Inhibition of TPPP3 attenuates β-catenin/NF-κB/COX-2 signaling in endometrial stromal cells and impairs decidualization

in Journal of Endocrinology
Correspondence should be addressed to A Dwivedi:
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Embryo implantation and decidualization are critical events that occur during early pregnancy. Decidualization is synchronized by the crosstalk of progesterone and the cAMP signaling pathway. Previously, we confirmed the role of TPPP3 during embryo implantation in mice, but the underlying role and mechanism of TPPP3 in decidualization has not yet been understood. The current study was aimed to investigate the role of TPPP3 in decidualization in vivo and in vitro. For in vivo experiments, decidual reaction was artificially induced in the uteri of BALB/c mice. TPPP3 was found to be highly expressed during decidualization, whereas in the uteri receiving TPPP3 siRNA, decidualization was suppressed and the expression of β-catenin and decidual marker prolactin was reduced. In human endometrium, TPPP3 protein was found to be predominantly expressed in the mid-secretory phase (LH+7). In the primary culture of human endometrial stromal cells (hESCs), TPPP3 siRNA knockdown inhibited stromal-to-decidual cell transition and decreased the expression of the decidualization markers prolactin and IGFBP-1. Immunofluorescence and immunoblotting experiments revealed that TPPP3 siRNA knockdown suppressed the expression of β-catenin, NF-κB and COX-2 in hESCs during decidualization. TPPP3 inhibition also decreased NF-kB nuclear accumulation in hESCs and suppressed NF-κB transcriptional promoter activity. COX-2 expression was significantly decreased in the presence of a selective NF-kB inhibitor (QNZ) implicating that NF-kB is involved in COX-2 expression in hESCs undergoing decidualization. TUNEL assay and FACS analysis revealed that TPPP3 knockdown induced apoptosis and caused loss of mitochondrial membrane potential in hESCs. The study suggested that TPPP3 plays a significant role in decidualization and its inhibition leads to the suppression of β-catenin/NF-κB/COX-2 signaling along with the induction of mitochondria-dependent apoptosis.


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    Effect of TPPP3 knockdown during artificial decidualization in mice. (A) Representation of stimulated decidualization procedures. (B) Gross morphology of un-stimulated (UH) or stimulated (SH) uterine horn. (C) Protein expressions of TPPP3, β-catenin and decidual marker prolactin were examined by Western blotting (left panel). Densitometric quantitation of protein expression levels is shown as fold changes (right panel). Data are presented as mean ± s.e.m. P values: aP < 0.001, bP < 0.01, cP < 0.05 and dP > 0.05 vs un-stimulated or non-decidual horn (number of animals per group=5). A full color version of this figure is available at

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    TPPP3 knockdown in hESCs seized stromal to decidual cells transformation. (A) Immunohistochemical staining of TPPP3 in the mid-secretory phase endometrium samples from fertile women and unexplained infertile patients (LH+7). Nonspecific rabbit IgG was used as a negative control. Brown represents positive staining. (B) Immunostaining with vimentin, a stromal cell biomarker and cytokeratin, an epithelial cell biomarker. Vimentin was visualized as green. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). (C) The TPPP3 was significantly increased during decidualization i.e. in the presence of MPA and db-cAMP which was suppressed in the cells transfected with TPPP3 siRNA. Data are presented as mean ± s.e.m. P values: aP < 0.001, bP < 0.01, cP < 0.05 and dP > 0.05 vs hESCs. (D) hESCs were transfected with TPPP3 siRNA or scrambled siRNA (control) on day 1 and day 6 and were cultured with db-cAMP and MPA for 9 days with medium changes at every 3 days. (E) TPPP3 knockdown in hESCs suppressed the mRNA level of decidualization markers prolactin and IGFBP-1. (F) Representative micrographs demonstrating the fluorescein isothiocyanate-labeled phalloidin to label F-actin filaments, and immunofluorescence was used to analyze the morphological transformation of hESCs during in vitro decidualization. (G) Co-localization of β-catenin and TPPP3 during in vitro decidualization. Each experiment was performed three times with three tissue samples.

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    Protein expression and nuclear translocation of NF-κB p65 during in vitro decidualization. (A) TPPP3 knockdown suppressed NF-κB p65 protein expression. (B) TPPP3 knockdown in decidual cells inhibits NF-κB p65 nuclear translocation. Representative micrographs demonstrating the distribution of NF-kB p65 are shown. Cell images were grasped using a confocal fluorescence microscope (×63). (C) Transcriptional activation of the NF-κB promoter in decidual cells analyzed after TPPP3 knockdown and then transiently transfected with pNF-kB-luc reporter plasmid. pRL-luc plasmid was used as an internal control, and normalized relative luciferase activity was determined. Data are presented as mean ± s.e.m. P values: aP < 0.001, bP < 0.01, cP < 0.05 and dP > 0.05 vs scrambled siRNA control. Each experiment was performed three times with three tissue samples. A full color version of this figure is available at

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    TPPP3 inhibition reduces COX-2 expression. (A) Protein expression of COX-2 was examined by Western blotting (left panel). Densitometric quantitation of protein expression levels is shown as fold changes (right panel). Data are presented as mean ± s.e.m. P values: aP < 0.001, bP < 0.01, cP < 0.05 and dP > 0.05 vs scrambled siRNA control. (B). Representative micrographs demonstrating the distribution of COX-2 are shown. NF-kB inhibitor QNZ inhibited COX-2 expression. Cell images were grasped using a Nikon fluorescence microscope (×40). Each experiment was performed three times with three tissue samples.

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    TPPP3 knockdown during in vitro decidualization in hESCs induce apoptosis and promote mitochondrial membrane potential (Δψm) loss. (A) TUNEL assay performed in scrambled siRNA or TPPP3 siRNA-transfected cells during in vitro decidualization. Cells were fixed and permeabilized, and the procedure was followed as described in the ‘Materials and Methods’ section. Representative figures stained for TUNEL showing a large number of TUNEL-positive cells in TPPP3 siRNA-transfected cells as compared with scrambled siRNA-transfected cells. Cell images were grasped using a Nikon fluorescence microscope at ×10. (B) Flow cytometric analysis of apoptosis in scrambled siRNA or TPPP3 siRNA-transfected cells stained with Annexin-V/PI (AV+/PI-intact cells; AV/PI+-nonviable/necrotic cells; AV+/PI and Av+/PI+-apoptotic cells). Representative images of flow cytometry of transfected cells are shown in the upper panel and the percentage of apoptosis with mean ± s.e.m. is shown in the lower panel. (C) Δψm was assessed by JC-1 staining using flow cytometry analysis. Representative images of flow cytometry of scrambled siRNA or TPPP3 siRNA-transfected cells are shown in the upper panel and the percentage loss of Δψm with mean ± s.e.m. (D) Effect of TPPP3 knockdown on apoptotic markers. Representative blots are shown (left panels) and densitometric quantitation of protein expression levels are shown as fold change (right panel). The experiments were performed three times. P values: aP < 0.001, bP < 0.01, cP < 0.05 and dP > 0.05 vs scrambled siRNA control. Each experiment was performed three times with three tissue samples.


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