TAZ as a novel regulator of oxidative damage in decidualization via Nrf2/ARE/Foxo1 pathway

Abstract

TAZ, as a crucial effector of Hippo pathway, is required for spermatogenesis and fertilization, but little is known regarding its physiological function in uterine decidualization. In this study, we showed that TAZ was localized in the decidua, where it promoted stromal cell proliferation followed by accelerated G1/S phase transition via Ccnd3 and Cdk4 and induced the expression or activity of stromal differentiation markers Prl8a2, Prl3c1 and ALP, indicating the importance of TAZ in decidualization. Knockdown of TAZ impeded HB-EGF induction of stromal cell proliferation and differentiation. Under oxidative stress, TAZ protected stromal differentiation against oxidative damage by reducing intracellular ROS and enhancing cellular antioxidant capacity dependent on the Nrf2/ARE/Foxo1 pathway. TAZ strengthened the transcriptional activity of Nrf2 which directly bound to the antioxidant response element (ARE) of Foxo1 promoter region. Additionally, silencing TAZ caused accumulation of intracellular ROS through heightening NOX activity whose blockade by APO reversed the disruption in stromal differentiation. Further analysis revealed that TAZ might restore mitochondrial function, as indicated by the increase in ATP level, mtDNA copy number and mitochondrial membrane potential with the reduction in mitochondrial superoxide. Additionally, TAZ modulated the activities of mitochondrial respiratory chain complexes I and III whose suppression by ROT and AA resulted in the inability of TAZ to defend against oxidative damage to stromal differentiation. Moreover, TAZ prevented stromal cell apoptosis by upregulating Bcl2 expression and inhibiting Casp3 activity and Bax expression. In summary, TAZ might mediate HB-EGF function in uterine decidualization through Ccnd3 and ameliorate oxidative damage to stromal cell differentiation via Nrf2/ARE/Foxo1 pathway.

Yap is essential for uterine decidualization through Rrm2/GSH/ROS pathway in response to Bmp2

“…As previously described 14 , 15 , uterine stromal cells from day 4 of pregnancy were isolated by enzymatic digestion followed by purity analysis with vimentin (a marker for stromal cells) and cytokeratin (a marker for epithelial cells) staining and then cultured with DMEM-F12 containing 10% heat-inactivated FBS (Clark, #25015). Meanwhile, stromal cells were treated with 10 nM of estradiol-17β (Sigma, #E1024, 10 nM) plus 1 μM of progesterone (Sigma, #850454, 1 μM) in DMEM-F12 containing 2% charcoal-treated FBS (Gibco, #12676-029) to in vitro decidualization which was verified as previously described 15 , 16 .…”

“…As previously described 14 , 15 , uterine stromal cells from day 4 of pregnancy were isolated by enzymatic digestion followed by purity analysis with vimentin (a marker for stromal cells) and cytokeratin (a marker for epithelial cells) staining and then cultured with DMEM-F12 containing 10% heat-inactivated FBS (Clark, #25015). Meanwhile, stromal cells were treated with 10 nM of estradiol-17β (Sigma, #E1024, 10 nM) plus 1 μM of progesterone (Sigma, #850454, 1 μM) in DMEM-F12 containing 2% charcoal-treated FBS (Gibco, #12676-029) to in vitro decidualization which was verified as previously described 15 , 16 . For further analysis, cells were treated with Yap inhibitor Verteporfin (100 nm, R&D systems, #5305) in the absence or presence of recombinant murine Bmp2 protein (rBmp2, 100 ng/ml, Peprotech, #120-02), glutathione reductase (GR) inhibitor 1, 3-bis (2-chloroethyl)-1-nitrosourea (BCNU, 20 μM, Sigma, #154-93-8), GSH (1 mM, Beyotime, #S0073) and mitochondria-targeted antioxidant Mito-TEMPO (MCE, 10 mΜ, #HY-112879) under the context of in vitro decidualization.…”

“…As previously described 15 , in situ hybridization was executed in frozen sections. After label of Yap cRNA probe with digoxigenin (DIG), cryosections were fixed with 4% paraformaldehyde and then hybridized for 16 h followed by the incubation of sheep anti-DIG antibody conjugated to alkaline phosphatase (1:5000, Roche, #11093274910) and visualization with 5-bromo-4-chloro-3-indolyl phosphate (BCIP, 0.4 mM, Amresco, #0885) and nitro blue tetrazolium (NBT, 0.4 mM, Amresco, #0329).…”

HB-EGF induces mitochondrial dysfunction via estrogen hypersecretion in granulosa cells dependent on cAMP-PKA-JNK/ERK-Ca2+-FOXO1 pathway

“…After total RNA extraction and cDNA synthetization, the expression levels of cytochrome P450 family 19 subfamily a member 1 (CYP19A1), FOXO1, Bcl2-associated X protein (BAX) and caspase 3 (CASP3) were measured by real-time PCR analysis using the corresponding primers (Table 1) as described previously [16].…”

“…(1:1000, Proteintech), BAX (1:1000, Proteintech), CASP3 (1:1000, Proteintech), histone H3 (1:5000, Proteintech) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, Proteintech) as described previously [16].…”