The Crosstalk Between Hedgehog Signaling Pathway And Autophagy

The evolutionarily conserved Hedgehog(Hh)signaling pathway plays an important role in tissue development,homeostasis and repair as a basic morphogenic factor and mitogen.The disorder of the Hh signaling pathway is the basis for the development of many genetic diseases.Holoprosencephaly,cyclopia,and limb abnormalities are the obvious features of damaged Hh pathway during embryonic development.In addition,abnormal activation of Hh signaling pathways can be found in many different types of tumors,including various solid tumors such as medulloblastoma(MB)and basal cell carcinoma(BCC).In recent years,Hedgehog signaling has also been found to be involved in the balance of sugar and lipid metabolism.However,the complex mechanism of the Hh pathway has not yet been clarified clearly.Autophagy is the main catabolic procedure activated by intracellular pressure.Intracellular pressure includes starvation and energy deficiency.The occurrence of autophagy starts with the production of autophagosomes.The autophagosome is a double membraned vesicle that expands continuously to engulf surrounding cytoplasmic components.Autophagosome formation is coordinated by a series of autophagy-related proteins.The mature autophagosomes are fused with lysosomes and acidified to form autolysosomes.The fusion of lysosomes and autophagosomes provides acid hydrolases that degrade proteins,lipids,carbohydrates,nucleic acids,and organelles to provide nutrients,which are then secreted to the cytoplasm by the lysosomal permease for reuse.Autophagy can also be induced by damaged organelles,aggregated proteins,and infected pathogens to maintain cell integrity or exert defense responses.Starvation induces the occurrence of autophagy and the growth of primary cilia,which is the regulatory center of the Hedgehog signaling pathway.The Hedgehog signaling pathway seems to share some functional interactions with autophagy.However,it is not clear whether there is a regulatory mechanism between the two pathways.To this end,we first explored the role of HH in autophagy and lipid metabolism.Treatment of normal mouse liver cells with SAG and GDC-0449 revealed elevated phosphorylation of AMPK and increased lipidation of LC3.SAG and GDC-0449 are agonist and antagonist of Smo in classical pathway respectively,but they play a consistent role in the regulation of autophagy in hepatocytes.At the same time,SAG and GDC-0449 did not affect the expression of Gli1 and Ptc1,suggesting the absence of classical Hh signaling in hepatocytes.We further knocked down the Smo and found that the effects of SAG and GDC-0449 disappeared,suggesting that the non-classical Hedgehog-Smo pathway is involved in the regulation of autophagy in hepatocytes.SAG and GDC-0449 promote lipid degradation and inhibit lipid production signals.Knockdown of Smo slows down the rate of lipid degradation rather than Sufu or Gli1,indicating that HH signaling regulates the metabolism of lipids via Smo.In summary,Hedgehog activates AMPK via Smo,promoting autophagy and lipid degradation.The activation of HH signaling pathway is dependent on the endocytic degradation of membrane receptors,and autophagy also has the function of degrading protein substrates to regulate cell homeostasis,so we consider whether autophagy involves in the regulation of HH signaling.We first used Atg5 small interfering RNA,autophagy inhibitor BafA1 and Crispr-Cas9 systems to inhibit autophagy respectively,and found that the mRNA and protein levels of Gli1 were inhibited.Cell imaging suggested that the membrane receptors Ptch1 and Smo partially colocalized with LC3-positive autophagosomes,but Sufu protein was not.The transport of Smo into primary cilia is the key point in the activation of the HH pathway.When treatment of MEFS with 3MA or BafA1 to block the autophagy pathway respectively,fluorescence intensity of Smo in primary cilia was reduced.We also had the same findings in Atg5-/-MEFs.However,inhibiting autophagy did not affect the growth of the primary cilia.Based on the above results,we hold the view that: 1.Hedgehog pathway activates AMPK via Smo,then promotes autophagy and lipid degradation.2.Blocking autophagy can inhibit classical Hedgehog signaling.Our studies provide a new perspective for the crosstalk between HH signaling pathway and autophagy,and new ideas for the treatment of related diseases in the future.