β-Arrestin1–Biased β1-Adrenergic Receptor Signaling Regulates MicroRNA ProcessingNovelty and Significance
Rationale: MicroRNAs (miRs) are small, noncoding RNAs that function to post-transcriptionally regulate gene expression. First transcribed as long primary miR transcripts (pri-miRs), they are enzymatically processed in the nucleus by Drosha into hairpin intermediate miRs (pre-miRs) and further processed in the cytoplasm by Dicer into mature miRs where they regulate cellular processes after activation by a variety of signals such as those stimulated by β-adrenergic receptors (βARs). Initially discovered to desensitize βAR signaling, β-arrestins are now appreciated to transduce multiple effector pathways independent of G-protein–mediated second messenger accumulation, a concept known as biased signaling. We previously showed that the β-arrestin–biased βAR agonist, carvedilol, activates cellular pathways in the heart.
Objective: Here, we tested whether carvedilol could activate β-arrestin–mediated miR maturation, thereby providing a novel potential mechanism for its cardioprotective effects.
Methods and Results: In human cells and mouse hearts, carvedilol upregulates a subset of mature and pre-miRs, but not their pri-miRs, in β1AR-, G-protein–coupled receptor kinase 5/6–, and β-arrestin1–dependent manner. Mechanistically, β-arrestin1 regulates miR processing by forming a nuclear complex with hnRNPA1 and Drosha on pri-miRs.
Conclusions: Our findings indicate a novel function for β1AR-mediated β-arrestin1 signaling activated by carvedilol in miR biogenesis, which may be linked, in part, to its mechanism for cell survival.
MicroRNAs (miRs), a class of ≈22 nucleotide noncoding RNAs, govern post-transcriptional repression of target mRNAs. Various roles of miRs in normal cardiac physiology have been reported, including the control of myocyte growth, contractility, and the maintenance of cardiac rhythm.1 Furthermore, gain- and loss-of-function studies of a selective group of miRs suggested that aberrant expression of miRs could be necessary and sometimes even sufficient for the pathogenesis of various heart diseases,2,3 pointing toward miRs as new regulatory mechanisms and potential therapeutic targets for heart disease to complement pharmacological approaches.1
In This Issue, see p 739
Editorial, see p 742
MiR biogenesis is regulated in a complex manner, involving numerous protein–protein and protein–RNA interactions.4 Both miR regulator and miR target availability often differ among cell types, tissues, and especially during disease initiation and progression, responding to different upstream signaling pathways to activate distinct downstream targets. It is understood that miRs are influenced at the transcriptional level but are also regulated during further downstream steps in which 2 RNase III enzymes, Drosha and Dicer, play dominant roles in the control of miR maturation. Several post-transcriptional regulatory mechanisms of miR maturation have been identified. For example, several proteins, including Smads and E2-ERα, modulate miR processing in a RNA helicase–dependent or –independent manner.5–7 Interestingly, a proteomic analysis assessing the global cellular interactions of the G-protein–coupled receptor (GPCR) signaling mediators, β-arrestin1 and β-arrestin2, identified that β-arrestins may play regulatory roles in miR processing.8
β-Arrestin1 and β-arrestin2 were initially discovered to desensitize GPCR signaling in response to agonist stimulation. However, it is now appreciated that β-arrestins can also transduce multiple effector pathways independent of G-protein signaling when receptors are stimulated by certain ligands, a concept known as biased signaling.9–13 The proposed mechanism for this signaling bias is based on the barcode hypothesis where unbiased and β-arrestin–biased ligands impart distinct patterns of receptor phosphorylation by specific GPCR kinases (GRKs), thus converting ligand-induced conformation of the receptor into selective β-arrestin functions.14–16 For example, ligands that promote GRK2/3-mediated receptor phosphorylation lead to desensitization and internalization, whereas ligands, such as β-adrenergic receptor (βAR) antagonist (ie, β-blocker) carvedilol (Carv), that promote GRK5/6-mediated receptor phosphorylation stimulate β-arrestin signaling.14–16 Indeed, Carv is 1 of 3 β-blockers approved for heart failure and has many documented actions, including antagonism of β1AR, β2AR, and α1AR, as well as antioxidant effects.17,18 We previously showed that Carv stimulates β-arrestin–mediated β1AR cardioprotective signaling without activating G-proteins, providing an additional mechanism for its clinical efficacy.9 However, our understanding of whether β-arrestin–biased signaling regulates nuclear processes remains limited.
We postulated that miR could, in part, explain how GPCR-mediated β-arrestin signaling pathways confer physiological outcomes such as antiapoptosis. Although β-arrestins are known to be involved in multiple cytoplasmic signaling networks,19,20 it is increasingly appreciated that β-arrestins also play important roles in the nucleus.21,22 Of the 2 nonvisual and ubiquitous arrestins, β-arrestin1 is thought to be the major isoform involved in nuclear signaling because, unlike β-arrestin2, it lacks a nuclear export signal.23 Here, we investigate whether stimulation of βARs by the β-arrestin–biased agonist, Carv, can regulate miR expression in both cultured cells and the heart. Out of 9 human and 1040 mouse miRs examined, we found that human miR-190 and 5 human/mouse miRs (125a-5p, 125b-5p, 150, 199a-3p, and 214) were upregulated by Carv stimulation and that this effect was absent in cells or mice lacking β1AR, GRK5/6, or β-arrestin1. Although Carv did not increase the expression of pri-miRs, it enhanced the expression of pre-miRs by promoting the interaction of β-arrestin1 with components of the nuclear Drosha microprocessor complex. Our data provide evidence that the biased β-blocker Carv stimulates β-arrestin1–mediated miR processing, which may be an important mechanism for its cardioprotective effects.
Details of cell culture, siRNA experiments, immunoprecipitation, immunoblotting, immunofluorescence staining, quantitative real-time RT-PCR, Northern blot, RNA-CHIP, treatment protocol for mice, βAR radioligand binding, miR microarray analysis, luciferase-based miR processing assay, and statistical analysis are provided in Online Data Supplement.
β-Arrestin–Biased βAR Ligand Carv Induces the Expression of Human miR-190 in HEK293 Cells
To test whether the β-arrestin–biased β-blocker Carv can regulate miR expression, we used HEK293 cells stably expressing the wild-type (WT) β1AR. WT β1AR cells were treated with 1 μmol/L of βAR agonist isoproterenol (Iso, unbiased agonist), β1AR antagonist metoprolol (Met, neutral unbiased β-blocker), or Carv (β-arrestin–biased β-blocker). We assessed the expression of 4 miRs (miR-1, -21, -190, and -221) based on their known association with GPCR signaling pathways.24–26 Among the 4 human miRs examined, only hsa (homo sapiens)-miR-190 was activated at 8 and 20 hours after Carv stimulation (Figure 1A; Online Figure I). In our Carv time-course experiments, 8 and 20 hours were the time points showing significant activation of miR-190 expression (Online Figure II). The increase in miR-190 was not seen with either Iso or Met stimulation, but Met pretreatment was able to block the increase with Carv (Figure 1A). In addition, Carv did not upregulate miR-190 expression in HEK293 cells overexpressing either β2AR or α1AR (Figure 1B). Lastly, treatment with 10 μmol/L of antioxidants (α-tocopherol and ebselen) failed to affect miR-190 expression (Online Figure III). Collectively, these results indicate a β1AR-mediated mechanism of Carv action.
Because Iso did not stimulate miR-190 expression (Figure 1A), we tested the effect of treatment with the activator of adenylyl cyclase, forskolin. Interestingly, the upregulation of miR-190 by Carv was blocked by pretreatment with 10 μmol/L forskolin (Online Figure IV), suggesting inhibition of miR-190 expression by Gαs protein–mediated signaling. Because Carv is dissolved in DMSO, we treated WT β1AR cells with DMSO (0.1% [v/v]) alone for 8 or 20 hours and found no activation of miR-190 expression (Online Figure V).
Increase in miR-190 Levels Elicited by β-Arrestin–Biased β1AR Stimulation Requires GRK5/6 Phosphorylation and β-Arrestin1
We next tested whether β-arrestin signaling is required for Carv-induced miR-190 expression. Because GRK-mediated phosphorylation of the receptor promotes the recruitment of β-arrestins to the ligand-activated receptor,9,14,27 we examined the involvement of GRK phosphorylation in Carv-mediated miR-190 activation. Cells stably expressing WT β1ARs or mutant GRK− β1ARs (those lacking GRK phosphorylation sites on the β1AR c-terminal tail) were treated with 1 μmol/L Carv. WT β1AR cells showed an increase in miR-190 expression on Carv stimulation, but GRK− β1AR cells lacked this effect (Figure 1C). To test which of the GRKs are involved in miR-190 upregulation, we performed knockdown experiments using siRNAs targeting the individual GRKs. WT β1AR cells transfected with either scrambled siRNA (Si-Control) or siRNAs individually targeting GRK2, GRK3, GRK5, or GRK6 were stimulated with 1 μmol/L Carv. Carv-mediated miR-190 upregulation was abrogated in cells transfected with siRNAs targeting GRK5 or GRK6, but not GRK2 or GRK3 (Figure 1D), consistent with the hypothesis that phosphorylation-specific GRK sites on the c-terminal tail of β1AR are required to promote Carv-mediated signaling.9,15,16 To test the role of β-arrestins in this process, we treated WT β1AR cells with Carv in the presence of siRNAs targeting β-arrestin1 (Si-βarr1), β-arrestin2 (Si-βarr2), or β-arrestin1/2 (Si-βarr1/2). Knockdown of β-arrestin1 abrogated the increase in miR-190 expression (Figure 1E), indicating its central role in this β1AR-mediated process.
Induction of miR-190 by β-Arrestin1–Biased β1AR Agonism Occurs at a Post-Transcriptional Step
To examine which step of miR biogenesis is regulated by β-arrestin1, we measured the expression of primary transcript (pri), premature (pre), and mature miR-190. WT β1AR cells were treated with 1 μmol/L Carv for 8 or 20 hours and the expression of pri-, pre-, or mature miR-190 was detected using QRT-PCR and Northern blot analysis. Although Carv increased the expression of pre- or mature miR-190, it did not increase the expression of pri-miR-190 at any of the time points examined (Figure 2A; Online Figure VI), suggesting that β-arrestin1 is involved in miR-190 processing. Supporting this idea, we observed that Carv-mediated upregulation of pre-miR-190 was prevented by treatment with siRNAs directed against β-arrestin1 or β-arrestin1/2 but not with siRNAs directed against β-arrestin2 (Figure 2B). Altogether, we demonstrate that Carv stimulation requires β-arrestin1 to activate human miR-190 processing.
Carv-Mediated β-Arrestin–Biased Agonism of βAR Induces Unique miR Signatures in Mouse Hearts
Based on our cell data, we hypothesized that in the mammalian heart β-arrestin1 may promote the processing of a specific subset of pri-miRs into pre-miRs by the nuclear Drosha microprocessor complex. We tested this hypothesis by performing miR microarray profiling in mouse hearts to identify miR signatures regulated by stimulation with Carv. We used 8- to 12-week-old WT mice and infused them with DMSO (vehicle control) or Carv (19 mg/kg per day) for 7 days based on our time-course experiments with 2 cardiac-enriched miRs (data not shown). Among 1040 mmu (mus musculus)-miRs that we profiled, 21 miRs were upregulated and 13 miRs were downregulated on stimulation with the β-arrestin–biased ligand Carv (Online Figure VII; Online Table I). Interestingly, we found that the expression level of miR-190, which was regulated by Carv in HEK293 cells, was not detectable in the mouse heart, and only ≈10% of profiled miRs were detectable in the hearts, indicating tissue- or cell type–specific miR expression patterns.
We next sought to validate the 11 miRs with a minimum intensity of 500 (Online Table I, top panel). Using Taqman miR QRT-PCR analysis, we found that only 5 miRs were verified to be upregulated by Carv (Online Table II, shown in red color). Time-course experiments from 1 to 7 days of Carv treatment showed that the relative expression levels of 5 regulated miRs were highest at 7 days after Carv treatment (Online Figure VIII). Importantly, Iso or Met did not significantly activate the expression of these 5 miRs (Online Figure IX), in agreement with our HEK293 cell data (Figure 1A). In summary, we found that the expression of 5 mouse miRs (125a-5p, 125b-5p, 150, 199a-3p, and 214) and human miR-190 is upregulated on stimulation with the β-arrestin–biased βAR ligand Carv.
β-Arrestin1 and GRK5/6 Phosphorylation of β1AR Post-Transcriptionally Induce In Vivo miR Expression by Promoting miR Processing
We next tested whether Carv-mediated induction of the 5 mouse miRs occurs post-transcriptionally and whether it requires β-arrestins, GRKs, and 2 βAR subtypes. We measured the expression level of the 5 verified miRs using QRT-PCR and Northern blot analysis in hearts from WT, β-arrestin1 knockout (KO), and β-arrestin2 KO mice infused with DMSO or Carv. The Carv-mediated activation of 5 miRs occurred in both WT (Figure 2C; Online Figure X) and β-arrestin2 KO mice (Figure 2C and 2F) but was not observed in hearts from β-arrestin1 KO mice (Figure 2C and 2F). Carv did not increase the expression of pri-miRs (Figure 2D; Online Figure X), although levels of pre-miRs were increased on Carv stimulation in WT (Figure 2E; Online Figure X) and β-arrestin2 KO mice (Figure 2E and 2F), and these increases were blunted in β-arrestin1 KO mice (Figure 2E and 2F). Although Carv stimulation of transgenic mice overexpressing WT β1ARs induced an increase in the expression of pre- and mature miRs, hearts overexpressing a receptor that lacks GRK phosphorylation sites (GRK− β1AR transgenic), or hearts lacking GRK5, GRK6, or β1AR, showed no induction of these 5 miRs (Figure 3A–3C; Online Figure XI). These in vivo data are consistent with cellular data and support the concept that Carv stimulates β1AR-mediated miR biogenesis in β-arrestin1– and GRK5/6-dependent manner.
To test whether the upregulation of the 5 miRs found in the in vivo experiments also occurs in Carv-stimulated WT β1AR cells, we performed QRT-PCR and Northern blot analysis after 20-hour treatment and showed the induction of 5 miRs (Online Figure XII), suggesting that the newly identified miR regulatory mechanism exists in both HEK293 cells and mouse hearts.
We next investigated whether the β1AR-mediated mechanism of miR regulation is confined to Carv. We measured the expression level of 6 identified pri-, pre- and mature miRs in the hearts from WT mice and WT β1AR cells treated with the βAR antagonist alprenolol, which has also been shown to be a weak β-arrestin–biased ligand of β1AR.9 Similar to Carv, alprenolol increased the levels of pre- and mature miRs without affecting the expression of pri-miRs in both alprenolol-treated mouse hearts and WT β1AR cells (Online Figure XIII). Taken together, these data indicate that β-arrestin1–biased signaling of β1AR stimulates the processing of a subset of miRs.
β-Arrestin1 Interacts With the Nuclear Drosha Microprocessor Complex in a Carv-Dependent Manner
Based on nuclear localization of β-arrestin123 and its potential interaction with 2 components of the nuclear Drosha microprocessor complex (DDX5 or hnRNPA1),8 we tested whether β-arrestin1 may regulate miR processing in the nucleus by interacting with the Drosha microprocessor complex. We performed coimmunoprecipitation experiments in the nuclear lysates of both WT β1AR cells transiently overexpressing tagged plasmids and mouse hearts without and with treatment of Carv. We observed that Carv induced a time-dependent association of β-arrestin1 with both hnRNPA1 (a RNA-binding protein involved in RNA helicase–independent miR processing)28 and Drosha in the nuclear lysates of WT β1AR cells overexpressing β-arrestin1 but not β-arrestin2 (Figure 4A and 4B; Online Figure XIVA–XIVD). We also demonstrate that β-arrestin1 colocalizes with endogenous Drosha and hnRNPA1 in the nucleus, by performing immunofluorescence staining on WT β1AR cells that contain overexpressed GFP–β-arrestin1 after stimulation with Carv (Online Figure XV).
The Carv-mediated association of β-arrestin1 with hnRNPA1 was markedly decreased by treatment with RNase A (single-stranded RNA nuclease) and RNase V1 (double-stranded RNA nuclease), whereas the interaction of β-arrestin1 with Drosha was not affected by these RNases, indicating that β-arrestin1 interacts with hnRNPA1 via RNA molecules (Figure 4C). Importantly, we demonstrate that Carv stimulated the nuclear interaction of β-arrestin1 with hnRNPA1 and Drosha in endogenous systems using WT, β-arrestin2 KO, and β1AR transgenic mouse hearts (Figure 4D and 4E; Online Figure XIVE), but was lost in Carv-treated β1AR KO hearts (Figure 4F). No interaction with β-arrestin1 was found with the 2 RNA helicases: DDX5 and DDX17. Taken together, our data suggest that Carv stimulation of β1AR promotes β-arrestin1 translocation to the nucleus where it interacts with hnRNPA1 and Drosha of the microprocessor complex to process a subset of pri-miRs.
Carv Induces the Association of β-Arrestin1 With Primary Transcripts of β-Arrestin1–Regulated miRs
To test whether the β-arrestin1–hnRNPA1–Drosha complex assembles specifically on pri-β1-miRs after Carv stimulation, we performed RNA chromatin immunoprecipitation (ChIP) analysis on WT β1AR cells cotransfected with pCMV-β1-miRs and tagged β-arrestin1, hnRNPA1, or Drosha along with siRNAs targeting β-arrestin1. The association of β-arrestin1 or Drosha with pri-β1-miRs was induced on Carv stimulation for 20 hours, whereas a RNA-binding protein, hnRNPA1, constitutively associated with pri-β1-miRs. Knockdown of β-arrestin1 abrogated the Carv-mediated increase in the association of β-arrestin1–Drosha complex with pri-β1-miRs (Figures 1E and 5A–5E). We detected a constitutive association of pri-miR-690 with β-arrestin1–hnRNPA1–Drosha complex while the nuclear interaction was not induced by Carv (Figure 5F), confirming that miR-690 is not regulated by Carv stimulation (Online Table II). Thus, the formation of β-arrestin1–hnRNPA1–Drosha complex is pri-miR–specific.
Carv Induces Drosha-Mediated miR Processing by β-Arrestin1
To demonstrate directly a role of β-arrestin1 in pri-miR processing by the Drosha microprocessor complex, we performed pri-miR processing assays in WT β1AR cells as described previously.29 We fused luciferase reporters to pri-miRs that are regulated by β-arrestin1 (β1-miRs; Figure 6A) and monitored the loss of luciferase activity as a measure of Drosha-dependent processing of pri-miRs into pre-miRs.29 Carv treatment resulted in a time-dependent 30% to 50% fall in luciferase activity because of cleavage of the pri-miRs (Figure 6B), which was prevented in the presence of siRNA targeting either β-arrestin1 or Drosha, but not β-arrestin2 (Figures 1E and 6C and 6D). Similar results were obtained for Carv-induced processing of the human pri-miR-190 by β-arrestin1 (Online Figure XVI). Taken together, these data indicate that β-arrestin1 can regulate the post-transcriptional processing of miRs through its nuclear interaction with pri-miRs and the Drosha microprocessor complex by stimulating β1AR with the biased ligand Carv.
In this study, we show an essential role of β-arrestin1 in miR processing after stimulation by the β-arrestin–biased βAR agonist Carv. We demonstrate that this process results from the stimulation of β1AR and requires β-arrestin1 to promote the processing of a subset of miRs in murine hearts and human cells. The molecular mechanism for this β-arrestin1–mediated miR processing function involves the formation of a nuclear complex of hnRNPA1 and Drosha with β-arrestin1 to activate RNA helicase–independent miR processing (Figure 7). Our working hypothesis for the mechanism by which β-arrestin1 enhances miR processing is that GRK5/6 phosphorylation of β1AR mediates the recruitment of β-arrestin1 to the ligand-occupied receptor, resulting in the translocation of β-arrestin1 to the nucleus where it interacts with a subset of pri-miRs and components of the Drosha microprocessor complex. Supporting this hypothesis is the miR processing data showing that knockdown of β-arrestin1 or Drosha prevented pri-miR processing of the 6 identified β1-miRs (Figure 6C; Online Figure XVI) and coimmunoprecipitation data showing that the interaction of β-arrestin1 with hnRNPA1 is sensitive to RNase treatment (Figure 4C).
Sequence motif detection analysis using 6 identified miRs suggests that β1-miRs may have potentially conserved sequence motifs in their stem regions, although additional profiling analyses in different tissues or cells will be required to definitely identify a β-arrestin1 sequence motif. This sequence analysis, together with the RNA-CHIP data (Figure 5), suggests that β-arrestin1 promotes miR processing by translocating to the nucleus and directly associating with pri-miRs. Although we think this to be the most plausible mechanism, it is possible that the activation of β1AR signaling pathways downstream of β-arrestin1 (eg, EGFR, ERK, or AKT) could regulate miR processing and thus indirectly exert regulatory effects on the activation of miR processing.30 It is also possible that the regulation of β-arrestin1 in the Drosha step is indirect through interaction with hnRNPA1 or other RNA-binding proteins, although our sequence analysis showed that β1-miRs have no consensus sequences for direct hnRNPA1 binding. Additional studies will be needed to further clarify the mechanism of β-arrestin1 in miR processing.
β-Arrestins not only desensitize G-protein signaling but also activate several signaling networks by scaffolding a diverse group of signaling proteins at the GPCRs.31,32 The important roles of β-arrestins in regulating cytoplasmic signaling networks are now well recognized.19,20 However, the role of β-arrestins in modulating nuclear function is less well studied.8,21,22 Our data identify a new nuclear function of β-arrestin1, the isoform of β-arrestins known to translocate to the nucleus,23 as a regulator of miR processing in β1AR. It is interesting that only β-arrestin1 regulates miR processing, whereas both β-arrestins are involved in β1AR-mediated cardioprotective signaling.9,27 This likely reflects the fact that β-arrestin1 lacks a nuclear export sequence allowing for its retention in the nucleus after activation and translocation.23 However, we cannot rule out the possibility that β-arrestin2 regulates other miR biogenesis steps (eg, miR degradation, nuclear export, and dicing) rather than Drosha processing, because we observed that the levels of pre-miR-150, pre-miR-214, and mature miR-214 are reduced in β-arrestin2 KO mouse hearts compared with WT (Figure 2C and 2E).
β-Arrestin1 functioning in the nucleus has recently been reported. β-Arrestin1 is a nuclear transcriptional regulator of endothelin type A receptor–mediated β-catenin signaling33 and has been shown to be important for tumor progression and stem cell regulation. β-Arrestin1 also functions as an important regulator of polycomb group proteins (transcriptional repressors), suggesting its involvement in epigenetic regulation22 as well as in the regulation of histone acetylation in human neuroblastoma cells after δ-opioid receptor stimulation.21
We previously showed that β1AR uses GRK5/6 and β-arrestins to promote cardiomyocyte survival pathways against chronic catecholamine stimulation in the absence of G-protein activation.27 Our subsequent study suggested that Carv functions as a β-arrestin–biased ligand to promote cardioprotective signaling,9 providing a possible mechanism for its clinical efficacy. Interestingly, a recent meta-analysis showed that Carv did not reduce patient readmissions compared with other β-blockers although it led to less sudden cardiac death and all-cause mortality in patients with acute myocardial infarction and those with heart failure.34 Therefore, identifying additional beneficial downstream signaling pathways activated by Carv should lead to a better understanding of how biased ligands exert their cardioprotective effects.
Consistent with our previous findings, we show that the unbiased agonist Iso and unbiased antagonist Met did not activate the expression of β1-miRs (Figure 1A; Online Figure IX) and that forskolin, which activates Gαs protein signaling, blocked Carv-mediated miR-190 activation (Online Figure IV). These data, in addition to the data in Figures 1D and 3B, are consistent with the receptor phosphorylation barcode hypothesis where distinct phosphorylation patterns of the c-terminal tail of the receptor encode for different function of β-arrestin.14 In particular, it was shown for β2AR that Carv induced a phosphorylation pattern distinct from that of Iso. Notably, Carv only induced an increase in phosphorylation on 2 GRK5/6 sites, whereas Iso triggered a change in the phosphorylation status of 13 sites, including PKA, GRK2, and GRK5/6 sites.14 This selective phosphorylation profile of Carv is consistent with β-arrestin–biased signaling induced by this ligand. Surprisingly, recent studies demonstrated that GRK2, which requires G-proteins for its activation, exerts a strong negative effect on β-arrestin–dependent signaling through its competition with GRK5 and GRK6 for receptor phosphorylation,14,16 which in turn mediates the balance between G-protein and β-arrestin signaling. These previous studies are again in agreement with our data showing that Iso did not activate the expression of β1-miRs and that Met or forskolin blocked Carv-mediated activation of β1-miRs.
Carv is a nonselective βAR antagonist with high affinity for both β1ARs and β2ARs. However, we show that only β1AR stimulation by Carv mediates miR processing. A possible mechanism for βAR subtype specificity is the requirement of a unique β-arrestin conformation for activating miR processing. This idea is supported by our recent study showing that the recruitment of β-arrestin to β1AR, but not β2AR, induces a β-arrestin conformation that promotes a stable complex between β1AR, β-arrestin, and CaMKII to activate signaling.35 Moreover, recent work has shown that catecholamine stimulation of β2ARs promotes DNA damage in a β-arrestin1–dependent manner.36 Taken together, our findings suggest that Carv-stimulated β1ARs require GRK5/6 to promote β-arrestin1–mediated miR processing in the nucleus and ultimately cardioprotective signaling.
Additional discussion of the therapeutic potential and the in vivo relevance of targeting the β-arrestin1–mediated miR regulatory mechanism in cardiac dysfunction is provided in the Online Data Supplement.
In conclusion, our data identify β-arrestin1 as an important mediator of Drosha function to regulate miR biogenesis in the heart and provide new insights into our understanding of how selective ligands for β1AR may modulate the metabolism of specific miRs. We postulate that the development of high-affinity, β1AR-biased ligands, which display better efficacy for this newly discovered β-arrestin1–mediated miR regulatory network, may provide a new class of drugs for the treatment of cardiovascular diseases.
We thank Drs David Fulton, David Stepp, David Pollock, Sangmi Kim, Ganesan Ramesh, and Ruth Caldwell for critically reviewing the article and sharing their equipment, and Weili Zou for excellent technical assistance.
Sources of Funding
This work was supported by Georgia Regents University (GRU: previously Georgia Health Sciences University) Departmental Start-Up Fund and American Heart Association (AHA) Greater Southeast Affiliate (GSA) Grant-in-Aid 12GRNT12100048 to I.K., GRU Diabetes and Obesity Discovery Institute Scholar Program to J.V., AHA National Scientist Development Grant 11SDG6960011 to H.S., AHA GSA Grant-in-Aid 13GRNT17080109 to J.A.J., and National Institutes of Health grant P01HL075443 to W.J.K. and H.A.R.
H.A. Rockman is a scientific cofounder of Trevena Inc, a company that is developing G-protein–coupled receptor–targeting drugs. The other authors have no conflicts to report.
In November 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.6 days.
This manuscript was sent to Paul C. Simpson, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.114.302766/-/DC1.
- Nonstandard Abbreviations and Acronyms
- β-arrestin1-regulated miR
- β-adrenergic receptor
- G-protein–coupled receptor
- GPCR kinase
- hairpin intermediate miR/premature miR
- long primary miR transcript
- Received October 2, 2013.
- Revision received December 9, 2013.
- Accepted December 13, 2013.
- © 2013 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
β-Arrestin–mediated β1-adrenergic receptor (β1AR) signaling confers cardiac protection.
The β-blocker carvedilol is a weak activator (biased ligand) of the β1AR/β-arrestin pathway.
A role for β-arrestin in activating cellular signaling networks is increasingly being recognized as an important modulator of normal physiology and disease.
What New Information Does This Article Contribute?
β-Arrestin1–biased β1AR signaling induced by the β-blocker carvedilol regulates the biogenesis of a subset of microRNAs in mouse heart and human cells.
Carvedilol-mediated microRNA processing requires G-protein–coupled receptor (GPCR) kinase 5/6, β-arrestin1, and the β1AR.
This effect of carvedilol is mediated by the formation of a nuclear complex of β-arrestin1 with Drosha and hnRNPA1, critical components for RNA helicase–independent microRNA processing.
β-Arrestin–biased agonism is an emerging concept in the GPCR signaling field in which unique ligand-activated conformational states can selectively stimulate GPCRs to signal through β-arrestin in the absence of G-protein activation. Because GPCRs are the target of ≈40% of all modern medicinal drugs, β-arrestin–biased ligands have been considered to have important therapeutic utility. Here, we report an essential role for β-arrestin1 in microRNA processing after stimulation by the β-arrestin–biased βAR agonist carvedilol. Although carvedilol did not increase the expression of primary microRNA transcripts, it enhanced the expression of premature microRNAs by promoting the interaction of β-arrestin1 with components of the nuclear Drosha microprocessor complex. We think that modulation of the β1AR/β-arrestin1/microRNA pathway by carvedilol could lead to the development of pharmacological strategies (ie, β-arrestin1–biased ligands), allowing effective modulation of microRNAs that may be important in regulating the progression of cardiac disease and cardiac remodeling.