An evolutionary-conserved function of mammalian notch family members as cell adhesion molecules.

Notch family members were first identified as cell adhesion molecules by cell aggregation assays in Drosophila studies. However, they are generally recognized as signaling molecules, and it was unclear if their adhesion function was restricted to Drosophila. We previously demonstrated that a mouse Notch ligand, Delta-like 1 (Dll1) functioned as a cell adhesion molecule. We here investigated whether this adhesion function was conserved in the diversified mammalian Notch ligands consisted of two families, Delta-like (Dll1, Dll3 and Dll4) and Jagged (Jag1 and Jag2). The forced expression of mouse Dll1, Dll4, Jag1, and Jag2, but not Dll3, on stromal cells induced the rapid and enhanced adhesion of cultured mast cells (MCs). This was attributed to the binding of Notch1 and Notch2 on MCs to each Notch ligand on the stromal cells themselves, and not the activation of Notch signaling. Notch receptor-ligand binding strongly supported the tethering of MCs to stromal cells, the first step of cell adhesion. However, the Jag2-mediated adhesion of MCs was weaker and unlike other ligands appeared to require additional factor(s) in addition to the receptor-ligand binding. Taken together, these results demonstrated that the function of cell adhesion was conserved in mammalian as well as Drosophila Notch family members. Since Notch receptor-ligand interaction plays important roles in a broad spectrum of biological processes ranging from embryogenesis to disorders, our finding will provide a new perspective on these issues from the aspect of cell adhesion

The Association of Notch2 and NF-κB Accelerates RANKL-Induced Osteoclastogenesis▿

Notch signaling plays a key role in various cell differentiation processes including bone homeostasis. However, the specific involvement of Notch in regulating osteoclastogenesis is still controversial. In the present study, we show that RANKL induces expression of Jagged1 and Notch2 in bone marrow macrophages during osteoclast differentiation. Suppression of Notch signaling by a selective γ-secretase inhibitor or Notch2 short hairpin RNA suppresses RANKL-induced osteoclastogenesis. In contrast, induction of Notch signaling by Jagged1 or by ectopic expression of intracellular Notch2 enhances NFATc1 promoter activity and expression and promotes osteoclastogenesis. Finally, we found that Notch2 and p65 interact in the nuclei of RANKL-stimulated cells and that both proteins are recruited to the NFATc1 promoter, driving its expression. Taken together, our results show a new molecular cross talk between Notch and NF-κB pathways that is relevant in osteoclastogenesis

The Association of Notch2 and NF-B Accelerates RANKL-Induced Osteoclastogenesis

Notch signaling plays a key role in various cell differentiation processes including bone homeostasis. However, the specific involvement of Notch in regulating osteoclastogenesis is still controversial. In the present study, we show that RANKL induces expression of Jagged1 and Notch2 in bone marrow macrophages during osteoclast differentiation. Suppression of Notch signaling by a selective gamma-secretase inhibitor or Notch2 short hairpin RNA suppresses RANKL-induced osteoclastogenesis. In contrast, induction of Notch signaling by Jagged1 or by ectopic expression of intracellular Notch2 enhances NFATc1 promoter activity and expression and promotes osteoclastogenesis. Finally, we found that Notch2 and p65 interact in the nuclei of RANKL-stimulated cells and that both proteins are recruited to the NFATc1 promoter, driving its expression. Taken together, our results show a new molecular cross talk between Notch and NF-kappa B pathways that is relevant in osteoclastogenesis

Enhanced adhesion of MCs by Notch ligands involved both Notch1 and Notch2 on MCs.

<p>An adhesion assay (60 min) for MCs on each OP9 cell in a 96-well plate (A and B) with or without 10 µg/ml of the indicated polyclonal Ab (pAb) and (C) with control pAb (20 µg/ml), anti-Notch2 pAb (10 µg/ml) plus control pAb (10 µg/ml) or anti-Notch2 pAb (10 µg/ml) plus anti-Notch1 pAb (10 µg/ml). Data represent the percentages of non-adherent MCs (mean ± SEM of triplicate cultures) (*p<0.05, the Student’s <i>t-</i>test). Cultures with pAbs contained (A and B) 1.0% PBS (vol/vol) and 38.5 µM NaN₃ and (C) 2.0% PBS (vol/vol) and 76.9 µM NaN₃, which had no effect on the adhesion of MCs.</p

MCs efficiently adhered to OP9-Dll1, -Dll4, -Jag1, and -Jag2, but not OP9-Dll3, than to OP9-Ctrl.

<p>(A and B) Flow cytometric analysis of the expression of (A) Dll1, Dll3, Dll4, Jag1, and Jag2 on OP9 stromal cells transduced with each Notch ligand gene, and (B) Notch receptors on MCs after staining with specific mAbs (open histograms) or isotype-matched control mAbs (filled histograms). (C) Total RNA was analyzed by RT-PCR for the expression of Notch receptors in MCs and OP9-Ctrl cells. (D) Relative expression levels of <i>Notch1</i> and <i>Notch2</i> to <i>Gapdh</i> in MCs were analyzed by quantitative RT-PCR. Data represent the mean ± SEM of 3 independent experiments. (E and F) An adhesion assay for MCs on each OP9 cell (E) in a 48-well plate for 60 min and (F) in 96-well plates with serial incubation times of 5, 15, 30, 60, and 120 min. Data represent the percentages of non-adherent MCs (mean ± SEM of triplicate cultures) (*p<0.05 significantly different from OP9-Ctrl at each time point, the Student’s <i>t-</i>test).</p