Cholesterol modulates TGF-β receptors heterocomplex formation and their signaling in hepatocytes

Cholesterol, is a membrane organizer essential for lipid raft formation and involved in multiple hepatic disorders. Cholesterol metabolism is involved in etiology of hypercholesteremia, atherosclerosis and more. Transforming growth factor-β (TGF-β) plays critical roles in different physiological processes and diseases (e.g., cancer and NAFLD/NASH). TGF-β signals through a complex of two Ser/Thr kinase recep­tors, type I and II (TβRI/II), stimulating the canonical Smad and several non-Smad signaling pathways. Despite numerous reports on TGF-β/cholesterol interdependence, the under­lying mechanisms remained elusive. To elucidate them, the effects of cholesterol depletion (CD) or enrichment (CE) on TβRI/TβRII complex formation/dynamics (+/- TGF-β1) were investigated. AML12 hepatocytes were transfected with differently-tagged TβRI/II and the effect of immobilizing TβRII by IgG crosslinking on the lateral diffusion of TβRI was measured employing FRAP. The effects on TGF-β1 signal­ing were measured using western-blotting analysis. To validate the efficacy of CD/CE, their effects on cholesterol metabolism genes were examined using qRT-PCR.

Patch/FRAP studies demonstrate TβRI/II complex formation prior to ligand bind­ing (Pre-Formed Comp­lexes; PFCs). TGF-β1 enhances TβRI/II complexes. PFCs were disrupted by CD, while TGF-β1 fully restored the complexes. CE augmented TβRI/II PFCs with minimal effect of TGF-β1 due to the already robust interactions. Of note, TGF-β1 Smad2/3 activation was unaffected by either CD/CE, indicating that this pathway doesn`t depend on rafts/caveolae (disrupted by CD). TGF-β1-signaling to PI3K/Akt was abrogated by CD indicating that it requires rafts and/or PFCs. However, this is insufficient for PI3K/Akt signaling, since it`s also lost following CE suggesting the potential involvement of other raft-resident proteins.