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Dual Promoters of Mouse μ-Opioid Receptor Gene1
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
234, 351–357 (1997)
RC976640
Dual Promoters of Mouse m-Opioid Receptor Gene1 Jane L. Ko,2 Sharon R. Minnerath, and Horace H. Loh Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455
Received April 8, 1997
Two transcriptional initiation sites, distal and proximal, located at approximately 500 bp apart in mouse m-opioid receptor gene were identified recently. Using deletional and transfection analyses, the distal or proximal promoter alone displayed similar activity in neuronal cells constitutively expressing m-opioid receptors (SH-SY-5Y). The presence of both promoters did not result in an increase in activity. However, when distal promoter linked with 3 *-downstream sequences without the proximal promoter region, the distal promoter activity was abolished. Transfection analysis using various cell lines further suggested that only the proximal promoter preferentially directed the neuronal subtype expression. To verify the physiological importance of each promoter, the ratio of m-receptor transcripts initiated by either promoter was examined by quantitative RT-PCR using mouse adult brain mRNA. We found that receptor mRNA was predominantly initiated by the proximal promoter. These studies suggested that the proximal promoter was the major functional promoter. q 1997 Academic Press
Opioids are used as potent clinical analgesics, but have serious limitations such as the induction of tolerance and dependence. Pharmacological studies have identified three major types of opioid receptors in mammalian brain, designated m, d and k (1). The cDNA of each of these receptors has now been cloned, and shown to have a predicted amino acid sequence characteristic of G-protein coupled receptors, with seven putative membrane domains (2-7). Expression of opioid receptors is mainly confined to the central nervous system (CNS), and each receptor type has a distinct distribution pattern (8-11). In addition, the regulation of the 1
This research was supported by NIH research grants DA-00546, DA-01583, DA-05695, KO5-DA-70554, and A & F Stark fund of the Minnesota medical foundation. 2 To whom correspondence should be addressed at Department of Pharmacology, University of Minnesota Medical School, 3-249 Millard Hall, 435 Delaware street S.E., Minneapolis, MN 55455. Fax: 612-625-8408. e-mail address: [email protected].
opioid receptor gene expression may be in response to fluctuating levels of various agents in certain brain regions (12-13). Study of the mechanism underlying the transcriptional regulation of opioid receptor genes may facilitate elucidation of the spatial expression and the modulation of expression in different physiological and pathological states. The recent isolation and partial analysis of genomic DNA of m-opioid receptor (14-15) has made it possible to study how the expression of this gene is regulated. The mouse m-opioid receptor gene is over 53 Kb in length, with its coding sequence divided by three introns. A cluster of four major transcription initiation sites in a region located between 291 and 268 bp upstream of the translation start site have been identified by our laboratory (14), and a second transcription initiation site located at 794 bp upstream of the translation start site was later reported (15). These results suggested that mouse m-opioid receptor gene was likely to be controlled by two promoters. The presence of dual or multiple promoters in some genes has been reported (16-20), and the activation of different promoter in each gene depended on certain physiological condition, such as specific tissues, specific cells, or different developmental stages. In this study, we investigate the expression of mouse m-opioid receptor gene under the control of two promoters. Using deletional analysis and transfection assay, the activity of each promoter was examined in various cultured cells expressing or not expressing the m-opioid receptors, and the physiological importance of each promoter was further verified by quantitative RT-PCR method using mouse brain mRNA. MATERIALS AND METHODS Plasmid construction. Promoter-luciferase fusion plasmids were constructed containing 4.7 Kb (04744 to 0249 bp, the translation start site was designated as /1) or shorter 5*-upstream regulatory sequences of the mouse m-opioid receptor gene. The pL4.7K construct (04744 to 0249 bp) was generated by ligation of the 4.4 Kb Sac I and BamH I DNA fragment (04744 to 0297 bp) of mouse m-opioid receptor gene and the PCR fragment containing sequence from the BamH I site to 0249 bp (0297 to 0249 bp) into the polylinker sites of a promoterless luciferase vector, pGL3-basic (Promega). The se0006-291X/97 $25.00
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Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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quence of the PCR fragment from the BamH I site to 0249 bp was confirmed by sequencing. The pL1.3K deletion construct (01326 to 0249 bp) was created by deletion of Sac I/EcoR I fragment (04474 to 01325 bp) from the pL4.7K plasmid. The pL1.3K/444 construct (01326 to 0444 bp) was generated by deletion of Pst I/Xho I fragment (0443 to 0249 bp) from the pL1.3K construct. The other constructs, designated pL800 (0800 to 0249 bp), pL621 (0621 to 0249 bp) and pL450 (0450 to 0249 bp), as well as pLup (01326 to 0775 bp), were generated by PCR, with all of the sense primers bearing the Kpn I site and the antisense primers bearing the Xho I site. The sequences of all the PCR fragments were cloned into PCR II vector (Invitrogen), confirmed by sequencing, and then subcloned into polylinker sites of pGL3-basic vector. The pL299 (0299 to 0249 bp) construct was generated by deletion of Kpn I/BamH I fragment (0450 to 0300 bp) from the pL450 plasmid. The 3*-ends of pL4.7K, pL1.3K, pL800, pL621, pL450 and pL299 constructs were all at position 0249 bp, which was 20 bp downstream from the proximal transcription initiation site. The 3*-end of the pLup construct was at position 0775, which was 20 bp downstream from the distal transcription initiation site. Cell culture. Human neuroblastoma SH-SY-5Y cells were grown in RPMI 1640 medium with 15% heat-inactivated fetal calf serum in an atmosphere of 5% CO2 and 95% air at 377 C. Mouse neuroblastoma neuro2a and NS20Y cell lines, as well as fibroblastoma of Chinese hamster ovary cells (CHO), were grown in Dulbecco’s modified Eagle’s medium with 10% heat-inactivated fetal calf serum and were incubated in 10% CO2 and 90% air at 377 C. Transient transfection and luciferase activity assay. Cells were transfected with the construct plasmids using the DOTAP (Boehringer Mannheim) lipofection method as described by the manufacturer. Briefly, cells in six-well plates with 30-50% confluency were transfected with an equimolar amount of each promoter-luciferase fusion plasmid. The amount of DNA used in each cell line was within the linear range of the relationship between the luciferase activity and the amount of DNA. All transfection experiments were repeated four times or more with similar results, utilizing the plasmids that were independently prepared at least twice. To correct the differences in transfection efficiency from dish to dish, one fifth molar ratio of pCH110 plasmid (Pharmacia) containing the b-galactosidase gene under the control of SV40 promoter was included in each transfection and used for normalization. Forty-eight hr. after transfection, cells were washed with phosphate-buffered saline and lysed with lysis buffer (Promega). The luciferase and b-galactosidase activities of each lysate were determined as described by the manufacturers (Promega and Tropix, respectively). Reverse transcription coupled polymerase chain reaction (RT-PCR). To detect different m-opioid receptor mRNA species transcribed from distal or proximal transcription initiation sites, quantitative reverse transcription coupled PCR (RT-PCR) was performed using mouse brain RNA. Mouse brain PolyA/ RNA was isolated by the oligo(dT)cellulose chromatography method (PolyATract mRNA isolation kit, Promega), and also purchased from Clontech for comparison. First strand cDNA was synthesized using 3 mg polyA/ RNA and 500 ng random hexamers (Promega) in the presence of reverse transcriptase (SuperScript II RNase H0 reverse transcriptase, Gibco BRL). To conduct quantitative analysis, the amount of amplified fragments was within a linear range of the relationship between the amount of RNA used and the amount of amplified fragments generated. Furthermore, the actin-specific primers (5*-TGGCCTTAGGGTTGCAGGGGG-3* and 5*-GTGGGCCGCTCTAGGCACCA-3*) were included in every PCR reaction for normalization (21). The PCR cycle was performed as 1 min at 947C, 1 min at 607C, and 2 min at 727C for 30 cycles. A pair of Actin primer was added at the cycle 20. Two sense primers were used, which started from the distal (D: 5*-GGGGGCACATGAAACAGGCTTCTTT-3*), or proximal transcription initiation sites (P: 5*-CTCAGAGAGTGGCGCTTTGGGATGC-3*). Two antisense primers, corresponding to the coding re-
gion of m-opioid receptor gene, were used: (1) ATG: 5*-CCTGGGCCGGCGCTGCTGTCCAG-3* (started from ATG in exon I), and (2) tm2: 5*-CGCTAAGGCGTCTGCCAGAGCAAG-3* (located at the 2nd transmembrane domain in exon II). Three additional antisense primers, corresponding to the region between the distal and proximal transcription initiation sites were also used: LC (5*-ATTATTGTTGTCCTTTACCCACAT-3*), 0.5P (5*-GCACCTTATACCTCAACACTCTTC-3*) and Pd (5*-GCATCCCCAAAGCGCCACTCTCTGAGT3 *). PCR products were analyzed on agarose gels and then subjected to Southern blot analysis. The blots were hybridized with m-opioid receptor specific probes, followed by hybridization with actin specific probes for normalization. The hybridizing signals were quantitized using a molecular Dynamic Storm 840 phosphoimager system. Two m-opioid receptor specific probes were used. Probe 1 was a 562 bp fragment located in the region between the distal and proximal transcription initiation sties, which was specifically used to detect the transcripts initiated by distal promoter only. Probe 2 was a 200 bp fragment (Hind III/Stu I fragment from pBluescript-MOR 1; generous gift from Dr. L. Yu), located at 5*-untranslated region of mopioid receptor. This 200 bp probe was mainly used for quantitation purpose.
RESULTS Identification and functional analysis of dual promoters in mouse m-opioid receptor gene using SH-SY5Y cells. Two transcriptional initiation sites (approximately 500 bp apart) in mouse m-opioid receptor gene were reported recently (14-15). These findings implicated that the expression of mouse m-opioid receptor gene might be under the control of two promoters. To test this, chimeric constructs (Fig. 1A) containing both promoters (pL1.3K construct), proximal promoter alone (pL800, pL621, and pL450 constructs), or distal promoter alone (pLup construct), together with the luciferase reporter gene (pGL3-basic vector, B), were tested using transient transfection assays in SH-SY-5Y, a neuroblastoma cell line constitutively expressing the m-opioid receptors (22-23). As shown in Fig. 1B, the luciferase activities were similar in magnitude among the constructs containing only the proximal promoter (pL800, pL621, and pL450 constructs), or only the distal promoter (pLup construct). These results suggested that the distal or the proximal promoter alone could function independently and possessed similar promoter activity in SH-SY-5Y cells. However, the construct containing both promoters (pL1.3K) did not produce greater luciferase activity than the constructs containing only one promoter. This data indicated that the presence of both promoters resulted in no additive effect in promoter activity. Moreover, the luciferase activity of pL299 construct (containing only 50 bp region of the proximal transcription initiation sites) was, in fact, not significantly greater than that of pGL3-basic construct. Comparison of the luciferase activities between pL450 and pL299 constructs, it suggested that the proximal promoter activity resided in 0450 to 0300 bp plus 0299 to 0249 bp region, which included the proximal transcription initiation sites.
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FIG. 1. Functional analysis of promoter activities of mouse m-opioid receptor gene in SH-SY-5Y cells. (A) A schematic diagram representing the mouse m-opioid receptor gene from nucleotide 01326 to 0249 relative to the translation start site (ATG), which was designated as /1. The distal and proximal transcription initiation sites, 0794 and 0268 bp, are indicated by arrows. The fragments in various lengths of the 5*-regulatory regions were inserted into the promoterless luciferase (open box) vector, pGL3-basic (B). The number on the left of each construct refers to the number of the nucleotide at the 5*-end of each inserted promoter fragment. A deletion is indicated by a broken line. The pGL3-control plasmid (C) containing SV40 promoter (black box) and enhancer (hatched box) was used as the positive control.; (B) Transient transfection and luciferase assay of the constructs with various fragments from mouse m-opioid receptor gene in SH-SY-5Y cells. To calibrate transfection efficiency, the promoter activity of each fusion gene was normalized according to the b-galactosidase activity from cotransfection of the internal control plasmid, pCH110, and was expressed as n-fold activation of the promoterless plasmid (pGL3-basic) activity in the cells. The histograms and n-fold activation show the mean { S.E. of the results from four or more independent transfection experiments with at least two different plasmid preparations.
Functional analysis of m-opioid receptor gene promoters in various cultured cells. Distribution of m-opioid receptors was mainly in the CNS with a neuron specific and neuron-subtype specific manner (8-11). The role of each promoter in gene expression was further examined using deletional and transfection assays in various cell lines. The m-opioid receptor expressed cell, SH-SY-5Y, was used as the positive control cell line. Neuro2a, a neuronal cell expressing no m-opioid receptors (24), was used as the negative cell line in comparison with SHSY-5Y cell for the neuronal subtype specificity. The CHO, a non-neuronal cell expressing no m-opioid receptors (2-5), was used in comparison with SH-SY-5Y cell for the neuron specificity. As shown in Fig. 2, the 5*-serial deletion constructs (pL4.7K, pL1.3K, pL800, pL621 and pL450), ranging from 04.7 Kb to 0450 bp, were examined. The positive control plasmid (C, pGL3-control contained the SV40 promoter and enhancer), and the negative control plasmids (pL299 construct contained 50 bp region of the proximal transcription initiation sites and displayed no activity) were also tested. The positive control plasmid produced similar luciferase activity (100-130 fold over that of the pGL3-basic construct) among three cell lines, and the negative control plasmid (pL299) produced no significant activity in all cell lines (Fig. 2). These 5*-deletion constructs (pL4.7K, pL1.3K,
pL800, pL621 and pL450), all containing the proximal promoter, consistently displayed approximately 3-5 times greater activities in SH-SY-5Y than those in neuro2a and CHO cells. High activities of these constructs in SH-SY-5Y and low activity in neuro2a and CHO cells correlated with the expression levels of m-opioid receptors in these three cell lines (m-receptors expressed in SH-SY-5Y and no m-receptor expressed in neuro2a and CHO cells). In contrast, the pLup construct, containing only the distal promoter, displayed high promoter activity in SH-SY-5Y (12.4 { 1.5 fold), and also in non-m-receptor expressing neuro2a (10.4 { 1.6 fold) cells, but not in CHO (2.9 { 0.2 fold) cells (Fig. 2). These results suggested that only the proximal promoter preferentially directed the neuronal subtype expression (SH-SY-5Y versus neuro2a cells). To confirm the differential neuronal subtype expression between these two promoters, we further tested the deletion constructs in a second neuroblastoma cell line (NS20Y), which does not express the m-but only the d-opioid receptor (25). As shown in Fig. 3, only the activity (13.4 fold) of pLup construct in NS20Y was as high as that obtained in neuro2a and SH-SY-5Y cells, while the 5*-deletion constructs containing the proximal promoter displayed low promoter activity in NS20Y cells as in neuro2a cells. These results thus corroborated our previous observation from neuro2a cells.
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FIG. 2. Functional analysis of the mouse m-opioid receptor gene promoter in various cell lines. A schematic diagram represented a series of 5*-deletion constructs of mouse m-opioid receptor gene, with various 5* ends ranging from 04.7 Kb to 0299 bp, and the 3* end at 0249 bp (equivalent to plus 20 nucleotides from proximal transcription start site). These constructs were inserted into the promoterless luciferase (open box) vector, pGL3-basic (B). The number on the left of each construct refers to the number of the nucleotide beginning of the 5*-regulatory sequence. A deletion is indicated by a broken line. pGL3-control plasmid (C) containing SV40 promoter (black box) and enhancer (hatched box) was used as the positive control. The promoter activity of each construct in SH-SY-5Y (gray columns), neuro2a (hatched columns) and CHO (open columns) cells was normalized to b-galactosidase activity. The histograms show the mean { S.E. of the results from four or more independent transfection experiments with at least two different plasmid preparations.
Negative regulatory sequences suppressed the expression initiated by the distal promoter. According to the results shown above, two points need to be further addressed. First, no additive promoter activity was observed in the presence of both promoters. Second, the distal promoter alone produced no neuronal subtype
FIG. 3. Functional analysis of the mouse m-opioid receptor gene promoter in NS20Y cells. The histograms show the n-fold activation in normalized luciferase activity over the promoterless vector pGL3basic activity. The data are presented as mean { S.E. from four transfection experiments with two different plasmid preparations. **Indicates that the value of pLup construct is significantly different from those of other deletion constructs (põ0.01, student t-test).
preferential expression, but when it was connected with the proximal promoter, the preferential expression was observed. To investigate the possible mechanism, the proximal promoter region was deleted from the pL1.3K construct, which contained both promoters. Because the proximal promoter activity was shown to reside in 0450 to 0248 bp region (Fig. 1), the pL1.3K/ 444 plasmid (from 01326 to 0444 bp) was then created. It contained only the distal promoter (01326 to 0775 bp), plus 332 bp fragment (0775 to 0444 bp) immediately 3*-downstream of the distal promoter (Fig. 2). Surprisingly, the activity of the pL1.3K/444 construct had no significant difference from that of pGL3basic in all the tested cells (1.2 { 0.3 fold in SH-SY5Y, 0.6 { 0.1 fold in neuro2a, 0.9 { 0.2 fold in CHO and 0.3 { 0.1 fold in NS20Y) (Fig. 2 and 3). Comparing these results with that of the pLup construct suggested that in the presence of 332 bp (0775 to 0444 bp) fragment located 3*-downstream of the distal transcription initiation site, the expression of reporter gene initiated by the distal promoter was abolished. Whereas, the expression of pL800 construct directed by the proximal promoter was still active in the presence of this 332 bp fragment (Fig. 1 and 2). The existence of negative regulatory element, located in 0775 to 0444 bp region and specific suppression of the distal promoter activity only, may explain why no additive effect was observed in the presence of both promoters (pL4.7K and pL1.3K constructs). Also, it
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may explain why in the absence of the proximal promoter and the 332 bp negative regulatory region, the distal promoter alone was active in non-m-opioid receptor expressed neuronal cells (neuro2a and NS20Y).
FIG. 4. Differential expression of m-opioid receptor mRNAs directed by the distal or proximal promoter of m-opioid receptor in mouse brain. (A) A schematic diagram represents the relative positions of the distal (D) and proximal (P) promoters in mouse m-opioid receptor gene. The gray boxes represent the coding regions divided into 4 exons. Positions and directions of the primers used in RT-PCR experiments are labeled by arrows with the arrow heads pointing to the 3*-end. The sizes of expected RT-PCR fragments amplified by each pair of primers are indicated by number. The broken line indicates the intron region, absence in receptor mRNA, nor in the RTPCR products. The m-opioid receptor specific probes (probe 1 and 2) for Southern blot analysis were indicated by the thick lines corresponding to the size and position in diagram.; (B) Analysis of the RT-PCR products by electrophoresis with ethidium bromide staining. Each lane represents the amount of receptor message obtained from 25 ng polyA RNA isolated from adult mouse brain. RT ‘‘/’’ or ‘‘0’’ indicates the presence or absence of reverse transcriptase in the first strand cDNA synthesis reaction. Hundred base pair ladder (Gibco BRL) was used as the DNA marker. A pair of actin-specific primers is included in every PCR reaction as an internal control for quantitation, and the actin transcript is indicated by white arrow.; (C) Quan-
Physiological role of m-opioid receptor gene promoters in brain. To test the physiological importance of each promoter in CNS, and to verify that the distal promoter activity was suppressed in vivo, the ratio of m-opioid receptor transcripts in mouse brain RNA directed by either the distal or the proximal promoters was examined using quantitative RT-PCR method. To perform the quantitative RT-PCR, a pair of actin-specific primers, located at different exons, was included in every PCR reaction as an internal control for the normalization purpose. The RT-PCR products were analyzed by electrophoresis and followed by Southern blot analysis. Each specific band was quantitized using the phosphoimager and the m-opioid receptor specific signal was normalized to the actin in each sample. Since there is no alternative splicing occurred at the 5*-untranslated region of m-opioid receptor gene, the only difference between the receptor mRNA initiated by proximal promoter or by distal promoter was the region between the distal and proximal transcription initiation sites. Therefore, if both promoters of m-opioid receptor gene were active, the RT-PCR fragment amplified from the proximal transcription initiation site, located at the common region of receptor mRNA initiated by both promoters, should represent the total transcripts directed by both promoters (Fig. 4A). The RTPCR fragment amplified from the distal transcription initiation site, represented the transcript directed only by the distal promoter. Consequently, the difference between the amount of these two RT-PCR fragments represented the transcripts directed only from the proximal promoter. As shown in Fig. 4B, receptor transcripts were clearly detected by using the primer pair of P (sense primer, starting at the proximal transcription initiation site 0268 bp) and tm2 (antisense primer, located at the 2nd transmembrane domain in exon II). Whereas, the receptor transcripts started from distal
titation analysis of PCR products by Southern blot. The blots were hybridized with the m-opioid receptor specific probe, stripped the signal, and then hybridized with actin specific probe. The hybridized signals were quantitized and each band was normalized using actin signal as internal control. To rule out the false signal from the genomic contamination, the exact same RT-PCR reactions were carried out in the presence or absence of the reverse transcriptase. The amounts of amplified RT-PCR products from primer pairs, D/LC, D/ 0.5P, D/Pd, D/ATG and P/ATG, were obtained by subtracting the signal from RT-PCR between the presence and absence of RT. The amount of P/tm2 product, probed with probe 2, was arbitrarily defined as 100%. The histograms show the percentage of normalized m-opioid receptor gene products. The data are presented as mean { S.E. from at least three different mouse brain RNA preparations.
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transcription initiation site were barely detected, by amplifying D/tm2 product using the primer pair of D (sense primer, beginning at the distal transcription initiation site) and tm2 (Fig. 4B). The amount of RT-PCR product (P/tm2), amplified by the primer pair of P and tm2 as well as probed with m-receptor-specific probe 2, was arbitrarily defined as 100% (Fig. 4C). Probe 2, a 200 bp fragment located at 5*-untranslated region of m-opioid receptor, was the probe used for quantitation (Fig. 4A). The difference between the amount of P/tm2 and D/tm2 RT-PCR products was merely equal to that of P/tm2 (Fig. 4C). These results suggested that the proximal promoter, but not the distal, was a functional promoter in the brain. To avoid the possibility that the low yield of D/tm2 (1154 bp) product was resulted from the secondary structure in receptor mRNA, the shorter RT-PCR fragment, representing the segment between distal initiation site and start codon (ATG) of coding region, was amplified by using sense primer D paired with 4 different antisense primers. As shown in Fig. 4A: primer ATG (including the start codon ATG), as well as primers LC, 0.5P, and Pd. The last three antisense primers annealed at the region between the distal and proximal transcription initiation sites. Because these primer pairs (D/ATG, D/LC, D/0.5P, and D/Pd) were located in the same exon, in order to correct the false PCR signal resulted from the genomic contamination, the RT-PCR reactions were performed in pairs; one in the presence and the other one in the absence of reverse transcriptase. The difference between each pair RT-PCR reactions then represented the true signal obtained from receptor mRNA. As shown in Fig. 4B and C, receptor transcripts started from distal transcription initiation site were still barely detected by using primer pair D/ATG. Whereas, receptor transcripts were clearly detected by amplifying with P/ATG primer pair (probed with probe 2 for quantitation). Furthermore, the amount of D/LC, D/0.5P and D/Pd amplified fragment from the region between the distal and proximal transcription initiation sites remained low (Fig. 4B and C), as detected using probe 1 (the position of probe was shown in Fig. 4A). These results demonstrated that the m-opioid receptor transcripts in the mouse brain were mainly directed by the proximal promoter with a ratio of approximately 20:1 (proximal:distal promoter). DISCUSSION The existence of both distal and proximal transcription initiation sites in mouse m-opioid receptor gene was previously reported (14-15). The present studies showed that these two TATA-lacking promoters alone can function independently and promote transcription of the luciferase gene in neuronal cells expressing m-
opioid receptor (SH-SY-5Y) with similar activities. Overall, these promoter activities of m-opioid receptor gene were weaker than SV40 promoter, but similar to the promoter activity reported from m4 muscarinic ACh receptor (26) and gp91-phox genes (27). In this study, the promoter activity of m-opioid receptor gene was not further enhanced in the presence of both promoters, which might result from the existence of negative regulatory element, located in 0775 to 0444 bp region. Upon inclusion of the 332 bp fragment of 3*-downstream region of the distal promoter, the expression of reporter gene utilizing the distal promoter (pL1.3K/444 construct) was completely abolished in all tested neuronal and non-neuronal cells (SH-SY5Y, neuro2a, NS20Y and CHO). Whereas, the proximal promoter together with the 332 bp fragment (pL800) was still active in SH-SY-5Y cells. Summarily, these data suggested that the proximal promoter might be the main functional promoter. This conclusion was further supported by the results from examining the receptor mRNA transcripts from these two promoters in vivo. Using quantitative RT-PCR method, we found that almost all of the receptor mRNA in mouse brain was directed by the proximal promoter. Furthermore, the deletion studies using various cultured cells shed some light on the possible physiological role of each promoter of m-opioid receptor gene. Constructs containing the proximal promoter always showed higher luciferase activities in SH-SY-5Y cells than those in neuro2a and NS20Y cells, which do not express m opioid receptors; on the other hand, the construct containing only the distal promoter showed similar activities in all these neuronal cell lines. The differential gene expression utilizing the proximal promoter, in cultured cells, correlated with the characteristics of expression and distribution patterns of m-opioid receptors in the CNS, since these receptors are expressed in restricted types of neurons, and not in all neurons (811). However, reporter gene expression utilizing the distal promoter did not show such a correlation. Taken together, these results suggested that the proximal promoter is the essentially functional promoter for m-opioid receptor gene expression in brain, which preferentially directed that neuronal subtype expression of m-opioid receptor gene. The opioid receptors were not exclusively expressed in the CNS, but also were reported in certain glia (28) and immune cells (29-31). Our studies suggest that mopioid receptor genes may possess other promoter specifying expression in a non-neuronal system; whether these utilize the distal or proximal promoter will be interesting to determine. ACKNOWLEDGMENTS We thank Dr. H. C. Towle for his kindness in providing many valuable suggestions and for critically reading this manuscript. We
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also thank Dr. L. B. Hersh for providing the SH-SY-5Y cells and Dr. A. Smith for helping to prepare the manuscript.
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15. Liang, Y., Mestek, A., Yu, L., and Carr, L. G. (1995) Brain Res. 679, 82–88. 16. Lee, H. S., Mccuaig, K. A., and White, J. H. (1996) M S-Medecine Sciences 12, 183–188. 17. Valdenaire, O., Vernier, P., Maus, M., Dumas Milne Edward, J. B., and Mallet, J. (1994) Eur. J. Biochem. 220, 577–584. 18. Suter, U., Snipes, G. J., Schoener-Scott, R., Welcher, A. A., Parcek, S., Lupski, J. R., Murphy, R. A., Shooter, E. M., and Patel, P. I. (1994) J. Biol. Chem. 269, 25795–25808. 19. Grandien, K., Backdahl, M., Ljunggren, O., Gustafsson, J. A., and Berkenstam, A. (1995) Endocrinology 136, 2223–2229. 20. Grossmann, M. E., Lindzey, J., Blok, L., Perry, J. E., Kumar, M. V., and Tindall, D. J. (1994) Biochem. 33, 14594–14600. 21. Wei, L. N., and Hsu, Y. C. (1994) Develop. Growth and Differ. 36, 187–196. 22. Prather, P. Y., Tsai, A. W., and Law, P. Y. (1994) J. Pharm. Exp. Ther. 270, 177–184. 23. Yu, V. C., Eiger, S., Duan, D. S., Lameh, J., and Sadee, W. (1990) J. Neurochem. 55, 1390–1396. 24. Chakrabarti, S., Law, P. Y., and Loh, H. H. (1995) Brain Res. 30, 269–278. 25. Law, P. Y., and Bergsbaken, C. (1995) J. Pharm. Exp. Ther. 272, 322–332. 26. Wood, A. C., Roopra, A., and Buckley, N. J. (1996) J. Biol. Chem. 271, 14221–14225. 27. Luo, W., and Skalnik, D. G. (1996) J. Biol. Chem. 271, 18203– 18210. 28. Ruzicka, B. B., Thompson, R. C., Watson, S. J., and Akil, H. (1996) J. Neurochem. 66, 425–528. 29. Wick, M. J., Minnerath, S. R., Roy, S., Ramakrishnan, S., and Loh, H. H. (1996) J. Neuroimmu. 64, 29–36. 30. Gaveriaux, C., Peluso, J., Simonin, F., Laforet, J., and Kieffer, B. (1995) FEBS letters 369, 272–276. 31. Sedqi, M., Roy, S., Ramakrishnan, S., and Loh, H. H. (1996) J. Neuroimmu. 65, 167–170.
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Dual Promoters of Mouse m-Opioid Receptor Gene1 Jane L. Ko,2 Sharon R. Minnerath, and Horace H. Loh Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455
Received April 8, 1997
Two transcriptional initiation sites, distal and proximal, located at approximately 500 bp apart in mouse m-opioid receptor gene were identified recently. Using deletional and transfection analyses, the distal or proximal promoter alone displayed similar activity in neuronal cells constitutively expressing m-opioid receptors (SH-SY-5Y). The presence of both promoters did not result in an increase in activity. However, when distal promoter linked with 3 *-downstream sequences without the proximal promoter region, the distal promoter activity was abolished. Transfection analysis using various cell lines further suggested that only the proximal promoter preferentially directed the neuronal subtype expression. To verify the physiological importance of each promoter, the ratio of m-receptor transcripts initiated by either promoter was examined by quantitative RT-PCR using mouse adult brain mRNA. We found that receptor mRNA was predominantly initiated by the proximal promoter. These studies suggested that the proximal promoter was the major functional promoter. q 1997 Academic Press
Opioids are used as potent clinical analgesics, but have serious limitations such as the induction of tolerance and dependence. Pharmacological studies have identified three major types of opioid receptors in mammalian brain, designated m, d and k (1). The cDNA of each of these receptors has now been cloned, and shown to have a predicted amino acid sequence characteristic of G-protein coupled receptors, with seven putative membrane domains (2-7). Expression of opioid receptors is mainly confined to the central nervous system (CNS), and each receptor type has a distinct distribution pattern (8-11). In addition, the regulation of the 1
This research was supported by NIH research grants DA-00546, DA-01583, DA-05695, KO5-DA-70554, and A & F Stark fund of the Minnesota medical foundation. 2 To whom correspondence should be addressed at Department of Pharmacology, University of Minnesota Medical School, 3-249 Millard Hall, 435 Delaware street S.E., Minneapolis, MN 55455. Fax: 612-625-8408. e-mail address: [email protected].
opioid receptor gene expression may be in response to fluctuating levels of various agents in certain brain regions (12-13). Study of the mechanism underlying the transcriptional regulation of opioid receptor genes may facilitate elucidation of the spatial expression and the modulation of expression in different physiological and pathological states. The recent isolation and partial analysis of genomic DNA of m-opioid receptor (14-15) has made it possible to study how the expression of this gene is regulated. The mouse m-opioid receptor gene is over 53 Kb in length, with its coding sequence divided by three introns. A cluster of four major transcription initiation sites in a region located between 291 and 268 bp upstream of the translation start site have been identified by our laboratory (14), and a second transcription initiation site located at 794 bp upstream of the translation start site was later reported (15). These results suggested that mouse m-opioid receptor gene was likely to be controlled by two promoters. The presence of dual or multiple promoters in some genes has been reported (16-20), and the activation of different promoter in each gene depended on certain physiological condition, such as specific tissues, specific cells, or different developmental stages. In this study, we investigate the expression of mouse m-opioid receptor gene under the control of two promoters. Using deletional analysis and transfection assay, the activity of each promoter was examined in various cultured cells expressing or not expressing the m-opioid receptors, and the physiological importance of each promoter was further verified by quantitative RT-PCR method using mouse brain mRNA. MATERIALS AND METHODS Plasmid construction. Promoter-luciferase fusion plasmids were constructed containing 4.7 Kb (04744 to 0249 bp, the translation start site was designated as /1) or shorter 5*-upstream regulatory sequences of the mouse m-opioid receptor gene. The pL4.7K construct (04744 to 0249 bp) was generated by ligation of the 4.4 Kb Sac I and BamH I DNA fragment (04744 to 0297 bp) of mouse m-opioid receptor gene and the PCR fragment containing sequence from the BamH I site to 0249 bp (0297 to 0249 bp) into the polylinker sites of a promoterless luciferase vector, pGL3-basic (Promega). The se0006-291X/97 $25.00
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Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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quence of the PCR fragment from the BamH I site to 0249 bp was confirmed by sequencing. The pL1.3K deletion construct (01326 to 0249 bp) was created by deletion of Sac I/EcoR I fragment (04474 to 01325 bp) from the pL4.7K plasmid. The pL1.3K/444 construct (01326 to 0444 bp) was generated by deletion of Pst I/Xho I fragment (0443 to 0249 bp) from the pL1.3K construct. The other constructs, designated pL800 (0800 to 0249 bp), pL621 (0621 to 0249 bp) and pL450 (0450 to 0249 bp), as well as pLup (01326 to 0775 bp), were generated by PCR, with all of the sense primers bearing the Kpn I site and the antisense primers bearing the Xho I site. The sequences of all the PCR fragments were cloned into PCR II vector (Invitrogen), confirmed by sequencing, and then subcloned into polylinker sites of pGL3-basic vector. The pL299 (0299 to 0249 bp) construct was generated by deletion of Kpn I/BamH I fragment (0450 to 0300 bp) from the pL450 plasmid. The 3*-ends of pL4.7K, pL1.3K, pL800, pL621, pL450 and pL299 constructs were all at position 0249 bp, which was 20 bp downstream from the proximal transcription initiation site. The 3*-end of the pLup construct was at position 0775, which was 20 bp downstream from the distal transcription initiation site. Cell culture. Human neuroblastoma SH-SY-5Y cells were grown in RPMI 1640 medium with 15% heat-inactivated fetal calf serum in an atmosphere of 5% CO2 and 95% air at 377 C. Mouse neuroblastoma neuro2a and NS20Y cell lines, as well as fibroblastoma of Chinese hamster ovary cells (CHO), were grown in Dulbecco’s modified Eagle’s medium with 10% heat-inactivated fetal calf serum and were incubated in 10% CO2 and 90% air at 377 C. Transient transfection and luciferase activity assay. Cells were transfected with the construct plasmids using the DOTAP (Boehringer Mannheim) lipofection method as described by the manufacturer. Briefly, cells in six-well plates with 30-50% confluency were transfected with an equimolar amount of each promoter-luciferase fusion plasmid. The amount of DNA used in each cell line was within the linear range of the relationship between the luciferase activity and the amount of DNA. All transfection experiments were repeated four times or more with similar results, utilizing the plasmids that were independently prepared at least twice. To correct the differences in transfection efficiency from dish to dish, one fifth molar ratio of pCH110 plasmid (Pharmacia) containing the b-galactosidase gene under the control of SV40 promoter was included in each transfection and used for normalization. Forty-eight hr. after transfection, cells were washed with phosphate-buffered saline and lysed with lysis buffer (Promega). The luciferase and b-galactosidase activities of each lysate were determined as described by the manufacturers (Promega and Tropix, respectively). Reverse transcription coupled polymerase chain reaction (RT-PCR). To detect different m-opioid receptor mRNA species transcribed from distal or proximal transcription initiation sites, quantitative reverse transcription coupled PCR (RT-PCR) was performed using mouse brain RNA. Mouse brain PolyA/ RNA was isolated by the oligo(dT)cellulose chromatography method (PolyATract mRNA isolation kit, Promega), and also purchased from Clontech for comparison. First strand cDNA was synthesized using 3 mg polyA/ RNA and 500 ng random hexamers (Promega) in the presence of reverse transcriptase (SuperScript II RNase H0 reverse transcriptase, Gibco BRL). To conduct quantitative analysis, the amount of amplified fragments was within a linear range of the relationship between the amount of RNA used and the amount of amplified fragments generated. Furthermore, the actin-specific primers (5*-TGGCCTTAGGGTTGCAGGGGG-3* and 5*-GTGGGCCGCTCTAGGCACCA-3*) were included in every PCR reaction for normalization (21). The PCR cycle was performed as 1 min at 947C, 1 min at 607C, and 2 min at 727C for 30 cycles. A pair of Actin primer was added at the cycle 20. Two sense primers were used, which started from the distal (D: 5*-GGGGGCACATGAAACAGGCTTCTTT-3*), or proximal transcription initiation sites (P: 5*-CTCAGAGAGTGGCGCTTTGGGATGC-3*). Two antisense primers, corresponding to the coding re-
gion of m-opioid receptor gene, were used: (1) ATG: 5*-CCTGGGCCGGCGCTGCTGTCCAG-3* (started from ATG in exon I), and (2) tm2: 5*-CGCTAAGGCGTCTGCCAGAGCAAG-3* (located at the 2nd transmembrane domain in exon II). Three additional antisense primers, corresponding to the region between the distal and proximal transcription initiation sites were also used: LC (5*-ATTATTGTTGTCCTTTACCCACAT-3*), 0.5P (5*-GCACCTTATACCTCAACACTCTTC-3*) and Pd (5*-GCATCCCCAAAGCGCCACTCTCTGAGT3 *). PCR products were analyzed on agarose gels and then subjected to Southern blot analysis. The blots were hybridized with m-opioid receptor specific probes, followed by hybridization with actin specific probes for normalization. The hybridizing signals were quantitized using a molecular Dynamic Storm 840 phosphoimager system. Two m-opioid receptor specific probes were used. Probe 1 was a 562 bp fragment located in the region between the distal and proximal transcription initiation sties, which was specifically used to detect the transcripts initiated by distal promoter only. Probe 2 was a 200 bp fragment (Hind III/Stu I fragment from pBluescript-MOR 1; generous gift from Dr. L. Yu), located at 5*-untranslated region of mopioid receptor. This 200 bp probe was mainly used for quantitation purpose.
RESULTS Identification and functional analysis of dual promoters in mouse m-opioid receptor gene using SH-SY5Y cells. Two transcriptional initiation sites (approximately 500 bp apart) in mouse m-opioid receptor gene were reported recently (14-15). These findings implicated that the expression of mouse m-opioid receptor gene might be under the control of two promoters. To test this, chimeric constructs (Fig. 1A) containing both promoters (pL1.3K construct), proximal promoter alone (pL800, pL621, and pL450 constructs), or distal promoter alone (pLup construct), together with the luciferase reporter gene (pGL3-basic vector, B), were tested using transient transfection assays in SH-SY-5Y, a neuroblastoma cell line constitutively expressing the m-opioid receptors (22-23). As shown in Fig. 1B, the luciferase activities were similar in magnitude among the constructs containing only the proximal promoter (pL800, pL621, and pL450 constructs), or only the distal promoter (pLup construct). These results suggested that the distal or the proximal promoter alone could function independently and possessed similar promoter activity in SH-SY-5Y cells. However, the construct containing both promoters (pL1.3K) did not produce greater luciferase activity than the constructs containing only one promoter. This data indicated that the presence of both promoters resulted in no additive effect in promoter activity. Moreover, the luciferase activity of pL299 construct (containing only 50 bp region of the proximal transcription initiation sites) was, in fact, not significantly greater than that of pGL3-basic construct. Comparison of the luciferase activities between pL450 and pL299 constructs, it suggested that the proximal promoter activity resided in 0450 to 0300 bp plus 0299 to 0249 bp region, which included the proximal transcription initiation sites.
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FIG. 1. Functional analysis of promoter activities of mouse m-opioid receptor gene in SH-SY-5Y cells. (A) A schematic diagram representing the mouse m-opioid receptor gene from nucleotide 01326 to 0249 relative to the translation start site (ATG), which was designated as /1. The distal and proximal transcription initiation sites, 0794 and 0268 bp, are indicated by arrows. The fragments in various lengths of the 5*-regulatory regions were inserted into the promoterless luciferase (open box) vector, pGL3-basic (B). The number on the left of each construct refers to the number of the nucleotide at the 5*-end of each inserted promoter fragment. A deletion is indicated by a broken line. The pGL3-control plasmid (C) containing SV40 promoter (black box) and enhancer (hatched box) was used as the positive control.; (B) Transient transfection and luciferase assay of the constructs with various fragments from mouse m-opioid receptor gene in SH-SY-5Y cells. To calibrate transfection efficiency, the promoter activity of each fusion gene was normalized according to the b-galactosidase activity from cotransfection of the internal control plasmid, pCH110, and was expressed as n-fold activation of the promoterless plasmid (pGL3-basic) activity in the cells. The histograms and n-fold activation show the mean { S.E. of the results from four or more independent transfection experiments with at least two different plasmid preparations.
Functional analysis of m-opioid receptor gene promoters in various cultured cells. Distribution of m-opioid receptors was mainly in the CNS with a neuron specific and neuron-subtype specific manner (8-11). The role of each promoter in gene expression was further examined using deletional and transfection assays in various cell lines. The m-opioid receptor expressed cell, SH-SY-5Y, was used as the positive control cell line. Neuro2a, a neuronal cell expressing no m-opioid receptors (24), was used as the negative cell line in comparison with SHSY-5Y cell for the neuronal subtype specificity. The CHO, a non-neuronal cell expressing no m-opioid receptors (2-5), was used in comparison with SH-SY-5Y cell for the neuron specificity. As shown in Fig. 2, the 5*-serial deletion constructs (pL4.7K, pL1.3K, pL800, pL621 and pL450), ranging from 04.7 Kb to 0450 bp, were examined. The positive control plasmid (C, pGL3-control contained the SV40 promoter and enhancer), and the negative control plasmids (pL299 construct contained 50 bp region of the proximal transcription initiation sites and displayed no activity) were also tested. The positive control plasmid produced similar luciferase activity (100-130 fold over that of the pGL3-basic construct) among three cell lines, and the negative control plasmid (pL299) produced no significant activity in all cell lines (Fig. 2). These 5*-deletion constructs (pL4.7K, pL1.3K,
pL800, pL621 and pL450), all containing the proximal promoter, consistently displayed approximately 3-5 times greater activities in SH-SY-5Y than those in neuro2a and CHO cells. High activities of these constructs in SH-SY-5Y and low activity in neuro2a and CHO cells correlated with the expression levels of m-opioid receptors in these three cell lines (m-receptors expressed in SH-SY-5Y and no m-receptor expressed in neuro2a and CHO cells). In contrast, the pLup construct, containing only the distal promoter, displayed high promoter activity in SH-SY-5Y (12.4 { 1.5 fold), and also in non-m-receptor expressing neuro2a (10.4 { 1.6 fold) cells, but not in CHO (2.9 { 0.2 fold) cells (Fig. 2). These results suggested that only the proximal promoter preferentially directed the neuronal subtype expression (SH-SY-5Y versus neuro2a cells). To confirm the differential neuronal subtype expression between these two promoters, we further tested the deletion constructs in a second neuroblastoma cell line (NS20Y), which does not express the m-but only the d-opioid receptor (25). As shown in Fig. 3, only the activity (13.4 fold) of pLup construct in NS20Y was as high as that obtained in neuro2a and SH-SY-5Y cells, while the 5*-deletion constructs containing the proximal promoter displayed low promoter activity in NS20Y cells as in neuro2a cells. These results thus corroborated our previous observation from neuro2a cells.
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FIG. 2. Functional analysis of the mouse m-opioid receptor gene promoter in various cell lines. A schematic diagram represented a series of 5*-deletion constructs of mouse m-opioid receptor gene, with various 5* ends ranging from 04.7 Kb to 0299 bp, and the 3* end at 0249 bp (equivalent to plus 20 nucleotides from proximal transcription start site). These constructs were inserted into the promoterless luciferase (open box) vector, pGL3-basic (B). The number on the left of each construct refers to the number of the nucleotide beginning of the 5*-regulatory sequence. A deletion is indicated by a broken line. pGL3-control plasmid (C) containing SV40 promoter (black box) and enhancer (hatched box) was used as the positive control. The promoter activity of each construct in SH-SY-5Y (gray columns), neuro2a (hatched columns) and CHO (open columns) cells was normalized to b-galactosidase activity. The histograms show the mean { S.E. of the results from four or more independent transfection experiments with at least two different plasmid preparations.
Negative regulatory sequences suppressed the expression initiated by the distal promoter. According to the results shown above, two points need to be further addressed. First, no additive promoter activity was observed in the presence of both promoters. Second, the distal promoter alone produced no neuronal subtype
FIG. 3. Functional analysis of the mouse m-opioid receptor gene promoter in NS20Y cells. The histograms show the n-fold activation in normalized luciferase activity over the promoterless vector pGL3basic activity. The data are presented as mean { S.E. from four transfection experiments with two different plasmid preparations. **Indicates that the value of pLup construct is significantly different from those of other deletion constructs (põ0.01, student t-test).
preferential expression, but when it was connected with the proximal promoter, the preferential expression was observed. To investigate the possible mechanism, the proximal promoter region was deleted from the pL1.3K construct, which contained both promoters. Because the proximal promoter activity was shown to reside in 0450 to 0248 bp region (Fig. 1), the pL1.3K/ 444 plasmid (from 01326 to 0444 bp) was then created. It contained only the distal promoter (01326 to 0775 bp), plus 332 bp fragment (0775 to 0444 bp) immediately 3*-downstream of the distal promoter (Fig. 2). Surprisingly, the activity of the pL1.3K/444 construct had no significant difference from that of pGL3basic in all the tested cells (1.2 { 0.3 fold in SH-SY5Y, 0.6 { 0.1 fold in neuro2a, 0.9 { 0.2 fold in CHO and 0.3 { 0.1 fold in NS20Y) (Fig. 2 and 3). Comparing these results with that of the pLup construct suggested that in the presence of 332 bp (0775 to 0444 bp) fragment located 3*-downstream of the distal transcription initiation site, the expression of reporter gene initiated by the distal promoter was abolished. Whereas, the expression of pL800 construct directed by the proximal promoter was still active in the presence of this 332 bp fragment (Fig. 1 and 2). The existence of negative regulatory element, located in 0775 to 0444 bp region and specific suppression of the distal promoter activity only, may explain why no additive effect was observed in the presence of both promoters (pL4.7K and pL1.3K constructs). Also, it
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may explain why in the absence of the proximal promoter and the 332 bp negative regulatory region, the distal promoter alone was active in non-m-opioid receptor expressed neuronal cells (neuro2a and NS20Y).
FIG. 4. Differential expression of m-opioid receptor mRNAs directed by the distal or proximal promoter of m-opioid receptor in mouse brain. (A) A schematic diagram represents the relative positions of the distal (D) and proximal (P) promoters in mouse m-opioid receptor gene. The gray boxes represent the coding regions divided into 4 exons. Positions and directions of the primers used in RT-PCR experiments are labeled by arrows with the arrow heads pointing to the 3*-end. The sizes of expected RT-PCR fragments amplified by each pair of primers are indicated by number. The broken line indicates the intron region, absence in receptor mRNA, nor in the RTPCR products. The m-opioid receptor specific probes (probe 1 and 2) for Southern blot analysis were indicated by the thick lines corresponding to the size and position in diagram.; (B) Analysis of the RT-PCR products by electrophoresis with ethidium bromide staining. Each lane represents the amount of receptor message obtained from 25 ng polyA RNA isolated from adult mouse brain. RT ‘‘/’’ or ‘‘0’’ indicates the presence or absence of reverse transcriptase in the first strand cDNA synthesis reaction. Hundred base pair ladder (Gibco BRL) was used as the DNA marker. A pair of actin-specific primers is included in every PCR reaction as an internal control for quantitation, and the actin transcript is indicated by white arrow.; (C) Quan-
Physiological role of m-opioid receptor gene promoters in brain. To test the physiological importance of each promoter in CNS, and to verify that the distal promoter activity was suppressed in vivo, the ratio of m-opioid receptor transcripts in mouse brain RNA directed by either the distal or the proximal promoters was examined using quantitative RT-PCR method. To perform the quantitative RT-PCR, a pair of actin-specific primers, located at different exons, was included in every PCR reaction as an internal control for the normalization purpose. The RT-PCR products were analyzed by electrophoresis and followed by Southern blot analysis. Each specific band was quantitized using the phosphoimager and the m-opioid receptor specific signal was normalized to the actin in each sample. Since there is no alternative splicing occurred at the 5*-untranslated region of m-opioid receptor gene, the only difference between the receptor mRNA initiated by proximal promoter or by distal promoter was the region between the distal and proximal transcription initiation sites. Therefore, if both promoters of m-opioid receptor gene were active, the RT-PCR fragment amplified from the proximal transcription initiation site, located at the common region of receptor mRNA initiated by both promoters, should represent the total transcripts directed by both promoters (Fig. 4A). The RTPCR fragment amplified from the distal transcription initiation site, represented the transcript directed only by the distal promoter. Consequently, the difference between the amount of these two RT-PCR fragments represented the transcripts directed only from the proximal promoter. As shown in Fig. 4B, receptor transcripts were clearly detected by using the primer pair of P (sense primer, starting at the proximal transcription initiation site 0268 bp) and tm2 (antisense primer, located at the 2nd transmembrane domain in exon II). Whereas, the receptor transcripts started from distal
titation analysis of PCR products by Southern blot. The blots were hybridized with the m-opioid receptor specific probe, stripped the signal, and then hybridized with actin specific probe. The hybridized signals were quantitized and each band was normalized using actin signal as internal control. To rule out the false signal from the genomic contamination, the exact same RT-PCR reactions were carried out in the presence or absence of the reverse transcriptase. The amounts of amplified RT-PCR products from primer pairs, D/LC, D/ 0.5P, D/Pd, D/ATG and P/ATG, were obtained by subtracting the signal from RT-PCR between the presence and absence of RT. The amount of P/tm2 product, probed with probe 2, was arbitrarily defined as 100%. The histograms show the percentage of normalized m-opioid receptor gene products. The data are presented as mean { S.E. from at least three different mouse brain RNA preparations.
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transcription initiation site were barely detected, by amplifying D/tm2 product using the primer pair of D (sense primer, beginning at the distal transcription initiation site) and tm2 (Fig. 4B). The amount of RT-PCR product (P/tm2), amplified by the primer pair of P and tm2 as well as probed with m-receptor-specific probe 2, was arbitrarily defined as 100% (Fig. 4C). Probe 2, a 200 bp fragment located at 5*-untranslated region of m-opioid receptor, was the probe used for quantitation (Fig. 4A). The difference between the amount of P/tm2 and D/tm2 RT-PCR products was merely equal to that of P/tm2 (Fig. 4C). These results suggested that the proximal promoter, but not the distal, was a functional promoter in the brain. To avoid the possibility that the low yield of D/tm2 (1154 bp) product was resulted from the secondary structure in receptor mRNA, the shorter RT-PCR fragment, representing the segment between distal initiation site and start codon (ATG) of coding region, was amplified by using sense primer D paired with 4 different antisense primers. As shown in Fig. 4A: primer ATG (including the start codon ATG), as well as primers LC, 0.5P, and Pd. The last three antisense primers annealed at the region between the distal and proximal transcription initiation sites. Because these primer pairs (D/ATG, D/LC, D/0.5P, and D/Pd) were located in the same exon, in order to correct the false PCR signal resulted from the genomic contamination, the RT-PCR reactions were performed in pairs; one in the presence and the other one in the absence of reverse transcriptase. The difference between each pair RT-PCR reactions then represented the true signal obtained from receptor mRNA. As shown in Fig. 4B and C, receptor transcripts started from distal transcription initiation site were still barely detected by using primer pair D/ATG. Whereas, receptor transcripts were clearly detected by amplifying with P/ATG primer pair (probed with probe 2 for quantitation). Furthermore, the amount of D/LC, D/0.5P and D/Pd amplified fragment from the region between the distal and proximal transcription initiation sites remained low (Fig. 4B and C), as detected using probe 1 (the position of probe was shown in Fig. 4A). These results demonstrated that the m-opioid receptor transcripts in the mouse brain were mainly directed by the proximal promoter with a ratio of approximately 20:1 (proximal:distal promoter). DISCUSSION The existence of both distal and proximal transcription initiation sites in mouse m-opioid receptor gene was previously reported (14-15). The present studies showed that these two TATA-lacking promoters alone can function independently and promote transcription of the luciferase gene in neuronal cells expressing m-
opioid receptor (SH-SY-5Y) with similar activities. Overall, these promoter activities of m-opioid receptor gene were weaker than SV40 promoter, but similar to the promoter activity reported from m4 muscarinic ACh receptor (26) and gp91-phox genes (27). In this study, the promoter activity of m-opioid receptor gene was not further enhanced in the presence of both promoters, which might result from the existence of negative regulatory element, located in 0775 to 0444 bp region. Upon inclusion of the 332 bp fragment of 3*-downstream region of the distal promoter, the expression of reporter gene utilizing the distal promoter (pL1.3K/444 construct) was completely abolished in all tested neuronal and non-neuronal cells (SH-SY5Y, neuro2a, NS20Y and CHO). Whereas, the proximal promoter together with the 332 bp fragment (pL800) was still active in SH-SY-5Y cells. Summarily, these data suggested that the proximal promoter might be the main functional promoter. This conclusion was further supported by the results from examining the receptor mRNA transcripts from these two promoters in vivo. Using quantitative RT-PCR method, we found that almost all of the receptor mRNA in mouse brain was directed by the proximal promoter. Furthermore, the deletion studies using various cultured cells shed some light on the possible physiological role of each promoter of m-opioid receptor gene. Constructs containing the proximal promoter always showed higher luciferase activities in SH-SY-5Y cells than those in neuro2a and NS20Y cells, which do not express m opioid receptors; on the other hand, the construct containing only the distal promoter showed similar activities in all these neuronal cell lines. The differential gene expression utilizing the proximal promoter, in cultured cells, correlated with the characteristics of expression and distribution patterns of m-opioid receptors in the CNS, since these receptors are expressed in restricted types of neurons, and not in all neurons (811). However, reporter gene expression utilizing the distal promoter did not show such a correlation. Taken together, these results suggested that the proximal promoter is the essentially functional promoter for m-opioid receptor gene expression in brain, which preferentially directed that neuronal subtype expression of m-opioid receptor gene. The opioid receptors were not exclusively expressed in the CNS, but also were reported in certain glia (28) and immune cells (29-31). Our studies suggest that mopioid receptor genes may possess other promoter specifying expression in a non-neuronal system; whether these utilize the distal or proximal promoter will be interesting to determine. ACKNOWLEDGMENTS We thank Dr. H. C. Towle for his kindness in providing many valuable suggestions and for critically reading this manuscript. We
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also thank Dr. L. B. Hersh for providing the SH-SY-5Y cells and Dr. A. Smith for helping to prepare the manuscript.
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