IFT122 Regulates Proliferation of mEPMCs Through the Shh Signaling Pathway by Primary Cilia.

Abstract

ObjectiveTo investigate how intraflagellar transport protein 122 (IFT122) regulates murine embryonic palate mesenchymal cell (mEPMC) proliferation via the Sonic Hedgehog (Shh) pathway and its dependency on primary cilia integrity, elucidating mechanisms underlying cleft palate pathogenesis.DesignIntraflagellar transport protein 122 was silenced in E14.5-derived mEPMCs using lentiviral shRNA. Primary cilia morphology (immunofluorescence), Shh signaling components (Smo, Gli3 via qPCR/Western blot), and proliferation markers (CCK-8 assay, Proliferating Cell Nuclear Antigen [PCNA], Cyclin D1) were analyzed. Rescue experiments employed the Smoothened agonist (SAG) to activate Shh signaling.Main Outcome MeasureCilia incidence and length; Smo/Gli3 mRNA and protein expression; Gli3A/Gli3R ratio; cell proliferation rates (OD values, PCNA, Cyclin D1); SAG-mediated rescue effects.ResultsIntraflagellar transport protein 122 knockdown reduced cilia incidence (10.2% vs 2.4%, P < .01) and length (7.8 μm vs 3.4 μm, P < .01), impaired Smo trafficking (mRNA↓42%, protein↓50%), suppressed Gli3A/Gli3R ratio (↓62%), and inhibited proliferation (PCNA/Cyclin D1↓40%-60%, P < .01). Smoothened agonist partially restored Smo expression (↑1.8×), Gli3 activation, and proliferation (P < .05), confirming cilia-dependent Shh regulation.ConclusionIntraflagellar transport protein 122 maintains primary cilia structure to enable Shh-driven mEPMC proliferation. Its deficiency disrupts cilia integrity and Shh signaling, linking ciliary dysfunction to cleft palate. Partial rescue by SAG validates the mechanism, highlighting therapeutic potential for targeting cilia-Shh axis defects.