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Originally published In Press as doi:10.1074/jbc.M601496200 on May 4, 2006

J. Biol. Chem., Vol. 281, Issue 27, 18277-18284, July 7, 2006
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MicroRNA-155 Regulates Human Angiotensin II Type 1 Receptor Expression in Fibroblasts*

Mickey M. Martin{ddagger}, Eun Joo Lee§, Jessica A. Buckenberger{ddagger}, Thomas D. Schmittgen§, and Terry S. Elton{ddagger}1

From the College of Pharmacy, Division of Pharmacology and §Pharmaceutics, {ddagger}Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio 43210

Received for publication, February 15, 2006 , and in revised form, May 2, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A large number of studies have demonstrated that the expression of the angiotensin II type 1 receptor (AT1R) is regulated predominantly by post-transcriptional mechanisms. Recently, it has been suggested that 10% of human genes may be regulated, in part, by a novel post-transcriptional mechanism involving microRNAs (miRNAs). miRNAs are small RNAs that regulate gene expression primarily through translational repression. The aim of this study was to determine whether miRNAs could regulate human AT1R expression. Luciferase reporter assays demonstrated that miR-155 could directly interact with the 3'-untranslated region of the hAT1R mRNA. Functional studies demonstrated that transfection of miR-155 into human primary lung fibroblasts (hPFBs) reduced the endogenous expression of the hAT1R compared with non-transfected cells. Additionally, miR-155 transfected cells showed a significant reduction in angiotensin II-induced extracellular signal-related kinase 1/2 (ERK1/2) activation. Furthermore, when hPFBs were transfected with an antisense miR-155 inhibitor, anti-miR-155, endogenous hAT1R expression and angiotensin II-induced ERK1/2 activation were significantly increased. Finally, transforming growth factor-beta1 treatment of hPFBs resulted in the decreased expression of miR-155 and the increased expression of the hAT1R. In summary, our studies suggest that miR-155 can bind to the 3'-untranslated region (UTR) of hAT1R mRNAs and translationally repress the expression of this protein in vivo. Importantly, the translational repression mediated by miR-155 can be regulated by physiological stimuli.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Classically, angiotensin II (Ang II),2 the biologically active component of the renin-angiotensin system has been shown to regulate a variety of physiological responses including fluid homeostasis, aldosterone production, renal function, contraction of vascular smooth muscle, and sympathetic nervous activity and thirst responses (1). However, Ang II also has non-hemodynamic actions that may play a central role not only in the etiology of hypertension but also in the pathophysiology of cardiac hypertrophy and remodeling, heart failure, vascular thickening, atherosclerosis, and glomerulosclerosis in humans (2–6).

Most of the known physiological and pathophysiological effects of Ang II are mediated via the angiotensin II type 1 receptor (AT1R) (1). A number of recent studies suggest that AT1R expression levels are predominantly regulated by post-transcriptional mechanisms (reviewed in Ref. 7). For example, Ang II (8), cAMP stimulating agents (9), and estrogens (10, 11) decrease rat AT1AR expression by stimulating rat AT1AR mRNA decay. In contrast, insulin (12), low density lipoprotein (13), and progesterone (11) up-regulate receptor expression by decreasing AT1AR mRNA decay rates. Additionally, rat AT1AR expression is regulated by translational mechanisms via cytosolic proteins that interact with the 5'-UTR of the receptor mRNA (10, 14–18). Finally, it has also been demonstrated that the human AT1R (hAT1R) 5'-UTR harbors an internal ribosome entry site, which allows this mRNA to be translated during physiological conditions when cap-dependent translation is inhibited (19).

Recently it has been demonstrated that microRNAs (miRNAs) may provide an additional post-transcriptional mechanism by which protein expression can be regulated. miRNAs are produced endogenously in mammalian cells by specific RNA gene transcription or from introns during pre-mRNA splicing (20–23). miRNAs are expressed as long hairpin-forming precursor RNAs that get processed to 21–23 nucleotide RNA molecules that regulate the stability or translational efficiency of target mRNAs (24–29). In animals, miRNAs usually control gene expression through partial complementary elements in the 3'-UTRs of their target mRNAs (26–30). The functional importance of miRNAs is evidenced by the many biological processes in which they are implicated, including developmental timing, cell proliferation, apoptosis, metabolism, cell differentiation, and morphogenesis (31, 32). Currently, it is not known whether miRNAs play a role in regulating components of the renin-angiotensin system. Therefore, in this study, we have examined the hypothesis that hAT1R expression can be regulated by miRNAs binding to specific target sequences harbored in the 3'-UTR of hAT1R mRNAs. Experimental analyses demonstrate, for the first time, that miR-155 can regulate the expression of the hAT1R in primary human lung fibroblasts.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—CHO cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 80 units/ml penicillin, 80 µg/ml streptomycin, and 0.0175 mg/ml L-proline (Sigma). Human pulmonary fibroblasts (hPFB) were established in primary culture from enzyme-dispersed tissue fragments adhered to Primaria® culture plates of human neonatal lung tissue obtained at autopsy and were the generous gift of Dr. Daren L. Knoell (Departments of Pharmacy and Internal Medicine, Ohio State University). Pulmonary fibroblasts were maintained in a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone), 80 units/ml penicillin, and 80 µg/ml streptomycin and FungizoneTM (Invitrogen). Cells were rendered quiescent by a 48 h exposure to HEPES-buffered Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum and penicillin-streptomycin-FungizoneTM. Cells were used for ~7–10 passages before replacement with fresh early passage stocks. All cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C.

Constructs—An 883-bp fragment encompassing the entire hAT1R 3'-UTR was PCR-amplified utilizing the following sense (5'-CATGTTCGAAACCTGTCCATAAAG-3') and antisense (5'-ATAAAATTATTTTATTTTAAAGTAAAT-3') primers using standard procedures and a proofreading polymerase (Platinum Pfu, Invitrogen). A full-length hAT1R cDNA clone (19) was used as template. The PCR product was subcloned into the pCRTM2.1 vector following the manufacturer's protocol (Invitrogen). Plasmid DNA was subsequently isolated from recombinant colonies and sequenced to ensure authenticity. The hAT1R 3'-UTR inserts were removed from the pCRTM2.1 plasmid by EcoRI digestion. The fragments were gel-purified, filled in, and blunt-end-ligated into a filled-in HindIII site which is located downstream of the firefly luciferase (f-luc) reporter gene (pMIR-REPORTTM, Ambion). The authenticity and orientation of the inserts relative to the luciferase gene were confirmed by sequencing. The resulting plasmids were designated, pMIR/883/5'->3' and pMIR/883/3'->5', respectively. Transformed bacterial cultures were grown, and each reporter construct was purified by using PureLinkTM Hipure plasmid Maxiprep kit (Invitrogen). The expression plasmid pMIR/883/5'->3'/{Delta}miR-155 was generated by utilizing the pMIR/883/5'->3' plasmid as template and deleting the miR-155 7-bp seed binding site (Fig. 1B) harbored in the hAT1R 3'-UTR using the QuikChange site-directed mutagenesis kit (Stratagene). Briefly, a forward mutagenic deletion primer (5'-CTTCACTACCAAATGGCTACTTTTCAGAAT-3') and a complementary reverse mutagenic deletion primer (5'-ATTCTGAAAAGTAGCCATTTGGTAGTGAAG-3'), where the desired sequence to be deleted (i.e. AGCATTA) was missing from the middle of the primer, were synthesized and utilized in a PCR experiment as described by the manufacturer. The amplification reaction was treated with DpnI restriction enzyme to eliminate the parental template and the remaining DNA was used for transformation. The deletion of the AGCAUUA miR-155 seed binding sequence was confirmed by dideoxy chain termination sequencing.

Transfection—The following partially double-stranded RNAs that mimic endogenous precursor miRNAs, hsa-miR-124a-1, hsa-miR-155, hsa-miR-365, and miRNA Negative Control #1 were obtained from Ambion. Additionally, the following anti-miR miRNA inhibitors (anti-miR-124a-1, anti-miR-155, anti-miR-365–1, and anti-miR Negative Control #1), designed to inhibit endogenous miRNAs, were also obtained from Ambion. These RNA-based inhibitors are chemically modified to increase their stability and to improve their activity. Transfection of CHO and hPFB cells with small RNAs was optimized utilizing Lipofectamine 2000 (Invitrogen) and a fluorescein-labeled double-stranded RNA oligomer designated BLOCK-iTTM fluorescent oligonucleotide (Invitrogen). CHO cells were transfected with the luciferase reporter constructs described above (1 µg), pRL-CMV (50 ng, Promega) and the appropriate miRNA precursor using Lipofectamine 2000. After 48 h, cells were washed and lysed with passive lysis buffer (Promega) and f-luc and Renilla luciferase (r-luc) activities were determined using the dual-luciferase reporter assay system (Promega) and a luminometer. The relative reporter activity was obtained by normalization to the r-luc activity. Alternatively, hPFB cells were cotransfected with pRL-CMV and either specific pre-miR mRNAs or anti-miR miRNA as described above. After 48 h, cells were either utilized for luciferase, real-time PCR, and radioreceptor binding or immunoassay assays.

AT1 Receptor Radioligand Binding Studies—Whole cell AT1 receptor binding was measured as described previously (33). Briefly, 48 h after transfection the cell medium was aspirated and replaced with monoiodinated 125I-[Sar1,Ile8]Ang II (2–3 x 105 cpm; Peptide Radioiodination Service, Oxford, MS) in Hanks' balanced salt solution, 20 mM HEPES, 0.1% bovine serum albumin). After incubation at room temperature for 60 min, unbound ligand was removed by washing each well twice with 1 ml of ice-cold phosphate-buffered saline. Bound ligand was recovered by dissolving the protein in each well with 1 ml 0.5 M NaOH, 0.01% SDS. Nonspecific binding was determined by performing the binding assay in the presence of 1 µM unlabeled Ang II. The quantity of 125I-[Sar1,Ile8]Ang II present in each sample was determined using a Cobra {gamma}-spectrophotometer (Packard Bell, Palo Alto, CA). Protein content in wells was assessed using the Bio-Rad protein assay dye reagent (Bio-Rad). Values presented represent specific (total minus nonspecific) binding.

Real-time PCR—miRNA certified FirstChoice human total RNA survey panel was purchased from Ambion, the RNA was subsequently treated with RNase-free DNase I, and cDNA was synthesized from 1 µg of total RNA using gene-specific primers to miR-155 precursor and 18 S rRNA as described (34). The expression of miR-155 precursor relative to 18 S rRNA was determined using SYBR green real-time quantitative PCR assay as described (35). Relative gene expression was calculated as 2–(CTmiR-155-CT18S rRNA). Relative gene expression was multiplied by 106 to simplify data presentation. An experiment was conducted to validate that the expression of the mature miR-155 correlated to the miR-155 precursor (data not shown). The TaqMan® microRNA assay (Applied Biosystems, Foster City, CA) was used to quantify the expression of the mature miR-155 (36). Alternatively, RNA was isolated from hPFBs using TRIzol (Invitrogen) and treated as described above; however, cDNA was synthesized using oligo(dT). The expression of hAT1R mRNA relative to 18 S rRNA was determined as described above. The hAT1R-specific primers used were as follows: sense primer, 5'-CACCATGTTTTGAGGTTGACTGAC-3'; antisense primer, 5'-CAGGCTAGGGAGATTGCATTTCTG-3'.

Immunoassay for Extracellular Signal-related Kinase (ERK)—Twenty-four hours after transfection the hPFB cells were washed and serum-starved for an additional 24 h. Serum-starved cells were stimulated with Ang II (1 µM) for 5 min, washed with phosphate-buffered saline, and lysed with a concentrated buffer solution containing 250 mM Tris, pH 6.8, 8% SDS, 40% glycerol, 200 mM dithiothreitol, and 0.04% bromphenol blue (300 µl/1 x 106 cells). An aliquot of the supernatant was separated by 10% SDS-PAGE. Following transfer to nitrocellulose membrane and blocking with 5% nonfat milk, the blot was incubated with an antibody (1:2000) specific for phospho-ERK1/2 (Cell Signaling, Beverly, MA). The immunoblot was then incubated with a secondary antibody conjugated with horseradish peroxidase and visualized with ECL and the autoradiograph was quantitated by densitometric analysis. The blots were subsequently stripped and reprobed with an ERK1/2-specific antibody (Cell Signaling) to normalize the level of phosphorylated ERK to total ERK.

Statistical Analysis—All data are reported as means ± S.E. When comparisons were made between two different groups, statistical significance was determined using Student's t test. When multiple comparisons were made, statistical significance was determined using one-way analysis of variance followed by Tukey's post-test. All statistical analyses were performed using the software package Prism 4.0b (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
MiR-155 Interacts with the 3'-UTR of the hAT1R mRNA—Bioinformatic approaches have been utilized to identify potential miRNA targets (reviewed in Ref. 37). An on-line search of the miRBase Target data base (38) demonstrated that 9–16 putative miRNA target sites were harbored in the 3'-UTR of the AT1R mRNA depending upon the species analyzed. For this study we have focused on three miRNAs (i.e. miR-124a, -155, and -365) that may target the hAT1R 3'-UTR since these sites are conserved, to various degrees, across species (Fig. 1). There is only a single target site for each miRNA studied, and the target site of miR-155 and miR-365 is overlapping (Fig. 1, A–C). To experimentally validate the computational data, the hAT1R 3'-UTR (i.e. 883 bp) was subcloned downstream of the f-luc open reading frame (Fig. 2A). This reporter construct (pMIR/883/5'->3') was cotransfected in the CHO cell line with pRL-CMV (to normalize for transfection differences) and a control non-targeting RNA oligonucleotide (miR-Control) or miR-124a, -155, or -365 precursor RNA oligonucleotides (Fig. 2B). Identical control experiments were also performed utilizing a reporter construct (pMIR/883/3'->5') in which the hAT1R3'-UTR was cloned in the opposite orientation. Interestingly, the relative luciferase activity was only markedly diminished (61.3 ± 4.2%) in cells cotransfected with the pMIR/883/5'->3' construct and miR-155 (50 nM final concentration, Fig. 2B). To test the potency of the miRNAs, pMIR/883/5'->3' was cotransfected into CHO cells with increasing concentrations of each specific RNA oligonucleotide and luciferase activities were determined. Dose response experiments demonstrated that relative luciferase activity was significantly decreased with as little as 1 nM miR-155 and a maximal decrease was obtained with a 50 nM concentration of this RNA (Fig. 2C). In contrast, increasing concentrations of miR-Control (Fig. 2C) or miR-124a or -365 had no effect on luciferase activity even when the concentration of these RNAs was 100 nM (data not shown).

To demonstrate that miR-155 interacts with a specific target sequence localized in the hAT1R 3'-UTR, an additional reporter construct was generated in which the 7 bp "seed" sequence (i.e. AGCAUUA), which is complementary to the 5'-end of miR-155 (Fig. 1B), was deleted using PCR. The resulting construct, pMIR/883/5'->3'/{Delta}miR-155, was cotransfected into CHO cells as described above and luciferase activity measured. Importantly, miR-155 could no longer decrease luciferase activity of the new reporter construct (Fig. 2D). Taken together, these results indicate that of the three potential miR targets identified in the hAT1R3'-UTR by bioinformatics analyses, only miR-155 can interfere with luciferase mRNA translation via direct interaction with the hAT1R3'-UTR.

miR-155 Decreases hAT1R Expression on Human Pulmonary Fibroblasts—We have previously demonstrated that hPFB endogenously express the hAT1R (33). Therefore, hPFBs were transfected with the appropriate miRNAs, and AT1R levels were quantitated by performing radioreceptor binding assays. AT1R binding assays demonstrated that only hPFBs transfected with miR-155 showed a significant reduction in the expression of hAT1Rs (48.3 ± 6.2%) compared with controls (Fig. 3A). To investigate whether miR-155 targeted hAT1R mRNA for degradation, real-time PCR experiments were performed on RNA isolated from transfected hPFB cells. These data demonstrated that cells transfected with miR-155 did not significantly decrease hAT1R steady state mRNA levels (Fig. 3B). To determine whether the reduction in hAT1R density also resulted in decreased Ang II-induced signal transduction, hPFBs were transfected with miRNA-124a or -155 and activated with 0.1 µM Ang II for 5 min and phospho-ERK1/2 levels were determined. These results demonstrated that only hPFB cells transfected with miR-155 exhibited decreased phospho-ERK1/2 levels (52.4% ± 5.3%, p < 0.001) compared with controls (Fig. 3, C and D). Taken together, these experiments suggest that miR-155 markedly decreased hAT1R expression by inhibiting the translation process and not by targeting hAT1R mRNA for degradation. Importantly, decreased hAT1R expression also resulted in a significant decrease in Ang II-induced signaling events.


Figure 1
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  		FIGURE 1.
The hAT1R3'-UTR harbors at least three putative miRNA binding sites. A, complementarity between miR-124a and the putative hAT1R3'-UTR site targeted (536–557 bp downstream from the hAT1R stop codon). Also shown are the conserved bases of the putative miR-124a target sequence present in the hAT1R3'-UTR. B, complementarity between miR-155 and the putative hAT1R3'-UTR site targeted (70–90 bp downstream). Also shown are the conserved bases of the putative miR-155 target sequence present in the hAT1R 3'-UTR. C, complementarity between miR-365 and the putative hAT1R site targeted (69–89 bp downstream). Also shown are the conserved bases of the putative miR-365 target sequence present in the hAT1R3'-UTR.

 
MiR-155 Is Expressed in Human Fibroblasts at Physiologically Important Levels—The hAT1R is expressed in a wide variety of human tissues (1, 33). Therefore, to begin to investigate the hypothesis that miR-155 may play a physiological role in regulating the expression of the hAT1R, we determined whether hPFB cells and human tissues expressed the miR-155 gene. Real-time PCR experiments demonstrated that miR-155 was expressed in hPFB cells and in all human tissues studied although at varying levels (Fig. 4). The TaqMan probes used to generate the data presented in Fig. 4 will fluoresce only in the presence of amplicon; therefore the data represent true miR-155 expression and not background. The data were classified as tissues with high (lung, spleen, and thymus), intermediate (colon, esophagus, ovary, small intestine, and trachea), low (adipose, bladder, brain, cervix, heart, kidney, prostate, skeletal muscle, and thyroid), and very low (liver, pancreas, placenta, and testes) miR-155 expression. The miR-155 expression in hPFB cells fell into the intermediate category (Fig. 4). The expression of miR-155 precursor paralleled that of mature miR-155 in both the tissues and hPFB cells (data not shown).


Figure 2
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  		FIGURE 2.
miR-155 can inhibit reporter activity. A, schematic representation of the f-luc reporter constructs utilized. The pMIR/883 plasmids contain the full-length 3'-UTR of the hAT1R in either the 5'->3' or 3'->5' orientation with respect to the f-luc open reading frame and were designated pMIR/883/5'->3' and pMIR/883/3'->5', respectively. The firefly luciferase start and stop codons are also shown. B, CHO cells were cotransfected with pMIR/883/5'->3' or pMIR/883/3'->5', pRL-CMV, and a 50 nM concentration of a given RNA oligonucleotide. Forty-eight hours after transfection luciferase activities were measured. f-luc activity was normalized to r-luc expression and the mean activities ± S.E. from five independent experiments are shown (*, p < 0.01 versus mock-transfected). C, CHO cells were cotransfected with pMIR/883/5'->3', pRL-CMV, and either miR-Control or miR-155 at the concentrations indicated, and luciferase activities were calculated as described above. The mean activities ± S.E. from five independent transfection experiments are shown (*, p < 0.01 versus miR-Control at each concentration shown). D, CHO cells were cotransfected with pMIR/883/5'->3'/{Delta}miR-155, pRL-CMV, and a 50 nM concentration of the appropriate miRNA as shown. Forty-eight hours after transfection luciferase activities were calculated as described above. The mean activities ± S.E. from five independent transfection experiments are shown.

 
To investigate whether the level of miR-155 expression in hPFBs is physiologically relevant, these cells were transfected with antisense RNA oligonucleotides (i.e. anti-miR-Control, anti-miR-124a, anti-miR-155, and anti-miR-365) complementary to each miRNA utilized in this study, and the cells were then subjected to radioreceptor binding assays. Importantly, these experiments demonstrated that only hPFBs transfected with anti-miR-155 showed an increase in hAT1R levels (32.5% ± 3.9%, p < 0.01, Fig. 5A). To investigate whether the antisense RNAs also modulated Ang II-induced signaling events, phospho-ERK1/2 levels were determined (Fig. 5, B and C). These experiments demonstrated that cotransfection of anti-miR-155 not only increased hAT1R expression but also enhanced Ang II-induced signaling via the hAT1R (31.2% ± 5.1%, p < 0.01, Fig. 5, B and C). Taken together, these results suggest that although the miR-155 gene is expressed at relatively low levels in hPFBs, the inhibition of the endogenously expressed miR-155 by anti-miR-155 resulted in enhanced levels of hAT1R, indicating that miR-155 plays a physiological role in regulating the expression of hAT1Rs in human fibroblasts.

TGF-beta1 Inversely Regulates hAT1R and miR-155 Expression in Human Fibroblasts—To determine whether physiological stimuli could modulate miR-155 expression, we utilized TGF-beta1 (4 ng/ml for 4 h) since it has been previously demonstrated that hAT1R protein expression levels were enhanced in TGF-beta1-treated fibroblasts (37). Thus, hPFBs were incubated with TGF-beta1 for the times indicated, and PCR and radioreceptor binding assays were performed. Importantly, TGF-beta1 treatment significantly decreased miR-155 expression levels (Fig. 6A). Although TGF-beta1 also up-regulated the expression of the hAT1R gene (Fig. 6B), the time course for the increased production of hAT1R mRNA did not correlate well with the time course for the enhanced synthesis of hAT1R protein levels since these values remained higher than expected at the 8 and 12 h time points (Fig. 6, C versus B). Therefore, the sustained increase in hAT1R protein levels cannot be accounted for by only a transcriptional mechanism and suggests that post-transcriptional mechanisms must also be involved.


Figure 3
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  		FIGURE 3.
miR-155 inhibits hAT1R expression and Ang II-mediated signaling in fibroblasts. hPFBs were either mock-transfected or transfected with the miRNAs as indicated; 48 h after transfection the cells were utilized as follows. A, AT1R radioreceptor binding assays were performed as described under "Experimental Procedures." The data have been normalized for protein and transfection differences and represent specific binding. The values are shown as percent of maximal specific binding of mock-transfected hPFBs and represent the mean ± S.E. from four independent experiments (*, p < 0.001 versus mock-transfected cells). B, real-time PCR experiments were performed as described previously under "Experimental Procedures" utilizing total RNA isolated from transfected cells. The relative gene expression of hAT1R mRNA was normalized to 18S rRNA expression and is expressed in arbitrary units. The mean of hAT1R steady state mRNA levels from four independent transfection experiments are shown. C, Ang II-induced phospho-ERK1/2 experiments were performed utilizing serum-starved, transiently transfected cells as described under "Experimental Procedures." A representative immunoblot is shown. Results are representative of four independent experiments. D, the quantitation of Ang II (1 µM for 5 min)-induced ERK1/2 phosphorylation was determined by densitometry. Values are expressed as a percent of the maximal phosphorylation of ERK1/2 in response to Ang II in mock-transfected cells and represent the mean ± S.E. from four independent transfection experiments (*, p < 0.001).

 


Figure 4
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  		FIGURE 4.
Mature miR-155 is expressed in human tissues. miRNA certified human total RNAs were reverse transcribed utilizing miR-155- and 18 S rRNA-specific primers, and PCR experiments were performed as described under "Experimental Procedures." Alternatively, total RNA was isolated from non-treated hPFBs and utilized as described above. The relative expression of the mature miR-155 gene was normalized to 18 S rRNA. The values shown were obtained from a single PCR experiment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Computational studies have suggested that ~10% of human genes (38) are targets of the 326 known human miRNAs (39). To date, however, relatively few target sites have been experimentally identified (Tar-Base; Ref. 40). An on-line search of the miRBase Target data base (38) suggested that all components of the renin-angiotensin system may be regulated by miRNAs (data not shown). A specific search of the AT1R gene demonstrated that 9–16 putative miRNA target binding sequences are present in the 3'-UTR of these mRNAs, depending upon which species are analyzed. The aim of this study was to begin to determine whether the identified miRNAs actually regulate the expression of the hAT1R. Therefore, three specific miRNAs (i.e. miR-124a, miR-155, and miR-365) were chosen and utilized to investigate whether they could mediate the translational repression of a luciferase/hAT1R 3'-UTR reporter construct and endogenous hAT1R expression. Interestingly, only miR-155 could efficiently reduce luciferase activity and hAT1R density (Figs. 2 and 3) suggesting that miR-155 could interact specifically with the 3'-UTR of the hAT1R mRNA and inhibit translation. At present it is not clear why miR-124a and -365 do not interact with hAT1R mRNAs. Current computational search strategies apply different assumptions about how to best identify functional target sites; therefore these approaches may only capture subsets of real targets and/or may include a number of background matches (41). It has recently been demonstrated that sites with as few as 7 bp of complementarity (designated as the seed sequence) to the miRNA 5' end are sufficient to confer regulation in vivo and are used in biologically relevant targets (30, 42). Additionally, it has been demonstrated that G:U wobble pairing in the seed sequence is highly detrimental to miRNA function despite its favorable contribution to RNA:RNA duplexes (30). Finally, it has been established that miRNAs that have weak 5' pairing need substantial 3' pairing to function (42). Therefore, based on these derived criteria, it might be expected that of the three miRNAs utilized in this study, only miR-155 should regulate hAT1R expression since this miRNA harbors a seed sequence of 7 nucleotides and does not contain a G:U base pair in this complementary region (Fig. 1).


Figure 5
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  		FIGURE 5.
Anti-miR-155 enhances hAT1R expression and Ang II-induced signaling in fibroblasts. hPFBs were transfected with the anti-miRNA oligonucleotides as indicated; 48 h after transfection the cells were utilized as follows. A, AT1R radioreceptor binding assays were performed as described under "Experimental Procedures." The data have been normalized for protein and transfection differences and data represent specific binding. The values are shown as percent of maximal specific binding of mock-transfected hPFBs and represent the mean ± S.E. from four independent transfection experiments (*, p < 0.01 versus mock-transfected cells). B, Ang II-induced phospho-ERK1/2 experiments were performed utilizing serum starved, transiently transfected cells as described under "Experimental Procedures." A representative immunoblot is shown. Results are representative of four independent experiments. C, the quantitation of Ang II (1 µM for 5 min)-induced ERK1/2 phosphorylation was determined by densitometry. Values are expressed as a percent of the maximal phosphorylation of ERK1/2 in response to Ang II in mock-transfected cells and represents the mean ± S.E. from four independent transfection experiments (*, p < 0.01).

 
While the luciferase reporter gene and miRNA transfection experiments were useful in confirming that miR-155 could regulate the expression of hAT1Rs, it is important to demonstrate that endogenous miR-155 can regulate the translatability of endogenously expressed hAT1R mRNA. Therefore, to investigate whether or not the expression levels of miR-155 in hPFB (Fig. 4) were physiologically relevant, these cells were transfected with antisense miRNA inhibitors (Fig. 5, A–C). For the first time, we have demonstrated that the inhibition of endogenously expressed miR-155, by anti-miR-155, markedly increased hAT1R expression (Fig. 5A). Importantly, the increased hAT1R expression levels also lead to enhanced Ang II-induced phospho-ERK1/2 activation (Fig. 5C). Taken together, these data clearly suggest that miR-155 regulates the in vivo expression of hAT1R in fibroblasts. Furthermore, our PCR data demonstrated that miR-155 was expressed at varying levels in a number of human tissues (Fig. 4) implying that miR-155 could regulate the expression of the hAT1R in any tissue or cell type where both RNAs are coexpressed. Since the vasoconstrictor response of Ang II is mediated via the AT1R expressed on vascular smooth muscle cells, we also investigated whether these cells expressed miR-155.3 Interestingly, human vascular smooth muscle cells expressed 5–6-fold more miR-155 versus fibroblasts. Therefore, this observation may suggest that, dependent upon the cell type, miR-155 translational repression of hAT1R expression may be more, or less, pronounced contingent upon the relative expression levels of both miR-155 and hAT1R mRNAs.


Figure 6
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  		FIGURE 6.
TGF-beta1 inversely regulates hAT1R and miR-155 expression in fibroblasts. hPFBs were grown to confluence, serum-starved for 24 h, and incubated with or without 4 ng/ml of TGF-beta for the times indicated. A, total RNA was isolated, and relative miR-155 expression was determined as described under "Experimental Procedures." The values shown represent the mean ± S.E. from at least three independent transfection experiments (*, p < 0.001) and are expressed as fold over mock-transfected values, which was set to "1." B, total RNA was isolated and relative hAT1R expression was determined as described under "Experimental Procedures." The values shown represent the mean ± S.E. from at least three independent transfection experiments (*, p < 0.001). C, AT1R radioreceptor binding assays were performed as described above. Values are expressed as fold increase over non-treated hPFBs and represent the mean ± S.E. from four independent transfection experiments (*, p < 0.001 versus non-treated cells).

 
To further demonstrate the physiological relevance of miR-155 in regulating hAT1R expression levels, we investigated whether TGF-beta1 stimulation also modulated miR-155 expression since it was previously demonstrated that this cytokine could regulate hAT1R synthesis in fibroblasts (37). Importantly, TGF-beta1 treatment markedly decreased miR-155 expression levels (Fig. 6A) suggesting that a miRNA mechanism was involved in the enhanced hAT1R protein levels (Fig. 6C). While it was clear from these data that hAT1R protein levels were also up-regulated by a TGF-beta1-mediated transcriptional mechanism (Fig. 6B), it was apparent that other mechanisms must be involved since the hAT1R protein levels remained high even though hAT1R mRNA levels returned to basal values (see the 12-h TGF-beta1 time point, Fig. 6, C versus B). Additional support for the involvement of a non-transcriptional mechanism in regulating hAT1R expression is based on a recently published study, which demonstrated that TGF-beta1-treated hPFBs predominantly synthesized a hAT1R mRNA splice variant (i.e. hAT1R-B, a mRNA comprised of exons 1, 2, and 4, which was up-regulated 6-fold) that harbored multiple upstream open reading frames (33). Importantly, the inclusion of these upstream open reading frames decreased hAT1R protein levels. Therefore, the hAT1R levels measured in TGF-beta1-treated hPFBs were much higher than expected (Fig. 6C). Since miR-155 inversely regulates hAT1R expression (Figs. 3A and 5A), the TGF-beta1-mediated down-regulation of miR-155 could explain, in part, the high hAT1R protein levels observed in the data shown in Fig. 6C. Taken together, our studies suggest that miR-155 can translationally repress the expression of hAT1R in vivo.

Although the miR-155 target site harbored in the 3'-UTR of AT1R mRNAs is highly conserved across species (Fig. 1B), the mRNA sequence, which is complementary to the miR-155 seed sequence, is not perfectly conserved in the mouse and rat AT1R genes. Therefore, we speculate that miR-155-mediated translational repression of the AT1R will only occur in humans, chimpanzees, and dogs.

As mentioned earlier, bioinformatics analysis suggested that hAT1R levels may be regulated by at least nine distinct miRNAs (data not shown). Importantly, all six of the miRNAs, not experimentally tested in this study, have the appropriate seed complementarity with distinct target sites in the hAT1R 3'-UTR, suggesting that they may also regulate hAT1R expression levels. This observation raises the possibility that the translatability of hAT1R mRNAs may be subject to combinatorial regulation by multiple miRNAs. In support of this hypothesis, Doench and Sharp (30) demonstrated that multiple miRNAs can simultaneously repress a given target mRNA, and these investigators demonstrated a cooperative regulation between coexpressed miRNAs in translationally repressing their target mRNA (30). Therefore, future studies will investigate whether multiple miRNAs can simultaneously regulate hAT1R expression.

In conclusion, AT1R expression can be regulated by a number of distinct transcriptional and post-transcriptional mechanisms (1, 7). Alternative splicing provides an additional mechanism by which the expression and function of the AT1R can be "fine-tuned" (14, 15, 43). Our study demonstrates, for the first time, that translational repression by miR-155 provides yet another mechanism by which AT1R expression can be modulated. Since AT1R activation initiates a cascade of pathological events, including altered vascular tone, endothelial dysfunction, structural remodeling, and vascular inflammation, all of which may significantly contribute to the development of cardiovascular and kidney disease (2–6), it has been speculated that the overproduction of the AT1R could lead to these health disorders. As a result of our current study, one possible scenario that would lead to abnormally high levels of AT1R would be if miR-155 levels were atypically low. Recently, it has been demonstrated that miRNAs are involved in the pathogenesis of solid tumors and support their function in either dominant or recessive fashion, by controlling the expression of protein-coding tumor suppressors and oncogenes (44). Therefore, future studies will begin to investigate the potential role of miRNAs in mediating cardiovascular disease.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Davis Heart and Lung Research Institute, The Ohio State University, DHLRI 515, 473 West 12th Ave., Columbus, OH 43210. Tel.: 614-292-1400; Fax: 614-247-7799; E-mail: terry.elton{at}osumc.edu.

2 The abbreviations used are: Ang II, angiotensin II; AT1R, angiotensin II type 1 receptor; h, human; UTR, untranslated region; miRNA, microRNA; CHO, Chinese hamster ovary; PFB, primary lung fibroblast; ERK, extracellular receptor kinase; TGF, transforming growth factor. Back

3 M. M. Martin, E. J. Lee, J. A. Buckenberger, T. D. Schmittgen, and T. S. Elton, unpublished data. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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