Cholesterol depletion enhances TGF-β Smad signaling by increasing c-Jun expression through a PKR-dependent mechanism

Transforming growth factor-beta (TGF-beta) plays critical roles in numerous physiological and pathological responses. Cholesterol, a major plasma membrane component, can have pronounced effects on signaling responses. Cells continually monitor cholesterol content and activate multilayered transcriptional and translational signaling programs, following perturbations to cholesterol homeostasis (e.g., statins, the commonly used cholesterol-reducing drugs). However, the cross-talk of such programs with ligand-induced signaling responses (e.g., TGF-beta signaling) remained unknown. Here, we studied the effects of a mild reduction in free (membrane-associated) cholesterol on distinct components of TGF-beta-signaling pathways. Our findings reveal a new regulatory mechanism that enhances TGF-beta-signaling responses by acting downstream from receptor activation. Reduced cholesterol results in PKR-dependent eIF2 alpha phosphorylation, which enhances c-Jun translation, leading in turn to higher levels of JNK-mediated c-Jun phosphorylation. Activated c-Jun enhances transcription and expression of Smad2/3. This leads to enhanced sensitivity to TGF-beta stimulation, due to increased Smad2/3 expression and phosphorylation. The phospho/total Smad2/3 ratio remains unchanged, indicating that the effect is not due to altered receptor activity. We propose that cholesterol depletion induces overactivation of PKR, JNK, and TGF-beta signaling, which together may contribute to the side effects of statins in diverse disease settings.