Regulatory Effects And Molecular Mechanism Of SENP2 And Extracellular Matrix On Sonic Hedgehog Signaling

Sonic Hedgehog(Shh)is an important morphogen in vertebrates.The signaling pathway mediated by Shh protein plays a central role in embryonic cell differentiation,tissue development and organ formation.Therefore,the study of the regulatory mechanism of Shh signaling is a great significance for understanding embryonic development,birth defects,and tumor development.Within the cell,precise regulation of the Gli family(Gli1,2,3)is an important factor in controlling the activity of the Shh signaling.Existing studies have shown that the Small Ubiquitin-like modifier(SUMO),a post-translational modification of proteins,can regulate the biological activity of Gli proteins by changing the SUMO modification level of each member of the Gli family.In previous work,our lab confirmed that the absence of the reverse-SUMO-modified protease SENP2 caused a disturbance in the response of the Shh signaling.Therefore,in the first part of the study,our work was mainly devoted to the analysis of the molecular mechanism of the SENP2 regulation of the Shh signaling.Using siSENP2 and SENP2 knocked-out MEFs cells,respectively,we found that the expression of Gli2 decreased significantly after SENP2 knockout.Further studies showed that the core member of Polycomb repressive complex 1(PRC1)CBX4 in the Polycomb group(PcG)family can regulate the expression of Gli2 gene.SENP2 can catalyze the de-SUMOylation of CBX4,which results in less occupancy of CBX4 in the Gli2 promoter region,and attenuates PcG-catalyzed inhibition of Gli2 expression by H3K27me3.This study provides a valuable basis for the regulation of Shh signaling.Outside the cell,the microenvironments in which the cells are located also plays an important role in regulating the life activities of cells.The coordination of cells and the microenvironments also affects phenotypic outcomes in biological or pathological processes.However,the conventional 2D cell culture system is not sufficient to study the effects of cellular microenvironments,especially to study the dynamic changes of cells or tissues during development or disease.Therefore,3D culture provides a cell growth microenvironment that is similar to the in vivo survival conditions.In our second part of the study,we designed a set of gelatin/polyacrylamide network IPN hydrogels with adjustable stiffness and cell adhesion properties,and used the matrix stiffness and adhesion performance as dynamic factors to determine the regulatory factors for the conduction output of the Shh signaling.We found that as the mass ratio of gelatin/polyacrylamide in the IPN hydrogel increases,there are two peaks in the Shh signaling response in MEFs cells.When the mass ratio of gelatin was low,the first peak of Gli1 appeared in the MEFs cells,which was regulated by the stiffness of the matrix.When the mass ratio of gelatin was further increased and the adhesion property was in the leading position,the expression of Gli1 induced by Shh increased rapidly for the second time.Finally,we also found that Integrin-β1 and focal adhesion kinase FAK serve as matrix stiffness and adhesion sensors,respectively,indicating the importance of precise regulation of Shh signaling through the cellular microenvironments.