Anti-Notch1 Anticorps [mN1A] (A86356)

Nodal and Notch signaling pathways play essential roles in vertebrate development. Through a yeast two-hybrid screening, we identified Notch3 as a candidate binding partner of the Nodal coreceptor Cripto-1. Coimmunoprecipitation analysis confirmed the binding of Cripto-1 with all four mammalian Notch receptors. Deletion analyses revealed that the binding of Cripto-1 and Notch1 is mediated by the Cripto-1/FRL-1/Cryptic domain of Cripto-1 and the C-terminal region of epidermal growth factor-like repeats of Notch1. Binding of Cripto-1 to Notch1 occurred mainly in the endoplasmic reticulum-Golgi network. Cripto-1 expression resulted in the recruitment of Notch1 protein into lipid raft microdomains and enhancement of the furin-like protein convertase-mediated proteolytic maturation of Notch1 (S1 cleavage). Enhanced S1 cleavage resulted in the sensitization to ligand-induced activation of Notch signaling. In addition, knockdown of Cripto-1 expression in human and mouse embryonal carcinoma cells desensitized the ligand-induced Notch signaling activation. These results suggest a novel role of Cripto-1 in facilitating the posttranslational maturation of Notch receptors.

Intramembrane proteolysis by presenilin-dependent gamma-secretase produces the Notch intracellular cytoplasmic domain (NCID) and Alzheimer disease-associated amyloid-beta. Here, we show that upon Notch signaling the intracellular domain of Notch-1 is cleaved into two distinct types of NICD species due to diversity in the site of S3 cleavage. Consistent with the N-end rule, the S3-V cleavage produces stable NICD with Val at the N terminus, whereas the S3-S/S3-L cleavage generates unstable NICD with Ser/Leu at the N terminus. Moreover, intracellular Notch signal transmission with unstable NICDs is much weaker than that with stable NICD. Importantly, the extent of endocytosis in target cells affects the relative production ratio of the two types of NICD, which changes in parallel with Notch signaling. Surprisingly, substantial amounts of unstable NICD species are generated from the Val-->Gly and the Lys-->Arg mutants, which have been reported to decrease S3 cleavage efficiency in cultured cells. Thus, we suggest that the existence of two distinct types of NICD points to a novel aspect of the intracellular signaling and that changes in the precision of S3 cleavage play an important role in the process of conversion from extracellular to intracellular Notch signaling.

BACKGROUND:

Basic fibroblast growth factor (bFGF) plays an important role in promoting wound healing and reducing scar, but the possible molecular mechanisms are still unclear. Our previous studies have found that activating the Notch1/Jagged1 pathway can inhibit the differentiation of epidermal stem cells (ESCs) to myofibroblasts (MFB). Herein, we document that bFGF reduces scar by inhibiting the differentiation of ESCs to MFB via activating the Notch1/Jagged1 pathway.

METHODS:

In in-vitro study, ESCs were isolated from 10 neonatal SD rats (1-3 days old), cultured in keratinocyte serum-free medium, and divided into six groups: bFGF group, bFGF + SU5402 group, bFGF + DAPT group, siJagged1 group, bFGF + siJagged1 group, and control group. Jagged1 of the ESCs in the siJagged1 group and bFGF + siJagged1 group was knocked down by small-interfering RNA transfection. Expression of ESC markers (CK15/CK10), MFB markers (α-SMA, Collagen I, Collagen III), and Notch1/Jagged1 components (Jagged1, Notch1, Hes1) was detected by FCM, qRT-PCR, and western blot analysis to study the relationships of bFGF, ESCs, and Notch1/Jagged1 pathway. In in-vivo study, the wound healing time and scar hyperplasia were observed on rabbit ear scar models. The quality of wound healing was estimated by hematoxylin and eosin staining and Masson staining. Expression of ESC markers, MFB markers and Notch1/Jagged1 components was elucidated by immunohistochemistry, immunofluorescence, and western blot analysis.

RESULTS:

The in-vitro study showed that bFGF could significantly upregulate the expression of ESC markers and Notch1/Jagged1 components, while downregulating the expression of MFB markers at the same time. However, these effects could be obviously decreased when we knocked down Jagged1 or added DAPT. Similarly, in in-vivo study, bFGF also exhibited its functions in inhibiting the differentiation of rabbit ESCs to MFB by activating the Notch1/Jagged1 pathway, which improved the wound healing quality and alleviated scar significantly.

CONCLUSION:

These results provide evidence that bFGF can reduce scar by inhibiting the differentiation of ESCs to MFB via the Notch1/Jagged1 pathway, and present a new promising potential direction for the treatment of scar.

Human cytomegalovirus (HCMV) infection of the developing fetus frequently results in major neural developmental damage. In previous studies, HCMV was shown to downregulate neural progenitor/stem cell (NPC) markers and induce abnormal differentiation. As Notch signaling plays a vital role in the maintenance of stem cell status and is a switch that governs NPC differentiation, the effect of HCMV infection on the Notch signaling pathway in NPCs was investigated. HCMV downregulated mRNA levels of Notch1 and its ligand, Jag1, and reduced protein levels and altered the intracellular localization of Jag1 and the intracellular effector form of Notch1, NICD1. These effects required HCMV gene expression and appeared to be mediated through enhanced proteasomal degradation. Transient expression of the viral tegument proteins of pp71 and UL26 reduced NICD1 and Jag1 protein levels endogenously and exogenously. Given the critical role of Notch signaling in NPC growth and differentiation, these findings reveal important mechanisms by which HCMV disturbs neural cell development in vitro. Similar events in vivo may be associated with HCMV-mediated neuropathogenesis during congenital infection in the fetal brain.

IMPORTANCE:

Congenital human cytomegalovirus (HCMV) infection is the leading cause of birth defects that primarily manifest as neurological disabilities. Neural progenitor cells (NPCs), key players in fetal brain development, are the most susceptible cell type for HCMV infection in the fetal brain. Studies have shown that NPCs are fully permissive for HCMV infection, which causes neural cell loss and premature differentiation, thereby perturbing NPC fate. Elucidation of virus-host interactions that govern NPC proliferation and differentiation is critical to understanding neuropathogenesis. The Notch signaling pathway is critical for maintaining stem cell status and functions as a switch for differentiation of NPCs. Our investigation into the impact of HCMV infection on this pathway revealed that HCMV dysregulates Notch signaling by altering expression of the Notch ligand Jag1, Notch1, and its active effector in NPCs. These results suggest a mechanism for the neuropathogenesis induced by HCMV infection that includes altered NPC differentiation and proliferation.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Protein phosphatase 2A (PP2A) is a heterotrimeric protein phosphatase consisting of a 36-kD catalytic C subunit (PP2Ac). This study aimed to explore the prognostic and biological significance of PP2Ac in human hepatocellular carcinoma (HCC). High PP2Ac expression was significantly (P < 0.01) associated with serum hepatitis B surface antigen positivity, serum hepatitis B e antigen positivity, liver cirrhosis, moderate to poor differentiation grade, advanced disease stage, intrahepatic metastasis, and early recurrence in HCC. Multivariate analysis revealed PP2Ac as an independent prognostic factor for overall survival. Enforced expression of hepatitis B virus X protein (HBx) and its carboxyl-terminal truncated isoform induced PP2Ac expression in HCC cells. Co-immunoprecipitation assay revealed a direct interaction between PP2Ac and HBx. Small interfering RNA-mediated knockdown of PP2Ac significantly inhibited in vitro cell proliferation, colony formation, migration, and invasion and reduced tumor growth in an xenograft mouse model. In contrast, overexpression of PP2Ac promoted HCC cell proliferation, colony formation, and tumorigenesis. Additionally, silencing of PP2Ac impaired the growth-promoting effects on HepG2 HCC cells elicited by overexpression of carboxyl-terminal truncated HBx. Gene expression profiling analysis showed that PP2Ac downregulation modulated the expression of numerous genes involved in cell cycle and apoptosis regulation. Collectively, PP2Ac upregulation has a poor prognostic impact on the overall survival of HCC patients and contributes to the aggressiveness of HCC. PP2Ac may represent a potential therapeutic target for HCC.

Notch/RBP-Jkappa and nuclear factor-kappaB (NFkappaB) complexes are key mediators of the progression of many cellular events through the activation of specific target gene transcription. Independent observations have shown that activation of Notch-dependent transcription generally correlates with inhibition of differentiation. In contrast, activated NFkappaB complexes are required for progression of differentiation in several systems. Although some interactions between both pathways have been observed, the physiological significance of their connection is unclear. We have now demonstrated that the increase in p65-NFkappaB protein levels enhances Notch-mediated activation of the Hes1 promoter up to three-fold. This effect does not require NFkappaB transcriptional activity, and it is independent of the previously described interaction between Notch and p50-NFkappaB. Furthermore, we show that p65-NFkappaB can modulate subcellular localization of the transcriptional corepressor N-CoR, abrogating N-CoR mediated repression of the Hes1 promoter. In addition, p65-NFkappaB is able to upregulate not only the Hes1 but also other promoters containing SRE and AP-1 sites, which are repressed by N-CoR. Thus, we conclude that p65-NFkappaB can regulate gene expression by a general mechanism that involves cytoplasmic translocation of the transcriptional corepressor protein N-CoR.