P4 is a key hormone, which contributes to the UF pathogenesis. However, the molecular mechanism by which P4 promotes the UF development is largely unknown. In this study, we used PrMyoN and PrMyoN cell model system and characterized the expression pattern of P4 response genes in response to P4 treatment, which may contribute to increased risk of UF development.
Previous study showed that cultured UF cells had an increased response to P4 compared to cultured normal myometrial cells [
30]. This study also showed that P4 receptor mRNA is highly expressed in UF cells as compared the cells from adjacent myometrium. In our study, we focused on P4 response in primary cells from normal myometrium (MyoN) and at-risk myometrium (MyoF). We demonstrated that the expression of PR was higher in PrMyoF as compared to PrMyoN cells. The differential response of PrMyoN and PrMyoF to P4 seems to be attractive. Among the genes we detected, we found two types of changes in response to P4 treatment, gain in induction and gain of repression respectively. These results suggested that the network of P4/PR signaling was varied between PrMyoN and PrMyoF and the primed PrMyoF turned out to be hyper-sensitive to P4, which might lead to increased risk of UF development.
In this study, the expression of 15 P4-responsive genes was examined in PrMyoF and PrMyoN cells using q-PCR analyses. Five of these genes including FOXO1A, CYP26a1, SCGB2A2, MMP11 and Bcl 2 showed significant up regulation in response to P4 treatment. The other seven genes exhibited a significant down regulation, these genes include CANP6, MT1E, ADHL5, Aldh1a1, KIK6, HHI, CIDEc. However, the expression of MT2A, MTG2 and calcitrone was not altered in response to P4 treatment.
Expression of genes control the apoptosis in response to progesterone
Apoptosis is a morphologic pattern of cell death [
31]. There are multiple genes responsible for regulating this process. Korsmeye [
32], reported that the
Bcl-2 proto-oncogene has the ability to block apoptotic cell death in multiple contexts. Increase in expression of
Bcl-2 in transgenic models will result in evasion of normal cell death mechanisms leading to accumulation of cells and tumor formation [
33].
Previous studies showed that
Bcl-2 protein expression was predominant in UF cells compared to that in normal myometrium cells [
34]. The expression of
Bcl-2 protein in normal myometrium cells was very low that raised the possibility that normal myometrium cells may be more susceptible to apoptotic cell death. In addition, UF cells exhibited increased expression of
Bcl-2 protein in response to P4 treatment. But the expression of
Bcl-2 protein in cultured normal myometrium cells was not affected by P4 treatment. Here in our study
Bcl-2 gene expression in at-risk myometrium tissues from the uterus with UFs was remarkably augmented by P4 treatment and this change was not found in normal myometrial cells.
Another gene that responsible also for apoptosis is
FOXO1A, it is a member of the
FOXO subfamily of Forkhead transcription factors [
35]. According to the previous study, activated
FOXO proteins induced expression of genes that encode for proteins involved in cell cycle inhibition [
36]. Our study showed that this gene exhibited hyper-response in PrMyoF cells after P4 treatment. Another study determined the progestin effect in
FOXO1 expression and its activity in the endometrium during endometrium menstrual cycle. They showed that progestin enhanced
FOXO1 mRNA levels in mid- and late-secretory endometrium [
37]. In addition,
FOXO1A was considered as a key transcription factor responsible for mediating apoptosis of decasualized human endometrial stromal cells (HESC) in response to progesterone withdrawal during the menstrual cycle by inducing the cell death. Moreover, this study explains the effect of admission of medroxyprogesterone acetate (MPA, a synthetic progestin) in enhancing the expression of
FOXO1A in differentiating human endometrial cells. MP also simultaneously induced cytoplasmic retention and inactivation of this gene. Withdrawal of the MPA from decidualized HESCs resulted in rapid nuclear accumulation of
FOXO1A gene, therefore leading to activation of apoptosis and cell death [
37]. Similar finding was observed in PrMyoF cells, where the expression of
FOXO1 was markedly increased in response to progesterone treatment, which provide a favorable condition for the pathogenesis of UFs.
SCGB2A2 (Secretoglobin family 2A member 2) was considered as uteroglobin-related protein, which controls cell cycle and DNA replication. It was originally detected by differential RNA expression levels in Breast Cancer biopsies [
38]. Previous studies demonstrated the effect of
SCGB overexpression on cell proliferation in other human diseases and ovarian carcinoma. The role of
SCGB2A2 in patho-physiology of the ovarian tumor was identified [
39]. The overexpression of
SCGB2A2 is positively correlated with the FIGO stage, the tumor grade and the mitotic index of the ovarian cancer [
40]. In our study, although no significant expression of
SCGB2A2 was observed in ProMyoF and PrMyoN cells, ProMyoF cells was remarkably augmented by P4, which was not the same with ProMyoN cells. This study suggests that P4 might promote the UF development by increased cell proliferation and enhancement of the DNA replication via
SCGB2A2.
The cell death-inducing DFF45-like effector (CIDE) family includes
CIDEa, CIDEb, and
CIDEc. It has been reported that the (CIDE) family plays an important role in lipid and fat metabolism [
40‐
42]. Previous studies have reported that
CIDEa, CIDEc were highly expressed in adipose tissue, and in skeletal muscle. I
CIDEc is capable of inducing apoptosis in mammalian cells [
43]. DFF45 is a subunit of the DNA fragmentation factor which is cleaved by active caspase-3 during apoptosis. The main function of
CIDEC is energy homeostasis, and its absence may result in insulin resistance, and resistance to diet-induced obesity [
44]. Here in our study this gene showed gain of repression in response to P4 as a marker of decrease in the apoptosis that might be involved in UF development.
Another gene that showed gain in regression in our study was
CANP6. It is calcium-activated cysteine proteinases. Calpains have been involved in many biological events including regulation of the cell cycle, apoptosis, cell adhesion and motility [
45,
46]. So the regression of this gene will decrease the apoptosis as well as down regulation of cell cycle all together will favor the development of UFs.
Expression of genes control the retinoic acid in response to progesterone
RA, is the natural metabolite of vitamin A. Previous studies showed that RA signaling played an important role in the female reproductive trace function [
47].
ADH5 and
ALDH1a1 are RA synthesis enzymes and
CYP26a1(cytochrome P450, family 26, subfamily a, polypeptide1) is a RA catabolizing enzyme. Previous studies demonstrated that the expression of these are altered during preganyc which may be related to progesterone signaling
. ADH5 expression was increased by 2.5 folds during pregnancy. The expression of
ALDH1a1 in the endometrial glandular compartment was increased on gestational early days until the implantation phase. The expression of
CYP26a1 was strongly detected in the uterine epithelium. Moreover, these studies indicated that early pregnancy needed the synthesis and degradation of RA to be balanced to allow RA signaling to prepare for implantation without harmful effects on the embryo [
48]. Our result has demonstrated that RA synthesis genes (
ADH5, ALDH1a1) show gain in repression in response to P4, and RA catabolic enzyme (
CYP26a1) were rapidly gain induction by the P4-PR axis. This might result in increasing retinoic acid catabolism and decrease in Vitamin A in the myometrium tissue. All this together will favorable the proliferation of the myometrium, which provide pro-fibroid condition to increase the risk of UF development.
Expression of other genes in response to progesterone
In human, over 20 functional Matrix metalloproteinases
MMPs have been identified [
48].
MMPs are zinc endopeptidases capable of releasing the growth factors that are bound to the extracellular matrix (ECM) [
49] regulating cell-matrix and cell-cell interactions. Matrix metalloproteinase 11(
MMP11) is responsible of serpins cleavage and so it stimulate the development of
tumor [
50,
51]. Our study showed that
MMP-11 mRNA was significantly increased in myometrium cells from uterus with UFs compared with myometrium cells of normal one. Also
gene expression of
MMP11 showed significant gain in induction after P4 treatment in PrMyoF cells. Previous study demonstrated increased expression of
MMP-11 in UFs as compared to myometrium.
KLK6 belonging to kallikreins gene family is a serine protease [
52‐
54]. It is down-regulated by the P4-PR axis.
KLk6 is responsible for regulation of the inflammatory process and vascular permeability, and edema [
55]. Previous studies in mouse graved uterus showed that this gene was upregulated by the P4-PR axis signaling suggesting the important role of this gene in the implantation of the embryo in the uterus [
27]. In our study, the repression of this gene in response to P4 may result in the formation of the UFs by losing the regulation of the inflammatory process.
Indian hedgehog
(HHI), one of the Hedgehog family of ligands, is a P4-regulated gene in the uterus [
56,
57]. It plays a role in down regulation of cellular division [
58]. In the human endometrium, the role of Hedgehog signaling in UFs is largely unknown [
59].
HHI gene shows a significant decrease in expression s between the early secretory to the mid secretory phase. It also plays a role in embryo implantation by regulation of stromal cell proliferation and inhibition of epithelial E2 signaling. In addition, Hedgehog signaling was involved in the women with endometriosis [
60‐
62]. and in endometrial cancer [
63]. Here in our study this gene showed a gain of repression in the PrMyoF cells in response to P4, suggesting that this change may be involved in the increased risk of UF pathogenesis.
Metallothioneins
(MTs) comprise a family of genes clustered on chromosome 16q that bind to heavy metal ions and minimize reactive oxygen species. Previous studies demonstrated low MT expression in endometrium of women with endometriosis [
29]. In our study
MT1E is the only one we detected that showed significant repression with P4 treatment in the PrMyoF cells.
The location of the Calcitonin gene is in non-neuronal tissues. Its define function remains unclear, but previous studies identified their role in cardiovascular system as it exhibited a potent vasodilator effect [
64]. There are many other researches done on calcitonin effect on the heart. These researchers show its role in prevention of ischemia as well as endotoxic shock [
65]. These shock can be done by the suppressor effect of calcitonin on some pro-inflammatory cytokines production [e.g., macrophage inflammatory protein-2 (
MIP-2) and keratinocyte chemoattractant (KC)] [
66,
67]. Moreover, calcitonin has a protective effect against ischemia [
68]. So the decrease in its expression may result in ischemia stimulation of the inflammatory reaction. However, in our study it showed gain in repression suggesting the complex role of this gene in response to P4 treatment.