Abstract
Intestinal epithelia self-renew constantly and generate differentiated cells such as secretary goblet cells. The intestine goblet cells secrete gel-forming mucins that form mucus to create a barrier of defense. We reported previously that loss of prolyl hydroxylase (PHD) 3 led to disruption of the intestinal epithelial barrier function. However, the underlying mechanism remains elusive. Here, we demonstrate that PHD3 controls the generation of intestine goblet cell. We found that genetic ablation of Phd3 in mice intestine epithelial cells reduced the amount of goblet cells. Mechanistically, PHD3 bounds the E3 ubiquitin ligase HUWE1 and prevented HUWE1 from mediating ubiquitination and degradation of ATOH1, an essential driver for goblet cell differentiation. The prolyl hydroxylase activity-deficient variant PHD3(H196A) also prevented ATOH1 destruction. A genetic intestine epithelial PHD3(H196A)-knockin had no effect on ATOH1 expression or goblet cell amount in mice, suggesting that the PHD3 prolyl hydroxylase activity is dispensable for its ability to control ATOH1 expression and goblet cell generation. In dextran sulfate sodium (DSS)-induced experimental colitis, PHD3-knockout rather than PHD3(H196A)-knockin sensitized the mice to DSS treatment. Our results reveal an additional critical mechanism underlying the regulation of ATOH1 expression and goblet cell generation and highlight that PHD3 plays a role in controlling intestine goblet cell generation in a hydroxylase-independent manner.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Schofield CJ, Ratcliffe PJ. Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol. 2004;5:343–54.
Kaelin WG Jr, Ratcliffe PJ. Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. Mol Cell. 2008;30:393–402.
Fong GH, Takeda K. Role and regulation of prolyl hydroxylase domain proteins. Cell Death Differ. 2008;15:635–41.
Metzen E, Berchner-Pfannschmidt U, Stengel P, Marxsen JH, Stolze I, Klinger M, et al. Intracellular localisation of human HIF-1α hydroxylases: implications for oxygen sensing. J Cell Sci. 2003;116:1319–26.
Köditz J, Nespe J, Wottawa M, Stiehl DP, Camenisch G, Franke C, et al. Oxygen-dependent ATF-4 stability is mediated by the PHD3 oxygen sensor. Blood. 2007;110:3610–7.
Fu J, Menzies K, Freeman RS, Taubman MB. EGLN3 prolyl hydroxylase regulates skeletal muscle differentiation and myogenin protein stability. J Biol Chem. 2007;282:12410–8.
Xie L, Xiao K, Whalen EJ, Forrester MT, Freeman RS, Fong G, et al. Oxygen-regulated 2-adrenergic receptor hydroxylation by EGLN3 and ubiquitylation by pVHL. Sci Signal. 2009;2:ra33.
Xue J, Li X, Jiao S, Wei Y, Wu G, Fang J. Prolyl hydroxylase-3 is down-regulated in colorectal cancer cells and inhibits IKKβ-independent of hydroxylase activity. Gastroenterology. 2010;138:606–15.
Luo W, Hu H, Chang R, Zhong J, Knabel M, O’Meally R, et al. Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell. 2011;145:732–44.
Xie L, Pi X, Mishra A, Fong G, Peng J, Patterson C. PHD3-dependent hydroxylation of HCLK2 promotes the DNA damage response. J Clin Investig. 2012;122:2827–36.
Fu J, Taubman MB. EGLN3 inhibition of NF-κB is mediated by prolyl hydroxylase-independent inhibition of IκB kinase γ ubiquitination. Mol Cell Biol. 2013;33:3050–61.
Deschoemaeker S, Di Conza G, Lilla S, MartÃn-Pérez R, Mennerich D, Boon L, et al. PHD1 regulates p53-mediated colorectal cancer chemoresistance. EMBO Mol Med. 2015;7:1350–65.
Rodriguez J, Herrero A, Li S, Rauch N, Quintanilla A, Wynne K, et al. PHD3 regulates p53 protein stability by hydroxylating proline 359. Cell Rep. 2018;24:1316–29.
Hiwatashi Y, Kanno K, Takasaki C, Goryo K, Sato T, Torii S, et al. PHD1 interacts with ATF4 and negatively regulates its transcriptional activity without prolyl hydroxylation. Exp Cell Res. 2011;317:2789–99.
Maloy KJ, Powrie F. Intestinal homeostasis and its breakdown in inflammatory bowel disease. Nature. 2011;474:298–306.
Fre S, Huyghe M, Mourikis P, Robine S, Louvard D, Artavanis-Tsakonas S. Notch signals control the fate of immature progenitor cells in the intestine. Nature. 2005;435:964–8.
Sancho R, Cremona CA, Behrens A. Stem cell and progenitor fate in the mammalian intestine: Notch and lateral inhibition in homeostasis and disease. EMBO Rep. 2015;16:571–81.
Yang Q, Bermingham NA, Finegold MJ, Zoghbi HY. Requirement of Math1 for secretory cell lineage commitment in the mouse intestine. Science. 2001;294:2155–8.
Pellegrinet L, Rodilla V, Liu Z, Chen S, Koch U, Espinosa L, et al. Dll1-and dll4-mediated notch signaling are required for homeostasis of intestinal stem cells. Gastroenterology. 2011;140:1230–40.
Jensen J, Pedersen EE, Galante P, Hald J, Heller RS, Ishibashi M, et al. Control of endodermal endocrine development by Hes-1. Nat Genet. 2000;24:36–44.
Shields JM, Christy RJ, Yang VW. Identification and characterization of a gene encoding a gut-enriched Kruppel-like factor expressed during growth arrest. J Biol Chem. 1996;271:20009–17.
Vandussen KL, Samuelson LC. Mouse atonal homolog 1 directs intestinal progenitors to secretory cell rather than absorptive cell fate. Dev Biol. 2010;346:215–23.
Elise S, Demitrack ES, Linda C, Samuelson LC. Notch regulation of gastrointestinal stem cells. J Physiol. 2016;594:4791–803.
Robinson A, Keely S, Karhausen J, Gerich ME, Furuta GT, Colgan SP. Mucosal protection by hypoxia-inducible factor prolyl hydroxylase inhibition. Gastroenterology. 2008;134:145–55.
Tambuwala MM, Cummins EP, Lenihan CR, Kiss J, Stauch M, Scholz CC, et al. Loss of prolyl hydroxylase-1 protects against colitis through reduced epithelial cell apoptosis and increased barrier function. Gastroenterology. 2010;139:2093–101.
Chen Y, Zhang HS, Fong GH, Xi QL, Wu GH, Bai CG, et al. PHD3 stabilizes the tight junction protein occludin and protects intestinal epithelial barrier function. J Biol Chem. 2015;290:20580–9.
Hollingsworth MA, Swanson BJ. Mucins in cancer: protection and control of the cell surface. Nat Rev Cancer. 2004;4:45–60.
Shroyer NF, Helmrath MA, Wang VY, Antalffy B, Henning SJ, Zoghbi HY. Intestine-specific ablation of mouse atonal homolog 1 (ATOH1) reveals a role in cellular homeostasis. Gastroenterology. 2007;132:2478–88.
Forget A, Bihannic L, Cigna SM, Lefevre C, Remke M, Barnat M, et al. Shh signaling protects Atoh1 from degradation mediated by the E3 ubiquitin ligase HUWE1 in neural precursors. Dev Cell. 2014;29:649–61.
Cheng YF, Tong M, Edge AS. Destabilization of Atoh1 by E3 ubiquitin ligase Huwe1 and casein kinase 1 Is essential for normal sensory hair cell development. J Biol Chem. 2016;291:21096–109.
Chen D, Kon N, Li M, Zhang W, Qin J, Gu W. ARF-BP1/Mule is a critical mediator of the ARF tumor suppressor. Cell. 2005;121:1071–83.
Bruick RK, McKnight SL. A conserved family of prolyl-4-hydroxylases that modify HIF. Science. 2001;294:1337–40.
Lee S, Nakamura E, Yang H, Wei W, Linggi MS, Sajan MP, et al. Neuronal apoptosis linked to EglN3 prolyl hydroxylase and familial pheochromocytoma genes:developmental culling and cancer. Cancer Cell. 2005;8:155–67.
Xu Y, Gao Q, Xue Y, Li X, Xu L, Li C, et al. Prolyl hydroxylase 3 stabilizes the p53 tumor suppressor by inhibiting the p53-MDM2 interaction in a hydroxylase- independent manner. J Biol Chem. 2019;294:9949–58.
Birchenough GM, Nyström EE, Johansson ME, Hansson GC. A sentinel goblet cell guards the colonic crypt by triggering Nlrp6-dependent Muc2 secretion. Science. 2016;352:1535–42.
Allaire JM, Morampudi V, Crowley SM, Stahl M, Yu H, Bhullar K, et al. Frontline defenders: goblet cell mediators dictate host-microbe interactions in the intestinal tract during health and disease. Am J Physiol Gastrointest Liver Physiol. 2018;314:G360–G377.
Van der Sluis M, De Koning BA, De Bruijn AC, Velcich A, Meijerink JP, Van Goudoever JB, et al. Muc2-deficient mice spontaneously develop colitis, indicating that Muc2 is critical for colonic protection. Gastroenterology. 2006;131:117–29.
Tytgat KM, Opdam FJ, Einerhand AW, Buller HA, Dekker J. Muc2 is the prominent colonic mucin expressed in ulcerative colitis. Gut. 1996;38:554–63.
Shaoul R, Okada Y, Cutz E, Marcon MA. Colonic expression of Muc2, MUC5AC, and TFF1 in inflammatory bowel disease in children. J Pediatr Gastroenterol Nutr. 2004;38:488–93.
Chan DA, Kawahara TL, Sutphin PD, Chang HY, Chi JT, Giaccia AJ. Tumor vasculature is regulated by PHD2-mediated angiogenesis and bone marrow-derived cell recruitment. Cancer Cell. 2009;15:527–38.
van Es JH, de Geest N, van de Born M, Clevers H, Hassan BA. Intestinal stem cells lacking the ATOH1 tumour suppressor are refractory to Notch inhibitors. Nat Commun. 2010;1:18.
Acknowledgements
This work was supported by China Ministry of Science and Technology (2017YFD0400206), National Natural Science Foundation of China (31470769, 31670785), and Shanghai Ministry of Science and Technology (16JC1406100).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Edited by R. Johnstone
Supplementary information
Rights and permissions
About this article
Cite this article
Xu, Ym., Gao, Q., Zhang, Jz. et al. Prolyl hydroxylase 3 controls the intestine goblet cell generation through stabilizing ATOH1. Cell Death Differ 27, 2131–2142 (2020). https://doi.org/10.1038/s41418-020-0490-7
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/s41418-020-0490-7
This article is cited by
-
Intestinal mucosal barrier repair and immune regulation with an AI-developed gut-restricted PHD inhibitor
Nature Biotechnology (2024)
-
PHD1-3 oxygen sensors in vivo—lessons learned from gene deletions
Pflügers Archiv - European Journal of Physiology (2024)
