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Notch receptor activation initiates cell fate decisions and is distinctive in its reliance on mechanical force and protein glycosylation. The 2.5-angstrom-resolution crystal structure of the extracellular interacting region of Notch1 complexed with an engineered, high-affinity variant of Jagged1 (Jag1) reveals a binding interface that extends ~120 angstroms along five consecutive domains of each protein. O-Linked fucose modifications on Notch1 epidermal growth factor–like (EGF) domains 8 and 12 engage the EGF3 and C2 domains of Jag1, respectively, and different Notch1 domains are favored in binding to Jag1 than those that bind to the Delta-like 4 ligand. Jag1 undergoes conformational changes upon Notch binding, exhibiting catch bond behavior that prolongs interactions in the range of forces required for Notch activation. This mechanism enables cellular forces to regulate binding, discriminate among Notch ligands, and potentiate Notch signaling.

Liver cancer comprises a group of malignant tumors, among which hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the most common. ICC is especially pernicious and associated with poor clinical outcome. Studies have shown that a subset of human ICCs may originate from mature hepatocytes. However, the mechanisms driving the trans-differentiation of hepatocytes into malignant cholangiocytes remain poorly defined. We adopted lineage tracing techniques and an established murine hepatocyte-derived ICC model by hydrodynamic injection of activated forms of AKT (myr-AKT) and Yap (YapS127A) proto-oncogenes. Wild-type, Notch1 flox/flox , and Notch2 flox/flox mice were used to investigate the role of canonical Notch signaling and Notch receptors in AKT/Yap-driven ICC formation. Human ICC and HCC cell lines were transfected with siRNA against Notch2 to determine whether Notch2 regulates biliary marker expression in liver tumor cells. We found that AKT/Yap-induced ICC formation is hepatocyte derived and this process is strictly dependent on the canonical Notch signaling pathway in vivo. Deletion of Notch2 in AKT/Yap-induced tumors switched the phenotype from ICC to hepatocellular adenoma-like lesions, while inactivation of Notch1 in hepatocytes did not result in significant histomorphological changes. Finally, in vitro studies revealed that Notch2 silencing in ICC and HCC cell lines down-regulates the expression of Sox9 and EpCAM biliary markers. Notch2 is the major determinant of hepatocyte-derived ICC formation in mice.

Abstract Context NOTCH signaling is activated in endometriotic lesions, but the exact mechanisms remains unclear. IL-6, which is increased in the peritoneal fluid of women with endometriosis, induces NOTCH1 through E-proteins including E2A and HEB in cancer. Objective To study the role of E-proteins in inducing NOTCH1 expression under the regulation of IL-6 in endometriosis. Setting and Design The expression of E-proteins and NOTCH1 was first investigated in endometrium of women with endometriosis and the baboon model of endometriosis. Regulation of E-proteins and NOTCH1 expression was examined after IL-6 stimulation and siRNA mediated inhibition of E2A or/and HEB in human endometriotic epithelial cells (12Z) in vitro, and subsequently following IL-6 treatment in the mouse model of endometriosis in vivo. Results E2A, HEB, and NOTCH1 were significantly upregulated in glandular epithelium (GE) of ectopic endometrium compared to eutopic endometrium in both women and the baboon model. IL-6 treatment upregulated the expression of NOTCH1 together with E2A and HEB in 12Z cells. Small interfering RNA inhibition of E2A and HEB or HEB alone decreased NOTCH1 expression. Binding efficiency of both E2A and HEB was significantly higher at the binding sites on the human NOTCH1 promoter after IL-6 treatment. Finally, IL-6 treatment resulted in a significantly increased number of endometriotic lesions along with increased expression of E2A, HEB, and NOTCH1 in GE of the lesions compared with the vehicle group in an endometriosis mouse model. Conclusions IL-6 induced NOTCH1 expression is mediated by E-proteins in the ectopic GE cells, which may promote endometriotic lesion development.

Notch signaling is an evolutionarily conserved pathway that regulates cell-cell interactions through binding of Notch family receptors to their cognate ligands. Notch signaling has an essential role in vascular development and angiogenesis. Recent studies have reported that Notch may be implicated in Alzheimer's disease (AD) pathophysiology. We measured the levels of soluble Notch1 (sNotch1) in the plasma samples from 72 dementia patients (average age 75.1 y), 89 subjects with amnestic mild cognitive impairment (MCI) (average age 73.72 y), and 150 cognitively normal controls (average age 72.34 y). Plasma levels of sNotch1 were 25.27% lower in dementia patients as compared to healthy control subjects. However, the level of Notch1 protein was significantly increased in human brain microvascular endothelial cells (HBMECs) after amyloid-beta treatment. Also, Notch1 mRNA level was significantly increased in HBMECs and iPSC-derived neuronal cells from AD patient compared to normal control. These results indicate that altered expression of Notch1 might be associated with the risk of Alzheimer's disease.

Store-operated Ca²⁺ entry (SOCE) is a major pathway for Ca²⁺ entry that regulates several cellular functions. SOCE remodeling mediated by changes in the expression and/or function of the Orai channels results in the reorganization of intracellular Ca 2+ homeostasis leading to a variety of pathologies, including cancer. Notably, a significant alteration of Orai function has been reported in breast cancer cells, where the dysregulation of the Notch1 signaling pathway plays a role in the development and progression of cancer hallmarks. Here, we have investigated the possible role of Notch1 in the regulation of the expression of Orai1 and Orai3 in different breast cancer cell lines. Expression of the active form of Notch1, as well as cell stimulation with the Notch1 agonist Jagged-1 (Jag-1), demonstrates a differential role of Notch1 in the regulation of Orai expression in non-tumoral breast epithelial cells and triple negative or luminal breast cancer cells. The role of Notch1 was confirmed using DAPT, a γ-secretase inhibitor that prevents activation of the Notch pathway. Modulation of Orai1 and Orai3 expression by Notch1 was paralleled by changes in SOCE. The effect in Orai expression mediated by activation of Notch1 signaling pathway was mimicked by the expression of HEY1 or the non-phosphorylatable HEY1-S68A mutant; by contrast, expression of the phosphomimetic HEY1-S68D mutant was without effect on Orai expression. Understanding the Notch1-HEY1-Orai axis might provide insights into the development of subtype-specific therapeutic strategies targeting breast cancer.

The Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear howthe 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand, Jagged1, to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical, and structural techniques on the full extracellular complex and targeted sites, we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network. The Notch1 membrane–proximal regulatory region individually binds to the established Notch1 epidermal growth factor (EGF) 8–EGF13 and Jagged1 C2–EGF3 activation sites aswell as to two additional Jagged1 regions, EGF8–EGF11 and cysteine-rich domain. XL-MS and quantitative interaction experiments show that the three Notch1-binding sites on Jagged1 also engage intramolecularly. These interactions, together with Notch1 and Jagged1 ectodomain dimensions and flexibility, determined by small-angle X-ray scattering, support the formation of nonlinear architectures. Combined, the data suggest that critical Notch1 and Jagged1 regions are not distal but engage directly to control Notch1 signaling, thereby redefining the Notch1–Jagged1 activation mechanism and indicating routes for therapeutic applications.

Diabetic foot ulcerations (DFUs) represent a major medical, social, and economic problem. Therapeutic options are restricted due to a poor understanding of the pathogenic mechanisms. The Notch pathway plays a pivotal role in cell differentiation, proliferation, and angiogenesis, processes that are profoundly disturbed in diabetic wounds. Notch signaling is activated upon interactions between membrane-bound Notch receptors (Notch 1–4) and ligands (Jagged 1–2 and Delta-like 1, 3, 4), resulting in cell-context-dependent outputs. Here, we report that Notch1 signaling is activated by hyperglycemia in diabetic skin and specifically impairs wound healing in diabetes. Local inhibition of Notch1 signaling in experimental wounds markedly improves healing exclusively in diabetic, but not in nondiabetic, animals. Mechanistically, high glucose levels activate a specific positive Delta-like 4 (Dll4)–Notch1 feedback loop. Using loss-of-function genetic approaches, we demonstrate that Notch1 inactivation in keratinocytes is sufficient to cancel the repressive effects of the Dll4–Notch1 loop on wound healing in diabetes, thus making Notch1 signaling an attractive locally therapeutic target for the treatment of DFUs.

Notch1 transactivates Notch3 to drive terminal differentiation in stratified squamous epithelia. Notch1 and other Notch receptor paralogs cooperate to act as a tumor suppressor in squamous cell carcinomas (SCCs). However, Notch1 can be stochastically activated to promote carcinogenesis in murine models of SCC. Activated form of Notch1 promotes xenograft tumor growth when expressed ectopically. Here, we demonstrate that Notch1 activation and epithelial–mesenchymal transition (EMT) are coupled to promote SCC tumor initiation in concert with transforming growth factor (TGF)-β present in the tumor microenvironment. We find that TGFβ activates the transcription factor ZEB1 to repress Notch3 , thereby limiting terminal differentiation. Concurrently, TGFβ drives Notch1-mediated EMT to generate tumor initiating cells characterized by high CD44 expression. Moreover, Notch1 is activated in a small subset of SCC cells at the invasive tumor front and predicts for poor prognosis of esophageal SCC, shedding light upon the tumor promoting oncogenic aspect of Notch1 in SCC. Notch receptors can exert different roles in cancer. In this manuscript, the authors reveal that Notch1 activation and EMT promote tumor initiation and cancer cell heterogeneity in squamous cell carcinoma, while the repression of Notch3 by ZEB1 limits Notch1-induced differentiation, permitting Notch1-mediated EMT.

Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.5 and no statistically discernable bipotency after E15.5. To gain insights into the mechanisms governing the switch from multipotency to unipotency, we used gain-of-function Notch1 mice and demonstrated that Notch activation cell autonomously dictates luminal cell fate specification to both embryonic and basally committed mammary cells. These functional studies have important implications for understanding the signals underlying cell plasticity and serve to clarify how reactivation of embryonic programs in adult cells can lead to cancer. Lilja et al. report that multipotent mouse embryonic mammary cells become lineage restricted as early as embryonic day 12.5 during development in a potency switch regulated by Notch1 signalling.

Senescent cells interact with the surrounding microenvironment achieving diverse functional outcomes. We have recently identified that NOTCH1 can drive ‘lateral induction’ of a unique senescence phenotype in adjacent cells by specifically upregulating the NOTCH ligand JAG1. Here we show that NOTCH signalling can modulate chromatin structure autonomously and non-autonomously. In addition to senescence-associated heterochromatic foci (SAHF), oncogenic RAS-induced senescent (RIS) cells exhibit a massive increase in chromatin accessibility. NOTCH signalling suppresses SAHF and increased chromatin accessibility in this context. Strikingly, NOTCH-induced senescent cells, or cancer cells with high JAG1 expression, drive similar chromatin architectural changes in adjacent cells through cell–cell contact. Mechanistically, we show that NOTCH signalling represses the chromatin architectural protein HMGA1, an association found in multiple human cancers. Thus, HMGA1 is involved not only in SAHFs but also in RIS-driven chromatin accessibility. In conclusion, this study identifies that the JAG1–NOTCH–HMGA1 axis mediates the juxtacrine regulation of chromatin architecture. Notch can drive senescence in a cell contact dependent manner. Here the authors show that NOTCH signalling can modulate chromatin structure autonomously and non-autonomously via the JAG1-NOTCH-HMGA1 interplay during senescence.