GSK3β functions as a mechanosensitive regulator that links flow stress to Shh signaling

Primary cilia sense laminar flow through physical deflection, yet how this mechanical cue is translated into signaling regulation remains unclear. Here, we demonstrate that flow-induced ciliary bending promotes the accumulation of GSK3β within cilia, leading to repression of Sonic Hedgehog (Shh) signaling. This response requires the tumor suppressor folliculin (FLCN). Loss of GSK3β results in ligand-independent ciliary accumulation of Smoothened (Smo) and aberrant Shh signaling. Mechanistically, GSK3β phosphorylates Smo at two sites in its C-terminal intracellular domain, and mutations at these sites enhance Smo retention within cilia. Shh stimulation induces inhibitory phosphorylation of GSK3β at T390, thereby relieving its negative regulation of Shh signaling. Together, these findings identify GSK3β as a mechanosensitive regulator that couples flow-induced ciliary bending to Shh pathway activity through control of Smo ciliary localization.