Regulation Of REGgamma In Bone Marrow Mesenchymal Stem Cell And The Mechanisms Involved

REGgamma,as a proteasome activator that directly degrades many important target proteins in an ATP and ubiquitin-independent manner,participates in the regulation of various physiological and pathological processes,such as the development of individuals,aging and cancer.Recent years,Mesenchymal stem cells,as the seed cells that have become more and more prevalent and mature in clinical cell therapy and tissue engineering applications,it plays an indispensable role in orthopedic diseases characterized by bone loss.However,studies on the relationship between REGγ and mesenchymal stem cells and how REGγ affect its pluripotency and fate decision still remains unknown.In our experiments,REGγ-deleted and wild-type mesenchymal stem cells(MSCs) were successfully isolated and cultured.We found that in the REGγ-deficient mesenchymal stem cells and the REGγ-knocked down mesenchymal stem cell line C3H10T1/2,the expression level of the stem cell pluripotency transcription factor SOX2 is down-regulated,then,the loss of the stemness drives its lineage-specific differentiation,but whether REGγ is directly interacting with SOX2 and relevant regulatory mechanisms are unclear.In the wild-type and REGγ-deficient mouse bone marrow-derived MSCs and the REGγ-knockdown C3H10T1/2,after in vitro adipogenic differentiation,oil red O staining revealed that when REGγ was knocked out or knocked down,the adipogenic differentiation was promoted.RT-PCR results showed that the expression of related adipogenic differentiation related transcription factors are up-regulated during the induction process compared with wild-type.In addition,after in vitro osteogenic differentiation of wild-type and REGγ-deficient MSCs,alizarin red S staining revealed that osteogenic differentiation of REGγ was impaired in the case of REGγ knockout,and RT-PCR results showed that compared with wild type,the expression of osteogenic differentiation related transcription factors are down-regulated.In terms of molecular mechanism,REGγ participates in the fate determination of mesenchymal stem cells by regulating PKA-FoxO1 and Wnt/β-catenin signaling pathways.Combine Western Blot and RT-PCR experiments confirmed that REGγ deletion increases PPARγ/C/ EBPα transcription level to increase its adipogenic differentiation capacity by activating PKA-FoxO1 signaling pathway;we also demonstrated that REGγ deficiency leads to attenuated osteogenic differentiation by inhibiting Wnt/β-catenin and the expression of important transcriptional regulators such as RUNX2/Osterix.So,the absence of REGγ promotes MSC adipogenic differentiation and impairs osteogenic differentiation.In summary,our experiments demonstrate for the first time that REGγ affects the stemness of mesenchymal stem cells and participates in the regulation of its adipogenic-osteogenic fate decision and homeostasis.This study provide new ideas and targets for the subsequent clinical study related to orthopedic diseases and drug research.