Ca2+ Controls the Epithelial Cell Quiescence-Proliferation Decision through Regulating IGF Signaling
Xin, Yi
2020
Abstract
Epithelial tissues renew rapidly and continuously by reactivating a pool of quiescent cells. How the quiescent cells are established, maintained, and reactivated is poorly defined. Recent studies suggest that the insulin-like growth factor (IGF)-PI3 kinase-AKT-mTOR signaling pathway plays a key role in regulating epithelial cell quiescence-proliferation decision but the underlying mechanism remains unclear. In my thesis work, I use a zebrafish model to investigate the IGF action in a group of Ca2+-transporting epithelial cells, known as Na+-K+-ATPase-rich (NaR) cells. When zebrafish are kept in normal and physiological [Ca2+] embryo rearing media, NaR cells are quiescent, characterized by a very slow division rate and undetectable Akt and Tor activity. When subjected to low [Ca2+] stress, the NaR cells exit the quiescent state and proliferate due to elevated IGF1 receptor-mediated Akt and Tor activity. To understand how the IGF signaling is activated exclusively in NaR cells under low [Ca2+] stress, I first investigated the role of Igfbp5a, a secreted protein belonging to the IGF binding protein (IGFBP) family. Zebrafish igfbp5a is specifically expressed in NaR cells and genetic deletion of igfbp5a blunted the low Ca2+ stress-induce IGF-Akt-Tor activity and NaR cell reactivation. Similarly, knockdown of IGFBP5 in human colon carcinoma cells resulted in reduced IGF-stimulated cell proliferation. Re-expression of zebrafish or human Igfbp5a/IGFBP5 in NaR cells restores NaR cell proliferation. Mechanistically, Igfbp5a acts by binding to IGFs using its ligand-binding domain and promoting IGF signaling in NaR cells. These results reveal a conserved mechanism by which a locally expressed Igfbp activates IGF signaling and promoting cell quiescence-proliferation transition under Ca2+-deficient states. NaR cells are functionally equivalent to human intestinal epithelial cells, and they contain all major molecular components of the transcellular Ca2+ transport machinery, including the epithelial calcium channel Trpv6. Ca2+ is a central intracellular second messenger controlling many aspects of cell biology. I next investigated the role of Trpv6 and intracellular [Ca2+]. I discovered that NaR cells are maintained in the quiescent state by Trpv6-mediated constitutive Ca2+ influx. Genetic deletion and pharmacological inhibition of Trpv6 promote NaR cell quiescence-proliferation transition. In zebrafish NaR cells and human colon carcinoma cells, Trpv6/TRPV6 elevated intracellular Ca2+ levels and activated PP2A, a group of conserved protein phosphatases, which down-regulates IGF signaling and promotes the quiescent state. Finally, chemical biology screens and genetic experiments identified CaMKK as a link between low Ca2+ stress and IGF signaling activation in NaR cells. Depletion of the ER Ca2+ store abolished NaR cell reactivation and IGF signaling. These results suggest that ER Ca2+ release in response to the low [Ca2+] stress activates CaMKK, which in turn increases IGF signaling and NaR cell reactivation. Taken together, the results of my thesis research provide new insights into the epithelial cell proliferation-quiescence regulation and have deepened our understanding of cellular quiescence regulation. These new findings may also contribute to the future development of strategies in improving wound healing and tissue regeneration.Subjects
IGF calcium
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Thesis
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