. Nomenclature and structure–function relationships of voltage-gated sodium channels. Pharmacol. Rev. 57, 397–409. Chong, J.A., Tapia-Ramirez, J., Kim, S., Toledo-Aral, J.J., Zheng, Y., Boutros, M.C., Altshuller, Y.M., Frohman, M.A., Kraner, S.D., Mandel, G., 1995. REST — a mammalian silencer protein that restricts sodiumchannel gene-expression to neurons. Cell 80, 949–957. Cox, J.J., Reimann, F., Nicholas, A.K., Thornton, G., Roberts, E., Springell, K., Karbani, G., Jafri, H., Mannan, J., Raashid, Y., Al-Gazali, L., Hamamy, H., Valente, E.M., Gorman, S., Williams, R., McHale, D.P., Wood, J.N., Gribble, F.M., Woods, C.G., 2006. An SCN9A channelopathy causes congenital inability to experience pain. Nature 444, 894–898. Cummins, T.R., Howe, J.R., Waxman, S.G., 1998. Slow closed-state inactivation: a novel mechanism underlying ramp currents in cells expressing the hNE/PN1 sodium channel. J. Neurosci. 18, 9607–9619. Cummins, T.R., Dib-Hajj, S.D., Waxman, S.G., 2004. Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy. J. Neurosci. 24, 8232–8236. Diss, J.K., Archer, S.N., Hirano, J., Fraser, S.P., Djamgoz, M.B., 2001. Expression profiles of voltage-gated Na+ channel alpha-subunit genes in rat and human prostate cancer cell lines. Prostate 48, 165–178. Diss, J.K.J., Fraser, S.P., Djamgoz, M.B.A., 2004. Voltage-gated Na+ channels: functional consequences of multiple subtypes and isoforms for physiology and pathophysiology. Eur. Biophys. J. 33, 180–193. Diss, J.K., Stewart, D., Pani, F., Foster, C.S., Walker, M.M., Patel, A., Djamgoz, M.B., 2005. A potential novel marker for human prostate cancer: voltage-gated sodium channel expression in vivo. Prostate Cancer Prostatic Dis. 8, 266–273. Diss, J.K.J., Faulkes, D.J., Walker, M.M., Patel, A., Foster, C.S., BudhramMahadeo, V., Djamgoz, M.B.A., Latchman, D.S., 2006. Brn-3a neuronal transcription factor functional expression in human prostate cancer. Prostate Cancer Prostatic Dis. 9, 83–91. Di Toro, R., Baiula, M., Spampinato, S., 2005. Expression of the repressor element-1 silencing transcription factor (REST) is influenced by insulinlike growth factor-I in differentiating human neuroblastoma cells. Eur. J. Neurosci. 21, 46–58. Djamgoz, M.B.A., Mycielska, M., Madeja, Z., Fraser, S.P., Korohoda, W., 2001. Directional movement of rat prostatic cancer cells in direct-current electric field: involvement of voltage-gated Na+ channel activity. J. Cell Sci. 114, 2697–2705. Drews, V.L., Lieberman, A.P., Meisler, M.H., 2005. Multiple transcripts of sodium channel SCN8A (Na(V)1.6) with alternative 5′- and 3′untranslated regions and initial characterization of the SCN8A promoter. Genomics 85, 245–257. Eng, S.R., Lanier, J., Fedtsova, N., Turner, E.E., 2004. Coordinated regulation of gene expression by Brn3a in developing sensory ganglia. Development 131, 3859–3870. Fertleman, C.R., Baker, M.D., Parker, K.A., Moffatt, S., Elmslie, F.V., Abrahamsen, B., Ostman, J., Klugbauer, N., Wood, J.N., Gardiner, R.M., Rees, M., 2006. SCN9A mutations in paroxysmal extreme pain disorder: allelic variants underlie distinct channel defects and phenotypes. Neuron 52, 767–774. Fraser, S.P., Salvador, V., Manning, E.A., Mizal, J., Altun, S., Raza, M., Berridge, R.J., Djamgoz, M.B., 2003. Contribution of functional voltage-gated Na+ channel expression to cell behaviours involved in the metastatic cascade in rat prostate cancer. I. Lateral motility. J. Cell. Physiol. 195, 479–487. Gershenzon, N.I., Ioshikhes, I.P., 2005. Synergy of human Pol II core promoter elements revealed by statistical sequence analysis. Bioinformatics 21, 1295–1300.

546

J.K.J. Diss et al. / Mol. Cell. Neurosci. 37 (2008) 537–547

Goldberg, Y.P., MacFarlane, J., MacDonald, M.L., Thompson, J., Dube, M.P., Mattice, M., Fraser, R., Young, C., Hossain, S., Pape, T., Payne, B., Radomski, C., Donaldson, G., Ives, E., Cox, J., Younghusband, H.B., Green, R., Duff, A., Boltshauser, E., Grinspan, G.A., Dimon, J.H., Sibley, B.G., Andria, G., Toscano, E., Kerdraon, J., Bowsher, D., Pimstone, S.N., Samuels, M.E., Sherrington, R., Hayden, M.R., 2007. Loss-of-function mutations in the Nav1.7 gene underlie congenital indifference to pain in multiple human populations. Clin. Genet. 71, 311–319. Goldin, A.L., Barchi, R.L., Caldwell, J.H., Hofmann, F., Howe, J.R., Hunter, J.C., Kallen, R.G., Mandel, G., Meisler, M.H., Netter, Y.B., Noda, M., Tamkun, M.M., Waxman, S.G., Wood, J.N., Catterall, W.A., 2000. Nomenclature of voltage-gated sodium channels. Neuron 28, 365–368. Gould, H.J., England, J.D., Soignier, R.D., Nolan, P., Minor, L.D., Liu, Z.P., Levinson, S.R., Paul, D., 2004. Ibuprofen blocks changes in Nav1.7 and 1.8 sodium channels associated with complete Freund’s adjuvantinduced inflammation in rat. J. Pain 5, 270–280. Grimes, J.A., Fraser, S.P., Stephens, G.J., Downing, J.E., Laniado, M.E., Foster, C.S., Abel, P.D., Djamgoz, M.B., 1995. Differential expression of voltage-activated Na+ currents in two prostatic tumour cell lines: contribution to invasiveness in vitro. FEBS Letts. 369, 290–294. Han, C., Rush, A.M., Dib-Hajj, S.D., Li, S., Xu, Z., Wang, Y., Tyrrell, L., Wang, X., Yang, Y., Waxman, S.G., 2006. Sporadic onset of erythermelagia: a gain-of-function mutation in Nav1.7. Ann. Neurol. 59, 553–558. Kasai, N., Fukushima, K., Ueki, Y., Prasad, S., Nosakowski, J., Sugata, K., Sugata, A., Nishizaki, K., Meyer, N.C., Smith, R.J., 2001. Genomic structures of SCN2A and SCN3A — candidate genes for deafness at the DFNA16 locus. Gene 264, 113–122. Klugbauer, N., Lacinova, L., Flockerzi, V., Hofmann, F., 1995. Structure and functional expression of a new member of the tetrodotoxin-sensitive voltage-activated sodium channel family from human neuroendocrine cells. EMBO J. 14, 1084–1090. Laniado, M.E., Lalani, E.N., Fraser, S.P., Grimes, J.A., Bhangal, G., Djamgoz, M.B., Abel, P.D., 1997. Expression and functional analysis of voltageactivated Na+ channels in human prostate cancer cell lines and their contribution to invasiveness in vitro. Am. J. Pathol. 150, 1213–1221. Leibovitch, B.A., Lu, Q., Benjamin, L.R., Liu, Y., Gilmour, D.S., Elgin, S.C., 2002. GAGA factor and the TFIID complex collaborate in generating an open chromatin structure at the Drosophila melanogaster hsp26 promoter. Mol. Cell. Biol. 22, 6148–6157. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-delta delta C(T) method. Methods 25, 402–408. McEvilly, R.J., Erkman, L., Luo, L., Sawchenko, P.E., Ryan, A.F., Rosenfeld, M.G., 1996. Requirement for Brn-3.0 in differentiation and survival of sensory and motor neurons. Nature 384, 574–577. Mora, G.R., Olivier, K.R., Mitchell, R.F., Jenkins, R.B., Tindall, D.J., 2005. Regulation of expression of the early growth response gene-1 (EGR-1) in malignant and benign cells of the prostate. Prostate 63, 198–207. Murthy, S., Marcelli, M., Weigel, N.L., 2003. Stable expression of full length human androgen receptor in PC-3 prostate cancer cells enhances sensitivity to retinoic acid but not to 1alpha,25-dihydroxyvitamin D3. Prostate 56, 293–304. Nassar, M.A., Stirling, L.C., Forlani, G., Baker, M.D., Matthews, E.A., Dickenson, A.H., Wood, J.N., 2004. Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain. Proc. Natl. Acad. Sci. U. S. A. 101, 12706–12711. Pahlman, S., Ruusala, A.I., Abrahamsson, L., Mattsson, M.E., Esscher, T., 1984. Retinoic acid-induced differentiation of cultured human neuroblastoma cells: a comparison with phorbolester-induced differentiation. Cell Differ. 14, 135–144. Sangameswaran, L., Fish, L.M., Koch, B.D., Rabert, D.K., Delgado, S.G., Ilnicka, M., Jakeman, L.B., Novakovic, S., Wong, K., Sze, P., Tzoumaka, E., Stewart, G.R., Herman, R.C., Chan, H., Eglen, R.M., Hunter, J.C., 1997. A novel tetrodotoxin-sensitive, voltage-gated sodium channel expressed in rat and human dorsal root ganglia. J. Biol. Chem. 272, 14805–14809.

Schade, S.D., Brown, C.B., 2000. Identifying the promoter region of the human brain sodium channel subtype II gene (SCN2A). Molec. Brain Res. 81, 187–190. Schulte, J.H., Schramm, A., Klein-Hitpass, L., Klenk, M., Wessels, H., Hauffa, B.P., Eils, J., Eils, R., Brodeur, G.M., Schweigerer, L., Havers, W., Eggert, A., 2005. Microarray analysis reveals differential gene expression patterns and regulation of single target genes contributing to the opposing phenotype of TrkA- and TrkB-expressing neuroblastomas. Oncogene 24, 165–177. Shang, L.L., Dudley, S.C., 2005. Tandem promoters and developmentally regulated 5′- and 3′-mRNA untranslated regions of the mouse Scn5a cardiac sodium channel. J. Biol. Chem. 280, 933–940. Smith, M.D., Latchman, D.S., 1996. The functionally antagonistic POU family transcription factors Brn-3a and Brn-3b show opposite changes in expression during the growth arrest and differentiation of human neuroblastoma cells. Int. J. Cancer 67, 653–660. Smith, M.D., Dawson, S.J., Latchman, D.S., 1997. Inhibition of neuronal process outgrowth and neuronal specific gene activation by the Brn-3b transcription factor. J. Biol. Chem. 272, 1382–1388. Smith, M.D., Ensor, E.A., Stohl, L., Wagner, J.A., Latchman, D.S., 1999. Regulation of NGFI-A (Egr-1) gene expression by the POU domain transcription factor Brn-3a. Brain Res. Mol. Brain Res. 74, 117–125. Strickland, I.T., Martindale, J.C., Woodhams, P.L., Reeve, A.J., Chessell, I.P., McQueen, D.S., 2007. Changes in the expression of Na(V)1.7, Na(V)1.8 and Na(V)1.9 in a distinct population of dorsal root ganglia innervating the rat knee joint in a model of chronic inflammatory joint pain. Eur. J. Pain (Electronic publication ahead of print). Suzuki, Y., Tsunoda, T., Sese, J., Taira, H., Mizushima-Sugano, J., Hata, H., Ota, T., Isogai, T., Tanaka, T., Nakamura, Y., Suyama, A., Sakaki, Y., Morishita, S., Okubo, K., Sugano, S., 2001. Identification and characterization of the potential promoter regions of 1031 kinds of human genes. Genome Res. 11, 677–684. Thomas, M.C., Chiang, C.M., 2006. The general transcription machinery and general cofactors. Crit. Rev. Biochem. Mol. Biol. 41, 105–178. Turner, E.E., Jenne, K.J., Rosenfeld, M.G., 1994. Brn-3.2: a Brn-3-related transcription factor with distinctive central nervous system expression and regulation by retinoic acid. Neuron 12, 205–218. Wada, A., 2006. Roles of voltage-dependent sodium channels in neuronal development, pain, and neurodegeneration. J. Pharmacol. Sci. 102, 253–268. Wallace, R.H., Scheffer, I.E., Barnett, S., Richards, M., Dibbens, L., Desai, R.R., Lerman-Sagie, T., Lev, D., Mazarib, A., Brand, N., Ben-Zeev, B., Goikhman, I., Singh, R., Kremmidiotis, G., Gardner, A., Sutherland, G.R., George, A.L., Mulley, J.C., Berkovic, S.F., 2001. Neuronal sodiumchannel alpha1-subunit mutations in generalized epilepsy with febrile seizures plus. Am. J. Hum. Genet. 68, 859–865. Waxman, S.G., 2001. Transcriptional channelopathies: an emerging class of disorders. Nat. Rev., Neurosci. 2, 652–659. Waxman, S.G., 2006. A channel sets the gain on pain. Nature 444, 831–832. Wernersson, J., Johansson, I., Larsson, U., Minth-Worby, C., Pahlman, S., Andersson, G., 1998. Activated transcription of the human neuropeptide Y gene in differentiating SH-SY5Y neuroblastoma cells is dependent on transcription factors AP-1, AP-2alpha, and NGFI. J. Neurochem. 70, 1887–1897. Wyse, B.D., Linas, S.L., Thekkumkara, T.J., 2000. Functional role of a novel cis-acting element (GAGA box) in human type-1 angiotensin II receptor gene transcription. J. Mol. Endocrinol. 25, 97–108. Yang, Y., Wang, Y., Li, S., Xu, Z., Li, H., Ma, L., Fan, J., Bu, D., Liu, B., Fan, Z., Wu, G., Jin, J., Ding, B., Zhu, X., Shen, Y., 2004a. Mutations in SCN9A, encoding a sodium channel alpha subunit, in patients with primary erythermalgia. J. Med. Genet. 41, 171–174. Yang, P., Kupershmidt, S., Roden, D.M., 2004b. Cloning and initial characterization of the human cardiac sodium channel (SCN5A) promoter. Cardiovasc. Res. 61, 56–65. Yiangou, Y., Facer, P., Chessell, I.P., Bountra, C., Chan, C., Fertleman, C., Smith, V., Anand, P., 2007. Voltage-gated ion channel Na(v)1.7

J.K.J. Diss et al. / Mol. Cell. Neurosci. 37 (2008) 537–547 innervation in patients with idiopathic rectal hypersensitivity and paroxysmal extreme pain disorder (familial rectal pain). Neurosci. Lett. 427, 77–82.

Further reading Angelsen, A., Sandvik, A.K., Syversen, U., Stridsberg, M., Waldum, H.L., 1998. NGF-beta, NE-cells and prostatic cancer cell lines. A study of

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neuroendocrine expression in the human prostatic cancer cell lines DU145, PC-3, LNCaP, and TSU-pr1 following stimulation of the nerve growth factor-beta. Scand. J. Urol. Nephrol. 32, 7–13. Merrill, R.A., Ahrens, J.M., Kaiser, M.E., Federhart, K.S., Poon, V.Y., Clagett-Dame, M., 2004. All-trans retinoic acid-responsive genes identified in the human SH-SY5Y neuroblastoma cell line and their regulated expression in the nervous system of early embryos. Biol. Chem. 385, 605–614.