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.

Promotion of In Vitro Hair Cell-like Cell Differentiation from Human Embryonic Stem Cells through the Regulation of Notch Signaling

The Notch signaling pathway plays an important role in otic neurogenesis by regulating the differentiation of inner ear hair cells and supporting cells. Notch-regulated differentiation is required for the regeneration of hair cells in the inner ear. The temporal expression pattern of Notch ligands and receptors during in vitro hair cell-like cell differentiation from human embryonic stem cells (hESCs) was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Subsequently, pAJ-U6-shRNA-CMV-Puro/GFP recombinant lentiviral vectors encoding short hairpin RNAs were used to silence JAG-1, JAG-2, and DLL-1, according to the temporal expression pattern of Notch ligands. Then, the effect of each ligand on the in vitro differentiation of hair cells was examined by RT-PCR, immunofluorescence, and scanning electron microscopy (SEM). The results showed that the individual deletion of JAG-2 or DLL-1 had no significant effect on the differentiation of hair cell-like cells. However, the simultaneous inhibition of both DLL-1 and JAG-2 increased the number of hair cell-like cells and decreased the number of supporting cells. JAG-2 and DLL-1 may have a synergistic role in in vitro hair cell differentiation.

Endothelial Upregulation of Mechanosensitive Channel Piezo1 in Pulmonary Hypertension

Piezo is a mechanosensitive cation channel responsible for stretch-mediated Ca2+ and Na+ influx in multiple types of cells. Little is known about the functional role of Piezo1 in the lung vasculature and its potential pathogenic role in pulmonary arterial hypertension (PAH). Pulmonary arterial endothelial cells (PAECs) are constantly under mechanic stretch and shear stress that are sufficient to activate Piezo channels. Here we report that Piezo1 is significantly upregulated in PAECs from patients with idiopathic PAH and animals with experimental pulmonary hypertension (PH) compared to normal controls. Membrane stretch by decreasing extracellular osmotic pressure or by cyclic stretch (18% CS) increases Ca2+-dependent phosphorylation (p) of AKT and ERK, and subsequently upregulates expression of Notch ligands, Jagged1/2 (Jag1 and Jag-2), and Delta like-4 (DLL4) in PAECs. siRNA-mediated downregulation of Piezo1 significantly inhibited the stretch-mediated pAKT increase and Jag-1 upregulation, while downregulation of AKT by siRNA markedly attenuated the stretch-mediated Jag1 upregulation in human PAECs. Furthermore, the mRNA and protein expression level of Piezo1 in the isolated pulmonary artery, which mainly contains pulmonary arterial smooth muscle cells (PASMCs), from animals with severe PH was also significantly higher than that from control animals. Taken together, our study suggests that membrane stretch-mediated Ca2+ influx through Piezo1 is an important trigger for pAKT-mediated upregulation of Jag-1 in PAECs. Upregulation of the mechanosensitive channel Piezo1 and the resultant increase in the Notch ligands (Jag-1/2 and DLL4) in PAECs may play a critical pathogenic role in the development of pulmonary vascular remodeling in PAH and PH.

Promotion of in Vitro Hair Cell-like Cell Differentiation from Human Embryonic Stem Cells through the Regulation of Notch Signaling

Background Notch signaling mediates the committed induced differentiation of ear sensory cells and promotes the formation of a precise arrangement of mosaics between hair cells and supporting cells. Embryonic stem cells (ESCs) are pluripotent stem cells which have the potential to differentiate into cell lines through three germ layers. Therefore, it is necessary to study the effects of regulating Notch receptors and ligand expression on the in vitro differentiation equilibrium of hair cells and supporting cells from ESCs.Methods and Results The temporal ex-pression pattern of Notch ligands and receptors during in vitro hair cell-like cell differentia-tion from human embryonic stem cells (hESCs) was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Subsequently, pAJ-U6-shRNA-CMV-Puro/GFP recombinant lentiviral vectors, encoding short hairpin RNAs, were used to silence JAG-1, JAG-2, and DLL-1, according to the temporal expression pattern of Notch ligands. Then the effect of each ligand on the in vitro differentiation of hair cells was examined by RT-PCR, immunofluorescence, and scanning electron microscopy (SEM).Conclusions Results showed that JAG-1 played an important role in regulating hESC differentiation to otic progenitors. The individual deletion of JAG-2 or DLL-1 had no significant effect on the differentiation of hair cell-like cells. Although the simultaneous inhibition of both DLL-1 and JAG-2 could increase the number of hair cell-like cells, it decreased the number of supporting cells.

P051 Macrophages as a source of Notch Ligands in Crohn’s disease: implications in fibrosis

Background Fibrosis constitute the main complications associated to Crohn’s disease (CD). Notch signalling has been implicated in lung, kidney, liver and cardiac fibrosis. Macrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their microenvironment. The aim of the present study is to analyze the role of Notch ligands derived from macrophages in the complications of CD. Methods We have analyzed: the mRNA expression of cytokines and Notch ligands in CD patients with fistulizing and stenting pattern, the mRNA and protein expression of macrophage markers and Notch ligands in macrophages treated with the main cytokines present in CD patients (IFNγ-, IL10-, IL4, TNFα-U937 treated cells), the mRNA expression of fibrosis markers of DLL4-HT29 treated cells. Results are expressed as fold induction (mean±SEM, n≥4). Statistical analysis was performed with one-way ANOVA followed by Newman-Keuls test. Correlations were analyzed with the Spearman coefficient. Results The mRNA expression of IFNγ, TNFα, IL4, IL6 and IL10 was significantly higher in intestinal samples from B2- and B3-CD patients (11.4±1.6, 6.9±1.3, 16.4±3.9, 4.1±0.4, and 5.0±1.0 respectively for B2, and 14.2±1.8, 9.6±2.6, 21.5±4.1, 6.3±1.0 and 9.2±1.7 respectively for B3) than in controls (1.0±0.09, 1.1±0.2, 1.2±0.1, 1.1±0.1 and 1.0±0.1 respectively). The mRNA expression of DLL3 and DLL4 was significantly higher in intestinal samples from B2 (6.1±1.3 and 9.3±2.4, respectively) than in B3 CD patients (4.4±1.0 and 1.8±0.7, respectively) and in controls (0.9±0.2 and 0.7±1.2 respectively). IFNγ-U937 treated cells increased significantly the mRNA expression of DLL3 and DLL4 (2,9±0,6 N=5 N=4 and 3,7±1,1 N=4, respectively) respect vehicle; IL1β increased significantly the expression of DLL4 (1,8±0,01 N=4) and TNFα increased significantly the expression of DLL3 (7,1±2,2 N=4), respect vehicle. DLL4-HT29 treated cells increased significantly fibrosis markers (VIMENTIN (4,6±0,6 N=6), α-SMA (20,9±8,2 N=6), SNAIL1 (1,8±0,4 N=6), SNAIL2 (8,3±1,3 N=6), ZEB1 (8,2±4,3 N=6) and ZEB2 (4,3±0,2 N=6), respect vehicle. Conclusion Macrophages may act as a source of Notch ligands who could act as fibrosis mediators in CD patients with a stenting (B2) behavior. The microenvironment rich in IFNγ could activate the fibrosis process in epithelial cells by favoring the expression of DLL4 in macrophages.

Oscillations of Delta-like1 regulate the balance between differentiation and maintenance of muscle stem cells

Cell-cell interactions mediated by Notch are critical for the maintenance of skeletal muscle stem cells. However, dynamics, cellular source and identity of functional Notch ligands during expansion of the stem cell pool in muscle growth and regeneration remain poorly characterized. Here we demonstrate that oscillating Delta-like 1 (Dll1) produced by myogenic cells is an indispensable Notch ligand for self-renewal of muscle stem cells in mice. Dll1 expression is controlled by the Notch target Hes1 and the muscle regulatory factor MyoD. Consistent with our mathematical model, our experimental analyses show that Hes1 acts as the oscillatory pacemaker, whereas MyoD regulates robust Dll1 expression. Interfering with Dll1 oscillations without changing its overall expression level impairs self-renewal, resulting in premature differentiation of muscle stem cells during muscle growth and regeneration. We conclude that the oscillatory Dll1 input into Notch signaling ensures the equilibrium between self-renewal and differentiation in myogenic cell communities.

Endothelial cell senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by pathological pulmonary artery remodeling. Endothelial cells (EC) injury including DNA damage is critically involved in the vascular remodeling in PAH, and persistent injury leads to cellular senescence in ECs. Here, we show that EC senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling. EC-specific progeroid mice that we recently generated showed exacerbated pulmonary hypertension after chronic hypoxia exposure, accompanied by the enhanced pulmonary arterial smooth muscle cells (PASMCs) proliferation in the distal pulmonary arteries. Mechanistically, we identified that senescent ECs highly expressed Notch ligands, and thus activated Notch signaling in PASMCs, leading to enhanced PASMCs proliferation and migration capacities. Consistently, pharmacological inhibition of Notch signaling attenuated the effects of senescent ECs on SMCs functions in vitro, and on the pulmonary hypertension in EC-specific progeroid mice in vivo. These data establish EC senescence as a crucial disease-modifying facor in PAH.

NOTCH Activation via gp130/STAT3 Signaling Confers Resistance to Chemoradiotherapy

Resistance of tumor cells to chemoradiotherapy represents a fundamental problem in clinical oncology. The underlying mechanisms are actively debated. Here we show that blocking inflammatory cytokine receptor signaling via STAT3 re-sensitized treatment-refractory cancer cells and abolished tumor growth in a xenograft mouse model when applied together with chemoradiotherapy. STAT3 executed treatment resistance by triggering the expression of RBPJ, the key transcriptional regulator of the NOTCH pathway. The mandatory RBPJ interaction partner, NOTCH intracellular domain, was provided by tumor cell-intrinsic expression of NOTCH ligands that caused tonic NOTCH proteolysis. In fact, NOTCH inhibition phenocopied the effect of blocking STAT3 signaling. Moreover, genetic profiling of rectal cancer patients revealed the importance of the STAT3/NOTCH axis as NOTCH expression correlated with clinical outcome. Our data uncovered an unprecedented signal alliance between inflammation and cellular development that orchestrated resistance to chemoradiotherapy. Clinically, our findings allow for biomarker-driven patient stratification and offer novel treatment options.