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R E L A X IN A N D R E L A T E D P E P T I D E S : F I F T H I N T E R NA T I O NA L C O N F ER E N C E

Relaxin Receptor LGR7 (RXFP1) Is Regulated

by Estrogen

Priya Maseelall, Jeff Gardner, Andrea Wojtczuk, Gerson Weiss,

and Laura T. GoldsmithNew Jersey Medical School, Newark, New Jersey, 07103 USA

Estrogen regulates LGR7 (RXFP1) mRNA expression in an in vitro model of human term

pregnancy cervix that utilizes lower uterine segment fibroblasts. LGR7 mRNA levels

were increased by estradiol to mean levels of 152% 5.9% above those in untreated

control cells. Therefore, estradiol may amplify relaxins actions in the cervix.

Key words: LGR7; RXFP1; estradiol

Introduction

The actions of relaxin in certain target tissues

appear to require exposure to estrogen, and in

certain cell types relaxin action is potentiated

by estrogen priming. The precise role of estro-

gen and the mechanisms utilized by estrogen in

the cellular response to relaxin have not been

well defined in any relaxin target tissue. One

possible mechanism by which estrogen may en-hance the response to relaxin is by upregulating

expression of the relaxin receptor.

We have previously used anin vitromodel of

human term pregnancy cervix, human lower

uterine segment fibroblasts, as a system for

studying of the effects of relaxin upon human

cervical function.1,2 We therefore used this sys-

tem to test the hypothesis that estrogen amplifi-

cation of the relaxin response in target tissues is

due to regulation of receptor expression. We de-

termined whether LGR7 mRNA is expressed

and if it is regulated by estrogen in ourin vitro

model of human lower uterine segment fibrob-

lasts.

Human lower uterine segment fibroblasts

at passages 1012 were plated in T75 tissue

Address for correspondence: Laura T. Goldsmith, Ph.D., Department

of Obstetrics, Gynecology, and Womens Health, 185 South Orange Ave.,

MSB E506, Newark, NJ 07103. goldsmit@umdnj.edu

culture flasks in Dulbeccos modified Eagles

medium supplemented with 10% fetal bovine

serum (Invitrogen, Carlsbad, CA, USA) and

antibiotics and maintained until reaching about

80% confluency. Medium was removed and

replicate flasks were incubated in complete

medium with or without 1 M 17-estradiol

for 72 h. Total cellular RNA was isolated from

the cells, and 1 g of total RNA from either

estrogen-treated or control (untreated) cells wasreverse transcribed into cDNA. RNAs isolated

from rhesus monkey myometrium and skele-

tal muscle were used as positive and negative

LGR7 mRNA expression controls, respectively.

To ensure reproducibility, the experiments were

repeated three times. Primers previously veri-

fied to have the specific nucleotide sequence of

the human LGR7 mRNA were used.3 PCRs

were performed in triplicate using 8 g of

cDNA and Platinum SYBR Green qPCR Su-

perMix reagents (Invitrogen) and the Rotor-

Gene 3000 real-time PCR system (Corbett

Research). LGR7 standard consisted of ampli-

fied PCR product of a known concentration,

2.65 fmol/L (330 pg/L), created from RNA

isolated from human endometrial glandular ep-

ithelial cells. Standard curves were generated

from serial dilutions of the LGR7 standard.

The cycle threshold value for each sample wasdetermined by the cycle number in which the

samples PCR amplification curve crossed the

Relaxin and Related Peptides: Fifth International Conference: Ann. N.Y. Acad. Sci. 1160: 9192 (2009).doi: 10.1111/j.1749-6632.2009.04048.x C2009 New York Academy of Sciences.

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92 Annals of the New York Academy of Sciences

threshold. LGR7 mRNA in each sample was

quantified by extrapolation of the cycle thresh-

old values from the standard curve. To assess

the purity, size, and identity of the products of

each PCR, melt curve analyses, electrophoretic

analysis, and nucleotide sequencing of PCRproducts were performed.

Human lower uterine segment fibroblasts ex-

press LGR7 mRNA. The expected amplicon of

192 bp was detected, and a single peak was seen

on melt analysis in all PCR products generated

from RNA of control and estrogen-treated cells

and the positive control tissue. As expected, no

LGR7 PCR products were detected in reac-

tions programmed by RNA from skeletal mus-

cle (negative control) and reactions which did

not include reverse transcriptase. Estrogen ap-

pears to regulate LGR7 mRNA expression.

LGR7 mRNA was increased by estradiol to

mean levels of 152% 5.9% (mean stan-

dard error from three experiments, each per-

formed using multiple reverse transcription-

PCRs) above those of control, untreated cells

(P= 0.04).

Recent findings have similarly demonstratedthat estrogen positively regulates LGR7 mRNA

expression in the neonatal porcine cervix.4

Since relaxins actions are often species specific,

it is important to independently address this in

several species. That we have demonstrated this

effect of estrogen in cells from adult humans

decreases the likelihood that this action of es-

trogen is unique to early development. Also,

few studies have addressed the issue of which

hormones and/or growth factors regulate re-

laxin receptor expression and other aspects of

relaxins cellular mechanisms of action. Estra-

diol regulation of the expression of the relaxin

receptor may be the first step in its modulationof relaxin action.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Demonstration of a relaxin receptor and relaxin

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