Abstract
The transcription factor NRF2 is a master regulator of cellular antioxidant and detoxification responses, but it also regulates other processes such as autophagy and pluripotency. In human embryonic stem cells (hESCs), NRF2 antagonizes neuroectoderm differentiation, which only occurs after NRF2 is repressed via a Primary Cilia-Autophagy-NRF2 (PAN) axis. However, the functional connections between NRF2 and primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae, remain poorly understood. For instance, nothing is known about whether NRF2 affects cilia, or whether cilia regulation of NRF2 extends beyond hESCs. Here, we show that NRF2 and primary cilia reciprocally regulate each other. First, we demonstrate that fibroblasts lacking primary cilia have higher NRF2 activity, which is rescued by autophagy-activating mTOR inhibitors, indicating that the PAN axis also operates in differentiated cells. Furthermore, NRF2 controls cilia formation and function. NRF2-null cells grow fewer and shorter cilia and display impaired Hedgehog signaling, a cilia-dependent pathway. These defects are not due to increased oxidative stress or ciliophagy, but rather to NRF2 promoting expression of multiple ciliogenic and Hedgehog pathway genes. Among these, we focused on GLI2 and GLI3, the transcription factors controlling Hh pathway output. Both their mRNA and protein levels are reduced in NRF2-null cells, consistent with their gene promoters containing consensus ARE sequences predicted to bind NRF2. Moreover, GLI2 and GLI3 fail to accumulate at the ciliary tip of NRF2-null cells upon Hh pathway activation. Given the importance of NRF2 and ciliary signaling in human disease, our data may have important biomedical implications.
Highlights
Primary cilia are microtubule-based plasma membrane protrusions that function as cell type-specific antennae by accumulating signal receptors and transducers
We find that fibroblasts lacking cilia have reduced autophagy and increased NRF2 activity, which we rescue with autophagy-activating mTOR inhibitors
Hmox[1] protein levels were increased in Ift88−/− mouse embryonic fibroblasts (MEFs), and the effect was clearer in sulforaphane-treated cells (Supplementary Fig. S1c)
Summary
Primary cilia are microtubule-based plasma membrane protrusions that function as cell type-specific antennae by accumulating signal receptors and transducers. Disruption of Hedgehog (Hh) signaling, a cilia-dependent pathway controlling multiple aspects of embryogenesis and adult stem cell function[7], explains many (but not all) ciliopathy symptoms and cilia-dependent cancers Hh ligands, such as Sonic Hedgehog (SHH), act in paracrine fashion by binding to Patched (PTCH1), a ciliary transmembrane protein. Ligand binding prevents PTCH1 from opposing the ciliary accumulation of Smoothened (SMO), a seven transmembrane protein whose activation displaces another G protein-coupled receptor, GPR161, from cilia This in turn lowers cAMP levels and PKA activity at the basal body, affecting phosphorylation of GLI2 and GLI3, the zinc finger transcription factors mediating Hh pathway output. Cilia in turn activate autophagy, resulting in the inactivation of NRF2, which antagonizes neuroectoderm differentiation Whether this so-called Primary Cilium-Autophagy-NRF2 (PAN) axis is conserved in other cell types, and whether mTOR is involved in it, has not been addressed[12]
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