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Decidualisation of human endometrial stromal cells is associated with increased expression and secretion of prorenin - PubMed

  • ️Thu Jan 01 2015

Decidualisation of human endometrial stromal cells is associated with increased expression and secretion of prorenin

Eugenie R Lumbers et al. Reprod Biol Endocrinol. 2015.

Abstract

Background: In pregnancy, the decidualised endometrium expresses high levels of prorenin and other genes of the renin-angiotensin system (RAS) pathway. In this study we aimed to determined if the RAS was present in endometrial stromal cells and if decidualisation upregulated the expression of prorenin, the prorenin receptor ((P)RR) and associated RAS pathways. Immortalised human endometrial stromal cells (HESCs) can be stimulated to decidualise by combined treatment with medroxyprogesterone acetate (MPA), 17β-estradiol (E2) and cAMP (MPA-mix) or with 5-aza-2'-deoxycytidine (AZA), a global demethylating agent.

Methods: HESCs were incubated for 10 days with one of the following treatments: vehicle, MPA-mix, a combination of medroxyprogesterone acetate (MPA) and estradiol-17β alone, or AZA. Messenger RNA abundance and protein levels of prorenin (REN), the (P)RR (ATP6AP2), angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AGTR1), vascular endothelial growth factor (VEGF), and plasminogen activator inhibitor-1 (PAI-1) were measured by real-time PCR and ELISA's, respectively. Promyelocytic zinc finger (PLZF) and phospho-inositol-3 kinase (PIK3R1) mRNA abundances were also measured.

Results: HESCs expressed the prorenin receptor (ATP6AP2), REN, AGT, ACE and low levels of AGTR1. MPA-mix and AZA stimulated expression of REN. Prorenin protein secretion was increased in MPA-mix treated HESCs. E2 + MPA had no effect on any RAS genes. MPA-mix treatment was associated with increased VEGF (VEGFA) and PAI-1 (SERPINE1) mRNA and VEGF protein.

Conclusions: An endometrial prorenin receptor/renin angiotensin system is activated by decidualisation. Since (P)RR is abundant, the increase in prorenin secretion could have stimulated VEGF A and SERPINE1 expression via Ang II, as both ACE and AGTR1 are present, or by Ang II independent pathways. Activation of the RAS in human endometrium with decidualisation, through stimulation of VEGF expression and secretion, could be critical in establishing an adequate blood supply to the developing maternal placental vascular bed.

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Figures

Fig. 1
Fig. 1

Prorenin/prorenin receptor activated pathways. Prorenin bound to the prorenin receptor ((P)RR) unfolds and can catalyse the formation of Ang I from AGT promoting the formation of downstream Ang peptides such as Ang II. Prorenin bound to the (P)RR activates ERK1/2 signalling, p38/HSP27 signalling, and causes translocation of PLZF to the nucleus which activates p85α-PI3K and down regulates (P)RR expression. The (P)RR can also activate the Wnt/β-catenin cascade

Fig. 2
Fig. 2

Effects of E2 + MPA, MPA-mix and AZA on the renin angiotensin system in HESCs. a REN mRNA levels were significantly higher in HESCs treated with MPA-mix compared with all other treatments (P < 0.0001). REN mRNA expression was also increased in AZA treated HESCs compared with control and E2 + MPA treated cells (P < 0.008 and P < 0.014, respectively). b AGT mRNA levels were significantly increased in MPA-mix treated cells compared with all other treatments (all P < 0.02). c Prorenin protein (ng/ml) secreted into media at 2 days. There was no difference between CON and treatments but MPA-mix had significantly more prorenin than E2 + MPA alone (P = 0.045). d Prorenin protein (ng/ml) secreted into media at 10 days. Prorenin levels were significantly increased in the supernatant at 10 days in MPA-mix treated samples compared with all other treatments (P < 0.022). AZA treatment was associated with increased prorenin levels in supernatant compared with E2 + MPA alone (P < 0.015). Different superscripts denote differences between groups. CON, treated with vehicle alone; E2 + MPA, treatment with 10 nM 17β-estradiol (E2) and 1 μM medroxyprogesterone acetate (MPA); E2 + MPA + cAMP, treatment as for E2 + MPA plus 0.5 mM cAMP

Fig. 3
Fig. 3

Effects on the expression of genes and proteins known to be responsive to stimulation by the prorenin receptor/prorenin angiotensin system. a and c Relative mRNA abundance of PAI-1 (SERPINE1) and its protein. Both E2 + MPA alone treatment and MPA-mix treatment stimulated expression of PAI-1(SERPINE1) in HESCs (P < 0.001). AZA had no effect. b and d MPA-mix treatment stimulated the expression of VEGF Aand its protein compared with all other treatments (all P < 0.001). Different superscripts denote differences between groups. CON, treated with vehicle alone; E2 + MPA, treatment with 10 nM 17β-estradiol (E2) and 1 μM medroxyprogesterone acetate (MPA); E2 + MPA + cAMP, E2 + MPA + cAMP, treatment as for E2 + MPA plus 0.5 mM cAMP

Fig. 4
Fig. 4

Effect of E2 + MPA, MPA-mix (E2, MPA + cAMP) and AZA on the expression of genes known to interact with prorenin/prorenin receptor that are independent of Ang II. a PLZF (Promyelocytic leukaemic zinc finger protein) mRNA was not detected in CON or AZA treated cells but was present in E2 + MPA and MPA-mix treated cells (P < 0.001 for both treatments compared with CON and AZA). Expression of PLZF in MPA-mix treated cells was much greater than in E2 + MPA treated cells (P < 0.001). b p85α-PI3kinase (PIK3R1) mRNA expression was significantly increased in MPA-mix treated HESCs compared with those treated with E2 + MPA alone (P = 0.007) although it was not greater then control (P < 0.07). Different superscripts denote differences between groups. CON, treated with vehicle alone; E2 + MPA, treatment with 10 nM 17β-estradiol (E2) and 1 μM medroxyprogesterone acetate (MPA); E2 + MPA + cAMP, treatment as for E2 + MPA plus 0.5 mM cAMP

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