The Investigation For Kruppel Like Factor 5 Interacting Proteins

https://openworks.mdanderson.org/sumexp21/1198/thumbnail.jp

IGF-II is regulated by microRNA-125b in skeletal myogenesis

miR-125b is identified as a myogenic miRNA that regulates skeletal muscle differentiation by targeting IGF-II under the control of mTOR signaling

Mammalian target of rapamycin regulates miRNA-1 and follistatin in skeletal myogenesis

mTOR induces MyoD-dependent miR-1 expression, leading to follistatin-mediated myocyte fusion

A Phosphatidylinositol 3-Kinase/Protein Kinase B-independent Activation of Mammalian Target of Rapamycin Signaling Is Sufficient to Induce Skeletal Muscle Hypertrophy

Overexpression of Rheb activates mTOR signaling via a PI3K/PKB-independent mechanism and is sufficient to induce skeletal muscle hypertrophy. The hypertrophic effects of Rheb are driven through a rapamycin-sensitive (RS) mechanism, mTOR is the RS element that confers the hypertrophy and the kinase activity of mTOR is necessary for this event

Regulation of skeletal myogenesis by the mTOR signaling network

Skeletal muscle is composed of post-mitotic multinucleated myofibers. During embryonic skeletal myogenesis, cells in somites commit to myogenic lineage and differentiate into myoblasts, which then fuse to form multinucleated myofibers. Post-natal growth, repair, and maintenance of skeletal muscle are dependent on muscle satellite cells, which in response to stimuli differentiate and fuse to form new myofibers. Developmental or post-natal failure of skeletal myogenesis results in diverse muscular dystrophies and atrophies, largely impairing life quality and sometimes directly causing death. The myogenic process is guided by various environmental cues and regulated by distinct signaling pathways, resulting in the activation of specific transcription factors and subsequent reprogramming of gene expression. The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase that controls a wide spectrum of cellular and developmental processes including regulation of skeletal myogenesis. In my dissertation work, I investigate the mechanisms of myogenic regulation, with a focus on the signaling network assembled by mTOR. Various processes of skeletal muscle differentiation and remodeling were known to be inhibited by the mTOR-specific inhibitor, rapamycin. In cultured myoblasts, the target of rapamycin – mTOR – had been reported to regulate differentiation at different stages through distinct mechanisms, including one that is independent of mTOR kinase activity. However, there had been no in vivo evidence to validate those mTOR myogenic mechanisms in vitro. In Chapter II, I show that rapamycin impairs injury-induced muscle regeneration. To validate the role of mTOR with genetic evidence and to probe the mechanism of mTOR function, I have generated and characterized transgenic mice expressing two mutants of mTOR under the control of human skeletal actin (HSA) promoter – rapamycin-resistant (RR) and RR/kinase-inactive (RR/KI) mTOR. My results show that muscle regeneration in rapamycin-administered mice is restored by RR-mTOR expression. In the RR/KI-mTOR mice, nascent myofiber formation during the early phase of regeneration proceeds in the presence of rapamycin, but growth of the regenerating myofibers is blocked by rapamycin. Igf2 mRNA levels increase drastically during early regeneration, which is sensitive to rapamycin in WT muscles but partially resistant to rapamycin in both RR- and RR/KI-mTOR muscles, consistent with mTOR regulation of Igf2 expression in a kinase-independent manner. Furthermore, systemic ablation of S6K1, a target of mTOR kinase, results in impaired muscle growth but normal nascent myofiber formation during regeneration. Therefore, mTOR regulates muscle regeneration through kinase-independent and kinaseiii dependent mechanisms at the stages of nascent myofiber formation and myofiber growth, respectively. MicroRNAs have emerged as key regulators of skeletal myogenesis, but our knowledge of the identity of the myogenic miRNAs and their targets remains limited. In Chapter III, I describe the identification and characterization of a novel myogenic microRNA – miR-125b. I find that the levels of miR-125b decline during myogenesis, and that miR-125b negatively modulates myoblast differentiation in culture and muscle regeneration in mice. My results identify the insulin-like growth factor 2 (Igf2), a critical regulator of skeletal myogenesis, as a direct and major target of miR-125b in both myocytes and regenerating muscles, revealing for the first time a microRNA mechanism controlling IGF-II expression. In addition, I provide evidence suggesting that miR-125b biogenesis is negatively controlled by kinase-independent mTOR signaling both in vitro and in vivo, as a part of a dual mechanism by which mTOR regulates the production of IGF-II – a master switch governing the initiation of skeletal myogenesis. In Chapter IV, in collaboration with a former graduate student in the lab, Dr. Yuting Sun, I find that expression of another microRNA, miR-1, is regulated by mTOR both in differentiating myoblasts and in mouse regenerating skeletal muscle. We have found that mTOR controls MyoD-dependent transcription of miR-1 through its upstream enhancer, most likely by regulating MyoD protein stability. Moreover, a functional pathway downstream of mTOR and miR-1 is delineated, in which miR-1 suppression of HDAC4 results in production of follistatin and subsequent myocyte fusion. Collective evidence strongly suggests that follistatin is the longsought mTOR-regulated fusion factor. In summary, these findings unravel yet another link between mTOR and microRNA biogenesis, and identify an mTOR-miR-1-HDAC4-follistatin pathway that regulates myocyte fusion during myoblast differentiation in vitro and skeletal muscle regeneration in vivo. The importance of the canonical mTOR complex 1 signaling components, including raptor, S6K1, and Rheb, had been suggested in muscle maintenance, growth, and metabolism. However, the role of those components in myogenic differentiation is not entirely clear. In Chapter V, I report the investigation of the functions of raptor, S6K1, and Rheb in the differentiation of C2C12 mouse myoblasts. I find that although mTOR knockdown severely impairs myogenic differentiation as expected, the knockdown of raptor, as well as Rheb, enhances differentiation. Consistent with a negative role for these proteins in myogenesis, overexpression of raptor or Rheb inhibits C2C12 differentiation. On the other hand, neither knockdown nor overexpression of S6K1 has any effect. Moreover, the enhanced differentiation elicited by raptor or Rheb knockdown is accompanied by increased Akt activation, elevated IRS1 iv protein levels, and decreased Ser307 (human Ser312) phosphorylation on IRS1. Finally, IRS1 knockdown eliminated the enhancement in differentiation elicited by raptor or Rheb knockdown, suggesting that IRS1 is a critical mediator of the myogenic functions of raptor and Rheb. In conclusion, the Rheb-mTOR/raptor pathway negatively regulates myogenic differentiation by suppressing IRS1-PI3K-Akt signaling. These findings underscore the versatility of mTOR signaling in biological regulations and implicate the existence of novel mTOR complexes and/or signaling mechanism in skeletal myogenesis. Lastly in Appendix B, I document the effort of an RNAi screening to search for novel myogenic regulators among secreted factors. A few distinct groups of cytokines and chemokines are found to either enhance or suppress myoblast differentiation and fusion when knocked down, suggesting that they may regulate myogenesis. Future characterization of these candidates will include assessing knockdown efficiency, identifying the exact processes that they regulate, and dissecting their regulatory pathways

MicroRNA-146b Promotes Myogenic Differentiation and Modulates Multiple Gene Targets in Muscle Cells

<div><p>MicroRNAs are established as crucial modulators of skeletal myogenesis, but our knowledge about their identity and targets remains limited. In this study, we have identified microRNA-146b (miR-146b) as a novel regulator of skeletal myoblast differentiation. Following up on a previous microRNA profiling study, we establish that the expression of miR-146b is up-regulated during myoblast differentiation in vitro and muscle regeneration in vivo. Inhibition of miR-146b led to reduced myoblast differentiation, whereas overexpression of miR-146b enhanced differentiation. Computational prediction combined with gene expression information has revealed candidates for miR-146b targets in muscles. Among them, the expression of Smad4, Notch1, and Hmga2 are significantly suppressed by miR-146b overexpression in myocytes. In addition, expression levels of Smad4, Notch1 and Hmga2 are decreased during myoblast differentiation and muscle regeneration, inversely correlating to the levels of miR-146b. Importantly, inhibition of endogenous miR-146b prevents the down-regulation of Smad4, Notch1 and Hmga2 during differentiation. Furthermore, miR-146b directly targets the microRNA response elements (MREs) in the 3′UTR of those genes as assessed by reporter assays. Reporters with the seed regions of MREs mutated are insensitive to miR-146b, further confirming the specificity of targeting. In conclusion, miR-146b is a positive regulator of myogenic differentiation, possibly acting through multiple targets.</p></div

Overexpression of miR-146b suppresses expression of Smad4, Hmga2 and Notch1 in myoblasts.

<p>(A) C2C12 myoblasts were transfected with 50 nM miRIDIAN miR-146b mimic or cel-miR-67 mimic as control. After 24 hours, cells were lysed for RNA isolation, followed by qRT-PCR to measure mRNA levels for the genes shown. Relative mRNA levels are shown with that of cel-miR-67 as 1. Data shown are the mean ± SD from three independent experiments. One-sample <i>t</i> test was performed. **<i>P</i><0.01. (B) Cells as described in A were lysed after 48 hours of transfection and subjected to Western blotting analysis. Representative results of three independent experiments are shown.</p

Overexpression of miR-146b promotes myoblast differentiation.

<p>(A) C2C12 myoblasts were transfected with 50 nM miRIDIAN miR-146b mimic for 1 day and then induced to differentiate for 3 days. A <i>C elegans</i> miRNA (cel-67) mimic was used as negative control. The differentiated cells were fixed and immunostained for MHC (green) and DAPI (red). (B) Fusion indexes for the cells described in A were quantified. In addition, cells transfected with 50 nM native miR-146b duplex, with an siRNA against EGFP (siEGFP) as control, were differentiated and quantified for fusion index. (C) Cells described in A were lysed and subjected to Western blot analysis. In A and C, representative results of at least three independent experiments are shown. Data in B is the mean ± SD from three independent experiments. Paired <i>t</i> test was performed to compare the data. *<i>P</i><0.05.</p

Smad4, Hmga2 and Notch1 are targets of miR-146b during myoblast differentiation.

<p>(A) C2C12 cells were induced to differentiate, and total RNA was isolated from the differentiating cells on various days as indicated (Diff. day) and subjected to analysis by qRT-PCR to determine the relative levels of mRNA for each gene, with that at day 0 as 1. (B) Regeneration of mouse TA muscles was induced by BaCl₂ injury. On day 3 and day 5 AI, the TA muscles were isolated for RNA extraction, followed by qRT-PCR assays to measure relative mRNA levels. Saline injection into contralateral TA muscles served as control (“−”) and was designated as 1. (C) C2C12 cells were transfected with 50 nM LNA-anti-miR-146b or LNA control for 1 day and then induced to differentiate for 3 days. Total RNA was isolated on day 0 and day 3 of differentiation and analyzed by qRT-PCR. (D) Cells as described in C were lysed and subjected to Western blotting analysis. Data shown are mean ± SD from three to four independent experiments (A & C) or at least three mice per time point (B), or representative results of three independent experiments (D). One-sample <i>t</i> test was performed to analyze data in A & B, and paired t test was performed for data in C. *<i>P</i><0.05; **<i>P</i><0.01.</p