|Abstract:||Cellular and developmental processes are regulated by extracellular and intracellular signals that are mediated by networks of signaling pathways. In recent years, microRNAs have also emerged as a class of critical modulators of the same processes. For my thesis studies, I focused on regulatory mechanisms underlying mammalian cell survival and differentiation. In particular, I investigated the regulation of mammalian target of rapamycin (mTOR), an evolutionarily conserved Ser/Thr kinase that integrates signals from nutrient availability, growth factors, differentiation inducers, and various types of stress, to control a wide range of biological processes. Separately, I also discovered a novel microRNA regulator of myogenic differentiation, microRNA-146b.
Emerging evidence implicates the deregulation of mTOR signaling in a variety of diseases including cancer and diabetes, underscoring the importance to fully understand the regulation of mTOR signaling. mTOR forms two distinct complexes known as mTORC1 and mTORC2. mTORC2 controls a wide range of cellular functions, but the regulation of its signaling remains incompletely understood. In Chapter 2, I identified XPLN, a RhoGEF, as an endogenous inhibitor of mTORC2 kinase activity towards Akt. Furthermore, I showed that the GEF activity of XPLN is dispensable for its regulation of mTORC2 and Akt, whereas an N-terminal 125-amino acid fragment of XPLN is both necessary and sufficient for the inhibition of mTORC2. XPLN negatively regulates myoblast differentiation and cell survival by suppressing mTORC2 and Akt, and could likely be an important player in many other aspects of biology and diseases involving mTORC2 and Akt.
Next, I set out to search for a mechanism by which XPLN could be regulated in cells in order to allow Akt activation following growth factor stimulation. In Chapter 3, I investigated the subcellular localization of XPLN, and found it to be localized throughout the cell but concentrated in the nucleus. I subsequently manipulated the location by tagging various localization signals to XPLN and examined the functional consequence. Furthermore, I studied the function and localization of alternative splice isoforms of XPLN.
Given the well-known hyperactivation of Akt in many human tumors, I probed a potential role of XPLN in cancer by analyzing its protein levels in various cancer cell lines, as described in Appendix A. Knockdown and overexpressed XPLN were also performed in cancer cells to probe the effects on Akt phosphorylation. Finally, as described in Appendix B, I am in the process of generating XPLN knockout mice using the TALEN technology, in order to facilitate future in vivo studies of XPLN.
In Chapter 4, I identified and characterized microRNA-146b (miR-146b) as a novel positive regulator of skeletal myogenesis. Inhibition of miR-146b led to reduced myoblast differentiation, whereas overexpression of miR-146b enhanced differentiation. In addition, miR-146b directly targets Smad4, Hmga2, and Notch1 in muscle cells. The expression of miR-146b and its target genes was inversely correlated during myoblast differentiation and muscle regeneration, suggesting that these genes most likely mediate the myogenic function of miR-146b.
In conclusion, my studies have uncovered novel regulators and mechanisms of mammalian cell survival and myogenic differentiation, and laid the foundation for future investigations.