Activation of SGK1 in Endometrial Epithelial Cells in Response to PI3K/AKT Inhibition Impairs Embryo Implantation

Background/Aims: High mobility group box 1 (Hmgb1) is associated with a variety of physiological processes including embryonic development, cell proliferation and differentiation, but little information is available regarding its biological role in decidualization. Methods: In situ hybridization, real-time PCR, RNA interference, gene overexpression and MTS assay were used to analyze the spatiotemporal expression of Hmgb1 in mouse uterus during the pre-implantation period, and explore its function and regulatory mechanisms during uterine decidualization. Results: Hmgb1 mRNA was obviously observed in uterine epithelium on day 2 and 3 of pregnancy, but its expression was scarcely detected on day 4 of pregnancy. With the onset of embryo implantation, abundant Hmgb1 expression was noted in the subluminal stromal cells around the implanting blastocyst at implantation sites. Meanwhile, the accumulation of Hmgb1 mRNA was visualized in the decidual cells. Hmgb1 advanced the proliferation of uterine stromal cells and induced the expression of prolactin family 8, subfamily a, member 2 (Prl8a2), a reliable differentiation marker for decidualization. In uterine stromal cells, cAMP analogue 8-Br-cAMP up-regulated the expression of Hmgb1, but the up-regulation was abrogated by protein kinase A (PKA) inhibitor H89. Silencing of Hmgb1 by specific siRNA impeded the induction of 8-Br-cAMP on Prl8a2. Further analysis evidenced that Hmgb1 was a critical mediator of Kruppel-like factor 5 (Klf5) function in stromal differentiation. Knockdown of bone morphogenetic protein 2 (Bmp2) prevented the up-regulation of Prl8a2 elicited by Hmgb1 overexpression, whereas addition of exogenous recombinant Bmp2 protein (rBmp2) reversed the repression of Hmgb1 siRNA on Prl8a2 expression. Conclusion: Hmgb1 may play an important role during mouse uterine decidualization.

Section: Discussionsupporting

confidence: 71%

High Mobility Group Box 1 Regulates Uterine Decidualization through Bone Morphogenetic Protein 2 and Plays a Role in Kruppel-Like Factor 5-Induced Stromal Differentiation

Wang

Yang

et al. 2018

“…The effect has been attributed in part to sustained increase of cytosolic Ca 2+ activity [47]. The present observation point to 1,25(OH) 2 D 3 sensitivity of the serine/threonine protein kinase AKT which supports cell survival and affects a variety of cellular processes including proliferation, survival, and protein translation [48]. Overexpression of AKT has been correlated with the development and metastasis of various cancers [38].…”

Section: Discussionmentioning

confidence: 70%

1α,25(OH) 2D3 Sensitive Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Ishikawa Cells

Zeng

Zhou

Zhang

et al. 2017

Self Cite

Background/Aims: Tumor cell proliferation is modified by 1,25-Dihydroxy-Vitamin D3 (1,25(OH)₂D₃), a steroid hormone predominantly known for its role in calcium and phosphorus metabolism. Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H⁺ ions and lactate, which is in part accomplished by Na⁺/H⁺ exchangers, such as NHE1 and monocarboxylate transporters, such as MCT4. An effect of 1,25(OH)₂D₃ on those transport processes has, however, never been reported. As cytosolic pH impacts on apoptosis, the study further explored the effect of 1,25(OH)₂D₃ on apoptosis and on the apoptosis regulating kinase AKT, transcription factor Forkhead box O-3 (FOXO3A) and B-cell lymphoma protein BCL-2. Methods: In human endometrial adenocarcinoma (Ishikawa) cells, cytosolic pH (pHi) was determined utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na⁺/H⁺ exchanger activity from Na⁺ dependent realkalinization after an ammonium pulse, NHE1 and MCT4 transcript levels using qRT-PCR, NHE1, MCT4, total & phospho AKT, total & phospho-FOXO3A and BCL-2 protein abundance by Western blotting, lactate concentration in the supernatant utilizing a colorimetric enzyme assay and cell death quantification using CytoTox 96^®, Annexin V and Propidium Iodide staining. Results: A 24 hours treatment with 1,25(OH)₂D₃ (100 nM) significantly increased cytosolic pH (pHi), significantly decreased Na⁺/H⁺ exchanger activity, NHE1 and MCT4 transcript levels as well as protein abundance and significantly increased lactate concentration in the supernatant. Treatment of Ishikawa cells with 1,25(OH)₂D₃ (100 nM) further triggered apoptosis, an effect paralleled by decreased phosphorylation of AKT and FOXO3A as well as decreased abundance of BCL-2. Conclusions: In Ishikawa cells 1,25(OH)₂D₃ is a powerful stimulator of glycolysis, an effect presumably due to cytosolic alkalinization. Despite stimulation of glycolysis, 1,25(OH)₂D₃ stimulates slightly but significantly suicidal cell death, an effect presumably in part due to decreased activation of AKT with decreased inhibition of pro-apoptotic transcription factor FOXO3A and downregulation of the anti-apoptotic protein BCL-2.

“…The following equations (9), (10), (11) and (12) respectively provide C 1 , C 2 , C 3 and C 4 appearing in equations (5), (6), (7) and (8), while l, m or n is one of the three roots, r, of the cubic equation (13) (c.f., Cardano's Formula for cubic equation):…”

Section: Marunaka Et Al: Enac Trafficking and Protein Tyrosine Kinasementioning

confidence: 99%

Action of Protein Tyrosine Kinase Inhibitors on the Hypotonicity-Stimulated Trafficking Kinetics of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells: Analysis Using a Mathematical Model

Marunaka

Taruno

Yamamoto

et al. 2018

Background/Aims: Epithelial Na₊ channels (ENaCs) play crucial roles in control of blood pressure by determining the total amount of renal Na₊ reabsorption, which is regulated by various factors such as aldosterone, vasopressin, insulin and osmolality. The intracellular trafficking process of ENaCs regulates the amount of the ENaC-mediated Na₊ reabsorption in the collecting duct of the kidney mainly by determining the number of ENaC expressed at the apical membrane of epithelial cells. Although we previously reported protein tyrosine kinases (PTKs) contributed to the ENaC-mediated epithelial Na₊ reabsorption, we have no information on the role of PTKs in the intracellular ENaC trafficking. Methods: Using the mathematical model recently established in our laboratory, we studied the effect of PTKs inhibitors (PTKIs), AG1296 (10 µM: an inhibitor of the PDGF receptor (PDGFR)) and AG1478 (10 µM: an inhibitor of the EGF receptor (EGFR)) on the rates of the intracellular ENaC trafficking in renal epithelial A6 cells endogenously expressing ENaCs. Results: We found that application of PTKIs significantly reduced the insertion rate of ENaC to the apical membrane by 56%, the recycling rate of ENaC by 83%, the cumulative time of an individual ENaC staying in the apical membrane by 27%, the whole life-time after the first insertion of ENaC by 47%, and the cumulative Na₊ absorption by 61%, while the degradation rate was increased to 3.8-fold by application of PTKIs. These observations indicate that PTKs contribute to the processes of insertion, recycling and degradation of ENaC in the intracellular trafficking process under a hypotonic condition. Conclusion: The present study indicates that application of EGFR and PDGFR-inhibitable PTKIs reduced the insertion rate (k_I), and the recycling rate (k_R) of ENaCs, but increased degradation rate (k_D) in renal A6 epithelial cells under a hypotonic condition. These observations indicate that hypotonicity increases the surface expression of ENaCs by increasing the insertion rate (k_I) and the recycling rate (k_R) of ENaCs associated with a decrease in the degradation rate but without any significant effects on the endocytotic rate (k_E) in EGFR and PDGFR-related PTKs-mediated pathways.