Skeletal ciliopathy variants of the dynein-2 DYNC2LI1 subunit impair osteogenic differentiation of mesenchymal stem cells.

Skeletal ciliopathies result from defects in primary cilia, which are crucial for embryonic development because they transduce extracellular signals, including Hedgehog. Selective transport of ciliary proteins is mediated by the intraflagellar transport (IFT) machinery, containing the IFT-A and IFT-B complexes and the kinesin-2 and dynein-2 motors. Biallelic loss-of-function variants in genes encoding dynein-2-specific subunits, including DYNC2LI1, cause skeletal ciliopathies. As mesenchymal stem cells (MSCs) differentiate into osteoblasts, we investigated the effects of pathogenic variants of DYNC2LI1 on osteogenic differentiation of the MSC-like line C3H10T1/2. Dync2li1-knockout cells expressing disease-causing DYNC2LI1 variants demonstrated defects in the retrograde ciliary protein trafficking, including Hedgehog pathway GPCRs, Smoothened and GPR161. Furthermore, Dync2li1-knockout cells expressing the pathogenic variants demonstrated impaired Hedgehog signaling, in particular, a reduced ratio of the GLI3 repressor form to total GLI3, resulting in impaired osteogenic differentiation of MSCs. By contrast, osteogenic differentiation via BMP signaling was derepressed in Dync2li1-knockout cells. This suggests that skeletal ciliopathies caused by DYNC2LI1 variants could be attributable in part to impaired osteogenic differentiation due to defects in Hedgehog signaling, resulting from defects in retrograde ciliary protein trafficking.