Novel renoprotective actions of erythropoietin: New uses for an old hormone

Erythropoietin (EPO) has been used widely for the treatment of anaemia associated with chronic kidney disease and cancer chemotherapy for nearly 20 years. More recently, EPO has been found to interact with its receptor (EPO-R) expressed in a large variety of non-haematopoietic tissues to induce a range of cytoprotective cellular responses, including mitogenesis, angiogenesis, inhibition of apoptosis and promotion of vascular repair through mobilization of endothelial progenitor cells from the bone marrow. Administration of EPO or its analogue, darbepoetin, promotes impressive renoprotection in experimental ischaemic and toxic acute renal failure, as evidenced by suppressed tubular epithelial apoptosis, enhanced tubular epithelial proliferation and hastened functional recovery. This effect is still apparent when administration is delayed up to 6 h after the onset of injury and can be dissociated from its haematological effects. Based on these highly encouraging results, at least one large randomized controlled trial of EPO therapy in ischaemic acute renal failure is currently underway. Preliminary experimental and clinical evidence also indicates that EPO may be renoprotective in chronic kidney disease. The purpose of the present article is to review the renoprotective benefits of different protocols of EPO therapy in the settings of acute and chronic kidney failure and the potential mechanisms underpinning these renoprotective actions. Gaining further insight into the pleiotropic actions of EPO will hopefully eventuate in much-needed, novel therapeutic strategies for patients with kidney disease.

New insights into growth hormone action

It has been 75 years since Evans and Long identified a somatic growth-promoting substance in pituitary extracts, yet it is only in the last 20 years that the molecular basis for this action has been established. Three key elements in this elucidation were the cloning of the GH receptor, the identification of Janus kinase (JAK) 2 as the receptor-associated tyrosine kinase, and the delineation of signal transduction and activators of transcription (STAT) 5a/b as the key transcription factor(s) activated by JAK2. The interaction between these three elements results in enhanced postnatal growth and is the subject of this review. We describe a new model for GH receptor activation based on subunit rotation within a constitutive dimer, together with the phenotype and hepatic transcript profile of mice with targeted knockins to the receptor cytoplasmic domain. These support a central role for STAT5a/b in postnatal growth.

Receptor Activity Modifying Proteins (RAMPs) interact with the VPAC2 Receptor and CRF1 receptors and modulate their function

Background and Purpose Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon-like peptide 1 receptor (GLP-1 receptor), the human vasoactive intestinal polypeptide/pituitary AC-Activating peptide 2 receptor (VPAC) and the type 1 corticotrophin releasing factor receptor (CRF) has been examined. Experimental Approach GPCRs were co-transfected with RAMPs in HEK 293S and CHO-K1 cells. Cell surface expression of RAMPs and GPCRs was examined by elisa. Where there was evidence for interactions, agonist-stimulated cAMP production, Ca mobilization and GTPγS binding to G, G, G and G were examined. The ability of CRF to stimulate adrenal corticotrophic hormone release in Ramp2 mice was assessed. Key Results The GLP-1 receptor failed to enhance the cell surface expression of any RAMP. VPAC enhanced the cell surface expression of all three RAMPs. CRF enhanced the cell surface expression of RAMP2; the cell surface expression of CRF was also increased. There was no effect on agonist-stimulated cAMP production. However, there was enhanced G-protein coupling in a receptor and agonist-dependent manner. The CRF: RAMP2 complex resulted in enhanced elevation of intracellular calcium to CRF and urocortin 1 but not sauvagine. In Ramp2 mice, there was a loss of responsiveness to CRF. Conclusions and Implications The VPAC and CRF receptors interact with RAMPs. This modulates G-protein coupling in an agonist-specific manner. For CRF, coupling to RAMP2 may be of physiological significance. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Acute and long-term effects of peroxisome proliferator-activated receptor-γ activation on the function and insulin secretory responsiveness of clonal beta-cells

Thiazolidinediones (TZDs) are used as antidiabetic therapy. The purpose of the present study was to examine whether the TZD rosiglitazone has direct actions on pancreatic beta-cells that contribute to its overall effects. Effects of acute and prolonged (48 h) exposure to rosiglitazone, as a model glitazone compound, were assessed in clonal pancreatic BRIN-BD11 beta-cells maintained in standard, glucotoxic and lipotoxic cultures. In acute 20-min incubations, rosiglitazone (0.2-100 M) did not alter basal or glucose-stimulated insulin secretion. However, rosiglitazone (6.25 M) enhanced (p

Cholecystokinin (CCK) receptor and CCK gene expression in human pituitary adenomas and in vitro effects of CCK peptides on GH and gonadotrophin secretion

There is growing evidence that cholecystokinin (CCK) affects growth and differentiation of anterior pituitary cells, via the CCK-B receptor. The possibility of an autocrine / paracrine role for CCK to modulate hormone secretion in human pituitary tumour cells is demonstrated here by RT-PCR and direct sequencing. In support of this conclusion, a neutralising antibody against the CCK peptide exhibited a dose dependent inhibition of hormone secretion by functionless pituitary adenomas. Total RNA was extracted from human pituitary adenomas, reverse transcribed into cDNA and subjected to PCR using primers specific for the gene for CCK, CCK-A and CCK-B receptors. PCR bands of the predicted length were observed in all tumours using human CCK gene and CCK-B receptor primers. Restriction digestion and direct sequence analysis provided further evidence that they represented both the human CCK peptide along with the CCK-A and/B receptor mRNA. CCK-33 and CCK octapeptide sulphate (CCK-8s) both powerfully stimulated phosphatidylinositol hydrolysis, providing evidence for functional activity of the CCK-A and/B receptors. A direct stimulatory effect of CCK peptides on both LH and FSH secretion is reported for the first time, whereas stimulatory effects on GH were blocked by antagonists to CCK. These results may indicate an autocrine role for CCK in the functioning and perhaps development of human pituitary tumours. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart.

Degradation, receptor binding, insulin secreting and antihyperglycaemic actions of palmitate-derivatised native and Ala8-substituted GLP-1 analogues

The hormone glucagon-like peptide-1(7-36)amide (GLP-1) is released in response to ingested nutrients and acts to promote glucose-dependent insulin secretion ensuring efficient postprandial glucose homeostasis. Unfortunately, the beneficial actions of GLP-1 which give this hormone many of the desirable properties of an antidiabetic drug are short lived due to degradation by dipeptidylpeptidase IV (DPP IV) and rapid clearance by renal filtration. In this study we have attempted to extend GLP-1 action through the attachment of palmitoyl moieties to the E-amino group in the side chain of the LyS26 residue and to combine this modification with substitutions of the Ala 8 residue, namely Val or amino-butyric acid (Abu). In contrast to native GLP-1, which was rapidly degraded, [Lys(pal) 26]GLP-1, [Abu8,Lys(pal)26]GLP-1 and [Val8,Lys-(pal)26]GLP-1 all exhibited profound stability during 12 h incubations with DPP IV and human plasma. Receptor binding affinity and the ability to increase cyclic AMP in the clonal β-cell line BRIN-BD11 were decreased by 86- to 167-fold and 15- to 62-fold, respectively compared with native GLP-1. However, insulin secretory potency tested using BRIN-BD11 cells was similar, or in the case of [Val8,Lys(pal)26]GLP-1 enhanced. Furthermore, when administered in vivo together with glucose to diabetic (ob/ob) mice, [Lys(pal)26]GLP-1, [Abu8,Lys(pal) 26]GLP-1 and [Val8,Lys(pal) 26]GLP-1 did not demonstrate acute glucose-lowering or insulinotropic activity as observed with native GLP-1. These studies support the potential usefulness of fatty acid linked analogues of GLP-1 but indicate the importance of chain length for peptide kinetics and bioavailability. Copyright © by Walter de Gruyter.

Identifying key residues important for CGRP binding to its receptor

The calcitonin gene related peptide (CGRP) is a 37 amino acid neuropeptide. Its receptor is a heterodimeric complex of calcitonin receptor-like receptor (CLR) – a family B G-protein coupled receptor – and a single-pass transmembrane protein, receptoractivity modifying protein 1 (RAMP1). Here, we identify residues, within the N-terminal extracellular domain (ECD) of CLR, potentially involved in ligand binding.Certain residues presumed to be possible sites of contact for the CGRP were picked from the CLR/RAMP1 ECD crystal structure (PDB 3N7S). Residues were mutated to alanine (A) bysite-directed mutagenesis (QuikChangeTM, Stratagene). Mutants were analysed for their ability to stimulate cAMP and cell surface expression as previously described [1]. All mutants showed reduced potency, though to varying degrees as indicated by their pEC50 values. W69A and D70Ashowed significant reduction in cell surface expression.These findings suggest that these residues are important for the interaction of CGRP with its receptor. W69A and D70A, part of the WDG motif of family B GPCRs, are thought to rather play a role in receptor stability [2]. The data is consistent with CGRP binding in agroove between CLR and RAMP1. This project was supported byAston School of Life and Health Sciences.References1. Barwell J, Conner A & Poyner D (2011) Extracellular loops 1and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function. Biochim Biophys Acta 1813, 1906–1916.2. Kumar S, Pioszak A, Zhang C et al. (2011) Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Cou-pled Receptors. PLoS One 6, e19682

Receptor activity-modifying proteins 2 and 3 generate adrenomedullin receptor subtypes with distinct molecular properties

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins (RAMP) 2 and 3, respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMPs 2 and 3 on the activation and conformation of the CLR subunit of AM receptors we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors and determined the effects on cAMP signalling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modelling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket, and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.

Le glucagon-like peptide-I : un facteur de croissance et une hormone anti-apoptotique pour la cellule pancréatique[bêta] : étude de la transduction du signal

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Quantifying Eukaryotic Gene Regulation in Hormone Response and Disease.

Quantifying the function of mammalian enhancers at the genome or population scale has been longstanding challenge in the field of gene regulation. Studies of individual enhancers have provided anecdotal evidence on which many foundational assumptions in the field are based. Genome-scale studies have revealed that the number of sites bound by a given transcription factor far outnumber the genes that the factor regulates. In this dissertation we describe a new method, chromatin immune-enriched reporter assays (ChIP-reporters), and use that approach to comprehensively test the enhancer activity of genomic loci bound by the glucocorticoid receptor (GR). Integrative genomics analyses of our ChIP-reporter data revealed an unexpected mechanism of glucocorticoid (GC)-induced gene regulation. In that mechanism, only the minority of GR bound sites acts as GC-inducible enhancers. Many non-GC-inducible GR binding sites interact with GC-induced sites via chromatin looping. These interactions can increase the activity of GC-induced enhancers. Finally, we describe a method that enables the detection and characterization of the functional effects of non-coding genetic variation on enhancer activity at the population scale. Taken together, these studies yield both mechanistic and genetic evidence that provides context that informs the understanding of the effects of multiple enhancer variants on gene expression.

JNK regulates Fas receptor mediated apoptosis in prostate cancer cell lines

Prostate Cancer is a disease that primarily affects elderly men. The incidence of prostate cancer has been progressively increasing in the western world over the last two decades. Life expectancy and diet are believed to be the main factors contributing to this increase in prevalence. Prostate cancer is a slowly progressing disorder and patients often live for over 10 years after initially being diagnosed with prostate cancer. However, patients with hormone refractory prostate cancer have a poor prognosis and generally do not survive for longer than 2 or 3 years. Hormone refractory prostate cancer is responsible for over 200,000 deaths each year and current chemotherapeutic regimens are only useful as palliative agents. The long-term survival rate is poor and chemotherapy does not significantly increase this. Cell lines derived from hormone refractory tumours usually display elevated resistance to many cytotoxic drugs. The Fas receptor is a membrane bound protein capable of binding to a ligand called Fas ligand. Engagement of Fas receptor with Fas ligand results in clustering of Fas receptor on the plasma membrane of cells. A number of proteins responsible for initiating apoptosis are recruited to the plasma membrane and are activated in response to elevated local concentrations. This series of events initiates a proteolysis cascade and that culminates in the degradation of structural and enzymatic processes and the repackaging of cellular constituents within membrane bound vesicles that can be endocytosed and recycled by surrounding phagocytic cells. The Fas receptor is believed to be a key mechanism by which immune cells can destroy damaged cells. Consequently, resistance to Fas receptor mediated apoptosis often correlates with tumour progression. It has been reported that prostate cancer cell lines display elevated resistance to Fas receptor mediated apoptosis and this correlates with the stage of tumour from which the cell lines were isolated. JNK, a stress-activated protein kinase, has been implicated both with increased survival and increased apoptosis in prostate cancer. Elevated endogenous JNK activity has been demonstrated to correlate with prostate cancer progression. It has been shown that endogenous JNK activity increases the expression of anti-apoptotic proteins and can increase the resistance of prostate cancer cell lines to chemotherapy. In addition, elevated endogenous JNK activity is required for improved proliferation and transformation of a number of epithelial tumours. However, prolonged JNK activation in response to cytotoxic stimuli can increase the sensitivity of cells to apoptosis. Prolonged JNK activity appears to induce the expression of a separate set of genes responsible for promoting apoptosis. Our group has recently shown that activation of JNK by chemotherapeutic drugs can sensitise DU 145 prostate carcinoma cells to Fas receptor mediated apoptosis. In order toidentify novel targets for treating hormone refractory prostate cancer we have investigated the role of JNK in Fas receptor mediated apoptosis. We have demonstrated that prolonged JNK activation is defective in DU 145 cells in response to Fas receptor activation alone. Co-administering anisomycin, a JNK agonist, greatly enhances the ability of DU 145 cells to undergo apoptosis by increasing the rate of Caspase 8 cleavage. We also investigated the role of endogenous JNK activity in Fas receptor mediated.

Le glucagon-like peptide-I : un facteur de croissance et une hormone anti-apoptotique pour la cellule pancréatique[bêta] : étude de la transduction du signal

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Attenuation of monocyte chemotaxis--a novel anti-inflammatory mechanism of action for the cardio-protective hormone B-type natriuretic peptide

B-type natriuretic peptide (BNP) is a prognostic and diagnostic marker for heart failure (HF). An anti-inflammatory, cardio-protective role for BNP was proposed. In cardiovascular diseases including pressure overload-induced HF, perivascular inflammation and cardiac fibrosis are, in part, mediated by monocyte chemoattractant protein (MCP)1-driven monocyte migration. We aimed to determine the role of BNP in monocyte motility to MCP1. A functional BNP receptor, natriuretic peptide receptor-A (NPRA) was identified in human monocytes. BNP treatment inhibited MCP1-induced THP1 (monocytic leukemia cells) and primary monocyte chemotaxis (70 and 50 %, respectively). BNP did not interfere with MCP1 receptor expression or with calcium. BNP inhibited activation of the cytoskeletal protein RhoA in MCP1-stimulated THP1 (70 %). Finally, BNP failed to inhibit MCP1-directed motility of monocytes from patients with hypertension (n = 10) and HF (n = 6) suggesting attenuation of this anti-inflammatory mechanism in chronic heart disease. We provide novel evidence for a direct role of BNP/NPRA in opposing human monocyte migration and support a role for BNP as a cardio-protective hormone up-regulated as part of an adaptive compensatory response to combat excess inflammation.

Gut hormone GPCRs: structure, function, drug discovery

Crystallization and determination of the high resolution three-dimensional structure of the β2-adrenergic receptor in 2007 was followed by structure elucidation of a number of other receptors, including those for neurotensin and glucagon. These major advances foster the understanding of structure-activity relationship of these receptors and structure-based rational design of new ligands having more predictable activity. At present, structure determination of gut hormone receptors in complex with their ligands (natural, synthetic) and interacting signalling proteins, for example, G-proteins, arrestins, represents a challenge which promises to revolutionize gut hormone endocrinonology.

Phocid seal leptin: tertiary structure and hydrophobic receptor binding site preservation during distinct leptin gene evolution

The cytokine hormone leptin is a key signalling molecule in many pathways that control physiological functions. Although leptin demonstrates structural conservation in mammals, there is evidence of positive selection in primates, lagomorphs and chiropterans. We previously reported that the leptin genes of the grey and harbour seals (phocids) have significantly diverged from other mammals. Therefore we further investigated the diversification of leptin in phocids, other marine mammals and terrestrial taxa by sequencing the leptin genes of representative species. Phylogenetic reconstruction revealed that leptin diversification was pronounced within the phocid seals with a high dN/dS ratio of 2.8, indicating positive selection. We found significant evidence of positive selection along the branch leading to the phocids, within the phocid clade, but not over the dataset as a whole. Structural predictions indicate that the individual residues under selection are away from the leptin receptor (LEPR) binding site. Predictions of the surface electrostatic potential indicate that phocid seal leptin is notably different to other mammalian leptins, including the otariids. Cloning the grey seal leptin binding domain of LEPR confirmed that this was structurally conserved. These data, viewed in toto, support a hypothesis that phocid leptin divergence is unlikely to have arisen by random mutation. Based upon these phylogenetic and structural assessments, and considering the comparative physiology and varying life histories among species, we postulate that the unique phocid diving behaviour has produced this selection pressure. The Phocidae includes some of the deepest diving species, yet have the least modified lung structure to cope with pressure and volume changes experienced at depth. Therefore, greater surfactant production is required to facilitate rapid lung re-inflation upon surfacing, while maintaining patent airways. We suggest that this additional surfactant requirement is met by the leptin pulmonary surfactant production pathway which normally appears only to function in the mammalian foetus.