The role of ERp29/FOS/EMT pathway in excessive apoptosis of placental trophoblast cells in intrahepatic cholestasis of pregnancy.

BACKGROUND: Abnormal bile acid metabolism leading to changes in placental function during pregnancy. To determine whether endoplasmic reticulum protein 29 (ERp29) can mediate the pregnancy effects of cholestasis by altering the level of trophoblast cell apoptosis. METHODS: ERp29 in serum of 66 intrahepatic cholestasis of pregnancy (ICP) pregnant women and 74 healthy were detected by ELISA. Subcutaneous injection of ethinyl estradiol (E2) was used to induce ICP in pregnant rats. Taurocholic acid (TCA) was used to simulate the ICP environment, and TGF-ß1 was added to induce the epithelial mesenchymal transformation (EMT) process. The scratch, migration, and invasion test were used to detect the EMT process. ERp29 overexpression/knockdown vector were constructed and transfected to verify the role of ERp29 in the EMT process. Downstream gene was obtained through RNA-seq. RESULTS: Compared with the healthy pregnant women, the expression levels of ERp29 in serum of ICP pregnancy women were significantly increased (P < 0.001). ERp29 in the placenta tissue of the ICP pregnant rats increased significantly, and the level of apoptosis increased. The placental tissues of the ICP had high expression of E-cadherin and low expression of N-cadherin, snail1, vimentin. After HTR-8/SVneo cells were induced by TCA, EMT was inhibited, while the ERp29 increased. Cell and animal experiments showed that, knockdown of ERp29 reduced the inhibition of EMT, the ICP progress was alleviated. Overexpression of FOS salvaged the inhibitory effects of ERp29 on cell EMT. DISCUSSION: The high level of ERp29 in placental trophoblast cells reduced FOS mRNA levels, inhibited the EMT process and aggravated the occurrence and development of ICP.

Dimerization of ER-resident molecular chaperones mediated by ERp29.

The lectin chaperones calnexin (CNX) and calreticulin (CRT) localized in the endoplasmic reticulum play important roles in glycoprotein quality control. Although the interaction between these lectin chaperones and ERp57 is well known, it has been recently reported that endoplasmic reticulum protein 29 (ERp29), a member of PDI family, interacts with CNX and CRT. The biochemical function of ERp29 is unclear because it exhibits no ERp57-like redox activity. In this study, we addressed the possibility that ER chaperones CNX and CRT are connected via ERp29, based on our observation that ERp29 exists as a dimer. As a result, we showed that CNX dimerizes through ERp29. These results endorse the hypothesis that ERp29 serves as a bridge that links two molecules of CNX. Also, we showed that similar complexes such as CNX-CRT were formed via ERp29.

ERp29 affects the migratory and invasive ability of human extravillous trophoblast HTR-8/SVneo cells via modulating the epithelial-mesenchymal transition.

Dysfunction of trophoblast metastasis into the endometrium is the main cause of pre-eclampsia (PE); however, the factors affecting this process are still unclear. In this study, we found that endoplasmic reticulum protein 29 (ERp29), one molecular chaperone of the endoplasmic reticulum, was aberrantly upregulated in the placenta of pre-eclamptic patients compared with healthy controls. Then, an in vitro study using human extravillous trophoblast HTR-8/SVneo cells showed that ERp29 upregulation could inhibit the migratory and invasive ability of HTR-8/SVneo cells, while ERp29 downregulation had the opposite effect. Mechanical experiments confirmed that ERp29 blocked trophoblast metastasis via inhibiting the process of epithelial-mesenchymal transition and affecting the Wnt/ß-catenin signaling pathway. In conclusion, this study revealed the important role of ERp29 in trophoblast metastasis and improved the mechanical understanding of PE occurrence.

miR-205-5p downregulation decreases gemcitabine sensitivity of breast cancer cells via ERp29 upregulation.

Breast cancer is the most common cancer in women worldwide, and the incidence and mortality rates are increasing every year. Dysregulation of microRNAs (miRNAs or miRs) is an important step in the initiation and development of breast cancer. Previous studies demonstrated that miR-205-5p is closely associated with occurrence and development of breast cancer; however, underlying mechanisms remain unclear. In the present study, reverse transcription-quantitative polymerase chain reaction assays were used to analyze miR-195-5p and endoplasmic reticulum protein 29 (ERp29) levels in breast cancer and matched normal tissues. Western blot analysis was performed to analyze ERp29 and heat shock protein 27 (HSP27) protein expression levels. Cell viability, flow cytometry and luciferase reporter assay were used to examine cell proliferation, apoptosis and direct miRNA-mRNA binding, respectively. The results revealed that miR-205-5p expression in breast cancer tissues and cell lines was decreased compared with normal tissues and a normal cell line. Overexpression of miR-205-5p significantly augmented cytotoxicity effects of gemcitabine treatment in MDA-MB-231 and BT549 cells. It was observed that miR-205-5p negatively regulated ERp29 expression in breast cancer cells. Dual luciferase assays confirmed that ERp29 was a target of miR-205-5p in breast cancer cells. Additionally, following the established gemcitabine-resistant MDA-MB-231 cells (MDA-MB-231/GEM), ERp29 and HSP27 expression was upregulated and miR-205-5p was downregulated compared with parental cells. Overexpression of miR-205-5p reversed gemcitabine resistance in MDA-MB-231/GEM cells. In conclusion, the present study indicated that miR-205-5p may inhibit gemcitabine resistance in breast cancer cells via inhibition of ERp29 expression.

ERp29 inhibition attenuates TCA toxicity via affecting p38/p53- dependent pathway in human trophoblast HTR-8/SVeno cells.

Intrahepatic cholestasis of pregnancy (ICP) is a liver disorder occurred in pregnant women, and the mechanism for such disease is still unclear. The bioinformatics analysis of our previous study has revealed the abnormal expression of endoplasmic reticulum protein 29 (ERp29) in placental tissue of ICP patients. In this study, the function of ERp29 was further explored using in vitro model of ICP. The results showed that up-regulation of ERp29 occurred in TCA (taurocholic acid)-treated human trophoblast HTR-8/SVeno cells, and ERp29 inhibition reversed TCA toxicity via attenuating G2/M arrest and cell apoptosis. Mechanical study revealed ERp29 inhibition suppressed phosphorylation and kinase activity of p38, thus subsequently affecting expression and phosphorylation of p53 (ser18) as well as the transcriptional activity of p53. The conduction of this study might confirm the important role of ERp29 in ICP and which would be helpful for the development of target therapeutic method for ICP.

High glucose regulates ERp29 in hepatocellular carcinoma by LncRNA MEG3-miRNA 483-3p pathway.

AIMS: Blood glucose dysregulation is an adverse factor in the prognosis of hepatocellular carcinoma (HCC). Endoplasmic reticulum (ER) is thought to be crucial component in the development of cancer and diabetes. This study aimed to investigate the mechanisms of poor outcomes in HCC patients with diabetes. MAIN METHODS: ER protein 29 (ERp29) was predicted by proteomics, immunohistochemistry, Western blot, Cell Counting Kit-8 (CCK-8) and cell scratch test were used to identify the expression and biological effects of ERp29 under high glucose (HG) in HCC cells. Bioinformatics found a competing endogenous RNAs (ceRNAs) regulatory network between microRNA-483-3p (miR-483-3p) and Long noncoding RNA (LncRNA MEG3), the above methods also were used to identify their expression, biological effects and their roles of HG on regulation of REp29 in HCC cells, Dual-luciferase reporter assay was carried out to study the interaction of ERp29 with miR-483-3p and miR-483-3p with MEG3. KEY FINDINGS: HG upregulated miR-483-3p expression in HCC cells and miR-483-3p overexpression suppressed ERp29 expression and also increased HCC cell proliferation and migration. Furthermore, we found that MEG3 was decreased in HCC cells incubated in medium with high glucose and knockdown of MEG3 downregulated ERp29 expression. Bioinformatics analysis found that MEG3 mediated its protective effects via binding to miR-483-3p. SIGNIFICANCE: Overall, our study established a novel regulatory network of LncRNA MEG3/miR483-3p/ERp29 in HCC which may be helpful in better understanding the effect of high glucose on poor prognosis of HCC and in exploring new diagnostic and therapeutic tools for managing HCC in patients with diabetes.

The chaperone ERp29 is required for tunneling nanotube formation by stabilizing MSec.

Tunneling nanotubes (TNTs) are membrane conduits that mediate long-distance intercellular cross-talk in several organisms and play vital roles during development, pathogenic transmission, and cancer metastasis. However, the molecular mechanisms of TNT formation and function remain poorly understood. The protein MSec (also known as TNFα-induced protein 2 (TNFAIP2) and B94) is essential for TNT formation in multiple cell types. Here, using affinity protein purification, mass spectrometric identification, and confocal immunofluorescence microscopy assays, we found that MSec interacts with the endoplasmic reticulum (ER) chaperone ERp29. siRNA-mediated ERp29 depletion in mammalian cells significantly reduces TNT formation, whereas its overexpression induces TNT formation, but in a strictly MSec-dependent manner. ERp29 stabilized MSec protein levels, but not its mRNA levels, and the chaperone activity of ERp29 was required for maintaining MSec protein stability. Subcellular ER fractionation and subsequent limited proteolytic treatment suggested that MSec is associated with the outer surface of the ER. The ERp29-MSec interaction appeared to require the presence of other bridging protein(s), perhaps triggered by post-translational modification of ERp29. Our study implicates MSec as a target of ERp29 and reveals an indispensable role for the ER in TNT formation, suggesting new modalities for regulating TNT numbers in cells and tissues.

ERp29 downregulation enhances lung adenocarcinoma cell chemosensitivity to gemcitabine by upregulating HSP27 phosphorylation.

The aim of the current study was to assess the underlying mechanism of endoplasmic reticulum protein 29 (ERp29) in lung adenocarcinoma chemosensitivity to gemcitabine. Western blot analysis was performed to detect ERp29 expression following lung adenocarcinoma cell treatment with gemcitabine. The effects of gemcitabine in combination with ERp29 siRNA on cell apoptosis, cell cycle and heat shock protein 27 (HSP27) expression were assessed. The results demonstrated that ERp29 expression was increased on exposure to gemcitabine. The apoptotic rate of lung adenocarcinoma cells were also increased following gemcitabine treatment and the combined application of gemcitabine and ERp29 siRNA synergistically increased apoptotic rates further. It was also revealed that gemcitabine and ERp29 siRNA synergistically increased the ratio of phosphorylated to total HSP27 protein. In addition, downregulation of HSP27 significantly reduced lung adenocarcinoma chemosensitivity to gemcitabine. These data indicate that ERp29 affects lung adenocarcinoma cell chemosensitivity to gemcitabine by regulating phosphorylated HSP27. ERp29 is a novel target, which may be used to enhance the therapeutic effect of lung adenocarcinoma treatment with gemcitabine.