ABSTRACT
We have evaluated the effectiveness of systemic adenovirally delivered mouse relaxin on reversing fibrosis in a transgenic murine model of fibrotic cardiomyopathy due to beta(2)-adrenergic receptor (beta(2)AR) overexpression. Recombinant adenoviruses expressing green fluorescent protein (Ad-GFP), rat relaxin (Ad-rRLN) and mouse relaxin (Ad-mRLN) were generated and Ad-rRLN and Ad-mRLN were demonstrated to direct the expression of bioactive relaxin peptides in vitro. A single systemic injection of Ad-mRLN resulted in transgene expression in the liver and bioactive relaxin peptide in the plasma. Ad-mRLN, but not Ad-GFP, treatment reversed the increased left ventricular collagen content in beta(2)AR mice to control levels without affecting collagen levels in other heart chambers or in the lung and kidney. Hence a single systemic injection of adenovirus producing mouse relaxin reverses cardiac fibrosis without adversely affecting normal collagen levels in other organs and establishes the potential for the use of relaxin gene therapy for the treatment of cardiac fibrosis.
Subject(s)
Adenoviridae/genetics , Cardiomyopathies/therapy , Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors , Relaxin/metabolism , Animals , Cardiomyopathies/genetics , Cardiomyopathies/metabolism , Collagen/metabolism , Disease Models, Animal , Feasibility Studies , Female , Fibrosis , Heart Ventricles/metabolism , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Rats , Receptors, Adrenergic, beta-2/genetics , Receptors, Adrenergic, beta-2/metabolism , Relaxin/blood , Relaxin/geneticsABSTRACT
Rodent models have been used for many years to probe the actions of relaxin. Identification of the orthologs of human leucine-rich repeat-containing g-protein-coupled receptor 7 (LGR7), the relaxin receptor, in mouse and rat will enable characterization of the response of LGR7 to relaxin in these species. Partial LGR7 homologous sequences from mouse and rat were discovered in the Celera and NCBI gene databases, amplified, cloned, and sequenced. At the protein level, mouse and rat LGR7 are 85.2% and 85.7% identical to human LGR7. Mouse and rat LGR7 were able to bind to and be activated by relaxin ligands.
Subject(s)
Receptors, G-Protein-Coupled/metabolism , Receptors, Peptide/metabolism , Animals , Cell Line , Cloning, Molecular , Female , Humans , Mice , Pregnancy , RNA, Messenger/genetics , Rats , Receptors, G-Protein-Coupled/genetics , Receptors, Peptide/genetics , Relaxin/metabolismABSTRACT
Human LGR8, initially discovered as a low-affinity relaxin receptor, has now been characterized as the INSL3 receptor. To investigate LGR8 function in the rat, an LGR8 ortholog was identified in the rat genome, and the full-length sequence was cloned and expressed. Rat LGR8 bound INSL3 with high affinity, clearly demonstrating that it is the rat INSL3 receptor. Interestingly, native rat relaxin did not activate rat LGR8, indicating that relaxin is not an endogenous ligand for rat LGR8. LGR8 mRNA expression was demonstrated in the gubernaculum at the time of testis descent and in the testis associated with germ cells.
Subject(s)
Insulin/metabolism , Proteins/metabolism , Receptors, Peptide/metabolism , Animals , Cloning, Molecular , In Situ Hybridization , Ligands , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Receptors, G-Protein-Coupled , Receptors, Peptide/genetics , Relaxin/metabolism , Testis/metabolismABSTRACT
1. Relaxin is an extracellular matrix (ECM)-remodelling hormone that is functionally important in reproductive tissues, brain, lung and heart. 2. Recently, the human relaxin receptor was identified as leucine-rich repeat-containing G-protein-coupled receptor 7 (LGR7). 3. Using human LGR7 as a template, we identified mouse and rat LGR7 orthologues in the Celera and National Centre for Biotechnology Information databases. 4. At the protein level, mouse and rat LGR7 share 85.2 and 85.7% identity with human LGR7, respectively. 5. Mouse LGR7 mRNA was detected in all tissues where relaxin binding is observed. 6. Mouse and rat LGR7 bound [33P]-relaxin with high affinity and, upon relaxin treatment, both receptors stimulated cAMP production in transfected HEK 293T cells. 7. These results indicate that mouse and rat LGR7 are the relaxin receptors in these species. 8. The actions of relaxin in rodents are well characterized, providing an established platform for research into the molecular pharmacology of the highly conserved relaxin receptor.
Subject(s)
Membrane Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Peptide/metabolism , Amino Acid Sequence , Animals , Blotting, Northern , Computational Biology , Cyclic AMP/biosynthesis , Humans , Ligands , Membrane Proteins/drug effects , Membrane Proteins/genetics , Mice , RNA, Messenger/biosynthesis , RNA, Messenger/chemistry , RNA, Messenger/genetics , Rats , Receptors, G-Protein-Coupled/drug effects , Receptors, G-Protein-Coupled/genetics , Receptors, Peptide/drug effects , Receptors, Peptide/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Species Specificity , Tissue DistributionABSTRACT
Biotin-avidin immobilization has been routinely used as a tool to study peptide-receptor and peptide-antibody interactions. Biotinylated peptides can also be employed to localize cells that express the peptides' receptor, and to analyse ligand-receptor binding. Insulin-like peptide 3 (INSL3) is a peptide hormone which contains A- and B-chains connected by two disulphide bonds and plays a role in testicular descent during sexual development. In order to study the interaction of INSL3 with its receptor LGR8, a G protein-coupled receptor, we chemically synthesized Nalpha-mono-biotinylated human INSL3 (B-hINSL3) and compared it structurally and biologically with hINSL3. Both peptides exhibited similar, but high, receptor binding affinities on human foetal kidney fibroblast 293T cells transfected human LGR8 based on a competition radioreceptor assay with 33P-labelled relaxin H2 (B33). The modified B-hINSL3 showed full biological activity as determined by the stimulation of gubernacular cell proliferation. The labelled B-hINSL3 contains a higher alpha-helix content, and this increased helical structure is accompanied by an increase in ability to stimulate cAMP accumulation in 293T cells expressing LGR8. Our results suggest that the N-terminal region of the A-chain is not involved in the interaction of INSL3 with its receptor. However, the introduction of biotin onto the N-terminus of the A-chain promoted conformational stability which, in turn, permitted better receptor activation.
