Noncanonical NF-κB activation and SDF-1 expression in human endothelial cells

NF-κB is a family of transcription factors regulated through two distinct signaling cascades, the classical and the Noncanonical NF-κB pathways. Noncanonical NF-κB plays important roles in the immune system, as it is necessary for lymphoid organogenesis and B-cell survival and differentiation, as well as osteoclastogenesis. In the last few years, there has been an increased number of studies focusing on both identifying the upstream events that regulate the noncanonical NF-κB pathway as well as determining the physiological roles of noncanonical NF-κB in normal and disease pathologies, such as cancer and autoimmune diseases. Dysregulation of noncanonical NF-κB has now been associated with the pathogenesis of several types of lymphomas and autoimmune diseases and is believed to contribute to chronic inflammatory diseases, including ulcerative colitis. These studies suggest that targeting the Noncanonical pathway, similar to classical NF-κB, may have some therapeutic potential in the future; however, there is still quite a bit about the regulation of the noncanonical signaling that remains to be defined. In this chapter we describe the use of HUVEC, as an in vitro model for examining noncanonical NF-κB signaling in response to different stimuli. We demonstrate two different methods to measure noncanonical NF-κB activation: the processing of p100 to p52, and noncanonical NF-κB-dependent gene expression of CXCL12. The first method examines a key regulatory requirement for noncanonical NF-κB activation, by which p100 undergoes proteolytic cleavage to relieve the inhibition of NF-κB dimers for nuclear translocation and activation of gene transcription. The latter demonstrates the downstream effects of activated noncanonical NF-κB in response to stimuli.