Relaxin Binds to and Elicits a Response from Cells of the Human Monocytic Cell Line, THP-1

Abstract

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...

Human Relaxin Gene 3 (H3) and the Equivalent Mouse Relaxin (M3) Gene

“…Demonstration of Relaxin Activity of Synthetic H3 RelaxinSynthetic H3 relaxin C-terminal amide derivatives were tested for relaxin activity in a relaxin receptor expressing cell line, THP-1 (25). H2 relaxin produces a dose-dependent increase in cAMP production from these cells (Fig.…”

“…THP-1 Cell Bioassay-The ability of H3 relaxin to induce cAMP production in the human monocytic cell line (THP-1) was compared with H1 and H2 relaxin following the procedure of Parsell and colleagues (25), with the following modifications: THP-1 cells that had been viability tested using trypan blue were resuspended in media and transferred to a 96-well plate at a density of 60,000 cells/well. Peptides (H1, H2, H3 relaxin, human INSL3, and bovine insulin) were added to the wells together with 1 M forskolin and 50 M isobutylmethylxanthine in RPMI media and incubated at 37°C for 30 min.…”

H2 relaxin overexpression increases in vivo prostate xenograft tumor growth and angiogenesis

“…To determine the amount of H2 relaxin peptide released into the cell culture medium, a recombinant human H2 relaxin-specific direct sandwich ELISA was performed as described in Parsell et al 21 Antibodies used for the ELISA experiments were goat anti-human H2 relaxin IgG and HRP anti-goat-IgG conjugate antibody (gift from Dr. E. Unemori, Connetics Corp, Palo Alto, CA). Supernatant samples consisted of complete conditioned medium harvested at days 2, 4 and 6 diluted 1:10 in assay diluent buffer.…”

“…Supernatant samples consisted of complete conditioned medium harvested at days 2, 4 and 6 diluted 1:10 in assay diluent buffer. 21 Cell proliferation assays were also performed 11 to observe whether H2 relaxin-expressing PC3 cells exhibited any proliferative growth advantage over controls. Briefly, PC3-Luc-H2/eGFP and PC3-Luc-eGFP cells were seeded at 5 3 10 3 cells/well in a 96-well plate.…”

“…Upon termination of the experiment (week 6), samples were diluted 1:10 in assay dilutent buffer to quantify H2 relaxin levels by ELISA, as described previously. 11,21 Prior to sacrifice, animals underwent whole-body noninvasive bioluminescence imaging (BLI) to examine PC3-Luc cell biodistribution. Briefly, mice were anaesthetized with 10 mg/kg ketamine hydrochloride (100 mg/mL, i.p.…”