Background Endothelial Notch signaling is a key player in regulating inflammation. While Notch1 has been extensively studied in the context of vascular inflammation, the role of endothelial Notch4, remains relatively unexplored. Growing evidence suggests that Notch4 plays critical roles in human endothelial inflammatory disorders. Polymorphisms in Notch4 are associated with endothelial dysfunction in preeclampsia, HIV-triggered glomerular injury, and severity of COVID-19 endothelial inflammation. Our recent work has shown that pharmacologic inhibition of endothelial Notch4 signaling using an anti-Notch4 antibody alters macrophage number and phenotype in mouse models of breast cancer, further supporting a distinct role for Notch4 in regulation of inflammatory targets. Our preliminary work has determined that Notch4 regulates an endothelial transcriptome distinct from Notch1, which is enriched for inflammatory genes such as CCL2 and IL6. However, all Notch proteins bind DNA via a common transcriptional co-factor, CSL, and the mechanism of how different Notch proteins regulate distinct transcriptional targets remains unclear. Better methods for detection of real-time and gene-specific activation of Notch signaling are critical for dissecting the roles of different Notch proteins in endothelium.

Methods To examine gene-specific Notch transcriptional activity, we used the NanoBiT protein-protein-interaction system, a complementation-based luciferase reporter system composed of two split luciferase fragments, LgBiT and SmBiT. The NanoBiT PPI system allows for detection of protein:protein interactions through two subunits, Large BiT (LgBit) and Small BiT (SmBit), fused to two proteins of interest. Interaction between target proteins allows the subunits to come together resulting in luminescent signal that can be quantified in live cells. Real-time quantification allows for dynamic monitoring, capturing transient interactions and providing temporal resolution that homogenate testing lacks. We designed fusions of the active intracellular domain of Notch4 (ICN4) or Notch1 (ICN1) with SmBiT, and CSL with LgBiT and expressed the fusion proteins in HeLa cells. The resulting interactions between fusion proteins resulted in luminescent signal that was quantified using the SpectraMax L Microplate Reader.

Results We validated that NanoBiT CSL and ICN fusion proteins were expressed at high levels and retained their ability to induce Notch pathway targets. Co-expression of CSL-LgBiT with either ICN4-SmBiT or ICN1-SmBiT resulted in robust induction of luminescent signal. Interaction between CSL and ICN4 consistently yielded a higher luminescent signal than ICN1, suggesting that Notch4 may more strongly interact with CSL.

Conclusion We have designed and tested a novel method for real time measurement of gene-specific Notch pathway activation. Our preliminary findings suggest that Notch4 plays a distinct role in inflammatory signaling, possibly due to its robust interaction with CSL. Our NanoBiT-based assay may be used to determine protective targets in inflammatory diseases.