Jakob Lerche Hansen

SNPs in MicroRNA Binding Sites in 3′-UTRs of RAAS Genes Influence Arterial Blood Pressure and Risk of Myocardial Infarction

BACKGROUNDnWe hypothesized that single nucleotide polymorphisms (SNPs) located in microRNA (miR) binding sites in genes of the renin angiotensin aldosterone system (RAAS) can influence blood pressure and risk of myocardial infarction.nnnMETHODSnUsing online databases dbSNP and TargetScan, we identified 10 SNPs in potential miR binding sites in eight RAAS-related genes, common in Caucasians. We genotyped a large case-control study on myocardial infarctions, the Study of Myocardial Infarctions LEiden (SMILE) for these 10 SNPs and found nine SNPs, in seven genes, to be prevalent. Functionality of each SNP in interfering with mRNA/miR binding was tested using a dual luciferase reporter gene system.nnnRESULTSnOf these nine SNPs, four SNPs, located in the arginine vasopressin 1A receptor (AVPR1A), bradykinin 2 receptor (BDKRB2), and thromboxane A2 receptor (TBXA2R) genes were associated with blood pressure. The rare allele of the AVPR1A SNP rs11174811, was associated with increased blood pressure whereas the rare alleles of the two linked BDKRB2 SNPs rs5225 and rs2069591 and of the TBXA2R SNP rs13306046 were associated with decreased blood pressure. Although not associated with blood pressure, the rare allele of the mineralocorticoid receptor (NR3C2) SNP rs5534, was associated with a twofold increased risk of myocardial infarction in men younger than 50 years. For all of these five SNPs, except rs2069591, we could demonstrate a reduction in miR-induced repression of gene expression.nnnCONCLUSIONSnCommon SNPs in miR binding sites of RAAS-related genes can influence both blood pressure and risk of myocardial infarction. These results may imply an important role for SNPs in miR target sites in human disease.

Angiotensin II type 1 receptor signalling regulates microRNA differentially in cardiac fibroblasts and myocytes

BACKGROUND AND PURPOSE The angiotensin II type 1 receptor (AT1R) is a key regulator of blood pressure and cardiac contractility and is profoundly involved in development of cardiac disease. Since several microRNAs (miRNAs) have been implicated in cardiac disease, we determined whether miRNAs might be regulated by AT1R signals in a Gαq/11‐dependent or ‐independent manner.

Cell-specific detection of microRNA expression during cardiomyogenesis by combined in situ hybridization and immunohistochemistry

MicroRNAs (miRNAs) regulate gene expression by mediating translational repression or mRNA degradation of their targets, and several miRNAs control developmental decisions through embryogenesis. In the developing heart, miRNA targets comprise key players mediating cardiac lineage determination. However, although several miRNAs have been identified as differentially regulated during cardiac development and disease, their distinct cell-specific localization remains largely undetermined, likely owing to a lack of adequate methods. We therefore report the development of a markedly improved approach combining fluorescence-based miRNA-in situ hybridization (miRNA-ISH) with immunohistochemistry (IHC). We have applied this protocol to differentiating embryoid bodies (EBs) as well as embryonic and adult mouse hearts, to detect miRNAs that were upregulated during EB cardiomyogenesis, as determined by array-based miRNA expression profiling. In this manner, we found specific co-localization of miR-1 to myosin positive cells (cardiomyocytes) of EBs, developing and mature hearts. In contrast, miR-125b and -199a did not localize to cardiomyocytes, as previously suggested for miR-199a, but were rather expressed in connective tissue cells of the heart. More specifically, by co-staining with α-smooth muscle actin (α-SMA) and collagen-I, we found that miR-125b and -199a localize to perivascular α-SMA− stromal cells. Our approach thus proved valid for determining cell-specific localization of miRNAs, and the findings we present highlight the importance of determining exact cell-specific localization of miRNAs by sequential miRNA-ISH and IHC in studies aiming at understanding the role of miRNAs and their targets. This approach will hopefully aid in identifying relevant miRNA targets of both the heart and other organs.

Characterization of G-Protein Coupled Receptor Kinase Interaction with the Neurokinin-1 Receptor Using Bioluminescence Resonance Energy Transfer

To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer2 (BRET2) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive mutants. We observed agonist induced interaction of both GRK5 and GRK2 with the activated NK-1 receptor. In saturation experiments, we observed GRK5 to interact with the activated receptor in a monophasic manner while GRK2 interacted in a biphasic manner with the low affinity phase corresponding to receptor affinity for GRK5. Agonist induced GRK5 interaction with the receptor was dependent on intact kinase-activity, whereas the high affinity phase of GRK2 interaction was independent of kinase activity. We were surprised to find that the BRET2 saturation experiments indicated that before receptor activation, the full-length NK-1 receptor, but not a functional C-terminal tail-truncated receptor, is preassociated with GRK5 in a relatively low-affinity state. We demonstrate that GRK5 can compete for agonist induced GRK2 interaction with the NK-1 receptor, whereas GRK2 does not compete for receptor interaction with GRK5. We suggest that GRK5 is preassociated with the NK-1 receptor and that GRK5, rather than GRK2, is a key player in competitive regulation of GRK subtype specific interaction with the NK-1 receptor.

Type II Turn of Receptor-bound Salmon Calcitonin Revealed by X-ray Crystallography

Calcitonin is a peptide hormone consisting of 32 amino acid residues and the calcitonin receptor is a Class B G protein-coupled receptor (GPCR). The crystal structure of the human calcitonin receptor ectodomain (CTR ECD) in complex with a truncated analogue of salmon calcitonin ([BrPhe22]sCT(8–32)) has been determined to 2.1-Å resolution. Parallel analysis of a series of peptide ligands showed that the rank order of binding of the CTR ECD is identical to the rank order of binding of the full-length CTR, confirming the structural integrity and relevance of the isolated CTR ECD. The structure of the CTR ECD is similar to other Class B GPCRs and the ligand binding site is similar to the binding site of the homologous receptors for the calcitonin gene-related peptide (CGRP) and adrenomedulin (AM) recently published (Booe, J. M., Walker, C. S., Barwell, J., Kuteyi, G., Simms, J., Jamaluddin, M. A., Warner, M. L., Bill, R. M., Harris, P. W., Brimble, M. A., Poyner, D. R., Hay, D. L., and Pioszak, A. A. (2015) Mol. Cell 58, 1040–1052). Interestingly the receptor-bound structure of the ligand [BrPhe22]sCT(8–32) differs from the receptor-bound structure of the homologous ligands CGRP and AM. They all adopt an extended conformation followed by a C-terminal β turn, however, [BrPhe22]sCT(8–32) adopts a type II turn (Gly28-Thr31), whereas CGRP and AM adopt type I turns. Our results suggest that a type II turn is the preferred conformation of calcitonin, whereas a type I turn is the preferred conformation of peptides that require RAMPs; CGRP, AM, and amylin. In addition the structure provides a detailed molecular explanation and hypothesis regarding ligand binding properties of CTR and the amylin receptors.

Therapeutic potential of functional selectivity in the treatment of heart failure

Adrenergic and angiotensin receptors are prominent targets in pharmacological alleviation of cardiac remodeling and heart failure, but their use is associated with cardiodepressant side effects. Recent advances in our understanding of seven transmembrane receptor signaling show that it is possible to design ligands with functional selectivity, acting as agonists on certain signaling pathways while antagonizing others. This represents a major pharmaceutical opportunity to separate desired from adverse effects governed by the same receptor. Accordingly, functionally selective ligands are currently pursued as next-generation drugs for superior treatment of heart failure.

A bioluminescence resonance energy transfer 2 (BRET2) assay for monitoring seven transmembrane receptor and insulin receptor crosstalk

Abstract The angiotensin AT1 receptor is a seven transmembrane (7TM) receptor, which mediates the regulation of blood pressure. Activation of angiotensin AT1 receptor may lead to impaired insulin signaling indicating crosstalk between angiotensin AT1 receptor and insulin receptor signaling pathways. To elucidate the molecular mechanisms behind this crosstalk, we applied the BRET2 technique to monitor the effect of angiotensin II on the interaction between Rluc8 tagged insulin receptor and GFP2 tagged insulin receptor substrates 1, 4, 5 (IRS1, IRS4, IRS5) and Src homology 2 domain-containing protein (Shc). We demonstrate that angiotensin II reduces the interaction between insulin receptor and IRS1 and IRS4, respectively, while the interaction with Shc is unaffected, and this effect is dependent on Gαq activation. Activation of other Gαq-coupled 7TM receptors led to a similar reduction in insulin receptor and IRS4 interactions whereas Gαs- and Gαi-coupled 7TM receptors had no effect. Furthermore, we used a panel of kinase inhibitors to show that angiotensin II engages different pathways when regulating insulin receptor interactions with IRS1 and IRS4. Angiotensin II inhibited the interaction between insulin receptor and IRS1 through activation of ERK1/2, while the interaction between insulin receptor and IRS4 was partially inhibited through protein kinase C dependent mechanisms. We conclude that the crosstalk between angiotensin AT1 receptor and insulin receptor signaling shows a high degree of specificity, and involves Gαq protein, and activation of distinct kinases. Thus, the BRET2 technique can be used as a platform for studying molecular mechanisms of crosstalk between insulin receptor and 7TM receptors.

A fat-tissue sensor couples growth to oxygen availability by remotely controlling insulin secretion

Organisms adapt their metabolism and growth to the availability of nutrients and oxygen, which are essential for normal development. This requires the ability to sense these environmental factors and respond by regulation of growth-controlling signals, yet the mechanisms by which this adaptation occurs are not fully understood. To identify novel growth-regulatory mechanisms, we conducted a global RNAi-based screen in Drosophila for size differences and identified 89 positive and negative regulators of growth. Among the strongest hits was the FGFR homologue breathless necessary for proper development of the tracheal airway system. Breathless deficiency results in tissue hypoxia (low oxygen), sensed primarily in this context by the fat tissue. The fat, in response, relays this information through release of one or more humoral factors that remotely inhibit insulin secretion from the brain, thereby restricting systemic growth. Thus our findings show that the fat tissue acts as an oxygen sensor that allows the organism to reduce its growth in adaptation to limited oxygen conditions.

Role of age, Rho‐kinase 2 expression, and G protein‐mediated signaling in the myogenic response in mouse small mesenteric arteries

The myogenic response (MR) and myogenic tone (MT) in resistance vessels is crucial for maintaining peripheral vascular resistance and blood flow autoregulation. Development of MT involves G protein‐coupled receptors, and may be affected by aging. Aims: (1) to estimate the mesenteric blood flow in myogenically active small mesenteric arteries; (2) to investigate the signaling from Gαq/11 and/or Gα12 activation to MT development; (3) to investigate the role of Rho‐kinase 2 and aging on MT in mesenteric resistance arteries. Methods: we used pressure myography, quantitative real‐time PCR, and immunolocalization to study small (<200 μm) mesenteric arteries (SMA) from young, mature adult, and middle aged mice. Results: Poiseuille flow calculations indicated autoregulation of blood flow at 60−120 mm Hg arterial pressure. Gαq/11 and Gα12 were abundantly expressed at the mRNA and protein levels in SMA. The Gαq/11 inhibitor YM‐254890 suppressed MT development, and the Phosholipase C inhibitors U73122 and ET‐18‐OCH3 robustly inhibited it. We found an age‐dependent increase in ROCK2 mRNA expression, and in basal MT. The specific ROCK2 inhibitor KD025 robustly inhibited MT in SMAs in all mice with an age‐dependent variation in KD025 sensitivity. The inhibitory effect of KD025 was not prevented by the L‐type Ca2+ channel activator BayK 8644. KD025 reversibly inhibited MT and endothelin‐1 vasoconstriction in small pial arteries from Göttingen minipigs. Conclusions: MT development in SMAs occurs through a Gαq/11/PLC/Ca2+‐dependent pathway, and is maintained via ROCK2‐mediated Ca2+ sensitization. Increased MT at mature adulthood can be explained by increased ROCK2 expression/activity.

SII Ang II Potentiates Insulin Receptor Signalling and Glycogen Synthesis in Hepatocytes

The angiotensin II type I receptor (AT1R) is involved in the regulation of cardiovascular function. Excessive activation of AT1R by angiotensin II (Ang II) leads to cardiovascular disease and may be involved in the development of insulin resistance and diabetes. Functionally selective Ang II analogues, such as the [Sar1, Ile4, Ile8]‐angiotensin II (SII Ang II) analogue, that only activate a subset of signalling networks have been demonstrated to have beneficial effects on cardiovascular function in certain settings, including lowering blood pressure and increasing cardiac performance. Here, we studied the effect of SII Ang II on insulin receptor (IR) signalling and glucose metabolism in primary rat hepatocytes. We show that long‐term pre‐treatment of hepatocytes with SII Ang II increased insulin‐stimulated glycogen synthesis, while Ang II and the AT1R antagonist losartan had no effect. Insulin‐stimulated suppression of hepatic glucose output was not affected by Ang II or SII Ang II. It is well known that insulin regulates glycogen synthesis and glucose output through Akt‐mediated phosphorylation of glycogen synthase kinase α/β (GSK3α/β) and forkhead box protein O1 (FOXO1), respectively. In line with this, we show that SII Ang II potentiated insulin‐stimulated phosphorylation of Akt and GSK3α/β, but not FOXO1. Furthermore, we demonstrate that the effect of SII Ang II on insulin‐stimulated signalling and glycogen synthesis was dependent on Src and Gαq, as inhibitors of these proteins abolished the potentiating effect of SII Ang II. Thus, our results demonstrate that SII Ang II may have a positive effect on IR signalling and glucose metabolism in hepatocytes.