Targeted Proteomic Profiling Revealed Roles of Small GTPases during Osteogenic Differentiation.

The small GTPase superfamily of proteins are crucial for numerous cellular processes, including early development. The roles of these proteins in osteogenic differentiation, however, remained poorly explored. In this study, we employed a high-throughput targeted proteomic method, relying on scheduled liquid chromatography-multiple-reaction monitoring (LC-MRM) coupled with synthetic stable isotope-labeled peptides, to interrogate systematically the temporal responses of the entire small GTPase proteome during the course of osteogenic differentiation of H9 human embryonic stem cells. Our results demonstrated that the method offers high quantification accuracy, reproducibility, and throughput. In addition, the quantification results revealed altered expression of a large number of small GTPases accompanied with osteogenic differentiation, especially those involved with autophagy. We also documented a previously unrecognized role of KRAS in osteogenesis, where it regulates the accumulation of extracellular matrix for mineralization through attenuating the activity of secreted matrix metalloproteinase 9 (MMP9). Together, this study represents a novel application of a state-of-the-art analytical method, i.e., targeted quantitative proteomics, for revealing the progressive reprogramming of the small GTPase proteome during osteogenic differentiation of human embryonic stem cells, and our results revealed KRAS as a new regulator for osteogenesis.

The FOXO signaling axis displays conjoined functions in redox homeostasis and stemness.

Previous views of reactive oxygen species (ROS) depicted them as harmful byproducts of metabolism as uncontrolled levels of ROS can lead to DNA damage and cell death. However, recent studies have shed light into the key role of ROS in the self-renewal or differentiation of the stem cell. The interplay between ROS levels, metabolism, and the downstream redox signaling pathways influence stem cell fate. In this review we will define ROS, explain how they are generated, and how ROS signaling can influence transcription factors, first and foremost forkhead box-O transcription factors, that shape not only the cellular redox state, but also stem cell fate. Now that studies have illustrated the importance of redox homeostasis and the role of redox signaling, understanding the mechanisms behind this interplay will further shed light into stem cell biology.