Molecular basis of senescence transmitting in the population of human endometrial stromal cells

Abstract

Hormone-regulated proliferation and differentiation of endometrial stromal cells (ESCs) determine overall endometrial plasticity and receptivity to embryos. Previously we revealed that ESCs may undergo premature senescence, accompanied by proliferation loss and various intracellular alterations. Here we focused on whether and how senescence may be transmitted within the ESCs population. We revealed that senescent ESCs may induce paracrine senescence in young counterparts via cell contacts, secreted factors and extracellular vesicles. According to secretome-wide profiling we identified plasminogen activator inhibitor -1 (PAI-1) to be the most prominent protein secreted by senescent ESCs (data are available via ProteomeXchange with identifier PXD015742). By applying CRISPR/Cas9 techniques we disclosed that PAI-1 secreted by senescent ESCs may serve as the master-regulator of paracrine senescence progression within the ESCs population. Unraveled molecular basis of senescence transduction in the ESCs population may be further considered in terms of altered endometrial plasticity and sensitivity to invading embryo, thus contributing to the female infertility curing.

Keywords: PAI-1; SASP; endometrial stromal cells; senescence.

Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

Figures

Figure 1

2D co-culturing with senescent ESCs negatively affects surrounding cells. (A) Experimental scheme of co-culturing of young mCherry-labeled ESCs with unlabeled young or senescent ones in 2D condition. (B and C) Growth curves of mCherry-labeled ESCs (co-cultured either with young or senescent cells) before and after reseeding, respectively. Cell number was determined by FACS at the indicated time points. (D) and (E) Cell size and autofluorescence of mCherry-labeled ESCs measured by FACS after 10 d of co-culturing. Forward scatter (FS) reflects the average cell size. Values are M ± S.D. (N=3). * – p<0.05, ** – p<0.01, *** – p<0.005 by Student’s t-test.

Figure 2

3D co-culturing with senescent ESCs negatively affects surrounding cells. (A) Experimental scheme of co-culturing of young mCherry-labeled ESCs with unlabeled young or senescent ones in 3D condition. (B) Representative photographs of spheroids formed from a mixture of unlabeled and mCherry-labeled ESCs. Scale bars of all images are 500 μm. (C–E) Growth curves, cell size and autofluorescence of mCherry-labeled ESCs, respectively. Cells were cultured in spheroids during 4 d, trypsinized and cultured for additional 5 d up to analysis by FACS. Forward scatter (FS) reflects the average cell size. Values are M ± S.D. (N=3 for (C) and (D), N=2 for (E)). ** – p<0.01, *** – p<0.005 by Student’s t-test.

Figure 3

SASP from senescent ESCs triggers senescence in young cells. Ctr – young ESCs cultured in standard conditions. CM-sen treated – ESCs exposed to condition medium from senescent cells. Sen – senescent ESCs. (A) Experimental scheme of ESCs CM-sen treatment. (B) and (C) Growth curves of ESCs before and after reseeding, respectively. Cell number was determined by FACS at the indicated time points. (D–F) Cell size, autofluorescence and intracellular ROS levels of ESCs determined by FACS after 9 d of CM-sen treatment. Forward scatter (FS) reflects the average cell size, DCF fluorescence reflects ROS levels by oxidation of H2DCF-DA. Values are M ± S.D. (N=3). * – p<0.05, ** – p<0.01, *** – p<0.005 by Student’s t-test. (G) SA-β-Gal staining of Ctr and CM-sen treated ESCs. After 7 d of treatment ESCs were reseeded and additionally cultured for 3 d in order to perform staining of non-confluent cultures. (H) Quantification of SA-β-Gal activity values (G). Values presented as M and 95 % C.I. (N=100). *** – p<0.005 by Mann-Whitney test. (I) Wound healing analysis of ESCs cultured in standard conditions or pre-exposed to CM-sen for 4 d. Cells’ monolayers were scratched and migration activity of cells were estimated at the indicated time points. Scale bars of all images are 500 μm. (J) Western blot analysis of ATM, H2A.X and p53 phosphorylation levels and p21 protein expression performed after 7 d of treatment. Representative results of the three experiments are shown in the Figure. GAPDH was used as loading control.

Figure 4

Soluble factors and extracellular vesicles secreted by senescent ESCs trigger senescence in young cells. Sen – senescent ESCs. Ctr – young ESCs cultured in standard conditions. SF-sen or EV-sen treated – young ESCs exposed to soluble factors and extracellular vesicles secreted by senescent ESCs, respectively. (A) Western blot analysis of CD63 and HSP70 total proteins amount in Sen and EV-sen lysates. (B) and (C) Growth curves and cell size of Ctr, SF-sen and EV-sen treated ESCs determined by FACS. Forward scatter (FS) reflects the average cell size evaluated after 6 d of exposure. Values are M ± S.D. (N=3). *** – p<0.005 by ANOVA with Tukey HSD versus Ctr. (D) Western blot analysis of p53 and Rb phosphorylation levels and p21 protein expression performed after 7 d of treatment. Representative results of the three experiments are shown in the Figure. GAPDH was used as loading control. (E) SA-β-Gal staining of Ctr, SF-sen and EV-sen treated ESCs. After 7 d of treatment ESCs were reseeded and additionally cultured for 3 d in order to perform staining of non-confluent cultures. (H) Quantification of SA-β-Gal activity values (E). Values presented as M and 95 % CI (N=100). *** – p<0.005 by ANOVA with Tukey HSD versus Ctr.

Figure 5

Proteomic analysis of ESCs secretome. CM-ctr and CM-sen – conditioned media from young or senescent ESCs, respectively. (A) Venn diagram presentation of all peptides identified within CMs by LC-MS/MS. (B) Volcano plot of proteins differentially secreted by Ctr and Sen ESCs. (C) and (D) Functional enrichment analysis in GO BP terms of up- and down-regulated proteins in CM-sen versus CM-ctr. Identified processes are organized in modules based on common parent GO terms presented in legends. To control the false discovery rate (FDR) to correct the p-value the Benjamini method was applied. Black line indicates threshold at p=0.05. (E) and (F) Levels of top up- and down-regulated proteins in CM-sen versus CM-ctr, respectively. Processes involving PAI-1 are marked with asterisk (*).

Figure 6

Altered PAI-1 secretion levels modulate SASP-induced senescence propagation within ESCs population. Ctr – young ESCs cultured in standard conditions. Sen – senescent ESCs. CM-ctr and CM-sen – conditioned media from young or senescent ESCs, respectively. SF-sen or EV-sen treated – young ESCs exposed to soluble factors and extracellular vesicles secreted by senescent ESCs, respectively. LV, KO and SAM – gene-modified ESCs with unaffected, down-regulated and overexpressed SERPINE1 gene. CM-sen LV, KO, SAM treated – young ESCs exposed to conditioned media from senescent gene-modified cells. (A, B) Western blot analysis and ELISA of PAI-1 composition in CM-ctr and CM-sen. For western blot CMs were collected from equal numbers of cells and in equal volumes of media. ELISA values presented as M ± S.D. (N=4). *** – p<0.005 by Student’s t-test. (C, D) Western blot of PAI-1 content in SF-sen and EV-sen obtained as described in Experimental procedures section. CD63 was used as EV marker protein. For western blot CMs and SF were collected from equal numbers of cells and in equal volumes of media. (E, F) PAI-1 expression levels in LV, KO and SAM estimated by RT-PCR and western blot, respectively. Values are M ± S.D. (N=3). *** – p<0.005 by ANOVA with Tukey HSD versus Ctr. (G) PAI-1 levels in LV, KO, SAM CM-sen by ELISA. Values are M ± S.D. (N=2). ** – p<0.01 by ANOVA with Tukey HSD versus Ctr. (H–J) Growth curves, cell size and autofluorescence of Ctr ESCs or LV, KO, SAM CM-sen treated ESCs by FACS. Cell size and autofluorescence after 6 d of treatment. Forward scatter (FS) reflects the average cell size. Values are M ± S.D. (N=3). * – p<0.05, ** – p<0.01, *** – p<0.005 by ANOVA with Tukey HSD versus Ctr of the same time point. (K) Western blot analysis of p53 and Rb phosphorylation levels and p21 protein expression performed after 6 d of treatment. Representative results of the three experiments are shown in the Figure. GAPDH was used as loading control.