Ca2+ Controls the Epithelial Cell Quiescence-Proliferation Decision through Regulating IGF Signaling

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.