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  • Here we used Drosophila and mouse models to address

    2022-01-13

    Here, we used Drosophila and mouse models to address the biological function of RALs in the adult intestine. Our results demonstrate a conserved in vivo role for RALs in ISC function during tissue homeostasis and regeneration. ISCs lacking RALs were at a disadvantage compared to wild-type neighbors. Importantly, we show that constitutive β-catenin activation through APC (3S,5S)-Atorvastatin sodium salt rescued the suppression of Wnt signaling following RAL loss, and that RALs promote Wnt signaling through control of Wnt signalosome internalization.
    Results
    Discussion We present a conserved in vivo role for RAL signaling in regulating ISC number, which impacts intestinal homeostasis and regeneration. RALs do so by promoting internalization of the Wnt pathway receptor complex at the cell surface, and activating canonical Wnt signaling.
    STAR★Methods
    Acknowledgments We thank Colin Nixon, the histology service, biological services, and all core services at CRUK Beatson Institute for their invaluable assistance. We thank Prof. Mariann Bienz and Dr. Melissa Gammons for SNAP-tag constructs. We thank Prof. Jim Norman for expertise in receptor internalization. We also thank Bruno Lemaitre, Gaiti Hasan, Hugo Bellen, Ginés Morata, Christian Ghiglione, the Vienna Drosophila RNAi Center, the Bloomington Drosophila Stock Center, and the Developmental Studies Hybridoma Bank for providing Drosophila lines and reagents. This work was supported by CRUK core funding to O.J.S. (A19196 and A21139) (J.J., M.C.H., K.A.P., B.W.M., R.A.R., A.D.C., and O.J.S.). J.J. and K.A.P. were funded as part of a collaborative research agreement with Novartis AG. M.N. is supported by a Leadership Fellowship from the University of Glasgow (to J.B.C.). Y.Y. is supported by CRUK core funding (A17196). J.B.C. is a Sir Henry Dale Fellow jointly funded by the Wellcome Trust and the Royal Society (grant number 104103/Z/14/Z).
    Introduction The Transforming Growth factor β (TGFβ) is the prototype member of a large superfamily of growth factors, also including the activins, the inhibins and the bone morphogenetic proteins among others, that play important roles during embryonic development and in adult life [1,2]. Non physiological activation or inhibition of the TGFβ signaling pathway has been considered as a causing factor for a plethora of human diseases including different types of cancer and fibrosis [2]. The pathway starts when TGFβ binds to type I and type II serine/threonine kinase receptors causing the formation of a heterotetramer and the subsequent phosphorylation of the type I receptor (also known as ALK5) by the type II receptor which is active even in the absence of the ligand. The activated type I receptor recruits and phosphorylates the cytoplasmic receptor-regulated Smad proteins (R-Smads) 2 and 3 which then interact physically with Smad4. The formation of heteromeric R-Smad/Smad4 complexes is necessary for their translocation to the nucleus where they bind to specific regulatory elements in the promoters of target genes and regulate their expression in a cell-type-specific manner through interactions with other transcription factors, coactivators or corepressors [3,4]. In addition to the classical TGFβ/Smad signaling pathway described above, accumulating evidence supports the operation of additional, Smad-independent, signaling pathways that are induced by TGFβ in different cell types such as the Ras-ERK1/2 pathway and the RhoA/B/ROCK/LIMK2/cofilin pathway regulating early actin cytoskeleton reorganization [[5], [6], [7], [8]]. Small GTPases of the Rho subfamily have been implicated in many physiological and pathological cellular processes including actin cytoskeleton organization, cell division, cell adhesion, cell cycle progression, vesicular trafficking and inflammation among others [9,10]. The three members of this subfamily, RhoA, RhoB and RhoC exhibit significant amino acid sequence identity (~85%) and are thought to interact with the same Rho effectors [9]. RhoB however has features that are not shared with the other Rho family members including its post-translational modifications by geranyl-geranyl, farnesyl and palmitate groups [11].