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  • br Materials and Methods br


    Materials and Methods
    Acknowledgements We thank the staff of beamlines ID29 and ID30A-1 of the European Synchrotron Radiation Facility. This work was supported by, the Marie Curie Career Integration Grant (PCIG13-GA-2013-630755), the Israel Science Foundation (Grant 1383/17) and the Israeli Cancer Association (Grant 20180069). Author Contributions: N.S. and R.W. designed the experiments, and N.S., and M.K performed all biochemical experiments. Cloning, expression and protein purification were carried out by N.S., E.C.K., B.M. and F.H. Complexes were prepared for crystallization, and crystals were grown by N.S. and P.P. M.I., A.L., P.P. and R.W. determined the crystal structures. N.S., P.P. and R.W. wrote the manuscript. Conflict of Interest Statement: The authors declare no conflicts of interest.
    Introduction The development of vertebrate endoskeletons initially began with osteoblast differentiation, which is mediated by such factors as extracellular signalling molecules (e.g., bone morphogenetic proteins (BMPs)) [1], topography [2], and bioactive elements [3]. These regulatory factors are crucial components of the extracellular matrix (ECM). Previous studies demonstrated that their unusual activities could induce various bone deformities and related orthopaedic disease [4], [5]. Mutations in genes were more likely to induce bone deformities in certain cases. For example, Noonan syndrome is an autosomal dominant disorder caused by dysfunction of the rat sarcoma (RAS)/mitogen-activated protein kinase (MAPK) signalling pathway with clinical features of skeletal dysplasia followed by many other diseases, including characteristic facial features, pectus abnormalities, cryptorchidism, lymphatic abnormalities and cardiac defects [6]. The pathway is essential for regulating a series of cellular responses (e.g., migration, proliferation, and differentiation) of osteogenic-related Cryptotanshinone (e.g., osteoblasts and mesenchymal stem cells (MSCs)) [7], [8], thereby sustaining bone homeostasis. However, there is limited information regarding abnormal osteoblast differentiation in Noonan syndrome. RAF-1 is a pivotal intermediate for the cellular responses of osteoblast differentiation [9], [10]. Yang et al. confirmed that sprouty homolog 2 (Spry2) expression was an early response to stimulation by fibroblast growth factor 1 (FGF1) in MC3T3-E1 cells and acted as a feedback inhibitor of FGF1-induced osteoblast responses, possibly through interaction with RAF-1 [9]. Thereafter, Li et al. applied integrated proteomics, statistical and network biology techniques to study proteome-level changes in bone tissue cells in response to two different conditions, normal loading and fatigue loading [10]. The study showed that the combination of a down-regulated anti-apoptotic factor, RAF-1, and an up-regulated pro-apoptotic factor, programmed cell death 8 (PDCD8), significantly increased the number of apoptotic osteocytes following fatigue loading [10]. Therefore, RAF-1 has the ability to abnormally activate osteoblast differentiation of MC3T3-E1 cells. Mutations in human RAF-1 that lead to the substitution of valine for leucine at amino acid 613 are associated with Noonan syndrome, which is characterized in part by cardiac hypertrophy [11]. A RAF-1 mouse mutant had enhanced mitogen-activated protein kinase kinase 1 (MKK1) and extracellular signal-regulated kinase 1 (ERK1) and ERK2 signalling [12]. Treatment of these mice with MAPK/ERK kinase (MEK) inhibitors attenuated many phenotypic abnormalities, including bone deformities [12]. Based on these findings, it is suggested that L613V might also abnormally activate osteoblast differentiationin Noonan syndrome. In short, RAF-1, especially RAF-1L613V, induced bone deformity upon the occurrence of Noonan syndrome. Therefore, it is necessary to seek a novel therapeutic agent for RAF-1 or L613V-induced bone deformity in Noonan syndrome. RAS can activate RAF-1 in the RAS/MAPK signalling pathway [13]. RAF-1 directly contributes to ERK activation but not to c-Jun N-terminal kinase (JNK) activation, whereas MEK kinase (MEKK) participates in JNK activation but causes ERK activation only after overexpression [13]. In MC3T3-E1 cells, after being activated by bone morphogenetic protein-2 (BMP-2) signalling, RAS becomes involved in osteoblastic determination, differentiation, and transdifferentiation under p38 MAPK and JNK regulation [14]. Therefore, we can hypothesize that RAF-1 has an amplification effect on osteoblast differentiation of MC3T3-E1 cells induced by BMP-2 because the RAS/MAPK signalling pathway has the same downstream signalling proteins as the BMP-2 signalling pathway, such as ERK and MEK.