Identification of Gli1‐interacting proteins during simvastatin‐stimulated osteogenic differentiation of bone marrow mesenchymal stem cells

Abstract

Simvastatin has been shown to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Our study aimed to illuminate the underlying mechanism, with a specific focus on the role of Hedgehog signaling in this process. BMSCs cultured with or without 10−7 mol/L simvastatin were subjected to evaluation of osteogenic differentiation capacity. Osteogenic markers such as type 1 collagen (COL1) and osteocalcin (OCN), as well as key molecules of Hedgehog signaling molecules, were examined by Western blot and real‐time polymerase chain reaction (PCR). Co‐immunoprecipitation and mass spectrometry assays were applied to screen for Gli1‐interacting proteins. Cyclopamine (Cpn) was used as a Hedgehog signaling inhibitor. Our results indicated that simvastatin increased alkaline phosphatase (ALP) activity; mineralization of extracellular matrix; mRNA expression of ALP, COL1, and OCN; and expression and nuclear translocation of Gli1. Contrasting effects were observed in Cpn‐exposed groups, but were partially rescued by the simvastatin treatment. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that Gli1‐interacting proteins were primarily associated with mitogen‐activated protein kinase (MAPK) (P = 7.04E−04), hippo, insulin, and glucagon signaling. Further, hub genes identified by protein‐protein interaction network analysis included Gli1‐interacting proteins such as Ppp2r1a, Rac1, Etf1, and XPO1/CRM1. In summary, the current study showed that the mechanism by which simvastatin stimulates osteogenic differentiation of BMSCs involves activation of Hedgehog signaling, as indicated by interactions with Gli1 and, most notably, the MAPK signaling pathway.

Research progress on the hedgehog signalling pathway in regulating bone formation and homeostasis

“…Contrasting effects were observed after the BMMSCs were exposed to cyclopamine (an Hh signalling inhibitor), indicating that simvastatin promotes BMMSC osteogenic differentiation, at least in part, via the Hh pathway. 15 During mandibular development, ciliary protein Ift88 was found to participate in chondrogenesis and osteogenesis at least partially via the Shh pathway. 16 Additionally, low‐level laser irradiation was also found to promote osteoblast proliferation via Hh signalling.…”

“…Then, the potential proteins interacting with Gli1 were explored using mass spectrometric analysis, and Hh signalling was found to interact with other pathways in terms of MAPK, Hippo, insulin or glucagon signalling by regulating the expression of the related molecules Ppp2r1a, Rac1, Etf1 and XPO1/CRM1. 15 …”