Induction of notch signaling by immobilization of jagged-1 on self-assembled monolayers.
Notch signaling is a key mechanism during mammal development and stem cell regulation. This study aims to target and control Notch signaling by ligands immobilization using self-assembled monolayers (SAMs) as model surfaces. Non-fouling substrates were prepared by immersion of gold substrates in (1-Mercapto-11-undecyl)tetra(ethylene glycol) thiol solutions. These surfaces were activated with N,N'-carbonyldiimidazole (CDI) at different concentrations (0, 0.03, 0.3, 3 and 30 mg/ml) and an anti-human IgG, Fc specific fragment antibody (Ab) was covalently bound to EG4-SAMs to guarantee the correct exposure of the Notch ligand Jagged-1/Fc chimera (Jag-1). The presence of Ab and Jag-1 was confirmed by radiolabeling, X-ray photoelectron spectroscopy (XPS), ellipsometry and ELISA. The biological activity of Jag-1-Ab-SAMs was assessed by real-time PCR for Hes-1 family gene expression, a Notch pathway target gene, in HL-60 cell line. Results have shown an increase of the amount of immobilized Ab with increasing surface activator concentrations. Jag-1 concentration also increases with Ab concentration. Interestingly, a higher Jagged-1 exposure and fold increase in Hes-1 expression were obtained for surfaces activated with the lowest concentration of CDI (0.03 mg/ml). These results illustrate the great importance of ligands orientation and exposure, when compared with density. This investigation brings new insights into Notch signaling mechanisms. In particular, Jag-1-Ab-SAMs have shown to be adequate model surfaces to study Notch pathway activation and may provide a basis to develop new interfaces in biomaterials to control Notch mechanism in different cell systems.
INEB, Biomaterials Division, NEWTherapies Group, R. Campo Alegre 823, 4150-180 Porto, Portugal. raquelg@ineb.up.pt
Raquel M
RM
Gonçalves
M Cristina L
MC
Martins
Graça
G
Almeida-Porada
Mário A
MA
Barbosa
D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006046:Gold / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D000072100:Jagged-1 Protein; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D056951:Photoelectron Spectroscopy; D011961:Receptors, Fc / Q000235:genetics / Q000378:metabolism; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000072099:Serrate-Jagged Proteins; D015398:Signal Transduction / Q000502:physiology*; D013499:Surface Properties
D002135:Calcium-Binding Proteins; D007074:Immunoglobulin G; D036341:Intercellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; D000072100:Jagged-1 Protein; D008565:Membrane Proteins; D011961:Receptors, Fc; D051880:Receptors, Notch; D011993:Recombinant Fusion Proteins; D000072099:Serrate-Jagged Proteins; D006046:Gold