RESUMEN
There are seven relaxin family peptides that are all structurally related to insulin. Relaxin has many roles in female and male reproduction, as a neuropeptide in the central nervous system, as a vasodilator and cardiac stimulant in the cardiovascular system, and as an antifibrotic agent. Insulin-like peptide-3 (INSL3) has clearly defined specialist roles in male and female reproduction, relaxin-3 is primarily a neuropeptide involved in stress and metabolic control, and INSL5 is widely distributed particularly in the gastrointestinal tract. Although they are structurally related to insulin, the relaxin family peptides produce their physiological effects by activating a group of four G protein-coupled receptors (GPCRs), relaxin family peptide receptors 1-4 (RXFP1-4). Relaxin and INSL3 are the cognate ligands for RXFP1 and RXFP2, respectively, that are leucine-rich repeat containing GPCRs. RXFP1 activates a wide spectrum of signaling pathways to generate second messengers that include cAMP and nitric oxide, whereas RXFP2 activates a subset of these pathways. Relaxin-3 and INSL5 are the cognate ligands for RXFP3 and RXFP4 that are closely related to small peptide receptors that when activated inhibit cAMP production and activate MAP kinases. Although there are still many unanswered questions regarding the mode of action of relaxin family peptides, it is clear that they have important physiological roles that could be exploited for therapeutic benefit.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Relaxina/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Regulación de la Expresión Génica , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/química , Receptores de Péptidos/efectos de los fármacos , Receptores de Péptidos/genética , Relaxina/química , Relaxina/genética , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
To study the specific actions of relaxin through RXFP1 in human cells, it would be advantageous to develop cell populations with permanent RXFP1 knockdown (KD). We have developed and assessed four microRNA against human RXFP1. One of the four designed microRNA displayed significant RXFP1 KD as assessed by reduced relaxin binding when co-transfected with human RXFP1 into HEK-293T cells. The selected microRNA sequence was subsequently retrovirally delivered into the human dermal fibroblast cell line BJ3 which natively expresses RXFP1. The RXFP1 KD BJ3 cells displayed diminished RXFP1 mRNA expression and complete loss of ability of relaxin treatment to reduce collagen deposition after TGF-beta1 stimulation. The retroviral expression of miRNA to successfully silence RXFP1 expression is an invaluable tool to investigate receptor specificity, signalling and possible off-target effects of newly developed relaxin analogs.
Asunto(s)
MicroARNs/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/genética , Retroviridae/genética , Fibroblastos/citología , Fibroblastos/fisiología , Técnicas de Silenciamiento del Gen , Vectores Genéticos/genética , Células HEK293 , HumanosRESUMEN
Relaxin-3 is a neuropeptide that is abundantly expressed by discrete brainstem neuron populations that broadly innervate forebrain areas rich in the relaxin-3 G-protein-coupled-receptor, RXFP3. Acute and subchronic central administration of synthetic relaxin-3 or an RXFP3-selective agonist peptide, R3/I5, increase feeding and body weight in rats. Intrahypothalamic injection of relaxin-3 also increases feeding. In this study, we developed a recombinant adeno-associated virus 1/2 (rAAV1/2) vector that drives expression and constitutive secretion of bioactive R3/I5 and assessed the effect of intrahypothalamic injections on daily food intake and body weight gain in adult male rats over 8 weeks. In vitro testing revealed that the vector rAAV1/2-fibronectin (FIB)-R3/I5 directs the constitutive secretion of bioactive R3/I5 peptide. Bilateral injection of rAAV1/2-FIB-R3/I5 vector into the paraventricular nucleus produced an increase in daily food intake and body weight gain (P<0.01, ~23%, respectively), relative to control treatment. In a separate cohort of rats, quantitative polymerase chain reaction analysis of hypothalamic mRNA revealed strong expression of R3/I5 transgene at 3 months post-rAAV1/2-FIB-R3/I5 infusion. Levels of mRNA transcripts for the relaxin-3 receptor RXFP3, the hypothalamic 'feeding' peptides neuropeptide Y, AgRP and POMC, and the reproductive hormone, GnRH, were all similar to control, whereas vasopressin and oxytocin (OT) mRNA levels were reduced by ~25% (P=0.051) and ~50% (P<0.005), respectively, in rAAV1/2-FIB-R3/I5-treated rats (at 12 weeks, n=9/8 rats per group). These data demonstrate for the first time that R3/I5 is effective in modulating feeding in the rat by chronic hypothalamic RXFP3 activation and suggest a potential underlying mechanism involving altered OT signalling. Importantly, there was no desensitization of the feeding response over the treatment period and no apparent deleterious health effects, indicating that targeting the relaxin-3-RXFP3 system may be an effective long-term therapy for eating disorders.
Asunto(s)
Dependovirus/genética , Trastornos de Alimentación y de la Ingestión de Alimentos/genética , Trastornos de Alimentación y de la Ingestión de Alimentos/terapia , Proteínas del Tejido Nervioso/genética , Péptidos/administración & dosificación , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Relaxina/genética , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/genética , Modelos Animales de Enfermedad , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/genética , Conducta Alimentaria , Fibronectinas/genética , Fibronectinas/metabolismo , Células HEK293 , Humanos , Hipotálamo/metabolismo , Masculino , Proteínas del Tejido Nervioso/administración & dosificación , Proteínas del Tejido Nervioso/agonistas , Oxitocina/metabolismo , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/genética , Relaxina/administración & dosificación , Relaxina/agonistasRESUMEN
Since its discovery in the 1920s, relaxin has enjoyed a reputation as a peptide hormone of pregnancy. However, relaxin and other relaxin family peptides are now associated with numerous non-reproductive physiologies and disease states. The new millennium bought with it the sequence of the human genome and subsequently new directions for relaxin research. In 2002, the ancestral relaxin gene RLN3 was identified from genome databases. The relaxin-3 peptide is highly expressed in a small region of the brain and in species from teleost to primates and has both conserved sequence and sites of expression. Combined with the discovery of the relaxin family peptide receptors, interest in the role of the relaxin family peptides in the central nervous system has been reignited. This review explores the relaxin family peptides that are expressed in or act upon the brain, the receptors that mediate their actions, and what is currently known of their functions.