Modulation of Notch signaling by antibodies specific for the extracellular negative regulatory region of NOTCH3

The Notch pathway regulates the development of many tissues and cell types and is involved in a variety of human diseases, making it an attractive potential therapeutic target. This promise has been limited by the absence of potent inhibitors or agonists that are specific for individual human Notch receptors (NOTCH1-4). Using an unbiased functional screening, we identified monoclonal antibodies that specifically inhibit or induce activating proteolytic cleavages in NOTCH3. Remarkably, the most potent inhibitory and activating antibodies bind to overlapping epitopes within a juxtamembrane negative regulatory region that protects NOTCH3 from proteolysis and activation in its resting autoinhibited state. The inhibitory antibodies revert phenotypes conveyed on 293T cells by NOTCH3 signaling, such as increased cellular proliferation, survival, and motility, whereas the activating antibody mimics some of the effects of ligand-induced Notch activation. These findings provide insights into the mechanisms of Notch autoinhibition and activation and pave the way for the further development of specific antibody-based modulators of the Notch receptors, which are likely to be of utility in a wide range of experimental and therapeutic settings.     

Calcium depletion dissociates and activates heterodimeric notch receptors

Notch receptors participate in a highly conserved signaling pathway that regulates morphogenesis in multicellular animals. Maturation of Notch receptors requires the proteolytic cleavage of a single precursor polypeptide to produce a heterodimer composed of a ligand-binding extracellular domain (N(EC)) and a single-pass transmembrane signaling domain (N(TM)). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of N(TM) (N(ICD)). In the current work, we show that the N(EC) and N(TM) subunits of Drosophila Notch and human Notch1 (hN1) interact noncovalently. N(EC)-N(TM) interaction was disrupted by 0.1% sodium dodecyl sulfate or divalent cation chelators such as EDTA, and stabilized by millimolar Ca(2+). Deletion of the Ca(2+)-binding Lin12-Notch (LN) repeats from the N(EC) subunit resulted in spontaneous shedding of N(EC) into conditioned medium, implying that the LN repeats are important in maintaining the interaction of N(EC) and N(TM). The functional consequences of EDTA-induced N(EC) dissociation were studied by using hN1-expressing NIH 3T3 cells. Treatment of these cells for 10 to 15 min with 0.5 to 10 mM EDTA resulted in the rapid shedding of N(EC), the transient appearance of a polypeptide of the expected size of N(ICD), increased intranuclear anti-Notch1 staining, and the transient activation of an Notch-sensitive reporter gene. EDTA treatment of HeLa cells expressing endogenous Notch1 also stimulated reporter gene activity to a degree equivalent to that resulting from exposure of the cells to the ligand Delta1. These findings indicate that receptor activation can occur as a consequence of N(EC) dissociation, which relieves inhibition of the intrinsically active N(TM) subunit.     

BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma

Melanoma is the most lethal form of skin cancer, and the incidence and mortality rates are rapidly rising. Epidemiologically, high numbers of nevi (moles) are associated with higher risk of melanoma . The majority of melanomas exhibit activating mutations in the serine/threonine kinase BRAF . BRAF mutations may be critical for the initiation of melanoma ; however, the direct role of BRAF in nevi and melanoma has not been tested in an animal model. To directly test the role of activated BRAF in nevus and melanoma development, we have generated transgenic zebrafish expressing the most common BRAF mutant form (V600E) under the control of the melanocyte mitfa promoter. Expression of mutant, but not wild-type, BRAF led to dramatic patches of ectopic melanocytes, which we have termed fish (f)-nevi. Remarkably, in p53-deficient fish, activated BRAF induced formation of melanocyte lesions that rapidly developed into invasive melanomas, which resembled human melanomas and could be serially transplanted. These data provide direct evidence that BRAF activation is sufficient for f-nevus formation, that BRAF activation is among the primary events in melanoma development, and that the p53 and BRAF pathways interact genetically to produce melanoma.     

Regulatory Mechanisms of Vitamin D<Subscript>3</Subscript> on Production of Nitric Oxide and Pro-inflammatory Cytokines in Microglial BV-2 Cells

Inhibition of pro-inflammatory functions of microglia has been considered a promising strategy to prevent pathogenic events in the central nervous system under neurodegenerative conditions. Here we examined potential inhibitory effects of nuclear receptor ligands on lipopolysaccharide (LPS)-induced inflammatory responses in microglial BV-2 cells. We demonstrate that a vitamin D receptor agonist 1,25-dihydroxyvitamin D₃ (VD3) and a retinoid X receptor agonist HX630 affect LPS-induced expression of pro-inflammatory factors. Specifically, both VD3 and HX630 inhibited expression of mRNAs encoding inducible nitric oxide synthase (iNOS) and IL-6, whereas expression of IL-1β mRNA was inhibited only by VD3. The inhibitory effect of VD3 and HX630 on expression of iNOS and IL-6 mRNAs was additive. Effect of VD3 and HX630 was also observed for inhibition of iNOS protein expression and nitric oxide production. Moreover, VD3 and HX630 inhibited LPS-induced activation of extracellular signal-regulated kinase (ERK) and nuclear translocation of nuclear factor κB (NF-κB). PD98059, an inhibitor of ERK kinase, attenuated LPS-induced nuclear translocation of NF-κB and induction of mRNAs for iNOS, IL-1β and IL-6. These results indicate that VD3 can inhibit production of several pro-inflammatory molecules from microglia, and that suppression of ERK activation is at least in part involved in the anti-inflammatory effect of VD3.     

Band 4.1-like proteins of the bovine lens. Effects of differentiation, distribution and extraction characteristics.

Bovine lens epithelium, cortex and nucleus were screened for the presence of red-cell-membrane band 4.1-like proteins by using an immunoblot method. Lens epithelial cells were found to contain proteins of Mr 78 000 and higher (approximately 150 000) that cross-reacted with anti-(protein 4.1) sera. Fibre cells of the superficial cortex were also found to contain these two proteins, as well as an additional protein of approx. 80 000 Mr. In contrast, deep layers of the cortex and the lens nucleus contained no detectable cross-reactive protein at these Mr values. Treatment of a crude membrane fraction prepared from superficial bovine cortices with a low-ionic-strength buffer resulted in release of the high-Mr band 4.1-like protein. The 80 000- and 78 000-Mr proteins remained with the membrane fraction in low-ionic-strength buffer, but were released into solution by high-ionic-strength-buffer treatment. We have also demonstrated that the human red-blood-cell membrane, like lens epithelial cells and fibre cells, also contains a high-Mr band 4.1-like protein that is released from membranes by low-ionic-strength-buffer treatment.     

Structural basis for autoinhibition of Notch

Notch receptors transmit signals between adjacent cells. Signaling is initiated when ligand binding induces metalloprotease cleavage of Notch within an extracellular negative regulatory region (NRR). We present here the X-ray structure of the human NOTCH2 NRR, which adopts an autoinhibited conformation. Extensive interdomain interactions within the NRR bury the metalloprotease site, showing that a substantial conformational movement is necessary to expose this site during activation by ligand. Leukemia-associated mutations in NOTCH1 probably release autoinhibition by destabilizing the conserved hydrophobic core of the NRR.