Integrin engagement triggers activation of
Integrin engagement triggers activation of several signaling pathway including Fak, which is recognized as the key signal transduction factor in the integrin signaling pathway that functions by activating downstream signaling in focal adhesion plaques. Whether changes in integrin-FAK signaling pathway would influence osteogenic differentiation in osteoblast-like cell line or not was still not known clearly, which was our main interest.
ILK is a protein kinase linked to downstream signaling of integrins that mediates response to Cordycepin changes by interacting with cytoplasmic domains of Integrin beta subunits. Yamaji S et al. found that the stimulation of ILK is dependent on Pi3 kinase action which was blocked by the lack of FAK. In our study, the increased expression levels of ITGB1 and ILK in control groups was attenuated in shRNA-Fak group during mechanical stretch, which suggested that both upstream and downstream expression of integrin signaling pathway was inhibited after Fak silencing. The inhibition of Fak induced the decrease of ILK probably in a PI3-kinase-dependent manner. And ILK is crucial for both focal adhesions and activation of the ITGB1 that the absence of ILK activation inhibits ITGB1 function, which is consistent with the decreased Itgb1 expression after Fak silencing in our study.
The increase of ALP activity and RUNX2/Runx2, Alp expression in both control groups demonstrated successful osteogenic differentiation of BMSCs after mechanical stretch. Alp expression is a well-established marker of BMSC transformation into osteoblasts which is triggered by cell contact with extracellular matrix (ECM). The dramatically decreased gene expression of Alp in the shRNA-Fak group was mostly associated with that the inhibition of Fak prevented osteogenic differentiation of BMSCs by decreasing the expression of extracellular signal-related kinase (ERK) dependent ALP activity. BMSCs osteogenesis in bone formation is mainly regulated by transcription of Runx2/Cbfa-1. The change of Runx2 vanished in the shRNA-Fak group was considered to be influenced via FAK-ERK1/2 downregulation. The decrease of Alp and Runx2 expression after Fak silencing indicated that the integrin-FAK signaling pathway is involved in the regulation of osteogenic differentiation of BMSC responding to mechanical stretch. The integrin signaling pathway influences this response as one pathway in a series of signaling pathways that together lead to osteogenesis. Signaling pathways such as Ras-Raf-MAPK/ERK, FAK-PI3K-Akt/Rac-JNK, and FAK-Rho-GTPase might be further activated to regulate and control cell functions after integrin-mediated activation of FAK.
In conclusion, silencing of the Fak gene inhibited the osteogenic differentiation of rat BMSCs induced by in vitro mechanical stretching model through integrin signaling pathway.
Conflicts of interest
Acknowledgements This study was supported by grants from National Natural Science Foundation of China (No. 81100779 and 81621062) and Sichuan Province Science and Technology Innovation Team Program (2017TD0016).
Esophageal squamous cell carcinoma (ESCC) along with oesophageal adenocarcinoma (EAC) is the major subtype of oesophageal cancer recognized, according to microanatomical categorization. In Asia, ESCC is the predominant form which accounts for 57% of diagnosed compared with 18% in the United States. It is also the fourth leading cause of cancer-related deaths in China. Eastern Asia has the highest incidence of esophageal cancer whereas it is lowest in Western Africa. Although the incidence rates from 2000 to 2011 decreased for ESCC and scientists have performed numerous studies to identify prognostic markers, the outcome for ESCC patients remains grim. Thus, understanding the detailed molecular mechanisms in ESCC progression and developing novel strategies for treatment are urgently needed to improve the survival rates for these sufferers.