220 Protein & Peptide Letters, 2011, 18, 220-229

The Relaxin Peptide FamilyStructure, Function and Clinical Applications

Linda Jiaying Chan1,2, Mohammed Akhter Hossain1,2, Chrishan S. Samuel1,3,

Frances Separovic2 and John D. Wade1,2,*,#

1
Florey Neuroscience Institutes, 2School of Chemistry and 3Department of Biochemistry and Molecular
Biology, The University of Melbourne, Victoria 3010, Australia

Abstract: The relaxin peptide family in humans consists of seven members, relaxin-1, -2 and -3 and insu-
lin-like (INSL) peptides 3, 4, 5 and 6. It is an offshoot of the large insulin superfamily. Each member con-
sists of two chains, commonly referred to as A and B, which are held together by two inter-chain disulfide
bonds and another intra-chain disulfide bond present within the A chain. The cysteine residues present in
each chain, together with the distinctive disulfide bonding pattern, are conserved across all members of the
superfamily. The chemical synthesis of these complex peptides poses a significant challenge. In the past, random combi-
nation of the two synthetic S-reduced chains under oxidizing conditions was utilized to form the three disulfide bonds.
Nowadays, with the aid of highly efficient solid phase peptide synthesis methodologies, in conjunction with selective S-
thiol-protecting groups, combination of individual A- and B- chains by sequential chemical formation of each of the three
disulfide bonds is now possible resulting in good yields of these peptides. The relaxin peptide family members bind to G-
protein coupled receptors (GPCRs) which have been classified as relaxin family peptide (RXFP) receptors. The various
unique receptor-ligand interactions are outlined in this review, together with the physiological roles of the relaxin peptide
family members and lastly their past and present clinical applications.
Keywords: Relaxin, structure, chemical synthesis, function, clinical applications.
#
Authors Profile: John Wade is the Head of Peptide and Protein Chemistry at the Florey Neurosciences Institutes, University
of Melbourne, an adjunct professor at the School of Chemistry and an NHMRC Principal Research Fellow. His expertise is in
peptide drug design and development with a focus on insulin-like peptides, particularly the relaxins.

1. RELAXIN PEPTIDE FAMILY
Relaxin was first discovered by Frederick Hisaw in 1926
following injection of serum from pregnant guinea pigs or
rabbits into virgin guinea pigs which induced relaxation of
the animals interpubic ligament [1]. It was not until between
the mid-1970s and 1980s when improved methods were
made available for the isolation and characterization of pro-
teins that the first relaxin primary structure was determined.
These, together with the emergence of recombinant DNA
technology, allowed comprehensive studies on the chemistry
and physiology of relaxin to be undertaken. To date, the pri-
mary structure of relaxin from more than twenty species has
been determined [2, 3].
Relaxin has the classic domain configuration of insulin
peptides in which the two peptide chains are held together by
three disulfide bonds. It shares approximately 25% sequence
identity with insulin. This established the concept of a super-
family of shared sequences and structural features which
clearly evolved from insulin during the evolution of verte-
brates [4, 5]. This relaxin peptide family in humans is now
known to comprise of seven members, relaxin-1, -2 and -3
and four other insulin-like peptides (INSL 3, 4, 5 and 6) Fig.
*Address correspondence to this author at the Florey Neuroscience Insti-
tutes, The University of Melbourne, Victoria, Australia 3010; Tel: +61 (3)
8344 7285; Fax: +61 (3) 9348 1707 or +61 (3) 9347 5826;
E-mail: john.wade@florey.edu.au
(1) [6, 7]. Two different genes, RLN1 and RLN2 encoding
two different forms of relaxin, relaxin-1 and relaxin-2 re-
spectively, are present in humans and other higher primates
which suggest ancestral duplication occurred during primate
evolution. The human RLN2 gene is the orthologue of the
RLN1 gene found in other mammalian species [8, 9]. The
RLN3 gene encoding for the final human relaxin member
(relaxin-3) discovered in 2002 is the ancestral peptide of the
relaxin family [10, 11]. All three relaxin-1, -2 and -3 pep-
tides were named as a relaxin due to a common and well-
characterized binding motif (Arg-X-X-X-Arg-X-X-Ile/Val)
that is present in the B chain [4, 5]. A summary of the relaxin
family peptides receptors, known in vivo functions and
therapeutic potential is provided in Table 1 and discussed
further below.
2. STRUCTURE OF THE RELAXIN FAMILY PEP-
TIDES
All relaxin family peptides are synthesized as a pro-
hormone which encompasses a signal sequence and a B-C-A
structure [12]. The signal sequence aids the secretion of the
pro-hormone while the interconnecting C-peptide which
links both A- and B-chains facilitates protein folding and the
formation of the three disulfide bridges. Subsequent enzy-
matic cleavage in vivo produces the mature active two-chain
(A-B) heterodimeric peptide which has only been demon-
strated for relaxin-1, -2, -3 and INSL3. However, the proc-
essing of INSL 4, 5 and 6 still remains unclear. The A- and

0929-8665/11 $58.00+.00 2011 Bentham Science Publishers Ltd.

The Relaxin Peptide Family Structure, Function and Clinical Applications Protein & Peptide Letters, 2011, Vol. 18, No. 3 221

Figure 1. Phylogenetic tree depicting the ancestral peptide of the relaxin family. *Relaxin-1 is found in humans and higher primates; Relaxin-
2 is found in humans and is equivalent to relaxin-1 in other species.

Table 1. The Human Relaxin Family: Names, Receptors, in vivo Functions and Therapeutic Potential

Past & Present Thera-

Relaxin Family Peptide Receptors In Vivo Functions
peutic Potential

RXFP1
Relaxin-1 Unknown NA
(LGR7)

Pregnancy processes:
Remodeling and growth of the uterus, cervix and vagina throughout
pregnancy. [29, 30]
Promotes endometrial thickening and vascularisation for embryo im-
plantation. [31]
Development of mammary nipple. [27, 37, 38] Heart failure symptoms
Non-pregnancy processes: Scleroderma
RXFP1 Increases sperm motility. [40] Pre-eclampsia
Relaxin-2
(LGR7) Control of blood pressure and fluid balance. [44] Cervical ripening
Release of hypothalamic hormones. [44] Orthodontic treatment
Cardiovascular adaptive changes such as increases in plasma volume, Fibrotic diseases
cardiac output and heart rate; and decreases in blood pressure and vascu-
lar resistance. [47]
Increases glomerular filtration rate and effective renal plasma flow in
kidney. [48] Anti-fibrotic effects in skin, lungs, heart and kidneys.
[54-57]

Neurotransmitter in the brain.

RXFP3 Anti-anxiety drugs, anti-
Relaxin-3 Potential in mediating stress responses and regulating appetite. [58, 60]
(GPCR135) obesity drugs
Anti-fibrotic effects [61]

Insulin-like peptide 3 (INSL3)
RXFP2
(Relaxin-like factor: RLF /
(LGR8)
Leydig cell insulin-like: Ley-IL)
Stimulates growth and development of the gubernaculums ligament
Cryptorchidism,
essential for testicular descent. [63]
fertility regulation
Survival of germ cells and ovarian follicles. [68, 70]

Insulin-like peptide 4 (INSL4)

(Early placental insulin-like Unknown Unknown NA
protein: EPIL)

Insulin-like peptide 5 (INSL5) RXFP4 Possible role in neuroendocrine signaling and/or fat and glucose metabo-
Orexigenic drugs
(Relaxin insulin-like factor 2) (GPCR142) lism.

Insulin-like peptide 6 (INSL6)

Unknown Required for the progression of spermatogenesis [75] Fertility regulation
(Relaxin insulin-like factor 1)
222 Protein & Peptide Letters, 2011, Vol. 18, No. 3 Chan et al.

B-chains are covalently linked by two inter-disulfide bonds

made by four conserved cysteine residues, two present in
each of the two chains respectively. An additional intra-
disulfide bond formed by two more conserved cysteine resi-
dues is present within the A chain Fig. (2) [13].
The primary sequences for each relaxin peptide family
member has been either determined or predicted. There is
very little sequence homology between members. The six
cysteine residues which form disulfide cross links between
the A- and B-chains, and a single glycine residue within the
B chain remain highly conserved across the relaxin family
members Fig. (3). Despite low primary sequence homology,
the relaxin members adopt similar tertiary structures, some
of which have been resolved either by X-ray crystallography
or NMR spectroscopy [14-17].
3. CHEMICAL SYNTHESIS OF THE RELAXIN FAM-
ILY PEPTIDES
The assembly of the individual relaxin A- and B-chains
has been achieved using standard Fmoc solid phase peptide
synthesis (SPPS) protocols. Automated peptide synthesizers
are used, either with or without microwave assistance. Indi-
vidual amino acid residues are activated in a mixture of 2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluo-
rophosphate (HBTU), diisopropylethylamine (DIPEA) and
N,N-dimethylformamide (DMF) and transferred into a reac-
tion vessel containing the deprotected resin for coupling.
Successive deprotection of the growing peptide chain is car-
ried out using piperidine in DMF. The cleavage of peptide
from the resin with the concomitant removal of side chain
protecting groups is done using a cleavage cocktail consist-

A) C peptide B)

A chain COOH
COOH
A chain
NH2
B chain
NH2
B chain

Figure 2. Structural representation of the pro-hormone states (A) of the relaxin family peptide hormones and the processed mature active
hormone (B) after excision of the C-peptide. Dotted lines indicate presence of disulfide bonds.

A chain 1 5 10 15 20

Relaxin-1 R P YVA L F EK C C L I G C TK R S L AKYC

Relaxin-2 Z L Y S ALANKC CHVGC TKRS L ARFC

Relaxin-3 D V L A G L S S S C C KWG C S K S E I S S L C

INSL3 A A A T N PARY C C L S G C TQQD L L T LC P Y

INSL4 R S GRHR FDP F C CE V I C DDGT S VKLC

INSL5 QDLQT L C C T DGC SMTD L S ALC

INSL6 GYS EKC CL TGC TKEE L S I AC

B chain

Relaxin-1 KWK D D V I K L C G R E L V R A Q I A I C GM S T W S

Relaxin-2 D S WM E E V I K L C G R E L V R A Q I A I C GM S T W S

Relaxin-3 R A A P YGV R L C GR E F I R A V I F T C GG RW

INSL3 P T P EMR E K L C GH H F V RA LV R VCGG P RWS T E A

INSL4 Z S L AAE L RGC GP R F G KH L L S YC PMP E KT F T TT P ~

INSL5 S KE SVRL C GL EY I RTV I Y I CAS S RW

INSL6 S D I S SARKL C GR Y L V KE I E K LCGHANWS FR

Figure 3. Primary sequences of the human relaxin peptide family. Positions of disulfide bond formation are indicated by grey boxes while
the highly conserved glycine residue is indicated by an arrow. *The conserved binding motif residues present within all relaxin peptides is
the Arg-X-X-X-Arg-X-X-Ile/Val sequence.
The Relaxin Peptide Family Structure, Function and Clinical Applications Protein & Peptide Letters, 2011, Vol. 18, No. 3 223

ing of trifluoroacetic acid (TFA), 3,6-dioxa-1,8-octanedithiol can bind to RXFP 1 and RXFP4 in addition to RXFP3. The
(DODT), anisole and triisopropylsilane (TIPS). The cleaved receptors for INSL4 and INSL6 remain unknown [23, 24].
peptide is then washed and precipitated with diethyl ether. Signaling pathways and the receptor-ligand binding and acti-
The individual crude relaxin A- and B-chains are then sub- vation of the relaxin family peptides have been reviewed
jected to further chemical steps as illustrated in Fig. (4) in extensively before [5, 25, 26].
order to achieve the sequential formation of the 3 disulfide
bonds [18]. 5. THE ROLES OF RELAXIN FAMILY PEPTIDES
5.1. Relaxin-1 and Relaxin-2
4. RELAXIN FAMILY PEPTIDE RECEPTORS
(RXFPS) Since human relaxin-2 (H2 relaxin) is the equivalent of
relaxin-1 in non-primate species, both will be simply referred
The relaxin family peptides interact with a class of recep-
tors known commonly as G-protein coupled receptors to as relaxin. Relaxin is mainly known as a reproductive
hormone which is produced by the corpus luteum and/or
(GPCRs). The receptor for relaxin is a leucine-rich repeat-
placenta in many species. There are varying effects of re-
containing GPCR (LGR) termed as LGR7 [19] while that of
laxin on the cervix, mammary glands, nipples, pubic sym-
INSL3 is known as LGR8 [20]. Both LGR7 and LGR8 have
physis and uterus of different species [6]. Relaxin mediates
been renamed as RXFP1 and RXFP2 respectively. RXFP1
various physiological processes of normal pregnancy and
and RXFP2 belong to class C LGRs and share 60% sequence
identity. Both possess a distinctive ectodomain which en- parturition, for example, in the relaxin knockout (KO) mouse
[27] and relaxin immunoneutralised rat [28], pup delivery
compasses a low density lipoprotein class A (LDLa) module
was prolonged and difficult thus significantly reducing their
present at the extreme N-terminus, ten leucine rich repeats
survival. It is also crucial for the remodeling and growth of
(LRRs) and a hinge region leading into the typical seven
the uterus, cervix and vagina throughout pregnancy. In pigs
transmembrane spanning regions of GPCRs. In contrast, the
and monkeys, the growth and remodeling was demonstrated
native receptors for relaxin-3 and INSL5 are type I GPCRs
unrelated to the LGRs which are GPCR135 and GPCR142 in the uterus [29, 30]. In the early stages of pregnancy in
humans and other primates, there is increasing evidence
respectively. GPCR135 is now termed as RXFP3 and
where relaxin is involved in promoting endometrial thicken-
GPCR142 as RXFP4. RXFP3 and RXFP4 are GPCRs which
ing and vascularisation to prepare the endometrium for em-
have a shortened N-terminus and lack the LRR and LDLa
bryo implantation [31]. There have been a number of studies
module present in RXFP1 and RXFP2 [21, 22]. The two
showing the association of relaxin with increased endo-
different classifications of type I GPCRs of the relaxin fam-
ily peptides have different effects in cells upon receptor acti- metrial angiogenesis, thickening and bleeding [30]. There
has been a suggested link between relaxin plasma levels in
vation. RXFP1 and RXFP2 activation allows coupling to G
s
women and embryo implantation where the highest plasma
and G
OB that activate and inhibit adenylate cyclase respec-
levels of relaxin present during the first trimester corre-
tively, causing an overall intracellular accumulation of
sponds to the time of embryo implantation [4]. Conversely,
cAMP. Conversely, RXFP3 and RXFP4 both couple to in-
the role of relaxin in pregnancy and other related processes is
hibitory G-proteins and inhibit forskolin-stimulated cAMP
production. There is also cross reactivity between relaxin probably facilitatory rather than mandatory for implantation
because this still occurs in humans and other primates that
family peptides and receptors. Fig. (5) For example, relaxin
lack ovaries. Moreover, women who became pregnant by
can also bind to RXFP2 in addition to RXFP1 and relaxin-3
ovum donation are able to maintain their pregnancies even

Figure 4. Schematic representation of the formation of the first intra-disulfide bond on the relaxin A chain, followed by the conversion of
side chain protecting groups on the A chain to facilitate the formation of the other 2 inter-disulfide bonds with the relaxin B chain.
224 Protein & Peptide Letters, 2011, Vol. 18, No. 3
Figure 5. Ligand-receptor interactions between the relaxin peptide family members and their respective receptors. Solid arrows represent
ligand binding to their respective cognate receptors. Broken arrows display cross-reactivity between certain relaxin peptide members and
receptors.
when some has low or no detectable levels of relaxin present
in their blood [32]. During pregnancy, there is growth and an
increase in the elasticity of the pubic joint cartilage and in
addition, relaxin aids in cervical ripening where the cervix is
softened to facilitate the passage of fetus at birth and reduce
the duration of labour. The effects of relaxin on the cervix
were seen in the rat and pig where there was an approximate
doubling of cervical weight [33-35]. The relaxin plateau in
early and mid-gestation weeks correlates with increases in
cervical length and diameter in women [36]. The levels of
relaxin tend to remain low throughout the third trimester.
The uterus is another target tissue for relaxin where the ef-
fects of relaxin on myometrial contractility vary between
species, for instance, relaxin inhibits uterine contractility in
rats, mice and pigs but not in sheep, cows or humans [6].
Relaxin also plays an important role in the development of
the mammary nipple in rats and mice. The requirement for
relaxin was first noticed in immunoneutralised rats where
treated rats had undeveloped nipples with dense collagen
fibre bundles and were unable to suckle their young [37]. A
similar observation was seen in the relaxin-KO mouse [27].
However, in relaxin-deficient pigs, nipple development is
relatively normal. The situation is reversed with respect to
mammary gland development. In pigs, but not in rats and
mice, relaxin is essential for the development of the mam-
mary gland [38].
Relaxin is also produced in the male reproductive tract
and is present in the seminal plasma [39]. Previous studies
have shown that relaxin affects sperm motility, for example,
the addition of porcine relaxin under physiological condi-
tions significantly increases the penetration of human sperm
into either human or bovine cervical mucous compared to
sperm treated with albumin or buffer. Synthetic human re-
laxin showed 10 to 100 times greater potency compared to
porcine relaxin [40]. Another feature of relaxin in males is its
ability to enhance fertility of oocytes. This was demonstrated
in the zona-free hamster egg penetration test where porcine
relaxin enhanced the penetration capability of human sper-
matozoa. In addition, porcine relaxin treatment showed en-
hanced fertilization of mouse oocytes by suboptimal concen-
trations of spermatozoa in vitro [41]. Since relaxin is a nor-
Chan et al.
mal constituent of seminal plasma, human relaxin could be
further developed as a therapeutic agent to assist reproduc-
tion or treat human infertility [40]. Consistent with this, re-
laxin-deficient mice displayed underdeveloped male repro-
ductive tracts and higher levels of collagen accumulation in
the prostate, testes and epididymis, and underwent impaired
male fertility as they aged [42].
Relaxin has other biological effects on other organ sys-
tems such as the brain, cardiovascular system and connective
tissue homeostasis [43]. Relaxin receptors are widely dis-
tributed in several regions of the brain including the circum-
ventricular organs (subfornical organ: SFO and organum
vasculosum of the lamina terminalis: OVLT) where both
have been shown to play a physiological role in the control
of blood pressure and fluid balance [44]. The presence of
other relaxin binding sites in the brain is found in the neu-
rosecretory magnocellular hypothalamic nuclei (paraven-
tricular and supraoptic nucleus) which are responsible for the
release of hypothalamic hormones such as vasopressin and
oxytocin [44]. Overall, these brain regions are readily acces-
sible to circulating relaxin which may be responsible for
plasma osmolality regulation. In previous studies, it has been
shown that rats in the second half of pregnancy display a
decline in plasma osmolality that is related to increased se-
rum relaxin levels, which does not occur in pregnant relaxin-
deficient rats. Likewise, a reduction in plasma osmolality in
wild-type mice during late pregnancy is not observed in re-
laxin KO mice. Studies have shown a link between relaxin
and increased water consumption in rats during the second
half of pregnancy. Intracerebroventricular or intravenous
relaxin also promotes drinking in non-pregnant rats [45, 46].
There is now strong evidence that relaxin has important
roles in cardiovascular adaptive changes that are associated
with pregnancy which include not only increases in plasma
volume, cardiac output and heart rate but also decreased
blood pressure and vascular resistance. In pregnant rats,
there is evidence of increased glomerular filtration rate and
effective renal plasma flow, while vascular resistance de-
creases in parallel to increases in plasma levels of relaxin
[47]. The administration of relaxin to both female and male
rats increases renal plasma flow and glomerular filtration
The Relaxin Peptide Family Structure, Function and Clinical Applications
rate. This accounts for the classic reduction in plasma osmo-
lality associated with pregnancy [48]. Vasodilation is a
common action in arterioles, capillaries and venules in vari-
ous tissue and organs in response to relaxin [49-51]. The
mechanisms suggested for the vasodilator actions of relaxin
are nitric oxide synthase (NOS) III activation through cAMP,
induction of NOS II [52] and extracellular matrix modifica-
tion of vessel walls [53].
Relaxin has also physiological roles in collagen biosyn-
thesis inhibition and promotion of collagen breakdown in
reproductive tissues but has also shown similar effects in
non-reproductive tissues. This suggests that relaxin has po-
tential as an effective treatment for fibrotic diseases. Fibrosis
is the excessive accumulation of extracellular matrix compo-
nents that includes collagens, glycoproteins such as fi-
bronectin, and proteoglycans [6]. Fibrosis is manifested in
organs such as the heart, lung, kidney and skin, where re-
laxin has been shown to act on to reduce the over-expression
of collagen. It does so by inhibiting transforming growth
factor (TGF)-
1-stimulated collagen synthesis, at the same
time increasing matrix metalloproteinase (MMP)-induced
collagen degradation and decreasing the actions of the tissue
inhibitors of MMPs (TIMPs). The anti-fibrotic actions of
relaxin have been demonstrated on TGF-
stimulated human
dermal fibroblasts [54], lung fibroblasts [55] and cardiac
fibroblasts [56] resulting in a reduction in the expression of
type I and type III collagen. In vivo rodent models have also
revealed anti-fibrotic effects in the lung, liver and kidney.
Alveolar thickening and collagen deposition were reduced in
mice with induced pulmonary fibrosis upon 2 weeks of ad-
ministering relaxin [55]. In male rats with induced hepatic
fibrosis, relaxin treatment reduced liver weight and collagen
levels after 28 days while in a model of induced renal fibro-
sis, serum creatinine and interstitial fibrosis decreased after
daily treatment with relaxin [57].
5.2. Relaxin-3
The most recently discovered member of the relaxin pep-
tide family, relaxin-3, is found primarily in the brain, espe-
cially in the ventromedial dorsal tegmental nucleus (also
known as the nucleus incertus) with projections into the hy-
pothalamus [58]. Relaxin-3 is a neurotransmitter in the brain
and has potential in mediating stress responses which in-
volve the activation of the corticotrophin releasing factor
system, stress related memory and control of hippocampal
theta rhythm [59] and in regulating appetite [60]. In rat
forced swim tests, there is increased relaxin-3 mRNA tran-
scription which suggests a role for relaxin-3 in stress and
anxiety. Moreover, feeding was increased in satiated rats
when relaxin-3 was injected into the paraventricular nucleus
which demonstrates a possible role of relaxin-3 in the modu-
lation of food intake [58]. The receptor for relaxin-3,
RXFP3, could be a significant therapeutic potential as a tar-
get for the design of anti-anxiety or anti-obesity drugs. Inter-
estingly, relaxin-3 has also been shown to demonstrate anti-
fibrotic properties [61] through a high affinity interaction
with RXFP1 [62].
5.3. INSL3
INSL3 was first termed as Leydig cell insulin-like pep-
tide (Ley-IL) because its mRNA was originally found in
Protein & Peptide Letters, 2011, Vol. 18, No. 3 225
Leydig cells of the testis. It has also been referred to as re-
laxin-like factor (RLF) due to its relaxin-like activity in the
mouse interpubic ligament bioassay. Like relaxin, INSL3 has
reproductive and non-reproductive functions. INSL3 pro-
duced by fetal Leydig cells is essential for the development
of the gubernacular ligament by promoting growth and dif-
ferentiation during testicular descent [63]. Studies have
shown that male INSL3-KO mice exhibit bilateral cryp-
torchidism where the testes are retained in the abdomen be-
cause of a poorly developed gubernaculum [64]. Cryp-
torchidism results in disrupted spermatogenesis and infertil-
ity [65]. Further studies have demonstrated the ability of
INSL3 to act directly on the gubernaculum to stimulate its
growth and development [66]. There is no clear established
link between INSL3 and testicular descent in humans due to
a low correlation between inactivating mutations of the
INSL3 gene and cryptorchidism [67]. INSL3 influences male
germ cells survival as shown in a study of male rats. Male
germ cell apoptosis induced by gonadotropin withdrawal was
suppressed with INSL3 treatment, suggesting the importance
of INSL3 as a paracrine factor in mediating gonadotropin
actions for the survival of germ cells [68].
INSL3 is expressed in the ovarian follicle and corpus
luteum in females and have shown a potential role in ovarian
physiology. A study in INSL3-KO mice has revealed a
higher apoptosis rate in the follicles and corpora lutea, pro-
longed estrous cycles and smaller litter sizes [69]. Addition-
ally, a study in bovine ovarian follicles has shown that the
loss of INSL3 expression in ovarian thecal cells accounts for
ovarian follicles degeneration [70]. These studies provide
evidences that INSL3 may have some protective anti-
apoptotic role in follicle selection processes. A recent study
in the rat has shown that INSL3 may play a role in oocyte
maturation where the treatment of rat ovarian cultures and
whole animals with INSL3 triggered meiotic progression in
arrested oocytes in preovulatory follicles [68]. From these
observations, the development of INSL3 agonists or antago-
nists provides significant clinical promise for use in both
male and female reproductive systems.
5.4. INSL4, INSL5 and INSL6
INSL4 was originally identified as a gene product spe-
cific to the placenta [71] and is exclusively found in higher
primates. It has been shown that INSL4 is produced in vari-
ous fetal tissues [72] but its physiological functions still re-
main unknown.
INSL5 is highly expressed in the brain and peripherial
tissues. It was first characterized by searching the expressed
sequence tag (EST) database while looking for insulin-like
sequences [73]. Possible roles of INSL5 could be neuroen-
docrine signaling and/or metabolism of fat and glucose.
INSL6 is predominantly found in the testis but there is
still discordance as to which cell type in the testis expresses
it [74]. Recently, INSL6-deficient mice were used to eluci-
date the role of INSL6 in germ cell development. Majority of
the INSL6-deficient males displayed impaired fertility while
females remained fertile [75]. The mature sperm count and
sperm motility were significantly reduced in the epididymis.
These results suggest that INSL6 is necessary for the pro-
gression of spermatogenesis. Further studies are required to
226 Protein & Peptide Letters, 2011, Vol. 18, No. 3
define the INSL6 receptor and its signaling cascade to attain
better understanding of its biological functions.
6. PAST AND PRESENT CLINICAL APPLICATIONS
OF RELAXIN
6.1. Heart Failure
Heart failure has significant impact on mortality and eco-
nomic costs. Despite ongoing research, new drug agents are
required for the treatment of heart failure. Relaxin plays an
active role in cardiovascular adaptive changes and is cur-
rently in phase III clinical trials for heart failure. Phase I
clinical trial for relaxin has been completed where a small
study was conducted using relaxin as a form of therapy in
chronic heart failure patients. The administration of relaxin
was found to have potential haemodynamic and neurohor-
monal effects, i.e. small increases in cardiac output and de-
creases in systemic vascular resistance. It was also found to
be safe and well-tolerated in patients [76]. Phase II clinical
trials has also recently concluded and it was observed that
relaxin administered to selected acute heart failure patients
was safe and showed favorable relief of heart failure symp-
toms such as shortness of breath and resolution of heart fail-
ure symptoms [77]. The current ongoing phase III clinical
trials are expected to conclude in 2011. These exciting clini-
cal developments highlight the therapeutic potential of re-
laxin which could be adopted and marketed as a cardiovascu-
lar drug for human use in the near future.
6.2. Systemic Sclerosis / Scleroderma
Systemic scleroderma (SSc, sceleroderma) is an autoim-
mune disease which causes fibrosis, inflammation and vas-
cular damage. There is currently no effective treatment in
reversing the fibrotic actions of the disease or to prevent dis-
ease progression. The anti-fibrotic and anti-inflammatory
effects of relaxin have been harnessed for the treatment of
SSc [78]. During the phase II clinical trial, relaxin was found
to be well-tolerated in patients and also clinically beneficial
in skin score compared to the placebo group [79]. A phase
III clinical trial was then conducted on a larger patient group
where recombinant human relaxin was given by continuous
subcutaneous infusion over a 24-week period. It was found
that there was no significant improvement in total skin score
or pulmonary function in patients in contrast to the placebo
group [80].
6.3. Pre-Eclampsia
Pre-eclampsia is a serious medical condition which af-
fects pregnant women. It is associated with a reduction of
blood flow to the placenta, leading to an increase in maternal
systemic vascular resistance causing hypertension, renal dys-
function and multiple organ failure [81]. Previous clinical
studies conducted have shown that with the administration of
relaxin in women is associated with heavier than usual men-
strual bleeding (menorrhagia and metrorrhagia). The increase
in blood flow could be an effect mediated by the inhibitory
actions of relaxin on platelet aggregation [82, 83]. Improve-
ments in renal function via increased blood flow could be
due to the fact that relaxin specifically upregulates the vascu-
lar endothelial growth factor (VEGF) in endometrial cells.
Hence, the administration of relaxin to women with pre-
Chan et al.
eclampsia could be beneficial in several ways such as in-
creasing placental blood flow, improving systemic haemo-
dynamic and renal function. The relaxin peptide was tested
in the United States on a group of women with severe pre-
eclampsia. The goal of this phase I safety study was to moni-
tor and evaluate the effects of relaxin on symptoms such as
hypertension, renal dysfunction and blood flow to the pla-
centa [81].
6.4. Cervical Ripening
Relaxin has been shown to induce cervical ripening in
rats and pigs; however some biological effects of relaxin are
species dependent. In a previous study conducted, intravagi-
nal recombinant human relaxin was used to ripen the cervix
in women with post-date pregnancies. There was neither any
evidence of cervical ripening nor elevation in relaxin serum
levels [84]. Thus, it was believed that the intravenous appli-
cation of relaxin could be a good candidate for cervical rip-
ening. A second study was conducted where relaxin was
administered intravenously over a period of 24 hours, yet
there was still no indication of cervical ripening. Other ef-
fects of relaxin such as decreases in serum creatinine and
blood urea nitrogen, increases in predicted creatinine clear-
ance were observed. A decrease in systolic blood pressure
was also recorded after the 24 hour period treatment [85].
Although relaxin did not promote cervical ripening in hu-
mans, the positive effects on blood pressure and renal func-
tion suggest a potential role of relaxin for the treatment of
hypertensive diseases during pregnancy.
6.5. Orthodontic Treatment
A major clinical issue with respect to the successful goals
of orthodontic treatment is the relapse of teeth movement.
Tooth movement is achieved by force systems which re-
model soft tissue systems that comprises of the periodontal
ligament (PDL) which connects the tooth to the alveolar
bone and gingival tissues [86]. The remodeling process in-
volves the synthesis and/or breakdown of collagen. The PDL
contains fibroblasts, nerves, blood vessels and a large abun-
dance of collagen (type I and III) [87]. A study has shown
that orthodontic corrections might be made easier or more
stable if a biological stimulus, like relaxin which could de-
grade or interfere with collagen metabolism, was used. The
effects of relaxin has been investigated on the release and
expression of collagen type I (Col-I) and matrix metallopro-
teinase 1 (MMP-1) by stretched human PDL (hPDL) cells in
vitro where relaxin decreases Col-I and increases MMP-1
[88]. In another study conducted, it was observed that human
relaxin is not involved in accelerating orthodontic tooth
movement but is able to reduce the level of PDL organisa-
tion, mechanical strength and increase tooth mobility in rats
at early time points [89]. This indicates a possible role of
relaxin in reducing the rate of the amount of tooth relapse
associated with orthodontic treatment. Consistent with these
studies in rats, the use of relaxin was also harnessed in pre-
venting relapse in a pilot study in dogs [86], but failed to
demonstrate efficacy in the remodelling and relapse of the
canine gingival in a follow-up larger study; due mainly to the
short half-life of relaxin after it was administered as a single
bolus injection and its inability to spread outside the injec-
tion site (unpublished findings).
The Relaxin Peptide Family Structure, Function and Clinical Applications
7. CONCLUSIONS
Over the years, the human relaxin peptide family has
provided a better and broader understanding of protein evo-
lution, structure and function, chemical syntheses of these
complex peptides and how receptor-ligand interactions lead
to downstream signalling upon activation. All relaxin pep-
tides have displayed great prospects for therapeutics that will
continue to be further investigated and developed over time.
These exciting developments and findings provide an avenue
for the design of novel drug agents such as small peptide
mimetics which will likely be therapeutically useful in hu-
mans in the near future.
ACKNOWLEDGEMENTS
LJC is the recipient of a Faculty of Science, University of
Melbourne, Postgraduate Research Scholarship, MAH is the
recipient of a John T. Reid Charitable Trusts Fellowship,
CSS is the recipient of a National Heart Foundation of
Australia / NHMRC RD Wright Fellowship and JDW is the
recipient of the NHMRC Principal Research Fellowship.
Several of the recent studies reported in this review were
funded by NHMRC Project grants 508995 and 509048 to
JDW. We thank Professor Geoffrey Tregear, Howard Florey
Institute, for his long-standing support.
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