Blockade of the Notch Signaling Pathway Promotes M2 Macrophage Polarization to Suppress Cardiac Fibrosis Remodeling in Mice With Myocardial Infarction

Myocardial infarction (MI) is regarded as a serious ischemic heart disease on a global level. The current study set out to explore the mechanism of the Notch signaling pathway in the regulation of fibrosis remodeling after the occurrence of MI. First, experimental mice were infected with recombination signal binding protein J (RBP-J) shRNA and empty adenovirus vector, followed by the establishment of MI mouse models and detection of cardiac function. After 4 weeks of MI, mice in the sh-RBP-J group were found to exhibit significantly improved cardiac function relative to the sh-NC group. Moreover, knockdown of RBP-J brought about decreased infarct area, promoted cardiac macrophages M2 polarization, reduced cardiac fibrosis, and further decreased transcription and protein expressions of inflammatory factors and fibrosis-related factors. Furthermore, downregulation of cylindromatosis (CYLD) using si-CYLD reversed the results that knockdown of RBP-J inhibited fibrogenesis and the release of inflammatory factors. Altogether, our findings indicated that the blockade of Notch signaling promotes M2 polarization of cardiac macrophages and improves cardiac function by inhibiting the imbalance of fibrotic remodeling after MI.

Ehrlichia SLiM ligand mimetic activates Notch signaling in human monocytes

Ehrlichia chaffeensis evades innate host defenses by reprogramming the mononuclear phagocyte through mechanisms that involve exploitation of multiple evolutionarily conserved cellular signaling pathways including Notch. This immune evasion strategy is directed in part by tandem repeat protein (TRP) effectors. Specifically, the TRP120 effector activates and regulates Notch signaling through interactions with the Notch receptor and the negative regulator, F-Box and WD repeat domain-containing 7 (FBW7). However, the specific molecular interactions and motifs required for E. chaffeensis TRP120-Notch receptor interaction and activation have not been defined. To investigate the molecular basis of TRP120 Notch activation, we compared TRP120 with endogenous canonical/non-canonical Notch ligands and identified a short region of sequence homology within the tandem repeat (TR) domain. TRP120 was predicted to share biological function with Notch ligands, and a function-associated sequence in the TR domain was identified. To investigate TRP120-Notch receptor interactions, colocalization between TRP120 and endogenous Notch-1 was observed. Moreover, direct interactions between full length TRP120, the TRP120 TR domain containing the putative Notch ligand sequence, and the Notch receptor LBR were demonstrated. To molecularly define the TRP120 Notch activation motif, peptide mapping was used to identify an 11-amino acid short linear motif (SLiM) located within the TRP120 TR that activated Notch signaling and downstream gene expression. Peptide mutants of the Notch SLiM or anti-Notch SLiM antibody reduced or eliminated Notch activation and NICD nuclear translocation. This investigation reveals a novel molecularly defined pathogen encoded Notch SLiM mimetic that activates Notch signaling consistent with endogenous ligands.

Activation of Notch3 in Renal Tubular Cells Leads to Progressive Cystic Kidney Disease

Background: Polycystic kidney disease (PKD) is a genetic disorder affecting millions of people worldwide that is characterized by fluid-filled cysts and leads to end-stage renal disease (ESRD). The hallmarks of PKD are proliferation and dedifferentiation of tubular epithelial cells, cellular processes known to be regulated by Notch signaling. Methods: We found increased Notch3 expression in human PKD and renal cell carcinoma biopsies. To obtain insight into the underlying mechanisms and the functional consequences of this abnormal expression, we developed a transgenic mouse model with conditional overexpression of the intracellular Notch3 (ICN3) domain specifically in renal tubules. We evaluated the alterations in renal function (creatininemia, BUN) and structure (cysts, fibrosis, inflammation) and measured the expression of several genes involved in Notch signaling and the mechanisms of inflammation, proliferation, dedifferentiation, fibrosis, injury, apoptosis and regeneration. Results: After one month of ICN3 overexpression, kidneys were larger with tubules grossly enlarged in diameter, with cell hypertrophy and hyperplasia, exclusively in the outer stripe of the outer medulla. After three months, mice developed numerous cysts in proximal and distal tubules. The cysts had variable sizes and were lined with a single- or multilayered, flattened, cuboid or columnar epithelium. This resulted in epithelial hyperplasia, which was observed as protrusions into the cystic lumen in some of the renal cysts. The pre-cystic and cystic epithelium showed increased expression of cytoskeletal filaments and markers of epithelial injury and dedifferentiation. Additionally, the epithelium showed increased proliferation with an aberrant orientation of the mitotic spindle. These phenotypic tubular alterations led to progressive interstitial inflammation and fibrosis. Conclusions: In summary, Notch3 signaling promoted tubular cell proliferation, the alignment of cell division, dedifferentiation and hyperplasia, leading to cystic kidney diseases and pre-neoplastic lesions.

Temperature sensitivity of Notch signaling underlies species-specific developmental plasticity and robustness in amniote brains

Ambient temperature significantly affects developmental timing in animals. The temperature sensitivity of embryogenesis is generally believed to be a consequence of the thermal dependency of cellular metabolism. However, the adaptive molecular mechanisms that respond to variations in temperature remain unclear. Here, we report species-specific thermal sensitivity of Notch signaling in the developing amniote brain. Transient hypothermic conditions increase canonical Notch activity and reduce neurogenesis in chick neural progenitors. Increased biosynthesis of phosphatidylethanolamine, a major glycerophospholipid components of the plasma membrane, mediates hypothermia-induced Notch activation. Furthermore, the species-specific thermal dependency of Notch signaling is associated with developmental robustness to altered Notch signaling. Our results reveal unique regulatory mechanisms for temperature-dependent neurogenic potentials that underlie developmental and evolutionary adaptations to a range of ambient temperatures in amniotes.

INPP5F translocates into cytoplasm and interacts with ASPH to promote tumor growth in hepatocellular carcinoma

Background Increasing evidence has suggested inositol polyphosphate 5-phosphatase family contributes to tumorigenesis and tumor progression. However, the role of INPP5F in hepatocellular carcinoma (HCC) and its underlying mechanisms is unclear. Methods The expression of INPP5F in HCC was analyzed in public databases and our clinical specimens. The biological functions of INPP5F were investigated in vitro and vivo. The molecular mechanism of INPP5F in regulating tumor growth were studied by transcriptome-sequencing analysis, mass spectrometry analysis, immunoprecipitation assay and immunofluorescence assay. Results High expression of INPP5F was found in HCC tissues and was associated with poor prognosis in HCC patients. Overexpression of INPP5F promoted HCC cell proliferation, and vice versa. Knockdown of INPP5F suppressed tumor growth in vivo. Results from transcriptome-sequencing analysis showed INPP5F not only regulated a series of cell cycle related genes expression (c-MYC and cyclin E1), but also promoted many aerobic glycolysis related genes expression. Further studies confirmed that INPP5F could enhance lactate production and glucose consumption in HCC cell. Mechanistically, INPP5F activated Notch signaling pathway and upregulated c-MYC and cyclin E1 in HCC via interacting with ASPH. Interestingly, INPP5F was commonly nuclear-located in cells of adjacent non-tumor tissues, while in HCC, cytoplasm-located was more common. LMB (nuclear export inhibitor) treatment restricted INPP5F in nucleus and was associated with inhibition of Notch signaling and cell proliferation. Sequence of nuclear localization signals (NLSs) and nuclear export signals (NESs) in INPP5F aminoacidic sequence were then identified. Alteration of the NLSs or NESs influenced the localization of INPP5F and the expression of its downstream molecules. Furthermore, we found INPP5F interacted with both exportin and importin through NESs and NLSs, respectively, but the interaction with exportin was stronger, leading to cytoplasmic localization of INPP5F in HCC. Conclusion These findings indicate that INPP5F functions as an oncogene in HCC via a translocation mechanism and activating ASPH-mediated Notch signaling pathway. INPP5F may serve as a potential therapeutic target for HCC patients.