Mechanisms of mTOR signal transduction regulating cell growth and differentiation

The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase that governs a myriad of cellular and developmental processes, including cell growth, proliferation and differentiation. In my thesis work, I investigate the signal transduction mechanisms of mTOR in the regulation of cell growth (Chapters II and III), as well as differentiation of skeletal myoblasts (Chapters IV and V). Tuberous sclerosis complex (TSC) is a genetic disorder characterized by widespread benign tumor formation in a variety of organs, through the loss of heterozygosity of either the gene TSC1 or TSC2. The tuberous sclerosis complex proteins TSC1/2 act as a GTPase activating protein (GAP) for the small G protein Rheb, which regulates cell growth by activating the mTORC1 pathway. How Rheb activates mTORC1 signaling is mysterious, and little is known about the effector(s) of Rheb. Previous work in our lab has identified the lipid second messenger phosphatidic acid (PA) as a crucial mediator for the mitogenic activation of mTORC1 signaling, likely through phospholipase D1 (PLD1). In Chapter II of my thesis, I examine the role of PLD in Rheb-induced mTORC1 signaling, and have uncovered a mechanism by which Rheb activates mTORC1. Decreasing PLD activity, either by using a PLD inhibitor or through specific knockdown of PLD1, inhibits Rheb-activated mTORC1 signaling. Overexpression of Rheb activates PLD1 in cells, whereas overexpression of TSC2 inhibits PLD1. Conversely, knockdown of Rheb inhibits PLD, and knockdown of TSC2 activates PLD in cells. Furthermore, Rheb binds and activates PLD1 in vitro in a GTP-dependent manner. Additional evidence suggests that maximal PLD activation and Rheb-PLD1 interaction require both mitogenic stimulation and amino acid sufficiency. Thus, we propose that Rheb activates mTORC1 by activating PLD1, and PLD1 is an effector of Rheb in the amino acid-sensing mTORC1 signaling pathway.;Previous work from our lab has demonstrated that PA activates mTORC1 signaling through direct binding with the FRB domain of mTOR. Despite the numerous studies in recent years that support PA's critical role in mTORC1 signaling, the molecular mechanism by which the lipid second messenger PA activates mTORC1 remains puzzling. In Chapter III, I study the relationship between the direct activator of mTORC1-PA, and a recently discovered direct inhibitor of mTORC1-FKBP38. The competition between PA and FKBP38 for mTOR binding has been observed. On one hand, PA disrupts the interaction between mTOR and FKBP38 in vitro and in cells. On the other, FKBP38 overexpression antagonizes mTORC1 activity stimulated by PA or mitogens. As a negative regulator of mTORC1, knockdown of FKBP38 leads to activation of mTORC1 in the absence or presence of mitogens, but not in the presence of exogenous PA, indicating that FKBP38 knockdown serves a similar function on mTOR as PA does. These observations altogether support a model that PA activates mTORC1 by displacing FKBP38.;Muscle development is a well-orchestrated process under the modulation of various pathways. In recent years microRNA has emerged as an important regulatory mechanism in skeletal myogenesis. Previous studies from our lab have shown that mTOR signaling is essential for skeletal myoblast differentiation at both initiation of myoblast differentiation stage and myotube/myofiber growth stage. To probe the potential interconnections between mTOR signaling and microRNA in the regulation of myogenesis, in chapter IV, a microRNA expression profiling with differentiating C2C12 cells in the presence and absence of rapamycin is performed. The results reveal a list of microRNAs that are either enhanced or inhibited by rapamycin in their expression. In addition, several microRNAs that have never been reported to be involved in myogenesis are also identified. These results, in combination with cDNA expression profiling that is ongoing, will potentially help dissect the regulatory network of skeletal myoblast differentiation.;In chapter V, among the list of microRNAs from the microarray analysis, we focus on the muscle-specific miR-1, which is up-regulated during differentiation. mTOR is essential for miR-1 expression in C2C12 cells and in vivo in regenerating myofibers. It regulates the transcription of miR-1 through its upstream enhancers, and this regulation is at least partially mediated by the myogenic transcription factor MyoD. To delineate the pathway downstream of mTOR-miR-1 in myogenesis, we have tested the reported miR-1 target in skeletal muscle-HDAC4, and separately, a myogenic factor that is reportedly inhibited by HDACs-follistatin. Anti-miR-1 LNA dampens follistatin mRNA level, which is rescued by HDAC inhibitor trichostatin A (TSA). Concurrently, anti-miR-1 LNA also inhibits myotube fusion, which could be rescued by either introducing recombinant follistatin, or inducing the expression of endogenous follistatin through TSA. These data strongly support an HDAC4-follistatin pathway as the functional target of miR-1 in myogenesis. Importantly, rapamycin enhances the expression of HDAC4 and decreases follistatin mRNA level during myoblast differentiation. Furthermore, myotube growth and maturation, which requires a late-stage myogenic fusion after nascent myotube formation, is rescued by either follistatin or TSA from rapamycin inhibition both in C2C12 cells and in skeletal muscle regeneration in vivo, suggesting that follistatin is most likely the long-sought fusion factor regulated by mTOR in muscle growth. Taken together, this study has revealed for the first time a link between mTOR signaling and microRNA, and identified a novel mTOR-miR-1-HDAC4-follistatin pathway that regulates myocyte fusion critical for myotube maturation and skeletal muscle growth.