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Identification of the pregnancy hormone relaxin as glucocorticoid receptor agonist

Medizinische Klinik m. S. Kardiologie, Angiologie, Pulmologie, Charité Berlin, Berlin, Germany

¹Correspondence: Charité Berlin, Campus Mitte, Medizinische Klinik m. S. Kardiologie, Angiologie und Pulmologie, Schumannstr. 20/21, Berlin 10117, Germany. E-mail: thomas.dschietzig{at}t-online.de

SPECIFIC AIMS

The insulin-like peptide relaxin is a central hormone of pregnancy, but it also produces anti-fibrotic, myocardial, renal, central nervous, and vascular effects. Recently, two G protein-coupled receptors, LGR7 and LGR8, have been identified as relaxin receptors. Prompted by reports on immunoregulatory effects of relaxin, we investigated possible interactions with human glucocorticoid receptor (GR).

PRINCIPAL FINDINGS

1. Relaxin blunts endotoxin-induced production of inflammatory cytokines by THP-1 cells in a GR-dependent manner
We analyzed the immunomodulatory properties of relaxin in THP-1 cells differentiated into macrophages. Endotoxin evoked a marked increase in secretion of IL-1, IL-6, and TNF- over 8, 24, and 48 h. Relaxin suppressed stimulated secretion of all cytokines—an effect that plateaued between 5 and 10 nmol/L, showed an estimated IC₅₀ of 0.8 nmol/L, and weakened at higher concentrations, although it was still present at 100 nmol/L. At maximum, stimulated IL-1, IL-6, and TNF- were decreased to 40% of the values in presence of endotoxin alone.

The glucocorticoid dexamethasone (100 nmol/L), used as positive control, also decreased stimulated cytokine secretion. We thereafter investigated whether the inhibitory effect of both dexamethasone and relaxin was sensitive to treatment with RU-486 (0.5 µmol/L), a well-established GR antagonist. RU-486 not only prevented the inhibitory effect of dexamethasone on endotoxin-induced cytokine secretion, but abolished the effect of relaxin.

These results indicated that relaxin exerted its inhibitory effect on stimulated cytokine secretion by interacting with the GR in a manner independent of endogenous glucocorticosteroids, since the charcoal-treated cell medium was steroid-free. Subsequent experiments were designed to investigate whether relaxin (1) activated the GR and/or (2) modulated its expression.

2. In three different cell lines, relaxin induced GR activation, nuclear translocation, and DNA binding as assessed in GRE-luciferase assays
In HeLa, 293 cells and THP-1 cells transiently transfected with the GRE-luciferase reporter gene construct, we established that relaxin was capable of activating GR and, consequently, of enhancing gene expression of the luciferase reporter in a time- and concentration-dependent fashion. In all cells, relative luciferase activity reached its maximum after 4 h of stimulation with 10 nmol/L of relaxin. Relaxin significantly activated gene expression of the reporter at concentrations as low as 500 pmol/L; the calculated EC₅₀ was 0.8 nmol/L. Maximum values amounted to 600 – 650% of baseline values and were already reached at 5 nmol/L of the peptide. At concentrations higher than 10 nmol/L, this effect of relaxin weakened, which resulted in a bell-shaped curve. GR antagonist RU-486 had no effect on baseline luciferase activity, but significantly and concentration-dependently suppressed activity stimulated by dexamethasone or by relaxin. Insulin, from 1nmol/L to 1 µmol/L, never evoked GR activation in this experimental setting.

As negative control, we transfected HeLa cells with a luciferase reporter gene construct containing the estrogen response element. Whereas estradiol (100 nmol/L) significantly increased luciferase activity, thereby indicating reliability of the system, it did not modulate baseline activity of luciferase.

3. Coimmunoprecipitation experiments revealed physical interaction of endogenous and exogenous relaxin with cytoplasmic and nuclear GR, as well as with Hsp70 and 90, which are known to be essential for GR agonist binding; labeled exogenous relaxin accumulated in the nucleus
We analyzed cytoplasmic extracts obtained from HeLa cells stimulated over 30 min. Following immunoprecipitation with the GR antibody, Western blot analysis clearly demonstrated coprecipitation of Hsp 70, Hsp 90, and relaxin (both under control conditions and after relaxin treatment (10 nmol/L)). Correspondingly, treatment with the relaxin antibody coprecipitated Hsp 70, Hsp 90, and the GR, both in controls and after relaxin administration. In contrast, relaxin-related peptide insulin and grp94, a chaperon of the endoplasmic reticulum, were not coprecipitated. Moreover, precipitation with the grp94 antibody likewise failed to yield bands for GR, relaxin, insulin, Hsp 70, or Hsp 90.

In nuclear extracts, a certain amount of coprecipitating GR and relaxin was always detectable under control conditions, which indicated the relevance of endogenous relaxin. After relaxin treatment over 30 min, we observed a marked increase in these signals. Again, insulin and grp94 were not coprecipitated, and grp94 precipitation did not reveal any interaction with GR, relaxin, and insulin.

We demonstrated that fluorescence-labeled exogenous relaxin entered intact (i.e., nonpermeabilized) cells and accumulated in the nucleus.

4. Relaxin competed with GR agonists for GR binding in vivo in whole cell assays and in vitro in fluorescence polarization assays
Relaxin, at concentrations between 0.5 and 5 nmol/L, proved to be a highly potent competitor at human GR (estimated IC₅₀=0.4 nmol/L) in a fluorescence polarization assay. At concentrations of 10 nmol/L and higher, this competition was almost completely reversed. Dexamethasone displaced fluorescent glucosteroid at concentrations higher than 5 nmol/L.

In competition binding experiments, in HeLa cells, employing labeled corticosterone and increasing concentrations of cold corticosterone or relaxin, rising corticosterone completely displaced the tracer. As in the polarization assay, relaxin exhibited high potency of displacing the tracer with an estimated IC₅₀ value of 1.2 nmol/L, but this displacement was abolished at higher concentrations.

5. Relaxin up-regulated GR gene and protein expression, as well as the number of functionally active GR sites
Relaxin exposition over 4 and 24 h markedly increased GR protein levels in HeLa, 293, and THP-1 cells. Treatment with dexamethasone over 24 h decreased expression of GR protein in these cells. Actions of relaxin and dexamethasone could be attributed to corresponding changes of GR gene expression and were found to be sensitive to administration of GR antagonist RU-486. In contrast to the relaxin-induced up-regulation of GR, relaxin exposition over 4 and 24 h did not increase protein or gene expression of estrogen receptors ER- and ER-ß.

Finally, we attempted to determine whether relaxin was capable of heightening the expression of functionally active GR receptors in HeLa and THP-1 cells. Toward this objective, we performed whole cell assays with labeled dexamethasone and corticosterone after pretreatment of the cells with 10 nmol/L relaxin over 4 or 24 h. In both cell types, exposition to relaxin approximately doubled the maximum number of glucocorticosteroid binding sites B_max without altering the apparent dissociation constant K_D.

6. In LGR7/8-free cells, relaxin-mediated activation of GR was preserved
We initially confirmed that spleen fibroblasts, in contrast to HeLa and THP-1, did not express relevant amounts of relaxin binding membrane receptors LGR7 and LGR8. In these cells, activation of GR by relaxin and dexamethasone as determined in the GRE-luciferase assay was well preserved. Similarly, relaxin evoked elevation of GR protein comparable to that observed in HeLa and THP-1 cells. In contrast, relaxin-induced up-regulation of endothelin type-B receptors (an ERK-1/2-mediated effect that is not sensitive to RU-486) was completely abrogated.

CONCLUSIONS AND SIGNIFICANCE

Discovered as pregnancy hormone at the beginning of the last century, relaxin is presently being recognized as one of the central mediators of body fluid and circulation homeostasis. Here, we demonstrate another surprising facet of relaxin: it (1) binds to and activates the human GR; (2) up-regulates, by using this pathway, GR expression at mRNA, protein, and functional levels; and (3) influences stimulated cytokine secretion in human macrophages in glucocorticoid-like fashion.

These findings are of great interest because glucocorticoids regulate a great variety of metabolic, behavioral, cardiovascular, and immune functions via the GR. Among these effects, their anti-inflammatory and immunosuppressive profile has attracted intensive attention and has rendered them the most widely employed drugs for treating chronic inflammatory and autoimmune diseases. Inhibition of cytokine production by different immunologically competent cells represents one of the major immunosuppressive mechanisms of glucocorticoids. The cytokines investigated in this study (IL-1, IL-6, and TNF-) are known to represent key mediators of the so-called acute-phase response of inflammation, and they are involved in a vast number of inflammatory diseases.

The question arises, however, as to the precise mode of GR binding of relaxin and as to the mechanism responsible for weakening of GR-relaxin binding and corresponding functional effects, luciferase activation and cytokine inhibition. Modes of relaxin and GR binding to their "classical" interaction targets are well defined. With regard to relaxin membrane receptors, LGR7 and LGR8, the structural components essential for receptor binding of relaxin reside in the B chain: two charged arginine residues and hydrophobic isoleucine. With respect to glucocorticoid binding to GR, recent crystallization of its ligand binding domain by Bledsoe and coworkers has revealed the presence of a binding pocket consisting of 11 -helices and 4 ß-strands folded into a three-layer helical sandwich. It is, however, unclear which mechanism accounts for relaxin binding to GR; this question is currently a matter of investigation. The bell-shaped curve of relaxin binding to GR, in turn, may be caused by the tendency of relaxin to form dimers at higher concentrations or by recruitment of additional binding sites that could induce negative cooperativity.

Because glucocorticoids have been shown to provide negative feedback to the expression of their own receptor, we investigated whether relaxin was able to regulate gene and protein expression of GR, as well as the number of functionally active GR sites. Whereas dexamethasone application led to down-regulation of GR protein, exposure to relaxin uniformly increased protein levels of GR. On the basis of recent reports on expression of GR isoforms in human tissues (i.e., studies that detected hardly any GR-ß protein), most if not all GR protein should represent GR-. This is corroborated by our finding that relaxin pretreatment in HeLa and THP-1 elevated the number of functionally active GR binding sites, as determined in whole cell assays using hot dexamethasone and corticosterone. Since GR-ß has repeatedly been reported not to bind glucocorticoids, this rise of glucocorticoid binding sites in all likelihood reflected GR- protein.

Disclosure of the dual use of relaxin’s own membrane receptors (LGR7 and LGR8) and a nuclear receptor (the GR) for relaxin signal transduction represents an unprecedented finding in terms of hormone signaling and opens a new field of investigation. To commence unraveling this signal network, we undertook initial experiments in spleen cells, which were known to express no relaxin binding membrane receptors. We established that essential characteristics of the relaxin-GR pathway (relaxin-induced GRE activation in the luciferase assay and GR up-regulation) were well preserved in these cells. It is therefore tempting to speculate that this new relaxin-GR pathway does not critically depend on the existence and involvement of relaxin binding membrane receptors. The precise mode of relaxin’s access to the GR, as well as possible interactions with the classical LGR7/8 pathway, remain to be investigated.

The experimental finding that relaxin, apart from acting via its membrane receptors, has GR-agonistic properties may significantly influence understanding of its pleiotropic physiological and pathophysiological role. There are a number of relaxin effects that may well involve GR signaling. First, relaxin appears to expedite maternal immunotolerance to fetal allograft during implantation and early pregnancy (a glucocorticoid-like mode of action would easily fit into this scheme). Second, relaxin has been demonstrated to suppress experimentally induced asthmoid reactions and cardiac anaphylaxis. Relaxin was also shown to promote differentiation of human activated T cells. With regard to central effects of relaxin, the peptide affects pituitary release of oxytocin, the precise effect depending on the distinct state of pituitary preactivation. A similar interaction (differential modulation of neurohypophyseal release of oxytocin) has been described for glucocorticoids and their influence on stress coping. Like relaxin, glucocorticoids are intricately linked to regulation of the central vasopressin system. Recombinant human relaxin, finally, is among the most promising drugs for treatment of scleroderma, a profile that could also indicate involvement of the relaxin-GR pathway.

In conclusion, we have shown that the hormone relaxin acts as a GR agonist and that this pathway is pivotal to its effects on cytokine secretion by human macrophages. These findings may impact on our understanding of the abundant physiological actions that relaxin exerts far beyond pregnancy. They may, moreover, deepen our insights into the general principles of hormone signaling.

View larger version (38K): [in this window] [in a new window]   Figure 1. Proposed dual mechanism of relaxin signaling via LGR7/8 and GR pathways.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1120fje;

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