Opposing Putative Roles for Canonical and Noncanonical NFκB Signaling on the Survival, Proliferation, and Differentiation Potential of Human Embryonic Stem Cells

The canonical and noncanonical NFκB signaling pathways regulate a variety of cellular activities; however, their functions in human embryonic stem cells (hESCs) have not been fully investigated. Expression studies during hESC differentiation indicated a significant increase in the expression of two key components of the canonical NFκB pathway (p50 and Ser529 phosphorylated form of p65) as well as a significant reduction in expression of key components of the noncanonical NFκB pathway [v-rel reticuloendotheliosis viral oncogene homolog B (RELB), p52, NIK]. Inhibition of canonical NFκB resulted in hESC apoptosis, changes in cell cycle distribution, and reduced hESC proliferation. In addition, inhibition of canonical NFκB was associated with significant changes in NANOG and OCT4 expression, suppression of differentiation toward all primitive extraembryonic and embryonic lineages with the exception of primitive ectoderm and ectodermal lineages. Inhibition of noncanonical NFκB via small interfering RNA-mediated downregulation of RELB resulted in reduced hESC proliferation and opposite changes to expression of key differentiation lineage markers genes when compared with downregulation of canonical NF-κB. Chromatin immunoprecipitation assays indicated binding of p65 and RELB to regulatory regions of key differentiation marker genes suggesting a direct transcriptional role for both branches of this pathway in hESC. These findings coupled with opposing trends in expression of key components during hESC differentiation, suggests a fine and opposing balance between the two branches of NFκB signaling pathways and their involvement in two distinct processes: the canonical pathway regulating hESC differentiation and the noncanonical pathway maintaining hESC pluripotency.