MUC4 encodes a large transmembrane mucin that is overexpressed in pancreatic adenocarcinomas. The molecular mechanisms responsible for that altered pattern of expression are unknown. TGF-b, a ...pleiotropic cytokine, regulates numerous genes involved in pancreatic carcino-genesis via activation of the Smads proteins and MUC4 promoter is rich in Smad-binding elements. Our aim was to study whether the regulation of MUC4 expression by TGF-b in pancreatic cancer cells was strictly dependent on Smad4 activity. Three pancreatic cancer cell lines, CAPAN-1 (MUC4 þ /Smad4À), CAPAN-2 (MUC4 þ / Smad4 þ) and PANC-1 (MUC4À/Smad4 þ), were used. By RT-PCR, transfection assays and immunohistochem-istry, we show that (i) both MUC4 mRNA and apomucin expression are upregulated by TGF-b, (ii) Smad2 positively cooperates with Smad4 to activate the promoter , (iii) activation of Smad4 by exogenous TGF-b induces Smad4 binding to the promoter, (iv) Smad7 and c-ski both inhibit activation by Smad4. When Smad4 is mutated and inactive, TGF-b activates MUC4 expression via MAPK, PI3K and PKA signaling pathways. Absence of expression in PANC-1 cells is due to histone deacetylation. Altogether, these results indicate that upregulation of MUC4 by TGF-b is restricted to well-differentiated pancreatic cancer cells, and point out a novel mechanism for TGF-b as a key molecule in targeting MUC4 overexpression in pancreatic adenocarcinomas.
Mucin production and secretion by specialized epithelial cells is a common mechanism used by mammals to protect the underlying mucosae against various injuries (pollutants, pathogens, pH). The ...expression of mucin genes is cell- and tissue-specific but is submitted to variations during cell differentiation, inflammatory process, and is altered during carcinogenesis. The molecular mechanisms responsible for the control of mucin transcription and expression are beginning to be understood as mucin gene promoters and regulatory regions are characterized. The four gel-forming mucin genes, MUC2-MUC5AC-MUC5B-MUC6, are clustered on the p15 arm of chromosome 11. Common regulatory mechanisms (PKA, PKC, PKG and Ca2+ signaling, Sp1/Sp3) may account for the capability of mucous-secreting cells to express several mucin genes simultaneously. In response to an insult or during carcinogenesis, the normal pattern of expression is altered and results from specific answers of the cell by activating different intracellular signaling pathways. 11p15 mucin genes are regulated at the transcriptional level by pro-inflammatory cytokines (IL-1beta, IL-6, TNF-alpha), pleiotropic cytokines (IL-4, IL-13, IL-9), bacterial exoproduct (LPS), growth factors (EGF, TGF-alpha), lipid mediator (PAF), retinoids and hormones. To date, the only downstream cascade known to activate mucin gene transcription is the Src/Ras/MAPK/pp90rsk cascade, which leads to the activation of the transcription factor NF-kappaB. Mucin gene transcription is also regulated by ATF-1, CREB and RAR-alpha transcription factors. Finally, repression of mucin transcription in cancer cells is under the control of the epigenetic mechanism of methylation. As transcriptional regulation of mucin genes begins to be unraveled, it becomes clear that many signaling pathways are involved. Our understanding of mucin gene transcriptional regulation, which awaits more data (identification of the signaling cascades and active cis-elements within promoters and introns), will most certainly lead to the use of mucin genes as molecular markers in cancer and molecular tools in human gene therapy, and to the synthesis of new therapeutic agents in inflammatory diseases of the epithelium.
Exocytosis and endocytosis are essential physiological processes and are of prime importance for brain function. Neurotransmission depends on the Ca2+-triggered exocytosis of synaptic vesicles (SVs). ...In neurons, exocytosis is spatiotemporally coupled to the retrieval of an equal amount of membrane and SV proteins by compensatory endocytosis. How exocytosis and endocytosis are balanced to maintain presynaptic membrane homeostasis and, thereby, sustain brain function is essentially unknown. We combine mouse genetics with optical imaging to show that the SV calcium sensor Synaptotagmin 1 couples exocytic SV fusion to the endocytic retrieval of SV membranes by promoting the local activity-dependent formation of the signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at presynaptic sites. Interference with these mechanisms impairs PI(4,5)P2-triggered SV membrane retrieval but not exocytic SV fusion. Our findings demonstrate that the coupling of SV exocytosis and endocytosis involves local Synaptotagmin 1-induced lipid signaling to maintain presynaptic membrane homeostasis in central nervous system neurons.
Display omitted
•Exocytosed Synaptotagmin 1 couples endocytosis to exocytic vesicle fusion•Exocytosed Synaptotagmin 1 promotes local presynaptic synthesis of PI(4,5)P2•Synaptotagmin 1 recruits the PI(4,5)P2 synthesizing enzyme PIPKIγ to endocytic sites•Nanoscale synthesis of PI(4,5)P2 via Synaptotagmin 1-PIPKIγ facilitates endocytosis
Synaptic exocytosis and endocytosis are of prime importance for brain function. How a neuronal synapse “knows” how many synaptic vesicles have fused is essentially unknown. Bolz et al. identify here a lipid-based mechanism for the coupling of exocytic vesicle fusion to synaptic vesicle endocytosis via Synaptotagmin 1.
Apoptosis of cells and their subsequent removal through efferocytosis occurs in nearly all tissues during development, homeostasis, and disease. However, it has been difficult to track cell death and ...subsequent corpse removal in vivo. We developed a genetically encoded fluorescent reporter, CharON (Caspase and pH Activated Reporter, Fluorescence ON), that could track emerging apoptotic cells and their efferocytic clearance by phagocytes. Using
expressing CharON, we uncovered multiple qualitative and quantitative features of coordinated clearance of apoptotic corpses during embryonic development. When confronted with high rates of emerging apoptotic corpses, the macrophages displayed heterogeneity in engulfment behaviors, leading to some efferocytic macrophages carrying high corpse burden. Overburdened macrophages were compromised in clearing wound debris. These findings reveal known and unexpected features of apoptosis and macrophage efferocytosis in vivo.
Infection with dengue virus (DENV) is widespread across tropical regions and can result in severe disease. Early diagnosis is important both for patient management and to differentiate infections ...that present with similar symptoms, such as malaria, chikungunya, and Zika. Rapid diagnostic tests that are used presently for point-of-care detection of DENV antigens lack the sensitivity of molecular diagnostics that detect viral RNA. However, no molecular diagnostic test for DENV is available for use in field settings. In this study, we developed and validated a reverse transcription-polymerase chain reaction (RT-PCR) for the detection of DENV adapted for use in field settings. Reverse transcription-polymerase chain reaction was performed directly from plasma samples without RNA extraction. The assay detected all four serotypes of DENV spiked into blood or plasma. Our RT-PCR does not cross-react with pathogens that cause symptoms that overlap with dengue infection. The test performed equally well in a conventional laboratory qPCR instrument and a small, low-cost portable instrument that can be used in a field setting. The lower limit of detection for the assay was 1 × 10
genome copy equivalents/mL in blood. Finally, we validated our test using 126 archived patient samples. The sensitivity of our RT-PCR was 76.7% (95% CI: 65.8-87.9%) on the conventional instrument, and 78.3% (95% CI: 65.8-87.9%) on the field instrument, when compared with the RealStar Dengue RT-PCR Kit 2.0. The molecular test described here is user-friendly, low-cost, and can be used in regions with limited laboratory capabilities.
PDF
