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Arginine Vasopressin Promoter Regulation Is Mediated by a Neuron-restrictive Silencer Element in Small Cell Lung Cancer¹

CRC Academic Unit of Clinical Oncology, University of Nottingham, City Hospital, Nottingham NG5 1PB [J. M. C., P. J. W.], and Veterinary Pathology, University of Edinburgh, Edinburgh EH9 1QH [C. E. F., J. P. Q.], United Kingdom

	ABSTRACT

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Arginine vasopressin (AVP) is often expressed in small cell lung cancer (SCLC), and a 65-bp AVP minimal promoter fragment is sufficient to restrict activity to SCLC in vitro. We now describe a motif with homology to the neuron-restrictive silencer element (NRSE) within this fragment. Electrophoretic mobility shift analysis demonstrated that multiple specific complexes are bound by this motif. These complexes are cross-competed with a characterized SCG10 NRSE probe and do not bind to the AVP probe with a specific mutation in the NRSE. The complexes vary in mobility between lung tumor cell lines, showing different levels of AVP expression, and some are differentially bound in SCLC. Overexpression of a neuron-restrictive silencer factor expression construct can silence reporter gene expression supported by the AVP promoter in SCLC, although this was dependent on both the level of endogenous AVP expression in the cells and putative enhancer elements in larger promoter constructs. Activation of the proximal AVP promoter in SCLC is therefore proposed to, at least partially, rely on modulation of normal repressor activity at the NRSE.

	Introduction

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SCLCs³ express both neural and epithelial markers. This neuroendocrine phenotype distinguishes SCLCs from the majority of other normal or neoplastic lung cell types and implies that SCLC transcriptional regulation may more closely resemble that of neuronal cells. AVP is frequently detected in SCLC (1) and can act as an autocrine growth factor in these tumors (2 , 3) . Elucidation of the molecular mechanisms mediating this abnormal gene expression may present opportunities for therapeutic intervention in this tumor type. AVP gene expression was reviewed recently (4) , but promoter characterization remains limited. We have described binding sites for several transcription factors in the rat AVP proximal promoter (5) and demonstrated that a small fragment of the human promoter retains SCLC-specific activity in vitro (6) . Repressors are known to be important in controlling neuron-specific gene expression (7) and have been implicated in the transcriptional regulation of several neuropeptides, such as PPT (8 , 9) . Because the physiological expression of AVP is largely restricted to the hypothalamus, disruption of a repressor mechanism in SCLC may in part account for the ectopic expression of this peptide. We have already located the major regulatory elements responsible for restricted expression to between -23 and +42 of the AVP promoter (6) and now describe a NRSE-like motif that spans the transcription initiation site. NRSE elements bind NRSF, a zinc finger repressor protein (10 , 11) that plays a role in restricting expression of numerous neuron-specific genes (12) , e.g., the m4 muscarinic acetylcholine receptor (13) . The location of the AVP NRSE-like motif is at a similar position to that reported previously in the PPT promoter (8) ,⁴ suggesting that the motif is of functional significance. The combination of an NRSE-like repressor and cell type-specific enhancers, such as E-boxes,⁵ may be required to achieve tissue-specific expression of AVP in SCLC. We report here that several sequence-specific DNA-binding proteins preferentially complex with the AVP NRSE-like motif in SCLC and that overexpression of wild-type NRSF can reduce reporter gene expression driven by the AVP promoter in SCLC cell lines in vitro. We suggest that these complexes may modulate the normal repressor function, thereby contributing to the pathological activation of the AVP promoter in SCLC.

	Materials and Methods

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Cell Culture.
The Lu-165 SCLC line (14) was a gift from Dr. T. Terasaki (National Cancer Center, Tokyo, Japan); NCI-H460 (NSCLC), NCI-H345, NCI-H69, and NCI-H711 (all SCLCs) originated from the American Type Culture Collection. Cells were routinely maintained in RPMI/10% bovine calf serum at 5% CO₂/37°C and used during exponential growth for experiments.

EMSA.
Whole-cell extracts were prepared from cell lines after three freeze/thaw cycles. The sample was suspended in an equal volume of buffer D (20 mM HEPES, 20% glycerol, 0.42 M NaCl, 0.2 mM EDTA, 0.25 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and 1.5 mM MgCl₂), and the overall salt concentration was adjusted to 0.42 M. Cells were Dounce homogenized, and debris was pelleted. The sequence of the AVP (NRSE-like motif) and the NRSE (from the SCG10 promoter) probes are shown in Fig. 1b . The mutated AVP probe has a two-base substitution, as described previously for other NRSE (AVPmut sense, 5'-tcgaGCAGAGGCAGCAGCACTTAGCCACCAAGCAG-3'). Double-stranded oligonucleotides were annealed by denaturing at 94°C and cooling slowly to room temperature. Oligonucleotides (100 ng) were labeled with [³²P]dCTP and Klenow (Boehringer); unincorporated label was removed using Chromaspin-10 columns (Clontech) according to the manufacturer’s instructions. Cell extract was incubated at room temperature for 15 min with 1 ng of probe in 100 mM NaCl, 200 ng/ml poly(deoxyinosinic-deoxycytidylic acid) (Sigma) and 100 ng per reaction of NS unlabeled competitor double-stranded oligonucleotide (NS sense: 5'-TCGAATCCCTTTAAATTTGCGAGC-3'). NS will bind the Ku antigen, often abundant in whole-cell extracts (15) . An excess of additional double-stranded competitor oligonucleotide was preincubated with the extract for 5 min, as indicated for individual experiments. For supershift experiments, NRSF antibody (16) was preincubated with extract for 10 min. Samples were analyzed on 4% polyacrylamide (29:1)/0.5x TBE gels, dried, and detected by phosphorimaging (Molecular Dynamics).

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. The human AVP NRSE-like motif. a, the human AVP promoter is shown in the center, aligned to the NRSE consensus sequence (12) above, and a motif from the rat PPT promoter below. Homology between these repressor elements and the AVP sequence are shown by vertical lines; the region of highest homology is boxed. b, the double-stranded oligonucleotide EMSA probes, representing the NRSE motif from the SCG10 promoter (17) , and the AVP NRSE-like motif shown in a. Bases that are identical to the NRSE core consensus motif are underlined.

Transfection and Reporter Gene Assays.
Single-cell suspensions were prepared either by manual disaggregation (SCLC) or trypsinization (NSCLC), and 7.5 x 10⁶ cells were transfected in a volume of 400 µl of RPMI/10% bovine calf serum as described previously (6) . Briefly, an Easiject electroporator (Flowgen) with settings of 260 V and 1050 µF was used to deliver 20 µg of each test plasmid, 20 µg of pSVGal (Promega), and the appropriate amounts of expression plasmids. Cells were transferred immediately to 20 ml of complete medium. Cell lysates were prepared 72 h after transfection in 500 µl of lysis buffer (Promega). These were assayed for protein content (ESL; Roche); CAT and -galactosidase reporter activity were determined using the respective ELISA kits (Roche) and converted to pg of enzyme/µg protein. -Galactosidase levels were used to control for transfection efficiency within each cell line.

	Results and Discussion

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The region of the human AVP promoter from -23 to +42 (p65) is sufficient to restrict activity to SCLC in vitro (6) . We now report that a motif with high homology to the NRSE consensus sequence (12) occurs within this region (Fig. 1a) . We have shown previously that the SCLC cell lines Lu-165 and NCI-H345 express high and low levels of AVP, respectively (6) , whereas the NSCLC line NCI-H460 has no detectable endogenous AVP expression. In each case, reporter gene expression from the -157 to +42 AVP promoter fragment (p199) was found to reflect the endogenous expression level (6) . These cell lines were therefore used to investigate the putative repressor binding site, related to the NRSE, within the minimal AVP promoter.

	Binding of Multiple Specific Complexes to the NRSE.

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In EMSA using whole-cell extracts from SCLC and NSCLC cell lines, the AVP NRSE-like motif (-8 to +23; Fig. 1b ) bound to four specific complexes (Fig. 2, a and b) . In addition, NS binding was apparent in all extracts; this is attributed to the Ku antigen, as confirmed by Western blotting of EMSA gels with a Ku antibody (data not shown). Ku binds DNA termini in a sequence-independent manner, producing random binding patterns on EMSA, which can be competed aberrantly (15) , e.g., in a non-concentration-dependent fashion (Fig. 2b) . In contrast, the multiple specific complexes (labeled A to D) were competed out with homologous double-stranded oligonucleotide but not with the NS oligonucleotide sequence (Fig. 2a) . These complexes differed between the three lung cancer cell lines, which demonstrate varying levels of endogenous AVP expression.

View larger version (86K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. EMSA showing binding of factors to NRSE-like motifs in lung tumor cell lines. Competition with increasing ratios of cold double-stranded oligonucleotides is shown for the AVP NRSE-like motif (AVP), SCG10 (NRSE), or NS probes. In all experiments, binding reactions were conducted with 100 ng NS competitor (NS) and 200 ng/ml poly(deoxyinosinic-deoxycytidylic acid). Specific bands are indicated with arrows or brackets; the non-specific Ku binding is also shown. a, the AVP NRSE-like motif binds several specific complexes (A–D), which vary between cell lines. Competition is shown with oligonucleotides at 50-, 200-, and 500-fold excess. Complexes B and D appear to be SCLC specific. Comp., competitor. b, the AVP NRSE-like motif, showing cross-competition by the characterized SCG10 NRSE. Competition is shown with 100-fold excess of the oligonucleotides. Comp., competitor. c, the SCG10 NRSE probe, showing competition with 10- and 100-fold excess of homologous probe or the AVP NRSE-related probe, and with 100-fold excess of NS. Comp., competitor. d, the AVP NRSE-like motif, showing a lack of competition for specific complexes by the mutated AVP probe (AVPmut) and a lack of binding to AVPmut itself. Competition is shown with 500-fold excess of the oligonucleotides. Comp., competitor.

All of the specific complexes were also competed away from the AVP probe using a well-characterized NRSE probe, derived from the SCG10 sequence (Fig. 1b) , as shown by EMSA in Fig. 2, b and d . These data indicate that the complexes bound by the AVP NRSE will also bind to a functional NRSE. When the SCG10 NRSE was used as a probe for EMSA, the binding of multiple specific complexes was again seen in the lung tumor cell extracts (Fig. 2c) , which were not competed out by excess NS oligonucleotide. These complexes are labeled with arrows and vary between the SCLC and NSCLC cell lines. With this probe, the most strongly bound complexes are evident in both cell lines, whereas the complex of highest mobility is preferentially bound in NSCLC and that of the lowest mobility is preferentially bound in SCLC. Excess of the AVP NRSE motif successfully cross-competed for the complexes bound to the labeled SCG10 NRSE probe, albeit in some cases with a lower affinity than the homologous probe (Fig. 2c) .

It is therefore likely that the proteins seen to bind the AVP probe are related to NRSF and may represent the same complexes that bind to the SCG10 NRSE, because there is evidence of multiple cross-competition; alternatively, they may represent other related or multiple complexes. We attempted to use an NRSF antibody (16) to confirm the identity of these proteins but, because we did not observe the super-shifting of proteins binding to the control SCG10 NRSE probe in HeLa extract, these experiments were not informative. However, specific mutation of the AVP probe, as described previously for the SCG10 and nicotinic receptor NRSEs (17 , 18) , altered the binding (Fig. 2d) . AVPmut failed to compete for binding of the specific complexes to the AVP NRSE probe, and when used as a probe itself, no complexes were observed.

NRSF is widely expressed during development in the mouse and chick embryo, being required to suppress neuronal gene expression in nonneuronal and undifferentiated neuronal tissue (16) . However, although NRSF is predominantly found in the nonneuronal cells of the adult, it is also expressed at low levels in cultured neuronal cells (19) and adult neurons (20) , where it may control differential expression patterns. The mechanism by which NRSF regulates transcription is therefore complex and not fully elucidated, although it has been suggested that NRSF may interact directly with the basic transcriptional machinery (20) . In addition, several splice variants of rat NRSF have now been reported (20) . Although the functional significance of these is not yet known, they showed differential expression patterns in vivo, implying a role for splicing in modulating repression. It therefore appears that the initial theory of NRSE as a dominant silencer element may be overly simplistic, and the mechanism of repression is likely to be highly complex, because the same element regulates numerous genes, which are expressed in different spatial and temporal patterns. The existence of multiple forms of NRSF, such as splice variants, may contribute to this complexity. The multiple complexes seen in EMSA (Fig. 2) could be modified forms of NRSF, e.g., differentially acetylated or phosphorylated proteins; closely related factors, such as splice variants; or multiple protein complexes containing NRSF.

	Modulation of Reporter Gene Expression by NRSF.

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The hypothesis that the NRSE is important in the regulation of AVP expression was supported by functional data, obtained by transient transfection assays in the lung tumor cell lines. The minimal AVP promoter construct (p65) is normally highly active in Lu-165 (SCLC, high AVP), with only 5% of this activity in NCI-H345 (SCLC, low AVP) and undetectable expression in the NSCLC line (Fig. 3a) . Cotransfection with the NRSF expression construct pCMV-HZ4 was carried out in this model to determine whether expression of wild-type NRSF silences the AVP promoter activity in these SCLC cell lines.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Cotransfection of AVP minimal promoter constructs with an NRSF expression plasmid. a, expression from the AVP p65 minimal promoter in lung tumor cell lines: Lu-165 (SCLC, high AVP); NCI-H345 (H345: SCLC, low AVP); and NCI-H460 (H460: NSCLC, no AVP). Values are shown as a percentage relative to the activity in Lu-165. b, reporter gene expression from cotransfection of p65 and pCMV-HZ4 into these lung cancer cell lines. All values are shown as a percentage of the Lu-165 control. c, titration of p65 repression by pCMV-HZ4 in NCI-H345; values are shown as a percentage of the unrepressed activity in NCI-H345 (control). For all data (a–c) n = 3; bars, SD. Statistical significance was determined by Students t test: **, P < 0.005.

We have described previously larger promoter fragments cloned from the AVP 5' region (6) , including p199 (-157 to +42), which contains putative enhancer binding sites, such as E-boxes. This construct has 5-fold higher activity than p65 in the NCI-H345 cell line (Fig. 4a) . When NRSF was cotransfected with this construct (at a ratio of 1:1), reporter gene expression was again down-regulated by 50%, despite the presence of other transcription factor binding sites. The activity of the p199 construct can be further elevated by cotransfection with an expression construct for the transcription factor USF-2.⁶ Although USF-2 conferred a 3-fold increase in the activity compared with p199 alone, again the introduction of NRSF significantly reduced activity by 50% (Fig. 4a) .

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. The effect of NRSF on AVP promoter constructs in SCLC cell lines. a, expression from the AVP promoter in NCI-H345 cells. pCMV-HZ4 was cotransfected (at a ratio of 1:1) with p65 or p199. The USF-2 expression construct pN4 was also cotransfected where indicated. Data are shown as a percentage of the control activity for p65; partial repression is seen in each example. b, expression from the p199 AVP promoter construct on cotransfection with pCMV-HZ4 in two other SCLC cell lines: NCI-H711 and NCI-H69. Values are shown as a percentage of control activity for p199 in NCI-H345; a higher ratio of the repressor is required in the line with higher reporter gene expression. For all data (a and b) n = 3; bars, SD. Statistical significance determined by Student’s t test: **, P < 0.005; *, P < 0.025; (*), P < 0.05.

The introduction of wild-type NRSF, at the ratios achievable in this model, silenced the AVP minimal promoter in those cell lines that show low endogenous AVP transcription and AVP promoter-dependent reporter gene expression. The observation that NRSF reduced activity of all of the constructs to 50% in NCI-H345 (Fig. 4b) , irrespective of the initial expression level, implies that wild-type NRSF is functioning as a dominant repressor. However, although NRSF turns off expression, the level of expression is determined by positively acting factors, and the relative levels of these factors may quantitatively control a regulatory switch. Whatever the mechanism for differential expression of AVP in SCLC, it is likely to result from a combination of positive and negative factors, and the NRSE constitutes a major negative regulator that is lost in SCLC.

Proposed Model of AVP Promoter Derepression in SCLC.
It was noted recently that regions of homology to the NRSE occur within both the AVP and oxytocin coding and intronic regions, which were hypothetically suggested to play a role in hypothalamic transcriptional regulation (4) . However, the AVP NRSE-like motif we describe here is adjacent to the transcriptional start site. This mimics the structural architecture of the PPT gene, where the restricted, neuron-specific expression is mediated by a dominant repressor element located at the same position (8) .⁴ We therefore predicted it would exert a major influence on transcription initiation. It was proposed recently that the NRSE can in fact have a dual function, as both silencer and enhancer, determined by the proximity of the motif to the TATA box (18) . Thus, the location of the NRSE-like motif in both the AVP and PPT promoters raises the possibility that, rather than acting solely as a repressor, this element could also mediate an enhancer function, as described recently for the nicotinic acetylcholine receptor 74 J2-subunit gene (18) and the L1 cell adhesion molecule gene (21) promoters. Bessis et al. (18) found activation of transcription via an NRSE in cultured neuroblastoma cells and also in a transgenic model, when the NRSE was located within the 5' untranslated region, or up to 50 bp from the TATA box. In contrast, no regulatory activity was found for NRSE motifs located in distal 5' promoters, such as the SCG10 gene. A recent report has, however, shown that NRSF alone was not sufficient to activate transcription in other neuroblastoma cell lines, whatever the location of the NRSE (22) .

Although this dual nature of the NRSE is controversial, by analogy the NRSE-like motif within the AVP promoter could function as an enhancer in SCLC. Cotransfection with wild-type NRSF did not activate the AVP promoter in NSCLC (Fig. 3b) ; therefore, NRSF alone had no enhancer function. However, the presence of several specific NRSE-binding complexes in SCLC indicates that factors other than wild-type NRSF are involved. The higher activity of p65 in Lu-165 (Fig. 3a) implies that an enhancer element is located between -23 and +42 of the promoter, which is strongly activated in these cells. The SCLC-specific, NRSF-related molecules we have shown to bind the AVP promoter, such as complexes B and D, may represent factors that act to up-regulate, rather than repress, transcription through this motif. Further characterization of these complexes, perhaps by protein purification, will be required to clarify their role.

The proximal AVP promoter contains multiple transcription factor binding sites, including several potential E-boxes (6) ⁵ and the NRSE-like motif described here. Although regulation of the AVP promoter is predicted to be highly complex, in view of the restricted expression pattern and its responsiveness to physiological stimuli, it appears from our data that the NRSE-like element in the AVP minimal promoter is important in regulating the expression of this gene. We have shown that this motif binds multiple specific complexes in lung cancer, as does a characterized NRSE from the SCG10 promoter. Cross-competition in EMSA demonstrated that these complexes are related to NRSF. The two NRSE sequences can bind differentially and preferentially to similar complexes, and interestingly, some of these complexes differ between SCLC and NSCLC. In addition, we have shown that wild-type NRSF can silence the AVP promoter in a SCLC cell line with low endogenous AVP expression. However, the location of the AVP NRSE at the transcription initiation site renders mutational analysis beyond the scope of this report.

These findings therefore suggest that AVP expression in SCLC is, at least partially, dependent on modulation of the normal repressor activity, and those complexes that differ between lung tumor cell lines may functionally affect AVP expression. Such a mechanism may mimic that which permits normal hypothalamic expression or constitute a novel mechanism for overcoming repression of certain genes in neuroendocrine tumors. Expression of proteins that block the binding of NRSF may allow an upstream enhancer element to function, leading to AVP expression in SCLC. Alternatively, one of the novel complexes may act as an enhancer, possibly through interaction with other molecules, and directly antagonize the repressor function though the NRSE. Protein purification will be required to determine the nature of these complexes, clarify the mechanisms of transcriptional regulation in the AVP promoter, and allow correlation of these proteins to the progression of SCLC. However, this represents the first study of this class of repressor in neuroendocrine SCLC tumors.

	ACKNOWLEDGMENTS

 
We thank Jodie Edgson and Julie Stanley for excellent technical assistance.

	FOOTNOTES

 
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.

¹ Supported by the Association for International Cancer Research, St. Andrews, Scotland, and the Cancer Research Campaign (to J. M. C. and P. J. W.), the Medical Research Council, and the Wellcome Trust (to C. E. F. and J. P. Q.).

² To whom requests for reprints should be addressed, at CRC Academic Unit of Clinical Oncology, City Hospital, Hucknall Road, Nottingham NG5 1PB, United Kingdom. Phone: 44 (0)115 969 1169, extension 47320; Fax: 44 (0)115 962 7923; E-mail: judy.coulson{at}nott.ac.uk

³ The abbreviations used are: SCLC, small cell lung cancer; AVP, arginine vasopressin; PPT, preprotachykinin; NRSE, neuron-restrictive silencer element; NRSF, neuron-restrictive silencer factor; NSCLC, non-small cell lung cancer; EMSA, electrophoretic mobility shift analysis; NS, nonspecific; CAT, chloramphenicol acetyltransferase; USF, upstream stimulatory factor.

⁴ A. Mackenzie, C. F. Fiskerstrand, B. Ebrahimi, and J. P. Quinn. Neuron-restrictive silencer factor (NRSF) is a dominant repressor of rat preprotachykinin-A promoter activity, submitted for publication.

⁵ J. M. Coulson, C. F. Fiskerstrand, P. J. Woll, and J. P. Quinn. E-box motifs within the human vasopressin promoter contribute to a major enhancer in small cell lung cancer. Biochem. J., in press.

⁶ J. M. Coulson, C. F. Fiskerstrand, P. J. Woll, and J. P. Quinn. E-box motifs within the human vasopressin promoter contribute to a major enhancer in small cell lung cancer. Biochem. J., in press.

Received 8/25/99. Accepted 9/ 2/99.

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