Characterization of the human somatostatin receptor type 4 promoter

Molecular and Cellular Endocrinology 188 (2002) 75 – 83 www.elsevier.com/locate/mce

Characterization of the human somatostatin receptor type 4 promoter Stephan Petersenn a,b,*, Anja C. Rasch a,*, S. Presch a, Frank U. Beil b, H.M. Schulte c a

IHF Institute for Hormone and Fertility Research, Uni6ersity of Hamburg, 22529 Hamburg, Germany b Department of Medicine, Uni6ersity of Hamburg, 20251 Hamburg, Germany c Endokrinologikum Hamburg, 22767 Hamburg, Germany Received 9 October 2001; accepted 20 November 2001

Abstract Somatostatin (SRIF) exerts inhibitory effects on virtually all endocrine and exocrine secretions. Five distinct SRIF receptor subtypes (sst 1–5) have been identified. In contrast to the other subtypes, very little is known about specific functions of sst4. We investigated structure and regulation of the human sst4 gene. A genomic clone containing the 5% region of the sst4 gene was isolated. 1.5 kb of the promoter was sequenced and putative transcription factor binding sites were identified. The transcription start site was located 88 nucleotides upstream of the translation start site. A − 984 sst4 promoter directed significant levels of luciferase expression in GH4 rat pituitary cells, Skut-1B endometrium cells, and BEAS-2B human bronchial epithelial cells, whereas only low activity was detected in JEG3 chorion carcinoma cells or COS-7 monkey kidney cells. A minimal −209 promoter allowed cell specific expression, its activity in COS-7 cells is not enhanced by co-transfection of the pituitary-specific transcription factor Pit-1. An enhancer element was localized between nt − 459 and − 984. We did not find any regulation of the sst4 promoter region analyzed by SRIF, forskolin, TPA, IGF-1, EGF, T3, glucocorticoids or 17b-estradiol. These studies identify the 5% region of the sst4 gene. Furthermore, specific activity of the promoter in various cell lines is demonstrated. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Somatostatin receptor; G-protein coupled receptor; Structure; Regulation

1. Introduction Somatostatin (SRIF) was originally discovered as a hypothalamic neuroendocrine hormone, which potently inhibits the secretion of growth hormone (GH) from the anterior pituitary. Later it became evident that SRIF exerts inhibitory effects on virtually all endocrine and exocrine secretions. SRIF was demonstrated to play an important role as an endogenous inhibitor of cell proliferation in various normal and neoplastic tissues (Reubi and Laissue, 1995). Attention has also focused on its function in certain areas of the CNS, including motor, sensory, behavioral, cognitive, and autonomic effects (Gillies, 1997). The various effects of * Corresponding authors. Present address: Division of Endocrinology, Medical Center, University of Essen, Hufelandstr. 55, 45122 Essen, Germany (S.P.). Tel.: +49-201-723-2822; fax: + 49-201-7235187. E-mail address: [email protected] (S. Petersenn).

SRIF are mediated through specific membrane receptors. Five distinct SRIF receptor subtypes (sst 1–5) have been identified (Patel, 1999). Based on binding similarities for the SRIF analog octreotide ssts can be viewed as subfamilies, with sst2, sst3 and sst5 comprising one family (SRIF1) with high affinity for octreotide and lanreotide, and sst1 and sst4 comprising another family (SRIF2) with virtually no binding affinity for this analog. The functional significance of endogenously expressed sst4 is currently unclear. Sst4 cDNAs from human (Demchyshyn et al., 1993; Rohrer et al., 1993; Xu et al., 1993; Yamada et al., 1993), rat (Bruno et al., 1992), and mouse (Schwabe et al., 1996) have been described. The amino acid alignment deduced from the human cDNA revealed that the human sst4 consists of 388 amino acids containing seven hydrophobic domains with the potential to serve as membranespanning helices. Transcripts for sst4 have been identified in the brain (Rohrer et al., 1993). By im-

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munohistochemistry, strong sst4 immunoreactivity was localized in specific areas, including the hippocampal formation, the cerebellar cortex, and the medulla (Selmer et al., 2000). In the pituitary, sst4 is transiently expressed during development, but is absent in the adult (Panetta and Patel, 1995). Mori et al. demonstrated intense expression of sst4 in the posterior iris epithelium and ciliary body of the rat eye by in-situ hybridization techniques (Mori et al., 1997). Reverse transcriptase polymerase chain reaction (RT-PCR) revealed the expression of sst4 in mouse, rat, and human lung tissue (Bruns et al., 1996), in human placenta (Caron et al., 1997), and in normal human testicular tissue (Baou et al., 2000). Specific agonists and antagonists will help to establish the physiological role of each receptor subtype in the future. Specificity of ssts might not only occur by interaction with selective ligands, but also involve regulation at the receptor level, or coupling to various signal transduction pathways. To gain a better understanding of factors controlling sst4 expression, we analyzed the transcriptional regulation of this subtype.

2. Material and methods

2.1. Plasmids Plasmid pGL2-Basic is a luciferase vector lacking eukaryotic promoter and enhancer sequences (Promega Corp., Madison, WI). pGL2-Control contains an SV40 promoter and an SV40 enhancer inserted into the structure of pGL2-Basic (Promega Corp.). pSV-b-GAL contains an SV40 promoter and an SV40 enhancer. Both promoter and enhancer drive transcription of the lacZ gene, which encodes the b-Galactosidase enzyme (Promega Corp.). Plasmid −344 hGH/luc contains 344 bp of the human GH promoter and includes two nucleotides of transcribed sequence.

2.2. Isolation of sst4 cDNA probe and screening of a genomic DNA library A DNA fragment containing residues 987– 1247 of the human sst4 gene (numbering of residues relative to the translation start codon) was amplified by PCR (95 °C 30 s, 65 °C 60 s, 72 °C 60 s, 40 cycles) from human lymphocyte genomic DNA using a GeneAmp PCR Kit (Perkin – Elmer, Norwalk, CT). Specific primers used were SR4S1 (5%-CTG-CTG-CCT-CCTGGA-AGG-TGC-TGG-3%) and SR4A1 (5%-GAGATG-CAG-TCT-TCG-GGG-CTG-TAG-3%). The PCR product was cloned into pCRII (hsst4/pCRII) using the TA-Cloning Kit (Invitrogen, San Diego, CA). hsst4/ pCRII was used to synthesize a digoxigenine-labeled probe by PCR using a GeneAmp PCR Kit (Perkin –

Elmer, Norwalk, CT), primers SR4S1 and SR4A1, and DIG-11-dUTP (Roche Diagnostics GmbH, Mannheim, Germany). An amplified human placenta lambda FIXRII genomic DNA library (Stratagene Corp., La Jolla, CA) was screened for a sst4 fragment by adapting a PCRbased method (Takumi and Lodish, 1994). Aliquots containing approximately 10 000 plaque-forming units (pfu) were distributed into each of the 96 wells of a microplate. Pools from each well in a column and from each well in a row were screened by PCR using primers SR4S1 and SR4A1. Screening of pools allowed identification of single wells, which were further investigated. Aliquots of positive wells were plated and screened with the digoxigenine-labeled probe. Hybridization and detection were performed using a DIG Luminescent Detection Kit (Roche Diagnostics GmbH) following the manufacturer protocol. The hybridized probe was immunodetected with anti-digoxigenine, and was then visualized with the chemiluminescense substrate CSPD*®. The light emission was recorded on Kodak XAR-5 film. Positive recombinant plaques were purified by replating twice and grown in liquid culture. Phage DNA was prepared with a QIAGEN Lambda Midi Kit (QIAGEN GmbH, Hilden, Germany) and digested with various restriction enzymes. Genomic fragments were mapped by hybridization with the digoxigenine-labeled probe described above. Subsequently genomic fragments were purified by QIAEX Gel Extraction Kit (QIAGEN GmbH) and subcloned into Bluescript SKII+ Vector (Stratagene Corp.).

2.3. Nucleotide sequence determination Double-stranded plasmid DNA was sequenced by fluorescent sequencing using dye-labeled terminators (ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit, PE Applied Biosystems, Warrington, GB) and Applied Biosystems instrumentation. Sequences were assembled using Lasergene computer software (DNASTAR, Madison, WI). The nucleotide sequence data reported in this paper has been submitted to GenBank and assigned the accession number AF443293. Transcription factor binding sites were identified using TFSEARCH on the Internet, which searches sequence fragments versus TFMATRIX, the transcription factor binding site profile database by E. Wingender, R. Knueppel, P. Dietze, and H. Karas (GBF-Braunschweig).

2.4. Determination of the transcription start site by 5 %-in6erse PCR An adapted inverse PCR method was used to clone 5% cDNA regions (Zeiner and Gehring, 1994). Briefly, mRNA obtained from lung tissue (right lobe, Clon-

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Tech, Palo Alto, CA) was reverse transcribed (51 °C, 30 min; 95 °C, 5 min) by use of 200 U Superscript reverse transcriptase (Life Technologies GmbH, Karlsruhe, Germamy) and 20 pmol antisense-primer SR4A2 (5%-TCC-ACG-CTG-AGC-ACG-GTG-AGA-CAG-3%, position +428). The second strand was synthesized with Escherichia coli DNA polymerase I and T4 DNA ligase, simultaneously RNA was degraded by RNase H (16 °C, 8 h; 75 °C, 5 min) (all enzymes from Life Technologies GmbH). Blunt ends were generated with T4 DNA polymerase (11 °C, 15 min; Life Technologies GmbH). cDNA ligation was performed with T4 DNA ligase (16 °C overnight). The reaction was used for 40 cycles of PCR (95 °C, 1 min; 62 °C, 1 min; 72 °C, 2 min) with sense-primer SR4S2 (5%-CCC-TTC-GGCTCC-GTG-CTG-TGC-3%, position + 337) and antisense-primer SR4A3 (5%-TCG-TCG-GCT-ACG-GCCAGG-TTG-AGC-3%, position + 281). PCR products were cloned into pCRII using the TA-Cloning Kit (Invitrogen). Transcription start sites were determined by sequencing analysis and comparison with genomic sequence and SR4A2 sequence.

2.5. Construction of Luciferase-expression 6ectors containing upstream sequence Upstream sequences were obtained by amplification of the genomic clone using SR4A4 (5%-GCT-GAGCGC-GGC-GGA-GCT-AG-3%, starting at position − 25) as antisense primer and SR4S3 (5%-CCC-GCGGAT-AAT-GAC-TGA-ACC-3%, starting at position − 984), SR4S4 (5%-AGA-AGT-TAA-AAG-GTC-CGGCAT-CTG-C-3%, starting at position − 808), SR4S5 (5%-GGA-TGT-CAA-GTG-CAA-GGC-AAG-3%, starting at position − 603), SR4S6 (5%-AAG-GGC-TGCTGG-GGA-AGA-CGG-3%, starting at position − 459), SR4S7 (5%-TGA-CAC-TGG-AGC-CGG-ACT-GGAGAC-3%, starting at position −363), SR4S8 (5%-ACTCCA-GGG-CTG-GGT-GAG-GCG-CTG-3%, starting at position − 266), and SR4S9 (5%-CCA-CAG-CGGTGC-GAG-CCA-GTC-3%, starting at position − 209) as sense primers (numbering of residues relative to the translation start codon). The PCR products were fractionated on a 1% agarose gel. Fragments of correct size were subsequently cloned into pCRII using the TACloning Kit (Invitrogen). DNA fragments were isolated by restriction digestion with KpnI and NotI, purified by QIAEX Gel Extraction Kit (QIAGEN GmbH) and inserted upstream of the Luciferase reporter gene into pGL2-Basic mammalian expression vector (Promega Corp.).

2.6. Cell culture studies Rat mammo-somatotroph pituitary GH4 cells, monkey kidney COS-7 cells, and human bronchial epithelial

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BEAS-2B cells (Wong et al., 1999) were grown in Dulbecco’s modified Eagle medium (Life Technologies GmbH) containing 10% fetal calf serum (Serva, Heidelberg, Germany). Human endometrium Skut-1B cells were grown in DMEM/HAM-F12 medium containing 10% fetal calf serum. 5× 105 cells/well were seeded in six-well plates for transfection. Experimental and control plasmids were mixed and transfected in triplicates by CaPO4-DNA co-precipitation. Transfections included 3 mg of reporter gene construct and 2 mg of pSV-b-GAL as an internal control of transfection efficiency. For co-transfection studies 1.5 mg of pCMVhPit1 was added. The total amount of DNA was maintained constant with non-specific DNA. After 16 h in the presence of DNA, cells were shocked for 2 min at room temperature with 15% glycerol in PBS, and then replaced with serum-free Dulbecco’s modified Eagle medium containing 3% BSA. Duration of treatment with various hormones was 48 h. Cells were harvested 64 h after transfection in lysis buffer (Promega Corp.). Luciferase assay and b-galactosidase assay were performed following the manufacturer protocol (Promega Corp.). Luciferase light units were normalized to the activity of b-galactosidase. Data are expressed as the mean9SEM. All experiments were repeated at least three times. Mean values were compared using unpaired Mann–Whitney rank sum analysis. A P-value of less than 0.05 was considered significant.

3. Results

3.1. Isolation of a genomic clone of the human sst4 gene Screening of  1 000 000 phage clones of a human genomic library by PCR resulted in the isolation of two positive clones. Phage DNA from these clones was prepared and digested with NotI restriction enzyme to release the insert. Southern analysis confirmed hybridization of the sst4 cDNA probe to an approximate 15 kb insert. The genomic fragment was subcloned into a pBluescript SKII+ vector and designated hsst4/SKII.

3.2. Analysis of the 5 %-flanking sequence and transcriptional start site The 15 kb Not1 fragment was shown to contain the 5% region and more than 1.2 kb of coding sequence of the sst4 gene. The nucleotide sequence of 1.5 kb of the 5%-flanking region of the sst4 gene was determined on both DNA strands (Fig. 1). RT-PCR analysis of human pituitary RNA did not detect any expression of sst4. Therefore, the transcription start site was investigated by 5%-Inverse PCR using total RNA extracted from human lung tissue (Fig. 2). We analyzed 12 indepen-

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dent clones, of which seven contained 88 nt of 5%-flanking region, determining a cytosine residue as a major transcription start site. The remaining clones contained various shorter length of 5%-flanking region, possibly indicating further transcription start sites or artificial early termination of reverse transcription. The sequence obtained by 5%-inverse PCR was identical to the genomic sequence, excluding introns in the 5%-utr identified. The proximal 5%-untranscribed region did not contain any potential TATA or CAAT boxes, but was highly GC-rich with 77% content within the first 300 nucleotides. A number of putative response elements

were found in the 5%-flanking region (Fig. 1). These include sites for the enhancer factor AP-1 at bp −975, − 720, − 365, − 246, for AP-2 at bp − 912 and − 507, and for AP-4 at − 1368, − 825, − 318, −208, − 132. Furthermore, consensus sequences for the nuclear factor NF-1 at bp − 891 and − 156, and for SP1 at bp − 286 were identified. In addition, the promoter region contains consensus motifs corresponding to inducible promoter elements that are known to bind transcription factors induced by exogenous stimuli. A putative binding site for the nuclear transcription factor ER was found at position − 569.

Fig. 1. Nucleotide sequence of 1.5-kb 5%-flanking region of the human sst4 gene. The translation start codon ATG is printed in bold, the translation start site is defined as + 1. Nucleotide positions are shown on the left side. Potential transcriptional regulatory sequences identified by a computer-assisted analysis are underlined. An arrow indicates the transcription start site.

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Fig. 2. Determination of the transcription start site of the human sst4 gene by 5%-Inverse PCR. An adapted 5%-inverse PCR method was used to clone 5% cDNA regions (Zeiner and Gehring, 1994). RNA obtained from human lung tissue was reverse transcribed. Following second strand synthesis and blunt end generation, the DNA strand was self-ligated and subjected to PCR-cloning. A major transcription start sites (seven of 12 clones) was determined at bp − 88 by sequencing analysis and comparison with genomic sequence. The positions of primer SR4A2 (horizontal arrow) and transcription start site (vertical arrow) are indicated.

3.3. Transient expression analysis of the 5 % flanking region To determine whether the sst4 5%-flanking region can direct cell-specific expression, a fragment containing 984 bp 5% of the translation start site was inserted into a transient expression vector, pGL2-Basic, which contains luciferase as the reporter gene. The resulting plasmid ( −984hsst4/luc) was transiently transfected into various cultured cell lines. Gene transfer studies were done by calcium-phosphate transfection, and luciferase enzyme activity was measured in light units as an indication of promoter activity. Cells were co-transfected with pSV-b-GAL as an internal control for transfection efficiency. As shown in Fig. 3, 984 bp of

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Fig. 4. Promoter activity of various deletion constructs of the sst4 5%-flanking region. The schematic diagram on the left represents a series of sst4 promoter – luciferase gene chimeric plasmids with variable 5% ends (from − 984 to −209) and the same 3% end ( − 25) (closed bars). Each construct was transiently transfected into human Skut-1B endometrium cells (open bars), human BEAS-2B bronchial epithelial cells (hatched bars), or GH4 rat pituitary cells (closed bars). Promoter activity (right panel) is expressed relative to the activity of the pGL2-Basic control.

the human sst4 promoter directed significant levels of luciferase expression in GH4 rat pituitary cells, Skut-1B endometrium cells, and BEAS-2B human bronchial epithelial cells, as compared with the promoter-less pGL2Basic luciferase vector. In contrast, we observed only low activity of − 984hsst4/luc in chorion carcinoma cells JEG3 or monkey kidney cells COS-7. Human sst4 5%-flanking region (984 bp) are sufficient to direct cellselective expression in transient transfection analysis. Relative activity of pGL2-Control containing a SV40 viral promoter in COS-7, JEG3, Skut-1B, BEAS-2B, and GH4 cells was 1100-, 400-, 386-, 148- and 27-fold, respectively.

3.4. Elements required for tissue-specific human sst4 promoter acti6ity are located within 209 bp of the translation start site

Fig. 3. Promoter activity of the sst4 5%-flanking region in various cell lines. The −984hsst4/luc construct was transfected in parallel with pGL2-Basic, which lacks promoter activity, into COS-7 monkey kidney cells, JEG3 chorion carcinoma cells, Skut-1B endometrium cells, BEAS-2B human bronchial epithelial cells, and GH4 rat pituitary cells. Cotransfection with CMV-b-Galactosidase was used to control for transfection efficiency. The luciferase activity of each construct was normalized with the b-Gal activity, and values were expressed as fold induction relative to the activity of the promoterless construct pGL2-Basic. Values represent the mean 9 SEM of at least three determinations.

To further analyze the 5%-flanking region of the human sst4 gene for constitutive promoter activity in GH4 rat pituitary cells, BEAS-2B human bronchial epithelial cells, and Skut-1B endometrium cells, varying lengths of 5%-flanking regions generated by PCR were placed upstream of the luciferase reporter gene (left panel of Fig. 4). Activity obtained with the construct containing 209 nucleotides 5% to the translation start site was 26-fold in GH4 cells (closed bars), 19-fold in BEAS-2B cells (hatched bars), and 14-fold in Skut-1B cells (open bars) compared to the promoterless control (right panel of Fig. 4). With constructs between −209 and −459 bp, the levels of luciferase activity were nearly constant for all three cell types, although levels for GH4 cells and BEAS-2B cells were generally higher

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than for Skut-1B cells. In all three cell types, the construct containing 984 bp 5% to the translation start site produced a significant higher activity compared with the other constructs (84-, 79-, 24-fold for GH4 cells, BEAS-2B cells, Skut-1B cells, respectively). These results suggest that the region between the transcription start site and − 209 contains elements sufficient for a minimal promoter, and that the region between − 459 and − 984 contains positive regulators of sst4 expression in GH4 rat pituitary cells, BEAS-2B human bronchial epithelial cells, and Skut-1B endometrium cells. The negative control, pGL2-Basic without any 5%-flanking region, caused low luciferase activity in all three cell lines (data not shown).

3.6. Hormonal regulation of the human sst4 promoter To investigate the hormonal regulation of the human sst4 5%-flanking region, we analyzed the effect of various agents on 984 bp of sst4 promoter. As shown in Fig. 6, treatment with 10 − 6 M forskolin, 10 − 10 M somatostatin, 10 − 7 M TPA, 13 nM IGF-1, EGF at 10 ng/ml, 10 − 7 M hydrocortisone, 10 − 9 M thyroid hormone (T3), and 10 − 9 M 17b-estradiol did not significantly influence activity of the human sst4 promoter in GH4 rat pituitary cells (upper panel) or BEAS-2B human bronchial epithelial cells (lower panel).

4. Discussion

3.5. The pituitary-specific transcription factor Pit-1 is not in6ol6ed in transcriptional acti6ation of the sst4 promoter in GH4 pituitary cells Significant transcriptional activity of the sst4 5%-flanking region in GH4 rat pituitary cells suggests an interaction between pituitary-specific factors and the sst4 promoter. The expression of the POU-domain transcription factor Pit-1 is strictly pituitary-specific and required for transcription of the GH gene. COS-7 monkey kidney cells do not produce any significant amount of the specific transcription factor Pit-1. Sst4 promoter constructs containing 209 or 984 bp 5% of the translation start site were used to determine the effect of Pit-1 co-transfection. As shown in Fig. 5, co-transfection of Pit-1 did not enhance the activity of either construct. In contrast, the activity of 344 bp of the human GH promoter was significantly enhanced by Pit-1 co-transfection. These results suggest that pituitary-specific transcription factors other than Pit-1 may be responsible for the significant transcriptional activity of the sst4 promoter in GH4 pituitary cells.

Fig. 5. Effect of Pit-1 co-transfection on the activity of the sst4 5%-flanking region. The − 209hsst4/luc and the − 984hsst4/luc were transiently cotransfected with (closed bars) or without (open bars) pCMV-hpit1 into COS-7 monkey kidney cells, as described in Section 2. Promoter activity is expressed relative to the activity of the pGL2-Basic control. The plasmid − 344hGH/luc was used as a positive control.

To gain insight into the specific functions of the ssts in various tissues, characterization of the 5% promoter elements and the gene structure is essential. For sst4, the putative promoter region of the rat gene has been cloned and characterized (Xu et al., 1995b). An analysis of the 5% flanking region of the human sst4 gene has not yet been described. We found very little homology between human and rat sst4 5%-flanking regions by computer-assisted nucleotide comparison. Thorough investigation with RNase protection analysis or primer extension analysis by us did not give any results regarding determination of transcription start sites, possibly indicating low abundance of the receptor. By 5%-Inverse PCR using total RNA extracted from human lung tissue, we located a major transcription start site 88 nucleotides upstream from the ATG initiating codon. Xu et al. identified multiple transcriptional initiation sites for the rat sst4 gene, the five major ones mapping between − 126 and −18 relative to the ATG initiation codon (Xu et al., 1995b). The differences for the transcription start sites may result from species- or tissuespecificity. Whereas Xu et al. used rat hippocampus tissue to study the rat sst4 promoter, we utilized RNA obtained from human lung tissue to analyze the human promoter. We did not identify any introns in the 5%-utr of the human sst4 by comparison of 5%-inverse PCR sequence and genomic sequence, as has been observed for other ssts (Kraus et al., 1998; Glos et al., 1998; Gordon et al., 1999). Similarly, Hauser et al. did not find any putative introns in the 5%-utr for the rat sst1 gene (Hauser et al., 1994). Therefore, members of the SRIF2-family may not possess tissue-specific promoters to provide another level of control and specificity. In our study, neither TATA or CAAT boxes nor the initiator consensus sequence PyPyANT/ApyPy were evident near the transcription start site of the human sst4 gene. However, the proximal 5% untranscribed region was highly GC-rich and contained a putative SP1 binding site. Such domains have been proposed to direct gene transcription primarily in housekeeping genes.

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Fig. 6. Hormonal regulation of the human sst4 promoter. A sst4 deletion construct −984hsst4/luc (open bars) and the promoter-less vector pGL2-Basic (closed bars) were transiently transfected into: (A) GH4 rat pituitary cells; or (B) BEAS-2B human bronchial epithelial cells. Regulation by various agents was tested by treatment with 10 − 6 M forskolin (For), 10 − 8 M somatostatin (SRIF), 10 − 7 M TPA, 13 nM IGF-1, EGF at 10 ng/ml, 10 − 7 M hydrocortisone (HC), 10 − 9 M triiodothyronine (T3), and 10 − 9 M 17b-estradiol (E2). Activity is expressed as fold induction relative to that driven by each construct transfected alone in the absence of treatment and represents the mean 9SEM of three independent experiments.

Similar to the human gene, the rat sst4 promoter was found to possess a high GC content, but lacked TATA and CCAAT promoter elements (Xu et al., 1995b). We observed significant transcriptional activity of the identified sst4 promoter in the rat pituitary cell line GH4, the human bronchial epithelial cell line BEAS-2B and the human endometrium cell line Skut-1B. Expression in the human pituitary occurs only transiently during development, but not in the adult (Panetta and Patel, 1995). However, sst4 expression was demonstrated in the rat pituitary (Reed et al., 1999). So far, no data has been published regarding the transcriptional activity of the rat sst4 promoter. Significant activity of the human sst4 promoter in rat pituitary GH4 cells suggests either the absence of specific transcription factors necessary for transcriptional activation of the sst4 gene or the presence of inhibiting transcription factors in the human pituitary. In placenta, binding studies are in agreement with the presence of sst4, but the physiological function remains to be elucidated (Caron et al., 1997). Binding studies also confirmed the presence of sst4 in

lung tissue (Schloos et al., 1997). At present, only limited information is available on the importance of SRIF for lung physiology. In guinea pig, SRIF was suggested to arrest lung liquid production at birth (Perks et al., 1992). SRIF was also found to inhibit the salbutamol-induced bronchorelaxation of canine bronchial muscles (Tamaoki et al., 1994). Airway mucus secretion in the rat stimulated by substance P and neurokinin A and B is inhibited by SRIF (Wagner et al., 1995). Our studies show that a minimal − 209 sst4 promoter contains element(s) that support gene expression in endometrial, pituitary, or bronchial epithelial cells. Furthermore, promoter activity in these cells is significantly enhanced by elements located between bp −459 and − 984. No putative binding sites for tissue-specific transcription factors were identified in these regions by searching the TFMATRIX database. The POU-domain transcription factor Pit-1 is specifically expressed in the pituitary and capable of activating both the prolactin and the GH promoter in non-pituitary cells (Bodner

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and Karin, 1987). However, co-transfection of a Pit-1 expression vector did not change activity of the human − 209 or the − 984 sst4 promoter constructs in our studies. Further analysis will identify the precise nature of transcription factors mediating the transcriptional activity of the sst4 promoter in pituitary, endometrial, and bronchial cells. Results from several systems suggest that the sst gene family may be under regulatory control. In the rat pituitary cell line GH3, treatment with SRIF significantly increased the mRNA levels of sst1, sst3, sst4 and sst5 after 24 and 48 h, whereas sst2 mRNA levels exhibited a biphasic response (Bruno et al., 1994). In our studies of rat pituitary GH4 cells or human bronchial epithelial BEAS-2B cells we did not observe any significant regulation of the human − 984 sst4 5%-flanking region. Regulation of the endogenous rat sst4 receptor in the rat pituitary cell line may differ from control of the human sst4 receptor. Alternatively, functional elements may be located 5% of the promoter region analyzed. Regulation by GH-releasing hormone (GHRH) has been observed for sst1, sst2 and sst5, but not for sst3 and sst4 (Park et al., 2000). Transcriptional activity of the human −984 sst4 promoter was not significantly altered by forskolin treatment in GH4 rat pituitary cells or BEAS-2B human bronchial epithelial cells which supports the results of the expression studies. GH secretagogues (GHS) were recently recognized to have profound effects on GH secretion. We used TPA as an activator of protein kinase C that is an essential element of the signaling cascade regulated by the GHSR to study transcriptional regulation of the human sst4 promoter by GHS. In agreement with the in-vivo situation (Park et al., 2000) we did not observe any significant changes of the − 984 promoter activity by TPA in GH4 pituitary cells or BEAS-2B bronchial epithelial cells. IGF-1 is a major feedback regulator of pituitary GH secretion, with defined actions occurring at both the hypothalamus and pituitary. Sst mRNA levels were not altered by IGF-1 infusion in-vivo, as was demonstrated in GH-deficient spontaneous dwarf rats (Park et al., 2000). Epidermal growth factor (EGF) is another growth factor that has been implicated in the hormonal control of GH (Johnson et al., 1980). Our transfection studies do not indicate any effects of IGF-1 or EGF on transcriptional regulation of sst4. SRIF binding and function in various endocrine target cells is influenced by glucocorticoid exposure. In-vitro, short time exposure of GH4 cells to dexamethasone increased sst1 and sst2 but decreased sst3 mRNA levels, whereas prolonged exposure resulted in opposite changes (Xu et al., 1995a). No data was presented regarding sst4. Our search of 1.5 kb of 5%flanking region of the human sst4 versus TFMATRIX did not identify any putative GRE by homology comparison. Similar, studies of a transient expression system

do not suggest regulation of sst4 by glucocorticoids. For thyroid hormone, expression of sst1 and sst5 was induced by incubation with T4 in TtT-97 murine thyrotrope tumor cells, whereas no significant effect was observed for sst2, sst3 or sst4 (James et al., 1997). We did not observe any significant regulation of the −984 sst4 promoter by thyroid hormone. In primary rat pituitary cells, estradiol significantly increased sst2 and sst3 but decreased sst1 mRNA levels, as demonstrated by quantitative RT-PCR analysis. A discrete, but not significant inhibition of mRNA levels corresponding to sst4 and sst5 was observed (Djordjijevic et al., 1998). A search of the 5%-flanking region of the sst4 gene versus TFMATRIX identified one putative estrogen receptor response element, which is located 569 bp upstream of the translation start site. However, in a transient expression system we observed no significant regulation of the − 984 sst4 promoter by estradiol, which supports the results of the expression studies. In summary, we characterized the 5%-flanking region of the human sst4 gene. Although TATA and CAAT boxes are absent, a GC-rich region was identified in the proximal promoter which may direct gene transcription. Tissue-specific activity of the 5%-sequence in pituitary, endometrial, and bronchial epithelial cells was demonstrated. However, neither in the pituitary nor in the bronchial epithelial cells we observed regulation of promoter activity by various agents. Further study is necessary and will provide insights into the mechanism that regulate the expression of the sst4 gene.

Acknowledgements We thank Dr Jo¨ rres of the Center for Pulmonary Disease, Groß Hansdorf, Germany for the BEAS-2B cells. This work is based in part on the doctoral study by A.R. performed at the Faculty of Biology, University of Hamburg, and was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) Schu 669/5-1.

References Baou, N., Bouras, M., Droz, J.P., Benahmed, M., Krantic, S., 2000. Evidence for a selective loss of somatostatin receptor subtype expression in male germ cell tumors of seminoma type. Carcinogenesis 21, 805 – 810. Bodner, M., Karin, M., 1987. A pituitary-specific trans-acting factor can stimulate transcription from the growth hormone promoter in extracts of nonexpressing cells. Cell 50, 267 – 275. Bruno, J.F., Xu, Y., Berelowitz, M., 1994. Somatostatin regulates somatostatin receptor subtype mRNA expression in GH3 cells. Biochem. Biophys. Res. Commun. 202, 1738 – 1743. Bruno, J.F., Xu, Y., Song, J., Berelowitz, M., 1992. Molecular cloning and functional expression of a brain-specific somatostatin receptor. Proc. Natl. Acad. Sci. USA 89, 11 151 – 11 155.

S. Petersenn et al. / Molecular and Cellular Endocrinology 188 (2002) 75–83 Bruns, C., Raulf, F., Hoyer, D., Schloos, J., Lubbert, H., Weckbecker, G., 1996. Binding properties of somatostatin receptor subtypes. Metabolism 45, 17 –20. Caron, P., Buscail, L., Beckers, A., Esteve, J.P., Igout, A., Hennen, G., Susini, C., 1997. Expression of somatostatin receptor SST4 in human placenta and absence of octreotide effect on human placental growth hormone concentration during pregnancy. J. Clin. Endocrinol. Metab. 82, 3771 –3776. Demchyshyn, L.L., Srikant, C.B., Sunahara, R.K., Kent, G., Seeman, P., Van Tol, H.H., Panetta, R., Patel, Y.C., Niznik, H.B., 1993. Cloning and expression of a human somatostatin-14-selective receptor variant (somatostatin receptor 4) located on chromosome 20. Mol. Pharmacol. 43, 894 –901. Djordjijevic, D., Zhang, J., Priam, M., Viollet, C., Gourdji, D., Kordon, C., Epelbaum, J., 1998. Effect of 17beta-estradiol on somatostatin receptor expression and inhibitory effects on growth hormone and prolactin release in rat pituitary cell cultures. Endocrinology 139, 2272 –2277. Gillies, G., 1997. Somatostatin: the neuroendocrine story. Trends Pharmacol. Sci. 18, 87 –95. Glos, M., Kreienkamp, H.J., Hausmann, H., Richter, D., 1998. Characterization of the 5%-flanking promoter region of the rat somatostatin receptor subtype 3 gene. FEBS Lett. 440, 33 – 37. Gordon, D.F., Woodmansee, W.W., Lewis, S.R., James, R.A., Wood, W.M., Ridgway, E.C., 1999. Cloning of the mouse somatostatin receptor subtype 5 gene: promoter structure and function. Endocrinology 140, 5598 –5608. Hauser, F., Meyerhof, W., Wulfsen, I., Schonrock, C., Richter, D., 1994. Sequence analysis of the promoter region of the rat somatostatin receptor subtype 1 gene. FEBS Lett. 345, 225 –228 published erratum appears in FEBS Lett. July 25;349(1):162. James, R.A., Sarapura, V.D., Bruns, C., Raulf, F., Dowding, J.M., Gordon, D.F., Wood, W.M., Ridgway, E.C., 1997. Thyroid hormone-induced expression of specific somatostatin receptor subtypes correlates with involution of the TtT-97 murine thyrotrope tumor. Endocrinology 138, 719 –724. Johnson, L.K., Baxter, J.D., Vlodavsky, I., Gospodarowicz, D., 1980. Epidermal growth factor and expression of specific genes: effects on cultured rat pituitary cells are dissociable from the mitogenic response. Proc. Natl. Acad. Sci. USA 77, 394 – 398. Kraus, J., Woltje, M., Schonwetter, N., Hollt, V., 1998. Alternative promoter usage and tissue specific expression of the mouse somatostatin receptor 2 gene. FEBS Lett. 428, 165 –170. Mori, M., Aihara, M., Shimizu, T., 1997. Differential expression of somatostatin receptors in the rat eye: SSTR4 is intensely expressed in the iris/ciliary body. Neurosci. Lett. 223, 185 –188. Panetta, R., Patel, Y.C., 1995. Expression of mRNA for all five human somatostatin receptors (hSSTR1-5) in pituitary tumors. Life Sci. 56, 333 – 342. Park, S., Kamegai, J., Johnson, T.A., Frohman, L.A., Kineman, R.D., 2000. Modulation of pituitary somatostatin receptor subtype (sst 1 – 5) messenger ribonucleic acid levels by changes in the growth hormone axis. Endocrinology 141, 3556 – 3563.

83

Patel, Y.C., 1999. Somatostatin and its receptor family. Front Neuroendocrinol. 20, 157 – 198. Perks, A.M., Kwok, Y.N., McIntosh, C.H., Ruiz, T., Kindler, P.M., 1992. Changes in somatostatin-like immunoreactivity in lungs from perinatal guinea pigs and the effects of somatostatin-14 on lung liquid production. J. Dev. Physiol. 18, 151 – 159. Reed, D.K., Korytko, A.I., Hipkin, R.W., Wehrenberg, W.B., Schonbrunn, A., Cuttler, L., 1999. Pituitary somatostatin receptor (sst)1– 5 expression during rat development: age-dependent expression of sst2. Endocrinology 140, 4739 – 4744. Reubi, J.C., Laissue, J.A., 1995. Multiple actions of somatostatin in neoplastic disease. Trends Pharmacol. Sci. 16, 110 – 115. Rohrer, L., Raulf, F., Bruns, C., Buettner, R., Hofstaedter, F., Schule, R., 1993. Cloning and characterization of a fourth human somatostatin receptor. Proc. Natl. Acad. Sci. USA 90, 4196 – 4200. Schloos, J., Raulf, F., Hoyer, D., Bruns, C., 1997. Identification and pharmacological characterization of somatostatin receptors in rat lung. Br. J. Pharmacol. 121, 963 – 971. Schwabe, W., Brennan, M.B., Hochgeschwender, U., 1996. Isolation and characterization of the mouse (Mus musculus) somatostatin receptor type-4-encoding gene (mSSTR4). Gene 168, 233 –235. Selmer, I., Schindler, M., Humphrey, P.P., Waldvogel, H.J., Faull, R.L., Emson, P.C., 2000. First localisation of somatostatin sst(4) receptor protein in selected human brain areas: an immunohistochemical study. Brain Res. Mol. Brain Res. 82, 114 – 125. Takumi, T., Lodish, H.F., 1994. Rapid cDNA cloning by PCR screening. Biotechniques 17, 443 – 444. Tamaoki, J., Tagaya, E., Yamauchi, F., Chiyotani, A., Konno, K., 1994. Pertussis toxin-sensitive airway beta-adrenergic dysfunction by somatostatin. Respir. Physiol. 95, 99 –108. Wagner, U., Fehmann, H.C., Bredenbroker, D., Yu, F., Barth, P.J., von Wichert, P., 1995. Galanin and somatostatin inhibition of neurokinin A and B induced airway mucus secretion in the rat. Life Sci. 57, 283 – 289. Wong, H.R., Ryan, M.A., Menendez, I.Y., Wispe, J.R., 1999. Heat shock activates the I-kappaBalpha promoter and increases I-kappaBalpha mRNA expression. Cell Stress Chaperones 4, 1 –7. Xu, Y., Berelowitz, M., Bruno, J.F., 1995a. Dexamethasone regulates somatostatin receptor subtype messenger ribonucleic acid expression in rat pituitary GH4C1 cells. Endocrinology 136, 5070 –5075. Xu, Y., Bruno, J.F., Berelowitz, M., 1995b. Characterization of the proximal promoter region of the rat somatostatin receptor gene, SSTR4. Biochem. Biophys. Res. Commun. 206, 935 – 941. Xu, Y., Song, J., Bruno, J.F., Berelowitz, M., 1993. Molecular cloning and sequencing of a human somatostatin receptor, hSSTR4. Biochem. Biophys. Res. Commun. 193, 648 – 652. Yamada, Y., Kagimoto, S., Kubota, A., Yasuda, K., Masuda, K., Someya, Y., Ihara, Y., Li, Q., Imura, H., Seino, S., et al., 1993. Cloning, functional expression and pharmacological characterization of a fourth (hSSTR4) and a fifth (hSSTR5) human somatostatin receptor subtype. Biochem. Biophys. Res. Commun. 195, 844 – 852. Zeiner, M., Gehring, U., 1994, Cloning of 5% cDNA regions by inverse PCR. Biotechniques 17, 1050 – 2, 1054.