Cloning and transcriptional analysis of the mouse receptor activity modifying protein-1 gene promoter
© Pondel and Mould; licensee BioMed Central Ltd. 2005
Received: 01 October 2004
Accepted: 24 March 2005
Published: 24 March 2005
Receptor activity modifying protein-1 (RAMP-1) is a single transmembrane-domain protein required for the functional expression of calcitonin gene-related peptide (CGRP) receptors. To date, little is known about the molecular mechanism(s) that activate/inhibit RAMP-1 gene expression. Such mechanism(s) are likely to play a major role in modulating the responsiveness of tissues to CGRP.
To initiate studies on the transcriptional regulation of the mouse RAMP-1 gene, RAMP-1 transcriptional initiation sites were mapped in a variety of tissues. Analysis of RAMP-1 expression in C2C12 myoblasts demonstrated that RAMP-1 mRNA is expressed at greatest levels in confluent myoblasts verses non-confluent and fused myoblasts. Transfection of confluent C2C12 myoblasts and NIH 3T3 cells with RAMP-1 promoter/luciferase deletion constructs revealed that 4.7 kb of RAMP-1 5' flanking region demonstrated optimal promoter activity while 343 bp of 5' flanking region was defined as a minimal RAMP-1 promoter. In non-RAMP-1 expressing HEK293 cells, constructs containing 4.7 kb to 782 bp of RAMP-1 5' flanking region were transcriptionally inactive. However, deletion of sequences -782 to -343 activated RAMP-1 promoter activity.
These findings suggest that tissue specificity of RAMP-1 gene expression is mediated by a negative acting transcription factor that represses RAMP-1 gene expression in non-RAMP-1 expressing tissues. This transcription factor is therefore likely to play an important role in modulating the responsiveness of tissues to CGRP.
The calcitonin gene-related peptide (CGRP) belongs to a family of related peptides that includes calcitonin (CT), adrenomedullin (AM) and amylin (AMY) [1, 2]. To date, CGRP is one of the most potent endogenous vasodilatory peptides discovered. CGRP mediates sensory neurotransmission and inhibits insulin action on carbohydrate metabolism . CGRP has been shown to modulate immune function by inhibiting the proliferation of T cells and synthesis of T cell-derived cytokines IL-2 and IFN-γ [3–6]. In the lung, CGRP mediates multiple effects some of which have potential implications in airway homeostasis . CGRP has also been shown to have cardioprotective effects in rats and humans [8, 9]. In skeletal muscle, CGRP potentiates muscle contraction , increases the numbers of acetylcholine receptors (AchR) [11–13] and their rate of desensitisation . In addition, CGRP locally increases the rate of blood flow following muscle contraction [15–17].
The effects of CGRP are mediated by CGRP receptors that are generated by a complex of proteins . CGRP receptors are formed by the interaction of two separate proteins. The first protein component is the calcitonin receptor like (CL) receptor. The CL receptor is a seven transmembrane-domain receptor but is inactive when expressed in cells alone . The second protein component required for CGRP receptor function is receptor activity modifying protein-1 (RAMP-1). RAMP-1 acts as a molecular chaperone and is required for the transportation of the CL receptor to the cell surface in addition to pharmacologic specificity .
RAMPs are a recently identified group of single transmembrane-domain accessory proteins. To date, three members of the RAMP family have been identified (RAMP-1, RAMP-2 and RAMP-3) . All share 30% sequence identity, differ in their tissue distributions and are comprised of approximately 160 amino acids that make up a large extracellular N-terminal domain, a single membrane-spanning domain and a short cytoplasmic domain [18, 20]. Recently, Christopoulos et al.  demonstrated that RAMPS interact with a number of Class II G protein-coupled receptors (GPCRs) in addition to the CL receptor. These include the vasoactive intestinal polypeptide/pituitary adenylate cyclase activating peptide receptor (VPAC1R), the glucagon and parathyroid hormone receptors (PTH1 and PTH2). VPAC1R/RAMP-2 heterodimers display a significant enhancement of agonist-mediated phosphoinositide hydrolysis compared with VPAC1R alone. This suggests that RAMPs may play a more general role in modulating cell signalling through other GPCRs than previously thought.
Despite the crucial role RAMP-1 plays in the generation of CGRP receptors, little is known about the molecular mechanism(s) regulating RAMP-1 gene expression. It is likely that such mechanism(s) play an important role in modulating the responsiveness of specific tissues to CGRP. To initiate studies on RAMP-1 gene regulation, we cloned and characterised the mouse RAMP-1 gene promoter. Analysis in three different RAMP-1 positive mouse tissues revealed multiple start sites of transcription. RT-PCR analysis of RAMP-1 mRNA in the C2C12 myoblast cell line demonstrated that endogenous RAMP-1 gene expression was greatest in confluent cultures compared to non-confluent or fused cells. Through the use of RAMP-1 promoter/luciferase constructs transfected into C2C12 myoblast cells and NIH 3T3 cells, we studied the effects RAMP-1 promoter deletions had on RAMP-1 transcriptional activity. We further demonstrated that the RAMP-1 promoter activity was tissue-specific and not expressed in the RAMP-1 negative cell line HEK293. Finally, we identified a repressor element in the RAMP-1 promoter which when deleted activates RAMP-1 promoter activity in HEK293 cells. This suggests the RAMP-1 gene is negatively regulated in non-RAMP-1 expressing cells.
Determination of RAMP-1 transcriptional initiation sites and PCR cloning the mouse RAMP-1 promoter
RAMP-1 5' RACE analysis. Total RNA was extracted from mouse brain, skeletal muscle and heart. RNA was treated with alkaline phosphatase, tobacco acid pyrophosphatase and ligated to a small adapter. Following reverse transcription, multiple rounds of PCR were performed and the PCR products cloned and sequenced. Transcriptional initiation sites are indicated relative to the start site of translation (ATG = 0).
Mouse RAMP-1 transcriptional start sites
RAMP-1 gene expression in C2C12 myoblasts and myotubes
RAMP-1 promoter deletional analysis in transiently transfected C2C12 myoblasts
To determine if the RAMP-1 promoter demonstrates tissue specificity in transfected cell lines, we compared the transcriptional activity of the RAMP-1 promoter in transfected C2C12 myoblasts and a cell line known to not express RAMP-1 mRNA (HEK293). Luciferase analysis demonstrated that constructs containing 4.7 kb to 782 bp of RAMP-1 5' flanking region were transcriptionally inactive in HEK293 cells. A construct containing 470 bp of RAMP-1 5' flanking region demonstrated modest transcriptional activity while a construct containing 343 bp of 5' flanking region demonstrated significant promoter activity. The results of these experiments suggest that the RAMP-1 promoter region between -782 and -343 contains a regulatory element that represses RAMP-1 promoter activity in a non-RAMP-1 expressing cell line.
Silencer activity of the RAMP-1 repressor element
Despite the important role RAMP-1 plays in CGRP cell signalling little is known about the transcriptional regulation of RAMP-1 gene. To date, no RAMP-1 gene promoter has been identified/cloned and RAMP-1 transcriptional initiation sites have not been determined in any RAMP-1 positive tissues. In the present study, we cloned the mouse RAMP-1 gene promoter and mapped multiple RAMP-1 transcriptional initiation sites in a variety of RAMP-1 positive tissues. Our data demonstrates that RAMP-1 transcriptional initiation occurs at similar sites and all within a 50 bp region in different RAMP-1 mRNA positive tissues.
To initiate studies on the transcriptional regulation of the mouse RAMP-1 gene during muscle differentiation, we examined RAMP-1 gene expression in C2C12 mouse myoblasts by semi-quantitative RT-PCR. Our results suggest that RAMP-1 gene expression is present in non-confluent, confluent and fused C2C12 cells. However RAMP-1 mRNA expression appears to be increased in confluent and fused C2C12 cells. Chakravarty et al.  demonstrated that a strong correlation exists between the level of RAMP-1 mRNA expression and CGRP binding. This suggests that the transcriptional activity of the RAMP-1 gene may control the responsiveness of tissues to CGRP. It will be important to determine if increased RAMP-1 mRNA expression in confluent and fused C2C12 cells results in significantly higher levels of CGRP receptor expression and increases in intracellular cAMP after treatment with CGRP. The role increased RAMP-1 mRNA gene expression plays in the process of C2C12 induced muscle differentiation is not yet clear. Nobel et al.  showed that CGRP causes increased creatine kinase activity in myoblast cultures. Creatine kinase activity is a well established marker of fused myoblasts (myotubes). It is reasonable to hypothesise that increased RAMP-1 gene expression in confluent C2C12s makes these cells more responsive to CGRP and thus may facilitate myoblast fusion.
Deletional mapping of the RAMP-1 gene promoter revealed that a 4.7 kb promoter fragment generates the highest levels of RAMP-1 promoter activity. However, a RAMP-1 promoter construct with only 343 bp of 5' region contains nearly 40% percent activity of the entire promoter. The delineation of such a small functional RAMP-1 promoter should facilitate the identification of DNA regulatory elements and the transcription factors they interact that activate RAMP-1 promoter activity in C2C12 cells.
Transfection of RAMP-1 promoter/luciferase constructs into RAMP-1 negative HEK293 cells demonstrated that a transcriptional control element present between -782 and -343 in the RAMP-1 promoter represses RAMP-1 promoter activity in the RAMP-1 negative-expressing cell line HEK293 cells. While this element appears to have modest silencing activity, our results suggest that full repressor activity of this element requires it to be in its native position within the RAMP-1 promoter. We hypothesise that negative-regulatory transcription factors, present in non-RAMP-1 expressing cells, bind to this element and repress RAMP-1 gene expression. Computer analysis of the 480 bp RAMP-1 repressor element has revealed potential binding sites for a variety of positive acting transcription factors such as MyoD, c-Myb, NF1 and Elk-1. Importantly, binding sites for two transcriptional repressors have also been identified: mammalian transcriptional repressor RBPJkappa/CBF (RBPJK) and bZIP domain, transcriptional repressor (E4BP4). Studies on the roles these transcription factors play in the repression of RAMP-1 gene expression are now underway.
Promoter deletional analysis demonstrated that both positive and negative acting transcription factors regulate the tissue specificity of the RAMP-1 gene. DNase 1 protection and gel-shift experiments with extracts from C2C12 and HEK293 cells are underway to identify specific DNA sequences and the potential transcription factors they bind that mediate the activity of the RAMP-1 gene in RAMP-1 expressing and non- expressing cells. Once specific sequences/factors binding to the RAMP-1 repressor element are isolated, the effects that mutating these sequences has on RAMP-1 promoter activity in transgenic mice will be tested. These experiments will enable us to test the role such sequences/factors play in regulating the tissue-specific expression and activity levels of the RAMP-1 gene in vivo. Such transcriptional mechanism(s) are likely to play an important role in modulating the responsiveness of tissues to CGRP and may prove useful targets for future drug development aimed at regulating CGRP receptor gene expression.
5' RACE analysis
5' RACE on total RNA from mouse heart, skeletal muscle and brain was performed employing a First Choice RLM-RACE kit (Ambion) according to manufacturers instructions. Briefly, 10 μg of total RNA from each tissue was treated with Calf Intestine Alkaline Phosphatase (CIP) for one hour at 37°C followed by extraction with phenol:chloroform and ethanol precipitation. The RNA was re-suspended in 11 μl of H20. 5 μl of this RNA was then treated with tobacco acid pyrophosphate (TAP) for one-hour at 37°C. A 5' RACE adapter (provided in kit) was ligated to 5 μl of the TAP treated RNA. 2 μl of ligated RNA was subjected to reverse transcription (RT). RT products (1 μl) were subjected to two rounds of nested PCR according to manufacturers instructions. The first round of PCR employed an outer primer binding to the 5' RACE adapter (5'GCTGATGGCGATGAATGAACACTG 3') and an outer RAMP-1 gene specific primer (5' CGGGACCCTGACTATGGGAC 3'). The second round of PCR employed an inner primer binding to the 5' RACE adapter (5'GCTGATGGCGATGAATGAACACTG 3') and an inner RAMP-1 gene specific primer (5' TCTTCATGGTCACTGCCTGC 3'). Following the second round of PCR, PCR products were purified on an agarose gel and ligated into the PCR cloning vector pGEM-T Easy (Promega). A total of 8–10 clones representing each tissue were purified and subjected to DNA sequencing.
Generation of RAMP-1 promoter/luciferase expression constructs
To generate RAMP-1 promoter/luciferase deletion constructs, PCR utilising an antisense primer corresponding to sequences -26 to -43 (see Fig. 1) and sense primers corresponding to -145 to -164; -324 to -343; -470 to -450; -760 to -782; -935 to -948; -1220 to -1243; -1574 to -1593; -1827 to -1846; -2032 to -2093; -2342 to -2365; -2533 to -2511; -4102 to -4079 and -4724 to -4702 was carried out as described in Jagger et al. . The PCR fragments were cloned into the vector pGEM-T Easy (Promega) and sequenced. The plasmids were then cut with Not 1 and the released RAMP-1 promoter fragment filled in with Klenow, purified and cloned into the luciferase expression vector pGL3 basic (Promega).
To generate a construct containing the RAMP-1 repressor element cloned at the 3' end of the luciferase gene, the RAMP-1 promoter/luciferase construct containing 343 bp of 5' flanking region was cut with Bam H1. A region between -343 and -782 of the mouse RAMP-1 promoter was generated by PCR as above and cloned into the Bam H1 cut -343 RAMP-1 promoter/luciferase construct in two orientations.
Tissue culture and RAMP-1/luciferase transfections
C2C12, HEK293 and NIH 3T3 cells were maintained in DMEM supplemented with 10% foetal calf serum, 100 μg/ml penicillin and 100 U/ml of streptomycin. To generate fused C2C12 myotubes, cells were allowed to become confluent and growth media was replaced with media containing only 1% foetal calf serum. For transient transfections in all the above cell lines, cells were transfected with 1.0–1.5 μg of specific RAMP-1/luciferase construct, 0.2 μg of a co-transfection control plasmid pRLTK (Promega) and Fugene 6 (Roche) according to manufacturers' instructions. Luciferase assays were performed employing a Dual Luciferase Reporter Assay System (Promega) and a Turner Design TD-20/20 luminometer.
Semi-quantitative RT-PCR on RAMP-1 mRNA in differentiating C2C12 myoblasts
Total RNA from non-confluent, confluent and fused C2C12 myoblasts was isolated as above and reverse transcriptase (RT) reactions performed as previously described . PCR amplification employing mouse RAMP-1 sense (5' CTATGGGGAGCTCACTTACTGC 3') and antisense (5'GCAGTCTTCCTTGGAGTTCAGAAT 3') oligos; mouse actin sense (5'GTTAACCAACTGGGACGACATGG 3') and anti-sense (5'GATCTTGATCTTCATGGTGC 3') mouse actin primers was performed as previously described . A total of 30, 35 and 40 cycles were employed. The PCR products were run on a 1% agarose gel and Southern blotted to nylon membranes. The blots were probed with an end-labelled 32P actin probe (5' CACACTGTGCCCATCTACGA3') or RAMP-1 probe (5'GCCCAATCCGGAAGTGGACA 3') followed by autoradiography and densitomitry.
calcitonin gene related peptide
receptor activity modifying protein
G protein coupled receptor
calcitonin receptor like
This work was supported by a grant from the Wellcome Trust.
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