Tumor susceptibility gene 101 is required for the maintenance of uterine epithelial cells during embryo implantation

Background The tumor susceptibility gene 101 (Tsg101), a component of the endosomal sorting complex required for transport (ESCRT) complex I, is involved in multiple biological processes involving endomembranous structures and the plasma membrane. The role of Tsg101 in the uterine epithelium was investigated in Tsg101 floxed mice crossed with Lactoferrin-iCre mice (Tsg101d/d). Methods Tsg101d/d mice were bred with stud male mice and the status of pregnancy was examined on days 4 and 6. Histological analyses were performed to examine the uterine architecture. Immunofluorescence staining of several markers was examined by confocal microscopy. Uterine epithelial cells (UECs) were isolated from Tsg101f/f and Tsg101d/d mice, and the expression of necroptosis effectors was examined by RT-PCR, western blotting, and immunofluorescence staining. UECs were also subjected to RNA expression profiling. Results Tsg101d/d female mice were subfertile with implantation failure, showing unattached blastocysts on day 6 of pregnancy. Histological and marker analyses revealed that some Tsg101d/d day 4 pregnant uteri showed a disintegrated uterine epithelial structure. Tsg101d/d UECs began to degenerate within 18 h of culture. In UECs, expression of necroptosis effectors, such as RIPK1, RIPK3, and MLKL were first confirmed. UECs responded to a stimulus to activate necroptosis and showed increased cell death. Conclusions Tsg101 deficiency in the uterine epithelium causes implantation failure, which may be caused by epithelial defects. This study provides evidence that UECs harbor a necroptotic machinery that responds to death-inducing signals. Thus, Tsg101 expression in the uterine epithelium is required for normal pregnancy in mice.

Tumor Susceptibility Gene 101 Is Required for the Maintenance of Uterine Epithelial Cells During Embryo Implantation

Background: The tumor susceptibility gene 101 (Tsg101), a component of the endosomal sorting complex required for transport (ESCRT) complex I, is involved in multiple biological processes involving endomembranous structures and the plasma membrane. The role of Tsg101 in the uterine epithelium was investigated in Tsg101 floxed mice crossed with Lactoferrin-iCre mice (Tsg101d/d).Methods: Tsg101d/d mice were bred with stud male mice and the status of pregnancy was examined on days 4 and 6. Histological analyses were performed to examine the uterine architecture. Immunofluorescence staining of several markers was examined by confocal microscopy. Uterine epithelial cells (UECs) were isolated from Tsg101f/f and Tsg101d/d mice, and the expression of necroptosis effectors was examined by RT-PCR, western blotting, and immunofluorescence staining. UECs were also subjected to RNA expression profiling.Results: Tsg101d/d female mice were subfertile with implantation failure, showing unattached blastocysts on day 6 of pregnancy. Histological and marker analyses revealed that some Tsg101d/d day 4 pregnant uteri showed a disintegrated uterine epithelial structure. Tsg101d/d UECs began to degenerate within 18 h of culture. In UECs, expression of necroptosis effectors, such as RIPK1, RIPK3, and MLKL were first confirmed. UECs responded to a stimulus to activate necroptosis and showed increased cell death. Conclusions: Tsg101 deficiency in the uterine epithelium causes implantation failure, which accompanies epithelial defects. This study provides evidence that UECs harbor a necroptotic machinery that responds to death-inducing signals. Thus, Tsg101 expression in the uterine epithelium is required for normal pregnancy in mice.

Isolation and Characterization of Human Adipocyte-derived Extracellular Vesicles Using Filtration and Ultracentrifugation

ABSTRACTExtracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte- derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances including, obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis including, transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing, microarray and utilized in functional in vitro/in vivo studies.SUMMARYWe describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Trastuzumab treatment in patients with breast cancer is associated with differential expression of the Wilms’ tumor susceptibility gene, WT1.

The mechanism of action underlying trastuzumab (Herceptin) function is ascribed to binding of the Fab region of trastuzumab to the extracellular domain of the human epidermal growth factor receptor (HER2) (1). The transcriptional responses that follow signals transduced through trastuzumab binding to HER2 are less well understood. We mined published microarray and multiplexed gene expression data (2, 3) to understand in an unbiased fashion genes most differentially expressed in the primary tumors of breast cancer patients treated with trastuzumab. We observed significantly increased and differential expression of the Wilms’ tumor susceptibility gene, WT1 in the tumors of breast cancer patients treated with trastuzumab.

Amyloid plaque deposition accelerates tau propagation via activation of microglia in a humanized APP mouse model

Microglia have an emerging role in development of tau pathology after amyloid plaque deposition in Alzheimer’s disease, although it has not been definitively shown. We hypothesize that plaque-associated activated microglia accelerate tau propagation via enhanced phagocytosis and secretion of tau. Here we show that the injection of adeno-associated virus expressing P301L tau mutant into the medial entorhinal cortex (MEC) in humanized APPNL-G-F knock-in mice induces exacerbated tau propagation in the dentate gyrus compared to wild type mice. Depletion of microglia dramatically reduces accumulation of phosphorylated tau (pTau) in the dentate gyrus as well as an extracellular vesicle (EV) marker, Tumor susceptibility gene 101, co-localized in microglia. Mac2+ activated microglia secrete significantly more EVs compared to Mac2− microglia in APPNL-G-F mice in vivo when injected with lentivirus expressing EV reporter gene mEmerald-CD9, suggesting enhanced EV secretion by microglial activation. Our findings indicate that amyloid plaque-mediated acceleration of tau propagation is dependent on activated microglia, which show enhanced EV secretion in vivo.

The Multifaceted Roles of the Tumor Susceptibility Gene 101 (TSG101) in Normal Development and Disease

The multidomain protein encoded by the Tumor Susceptibility Gene 101 (TSG101) is ubiquitously expressed and is suggested to function in diverse intracellular processes. In this review, we provide a succinct overview of the main structural features of the protein and their suggested roles in molecular and cellular functions. We then summarize, in more detail, key findings from studies using genetically engineered animal models that demonstrate essential functions of TSG101 in cell proliferation and survival, normal tissue homeostasis, and tumorigenesis. Despite studies on cell lines that provide insight into the molecular underpinnings by which TSG101 might function as a negative growth regulator, a biologically significant role of TSG101 as a tumor suppressor has yet to be confirmed using genuine in vivo cancer models. More recent observations from several cancer research teams suggest that TSG101 might function as an oncoprotein. A potential role of post-translational mechanisms that control the expression of the TSG101 protein in cancer is being discussed. In the final section of the review, we summarize critical issues that need to be addressed to gain a better understanding of biologically significant roles of TSG101 in cancer.