Notch2 activation ameliorates nephrosis.

Activation of Notch1 and Notch2 has been recently implicated in human glomerular diseases. Here we show that Notch2 prevents podocyte loss and nephrosis. Administration of a Notch2 agonistic monoclonal antibody ameliorates proteinuria and glomerulosclerosis in a mouse model of nephrosis and focal segmental glomerulosclerosis. In vitro, the specific knockdown of Notch2 increases apoptosis in damaged podocytes, while Notch2 agonistic antibodies enhance activation of Akt and protect damaged podocytes from apoptosis. Treatment with triciribine, an inhibitor of Akt pathway, abolishes the protective effect of the Notch2 agonistic antibody. We find a positive linear correlation between the number of podocytes expressing activated Notch2 and the number of residual podocytes in human nephrotic specimens. Hence, specific activation of Notch2 rescues damaged podocytes and activating Notch2 may represent a novel clinical strategy for the amelioration of nephrosis and glomerulosclerosis.

Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes.

INTRODUCTION: Osteoclastogenesis plays an important role in the bone erosion of rheumatoid arthritis (RA). Recently, Notch receptors have been implicated in the development of osteoclasts. However, the responsible Notch ligands have not been identified yet. This study was undertaken to determine the role of individual Notch receptors and ligands in osteoclastogenesis. METHODS: Mouse bone marrow-derived macrophages or human peripheral blood monocytes were used as osteoclast precursors and cultured with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) to induce osteoclasts. Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining. K/BxN serum-induced arthritic mice and ovariectomized mice were treated with anti-mouse Delta-like 1 (Dll1) blocking monoclonal antibody (mAb). RESULTS: Blockade of a Notch ligand Dll1 with mAb inhibited osteoclastogenesis and, conversely, immobilized Dll1-Fc fusion protein enhanced it in both mice and humans. In contrast, blockade of a Notch ligand Jagged1 enhanced osteoclastogenesis and immobilized Jagged1-Fc suppressed it. Enhancement of osteoclastogenesis by agonistic anti-Notch2 mAb suggested that Dll1 promoted osteoclastogenesis via Notch2, while suppression by agonistic anti-Notch1 mAb suggested that Jagged1 suppressed osteoclastogenesis via Notch1. Inhibition of Notch signaling by a gamma-secretase inhibitor suppressed osteoclastogenesis, implying that Notch2/Dll1-mediated enhancement was dominant. Actually, blockade of Dll1 ameliorated arthritis induced by K/BxN serum transfer, reduced the number of osteoclasts in the affected joints and suppressed ovariectomy-induced bone loss. CONCLUSIONS: The differential regulation of osteoclastogenesis by Notch2/Dll1 and Notch1/Jagged1 axes may be a novel target for amelioration of bone erosion in RA patients.

Regulation of experimental autoimmune uveoretinitis by anti-delta-like ligand 4 monoclonal antibody.

PURPOSE: To investigate the involvement of δ-like ligand (Dll)4 in the development of experimental autoimmune uveoretinitis (EAU) in B10.RIII mice. METHODS: B10.RIII mice were immunized with interphotoreceptor retinoid binding protein (IRBP) peptide 161-180 in complete Freund's adjuvant together with intraperitoneal injection of Bordetella pertussis toxin. mRNA expressions of Notch receptors and their ligands in the eye were evaluated. To investigate the involvement of Dll in EAU, anti-Dll1, anti-Dll4, or control antibody (Ab) was intraperitoneally injected during both the induction and the effector phases or only the effector phase. Alternatively, mice were intraperitoneally injected with γ-secretase inhibitor (GSI) or the control vehicle during the induction phase. Fourteen days after immunization, the eyes and spleens were harvested. The eyes were used for histologic and/or cytokine mRNA expression analysis, whereas the spleens were used for flow cytometric analysis, and antigen-recall proliferation and cytokine assays. RESULTS: Expression of Notch1, 2, 4, and Dll4 in the eye were upregulated by EAU induction. Anti-Dll4 Ab treatment during both the induction and effector phases, but not only the effector phase, significantly reduced the severity of EAU. IFN-γ, IL-12p35, IL-17A, and TGF-ß mRNA expression in the eye were significantly attenuated by treatment with anti-Dll4 Ab. Splenocytes from anti-Dll4 Ab-treated mice showed significantly less proliferation and IL-17 production on antigen stimulation. Also, the severity of EAU was significantly reduced by γ-secretase inhibitor treatment during the induction phase. CONCLUSIONS; Dll4-mediated Notch signaling during the sensitization is critical for the development of EAU. This can be a novel prophylactic target for autoimmune uveitis.