RESUMO
The peptide hormone amylin reduces food intake and body weight and is an attractive candidate target for novel pharmacotherapies to treat obesity. However, the short half-life of native amylin and amylin analogs like pramlintide limits these compounds' potential utility in promoting sustained negative energy balance. Here, we evaluate the ability of the novel long-acting amylin/calcitonin receptor agonist ZP5461 to reduce feeding and body weight in rats, and also test the role of calcitonin receptors (CTRs) in the dorsal vagal complex (DVC) of the hindbrain in the energy balance effects of chronic ZP5461 administration. Acute dose-response studies indicate that systemic ZP5461 (0.5-3 nmol/kg) robustly suppresses energy intake and body weight gain in chow- and high-fat diet (HFD)-fed rats. When HFD-fed rats received chronic systemic administration of ZP5461 (1-2 nmol/kg), the compound initially produced reductions in energy intake and weight gain but failed to produce sustained suppression of intake and body weight. Using virally mediated knockdown of DVC CTRs, the ability of chronic systemic ZP5461 to promote early reductions in intake and body weight gain was determined to be mediated in part by activation of DVC CTRs, implicating the DVC as a central site of action for ZP5461. Future studies should address other dosing regimens of ZP5461 to determine whether an alternative dose/frequency of administration would produce more sustained body weight suppression.
Assuntos
Agonistas dos Receptores da Amilina/farmacologia , Depressores do Apetite/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Receptores da Calcitonina/agonistas , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/efeitos dos fármacos , Rombencéfalo/efeitos dos fármacos , Nervo Vago/efeitos dos fármacos , Aumento de Peso/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Ingestão de Energia/efeitos dos fármacos , Masculino , Ratos Sprague-Dawley , Receptores da Calcitonina/genética , Receptores da Calcitonina/metabolismo , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/genética , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismo , Rombencéfalo/metabolismo , Transdução de Sinais , Fatores de Tempo , Nervo Vago/metabolismoRESUMO
Based upon its interactions with amyloid ß peptide (Aß), the amylin receptor, a class B G protein-coupled receptor (GPCR), is a potential modulator of Alzheimer's disease (AD) pathogenesis. However, past pharmacological approaches have failed to resolve whether activation or blockade of this receptor would have greater therapeutic benefit. To address this issue, we generated compound mice expressing a human amyloid precursor protein gene with familial AD mutations in combination with deficiency of amylin receptors produced by hemizygosity for the critical calcitonin receptor subunit of this heterodimeric GPCR. These compound transgenic AD mice demonstrated attenuated responses to human amylin- and Aß-induced depression of hippocampal long-term potentiation (LTP) in keeping with the genetic depletion of amylin receptors. Both the LTP responses and spatial memory (as measured with Morris water maze) in these mice were improved compared to AD mouse controls and, importantly, a reduction in both the amyloid plaque burden and markers of neuroinflammation was observed. Our data support the notion of further development of antagonists of the amylin receptor as AD-modifying therapies.
Assuntos
Doença de Alzheimer/genética , Aprendizagem em Labirinto/fisiologia , Receptores da Calcitonina/genética , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/genética , Memória Espacial/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/psicologia , Precursor de Proteína beta-Amiloide/genética , Animais , Células Cultivadas , Endotélio Vascular/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Receptores da Calcitonina/deficiência , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/deficiênciaRESUMO
Amylin, a pancreatic hormone and neuropeptide, acts principally in the hindbrain to decrease food intake and has recently been shown to act as a neurotrophic factor to control the development of area postrema â nucleus of the solitary tract and arcuate hypothalamic nucleus â paraventricular nucleus axonal fiber outgrowth. Amylin is also able to activate ERK signaling specifically in POMC neurons independently of leptin. For investigation of the physiological role of amylin signaling in POMC neurons, the core component of the amylin receptor, calcitonin receptor (CTR), was depleted from POMC neurons using an inducible mouse model. The loss of CTR in POMC neurons leads to increased body weight gain, increased adiposity, and glucose intolerance in male knockout mice, characterized by decreased energy expenditure (EE) and decreased expression of uncoupling protein 1 (UCP1) in brown adipose tissue. Furthermore, a decreased spontaneous locomotor activity and absent thermogenic reaction to the application of the amylin receptor agonist were observed in male and female mice. Together, these results show a significant physiological impact of amylin/calcitonin signaling in CTR-POMC neurons on energy metabolism and demonstrate the need for sex-specific approaches in obesity research and potentially treatment.
Assuntos
Metabolismo Energético/fisiologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Neurônios/fisiologia , Pró-Opiomelanocortina/metabolismo , Receptores da Calcitonina/metabolismo , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismo , Tecido Adiposo Marrom , Animais , Masculino , Camundongos , Atividade Motora , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/genética , Transdução de Sinais/fisiologia , alfa-MSH/genética , alfa-MSH/metabolismoRESUMO
Alzheimer'sdisease (AD) is a progressive neurodegenerative disorder, characterized by senile plaques constituting extracellular deposits of ß-amyloid (Aß) fibrils. Since Aß accumulation in the brain is considered an early event preceding, by decades, cognitive dysfunction, disease-modifying treatments are aimed at facilitating clearance of this protein from the brain or ameliorating its toxic effects. Recent studies have identified the amylin receptor as a capable mediator of the deleterious actions of Aß and furthermore, administration of amylin receptor-based peptides has been shown to improve spatial memory and learning in transgenic mouse models of AD. Here, by discussing available evidence, we posit that the amylin receptor could be considered a potential therapeutic target for AD, and present the rationale for using amylin receptor antagonists to treat this debilitating condition.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos/uso terapêutico , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/antagonistas & inibidores , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Camundongos , Camundongos Transgênicos , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/genéticaRESUMO
BACKGROUND: The pancreatic- and brain-derived hormone amylin promotes negative energy balance and is receiving increasing attention as a promising obesity therapeutic. However, the neurobiological substrates mediating amylin's effects are not fully characterized. We postulated that amylin acts in the lateral dorsal tegmental nucleus (LDTg), an understudied neural processing hub for reward and homeostatic feeding signals. METHODS: We used immunohistochemical and quantitative polymerase chain reaction analyses to examine expression of the amylin receptor complex in rat LDTg tissue. Behavioral experiments were performed to examine the mechanisms underlying the hypophagic effects of amylin receptor activation in the LDTg. RESULTS: Immunohistochemical and quantitative polymerase chain reaction analyses show expression of the amylin receptor complex in the LDTg. Activation of LDTg amylin receptors by the agonist salmon calcitonin dose-dependently reduces body weight, food intake, and motivated feeding behaviors. Acute pharmacological studies and longer-term adeno-associated viral knockdown experiments indicate that LDTg amylin receptor signaling is physiologically and potentially preclinically relevant for energy balance control. Finally, immunohistochemical data indicate that LDTg amylin receptors are expressed on gamma-aminobutyric acidergic neurons, and behavioral results suggest that local gamma-aminobutyric acid receptor signaling mediates the hypophagia after LDTg amylin receptor activation. CONCLUSIONS: These findings identify the LDTg as a novel nucleus with therapeutic potential in mediating amylin's effects on energy balance through gamma-aminobutyric acid receptor signaling.
Assuntos
Agonistas dos Receptores da Amilina/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Transdução de Sinais/fisiologia , Área Tegmentar Ventral/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Calcitonina/farmacologia , Condicionamento Operante/efeitos dos fármacos , Condicionamento Operante/fisiologia , Ingestão de Alimentos/efeitos dos fármacos , Preferências Alimentares/efeitos dos fármacos , GABAérgicos/farmacologia , Masculino , Motivação/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fosfopiruvato Hidratase/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Modificadoras da Atividade de Receptores/genética , Proteínas Modificadoras da Atividade de Receptores/metabolismo , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/antagonistas & inibidores , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/genética , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Findings from Alzheimer's disease (AD) mouse models showed that amylin treatment improved AD pathology and enhanced amyloid-ß (Aß) brain to blood clearance; however, the mechanism was not investigated. Using the Tg2576 AD mouse model, a single intraperitoneal injection of amylin significantly increased Aß serum levels, and the effect was abolished by AC253, an amylin receptor antagonist, suggesting that amylin effect could be mediated by its receptor. Subsequent mechanistic studies showed amylin enhanced Aß transport across a cell-based model of the blood-brain barrier (BBB), an effect that was abolished when the amylin receptor was inhibited by two amylin antagonists and by siRNA knockdown of amylin receptor Ramp3. To explain this finding, amylin effect on Aß transport proteins expressed at the BBB was evaluated. Findings indicated that cells treated with amylin induced LRP1 expression, a major receptor involved in brain Aß efflux, in plasma membrane fraction, suggesting intracellular translocation of LRP1 from the cytoplasmic pool. Increased LRP1 in membrane fraction could explain, at least in part, the enhanced uptake and transport of Aß across the BBB. Collectively, our findings indicated that amylin induced Aß brain to blood clearance through amylin receptor by inducing LRP1 subcellular translocation to the plasma membrane of the BBB endothelium.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar/fisiologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Técnicas de Cultura de Células , Células Cultivadas , Fármacos do Sistema Nervoso Central/administração & dosagem , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos Transgênicos , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/antagonistas & inibidores , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/genética , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismo , Receptores de LDL/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity.
Assuntos
Peptídeos/metabolismo , Receptores da Calcitonina/metabolismo , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismo , Regulação Alostérica/fisiologia , Motivos de Aminoácidos , Células HEK293 , Humanos , Peptídeos/química , Peptídeos/genética , Estrutura Terciária de Proteína , Proteína 1 Modificadora da Atividade de Receptores/química , Proteína 1 Modificadora da Atividade de Receptores/genética , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Proteína 2 Modificadora da Atividade de Receptores/química , Proteína 2 Modificadora da Atividade de Receptores/genética , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Receptores da Calcitonina/química , Receptores da Calcitonina/genética , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/química , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/genéticaRESUMO
Amylin is a pancreatic ß-cell hormone that acts as a satiating signal to inhibit food intake by binding to amylin receptors (AMYs) and activating a specific neuronal population in the area postrema (AP). AMYs are heterodimers that include a calcitonin receptor (CTR) subunit [CTR isoform a or b (CTRa or CTRb)] and a member of the receptor activity-modifying proteins (RAMPs). Here, we used single-cell quantitative polymerase chain reaction to assess co-expression of AMY subunits in AP neurons from rats that were injected with amylin or vehicle. Because amylin interacts synergistically with the adipokine leptin to reduce body weight, we also assessed the co-expression of AMY and the leptin receptor isoform b (LepRb) in amylin-activated AP neurons. Single cells were collected from Wistar rats and from transgenic Fos-GFP rats that express green fluorescent protein (GFP) under the control of the Fos promoter. We found that the mRNAs of CTRa, RAMP1, RAMP2 and RAMP3 were all co-expressed in single AP neurons. Moreover, most of the CTRa+ cells co-expressed more than one of the RAMPs. Amylin down-regulated RAMP1 and RAMP3 but not CTR mRNAs in AMY+ neurons, suggesting a possible negative feedback mechanism of amylin at its own primary receptors. Interestingly, amylin up-regulated RAMP2 mRNA. We also found that a high percentage of single cells that co-expressed all components of a functional AMY expressed LepRb mRNA. Thus, single AP cells expressed both AMY and LepRb, which formed a population of first-order neurons that presumably can be directly activated by amylin and, at least in part, also by leptin.
Assuntos
Área Postrema/metabolismo , Neurônios/metabolismo , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismo , Receptores para Leptina/metabolismo , Animais , Área Postrema/citologia , Retroalimentação Fisiológica , Feminino , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Leptina/farmacologia , Masculino , Neurônios/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/genética , Receptores para Leptina/genéticaRESUMO
Amylin is co-localized and co-secreted with insulin, however its direct effects on adipocytes are unexplored. In 3T3-L1 preadipocytes, amylin increased thymidine incorporation (174%; p<0.05) and Myc mRNA expression (378%; p<0.01). Amylin supplementation during differentiation enhanced triglyceride accumulation (272%; p<0.001). In 3T3-L1 adipocytes, amylin increased fatty acid uptake (238%; p<0.01) and further potentiated the effects of insulin (insulin 158%; p<0.01, amylin+insulin 335%; p<0.001 vs CTL, p<0.001 vs insulin). By contrast, amylin inhibited glycerol release in 3T3-L1 adipocytes (-50%; p<0.05) and primary adipocytes (-34%; p<0.05). Amylin stimulated cytokine secretion (monocyte chemotactic protein-1+166%, keratinocyte-derived chemokine+174%; both p<0.05) and mRNA expression of PPARγ (163%; p<0.01), C/EBPß (121%, p<0.05), DGAT1 (157%; p<0.01), FABP4 (122%; p<0.01), and CD36 (122%; p<0.05). In human adipose tissue, mRNA expression of amylin receptor genes (CALCR and RAMP3) correlated with numerous lipid and insulin signaling genes, plasma glucose and HOMA. Altogether amylin directly stimulates fat cells, potentiates the effects of insulin and may influence insulin resistance.
Assuntos
Adipócitos/metabolismo , Ácidos Graxos/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Células 3T3-L1 , Adipocinas/genética , Adipocinas/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Esterificação/efeitos dos fármacos , Ácidos Graxos não Esterificados/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glicerol/metabolismo , Humanos , Insulina/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/genética , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismoRESUMO
Deposition of amyloid-beta (Aß) protein, a 39-43 amino acid peptide, in the brain is a major pathological feature of Alzheimer's disease (AD). We have previously provided evidence that in primary cultures of rat basal forebrain and human fetal neurons (HFNs), neurotoxic effects of oligomeric Aß are expressed through the amylin receptor. In this study, we utilized RT-PCR arrays to compare RNA expression levels of 84 markers for pro and anti- apoptotic signalling pathways following exposure of HFNs to either Aß(1-42) (20 µM) or human amylin (2 µM). Oligomeric Aß(1-42) or human amylin was applied to HFNs alone or after pre-treatment of cultures with the amylin receptor antagonist, AC253. Changes in RNA levels were then quantified and compared to each other in order to identify increases or decreases in gene expression of apoptotic markers. Applications of Aß(1-42) or human amylin, but not the inactive inverse sequence Aß(42-1) or rat amylin, resulted in a time-dependent marked increase in mediators of apoptosis including a 10- to 30-fold elevations in caspases 3, 6, 9, BID and XIAP levels. Amylin receptor antagonists, AC253 (10 µM) or AC187 (10 µM), significantly attenuated the induction of several pro-apoptotic mediators up-regulated following exposure to Aß(1-42) or human amylin and increased the expression of several anti-apoptotic markers. These data allow us to identify key elements in the Aß-induced apoptosis that are blocked by antagonism of the amylin receptor and further support the potential for amylin receptor blockade as a potential therapeutic avenue in AD.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas Reguladoras de Apoptose/genética , Apoptose , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Neurônios/citologia , Neurônios/metabolismo , Ratos , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/antagonistas & inibidores , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/genéticaRESUMO
Disruption of neurotoxic effects of amyloid ß protein (Aß) is one of the major, but as yet elusive, goals in the treatment of Alzheimer's disease (AD). The amylin receptor, activated by a pancreatic polypeptide isolated from diabetic patients, is a putative target for the actions of Aß in the brain. Here we show that in primary cultures of human fetal neurons (HFNs), AC253, an amylin receptor antagonist, blocks electrophysiological effects of Aß. Pharmacological blockade of the amylin receptor or its down-regulation using siRNA in HFNs confers neuroprotection against oligomeric Aß-induced caspase-dependent and caspase-independent apoptotic cell death. In transgenic mice (TgCRND8) that overexpress amyloid precursor protein, amylin receptor is up-regulated in specific brain regions that also demonstrate an elevated amyloid burden. The expression of Aß actions through the amylin receptor in human neurons and temporospatial interrelationship of Aß and the amylin receptor in an in vivo model of AD together provide a persuasive rationale for this receptor as a novel therapeutic target in the treatment of AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Apoptose , Neurônios/fisiologia , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/fisiologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/toxicidade , Animais , Encéfalo/metabolismo , Citoproteção , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/antagonistas & inibidores , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/genéticaRESUMO
Lysyl oxidase-like protein-2 (LOXL2) induces epithelial to mesenchymal transition and promotes invasiveness. To understand the mechanisms involved, we examined the effect of LOXL2 overexpression in MCF-7 cells on gene expression. We found that LOXL2 up-regulated the expression of receptor activity modifying protein-3 (RAMP3). Expression of RAMP3 in MDA-MB-231 cells in which LOXL2 expression was inhibited restored vimentin expression, invasiveness, and tumor development. Inhibition of RAMP3 expression in MDA-MB-231 cells mimicked the effects produced by inhibition of LOXL2 expression and was accompanied by inhibition of p38 phosphorylation. LOXL2 overexpression in these cells did not restore invasiveness, suggesting that RAMP3 functions downstream to LOXL2. LOXL2 and RAMP3 are strongly coexpressed in human colon, breast, and gastric carcinomas but not in normal colon or gastric epithelial cells. RAMP3 associates with several G-protein-coupled receptors forming receptors for peptides, such as adrenomedullin and amylin. We hypothesized that RAMP3 could function as a transducer of autocrine signals induced by such peptides. However, the proinvasive effects of RAMP3 could not be abrogated following inhibition of the expression or activity of these peptides. Our experiments suggest that the protumorigenic effects of LOXL2 are partially mediated by RAMP3 and that RAMP3 inhibitors may function as antitumorigenic agents. -