Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells

Notch signaling is a conserved cell fate regulator during development and postnatal tissue regeneration. Using skeletal muscle satellite cells as a model and through myogenic cell lineage-specific NICD(OE) (overexpression of constitutively activated Notch 1 intracellular domain), here we investigate how Notch signaling regulates the cell fate choice of muscle stem cells. We show that in addition to inhibiting MyoD and myogenic differentiation, NICD(OE) upregulates Pax7 and promotes the self-renewal of satellite cell-derived primary myoblasts in culture. Using MyoD(-/-) myoblasts, we further show that NICD(OE) upregulates Pax7 independently of MyoD inhibition. In striking contrast to previous observations, NICD(OE) also inhibits S-phase entry and Ki67 expression and thus reduces the proliferation of primary myoblasts. Overexpression of canonical Notch target genes mimics the inhibitory effects of NICD(OE) on MyoD and Ki67 but not the stimulatory effect on Pax7. Instead, NICD regulates Pax7 through interaction with RBP-Jκ, which binds to two consensus sites upstream of the Pax7 gene. Importantly, satellite cell-specific NICD(OE) results in impaired regeneration of skeletal muscles along with increased Pax7(+) mononuclear cells. Our results establish a role of Notch signaling in actively promoting the self-renewal of muscle stem cells through direct regulation of Pax7.     

Rnd3 Regulates Lung Cancer Cell Proliferation through Notch Signaling

Rnd3/RhoE is a small Rho GTPase involved in the regulation of different cell behaviors. Dysregulation of Rnd3 has been linked to tumorigenesis and metastasis. Lung cancers are the leading cause of cancer-related death in the West and around the world. The expression of Rnd3 and its ectopic role in non-small cell lung cancer (NSCLC) remain to be explored. Here, we reported that Rnd3 was down-regulated in three NSCLC cell lines: H358, H520 and A549. The down-regulation of Rnd3 led to hyper-activation of Rho Kinase and Notch signaling. The reintroduction of Rnd3 or selective inhibition of Notch signaling, but not Rho Kinase signaling, blocked the proliferation of H358 and H520 cells. Mechanistically, Notch intracellular domain (NICD) protein abundance in H358 cells was regulated by Rnd3-mediated NICD proteasome degradation. Rnd3 regulated H358 and H520 cell proliferation through a Notch1/NICD/Hes1 signaling axis independent of Rho Kinase.     

Gamma-secretase-regulated proteolysis of the Notch receptor by mitochondrial intermediate peptidase

Notch is a transmembrane receptor that controls a diverse array of cellular processes including cell proliferation, differentiation, survival, and migration. The cellular outcome of Notch signaling is dependent on extracellular and intracellular signals, but the complexities of its regulation are not well understood. Canonical Notch signaling involves ligand association that triggers sequential and regulated proteolysis of Notch at several sites. Ligand-dependent proteolysis at the S2 site removes the bulk of the extracellular domain of Notch. Subsequent γ-secretase-mediated intramembrane proteolysis of the remaining membrane-tethered Notch fragment at the S3 site produces a nuclear-destined Notch intracellular domain (NICD). Here we show that following γ-secretase cleavage, Notch is proteolyzed at a novel S5 site. We have identified this S5 site to be eight amino acids downstream of the S3 site. Biochemical fractionation and purification resulted in the identification of the S5 site protease as the mitochondrial intermediate peptidase (MIPEP). Expression of the MIPEP-cleaved NICD (ΔNICD) results in a decrease in cell viability and mitochondria membrane potential. The sequential and regulated proteolysis by γ-secretase and MIPEP suggests a new means by which Notch function can be modulated.     

YTHDF2 Suppresses Notch Signaling through Post-transcriptional Regulation on Notch1

YTH domain family 2 (YTHDF2) is an N6-methyladenosine (m⁶A) binding protein promoting mRNA degradation in various biological processes. Despite its essential roles, the role of YTHDF2 in determining cell fates has not been fully elucidated. Notch signaling plays a vital role in determining cell fates, such as proliferation, differentiation, and apoptosis. We investigated the effect of YTHDF2 on Notch signaling. Our results show that YTHDF2 inhibits Notch signaling by downregulating the Notch1, HES1, and HES5 mRNA levels. Analyzing YTHDF2 deletion mutants indicates that the YTH domain is critical in regulating the Notch signal by directly binding m⁶A of Notch1 mRNA. Recently, YTHDF2 nuclear translocation was reported under heat shock conditions, but its physiological function is unknown. In our study, the YTH domain is required for YTHDF2 nuclear translocation. In addition, under heat shock stress, the Notch signal was significantly restored due to the increased expression of the Notch1 targets. These results suggest that YTHDF2 in the cytoplasm may act as an intrinsic suppressor in Notch signaling by promoting Notch1 mRNA degradation under normal cellular conditions. Conversely, upon the extracellular stress such as heat shock, YTHDF2 nuclear translocation resulting in reduced Notch1 mRNA decay may contribute to the increasing of Notch intracellular domain (NICD) regulating the survival-related target genes.     

Dicer is required for the maintenance of notch signaling and gliogenic competence during mouse retinal development

MicroRNAs (miRNAs) are 19-25 nucleotide RNAs that regulate messenger RNA translation and stability. Recently, we performed a conditional knockout (CKO) of the miRNA-processing enzyme Dicer during mouse retinal development and showed an essential role for miRNAs in the transition of retinal progenitors from an early to a late competence state (Georgi and Reh [2010]: J Neurosci 30:4048-4061). Notably, Dicer CKO progenitors failed to express Ascl1 and generated ganglion cells beyond their normal competence window. Because Ascl1 regulates multiple Notch signaling components, we hypothesized that Notch signaling is downregulated in Dicer CKO retinas. We show here that Notch signaling is severely reduced in Dicer CKO retinas, but that retinal progenitors still retain a low level of Notch signaling. By increasing Notch signaling in Dicer CKO progenitors through constitutive expression of the Notch intracellular domain (NICD), we show that transgenic rescue of Notch signaling has little effect on the competence of retinal progenitors or the enhanced generation of ganglion cells, suggesting that loss of Notch signaling is not a major determinant of these phenotypes. Nevertheless, transgenic NICD expression restored horizontal cells, suggesting an interaction between miRNAs and Notch signaling in the development of this cell type. Furthermore, while NICD overexpression leads to robust glial induction in control retinas, NICD overexpression was insufficient to drive Dicer-null retinal progenitors to a glial fate. Surprisingly, the presence of transgenic NICD expression did not prevent the differentiation of some types of retinal neurons, suggesting that Notch inactivation is not an absolute requirement for the initial stages of neuronal differentiation.     

Distinct roles for the NK cell-activating receptors in mediating interactions with dendritic cells and tumor cells

Hyperproduction of goblet cells and mucin in the airway epithelium is an important feature of airway inflammatory diseases. We investigated the involvement of Notch signaling in MUC5AC expression in NCI-H292 cells, a human lung carcinoma cell line. Epidermal growth factor (EGF) stimulated generation of the Notch intracellular domain (NICD) in a RBP-Jκ-dependent manner. Treatment with γ-secretase inhibitors L-685,458 or DAPT or introduction of small interfering RNA directed against Notch1 reduced EGF-induced MUC5AC expression. The inhibitory effect of L-685,458 on EGF-induced MUC5AC mRNA and protein expression was also observed in primary human bronchial epithelial cells. Blockage of Notch signaling with L-685,458 or Notch siRNA resulted in a decrease in EGF-induced phosphorylation of ERK. These results suggested that ERK activation is necessary for the regulation of EGF receptor (EGFR)-mediated MUC5AC expression by Notch signaling. Conversely, forced expression of NICD induced both EGFR and ERK phosphorylation with MUC5AC expression even in the absence of EGF. Treatment of the NICD-expressing cells with EGF further augmented ERK phosphorylation in an additive manner. The ERK phosphorylation induced by exogenous NICD was inhibited by treatment with an Ab that antagonizes EGFR activity as well as by inhibitors of EGFR and ERK, implying that Notch signaling induces MUC5AC expression by activating the EGFR pathway. Collectively, these results suggest that MUC5AC expression is regulated by a bidirectional circuit between Notch and EGFR signaling pathways.     

Analysis of Notch function in presomitic mesoderm suggests a gamma-secretase-independent role for presenilins in somite differentiation

The role of Notch signaling in general and presenilin in particular was analyzed during mouse somitogenesis. We visualize cyclical production of activated Notch (NICD) and establish that somitogenesis requires less NICD than any other tissue in early mouse embryos. Indeed, formation of cervical somites proceeds in Notch1; Notch2-deficient embryos. This is in contrast to mice lacking all presenilin alleles, which have no somites. Since Nicastrin-, Pen-2-, and APH-1a-deficient embryos have anterior somites without gamma-secretase, presenilin may have a gamma-secretase-independent role in somitogenesis. Embryos triple homozygous for both presenilin null alleles and a Notch allele that is a poor substrate for presenilin (N1(V-->G)) experience fortuitous cleavage of N1(V-->G) by another protease. This restores NICD, anterior segmentation, and bilateral symmetry but does not rescue rostral/caudal identities. These data clarify multiple roles for Notch signaling during segmentation and suggest that the earliest stages of somitogenesis are regulated by both Notch-dependent and Notch-independent functions of presenilin.     

Among γ-secretase substrates Notch1 alone is sufficient to block neurogenesis but does not confer self-renewal properties to neural stem cells

Notch signaling pathway enhances neural stem cell characters and regulates cell fate decisions during neural development. Interestingly, besides Notch, other γ-secretase substrates such as APP, LRP2, and ErbB4 have also proven to have biological functions in neural development. We designed a unique experimental setting, combining gain-of- (expression of Notch intracellular domain, NICD) and loss-of-function (γ-secretase inhibition) methods, and were able to examine the function of Notch alone by excluding the activity of other γ-secretase substrates. Here, we show that the frequency and size of neurospheres generated from embryonic neural stem cells (NSCs) significantly decreased by 62.7% and 37.2%, respectively, in the presence of γ-secretase inhibitor even when NICD was expressed. Under the condition of differentiation, however, the γ-secretase inhibitor treatment did not influence the promotion of astrogenesis at the expense of neurogenesis by NICD. These results indicate that other γ-secretase substrate(s) along with Notch are important in the maintenance of the stemness of NSCs, but that Notch alone can sufficiently inhibit neurogenesis without the action of the other γ-secretase substrates during differentiation.