Il Man Kim

β-Blockers alprenolol and carvedilol stimulate β-arrestin-mediated EGFR transactivation

Recent evidence suggests that binding of agonist to its cognate receptor initiates not only classical G protein-mediated signaling, but also β-arrestin-dependent signaling. One such β-arrestin-mediated pathway uses the β1-adrenergic receptor (β1AR) to transactivate the EGFR. To determine whether β-adrenergic ligands that do not activate G protein signaling (i.e., β-blockers) can stabilize the β1AR in a signaling conformation, we screened 20 β-blockers for their ability to stimulate β-arrestin-mediated EGFR transactivation. Here we show that only alprenolol (Alp) and carvedilol (Car) induce β1AR-mediated transactivation of the EGFR and downstream ERK activation. By using mutants of the β1AR lacking G protein-coupled receptor kinase phosphorylation sites and siRNA directed against β-arrestin, we show that Alp- and Car-stimulated EGFR transactivation requires β1AR phosphorylation at consensus G protein-coupled receptor kinase sites and β-arrestin recruitment to the ligand-occupied receptor. Moreover, pharmacological inhibition of Src and EGFR blocked Alp- and Car-stimulated EGFR transactivation. Our findings demonstrate that Alp and Car are ligands that not only act as classical receptor antagonists, but can also stimulate signaling pathways in a G protein-independent, β-arrestin-dependent fashion.

beta-Arrestin-biased agonism of the angiotensin receptor induced by mechanical stress.

Mechanical stress on hearts triggers β-arrestin–dependent cell survival signaling through the angiotensin receptor. An Arrestin Stretch Activation of G protein–coupled receptors (GPCRs) can selectively trigger distinct signaling cascades through a process known as biased agonism. Mechanical stress has been linked to activation of the angiotensin type I receptor (AT1R) in a manner that does not require its ligand (angiotensin II). Rakesh et al. found that, in cells and ex vivo heart preparations, mechanical stress activated a signaling pathway that required neither angiotensin II nor G proteins. Instead, β-arrestin was recruited to AT1R, the complex was internalized, and β-arrestin activated an antiapoptotic signaling pathway through extracellular signal–regulated kinase (ERK) and Akt. Treating mice with the angiotensin receptor blocker losartan led to increased cardiomyocyte apoptosis, leading the authors to suggest that these drugs may block β-arrestin–mediated protective signaling in response to mechanical stress. β-Arrestins, which were originally characterized as terminators of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptor (GPCR) signaling, also act as important signal transducers. An emerging concept in GPCR signaling is β-arrestin–biased agonism, in which specific ligand-activated GPCR conformational states selectively signal through β-arrestins, rather than through G proteins. Here, we show that mechanical stretch induced β-arrestin–biased signaling downstream of angiotensin II type I receptors (AT1Rs) in the absence of ligand or G protein activation. Mechanical stretch triggered an AT1R-mediated conformational change in β-arrestin similar to that induced by a β-arrestin–biased ligand to selectively stimulate receptor signaling in the absence of detectable G protein activation. Hearts from mice lacking β-arrestin or AT1Rs failed to induce responses to mechanical stretch, as shown by blunted extracellular signal–regulated kinase and Akt activation, impaired transactivation of the epidermal growth factor receptor, and enhanced myocyte apoptosis. These data show that the heart responds to acute increases in mechanical stress by activating β-arrestin–mediated cell survival signals.

Crosstalk between Long Noncoding RNAs and MicroRNAs in Health and Disease

Protein-coding genes account for only a small part of the human genome; in fact, the vast majority of transcripts are comprised of non-coding RNAs (ncRNAs) including long ncRNAs (lncRNAs) and small ncRNAs, microRNAs (miRs). Accumulating evidence indicates that ncRNAs could play critical roles in regulating many cellular processes which are often implicated in health and disease. For example, ncRNAs are aberrantly expressed in cancers, heart diseases, and many other diseases. LncRNAs and miRs are therefore novel and promising targets to be developed into biomarkers for diagnosis and prognosis as well as treatment options. The interaction between lncRNAs and miRs as well as its pathophysiological significance have recently been reported. Mechanistically, it is believed that lncRNAs exert “sponge-like” effects on various miRs, which subsequently inhibits miR-mediated functions. This crosstalk between two types of ncRNAs frequently contributes to the pathogenesis of the disease. In this review, we provide a summary of the recent studies highlighting the interaction between these ncRNAs and the effects of this interaction on disease pathogenesis and regulation.

MicroRNA-150 protects the mouse heart from ischaemic injury by regulating cell death

AIMS Cardiac injury is accompanied by dynamic changes in the expression of microRNAs (miRs). For example, miR-150 is down-regulated in patients with acute myocardial infarction, atrial fibrillation, dilated and ischaemic cardiomyopathy as well as in various mouse heart failure (HF) models. Circulating miR-150 has been recently proposed as a better biomarker of HF than traditional clinical markers such as brain natriuretic peptide. We recently showed using the β-arrestin-biased β-blocker, carvedilol that β-arrestin1-biased β1-adrenergic receptor cardioprotective signalling stimulates the processing of miR-150 in the heart. However, the potential role of miR-150 in ischaemic injury and HF is unknown. METHODS AND RESULTS Here, we show that genetic deletion of miR-150 in mice causes abnormalities in cardiac structural and functional remodelling after MI. The cardioprotective roles of miR-150 during ischaemic injury were in part attributed to direct repression of the pro-apoptotic genes egr2 (zinc-binding transcription factor induced by ischaemia) and p2x7r (pro-inflammatory ATP receptor) in cardiomyocytes. CONCLUSION These findings reveal a pivotal role for miR-150 as a regulator of cardiomyocyte survival during cardiac injury.

β-Arrestin1–Biased β1-Adrenergic Receptor Signaling Regulates MicroRNA Processing

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 &bgr;-adrenergic receptors (&bgr;ARs). Initially discovered to desensitize &bgr;AR signaling, &bgr;-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 &bgr;-arrestin–biased &bgr;AR agonist, carvedilol, activates cellular pathways in the heart. Objective: Here, we tested whether carvedilol could activate &bgr;-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 &bgr;1AR-, G-protein–coupled receptor kinase 5/6–, and &bgr;-arrestin1–dependent manner. Mechanistically, &bgr;-arrestin1 regulates miR processing by forming a nuclear complex with hnRNPA1 and Drosha on pri-miRs. Conclusions: Our findings indicate a novel function for &bgr;1AR-mediated &bgr;-arrestin1 signaling activated by carvedilol in miR biogenesis, which may be linked, in part, to its mechanism for cell survival.

Gene Deletion Screen for Cardiomyopathy in Adult Drosophila Identifies a New Notch Ligand

Rationale: Drosophila has been recognized as a model to study human cardiac diseases. Objective: Despite these findings, and the wealth of tools that are available to the fly community, forward genetic screens for adult heart phenotypes have been rarely performed because of the difficulty in accurately measuring cardiac function in adult Drosophila. Methods and Results: Using optical coherence tomography to obtain real-time analysis of cardiac function in awake Drosophila, we performed a genomic deficiency screen in adult flies. Based on multiple complementary approaches, we identified CG31665 as a novel gene causing dilated cardiomyopathy. CG31665, which we name weary (wry), has structural similarities to members of the Notch family. Using cell aggregation assays and &ggr;-secretase inhibitors we show that Wry is a novel Notch ligand that can mediate cellular adhesion with Notch expressing cells and transactivates Notch to promote signaling and nuclear transcription. Importantly, Wry lacks a DSL (Delta-Serrate-Lag) domain that is common feature to the other Drosophila Notch ligands. We further show that Notch signaling is critically important for the maintenance of normal heart function of the adult fly. Conclusions: In conclusion, we identify a previously unknown Notch ligand in Drosophila that when deleted causes cardiomyopathy. Our study suggests that Notch signaling components may be a therapeutic target for dilated cardiomyopathy.

Regulation of Metastasis by microRNAs in Ovarian Cancer.

Ovarian cancer (OC) is the second most common and the most fatal gynecologic cancer in the United States. Over the last decade, various targeted therapeutics have been introduced but there has been no corresponding improvement in patient survival mainly because of the lack of effective early detection methods. microRNAs (miRs) are small, non-coding RNAs that regulate gene expression post-transcriptionally. Accumulating data suggest central regulatory roles of miRs in modulating OC initiation, progression, and metastasis. More recently, aberrant miR expression has been also associated with cancer stem cell (CSC) phenotypes and development of CSC chemo-resistance. Here, we review recent advances on miRs and OC metastasis and discuss the concept that miRs are involved in both CSC transformation and subsequent OC metastasis. Finally, we describe the prevalence of circulating miRs and assess their potential utilities as biomarkers for OC diagnosis, prognosis, and therapeutics.

Biased G Protein-Coupled Receptor Signaling: New Player in Modulating Physiology and Pathology

G protein-coupled receptors (GPCRs) are a family of cell-surface proteins that play critical roles in regulating a variety of pathophysiological processes and thus are targeted by almost a third of currently available therapeutics. It was originally thought that GPCRs convert extracellular stimuli into intracellular signals through activating G proteins, whereas β-arrestins have important roles in internalization and desensitization of the receptor. Over the past decade, several novel functional aspects of β-arrestins in regulating GPCR signaling have been discovered. These previously unanticipated roles of β-arrestins to act as signal transducers and mediators of G protein-independent signaling have led to the concept of biased agonism. Biased GPCR ligands are able to engage with their target receptors in a manner that preferentially activates only G protein- or β-arrestin-mediated downstream signaling. This offers the potential for next generation drugs with high selectivity to therapeutically relevant GPCR signaling pathways. In this review, we provide a summary of the recent studies highlighting G protein- or β-arrestin-biased GPCR signaling and the effects of biased ligands on disease pathogenesis and regulation.

Carvedilol-responsive microRNAs, miR-199a-3p and -214 protect cardiomyocytes from simulated ischemia-reperfusion injury

The nonselective β-adrenergic receptor antagonist (β-blocker) carvedilol has been shown to protect against myocardial injury, but the detailed underlying mechanisms are unclear. We recently reported that carvedilol stimulates the processing of microRNA (miR)-199a-3p and miR-214 in the heart via β-arrestin1-biased β1-adrenergic receptor (β1AR) cardioprotective signaling. Here, we investigate whether these β-arrestin1/β1AR-responsive miRs mediate the beneficial effects of carvedilol against simulated ischemia/reperfusion (sI/R). Using cultured cardiomyocyte cell lines and primary cardiomyocytes, we demonstrate that carvedilol upregulates miR-199a-3p and miR-214 in both ventricular and atrial cardiomyocytes subjected to sI/R. Overexpression of the two miRs in cardiomyocytes mimics the effects of carvedilol to activate p-AKT survival signaling and the expression of a downstream pluripotency marker Sox2 in response to sI/R. Moreover, carvedilol-mediated p-AKT activation is abolished by knockdown of either miR-199a-3p or miR-214. Along with previous studies to directly link the cardioprotective actions of carvedilol to upregulation of p-AKT/stem cell markers, our findings suggest that the protective roles of carvedilol during ischemic injury are in part attributed to activation of these two protective miRs. Loss of function of miR-199a-3p and miR-214 also increases cardiomyocyte apoptosis after sI/R. Mechanistically, we demonstrate that miR-199a-3p and miR-214 repress the predictive or known apoptotic target genes ddit4 and ing4, respectively, in cardiomyocytes. These findings suggest pivotal roles for miR-199a-3p and miR-214 as regulators of cardiomyocyte survival and contributors to the functional benefits of carvedilol therapy.

MicroRNA-150 deletion in mice protects kidney from myocardial infarction induced acute kidney injury

Despite greater understanding of acute kidney injury (AKI) in animal models, many of the preclinical studies are not translatable. Most of the data were derived from a bilateral renal pedicle clamping model with warm ischemia. However, ischemic injury of the kidney in humans is distinctly different and does not involve clamping of renal vessel. Permanent ligation of the left anterior descending coronary artery model was used to test the role of microRNA (miR)-150 in AKI. Myocardial infarction in this model causes AKI which is similar to human cardiac bypass surgery. Moreover, the time course of serum creatinine and biomarker elevation were also similar to human ischemic injury. Deletion of miR-150 suppressed AKI which was associated with suppression of inflammation and interstitial cell apoptosis. Immunofluorescence staining with endothelial marker and marker of apoptosis suggested that dying cells are mostly endothelial cells with minimal epithelial cell apoptosis in this model. Interestingly, deletion of miR-150 also suppressed interstitial fibrosis. Consistent with protection, miR-150 deletion causes induction of its target gene insulin-like growth factor-1 receptor (IGF-1R) and overexpression of miR-150 in endothelial cells downregulated IGF-1R, suggesting miR-150 may mediate its detrimental effects through suppression of IGF-1R pathways.