Mechanosensitive ion channel PIEZO1 enhances endometrial decidualization through BECN1-dependent autophagy

Mechanosensitive ion channel PIEZO1 enhances endometrial decidualization through BECN1-dependent autophagy | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var 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Xiao-Zheng Liu , Shan-Shan Song , Ying-Nan Liu , Aftab Shaukat , Yong Song , Samantha Hrbek , John Lydon , View ORCID Profile Bin Guo , Hong-Lu Diao , Zeng-Ming Yang , Asgerally Fazleabas , Ren-Wei Su doi: https://doi.org/10.1101/2025.08.05.668790 Jin-Wen Kang 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jin-Wen Kang Yao Wu 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yu Zhang 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hui-Xia Li 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Guang-Ya Li 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yao-Feng Yang 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xiao-Qing Huang 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jia-Ying Yu 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chen Liang 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rui Zhang 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xiao-Zheng Liu 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shan-Shan Song 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ying-Nan Liu 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Aftab Shaukat 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yong Song 2 Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University , Grand Rapids, MI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Samantha Hrbek 2 Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University , Grand Rapids, MI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site John Lydon 3 Department of Molecular and Cellular Biology, Baylor College of Medicine , Houston, TX USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bin Guo 4 College of Veterinary Medicine, Jilin University , Changchun, Jilin, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Bin Guo Hong-Lu Diao 5 Reproductive Medicine Center, Renmin Hospital, Hubei University of Medicine , Shiyan, Hubei, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: renweisu{at}scau.edu.cn fazleabas{at}msu.edu yangzm{at}gzu.edu.cn hldiao{at}hbmu.edu.cn Zeng-Ming Yang 6 College of Animal Science, Guizhou University , Guiyang, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: renweisu{at}scau.edu.cn fazleabas{at}msu.edu yangzm{at}gzu.edu.cn hldiao{at}hbmu.edu.cn Asgerally Fazleabas 2 Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University , Grand Rapids, MI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: renweisu{at}scau.edu.cn fazleabas{at}msu.edu yangzm{at}gzu.edu.cn hldiao{at}hbmu.edu.cn Ren-Wei Su 1 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: renweisu{at}scau.edu.cn fazleabas{at}msu.edu yangzm{at}gzu.edu.cn hldiao{at}hbmu.edu.cn Abstract Full Text Info/History Metrics Preview PDF Abstract The mechanosensitive ion channel PIEZO1 plays critical roles in physiological and pathological processes in response to various types of mechanical forces, including shear stress, stretch, and extracellular matrix (ECM) stiffness. Decidualization is crucial for a successful pregnancy, characterized by the differentiation of fibroblastic endometrial stromal cells into round, secretory decidual cells, along with the rapid remodeling of the ECM. Herein, we report that PIEZO1 plays a crucial role in enhancing decidualization in response to extracellular matrix (ECM) stiffness and cell contraction. Uterine-specific knockout of Piezo1 using Pgr-Cre in mice results in subfertility due to decidualization impairment in mid-late pregnancy. Silencing of PIEZO1 in human endometrial stromal cells also results in impaired decidualization. Treatment with the PIEZO1 agonist Yoda1 enhances decidualization in both in vivo and in vitro models. Stromal cells growing on ECM with 25 kPa stiffness display a better decidualization response than cells seeded on softer 2 kPa surface or harder surface of the regulator petri dish, and this difference is abolished by null of Piezo1. Consistent with PIEZO1 as a Ca 2+ modulator, blocking of intracellular Ca 2+ or pCaMKII significantly inhibits Yoda1-enhanced decidualization. Further investigation reveals that BECN1-dependent autophagy acts as the downstream of PIEZO1. Silencing of Beclin1 abolishes Yoda1-induced decidualization, while Tat-BECN1 fully rescues impaired decidualization caused by the lack of PIEZO1. Finally, the lower expression of PIEZO1 is associated with impaired decidualization in the endometrium of endometriotic baboons. In conclusion, we have uncovered a novel mechanism of decidualization that is regulated by PIEZO1-mediated mechanotransduction, providing further insight into decidualization studies. Download figure Open in new tab Introduction Decidualization is a highly regulated and essential process during pregnancy in primates and rodents, which defines as the trans-differentiation of fibroblastic endometrial stromal cells into specialized epithelial-like secretory decidual cells, accompany with rapid extracellular matrix (ECM) remodeling [ 1 ]. Disrupted decidualization is associated with several pregnancy-related complications, such as recurrent implantation failure, recurrent pregnancy loss, preeclampsia, preterm birth, and infertility/subfertility caused by endometriosis. Therefore, decidualization is considered “the primary driver of pregnancy health” [ 2 – 6 ]. Over the past decades, numerous factors, including two dominant ovarian hormones, estrogen, and progesterone, growth factors, cytokines, endocrine factors, embryonic signals, kinases and transcriptional factors, and the implanting embryo itself, have been identified as key regulators of decidualization [ 1 , 7 , 8 ]. In addition to these factors, the morphological alterations of endometrial stromal cells during decidualization and the changes in local stiffness and tissue tension caused by ECM remodeling may also be one of the regulators. Although mechanical forces are increasingly recognized as significant contributors during pregnancy, whether or not endometrial stromal cells integrate mechanical force signals from their in vivo microenvironment and the underlying mechanism of mechanical forces driving decidualization is still not understood (reviewed in [ 9 ]). Cells convert mechanical stimulations, like hydrostatic pressure (HP), fluid shear stress (FSS), extracellular matrix (ECM) stiffness, and tensile force (TF) into cellular signals, known as mechanotransduction, which represents an indispensable biological function with evolutionary conservation [ 10 ]. Mechanically activated channels transfer force sensitivity to cells and organisms by allowing the passage of ions across the membrane in response to a mechanical stimulus, which triggers the electrochemical signals intercellularly [ 11 ]. In 2010, the mechanosensitive ion channel proteins PIEZO1 and PIEZO2 were identified as crucial mechanotransducers responsible for converting mechanical force into electrochemical signals [ 12 ]. PIEZO channels exhibit remarkable sensitivity to mechanical force, through interactions with lipid membranes, the cytoskeleton, or ECM [ 13 , 14 ]. Over the past decade, PIEZO1 has emerged as a mechanosensitive ion channel with critical roles in numerous physiological and pathological processes, including cardiovascular [ 15 ], bone homeostasis [ 16 ], pressure-induced pancreatitis [ 17 ], lung functions [ 18 ], immune functions [ 19 – 21 ], and intestinal stem cell fate decision [ 22 ]. Previous studies have shown that during pregnancy, the distending forces from the growing deciduoma, fetus, and placenta progressively enhance tissue mechanics[ 23 ]. Using atomic force microscopy, researchers have observed significantly higher elastic modulus in ex vivo tissue samples obtained from the decidua basalis, the endometrial site of placental invasion, when compared to endometrial tissue samples from non-pregnant women (1250 Pa vs. 250 Pa), suggesting that invading extravillous may enhance decidual stiffness by remodeling vasculature and ECM [ 24 ]. Furthermore, studies have reported that the cycling mechanical stretch (occurring twice per minute) triggers the up-regulation of IGFBP1 , a well-known decidualization marker, in an in vitro cultured decidualization model [ 25 ]. However, when the stretch frequency is increased (six times per minute), it adversely affects decidualization [ 26 ]. Another study indicates that stretch force leads to elevated cAMP levels and subsequent downstream signaling which are critical for decidualization, as well as the expression of αSMA, an early decidualization marker [ 27 ]. These investigations strongly demonstrate that endometrial stromal cell decidualization may be regulated by mechanical forces from its in vivo microenvironment. However, the specific molecular mechanism involved in the regulation of mechanical force-induced decidualization remains poorly understood. Autophagy is an evolutionarily conserved biological process in eukaryotic cells that plays an important role in regulating cell metabolic homeostasis [ 28 ]. Three critical protein complexes contribute to the initiation and nucleation of autophagy: the ULK1 initiation complex which is negatively regulated by mTOR signaling, the PI3K III nucleation complex, and the PI3P-binding complex [ 29 ]. Autophagy has been shown to be enhanced during the decidualization process[ 30 ]. Inhibiting autophagy impairs decidualization in both mouse models and human cells[ 30 – 34 ]. Clinically, autophagy is decreased in endometrial stromal cells of patients with recurrent miscarriage and is associated with impaired decidualization capability [ 35 ]. On the other hand, mechanical cues, such as microgravity, stretch, and ECM stiffness are reported to affect autophagy [ 36 – 38 ]. However, whether autophagy acts as a mediator of mechanical cues, especially PIEZO1-mediated mechanotransduction, in regulating decidualization is still largely unknown. In this study, we identified that PIEZO1 is functionally expressed during embryo implantation and decidualization. Uterine-specific knockout of Piezo1 in mice resulted in significantly reduced fertility due to impaired decidualization during mid-late pregnancy compared to control mice. Furthermore, we demonstrated that specific mechanical cues, cell contraction, and ECM stiffness, regulate decidualization in both mice and human cells through PIEZO1. Detailed mechanistic analyses revealed dual downstream signaling pathways: Ca 2+ -CaMKII-ERK and Ca 2+ -CaMKII-BECN1-autophagy. Importantly, we uncovered that PIEZO1 may serve as a potential therapeutic target in impaired decidualization and endometriosis, offering new insights into its pathophysiology and treatment strategies. Results Spatiotemporal expression of functional PIEZO1 in the endometrium We initially examined the expression pattern of PIEZO1 protein in the endometrium during early pregnancy using Piezo1 tdTomato mice, which carry a Piezo1 gene fused with a sequence encoding tdTomato. Application of both anti-RFP and anti-PIEZO1 antibodies immunostaining revealed that the robust spatiotemporal expression of PIEZO1-tdTomato fused protein in the decidualized zones from 4.5 to 7.5 days post-coitum (dpc) ( Fig. 1A ; Fig. S1A). In an artificial decidualization mouse model, PIEZO1 expression was significantly up-regulated in the decidualized horn compared to the non-stimulated control horn ( Fig. 1B-D ). Furthermore, we wondered if the expression of PIEZO1 was correlated with endometriosis, a gynecological disease that negatively affects decidualization [ 4 ]. We used a well-established non-human primate endometriotic model in which endometriosis was artificially induced by the intraperitoneal inoculation of menstrual tissue in baboons [ 39 ]. The results showed that the mRNA levels of PIEZO1 were significantly down-regulated in the eutopic endometrium of baboons 15 months after the induction of disease compared to the disease-free endometrium from the same animal prior to the induction of endometriosis ( Fig. 1E ), which correlated with the impaired decidualization associated with this disease [ 4 ]. Moreover, applying mechanical stretching or treating with Yoda1, an agonist of PIEZO1 protein, to ex vivo cultured endometrial tissue up-regulated Piezo1 mRNA levels within 5 minutes, suggesting a rapid self-regulation of Piezo1 expression in response to mechanical stimulation ( Fig1. F&G ). Download figure Open in new tab Figure 1. Expression of functional PIEZO1 in mouse and baboon endometrium. (A) Expression of PIEZO1-tdTomato fusion protein in implantation sites at 4.5, 5.5, 6.5, and 7.5 dpc in Piezo1 tdTomato mice detected by anti-RFP antibody. S, stromal cells; IS, implantation sites; GE, glandular epithelial cells; (B) Expression of PIEZO1 in non-stimulated (NON-S) and artificial decidualized (S) uterine horns of WT mice, detected by anti-PIEZO1 antibody; (C) qPCR detected the mRNA level of Piezo1 in non-stimulated (NON-S) and artificial decidualized (S) uterine horns of WT mice; (D) qPCR detected mRNA level of decidualization marker Prl8a2 in non-stimulated (NON-S) and artificial decidualized (S) uterine horns of WT mice; (E) Expression of PIEZO1 in eutopic baboon endometrium before (Pre) and 15 months after induction of endometriosis. (F) qPCR detected the mRNA level of Piezo1 in ex vivo cultured uterine tissue of WT mice after mechanical stretching (0, 5, 30, and 60 min); (G) qPCR detected the mRNA level of Piezo1 in ex vivo cultured uterine tissue of Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice after treating with Yoda1 (0, 5, 30, and 60 min); (H) Intracellular Ca 2+ levels of mESCs isolated from Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice with (Pressure) or without 25 kPa of hydrostatic pressure detected by Fluo-4 Ca 2+ probe (Green); (I) The quantification of fluorescence in F; Scale bar = 100 μm in A&B; Scale bar = 50 μm in H; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. To assess the functional activity of PIEZO1 in medicating Ca 2+ influx in uterine cells, we crossed Piezo1 f/f mice with Pgr Cre/+ mice to generate a uterine-specific Piezo1 knockout mouse model ( Pgr Cre/+ Piezo1 f/f , referred to as Piezo1 d/d ). The littermates without Pgr-Cre ( Pgr +/+ Piezo1 f/f , referred to as Piezo1 f/f ) were used as a control (Fig. S1B). The absence of PIEZO1 in the endometrium was verified through qPCR and immunohistochemistry (Fig. S1C&D). Next, we applied a hydrostatic pressure of 25 kPa onto in vitro cultured mouse stromal cells (mESCs). We detected increased cytoplastic Ca 2+ levels using the fluo-4 AM Ca 2+ probe in mESCs isolated from Piezo1 f/f mice but not in mESCs from the Piezo1 d/d mice, confirming the functioning of PIEZO1 protein in these cells ( Fig. 1H&I ). Collectively, these findings confirm the expression of functional PIEZO1 in the mouse endometrium, specifically in stromal cells during decidualization. Piezo1 is critical for decidualization in the endometrium Next, we investigated the impact of PIEZO1 on mouse pregnancy by mating the Piezo1 f/f and Piezo1 d/d females with fertile wild-type males. A four-month fertility test showed significantly smaller litter size and number of litters/mouse in the Piezo1 d.d mice compared to Piezo1 f/f mice ( Figure 2A ). Notably, the absence of PIEZO1 did not exert any influence on the number and weight of implantation sites at 7.5 dpc during early pregnancy ( Fig. 2B-D ). However, at 11.5 and 14.5 dpc, a considerable number of embryos were absorbed in the Piezo1 d/d uterus, suggesting the presence of a developmental disorder during mid-late pregnancy ( Fig. 2B-D ). The weight of the remaining fetus, not the placentas, was significantly lower in the Piezo1 d.d mice compared to Piezo1 f/f mice at 14.5 dpc (Fig. S1E&F). We, therefore, conducted a comparison of the decidua between Piezo1 f/f and Piezo1 d/d mice at 11.5 and 14.5 dpc. The histological staining revealed a significantly thinner decidua basalis in the uterine tissue lacking PIEZO1 expression at both time points ( Fig. 3A ). Moreover, qPCR results demonstrated significantly reduced levels of decidualization markers Prl8a2 , Prl3c1 , Bmp2 , and Wnt4 in the decidua tissue from the Piezo1 d/d mice, indicating an impaired decidualization status in these mice ( Fig. 3B ). More importantly, treatment of Yoda1 in wildtype mice significantly up-regulated the expression of decidualization markers at 11.5 dpc ( Fig. 3C ). Download figure Open in new tab Figure 2. The pregnancy disorder of Piezo1 d/d mice (A) Litter size and number of litters per mouse of Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice in a 4-mouth fertility test; (B) Morphology of uterine horns of Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice at 7.5, 11.5 and 14.5 dpc; (C) Number of implantation sites of Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice at 7.5, 11.5 and 14.5 dpc; (D) Average weight of implantation sites from each mouse of Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice at 7.5, 11.5 and 14.5 dpc; Scale bar = 1 cm; ns p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Download figure Open in new tab Figure 3. Critical roles of PIEZO1 to endometrial decidualization in mice and humans. (A) Histological images of decidua (Dec), junction zone (JZ), and labyrinth (Lab) of Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice at 11.5 and 14.5 dpc; (B) mRNA expression of decidualization marker genes in decidua of Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice at 11.5 and 14.5 dpc; (C) mRNA expression of decidualization marker genes in the decidua of WT mice with (Yoda1) or without (Vehicle) Yoda1 treatment; (D) Morphology of stimulated (S) and non-stimulated (NON-S) uterine horns of artificially decidualized Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice at 2 and 4 days after stimulation (E) Ratio of the uterine wet weight of artificially decidualized Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice 6 hours, 1, 2, and 4 days after stimulation; (F) mRNA expression of decidualization marker genes in deciduma of Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice 2 days after stimulation; (G) mRNA expression of decidualization marker genes in deciduma of Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice 4 days after stimulation; Scale bar = 500 μm in A; Scale bar = 1 cm in D; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. To further validate the crucial role of PIEZO1 in regulating decidualization, while isolating the influence of embryo and ovaries, we employed an artificial decidualization model that induces decidualization through a single mechanical stimulation in ovariectomized mice primed with exogenous E 2 and P 4 [ 40 ]. One day after mechanical stimulation, the levels of Prl8a2 were notably lower in Piezo1 d/d mice compared to Piezo1 f/f mice, despite no differences observed in the weight of deciduoma ( Fig. 3D , Fig. S2G). Subsequently, at 2 and 4 days post-stimulation, the weight of the stimulated horn in the Piezo1 f/f mice increased more than 6.2 and 20.4 times, respectively, in comparison to the non-stimulated control horn ( Fig. 3D &E). In contrast, the deciduoma showed only approximately 3.9 and 7.2 times greater weight compared to the control horns in the Piezo1 d/d mice, respectively ( Fig. 3D &E). Similarly, the expression levels of decidualization markers were significantly lower in the stimulated horns of the Piezo1 d/d mice than that of the Piezo1 f/f mice, indicating the impaired artificial decidualization in the absence of PIEZO1 ( Fig. 3F &G). Collectively, these findings strongly suggest that the absence of PIEZO1 disrupts the development, but not the initiation of decidualization, consistent with our observation during the natural pregnancy. Next, we investigated whether PIEZO1 plays a favorable role in the in vitro decidualization process of mESCs. When exposed to the well-established cocktail of E 2 +P 4 to induce decidualization, mESCs derived from Piezo1 d/d mice exhibited a notable reduction in the expression of decidualization markers Prl8a2 and Prl3c1 compared to mESCs from Piezo1 f/f mice ( Fig. 4A &B). Co-treatment with Yoda1 significantly enhanced the decidualization response in mESCs isolated from Piezo1 f/f mice ( Fig. 4A &B). This Yoda1-enhanced decidualization effect was abolished in mESCs isolated from Piezo1 d/d mice ( Fig. 4A &B). Furthermore, in human endometrial stromal cells (HESCs), silencing PIEZO1 expression by siRNA resulted in a significantly decreased expression of decidualization markers FOXO1 , PRL , and IGFBP1 following treatment with the decidualization-inducing cocktail MPA+cAMP ( Fig. 4C-E ). Notably, co-treatment with Yoda1 significantly enhanced the expression of all three decidualization marker genes in siCtrl-transfected but no such effect was observed in siPIEZO1-transfected HESCs ( Fig. 4C-E ). These findings demonstrate the conserved role of PIEZO1 in supporting successful decidualization across humans and mice. Download figure Open in new tab Figure 4. Critical roles of Yoda1/PIEZO1 to in vitro decidualization in mESCs and HESCs. (A&B) mRNA expression of decidualization marker genes in 2-day in vitro decidualized mESCs (E 2 +P 4 treatment) isolated from Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice with or without co-treatment with Yoda1; (C-E) mRNA expression of decidualization marker genes in 2-day in vitro decidualized, scrambled (Ctrl) or PIEZO1 siRNA (P1-KD) transfected HESCs (MPA+cAMP treatment), with or without co-treatment with Yoda1; ns p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Stromal cell contraction and ECM stiffness affect decidualization via activation of PIEZO1 Previous studies have demonstrated that stromal cells exhibit a myofibroblastic phenotype, termed fibroblast activation, during the early stage of decidualization in human, baboon, and mouse models, which is characterized by the increased expression of the cytoskeletal protein α smooth muscle actin (αSMA), indicating enhanced contraction capability of these cells [ 41 – 43 ]. Consistent with this, our results revealed that decidualization resulted in significantly increased contractility, with the gel area reduced by about 50.8% in mESCs derived from Piezo1 f/f mice, compared to a reduction of approximately 23.1% in non-decidualized cells ( Fig. 4A &B). Interestingly, mESCs driven from Piezo1 d/d mice exhibited a notably lower contraction during decidualization, with the gel area decreasing by 39.9%, accompanied with their impaired decidualization status ( Fig. 5A &B). However, treatment with butanedione monoxime (BDM), an inhibitor of cell contraction, effectively suppressed the decidualization-induced contraction of Piezo1 f/f mESCs but did not on Piezo1 d/d mESCs ( Fig. 5A &B). Moreover, BDM treatment significantly impaired the in vitro decidualization of cells isolated from Piezo1 f/f mice, but not cells from Piezo1 d/d mice ( Fig. 5C ). Collectively, these findings suggest that the enhanced contractility of stromal cells during decidualization activates PIEZO1, thereby promoting decidualization in mice, establishing a positive feedback loop. Download figure Open in new tab Figure 5. Cell contraction and ECM stiffness affect decidualization through PIEZO1. (A) Collagen contraction assay of in vitro decidualized mESCs (E 2 +P 4 treatment, EP) isolated from Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice, the vehicle was used as control (CON), butanedione monoxime (BDM) was used as an inhibitor of cell contraction; (B) The quantification of contraction area (%) in A; (C) mRNA expression of decidualization marker genes in 2-day in vitro decidualized mESCs (E 2 +P 4 treatment) isolated from Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice with or without co-treatment with BDM; (D) mRNA expression of decidualization marker genes in 2-day in vitro decidualized mESCs (E 2 +P 4 treatment) isolated from Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice cultured on the surface with different stiffness. Scale bar = 1 cm; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. The elasticity or stiffness of ECM influences numerous fundamental cellular processes, including proliferation, differentiation, and organoid formation [ 44 ]. The endometrium undergoes ECM rapid remodeling during decidualization [ 8 ]. Therefore, we examined the impact of different ECM stiffness on the process of decidualization. We seeded the mESCs on Softwell ® plates with varying stiffness (2 kPa and 25 kPa), coated with type I collagen (COL I), while a regular petri dish, coated with COL I, served as high-stiffness control (∼ 10 7 kPa). Piezo1 f/f mESCs cultured on softer substrates (2 kPa and 25 kPa) exhibited considerably higher expression levels of decidualization markers ( Prl8a2 and Prl3c1 ) in response to in vitro decidualization, compared to those on high-stiffness control ( Fig. 5D ). In contrast, Piezo1 d/d mESCs did not show any difference in decidualization markers across substrates with different stiffness, suggesting that ECM stiffness effectively regulates the decidualization response through PIEZO1 ( Fig. 5D ). These collective findings suggest that the contractility of stromal cells is activated during decidualization, interacts with ECM stiffness to activate PIEZO1, and eventually enhances decidualization. PIEZO1 regulates decidualization through the Ca 2+ -CaMKII cascade Next, we investigated whether PIEZO1 influences stromal cell decidualization through its primary role as a Ca 2+ ion channel modulator. The Fluo-4 AM fluorescence analysis showed a significant increase in cytoplastic Ca 2+ levels in Piezo1 f/f mESCs under in vitro decidualization conditions, whereas Piezo1 d/d mESCs exhibited no such response, indicating that decidualization activates PIEZO1-mediated Ca 2+ influx ( Fig. 6A &B). Furthermore, upon treatment with Yoda1, a robust higher cytoplastic Ca 2+ level was observed in the Piezo1 f/f cells during in vitro decidualization, while Piezo1 d/d cells failed to respond ( Fig. 6A &B). To explore the relationship between Yoda1-induced Ca 2+ level and the previously mentioned Yoda1-enhanced decidualization, we employed BAPTA-AM, a cell-permeating Ca 2+ chelator, to block the intracellular Ca 2+ . The results demonstrated that BAPTA-AM significantly abolished Yoda1-enhanced decidualization in Piezo1 f/f mESCs ( Fig. 6C ). Similarly, BAPTA-AM played the same role in HESCs ( Fig. 6D ). These findings indicate that PIEZO1 regulates decidualization by mediating Ca 2+ influx in both mESCs and HESCs. Download figure Open in new tab Figure 6. PIEZO1 enhances decidualization through the Ca 2+ -CamKII cascade. (A) Intracellular Ca 2+ levels of in 2-day in vitro decidualized mESCs isolated from Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice co-treated with Yoda1, detected by Fluo-4 Ca 2+ probe (Green); (B) The quantification of fluorescence in A; (C) mRNA expression of decidualization marker Prl8a2 in 4-day in vitro decidualized mESCs (E 2 +P 4 treatment) isolated from Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice, co-cultured with Yoda1 and BAPTA-AM; (D) mRNA expression of decidualization marker genes in 2-day in vitro decidualized HESCs (MPA+cAMP treatment), co-cultured with Yoda1 and BAPTA-AM; (E) Immunostaining of pCaMKII in artificial decidualized uterine horns of Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice 4 days after stimulation; (F) Expression of pCaMKII in 2-day in vitro decidualized mESCs (E 2 +P 4 treatment, EP) co-treated with Yoda1 (Y), BAPTA-AM (B), KN93 (K), or U0126 (U), the vehicle was used as control; (G) Expression of pCaMKII in 2-day in vitro decidualized HESCs (MPA+cAMP treatment, Mc) co-treated with Yoda1 (Y), BAPTA-AM (B), KN93 (K), or U0126 (U); (H) mRNA expression of decidualization marker genes in 2-day in vitro decidualized mESCs (E 2 +P 4 treatment), co-cultured with Yoda1 and KN93; (I) mRNA expression of decidualization marker genes in 2-day in vitro decidualized HESCs (MPA+cAMP treatment), co-cultured with Yoda1 and KN93; Scale bar = 5 μm in A; Scale bar = 100 μm in E; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Calcium/calmodulin-dependent kinase II (CaMKII) functions as a downstream target of the Ca 2+ -calmodulin complex and plays a pivotal role in regulating many cellular functions [ 45 ], which retains its ability to undergo Ca 2+ -and calmodulin-dependent auto-phosphorylation at Thr286 or Thr287 [ 46 , 47 ]. Inhibiting CaMKII activation down-regulates the expression of decidualization marker genes PRL and IGFBP1 in HESCs [ 48 ]. Therefore, we hypothesized that CaMKII acts as a downstream mediator of Ca 2+ influx induced by PIEZO1. Firstly, we evaluated the phosphorylation level of CaMKII in in vivo artificial decidualization model. The results revealed the increased expression of phosphorylated CaMKII (pCaMKII) in the secondary decidual zone (SDZ) of mechanically stimulated uterine horns of Piezo1 f/f mice 4 days after stimulation but was significantly lower in Piezo1 d/d mice ( Fig. 6E ). Moreover, compared to the vehicle-treated control group, the pCaMKII level was significantly up-regulated in both Piezo1 f/f mESCs and HESCs under in vitro decidualization conditions and further enhanced by co-treatment with Yoda1 ( Fig. 6F &G). In addition, when intracellular Ca 2+ was chelated by BAPTA-AM, the Yoda1-induced pCaMKII phosphorylation was inhibited ( Fig. 6F &G). These findings strongly suggest a close association between pCaMKII levels and Yoda1/PIEZO1 enhanced decidualization. To provide further evidence for the necessity of elevated pCaMKII in Yoda1-enhanced decidualization, we treated mESCs and HESCs with KN93, a pCaMKII inhibitor, in combination with Yoda1 and the decidualization-inducing cocktail. Remarkably, the expression of decidualization markers induced by Yoda1 treatment was significantly reversed by KN93, indicating that pCaMKII mediated Yoda1-enhanced decidualization ( Fig. 6H &I). Taken together, our data provide evidence that Yoda1/PIEZO1 governs decidualization through the Ca 2+ -CaMKII cascade. PIEZO1 promotes decidualization via ERK1/2 MAPK signaling Next, we employed RNA-Seq to analyze decidualized and non-decidualized uterine horns from both Piezo1 f/f and Piezo1 d/d mice 4 days after artificial decidualization. Differentially expressed genes (DEGs) meeting the criteria of a fold change greater than 2.0 and an adjusted p-value lower than 0.05 were selected from four distinct comparisons as depicted in Fig. S2A. In non-decidualized uterine horns, Piezo1 knockout resulted in only 78 significantly up-regulated genes and 47 down-regulated genes (Comparison 1, Fig. S2B), whereas decidualized horns exhibited significant changes, with 1364 up-regulated and 551 down-regulated genes (Comparison 2, Fig. S2C). These data suggest that the absence of Piezo1 resulted in minimal changes in non-decidualized uterine horns but significant alterations in gene expression during the decidualization. Further analysis compared the differences of decidualization on gene expression in Piezo1 f/f and Piezo1 d/d mice, which were shown in comparison 3 and 4, respectively, provided additional insights. (Fig. S2D&E). By overlapping of DEGs of comparisons 2 and 3 visualized in a Venn diagram (Fig. S2F), we identified 1474 genes that were significantly altered by both decidualization and the absence of Piezo1 (Fig. S2G). KEGG pathway analysis of these 1474 DEGs highlighted multiple affected signaling pathways, including the calcium signaling pathway, which aligns with the primary function of PIEZO1 as previously described ( Fig. 7A ). Enriched pathways also included cAMP, Wnt, Rap1, and its downstream MAPK signaling pathways ( Fig. 7A ), all known to be critical for successful decidualization [ 48 , 49 ]. Download figure Open in new tab Figure 7. PIEZO1 enhances decidualization through MAPK signaling. (A) KEGG pathway analysis of DEGs between artificial decidualized uteri from Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice 4 days after stimulation; (B) Expression of phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2 (t-ERK1/2), CYCLIN D1, and CDK4 in artificial decidualized uteri ( in vivo , 4 days) and in vitro decidualized mESCs ( in vitro , 2 days) of Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice; (C) Immunostaining of p-ERK1/2 in artificial decidualized uterine horns of Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice 4 days after stimulation; (D) Expression of p-ERK1/2 in 2-day in vitro decidualized mESCs (E 2 +P 4 treatment, EP) co-treated with Yoda1 (Y), BAPTA-AM (B), KN93 (K), or U0126 (U); (E) Expression of pERK1/2 in 2-day in vitro decidualized HESCs (MPA+cAMP treatment, Mc) co-treated with Yoda1 (Y), BAPTA-AM (B), KN93 (K), or U0126 (U); (F) mRNA expression of decidualization marker genes in 2-day in vitro decidualized mESCs (E 2 +P 4 treatment), co-cultured with Yoda1 and U0126; (G) mRNA expression of decidualization marker genes in 2-day in vitro decidualized HESCs (MPA+cAMP treatment), co-cultured with Yoda1 and U0126; Scale bar = 500 μm; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Previous study shows that the ERK1/2 MAPK pathway is required for endometrial decidualization[ 50 ]. Several studies have shown that calcium signaling induces cell differentiation through CaMKII and MAPK/ERK pathways [ 51 – 53 ]. Therefore, we further investigated the potential involvement of the ERK1/2 MAPK signaling pathway as a mediator of decidualization failure resulting from the absence of Piezo1 . First, we examined the change in phosphorylated ERK1/2 levels between Piezo1 f/f and Piezo1 d/d mice during decidualization. Western blot confirmed a significant decrease in pERK1/2 levels in both in vivo and in vitro decidualized tissue/cells of Piezo1 d/d mice compared to those of Piezo1 f/f mice, which correlated with the reduced expression of cell cycle regulators CyclinD1 and CDK4 ( Fig. 7B ). Immunostaining further revealed distinct localization patterns of pERK1/2 between Piezo1 f/f and Piezo1 d/d mice. In Piezo1 f/f decidualized horns, pERK1/2 showed higher expression in the SDZ of Piezo1 f/f mice, whereas pERK1/2 was predominantly localized in the primary decidual zone (PDZ) in Piezo1 d/d decidualized horns at 4 days post-stimulation ( Fig. 7C ). Additionally, we investigated whether Yoda1 enhanced decidualization via pERK1/2 activation. Results from the western blot and immunostaining demonstrated a significant induction of pERK1/2 levels and nuclear localization upon decidualization, which was further enhanced by Yoda1 treatment in decidualized stromal cells ( Fig. 7D , S2H). Similarly, the pERK1/2 level was also induced by decidualization and further enhanced by Yoda1 in HESCs ( Fig. 7E ). Conversely, the induction was attenuated by MEK/ERK inhibitor U0126, accompanied by the decreased expression of decidualization markers in both mESCs and HESCs, indicating the necessity of pERK1/2 activation for Yoda1/PIEZO1-induced decidualization ( Fig. 7F &G). Furthermore, BAPTA-AM or KN93 treatment significantly decreased pERK1/2 levels, indicating that ERK/MAPK signaling acts downstream of the Ca 2+ -CaMKII cascade during Yoda1/PIEZO1-enhanced decidualization ( Fig. 7D &E). Interestingly, the Yoda1-induced pCaMKII level decreased by U0126 in HESCs but not mESCs, suggesting a feedback loop between Ca 2+ /CamKII and ERK/MAPK signaling in humans ( Fig. 7F &G). All these data collectively prove that the activation of PIEZO1 enhances decidualization through the Ca 2+ -CaMKII-ERK pathway. Autophagy mediates Yoda1/PIEZO1 enhanced decidualization depending on pBECN1 Multiple studies have shown that the intracellular Ca 2+ , pCaMKII, and ERK1/2 MAPK signaling regulate autophagy by regulating the phosphorylation of BECN1, a core protein of the class III PI3K nucleation complex [ 54 – 58 ]. In our KEGG pathway analysis, the autophagy pathway is one of the top 15 significantly changed cell processes (Fig. S3A). Therefore, we hypothesized that autophagy might mediate Yoda1/PIEZO1 enhanced decidualization. We first analyzed the expression of autophagy-related proteins in our in vivo and in vitro models. The results showed that the level of LC3A/B, the protein that contributes to the formation of autophagosome, significantly decreased in the decidualized tissue of Piezo1 d/d mice compared to that of Piezo1 f/f mice ( Fig. 8A ). Additionally, transmission electron microscopy (TEM) showed that the number of autophagic vacuoles was markedly increased in in vitro decidualized Piezo1 f/f mESCs compared to non-decidualized cells, but not Piezo1 d/d cells ( Fig. 8B &C). In addition, LC3-GFP-RFP adenoviral transfection was employed to measure autophagic flux. We observed significantly fewer autophagosomes (yellow dots) and autolysosomes (free red dots) in decidualized stromal cells from Piezo1 d/d mice than that of Piezo1 f/f mice ( Fig. 8D , Fig. S3B). The protein level of LC3A/B was increased by in vitro decidualization and further enhanced by the Yoda1 treatment in both mESCs and HESCs ( Fig. 8E &F). These data indicate that autophagy is enhanced in the stromal cells during decidualization, mediated by PIEZO1. Download figure Open in new tab Figure 8. BECLIN1-dependent autophagy mediates Yoda1/PIEZO1 enhanced decidualization. (A) Expression of LC3A/B in artificial decidualized uteri of Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice 2 or 4 days after stimulation; (B) Representative TEM images of 2-day in vitro decidualized mESCs (E 2 +P 4 treatment, EP) isolated from Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice, vehicle was used as control (CON), red arrows represent autophagosomes; (C) The quantification of autophagosomes in B; (D) The number of autophagosomes and autolysosomes in 2-day in vitro decidualized mESCs (E 2 +P 4 treatment, EP) isolated from Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice, detected by LC3-GFP-RFP vector, representative images shown in figure S4A; (E) Expression of LC3A/B in 2-day in vitro decidualized mESCs (E 2 +P 4 treatment, EP) co-treated with Yoda1 (Y), BAPTA-AM (B), KN93 (K), or U0126 (U); (F) Expression of LC3A/B in 2-day in vitro decidualized HESCs (MPA+cAMP treatment, Mc) co-treated with Yoda1 (Y), BAPTA-AM (B), KN93 (K), or U0126 (U); (G) Expression of p-BECN1 in artificial decidualized uteri of Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice 2 or 4 days after stimulation; (H) Expression of p-BECN1 in 2-day in vitro decidualized mESCs (E 2 +P 4 treatment, EP) co-treated with Yoda1 (Y), BAPTA-AM (B), KN93 (K), or U0126 (U); (I) Expression of p-BECN1 in 2-day in vitro decidualized HESCs (MPA+cAMP treatment, Mc) co-treated with Yoda1 (Y), BAPTA-AM (B), KN93 (K), or U0126 (U); (J) mRNA expression of decidualization marker Prl8a2 in 2-day in vitro decidualized, scrambled (siScra) or Beclin1 siRNA (siBeclin1) transfected mESCs (E 2 +P 4 treatment) isolated from Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice, co-cultured with or without Yoda1; (K) mRNA expression of decidualization marker Prl8a2 in 2-day in vitro decidualized mESCs (E 2 +P 4 treatment) isolated from Piezo1 f/f ( P1 f/f ) and Piezo1 d/d ( P1 d/d ) mice, co-cultured with different dose of Tat-BECLIN1; (L) mRNA expression of decidualization marker genes in 2-day in vitro decidualized HESCs (MPA+cAMP treatment), co-cultured with different doses of Tat-BECLIN1; Scale bar = 500 nm; ns p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Next, we further explored if Yoda1/PIEZO1 enhances decidualization by regulating autophagy through Ca 2+ /pCaMKII and pERK1/2 controlled BECN1-dependent manners. BECN1 has been shown to be phosphorylated at its Ser90 site by pCaMKII and subsequently promotes autophagy and cell differentiation in Hela cells [ 55 ]. Western blot analysis revealed that the level of p-BECN1 (Ser90) was significantly decreased in Piezo1 d/d mice compared to the Piezo1 f/f mice, following artificial decidualization ( Fig. 8G ). On the other hand, the Yoda1 treatment remarkably enhanced the p-BECN1 level in decidualized mESCs and HESCs, and was abolished by co-treatment with BAPTA-AM or KN93, suggesting Yoda1-induced p-BECN1 via Ca 2+ -pCaMKII cascade in both mouse and humans ( Fig. 8H &I). However, the MEK/ERK inhibitor U0126 neutralized the Yoda1-enhanced LC3A/B levels, but was not able to affect Yoda1-induced p-BECN1 levels in mESCs, suggesting ERK/MAPK pathway-regulated autophagy in a BECIN1 Ser90 phosphorylation independent manner in mice ( Fig. 8H ). In contrast, U0126 inhibited both LC3A/B and pBECN1 in HESCs, consistent with its inhibition in pCaMKII level in these cells, suggesting difference in roles of ERK/MAPK signaling in regulating autophagy between humans and mice ( Fig. 8I ). When Becn1 is silenced by siRNA, the decidualization-induced and Yoda1-enhanced autophagy was completely inhibited, evidenced by the expression level of LC3A/B (Fig. S3D). Most importantly, the Yoda1-enhanced decidualization marker expression was abolished by the silencing of Becn1 ( Fig. 8J ). Conversely, the autophagy activator Tat-BECN1, a cell membrane permeable peptide derived from a region of BECN1 protein [ 59 ], was able to rescue the impaired decidualization in Piezo1 d/d mESCs and si PIEZO1- treated HESCs ( Fig. 8K &L). Interestingly, the level of p-mTOR was significantly decreased in decidualized Piezo1 d/d stromal cells compared to that of Piezo1 f/f cells under the same condition, suggesting an enhanced ULK1 complex activation in the absence of PIEZO1, which we considered as feedback due to inhibited autophagy process in these mice (Fig. S3C). Nevertheless, these data collectively indicates that autophagy mediates the Yoda1/PIEZO1 enhanced decidualization through the Ca 2+ -CaMKII-pBECN1 cascade in both mouse and human endometrium. Discussion The decidua, is a key compartment of endometrium in humans, non-human primates, and rodents, which plays a critical role in various processes associated with pregnancy, including facilitating embryo implantation, selecting healthy embryos, and controlling trophoblast invasion[ 1 ]. Herein, we report PIEZO1 mediates mechanical force as a novel regulator of decidualization, in response to its microenvironment created by the contraction of stromal cells and the stiffness of ECM. Conditional knockout of Piezo1 in the mouse uterus leads to impairment of decidualization in both natural pregnancy and artificial decidualization models. On the other hand, the PIEZO1 agonist Yoda1 also enhanced decidualization during pregnancy. In vitro cell culture experiments demonstrates that the role of PIEZO1 in decidualization is similar between humans and mice. Mechanistically, upon PIEZO1 channel activation by cell contraction and ECM stiffness, intracellular Ca 2+ influx is generated which subsequently phosphorylates CaMKII. Further investigation demonstrated that PIEZO1-mediated Ca 2+ -CaMKII regulates decidualization through BECN1-dependent autophagy. In this study, we first assessed the upstream mechanical cues that induce PIEZO1 activation. Cells exert intrinsic forces on their environment, that is, on the ECM and neighboring cells, through mechanisms such as actomyosin contractility and cytoskeletal assembly [ 60 ]. On the other hand, cell contraction can induce long-range stress stiffening in the ECM [ 61 ]. Multiple studies reveal that such mechanical forces direct stem cell behaviors in development and regeneration (reviewed in [ 60 ]. During decidualization, the endometrial stromal cells undergo differentiation, associated with rapid remodeling of the cytoskeleton and the surrounding ECM [ 8 , 43 ]. Our data also shows that the contractility of decidualized stromal cells is stronger than non-decidualized ones. In humans, the stiffness of decidua basalis is significantly higher than that of non-decidualized endometrium [ 24 ]. These observations indicate that the decidualization of stromal cells can largely change their mechanical microenvironment, which in turn enhances decidualization via the activation of PIEZO1 proteins. Such inference may explain why the null phenotype of PIEZO1 impairs decidualization at a late stage, but not during the initiation of the process. Similarly, the decidua basalis at 11.5 dpc and 14.5 dpc are impaired by the absence of PIEZO1, but 7.5 dpc is not significantly affected. In addition, the invasion of trophoblastic cells can be another contributor to the changes of tissue stiffness in the decidua basalis [ 24 ]. Therefore, the fact that expression of Piezo1 is up-regulated by stretch and Yoda1 treatment suggests positive feedback on PIEZO1 and its function during this process. The endometrium undergoes ECM remodeling during decidualization, suggesting ECM may play an important role in the process [ 8 ]. The average stiffness of the human decidua basalis is much higher than endometrium from non-pregnant women [ 24 ]. Our study reports that mESCs growing on ECM of 25 kPa stiffness display a better decidualization response compare to the hard surface of regulator petri dishes, which confirms the importance of ECM stiffness in regulating decidualization. Similarly, a recent study showed significantly increased PRL secretion from decidualizing HESCs cultured in 3-D in soft PEG gels compared to cells cultured in 2D on the hard surface of regulator petri dishes [ 62 ]. More importantly, we demonstrate that PIEZO1 is the key modulator of ECM stiffness regulation on decidualization response. On the other hand, the mESCs growing on ECM with 2 kPa stiffness display a weakened decidualization response than mESCs on 25 kPa surface, which supports the finding that PIEZO channels are more prone to activation on rigid substrates with stiffness ranging from 11 to 30 kPa [ 22 ]. However, the average stiffness of the human decidua basalis is 1.25 kPa, and in some cases up to 6 kPa, which is lower than the PIEZO channel activation range and the stiffness used in our study [ 24 ], which may be due to the limitation of AFM measurements in the study, which measures the stiffness of tissue rather than the ECM. However, the fact that cells grown on 2 kPa and hard surface regulator petri dishes also display impaired decidualization when knocking out Piezo1 , suggests that this channel can be activated within a large range of stiffness. Nevertheless, our data show that a certain stiffness is beneficial for decidualization through activation of PIEZO1. In addition, an in vitro study using a microfluidic model reported that hemodynamic forces activate PIEZO1 in endothelial cells, inducing endothelial-derived prostaglandin E2 and prostacyclin which subsequently enhances decidualization via a paracrine response, providing a different cascade through which the PIEZO1 channel contributes to decidualization [ 63 ]. It is well established that the PIEZO1 mechanotransduction mechanism permits a calcium influx that further modulates downstream intracellular pathways [ 64 ]. Indeed, using a Fluo-4 AM Ca 2+ probe, we showed that PIEZO1 activation by Yoda1 induces intracellular Ca 2+ influx in 2D monolayers, and was associated with the upregulation of p-CaMKII. Moreover, by using p-CaMKII as a marker, we observed the Ca 2+ influx in vivo during decidualization. Previous studies reported that Ca 2+ -CaMK plays an important role in the decidualization process since blocking Ca 2+ influx with Gd 3+ or inhibiting CaMK result in impaired decidualization of HESCs [ 48 , 65 ]. In addition, the decidualization enhanced by Yoda1 is completely abolished by BAPTA-AM and KN93, suggesting that the PIEZO1-mediated decidualization is through its regulatory function as a Ca 2+ ion channel modulator. Consistent with our data, other studies showed increased intracellular Ca 2+ upon in vitro decidualization of HESCs [ 65 , 66 ]. However, another study showed that L-type voltage-dependent Ca 2+ channel (VDCC) mediated Ca 2+ influx induced by ionophores inhibits cAMP-promoted decidualization [ 67 ], suggesting that the level of intracellular Ca 2+ plays dual roles, by enhancing decidualization at low levels but inhibiting decidualization at high levels, which can explain the phenomenon that low frequency of cycling mechanical stretch enhances decidualization while a high-frequency stretch impairs it [ 25 , 26 ]. In 1992, spontaneous Ca 2+ influx was observed in adherent cultured human decidual cells regardless of any external stimulation [ 68 ]. We infer that this Ca 2+ influx may be generated by the contraction and higher expression of PIEZO1 in decidual cells as we report in this study. Other Ca 2+ mediators have also been reported to contribute to the increase in intracellular Ca 2+ , such as IP 3 a sensitive Ca 2+ channel modulator [ 66 ], and TRPC1[ 65 ] during in vitro decidualization of HESCs. Inhibition of autophagy via the AMPK-mTOR signaling pathway or by knocking down ATG7 or ATG5 genes negatively affects the decidualization of human endometrial cells [ 30 , 31 ]. Similarly, in mice, autophagy inhibitors significantly inhibit artificially induced decidualization [ 32 ], and mice lacking autophagy proteins FIP200 and Atg16L1 display embryo implantation and decidualization failure [ 33 , 34 ]. In addition, the decidualization process is inhibited under microgravity conditions, associated with decreased autophagy flux [ 36 ]. These studies indicate that the process of cellular autophagy is highly related to the decidualization of endometrial stromal cells. Furthermore, autophagy is closely related to the cellular tension and the Ca 2+ influx [ 37 ]. The intracellular Ca 2+ chelation agent BAPTA-AM inhibits autophagy flux [ 54 ]. In our study, the absence of PIEZO1 leads to impaired decidualization of mice and human stromal cells along with an inhibited autophagy flux. Detailed investigation revealed a Yoda1/PIEZO1-Ca 2+ /CaMKII-BECN1 cascade, but not mTOR signaling, controlling autophagy and decidualization is, consistent with a previous study which reported that CaMKII promotes autophagy and differentiation by mediating the phosphorylation of BECN1 at Ser90 in Hela cells [ 55 ]. This BECN1-dependent autophagy is necessary and sufficient for the PIEZO1-regulated decidualization, as the silencing of Beclin1 abolished Yoda1-induced decidualization and Tat-BECN1 treatment fully rescued Piezo1 knocking out caused decidualization failure. As for the decreased mTOR signaling in our Piezo1 d/d mice, we speculate that this is negative feedback caused by the inhibition of autophagy. The limitation of this study is its relatively weak link to clinical diseases. Many gynecological diseases such as endometriosis, adenomyosis, recurrent pregnancy loss, and preeclampsia are associated with decidualization impairment or failure. Herein, we demonstrate that PIEZO1 mediated decidualization through autophagy is consistent between mice and human cells. We also report that the expression of PIEZO1 is significantly decreased in the endometrium of baboons with induced endometriosis, suggesting a possibility that PIEZO1 may contribute to the decidualization impairment in endometriosis. However, we observed a rapid increase in Piezo1 mRNA expression in response to the stretch in mice, which implies variation among samples collected under different mechanical conditions, providing difficulties for comparing PIEZO1 levels in human biopsies. In summary, in this study, we have provided evidence for the important roles of the mechanical microenvironment in regulating endometrial decidualization mediated by its receptor PIEZO1 through Ca 2+ /CaMKII and autophagy cascade in both mice and humans. Our findings have uncovered a novel mechanism that regulates decidualization. Methods Sex as a biological variable Our study exclusively examined female mice because the disease modeled is only relevant in females. Animals Pgr Cre/+ mice were kindly provided by Dr. FJ DeMayo and Dr. JP Lydon, Piezo1 f/f (Strain #029213) and Piezo1 tdTomato (Strain #029213) mice were obtained from The Jackson Laboratory. The mice were housed in SPF-level facility of experimental animal center of South China agricultural University, with a cycle of 12 hours light (07:00-19:00) and 12 hours dark (19:00-07:00), allowing free access to water and food. All experimental procedures adhered to the guidelines established by the Institutional Animal Care and Use Committee (IACUC) of South China Agricultural University. The Pgr Cre/+ mice were mated with Piezo1 f/f mice to generate uterine specific Piezo1 knockout Pgr Cre/+ Piezo1 f/f ( Piezo1 d/d ) mice, and the Pgr +/+ Piezo1 f/f ( Piezo1 f/f ) mice were used as control. 6 to 8-week-old females were caged with fertile wildtype male to induce pregnancy, the next day with vaginal plug observation was counted as 0.5 dpc. Uteri from Piezo1 d/d and Piezo1 f/f mice at various stages of pregnancy (0.5-7.5 dpc), along with developing fetuses and placentas at 11.5 dpc and 14.5 dpc, were collected, and histomorphological images were obtained. Artificial decidualization model was performed as described in [ 40 ]. Briefly, ovariectomized 6-week-old mice were allowed to recover for 2 weeks, and then subcutaneously injected with 0.1 mL of E 2 (1 μg/mL) daily at 9:00 a.m. for three consecutive days (Days 1-3), followed by a 2-day rest. On Day 6, at 8:00 a.m., a progesterone-containing silicone tube (1 cm, 250 mg/mL P 4 , Dow Corning) was implanted subcutaneously. At the same time, mice were subcutaneously injected with 0.1 mL of E 2 (67 ng/mL) daily for three consecutive days. On Day 8, at 3:00 p.m. (6 hours after the final E 2 injection), artificial decidualization was induced by scratching the antimesometrial side of one uterine horn six times to induce decidualization, the contralateral horn serving as control. Uterine tissues were collected at 6 hours, 1 day, 2 days, and 4 days post-decidualization for further analysis. All experimental procedures regarding baboons were approved by the Institutional Animal Care and Use Committee (IACUC) of the Michigan State University. Endometriosis was experimentally induced in adult female baboons (Papio anubis) by i.p. inoculation with menstrual endometrium on two consecutive menstrual cycles, as previously described [ 69 ]. This model allows us to study the progression of endometriosis by collecting EUE from these animals at various time points following the induction of the disease. Measurement of Serum E2 and P4 Levels The mouse serum collected was stored at -80°C. The estrogen and progesterone concentration in the mouse serum samples was measured by Shanghai Yanhui Biotechnology Co., Ltd. Histology and immunostaining Tissues fixed in 4% paraformaldehyde (PFA) were subjected to dehydration and embedded in paraffin. The paraffin-embedded tissues were sectioned to a thickness of 5 μm. The sections were dewaxed using xylene, rehydrated with a gradient of alcohol, stained with eosin and hematoxylin, dehydrated again and finally mounted with neutral gum. Immunostaining was conducted according to the previously described protocol [ 70 ]. Briefly, paraffin sections (5 μm) were deparaffinized, rehydrated, and subjected to antigen retrieval by boiling in 10 mM citrate buffer for 10 minutes. Endogenous horseradish peroxidase (HRP) activity was inhibited using a 3% H2O2 solution in methanol. After washing three times with PBS, the sections were incubated at 37°C for 1 hour in 10% horse serum for blocking, followed by overnight incubation with each primary antibody at 4°C. The primary antibodies utilized in this study were anti-PIEZO1 (ab128245, Abcam, Cambridge, UK), anti-RFP (600401376, Rockland), anti-pCaMKII (af3493, Affinit), anti-pmTOR (2971s, Cell Signaling Technology), anti-CDK4 (12790T, Cell Signaling Technology), anti-Cyclin D1 (2978T, Cell Signaling Technology), anti-pERK1/2 (4370, Cell Signaling Technology), anti-tERK1/2 (4695, Cell Signaling Technology). After washing, the sections were incubated with biotinylated rabbit anti-goat IgG antibody (1:200, Zhongshan Golden Bridge, Beijing, China) and streptavidin-HRP complex (1:200, Zhongshan Golden Bridge). Positive signals were visualized using the DAB Horseradish Peroxidase Color Development Kit (Zhongshan Golden Bridge) according to the manufacturer’s protocol. The nuclei were counterstained with hematoxylin. For immunofluorescence, sections were incubated with secondary antibody Alexa Fluor Goat anti-Rabbit 488 (Invitrogen, A11008). DAPI was used for counterstaining for immunofluorescence, and Images were captured using a confocal microscope (Leica, TCS SP8, Germany). Primary culture and treatment of mESCs Endometrial stromal cells were isolated from the uteri of mice at 3.5 dpc. The uteri were longitudinally sectioned, rinsed in Hanks’ balanced salt solution (HBSS), and incubated with 1% (w/v) trypsin and 6 mg/ml dispase in 3.5 mL HBSS for 1 hour at 4 °C, followed by 1 hour at room temperature and 10 minutes at 37 °C. The uterine tissues were then washed with HBSS to remove epithelium and subsequently incubated in 6 mL of HBSS containing 0.15 mg/ml Collagenase I (Invitrogen, 17100–017) at 37 °C for 35 minutes. Primary endometrial stromal cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS). Primary endometrial stromal cells were treated with 10 nM of E 2 and 1 μM of P 4 in DMEM/F12 medium containing 2% charcoal-treated fetal bovine serum (cFBS, Biological Industries) to induce in vitro decidualization for 48 hours. Stromal cells were treated with Yoda1 (800nM, HY-18723, MedChemExpress) for 48-hour during decidualization period. Inhibitors and recombinant protein, including BAPTA-AM (10 μM, S7534, Selleck, Shanghai, China), KN93 (5 μM, S6787, Selleck, Shanghai, China), U0126 (25 μM, S1102, Selleck, Shanghai, China), and Tat-BECLIN1 (2 μM, S8595, Selleck, Shanghai, China), were applied 1 hour prior to Yoda1, respectively. Culture and treatment of HESCs Human endometrial stromal cells (ATCC, CRL-4003TM) were seeded in 12-well plates and cultured in DMEM/F12 supplemented with 10% charcoal-treated fetal bovine serum (cFBS, Biological Industries) until approximately 80% confluence at 37 °C and 5% CO 2 . in vitro decidualization was performed as previously described [ 40 ]. Briefly, HESCs were induced with 500 μM dibutyryl cyclic adenosine monophosphate (db-cAMP, D0627, Merck) and 1 μM medroxyprogesterone acetate (MPA, M1629, Merck) for the indicated durations. The treatment with Yoda1 and all inhibitors was consistent with those used for mESCs. RNA silencing The siRNAs targeting human PIEZO1 ( siPIEZO1 , 5′-GGGACTGCCTCATTCTGTA-3′) and mouse Beclin1 ( siBeclin1 , 5′-GGCACAATCAATAATTTCA-3′) were designed and synthesized by Ribobio Co., Ltd. (Guangzhou, China). Following the manufacturer’s protocol, mESCs or HESCs were transfected with each hPIEZO1 siRNA or siBeclin1 siRNA using Lipofectamine 2000 Transfection Reagent (Invitrogen, Grand Island, NY) for 6 hours and 24 hours, respectively. A scramble sequence (siScra, siN0000001-1-5, Ribobio) was used as negative control. Each experiment was repeated at least three times. Gel-based cell contraction assay The gel-based cell contraction assay was performed following the manufacturer’s instructions (Cell Biolabs, CBA-201). Briefly, mESCs were separated and counted prior to the experiment. Subsequently, 4.77 mL of collagen solution was added to a 10 mL aseptic centrifuge tube, followed by the addition of 1.23 mL of 5× DMEM solution. After mixing, 170 μL of neutralizing reagent was added in four separate increments. The counted cells were taken in volumes ranging from 0.75 mL to 3 mL and mixed immediately. At this stage, the concentration of cells in the mixture is approximately 10 6 cells/mL. Following the mixing step, 0.5 mL from each sample was added to 24-well plate and incubated for 1 hour, after which the preheated decidualization treatment cocktail was added and cultured for 48 hours. Photographs were then taken, and the area of gel shrinkage was analyzed using Image J software. Western Blot Tissues and cells were lysed using RIPA Lysis Buffer (Yamei, China), and protein concentrations were quantified with the Pierce™ BCA Protein Assay Reagent (Thermo, USA). A total of 10 μg of protein was loaded onto a 10% SDS-PAGE gel and subsequently transferred to a nitrocellulose membrane, blocked for 1 hour, and then incubated with primary antibody overnight at 4 °C. The primary antibodies used in this study included phosphorylated CaMKII (pCaMKII, 1:1000, af3493, Affinit), phosphorylated ERK1/2 (p-ERK1/2, 1:1000, 4370, Cell Signaling Technology), total ERK1/2 (t-ERK1/2, 1:1000, 4695, Cell Signaling Technology), Cyclin D1 (1:1000, 2978, Cell Signaling Technology), CDK4 (1:1000, 23972, Cell Signaling Technology), LC3A/B (1:1000, 4108, Cell Signaling Technology), phosphorylated BECN1 (p-BECN1, 1:1000, PA5-112018, Thermo Fisher Scientific), total BECN1 (t-BECN1, 1:1000, 3495s, Cell Signaling Technology), phosphorylated mTOR (p-mTOR, 1:1000, 2971, Cell Signaling Technology), TUBULIN (1:1000, 2144 S, Cell Signaling Technology), and GAPDH (1:1000, sc-32233, Santa Cruz Biotechnology). After the membranes were incubated with an HRP-conjugated secondary antibody (1:5000, Invitrogen) for 1 hour, the signals were detected using an ECL Chemiluminescent Kit (Millipore, USA). Real-time qPCR Total RNA was isolated using a Trizol RNA reagent (Takara, Berkeley, CA, USA) and reverse-transcribed into cDNA using the HiScript II Reverse Transcriptase kit (Vazyme, Nanjing, China). For real-time PCR, the cDNA was amplified with a ChamQTM Universal SYBR® qPCR Master Mix (Vazyme) on the RotorGene Q system (Bio-Rad, Hercules, CA, USA). Data was analyzed using the 2^-△△Ct method and normalized to the levels of Rpl19 (mouse) or GAPDH (human). The corresponding primer sequences for each gene are provided in Table S1. Each experiment was repeated at least three times. Intracellular calcium concentration assay Fluo-4 AM was obtained from Beyotime (S1060, Shanghai, China) and used according the manufacturer’s instructions. Briefly, the cells were washed three times with preheated HBSS free of penicillin and streptomycin and then incubated in HBSS containing Fluo-4-AM at a ratio of 1:1000 for 40 min. The media was then incubated in 2% CFBS medium for an additional 30 minutes. Finally, cells were fixed with 4% PFA at room temperature for 20 min, and then stained with DAPI. Images were captured using a Confocal microscope (Leica, TCS SP8, Germany), and the fluorescence values were analyzed using ImageJ software. Transmission electron microscope imaging Specimens were prepared for TEM analysis as follows: Cells were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature for 2 hours, then transferred to fresh 2.5% glutaraldehyde buffer overnight at 4°C. On the second day, cells were post-fixed in 1% OsO4 for 2 hours, dehydrated with a graded ethanol series, and embedded in epoxy resin. Ultrathin sections (80 nm) were prepared using an ultramicrotome (Leica EM FC7), placed on a copper grid, and observed under a transmission electron microscope (FEI/Talos L120C). Autophagosomes and autolysosomes measurement The cells were then transduced with LC3-GFP-RFP adenovirus-containing medium (8 × 107 PFU/mL) for 4 hours with 2% cFBS. After the transduction, the medium was replaced with a normal 2% cFBS for 6 hours. Following incubation, the cells were fixed with 4% PFA at room temperature for 20 minutes, and the nuclei were stained with DAPI. Images were captured using a Confocal microscope (Leica, TCS SP8, Germany). Autolysosomes were observed as yellow dots, while autophagosomes appeared as free red dots. RNA-Seq and Data Analysis The Trizol RNA Reagent (Takara, Dalian, China) was utilized to extract total RNA from the decidual uteri. The concentration and integrity of the RNA were measured using the ND-1000 Nanodrop and the Agilent 2100 TapeStation (Novogene Bioinformatic Technology, Beijing, China), respectively. The quality control parameters employed in this study were: A260/A280 ratio ⩾ 1.8, A260/A230 ratio ⩾ 2.0, and RNA integrity number ⩾ 8.0. The TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA) was utilized to generate cDNA libraries. RNA sequencing was conducted on an Illumina HiSeq 2500 system. Raw data were processed using an in-house computational pipeline. Differentially expressed genes were selected based on the criteria of fold change > 2 and a false discovery rate (FDR) < 0.05. The RNA-seq raw data were deposited in the Gene Expression Omnibus (GEO) under the accession numbers GSE285591 and GSE286131. GO and KEGG analyses were performed using the DAVID online tools. The cutoff for the false discovery rate (FDR) was established at 0.05. Statistics The data were analyzed using GraphPad Prism 8.0. 2-tails Student’s t-test was employed to compare differences between two groups, while comparisons among multiple groups were performed using one-or two-Way ANOVA followed by Tukey test. All experiments were repeated independently at least three times. In the mouse study, each group consisted of at least three mice. Data are presented as mean±SEM unless stated otherwise. p-value of <0.05 was considered significant. Study approval All animal welfare, procedures, and experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of South China Agricultural University, and Michigan State University. Author contributions RWS, KJW, YW, ATF, ZMY, and HLD conceived the study; JWK, YW, YZ, HXL, GYL, YFY, XQH, JYY, CL, RZ, XZL, SSS, YNL, SH, and YS performed experiments; JPL and FJD provide key resources for mice experiments; RWS, JWK, and YW analyzed the data; RWS, JWK, and YW wrote the draft of manuscript; All authors contributed to the reviewing and editing of the manuscript. Declaration of Interests The authors declare no competing interests. Conflict of interest The authors have declared that no conflict of interest exists. 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OpenUrl CrossRef PubMed 70. ↵ Kang , J.W. , et al. , Aberrant activated Notch1 promotes prostate enlargement driven by androgen signaling via disrupting mitochondrial function in mouse . Cell Mol Life Sci , 2024 . 81 ( 1 ): p. 155 . OpenUrl PubMed View the discussion thread. Back to top Previous Next Posted August 07, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Mechanosensitive ion channel PIEZO1 enhances endometrial decidualization through BECN1-dependent autophagy Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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Share Mechanosensitive ion channel PIEZO1 enhances endometrial decidualization through BECN1-dependent autophagy Jin-Wen Kang , Yao Wu , Yu Zhang , Hui-Xia Li , Guang-Ya Li , Yao-Feng Yang , Xiao-Qing Huang , Jia-Ying Yu , Chen Liang , Rui Zhang , Xiao-Zheng Liu , Shan-Shan Song , Ying-Nan Liu , Aftab Shaukat , Yong Song , Samantha Hrbek , John Lydon , Bin Guo , Hong-Lu Diao , Zeng-Ming Yang , Asgerally Fazleabas , Ren-Wei Su bioRxiv 2025.08.05.668790; doi: https://doi.org/10.1101/2025.08.05.668790 Share This Article: Copy Citation Tools Mechanosensitive ion channel PIEZO1 enhances endometrial decidualization through BECN1-dependent autophagy Jin-Wen Kang , Yao Wu , Yu Zhang , Hui-Xia Li , Guang-Ya Li , Yao-Feng Yang , Xiao-Qing Huang , Jia-Ying Yu , Chen Liang , Rui Zhang , Xiao-Zheng Liu , Shan-Shan Song , Ying-Nan Liu , Aftab Shaukat , Yong Song , Samantha Hrbek , John Lydon , Bin Guo , Hong-Lu Diao , Zeng-Ming Yang , Asgerally Fazleabas , Ren-Wei Su bioRxiv 2025.08.05.668790; doi: https://doi.org/10.1101/2025.08.05.668790 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Physiology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41937) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15156) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)