Our data provide mechanistic insight as to how quercetin enhances the decidualization response of ME-eSCs. Decidualization refers to the differentiation of fibroblast-like eSCs into enlarged specialized decidual stromal cells that produce the growth factors needed to create a nutrient-rich uterine environment that is critical for implantation and successful pregnancy in humans (25). In mice and most other eutherian mammals this process occurs post-implantation. By contrast, in the 4% of mammals that menstruate, including humans, decidualization occurs spontaneously during each secretory phase of the menstrual cycle.
It is well-established that progesterone raises [cAMP]i levels to trigger decidualization in vivo and this process can be recapitulated with cultured eSCs using cell-permeable cAMP ± MPA (25). Using this model system, numerous studies report decidualization defects with uterine biopsy-derived eSCs (18, 19, 30) and ME-eSCs (16, 17) in the setting of endometriosis. Here, we report that quercetin enhanced the decidualization response regardless of whether ME-eSCs were obtained from endometriosis cases or unaffected healthy controls (Fig. 3) or if induced with cAMP ± MPA or PGE2, and quercetin treatment brought decidualization responses of endometriosis-eSCs to levels found in vehicle-treated control-eSCs. However, quercetin did not increase [cAMP]i levels (Fig. 4), nor did it appear to mediate decidualization through antioxidant activity (Fig. 5).
Previous studies have linked quercetin’s effect on decidualization to its anti-proliferative effect (4, 5, 20). However, not all agents that inhibit eSC proliferation enhance decidualization responses, suggesting that this effect is likely mediated through specific signaling pathways. Our data show that there are variable expression levels of pAKT among subjects, even among controls. This may reflect their decidualization capacity – but requires future studies. Regardless, we now show that quercetin rapidly reduces the phosphorylation of AKT (S473/T308), as well as ERK1/2 (multi), PRAS40 (T246), and WNK1 (T60). ERK1/2 and AKT pathways converge to inhibit p53. Accordingly, we found that quercetin increased phosphorylation of p53 (S46) and increased expression of total p53 protein.
These data are consistent with prior studies revealing AKT dephosphorylation during decidualization and increased phospho-AKT expression by stromal cells in the eutopic endometrium of endometriosis cases and endometriosis lesions (31–33). We propose that persistent phospho-AKT activity may explain decidualization defects observed in the setting of endometriosis that can be corrected with quercetin, since quercetin rapidly reduced AKT phosphorylation by eSCs (within 30 min, Fig. 6E). Likewise, the AKT inhibitor, MK-2206, significantly increased decidualization (Fig. 6F-I). AKT is a pro-survival factor with potent apoptosis-inhibiting activity whose function in regulating transcription factors and other proteins is mediated, in part, by its phosphorylation status. Thus, our findings are consistent with the 'AKT inhibitory action' of quercetin on eSCs (5) and other cells (34) and may explain the connection between inhibition of AKT signaling and increased decidualization and fertility (35, 36).
A prior study reported that quercetin reduced TP53 mRNA expression by primary eSCs while promoting decidualization (20). By contrast, we observed that quercetin increased p53 phosphorylation (Fig. 6A-B) and total p53 protein levels (Fig. 7A-B). We did not assess TP53 mRNA expression and TP53 mRNA expression may not correlate with p53 protein expression and activation. Our data support that the effects of quercetin on p53 expression and activation are related to the convergence of the AKT and ERK1/2 pathways. AKT phosphorylates numerous proteins, including 'with no lysine kinase-1' or WNK1 (T60) (37) and 'proline-rich AKT substrate of 40 kDa' (PRAS40), which in turn enhances activation of PI3K/AKT signaling (38). Accordingly, we found that quercetin reduced the phosphorylation of PRAS40 and WNK1 and by eSCs within 30 min (Fig. 6E). Interestingly, PRAS40 also plays important roles in cellular senescence and p53 regulation (39). Specifically, phospho-PRAS40 decreases p53 expression level (39) and inhibits pro-apoptotic gene expression (40). Our data suggest that quercetin blocks signaling through this pathway and therefore may enhance p53 stability. Additionally, we found that quercetin-treated eSCs show a reduction in ERK1/2 phosphorylation (Fig. 6A-D). ERK1/2 is a parallel signaling pathway to the AKT pathway and downstream of receptor tyrosine kinase activation. Here again, inhibition of ERK phosphorylation would lead to a release of suppression of p53. These relationships are summarized in Fig. 10.
Quercetin, a member of a large family of flavonoids found in fruits, vegetables, tea, seeds, nuts, and medicinal botanicals, has been reported to have antioxidant, anti-inflammatory, and immunomodulatory activities throughout the body, including the female reproductive tract (41, 42). However, it is the senolytic property of quercetin that has garnered the most attention in recent years. Indeed, recent studies describe the inhibitory effects of eSC senescence and the senescence-associated secretory phenotype (SASP) on decidualization in vitro (43) and this provides a functional link between dysfunctional decidualization, senescence and reduced female fertility (20, 44). Therefore, we searched for evidence that quercetin induces apoptosis of senescent-like eSCs. As shown in Fig. 9, inhibition of apoptosis using Z-VAD, a pan-caspase inhibitor, inhibits decidualization. However, quercetin selectively induces apoptosis eSCs (Fig. 9C-D) and specifically eliminates senescent-like eSCs (Fig. 9E-F). This activity is accompanied by an increase in decidualization and as previously reported, senescent-like eSCs inhibit decidualization (21). Thus, apoptotic elimination of senescent-like eSCs may be one mechanism by which quercetin enhances decidualization.
More direct proof of this hypothesis will require isolation and further experiments with purified senescent/senescent-like stromal cells. The hallmarks of cellular senescence are cell cycle arrest caused by increased expression of p16 and p21, changes in the nuclear membrane (loss of lamin B1), the production of SASP factors, and the expression of SA-β-gal. However, there are no global characteristics that identify senescent/senescent-like-like cells in all tissues, and that can be easily applied at the single cell level. This is a major challenge for studies of cellular senescence. For example, it appears that the ‘senescence inducer’ (e.g. replicative exhaustion, oxidative stress, ER or mitochondrial stress) influences the senescence phenotype and the ‘senescence inducer’ in the endometrium has not been defined. Also, markers of senescence tend to be different in various cell types and there is no standard gene or protein expression pattern that can definitively identify these cells across tissue types. However, increased p16 and p21 expression and reduction of lamin B1 protein are consistent with a senescence phenotype in a subset of our stromal cells as shown in Fig. 8D after exposure to H202 (a common method to induce senescence) and the expression of SA-β-gal by larger eSCs shown in Fig. 9E-F.
In a larger context, the roles of senescence in aging, cell differentiation, as well as tissue injury and repair have been widely reported (45–47). Senolytic agents have been developed to treat aging-related and other conditions by selectively clearing senescent cells (i.e., growth arrested, viable cells that have undergone metabolic and gene expression changes) (3). In the context of reproduction, quercetin supplementation improves fecundity in young female mice (48) and increases pregnancy rates in a small cohort of patients with PCOS (49). Additionally, quercetin administration significantly reduces the growth of endometrial implants in rats (4) and mice (5, 6). However, in these studies there is no or limited direct evidence that the effects of quercetin are mediated by its senolytic activity.
Although it is relatively non-toxic, the main disadvantage of quercetin is its low bioavailability (50). The dose used herein is below ranges reported for many in vitro studies (reporting up to 120 µM (51–53)). Furthermore, it is unclear how to translate the in vitro dose used for cell culture studies to humans. Nonetheless, numerous efforts are underway to improve quercetin’s solubility and bioavailability. One example is the food-grade lecithin-based formulation of quercetin, known as Quercetin Phytosome®, which exhibits enhanced solubility (54). When given orally and compared to non-modified quercetin, Quercetin Phytosome® facilitates the achievement of up to 20 times higher plasma levels of quercetin, without notable adverse effects (54). This form of quercetin and the development of new senolytics with higher bioavailability will permit future in vivo studies to test the effects of quercetin on decidualization capacity of ME-eSCs in controls or affected cases. Also, the use of non-invasive sampling methods for collecting ME-eSCs from various populations is critical for advancing our understanding of senolytics in the setting of endometriosis and other uterine health disorders.