T?R? Regulates The Proliferation Of Metanephric Mesenchyme Cells Through Six2 In Vitro

During the early kidney development,the self-renew and differentiation of metanephric mesenchyme(MM)cells play an important role in the development of kidney and Six2-positive nephron progenitor cells vitally give rise to renal vesicle,comma-shaped body,S-shaped body,renal tubules etc.A lot of molecular proteins and signaling pathways involved in this process regulate the dynamic balance between the self-renew and differentiation of MM cells.The TGF? family signaling pathways play an important role in the regulatory cellular networks and exert specific effects on the developmental programs during embryo development.However,the function of TGF? signaling pathways on the early kidney development remains unclear.In this work,we aim to detect the underlying role of TGF? type ? receptor(T?R?)in vitro,which has a similar expression pattern with the crucial regulator Six2 during the early kidney development.Firstly,the EdU assay showed knock down of T?R? significantly decreased the proliferation ratio of metanephric mesenchyme(MM)cells.Besides,Real-time PCR,Western Blot together withImmunofluorescence found the mRNA and protein levels of Six2 declined after the T?R? knock down.And Six2 could partially rescue the proliferation phenotype caused by the depletion of T?R?.The treatment of TGF?I could upregulate the expression of Six2 and p-Smad3,while SB431542,the selective inhibitor of p-Smad3,could suppress both the expression of Six2 and the proliferation of MM cells.Moreover,bioinformatics analysis and Luciferase assay indicated Smad3 could transcriptionally target Six2,and the Real-time PCR together with western blot assays demonstrated that Smad3 could significantly up-regulate the expression of Six2 both at mRNA and protein levels.Further,the EdU assay showed Smad3 could also rescue the inhibition of proliferation caused by the knock down of T?R?.Taken together,these findings delineate the important function of TGF? signaling pathway in the early development of kidney and T?R? could promote the expression of Six2 through Smad3-mediated transcriptional regulation and in turn activate the proliferation of MM cells.