Cardiac fibrosis is a key component of heart disease and involves the proliferation and differentiation of matrix-producing fibroblasts. The effects of an antifibrotic peptide hormone, relaxin, in inhibiting this process were investigated. We used rat atrial and ventricular fibroblasts, which respond to profibrotic stimuli and express the relaxin receptor (LGR7), in addition to two in vivo models of cardiac fibrosis. Cardiac fibroblasts, when plated at low density or stimulated with TGF-beta or angiotensin II (Ang II), accelerated fibroblast differentiation into myofibroblasts, as demonstrated by significantly increased alpha-smooth muscle actin expression, collagen synthesis, and collagen deposition (by up to 95% with TGF-beta and 40% with Ang II; all P < 0.05). Fibroblast proliferation was significantly increased by 10(-8) m and 10(-7) m Ang II (63-75%; P < 0.01) or 0.1-1 microg/ml IGF-I (27-40%; P < 0.05). Relaxin alone had no marked effect on these parameters, but it significantly inhibited Ang II- and IGF-I-mediated fibroblast proliferation (by 15-50%) and Ang II- and TGF-beta-mediated fibroblast differentiation, as detected by decreased expression of alpha-smooth muscle actin (by 65-88%) and collagen (by 60-80%). Relaxin also increased matrix metalloproteinase-2 expression in the presence of TGF-beta (P < 0.01) and Ang II (P < 0.05). Furthermore, relaxin decreased collagen overexpression when administered to two models of established fibrotic cardiomyopathy, one due to relaxin deficiency (by 40%; P < 0.05) and the other to cardiac-restricted overexpression of beta2-adrenergic receptors (by 58%; P < 0.01). These coherent findings indicate that relaxin regulates fibroblast proliferation, differentiation, and collagen deposition and may have therapeutic potential in diseased states characterized by cardiac fibrosis.
Relaxin is a 6-kDa peptide of the insulin family that is present at increased levels in the circulation during pregnancy. Its functions at that time are thought to include maintenance of myometrial quiescence, regulation of plasma volume, and release of neuropeptides, such as oxytocin and vasopressin. The protein also promotes connective tissue remodeling, which allows cervical ripening and separation of the pelvic symphysis in various mammalian species. In this report, we provide evidence for a novel target of relaxin, the human monocytic cell line, THP-1. Relaxin bound with high affinity (K d ؍ 102 pM) to a specific receptor on THP-1 cells. Receptor density was low (ϳ275 receptors/cell), but binding of relaxin triggered intracelluar signaling events. Receptor density was not modulated by pretreatment with estrogen, progesterone, or a number of other agents known to induce differentiation of THP-1 cells. Cross-linking studies showed radiolabeled relaxin bound primarily to cell surface proteins with an apparent molecular mass of >200 kDa. Other members of the insulin-like family of proteins (insulin, insulin-like growth factors I and II, and relaxin-like factor) were unable to displace the binding of relaxin to THP-1 cells, suggesting that a distinct receptor for relaxin exists on this monocyte/macrophage cell line.Relaxin is a 6-kDa polypeptide with basic tertiary structural fold nearly identical to that of insulin (1). However, analysis of chemically synthesized relaxin derivatives has shown that the receptor-binding residues of the two proteins are discrete (2) and, although information is very limited (3, 4), the intracellular signaling events triggered when relaxin binds its receptor appear to be different from those triggered by insulin receptor binding. Consequently, it is not surprising that the physiological functions of relaxin are quite distinct from those of insulin and the other members of the insulin superfamily. Discovered in the 1920s, relaxin has been classically thought of as a "hormone of pregnancy" since it is present in the circulation at increased levels during pregnancy, when its synthesis is primarily directed by the corpus luteum. It serves to promote connective tissue remodeling, which allows cervical ripening and separation of the pelvic symphysis, and also acts to maintain quiescence of the myometrium (5). In addition, relaxin binding sites identified in specific areas of the brain (6) and heart (7) point to a role for it in cardiovascular function, perhaps in setting the critical thresholds for plasma volume in pregnancy (8). Relaxin is likely to play an important role in male reproductive functions as well. The protein is synthesized in the prostate, and has been shown, in vitro, to increase the motility and egg penetrating ability of sperm (9). Relaxin probably possesses non-reproductive functions as well. The identification of sites of relaxin mRNA synthesis, as well as relaxin binding sites, in the brain and heart of both males and females suggests that in those organs, the pro...
The infiltration and activation of monocytes is a hallmark of chronic inflammation, including that associated with a variety of disease states such as rheumatoid arthritis, atherosclerosis, and various autoimmune conditions. Recently, a family of small molecular mass proteins has been described which appear to have inflammatory properties, including chemoattractant effects on monocytes. We report here on the molecular cloning, characterization, and functional expression of mu RANTES, a new murine member of this family. mu RANTES expressed in a mammalian expression system is an approximately 8-kDa protein exhibiting immune cross-reactivity with a rabbit polyclonal antiserum generated against human RANTES. Boyden chamber chemotaxis experiments reveal some lack of species specificity in monocyte chemoattractant potential, as recombinant mu RANTES attracts human monocytes in a dose-dependent fashion in vitro. mu RANTES and its human homolog share approximately 85% amino acid identity, a higher level of conservation than that seen with any other species homologs in this cytokine family, and second only to transforming growth factor-beta among reported immune cytokines.
The Duffy blood group antigen has been postulated to be a receptor on red blood cells (RBCs) for the malarial parasite Plasmodium vivax and a promiscuous receptor for the chemokine superfamily of inflammatory proteins. Recently, the Duffy antigen glycoprotein D cDNA has been cloned (Chaudhuri et al: Proc Natl Acad Sci USA 90:10793, 1993). We have analyzed the binding properties of the cloned Duffy antigen. Duffy- antigen cDNAs expressed in human embryonic kidney cells produced cell- surface proteins that reacted with two known anti-Duffy monoclonal antibodies. Direct ligand binding and displacement experiments using recombinant chemokine proteins also show that the cloned Duffy protein is the RBC chemokine receptor. Radiolabeled chemokines of both the C-C (RANTES and MCP-1) and C-X-C (IL-8 and MGSA/gro) subclasses bound reversibly to transfected cells with dissociation constants in the nanomolar range. Chemokines of either class displaced heterologous chemokines, indicating that they were competing for a single site on the transfected cells. Although the chemokines bound to the transfected cells with high affinity, there was no evidence for signal transduction, as measured by transient increases in intracellular calcium ion concentration, through the Duffy antigen/RBC chemokine receptor in transfected cells. Lastly, we have performed a computer analysis on the amino acid structure of the Duffy antigen/RBC chemokine receptor. Although the cloned Duffy antigen has been postulated to be a nine-transmembrane-spanning receptor, our analysis suggests that the molecule most likely belongs to the seven-transmembrane-spanning receptor superfamily and is therefore similar to other chemokine receptors previously identified.
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