Transforming growth factor beta (TGFβ) signaling is regulated at the pocket region of primary cilia

Abstract

TGFb signaling extensively cross-talks with Hh, Wnt and RTK signaling to control cell proliferation, migration and differentiation, and when aberrantly regulated leads to developmental defects and cancer. TGFb signaling is acti-vated through the internalization of TGFb receptors via clathrin-dependent endocytosis (CDE), at which the recep-tor activates Smad transcription factors. Here we investi-gated the relationship between TGFb signaling and primary cilia in fibroblasts and in EC and human embryo-nic stem cells during their differentiation into cardiomyo-cytes and neurons using transcriptomics, imaging and molecular biology tools. During cardiomyocyte differentia-tion, expression of TGFb receptors and Smad proteins were up-regulated and targeted to the pocket region of primary cilia, at which the receptor colocalized with cla-thrin-coated pits and vesicles to activate Smad2/3. This activation was blocked by receptor antagonists or by Ift20 knockdown. In contrast, neuronal differentiation was asso-ciated with a loss of ciliary TGFb signaling. In mouse embryonic fibroblasts (MEFs) and human foreskin fibro-blasts (hFFs), TGFb stimulation increased the targeting of TGFb receptors to the ciliary pocket region followed by activation of Smad signaling to promote cell cycle entry. In Tg737orpk MEFs there was a major reduction in TGFb-induced Smad2/3 phosphorylation, and this was associated with reduced activity of clathrin-dependent endocytosis at stumpy primary cilia. Similarly, inhibition of CDE blocked activation of Smad2/3 at the ciliary pocket region in hFFs. Our results suggest that the ciliary pocket region functions as a unique site for regulation of TGFb signaling and potentially in cross-talking with other signaling pathways during development and in tissue homeostasis.