Maintenance of Taste Organs Is Strictly Dependent on Epithelial Hedgehog/GLI Signaling.

For homeostasis, lingual taste papilla organs require regulation of epithelial cell survival and renewal, with sustained innervation and stromal interactions. To investigate a role for Hedgehog/GLI signaling in adult taste organs we used a panel of conditional mouse models to manipulate GLI activity within epithelial cells of the fungiform and circumvallate papillae. Hedgehog signaling suppression rapidly led to taste bud loss, papilla disruption, and decreased proliferation in domains of papilla epithelium that contribute to taste cells. Hedgehog responding cells were eliminated from the epithelium but retained in the papilla stromal core. Despite papilla disruption and loss of taste buds that are a major source of Hedgehog ligand, innervation to taste papillae was maintained, and not misdirected, even after prolonged GLI blockade. Further, vimentin-positive fibroblasts remained in the papilla core. However, retained innervation and stromal cells were not sufficient to maintain taste bud cells in the context of compromised epithelial Hedgehog signaling. Importantly taste organ disruption after GLI blockade was reversible in papillae that retained some taste bud cell remnants where reactivation of Hedgehog signaling led to regeneration of papilla epithelium and taste buds. Therefore, taste bud progenitors were either retained during epithelial GLI blockade or readily repopulated during recovery, and were poised to regenerate taste buds once Hedgehog signaling was restored, with innervation and papilla connective tissue elements in place. Our data argue that Hedgehog signaling is essential for adult tongue tissue maintenance and that taste papilla epithelial cells represent the key targets for physiologic Hedgehog-dependent regulation of taste organ homeostasis. Because disruption of GLI transcriptional activity in taste papilla epithelium is sufficient to drive taste organ loss, similar to pharmacologic Hedgehog pathway inhibition, the findings suggest that taste alterations in cancer patients using systemic Hedgehog pathway inhibitors result principally from interruption of signaling activity in taste papillae.

Primary Cilia on Horizontal Basal Cells Regulate Regeneration of the Olfactory Epithelium.

The olfactory epithelium (OE) is one of the few tissues to undergo constitutive neurogenesis throughout the mammalian lifespan. It is composed of multiple cell types including olfactory sensory neurons (OSNs) that are readily replaced by two populations of basal stem cells, frequently dividing globose basal cells and quiescent horizontal basal cells (HBCs). However, the precise mechanisms by which these cells mediate OE regeneration are unclear. Here, we show for the first time that the HBC subpopulation of basal stem cells uniquely possesses primary cilia that are aligned in an apical orientation in direct apposition to sustentacular cell end feet. The positioning of these cilia suggests that they function in the detection of growth signals and/or differentiation cues. To test this idea, we generated an inducible, cell type-specific Ift88 knock-out mouse line (K5rtTA;tetOCre;Ift88(fl/fl)) to disrupt cilia formation and maintenance specifically in HBCs. Surprisingly, the loss of HBC cilia did not affect the maintenance of the adult OE but dramatically impaired the regeneration of OSNs following lesion. Furthermore, the loss of cilia during development resulted in a region-specific decrease in neurogenesis, implicating HBCs in the establishment of the OE. Together, these results suggest a novel role for primary cilia in HBC activation, proliferation, and differentiation. SIGNIFICANCE STATEMENT: We show for the first time the presence of primary cilia on a quiescent population of basal stem cells, the horizontal basal cells (HBCs), in the olfactory epithelium (OE). Importantly, our data demonstrate that cilia on HBCs are necessary for regeneration of the OE following injury. Moreover, the disruption of HBC cilia alters neurogenesis during the development of the OE, providing evidence that HBCs participate in the establishment of this tissue. These data suggest that the mechanisms of penetrance for ciliopathies in the OE extend beyond that of defects in olfactory sensory neurons and may include alterations in OE maintenance and regeneration.

SUMOylation regulates ciliary localization of olfactory signaling proteins.

Cilia are evolutionarily conserved organelles found on many mammalian cell types, including neuronal populations. Although neuronal cilia, including those on olfactory sensory neurons (OSNs), are often delineated by localization of adenylyl cyclase 3 (AC3, also known as ADCY3), the mechanisms responsible for targeting integral membrane proteins are largely unknown. Post-translational modification by small ubiquitin-like modifier (SUMO) proteins plays an important role in protein localization processes such as nuclear-cytosolic transport. Here, we identified through bioinformatic analysis that adenylyl cyclases harbor conserved SUMOylation motifs, and show that AC3 is a substrate for SUMO modification. Functionally, overexpression of the SUMO protease SENP2 prevented ciliary localization of AC3, without affecting ciliation or cilia maintenance. Furthermore, AC3-SUMO mutants did not localize to cilia. To test whether SUMOylation is sufficient for cilia entry, we compared localization of ANO2, which possesses a SUMO motif, and ANO1, which lacks SUMOylation sites and does not localize to cilia. Introduction of SUMOylation sites into ANO1 was not sufficient for ciliary entry. These data suggest that SUMOylation is necessary but not sufficient for ciliary trafficking of select constituents, further establishing the link between ciliary and nuclear import.

DNA polymerase ζ is a major determinant of resistance to platinum-based chemotherapeutic agents.

Oxaliplatin, satraplatin, and picoplatin are cisplatin analogs that interact with DNA forming intrastrand and interstrand DNA cross-links (ICLs). Replicative bypass of cisplatin DNA adducts requires the cooperative actions of at least three translesion DNA synthesis (TLS) polymerases: Polη, REV1, and Polζ. Because oxaliplatin, satraplatin, and picoplatin contain bulkier chemical groups attached to the platinum core compared with cisplatin, we hypothesized that these chemical additions may impede replicative bypass by TLS polymerases and reduce tolerance to platinum-containing adducts. We examined multiple responses of cancer cells to oxaliplatin, satraplatin, or picoplatin treatment under conditions where expression of a TLS polymerase was limited. Our studies revealed that, although Polη contributes to the tolerance of cisplatin adducts, it plays a lesser role in promoting replication through oxaliplatin, satraplatin, and picoplatin adducts. REV1 and Polζ were necessary for tolerance to all four platinum analogs and prevention of hyperactivation of the DNA damage response after treatment. In addition, REV1 and Polζ were important for the resolution of DNA double-stranded breaks created during replication-associated repair of platinum-containing ICLs. Consistent with ICLs being the predominant cytotoxic lesion, depletion of REV1 or Polζ rendered two different model cell systems extremely sensitive to all four drugs, whereas Polη depletion had little effect. Together, our data suggest that REV1 and Polζ are critical for promoting resistance to all four clinically relevant platinum-based drugs by promoting both translesion DNA synthesis and DNA repair.