TEM 1237 No.

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Review
Progesterone-Mediated
Non-Classical Signaling
Deepika Garg,1 Sinnie Sin Man Ng,2 K. Maravet Baig,2,3
Paul Driggers,2 and James Segars2,*
Progesterone is essential for pregnancy maintenance and menstrual cycle
Trends
regulation. Hormone action has been primarily ascribed to the well-character-
Progesterone modulates a wide range
ized classical signaling pathway involving ligand binding, activation of nuclear of physiological processes through
progesterone receptors (PRs), and subsequent activation of genes containing non-classical signaling pathways via
various non-nuclear progesterone
progesterone response elements (PREs). Recent studies have revealed pro- receptors including membrane-bound
gesterone actions via non-classical signaling pathways, often mediated by progesterone receptors, PGRMC1,
non-genomic signaling. Progesterone signaling, in conjunction with growth and GABA-A receptors.

factor signaling, impacts on the function of growth factors and regulates Non-classical signaling includes growth
important physiological actions such as cell growth and remodeling, as well receptor signaling pathways such as
alterations in intracellular cAMP levels
as apoptosis. This review focuses on non-classical progesterone signaling
and modulation of CaMKII activity.
pathways, both including and excluding PR, and highlights how research in
this area will provide a better understanding of progesterone actions and may Activation of tyrosine kinase SRC, the
p44/p42 MAPK pathway, and p38
inform novel therapeutic strategies. MAPK activation via progesterone
are other non-classical signaling
Progesterone and Its Actions pathways.
Progesterone is a steroid hormone synthesized by the placenta, ovaries, and adrenal glands,
Recent discovery of integration of
and is essential for reproductive functions such as ovulation and the initiation and maintenance mechanical and endocrine signals dur-
of pregnancy [1]. Notably, the physiological roles of progesterone include regulation of other ing labor has emphasized the crucial
tissues including the brain, breast, and bone [2–4]. Progesterone effects are primarily trans- role of non-classical progesterone
actions.
duced through the classical signaling pathway via ligand binding to nuclear progesterone
receptors (PRs, see Glossary). These ligand-bound PRs subsequently bind to gene regulatory Remarkable progress in the identifica-
regions including progesterone response elements (PREs) to initiate transcription of pro- tion of progesterone effects via non-
gesterone-responsive genes [5] (Box 1). Interestingly, recent data have revealed that proges- classical signaling has provided a foun-
dation upon which to develop new
terone also acts through non-classical signaling pathways [6] (Figure 1).
therapeutic targets for novel pharma-
cological interventions in diverse phy-
These non-classical pathways can be dependent or independent of transcriptional or genomic siological processes.
regulation [7]. In addition to the modulation of gene transcription by PR, various second
messengers and signal transduction pathways play an important role to exert rapid hormonal
effects mediated by non-classical signaling [8,9]. Accumulating evidence suggests that rapid
progesterone responses are mediated by activation of cell membrane receptors, cytoplasmic 1
Department of Obstetrics and
PR, or receptor-independent intracellular signaling cascades [10,11]. Moreover, criteria have Gynecology, Maimonides Medical
been established to better distinguish non-classical effects; these are as follows: (i) rapidity of Center, Brooklyn, New York, NY
11219, USA
action incompatible with transcriptional activation followed by translation; (ii) insensitivity to 2
Department of Gynecology and
inhibitors of transcription or translation; (iii) lack of response to nuclear receptor antagonists; (iv) Obstetrics, Division of Reproductive
presence in cells lacking nuclei such as platelets and erythrocytes; and (iv) induction by cell- Sciences and Women’s Health
Research, Johns Hopkins School of
impermeable molecular conjugates [12]. Although non-classical progesterone-dependent Medicine, Baltimore, MD 21205, USA
actions may be rapid, the downstream consequences can be intense and sustained. This 3
Department of Biochemistry and
review summarizes current understanding of several non-classical progesterone receptor- Molecular Biology, Virginia
Commonwealth University School of
dependent/independent and non-genomic signaling pathways for the cellular and molecular Medicine, Richmond, VA 23298, USA
actions of progesterone (Figure 2).

Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy http://dx.doi.org/10.1016/j.tem.2017.05.006 1
© 2017 Published by Elsevier Ltd.
 TEM 1237 No. of Pages 13

Box 1. Progesterone signaling: The Classical Pathway

Classical progesterone actions are mediated by nuclear progesterone receptors (nPRs). These nPRs have two well- *Correspondence:
described receptor isoforms: PR-A (94 kDa) and PR-B (120 kDa) [468_TD$IF][86]. Both isoforms are encoded by a single gene but jsegars2@jhmi.edu (J. Segars).
are transcribed from different promoters to generate distinct subclasses of PR mRNAs [469_TD$IF][87]. The PR-A and PR-B
isoforms share transcription-activating function (AF) domains AF-1 and AF-2; the PR-B isoform contains a specific N-
terminal segment AF-3 containing the B-upstream segment (BUS) that is absent from the PR-A isoform. Both PR-A and
PR-B both contain the C-terminal ligand-binding domain (LBD) and the hinge region (H). A consensus SUMO-
conjugation motif is present in the N-terminal domain (NTD) of PR-A and PR-B, and sumoylation modulates
ligand-dependent transcriptional activity on promoters containing multimerized synthetic PREs [470_TD$IF][88]. The NTD encodes
a proline-rich sequence that has a binding site for the SH3 domain of SRC tyrosine kinase, a protein contributing to
extranuclear progesterone signaling [471_TD$IF][89].

A third N-terminally truncated isoform, termed PR-C (45–50 kDa), has been identified as the product of an mRNA
expressed in breast cancer cells formed by translation initiation at methionine residue 595 [472_TD$IF][90]. This receptor has ligand-
binding properties but lacks DNA-binding activity. Upregulation of PR-C expression in the endometrium at parturition
may antagonize the actions of PR-A and PR-B during parturition by sequestration of progesterone. It has been
suggested that, during labor, interleukin-1b (IL-1b) induces recruitment of nuclear factor kB (NF-kB) to the PR promoter
to induce expression of PR-C [473_TD$IF][91].

PR is rapidly transported between the cytoplasm and the nucleus. The unbound receptor exists in both subcellular
compartments and resides in complexes with heat-shock protein [47_TD$IF](HSP) chaperone molecules such as HSP70 and
HSP90 [475_TD$IF][92]. Ligand binding to PR induces dissociation from HSPs, retention of dimerized PR in the nucleus, and
activation of transcription. Numerous transcription factors (AP1, SP1, STATs) are involved in the action of PR, and may
either activate or inhibit transcriptional activities [476_TD$IF][93,94]. Post-translational modifications of PR such as N-terminal serine
phosphorylation and lysine acetylation can also alter PR transcriptional activity and affect promoter selectivity [47_TD$IF][95].

Progesterone Signaling: Non-Classical Pathways

Membrane-Associated Receptors and Progesterone Signaling Pathways
Specific membrane-bound PRs (mPRs), also called PAQRs (progestin and adipoQ receptors),
have been implicated in non-genomic progesterone action and participate in the regulation of

mPRα,mPRβ,
Progesterone and mPRγ
signaling
pathways Membrane-
associated PGRMC1
receptors (mPRs)

GABA-A

Intracellular cAMP/PKA
growth factors signaling
Classical Non-classical
signaling signaling
(Box 1)
c-SRC/MAPK Ca2+/PKC
pathway signaling

PI3K/AKT
PR-A PR-B pathway
receptors receptors
Integraon of
endocrine and
mechanical
signals

Figure 1. Progesterone Signaling Pathways: Classical and Non-Classical Pathways. Comparison of gene
activation mediated by nuclear progesterone receptors is compared to ‘non-classical' progesterone-mediated cellular
events. Non-classical signaling is complex, but can be considered to involve five overlapping but distinct mechanisms.

2 Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy

 TEM 1237 No. of Pages 13

membrane-associated progesterone signaling events [13]. Zhu et al. [14] demonstrated the Glossary
presence of mPR by northern blotting of RNA isolated from the ovaries of spotted seatrout. This Epidermal growth factor (EGF): a
mPR was required for oocyte maturation. Initially, three mPR isoforms (mPRa, mPRb, and low molecular weight polypeptide;
mPRg) with a Kd for progesterone of 3–7 nM were identified in a fish model and in other binding to its receptor regulates
cellular growth, proliferation, and
vertebrate species [15] (Figure 2). mPRs have been described as having seven transmembrane differentiation. EGF stimulates the
domains, similarly to G protein-coupled receptors (GPCRs), and are expressed in various tyrosine kinase activity of its receptor
tissues including the uterus, ovary, kidney, central nervous system, and intestine [16,17]. which, in turn, induces a signal
transduction cascade and results in
Recently, two additional subtypes of mPR (mPRd and mPRe) were identified that exhibited
a variety of cellular changes that
high-affinity binding of progesterone (Kd = 2.71 and 2.85 nM, respectively), were abundant in ultimately cause DNA synthesis and
neural tissues, and unlike the other mPR subtypes activated Gas [18]. cellular proliferation.
Extracellular matrix (ECM): an
assembly of extracellular molecules
Conflicting results have been reported by various researchers regarding the expression of
produced by cells that provides
mPRs solely on the cell membrane [19,20]. Thomas and Pang [21] suggested that the mechanical and biochemical support
neuroprotective effects of progesterone and the neurosteroid allopregnanolone were mediated to the surrounding cellular structures.
by membrane-associated PRs[480_TD$IF]. Moreover, studies suggested that [481_TD$IF]antiproliferative and It also helps in tissue segregation,
mediates intercellular communication,
[482_TD$IF]antiapoptotic effects of progesterone on cancer cells were mediated via activation of mPRs
and stores and releases particular
[22,23]. In conclusion, progesterone acts as neuroprotective hormone and modulates the growth factors.
cancer growth via non-classical mechanism which is regulated by mPRs. Germinal vesicle breakdown
(GVBD): a process of dissolution of
the oocyte nucleus arrested in
Lu et al. [24] described the role of non-classical progesterone signals in maintaining pregnancy meiosis I prophase, leading to arrest
by altering functions in murine macrophages that lack classical nuclear progesterone recep- in meiosis II at the metaphase (MII)
tors. Notably, macrophages infiltrate the decidua in early pregnancy and at the beginning of stage. It occurs before ovulation in
labor [25]. Progesterone binds to mPR in macrophages and upregulates mRNA expression of response to the luteinizing hormone
surge.
cyclooxygenase 2 (COX2), tumor necrosis factor (TNF), and prostaglandin-endoperoxide Phosphatidylinositol 3-kinase
synthase 2 (PTGS2), leading to activation of mitogen-activated protein kinase (MEK1/2) (PI3K): a family of signal transducer
and protein kinase A (PKA), thus contributing to the inflammatory responses in macrophages enzymes that are regulate diverse set
of cellular functions including growth,
with subsequent regulation of labor [24]. This could contribute to early pregnancy failure, pre-
motility, survival, proliferation, and
term labor, and onset of labor at term even in the presence of adequate progesterone levels – differentiation.
specifically via a progesterone-induced inflammatory response mediated by non-classical Progesterone receptors (PRs):
signals operating through activation of mPR in macrophages. members of the nuclear steroid
hormone receptor family that are
stimulated by progesterone. Upon
Similarly, progesterone alters signal transduction pathways by activating mitogen-activated stimulation, PR induces transcription
protein kinases (MAPKs) and inhibiting adenylyl cyclase and cAMP formation via mPR. Upon of progesterone-responsive genes
progesterone binding, mPR participates in various signal transduction cascades, including that mediate several of the
physiological effects of progesterone.
stimulation of extracellular signal-regulated kinases 1/2 (ERK1/2 or p42/44) or p38 MAPKs, and
Progesterone response elements
stimulation of intracellular Ca2+[479_TD$IF] mobilization [26]. Ashley et al. demonstrated that ovine mPR is (PRE): DNA sequences in
stimulated by progesterone that induced mobilization of intracellular Ca2+ from the endoplas- progesterone-responsive genes that
mic reticulum (ER) to Ca2+-free medium and plays an important role in the ovine estrous cycle are bound by the progesterone
receptor–ligand complex.
[26,27]. Their findings suggest that progesterone-induced activation of mPR can also induce Ventral tegmental area (VTA): a
intracellular growth factors and stimulate transduction pathways without involving the classical group of dopaminergic neurons in
nuclear receptors and transcription pathways. midbrain that are located close to
the midline and project into various
other areas of brain. They regulate
Another mPR, progesterone receptor membrane component 1 (PGRMC1), was isolated from cognition, orgasm, motivation, drug
neural tissue and was distinct from known mPRs and nuclear PR [28] (Figure 2). PGRMC1 addiction, emotions, and psychiatric
comprises a single membrane-spanning domain and is primarily located in endomembranes disorders.
Ventromedial nucleus of the
(endoplasmic reticulum and Golgi apparatus) of porcine hepatocytes. Its localization was
hypothalamus (VMN): a nucleus of
supported by transfection studies in HEK cells [29]. PGRMC1 has three binding interfaces the hypothalamus with four
for SRC homology (SH) domains (two for SH2 and one for SH3), suggesting that PGRMC1 is subdivisions that regulates feeding,
involved in progesterone-mediated MAPK activation [30]. Through a PGRMC1-dependent fear, satiety, thermoregulation, and
sexual activity.
mechanism, progesterone inhibited T cell factor/lymphoid enhancer factor (TCF/LEF) activity
[31]. The ability of progesterone to suppress TCF/LEF activity was dependent upon the
sumoylation status of PGRMC1. PGRMC1 protein with mutated sumoylation sites diminished

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Membrane-associated PR

EGF Ca2+
EGFR

PR PKG
SRC NH2
P P
P P cAMP N N

PR p38 N N
PKC Mechanical
ERK O
stretching
PKA
O
O P O PR
OH
O
?
P PR
ER P P
p21 Facilitates
acrosome
p27 reacon
Stabilizaon
of mRNA

Oocyte Growth Producon of Producon of

development regulaon in inflammatory ECM protein
breast cancer cytokines
cells Nucleus

Figure 2. Progesterone-Mediated Non-Classical Signaling pathways. Key pathways involved in non-classical progesterone signaling are illustrated sche-
matically. Non-classical signaling has been shown to affect several processes including oocyte development, the growth of breast cancer cells, production of
inflammatory cytokines, and extracellular matrix (ECM) protein production induced by mechanical stimulation of myocytes. P indicates phosphorylation; the question
mark indicates that the mechanism is currently unknown. Abbreviations: EGF, epidermal growth factor; EGFR, EGF receptor; ER, estrogen receptor; ERK, extracellular
signal-regulated kinase; p38, p38 mitogen activated protein kinase; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; PR, progesterone receptor.

the ability of progesterone to suppress TCF/LEF activity [31]. These actions of PGRMC1 are
independent of the classical nuclear PRs and suggest non-classical progesterone effects.

A role for PGRMC1 has been suggested in the regulation of progesterone-induced neuronal
functions. Bashour [479_TD$IF]and Wray [32] showed that the inhibitory action of progesterone on GnRH
neurons was mediated through PGRMC1 because this action was blocked by AG-205, a
PGRMC1 ligand inhibitor, and not by RU486, an antagonist of classical nuclear PR signaling. In
addition, progesterone-induced brain-derived neurotrophic factor (BDNF) release in glial cells
was mediated by ERK5 signaling via PGRMC1 [33]. Furthermore, a role for PGRMC1 was
reported in reproductive biology. The serpin 1 mRNA binding protein 1 (SERBP1, also known
as CGI-55, RDA288, and PAIRBP1) associates with PGRMC1 to facilitate the antiapoptotic
effects of progesterone in granulosa cells via regulation of intracellular Ca2+[483_TD$IF] levels and activation
of protein kinase G [34]. Moreover, diminished PGRMC1 expression was present in chorion and
amnion of preterm pre-labor rupture of membranes (PPROM), suggesting a role in maintaining
progesterone-mediated fetal membrane integrity during pregnancy by non-classical proges-
terone signaling because fetal membranes lack classical PR [35].

Recent studies have focused on the role of PGRMC1 and mPR in non-classical progesterone
signaling [36]. Salazar et al. [37] examined the role of progesterone and its derivative, the mPR
agonist medroxyprogesterone acetate (MPA), in breast cancer. Studies identified the presence
of non-classical progestin receptors in nuclear PR-negative non-tumorigenic breast epithelial

4 Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy

 TEM 1237 No. of Pages 13

cell lines. Five isoforms of mPRs and PGRMC1 were detected in MCF10A breast epithelial cells,
and all progestin receptors except for mPRb were detected in MCF12A breast epithelial cells.
ERK and JNK phosphorylation were detected in both cell lines following treatment with
progesterone or its metabolites MPA and mPR-Agonist (mPR-Ag) [37]. AKT phosphorylation
was promoted by progesterone and MPA in the MCF12A cell line only, and no activation was
seen in MCF10A cells. Induction of p38 MAPK phosphorylation varied between progestins and
cell lines, suggesting that non-classical progesterone actions via cell membrane receptors are
present but may differ between breast epithelial cell lines[48_TD$IF].

The membrane-associated GABA type A (GABA-A) receptors also participate in progesterone-

mediated signaling [38] (Figure 1[485_TD$IF]). The progesterone metabolite allopregnanolone (3a-hydroxy-
5a-pregnan-20-one) acts as a positive modulator of GABA-A receptors and thus as an
inhibitory steroid. By contrast, pregnenolone sulfate (PREGS) is a negative modulator of the
GABA-A receptor and a positive modulator of the N-methyl-D-aspartate (NMDA) receptor, thus
acting as an excitatory steroid [486_TD$IF][39]. Allopregnanolone stimulates GABA-A receptor-activated
voltage-gated L-type Ca2+ channels and accelerates myelination in the rat hippocampus, as
well as human neural stem cell (hNSC) proliferation, revealing a role of ion-gated channel
receptors in non-classical progesterone actions in neuronal tissues [40]. Activation of mem-
brane-associated signaling complexes by progesterone and its metabolites thus activates
cytoplasmic signaling modules that lead to various progesterone mediated non-classical
actions in neuronal tissues, endothelium, cancer cells, and in the maintenance of pregnancy.

Progesterone and Intracellular Signaling Pathways and Proteins

Activation of second messenger systems and intracellular signaling pathways mediates non-
genomic progesterone actions. Progesterone facilitates the acrosome reaction in human
sperm by causing rapid influx of extracellular Ca2+[487_TD$IF] and efflux of intracellular Cl
through
activation of a sperm calcium channel called CatSper [41,42]. Ca2+[48_TD$IF] increases intracellular
cAMP that leads to phosphorylation of spermatozoa proteins [489_TD$IF][42]. The progesterone-mediated
rise in Ca2+ correlates with the fertilization rate in oligozoospermic subjects [43]. Furthermore,
the progesterone-facilitated acrosomal reaction in sperm could not be blocked by mifepristone
(RU486), consistent with non-classical progesterone signaling [491_TD$IF][44]. This conclusion is sup-
ported by the preserved fertility in male progesterone receptor knockout mice [492_TD$IF][45].

Recently, mPRa have also been shown to mediate progesterone actions in sperm fertilization
[493_TD$IF][46,47]. Lishko et al. [49_TD$IF][48] revealed the presence of an elusive non-genomic PR in human sperm
that was associated with progesterone-induced Ca2+[490_TD$IF] channel activation and successful
fertilization. Furthermore, Miller et al. [495_TD$IF][49] identified a progesterone receptor in human sperm
that was activated by a/b hydrolase domain-containing protein 2 (ABHD2) enzymes that bind
progesterone. ABHD2 expression was high in human sperm and acted as a lipid hydrolase
depleting endocannabinoid 2-arachidonoylglycerol (2AG) from the plasma membrane, and
thereby inhibiting the sperm calcium channel (CatSper). Removal of 2AG by ABHD2 led to Ca2+
influx and subsequent sperm activation. These progesterone-induced actions are extremely
rapid and appear to involve membrane-associated receptor-induced signaling complexes.

In addition, progesterone promotes oocyte maturation in Xenopus laevis by inducing resump-

tion of meiotic division in oocytes arrested in G2 phase through inhibition of adenylate cyclase
and subsequent reduction of intracellular cAMP levels [50]. This action is mediated by a rise in
intracellular Ca2+[496_TD$IF] that leads to an abrupt rise in intracellular pH and a reduction in membrane
conductance. During oocyte maturation transcription is suppressed and transcription inhibitors
do not alter the rate of oocyte maturation, suggesting that rapid non-genomic signal trans-
duction pathways contribute to oocyte maturation [497_TD$IF][51]. In addition, mPRb has been found to be
associated with resumption of meiosis during Xenopus oocyte maturation [498_TD$IF][52].

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Petralia et al. [49_TD$IF][53] demonstrated that progesterone-facilitated lordosis in rats and hamsters was
due to activation of dopamine type 1 receptors (D1) in the ventral tegmental area (VTA) and
subsequent increases in cAMP. These investigators infused rats and hamsters with the cAMP
analog 8-bromo-cAMP, or with the adenylyl cyclase inhibitor 20 ,50 -dideoxyadenosine, and
observed that in estradiol- and progesterone-primed rats and hamsters 8-bromo-cAMP
increased lordosis while 20 ,50 -dideoxyadenosine diminished lordosis. A role of mPRb was
also apparent in progesterone-facilitated lordosis because this action was lacking in mPRb
knockdown animals [50_TD$IF][54].

Balasubramanian et al. [501_TD$IF][55] explored progesterone-induced rapid and non-classical signaling in

ventromedial nucleus of the hypothalamus (VMN) of Sprague–Dawley rats. They reported
unchanged total PKC activity (Ca2+-dependent) as well as rapidly induced (within 30 minutes)
basal PKC activity (Ca2+-independent) following progesterone treatment that was inhibited by
30 minutes of preadministration of a PKC inhibitor. The authors suggested that non-classical
pathways were responsible for rapid changes in progesterone-mediated PKC activity rather
than changes in PKC phosphorylation. The same investigators examined progesterone-medi-
ated rapid changes in calcium and calmodulin-dependent protein kinase II (CaMKII) in the VMN
and preoptic area (POA) of female Sprague–Dawley rats and noted activation of CaMKII basal
activity at 30 minutes in VMN that was inhibited by KN-93, a CaMKII-specific inhibitor [56]. They
demonstrated no change in phosphorylation of Thr286 on CaMKII or in CaMKII protein levels,
revealing progesterone-induced rapid modulation in CaMKII activity in rats. These examples
suggest that there are progesterone-sensitive intracellular signaling cascades that are capable
of modulating several secondary messenger molecules [503_TD$IF](Table 1).

Progesterone Action in Major Signaling Pathways

Association of Progesterone with Tyrosine Kinase SRC
Progesterone actions originating at the plasma membrane are capable of stimulating several
cytoplasmic signaling cascades. PR[504_TD$IF]-B can activate the cytoplasmic tyrosine kinase SRC/RAS/
ERK pathway [50_TD$IF][57], and ligand binding to PR facilitates association between the SRC-homology
3 (SH3) domain of SRC tyrosine kinases and proline-rich regions in cytoplasm-localized
receptors [506_TD$IF][58]. This interaction stimulates rapid activation of the RAS/RAF1/MAPK pathway.
Myristoylation of SRC at its N [507_TD$IF]terminus promotes attachment to the inner surface of the plasma
membrane [508_TD$IF][59]. Thus, activation of SRC by progesterone could represent a plasma-membrane
effect.

In PR-null T47D-Y breast cancer cells, cyclin D1 is a key downstream target for PR-[509_TD$IF]B-mediated
MAPK activation. Biphasic regulation of cell cycle-dependent protein kinase by progesterone
leads to proliferative growth followed by arrest in breast cancer cells, loss of cyclins A and B,
and decreases in cyclins D1, D3, and E, with successive induction of the cyclin-dependent
kinase (CDK) inhibitors, p21 and p27(KIP1) [510_TD$IF][60]. Béguelin et al. [51_TD$IF][61] highlighted progestin-
induced cyclin D1 activation in breast cancer cells driven by ERBB2, a STAT3 coactivator that
stimulates the proliferation of breast cancer cells. Furthermore, progesterone-induced cyclin
D1 promoter activity was associated with AP-1, STAT3, PR, and ERBB2 functioning as an
enhanceosome in the regulation of breast cancer [512_TD$IF][62].

Recently, Nolan et al. [513_TD$IF][63] identified tumor necrosis factor superfamily member 11 (TNFSF11/
RANKL) and its receptor TNFRSF11A (also known as RANK) in progesterone-mediated
signaling in BRCA1 mutation carriers. The authors found two subsets of BRCA1 mutation
carriers: RANK+[502_TD$IF] and RANK
, of which RANK+ BRCA1 mutation carriers were associated with
the cellular and nuclear changes similar to the basal type of breast cancer. Treatment with
denosumab, a RANKL signaling inhibitor, in precancerous BRCA1mut/+ tissue led to inhibition of
progesterone-induced proliferation, suggesting involvement of progesterone-induced RANKL

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 TEM 1237 No. of Pages 13

Table 1. Non-Classical Progesterone Actions

Authors Organism/tissue/cell Signaling pathway Outcomes measured Physiological [426_TD$IF]Refs
characterstics outcome

Sumigama et al., Rhesus macaque Ca2+ influx Progesterone induced CatSper, (a Ca2+[342_TD$IF] [427_TD$IF]Acrosome reaction, [42]
2015 spermatozoa channel of the sperm that is pH-dependent) sperm capacitation
and spermatozoa
‘priming’

Zhu et al., 2003 Spotted seatrout Presence of a progestin [428_TD$IF](i) Cloning of a cDNA encoding protein [432_TD$IF]Oocyte maturation [43_TD$IF][14]
ovaries membrane receptor (40 kDa) rich in cysteine and leucine, has
properties of G protein-coupled membrane
receptors and fulfills the criteria for steroid
membrane receptor. It is present in plasma
membranes of reproductive tissues and has
a progestin[429_TD$IF]-specific single binding site
[430_TD$IF](ii) Progesterone induced reduced
production of cAMP, activation of [431_TD$IF]ERK1 and
ERK2, activation of MAP kinase

Baldi et al., 1995 Human spermatozoa Influx of extracellular Ca2+[43_TD$IF],
efflux of intracellular Cl
[435_TD$IF],
and cyclic AMP (cAMP)
[436_TD$IF](i) Tyrosine phosphorylation in sperm was Acrosome reaction [439_TD$IF][45]
induced by progesterone with subsequent
increase in tyrosine kinase(s)
[437_TD$IF](ii) Progesterone altered phospholipid
metabolism in sperm memrane and increase
in platelet-activating factor (PAF) formation
[438_TD$IF](iii) Not altered by RU486 or by bicuculline
and picrotoxin (GABA-A antagonists)

Shynlova et al., Myometrium of Inhibition of pro- [41_TD$IF](i) Elevated progesterone in late gestation Inhibition of term [45_TD$IF][81]
2008 pregnant wistar rats inflammatory chemokine inhibited [42_TD$IF]CCL2 mRNA gene expression and pre-term labor
such as MCP-1 [40_TD$IF](CCL2 [43_TD$IF](ii) 25% mechanical stretch of smooth
mRNA and proteins) muscle cells from rat myometrium lead to
increase in [42_TD$IF]CCL2 protein which was
inhibited by progesterone pre-treatment
[4_TD$IF](iii) Administration of RU486 (anti-progestin)
induced an elevation in [42_TD$IF]CCL2 mRNA

Shynlova et al., Myometrium of wistar Maintainance of insulin-like [436_TD$IF](i) Elevated IGFBP-6 gene expression during [48_TD$IF]Maintenance of [49_TD$IF][76]
2007 rats growth factor (IGF) and synthetic phase and parallels with pregnancy
IGF-signaling by affecting progesterone
binding proteins (IGFBP1- [46_TD$IF](ii) During late gestation, administration of
6) progesterone maintained IGFBP-6 mRNA
levels
[47_TD$IF](iii) RU486 treatment induced decrease in
IGFBP-6 gene expression

Lu et al., 2015 Mouse RAW 267.4 Alteration in macrophages [436_TD$IF](i) Progesterone induced activation of mPR [453_TD$IF]Maintenance of [24]
macrophage cell (lack nPR) induced lead to a [450_TD$IF]proinflammatory shift pregnancy
inflammatory response by (ii) Increased expression of cyclooxygenase
activation of membrane- 2 (COX2), IL[451_TD$IF]-1b, and TNF
associated PR (iii) Downregulation of oxytocin receptor and
mPRa
[452_TD$IF](iv) Activation of PKA and MEK1/2 pathway

Bashour and Wray Mice Presence of a progestin [436_TD$IF](i) Progesterone induced significant Neuroprotective [45_TD$IF][32]
2012 membrane receptor decrease in the activity of GnRH neurons by effects
(PGRMC-1) direct action or by stimulation of PKG
pathway
[430_TD$IF](ii) RU486 could not bock this progesterone
mediated activity
47_TD$IF]([ iii) AG-205, (PGRMC-1 ligand and inhibitor)
blocked the progesterone mediated GnRH
inhibition

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 TEM 1237 No. of Pages 13

Table 1. (continued)
Authors Organism/tissue/cell Signaling pathway Outcomes measured Physiological [426_TD$IF]Refs
characterstics outcome

Wang et al., 2005 Sprague Dawley rats
[45_TD$IF]Human embryonic brain
cortical stem cells
Elevation in intracellular
Ca2+[456_TD$IF] by GABA-A receptor
activated voltage-gated L-
type calcium channel
(VGLCC)
[457_TD$IF](i) Neuroactive progesterone metabolite: Promotion of [458_TD$IF][41]
allopregnanolone, increases proliferation of neurogenesis and
neuroprogenitor cells (NPCs) in rat restoration of
hippocampus and human neural stem cells neurons in brain
(hNSCs) in the cerebral cortex
[430_TD$IF](ii) Increased expression of mitosis
promoting genes and inhibition of cell
proliferation repression genes

Petralia and Frye Rats and hamsters Increase in cAMP Progesterone facilitates increase in cAMP by Lordosis [459_TD$IF][54]
2006 altering adenylyl cyclase through activation
of dopamine type 1 receptors (D1) in the
ventral tegmental area (VTA)

Lange et al., 1998 T47D-Y breast cancer
cells: Monoclonal PR-
negative and stably
expressing PR B-
receptors T47D-YB
cells
Potentiation of epidermal [460_TD$IF](i) Upregulation of EGFR, c-ErbB2 and c- Increase breast [465_TD$IF][67]
growth factor (EGF)- ErbB3 receptors [46_TD$IF]cancer cells
stimulated signaling [461_TD$IF](ii) Elevation in Stat5 protein levels sensitivity to the
pathways [462_TD$IF](iii) Stimulation of EGF-activated p42/p44/ peptide growth
MAPK, p38 MAP kinase, and JNK activities factors
[463_TD$IF](iv) Upregulation of cyclin D1, cyclin E, and
p21

Béguelin et al., 2010 Female BALB/c mice
with C4HD tumor line
Stat3/ErbB-2 [46_TD$IF](i) Nuclear translocation of ErB-2 induced by Proliferation of [467_TD$IF][62]
transcriptional complex PR breast cancer cells
drives cyclin D1 promoter (ii) ErbB-2 acts as a Stat3 coactivator
activation (iii) Recruitment of PR with Stat3 and ErbB-2
to the cyclin D1 promoter

signaling in the development of breast cancer in BRCA1 mutation carriers [63]. Ballare et al. [514_TD$IF][64]
proposed that progesterone-induced SRC/MAPK activation is mediated by the indirect action
of the SH2 domain of SRC with phosphotyrosine 537 of estrogen receptor a (ERa). In these
experiments, progesterone-mediated SRC/MAPK activation was dependent on the interaction
of unliganded ERa with the proline-rich sequence domain of PR.

Progesterone and the p44/p42 MAPK Pathway

Progesterone stimulates the p44/p42 MAPK pathway to influence cell functions. Bagowski
et al. [51_TD$IF][65] demonstrated that progesterone induces non-genomic signaling and oocyte matu-
ration via classical PR in Xenopus laevis. The researchers found that p42 MAPK was associated
with PR, and a constitutively active MAPK mutant phosphorylated PR in vitro. Progesterone-
induced maturation was delayed by a PI3K inhibitor. Their findings demonstrated that the
activity of p42 MAPK and PI3K is required for oocyte maturation via nPRs.

In addition, PR-null T47D-Y breast cancer cells treated with R5020 showed activation of
epidermal growth factor (EGF) signaling. Progesterone potentiated the effects of EGF by
upregulating the EGFR, ERB2, and ERBB3 receptors, and augmented EGF-stimulated tyrosine
phosphorylation. The authors concluded that breast cancer cells were primed by progesterone
for growth signals by EGF-stimulated p44/p42 MAPK [516_TD$IF][66]. The synergy between EGF and
progesterone was documented with a sixfold increase in EGF mRNA levels [517_TD$IF][67]. Progesterone
attenuated its own effects on breast cancer cells by autoinhibition [516_TD$IF][66]. Notably, crosstalk
between progesterone, growth factors, and their receptors may explain the responsiveness of
breast cancer cells to growth factors even after these cells become resistant to steroid
hormones.

8 Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy

 TEM 1237 No. of Pages 13

Furthermore, fluctuations in [518_TD$IF]nuclear PR mRNA and protein levels accompanied progesterone

treatment of breast cancer cells. These fluctuations can be explained by MAPK-induced
phosphorylation of PR Ser294 leading to its degradation by the 26S proteasome [519_TD$IF][68]. Inhibitors
of p42 and p44 MAPKs blocked this PR degradation pathway [519_TD$IF][68].

p38 MAPK Activation and Progesterone

Meiotic maturation involves resumption of meiosis in oocytes arrested in the G2 phase of
meiosis I. Progesterone induces M phase entry and meiotic maturation of Xenopus oocytes by
activation of p38 MAPKs. Perdiguero et al. [520_TD$IF][69] injected a constitutively active mutant of the p38
activator mitogen-activated protein kinase kinase 6 (MKK6-DD) in oocytes and found increased
germinal vesicle breakdown (GVBD) upon progesterone stimulation, with GVBD proceeding
3–4 h earlier than in control oocytes. While >80% GVBD was observed in MKK6-DD-injected
oocytes, <10% of control-injected oocytes achieved GVBD. Coexpression of p38 and MKK6-
DD was sufficient to trigger maturation. Inhibition of p38a/b MAPK with SB203580 did not alter
the ability of MKK6-DD to increase progesterone-induced maturation, suggesting that the
effect was dependent on different p38 MAPK isoform(s). Activation of endogenous p38 MAPKs
in progesterone-stimulated oocytes was confirmed by p38 phosphospecific antibodies [520_TD$IF][69].
Moreover, progesterone stimulation of MKK6-DD-injected oocytes showed accelerated acti-
vation of Xp42Mpk1 MAPK and CDC2–cyclin B. Injection of the catalytically inactive mutant
MKK6-DA affected progesterone-induced maturation, supporting the role of Xp38g/SAPK3
signaling pathway in progesterone-induced oocyte maturation [520_TD$IF][69].

The T47D-YB breast cancer cell line constitutively expresses nuclear receptor PR-B. The
proliferative effects of EGF on these cells were enhanced by pretreatment with progesterone
[510_TD$IF][60]. In T47D-YB cells, treatment with progesterone accelerated the first mitotic cell cycle, but
induced arrest in late G1 phase that was followed by induction of inhibitors of CDK, p21, and
KIP1. Lange et al. [516_TD$IF][66] described this effect of progesterone in breast cancer cells as a priming
agent that activates EGF-stimulated p38 MAPK activity. In addition to stimulation of rapid
membrane-associated signaling, evidence from these examples indicates that non-classical
progesterone actions take place via independent progesterone-induced modulation of cyto-
plasmic signaling cascades. Collectively, these observations, and others not cited, suggest that
progesterone-induced cytoplasmic signaling pathways might impact on significant physiologi-
cal processes.

Integration of Endocrine and Mechanical Signals and Progesterone Effects

As previously mentioned, progesterone is an essential hormone for establishing and main-
taining pregnancy [521_TD$IF][70]. A new model integrating mechanical and endocrine signals has been
recently proposed for phenotypic changes in myometrium during pregnancy and labor in the rat
[52_TD$IF][71]. According to this model, the mechanical tension of the uterus caused by a growing fetus
works in conjunction with endocrine regulation via the fetal hypothalamic–pituitary–adrenal–
placental axis and progesterone actions on the myocyte. Mechanical signaling induces bio-
chemical and molecular alterations within the myometrium, leading to myometrial growth and
remodeling [523_TD$IF][72]. Progesterone plays an important role in maintenance of pregnancy by
integrating mechanical signals through extracellular matrix (ECM) proteins, altering gene
expression, growth factors, binding proteins, and inflammatory cascades that include non-
classical progesterone signaling. Decreased progesterone levels at term and increased
mechanical tension from the growing fetus causes changes in myocyte ECM proteins
(decreased fibrillar collagens and increased basement membrane components) and their
integrin receptors, without involving the classical nuclear PR.

Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy 9

 TEM 1237 No. of Pages 13

During pregnancy the myometrial phenotype evolves over a series of phases, including an Outstanding Questions
early proliferative phase, an intermediate synthetic phase of cellular hypertrophy and matrix What progesterone-mediated non-
expansion, a third phase during which myocytes acquire a contractile phenotype, and a final classical signals play a role in the inte-
gration of mechanical stimulation and
phase of highly active cells supporting labor. The early proliferative phase is associated with phenotypic modulation of uterine myo-
upregulation of [482_TD$IF]antiapoptotic factors such as BCL2 and altered expression of insulin-like cytes during pregnancy and labor?
growth factor (IGF) family proteins. Investigators suggest that myometrial hyperplasia during
the proliferative phase is regulated by the phosphatidylinositol 3-kinase (PI3K)–AKT– What are the other various key medi-
ators of progesterone non-classical
mTOR signaling pathway activated by estrogen [524_TD$IF][73]. The synthetic phase was characterized
pathways such as non-nuclear pro-
by increased gene expression of ECM proteins collagens (I and III) and elastin, in addition to gesterone receptors, ECM proteins,
other structural proteins. Progesterone maintained collagen III mRNA levels during preg- or growth factors that play a role in
nancy, and reduced fibronectin and laminin mRNA levels that were elevated during labor [52_TD$IF][74]. the mechanical response of the cells?
Elevated serum progesterone levels prevented a reduction in IGF binding protein 6 (IGFBP-6)
How can the findings of progesterone-
mRNA levels, hence increasing serum IGFBP-6 levels to maintain pregnancy [526_TD$IF][75]. During the
mediated actions via non-classical sig-
contractile phase there are dramatic changes, with increased expression of basement nals be successfully translated to the
membrane matrix proteins such as collagen IV, laminin b2, and fibronectin (FN) associated prevention, diagnosis, and treatment
with a reduction in progesterone levels [523_TD$IF][72]. In the labor phase, increased myometrial tension of different physiological disorders?

exerted by the growing fetus and reduction of progesterone induce the expression of
contraction-associated proteins [CAPs; e.g., the gap junction protein connexin 43 (Cx43),
sodium channel, prostaglandin F receptor, and oxytocin receptor] to enhance myometrial
contractility [527_TD$IF][76,77]. In addition, the labor phase is associated with changes in focal adhesion
signaling via reduction of phosphorylation of focal adhesion kinase (FAK)/paxillin, thereby
altering the interaction between myometrial smooth muscles and the underlying matrix. A role
of mPR signaling leading to progesterone withdrawal and thus shifting the balance towards
the contractile phase of the labor has been shown in the initiation of the labor [528_TD$IF][78]. Further
elucidation of the molecular biology behind progesterone-mediated mechanical responses of
myocytes may supplement knowledge of non-classical progesterone pathways, and possibly
guide prevention and treatment of pre-term labor.

Furthermore, myometrial cells release the proinflammatory chemokine MCP-1 (also known as
C-C chemokine motif ligand 2, CCL2). MCP-1 plays an important role in uterine inflammation
for the promotion of labor [529_TD$IF][79]. Notably, progesterone suppresses MCP-1 expression as
evidenced by the inhibition of myometrial MCP-1 during pregnancy. Therefore, the local
production of MCP-1 in the uterus is tightly regulated by progesterone and mechanical
stretching of the uterus during gestation [530_TD$IF][80]. Mechanical stretch of the uterine wall due to
the growing fetus contributes to increased expression of proinflammatory cytokines (IL-6, IL-
1a) and various chemokines (CCL2, CXCL1, and CXCL2). Application of static mechanical
stretch to immortalized human myometrial tissue led to significant increase in CCL2 [531_TD$IF][80–82].
Conflicting results have been shown in different studies regarding progesterone-mediated
antagonism of stretch-induced CCL2 production and modulation of the inflammatory response
[532_TD$IF][83].

Taken together, these data suggest that progesterone inhibits labor, uterine proliferative
growth, and the inhibition of CAP gene expression during gestation [53_TD$IF][84]. Progesterone actions
via non-classical signaling pathways may provide future directions in the prevention of pre-term
labor by targeting these pathways [534_TD$IF][85].

Concluding Remarks and Future Perspectives

Recent studies have emphasized both the complexity and necessity of progesterone action.
Pregnancy maintenance, ovulation, and proper brain function are a few noteworthy examples
of physiological progesterone action. Importantly, progesterone action is often transduced
through non-classical signaling pathways including mPRs, intracellular signaling, mechanical
signaling, and other key membrane receptors to accomplish these physiological effects.

10 Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy

 TEM 1237 No. of Pages 13
Although recent studies have begun to elucidate non-classical signaling pathways, current
understanding of these pathways remains incomplete. Future research efforts should focus on
illuminating the precise signaling events in these non-classical pathways, and by doing so may
provide novel therapeutic targets and strategies (see Outstanding Questions).
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