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RELAXIN AND RELATED PEPTIDES: FIFTH INTERNATIONAL CONFERENCE

Relaxin Receptor LGR7 (RXFP1) Is Regulated
by Estrogen
Priya Maseelall, Jeff Gardner, Andrea Wojtczuk, Gerson Weiss,
and Laura T. Goldsmith
New 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 relaxin’s 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 estrogen 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 enhance the response to relaxin is by upregulating
expression of the relaxin receptor.
We have previously used an in vitro model 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 system to test the hypothesis that estrogen amplification of the relaxin response in target tissues is
due to regulation of receptor expression. We determined whether LGR7 mRNA is expressed
and if it is regulated by estrogen in our in vitro
model of human lower uterine segment fibroblasts.
Human lower uterine segment fibroblasts
at passages 10–12 were plated in T75 tissue

Address for correspondence: Laura T. Goldsmith, Ph.D., Department
of Obstetrics, Gynecology, and Women’s Health, 185 South Orange Ave.,
MSB E506, Newark, NJ 07103. goldsmit@umdnj.edu

culture flasks in Dulbecco’s modified Eagle’s
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 μM 17β-estradiol
for 72 h. Total cellular RNA was isolated from
the cells, and 1 μg of total RNA from either
estrogen-treated or control (untreated) cells was
reverse transcribed into cDNA. RNAs isolated
from rhesus monkey myometrium and skeletal muscle were used as positive and negative
LGR7 mRNA expression controls, respectively.
To ensure reproducibility, the experiments were
repeated three times. Primers previously verified to have the specific nucleotide sequence of
the human LGR7 mRNA were used.3 PCRs
were performed in triplicate using 8 μg of
cDNA and Platinum SYBR Green qPCR SuperMix reagents (Invitrogen) and the RotorGene 3000 real-time PCR system (Corbett
Research). LGR7 standard consisted of amplified PCR product of a known concentration,
2.65 fmol/μL (330 pg/μL), created from RNA
isolated from human endometrial glandular epithelial cells. Standard curves were generated
from serial dilutions of the LGR7 standard.
The cycle threshold value for each sample was
determined by the cycle number in which the
sample’s PCR amplification curve crossed the

Relaxin and Related Peptides: Fifth International Conference: Ann. N.Y. Acad. Sci. 1160: 91–92 (2009).
C 2009 New York Academy of Sciences.
doi: 10.1111/j.1749-6632.2009.04048.x 

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A. D. J. That we have demonstrated this effect of estrogen in cells from adult humans decreases the likelihood that this action of estrogen is unique to early development. Tseng. Wotjczuk. Human lower uterine segment fibroblasts express LGR7 mRNA. et al. et al. Relaxin (RLX) and estrogen affect estrogen receptor.E. vascular endothelial growth factor.E. and nucleotide sequencing of PCR products were performed. Estradiol regulation of the expression of the relaxin receptor may be the first step in its modulation of relaxin action. Palejwala. Reprod. Annals of the New York Academy of Sciences few studies have addressed the issue of which hormones and/or growth factors regulate relaxin receptor expression and other aspects of relaxin’s cellular mechanisms of action. but TGFβ1 inhibits. melt curve analyses. Recent findings have similarly demonstrated that estrogen positively regulates LGR7 mRNA expression in the neonatal porcine cervix. The expected amplicon of 192 bp was detected. Also. and RLX receptor expression in the neonatal porcine uterus and cervix. size. References 1.. M. G. S. A. A. LGR7 mRNA was increased by estradiol to mean levels of 152% ± 5. et al. 3. electrophoretic analysis. 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. To assess the purity. Wiley. Endocrinology 142: 3405–3413.. . Reproduction 135: 705–712. 1998. D. As expected.9% (mean ± standard error from three experiments. Disparate effects of relaxin and TGFβ1: Relaxin increases. Endocrinology 139: 1208– 1212.04). 4. untreated cells (P = 0. J. 2. Chen. Stein. no LGR7 PCR products were detected in reactions programmed by RNA from skeletal muscle (negative control) and reactions which did not include reverse transcriptase. Tang & L. W. and identity of the products of each PCR. Demonstration of a relaxin receptor and relaxin stimulated tyrosine phosphorylation in human lower uterine segment fibroblasts. Mazella. Hum.4 Since relaxin’s actions are often species specific. 2004. 2001 Relaxin positively regulates matrix metalloproteinase expression in human lower uterine segment fibroblasts using a tyrosine kinase signalling pathway. S. 19: 1513– 1518.92 threshold.. 2008. LGR7 mRNA in each sample was quantified by extrapolation of the cycle threshold values from the standard curve. Conflicts of Interest The authors declare no conflicts of interest. Stein. it is important to independently address this in several species. each performed using multiple reverse transcriptionPCRs) above those of control. the relaxin receptor and the production of IGFBP-1 in human endometrial stromal/decidual cells. Palejwala.. Estrogen appears to regulate LGR7 mRNA expression. Weiss. Yan.