Hye-Jin Lee

Regulation of Myoblast Motility and Fusion by the CXCR4-associated Sialomucin, CD164

Myoblast fusion is fundamental to the development and regeneration of skeletal muscle. To fuse, myoblasts undergo cell-cell recognition and adhesion and merger of membranes between apposing cells. Cell migration must occur in advance of these events to bring myoblasts into proximity, but the factors that regulate myoblast motility are not fully understood. CD164 is a cell surface sialomucin that is targeted to endosomes and lysosomes via its intracellular region. In hematopoietic progenitor cells, CD164 forms complexes with the motility-stimulating chemokine receptor, CXCR4, in response to the CXCR4 ligand, CXCL12/SDF-1 (Forde, S., Tye, B. J., Newey, S. E., Roubelakis, M., Smythe, J., McGuckin, C. P., Pettengell, R., and Watt, S. M. (2007) Blood 109, 1825–1833). We have previously shown that CD164 stimulates myotube formation in vitro. We report here that CD164 is associated with CXCR4 in C2C12 myoblasts. Cells in which CD164 levels are increased or decreased via overexpression or RNA interference-mediated knockdown, respectively, show enhanced or reduced myotube formation and cell migration, the latter both basally and in response to CXCL12/SDF-1. Furthermore, expression of CD164 cytoplasmic tail mutants that alter the endosome/lysosome targeting sequence and, consequently, the subcellular localization in myoblasts, reveals a similar correlation between cell motility and myotube formation. Finally, Cd164 mRNA is expressed in the dorsal somite (the early myogenic compartment of the mouse embryo) and in premuscle masses. Taken together, these results suggest that CD164 is a regulator of myoblast motility and that this property contributes to its ability to promote myoblast fusion into myotubes.

TGFβ mediates activation of transglutaminase 2 in response to oxidative stress that leads to protein aggregation

Transglutaminase 2 (TGase2) is a ubiquitously expressed enzyme that catalyzes irreversible post‐translational modification of protein, forming cross‐linked protein aggregates. We previously reported that intracellular TGase2 is activated by oxidative stress. To elucidate the functional role of TGase2 activation in cells under the oxidatively stressed condition, we identified the mediator that activates TGase2. In this study, we showed that low levels of oxidative stress trigger the release of TGFβ, which subsequently activates TGase2 through the nuclear translocation of Smad3. Analysis of substrate proteins reveals that TGase2‐mediated protein modification results in a decrease of protein solubility and a collapse of intermediate filament network, which leads to aggregation of proteins. We confirm these results using lens tissues from TGase2‐deficient mice. Among several antioxidants tried, only N‐acetylcysteine effectively inhibits TGFβ‐mediated activation of TGase2. These results indicate that TGFβ mediates oxidative stress‐induced protein aggregation through activation of TGase2 and suggest that the formation of protein aggregation may not be a passive process of self‐assembly of oxidatively damaged proteins but may be an active cellular response to oxidative stress. Therefore, TGFP‐TGase2 pathway may have implications for both the pathogenesis of age‐related degenerative diseases and the development of pharmaceutics.—Shin, D.‐M., Jeon, J.‐H., Kim, C.‐W., Cho, S.‐Y., Lee, H.‐J., Jang, G.‐Y., Jeong, E. M., Lee, D.‐S., Kang, J.‐H., Melino, G., Park, S.‐C., Kim, I.‐G. TGFβ mediates activation of transglutaminase 2 in response to oxidative stress that leads to protein aggregation. FASEB J. 22, 2498–2507 (2008)

Neogenin regulates skeletal myofiber size and focal adhesion kinase and extracellular signal-regulated kinase activities in vivo and in vitro.

A variety of signaling pathways participate in the development of skeletal muscle, but the extracellular cues that regulate such pathways in myofiber formation are not well understood. Neogenin is a receptor for ligands of the netrin and repulsive guidance molecule (RGM) families involved in axon guidance. We reported previously that neogenin promoted myotube formation by C2C12 myoblasts in vitro and that the related protein Cdo (also Cdon) was a potential neogenin coreceptor in myoblasts. We report here that mice homozygous for a gene-trap mutation in the Neo1 locus (encoding neogenin) develop myotomes normally but have small myofibers at embryonic day 18.5 and at 3 wk of age. Similarly, cultured myoblasts derived from such animals form smaller myotubes with fewer nuclei than myoblasts from control animals. These in vivo and in vitro defects are associated with low levels of the activated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), both known to be involved in myotube formation, and inefficient expression of certain muscle-specific proteins. Recombinant netrin-2 activates FAK and ERK in cultured myoblasts in a neogenin- and Cdo-dependent manner, whereas recombinant RGMc displays lesser ability to activate these kinases. Together, netrin-neogenin signaling is an important extracellular cue in regulation of myogenic differentiation and myofiber size.

Cdo promotes neuronal differentiation via activation of the p38 mitogen-activated protein kinase pathway.

Neural basic helix‐loop‐helix transcription factors (bHLHs) control many aspects of neurogenesis, such as proliferation, fate determination, and differentiation. We have previously shown that the promyogenic cell surface receptor Cdo modulates the Cdc42 and p38 mitogen‐activated protein kinase (MAPK) pathways via a direct association with two scaffold‐type proteins, JLP and Bnip‐2, to regulate activities of myogenic bHLH factors and myogenic differentiation. We report here that Cdo uses similar regulatory mechanisms to promote neuronal differentiation. Expression of JLP, a scaffold protein for p38MAPK, and Bnip‐2, a regulator of Cdc42, is increased during differentiation of C17.2 neural precursor cells and P19 embryonal carcinoma cells. These molecules regulate Cdc42 and p38MAPK activities, which increase in a Cdo‐dependent manner during neuronal differentiation of C17.2 cells and retinoic acid‐treated P19 cells. Furthermore, enhancement or reduction of Cdc42 and p38MAPK activities enhances or reduces, respectively, neuronal differentiation of these cell lines. Cdc42 and p38MAPK activities also promote heterodimerization of neurogenin1 and E47, suggesting that one way they promote neurogenesis is via regulation of neural bHLH factor activities. These results imply that a conserved intracellular signaling mechanism initiated by Cdo regulates the activities of tissue‐specific bHLH factors and therefore functions as a key regulator of differentiation of several different cell lineages.—Oh, J.‐E., Bae, G.‐U., Yang, Y.‐J., Yi, M.‐J., Lee, H.‐J., Kim, B.‐G., Krauss, R. S., Kang, J.‐S. Cdo promotes neuronal differentiation via activation of the p38 mitogen‐activated protein kinase pathway. FASEBJ. 23, 2088–2099 (2009)

Cdo Binds Abl To Promote p38α/β Mitogen-Activated Protein Kinase Activity and Myogenic Differentiation

ABSTRACT The p38 mitogen-activated protein kinase (MAPK) pathway is required for differentiation of skeletal myoblasts, but how the pathway is activated during this process is not well understood. One mechanism involves the cell surface receptor Cdo (also known as Cdon), which binds to Bnip-2 and JLP, scaffold proteins for Cdc42 and p38, respectively; formation of these complexes results in Bnip-2/Cdc42-dependent activation of p38. It has been reported that the tyrosine kinase Abl promotes myogenic differentiation in a manner dependent on its cytoplasmic localization, but the cytoplasmic signaling proteins with which it interacts to achieve this effect are unidentified. We report that Abl associates with both Cdo and JLP during myoblast differentiation. Abl binds a proline-rich motif in Cdo via its SH3 domain, and these regions of Abl and Cdo are required for their promyogenic effects. Cdo is important for full Abl kinase activity, and Abl is necessary for full activation of p38 MAPK, during myogenic differentiation. As seen with myoblasts depleted of Cdo, the diminished differentiation displayed by Abl-depleted cells is rescued by the expression of an activated form of the immediate upstream p38-activating kinase MAPK kinase 6. Abls promyogenic effect is therefore linked to a multiprotein cell surface complex that regulates differentiation-dependent p38 activation.

Phosphorylation of Stim1 at serine 575 via netrin-2/Cdo–activated ERK1/2 is critical for the promyogenic function of Stim1

A functional link is identified between Cdo and Stim1 that leads to NFATc3 activation. Stim1 is required for muscle differentiation via activation of the calcineurin/NFAT pathway. The netrin-2–mediated activation of NFATc3 is coincident with an interaction between Cdo and Stim1 via ERK-mediated phosphorylation of Stim1 at Ser-575.

Gas1 cooperates with Cdo and promotes myogenic differentiation via activation of p38MAPK

Skeletal myogenesis is a multistep process that involves cell cycle exit, expression of muscle-specific genes and formation of multinucleated myotubes. Growth arrest specific gene 1 (Gas1) is a GPI-linked membrane protein and originally identified as a growth arrest-linked gene in fibroblasts. Promyogenic cell surface protein, Cdo functions as a component of multiprotein complexes that include other cell adhesion molecules, like Cadherins to mediate cell contact signaling. Here we report that Gas1 and Cdo are coexpressed in muscle cells and form a complex in differentiating myoblasts. Interestingly, Cdo(-/-) myoblasts display defects in Gas1 induction during differentiation. Overexpression or depletion of Gas1 enhances or decreases myogenic differentiation, respectively. During myoblast differentiation, Gas1 depletion causes defects in downregulation of Cdk2 and Cyclin D1 and up-regulation of miR-322, a negative regulator of Cdk2 activities. Furthermore overexpression or knockdown of Gas1 either enhances or decreases activation of p38MAPK that functions downstream of Cdo. Additionally, Gas1 overexpression in Cdo-depleted C2C12 cells restores p38MAPK activities and differentiation abilities. These data suggest that Gas1 promotes myogenic differentiation through regulation of cell cycle arrest and is critical to activate p38MAPK, most likely via association with Cdo/Cadherin multiprotein complexes.

Promyogenic function of Integrin/FAK signaling is mediated by Cdo, Cdc42 and MyoD

The Integrin-mediated cell adhesion to the extracellular matrix is implicated in the control of proliferation, survival, migration and differentiation of myoblasts. Focal adhesion kinase (FAK) mediates signals from Integrins and plays an essential role in myotube formation. Cdo forms a multiprotein complex that includes other cell adhesion molecules like Cadherins and Boc. Multiple signals emanate from such complexes, including Cdc42 and p38MAPK pathways to activate MyoD. Here we show that C2C12 myoblasts cultured in suspension or on Poly-L-Lysine (PLL), a well known Integrin-independent substratum, failed to express Cdo and MyoD, while the expression of Cadherins and Boc was unchanged. In addition, the activation of Akt and p38MAPK as well as the expression of Cdc42 was affected in these cells. Overexpression of FAK rescued MyoD and Cdo expression as well as myotube formation of C2C12 cells on PLL. Furthermore, reintroduction of Cdo induced enhanced myotube formation on PLL and increased the expression of myogenic markers. Inhibition of ROCK or overexpression of Cdc42-V12 in C2C12 cells upregulated Cdc42 and MyoD expression and rescued defective myoblast differentiation. Taken together, these data indicate that the Integrin/FAK signaling pathway is required for myoblast differentiation by regulating the expression of the promyogenic factors, Cdo, MyoD and Cdc42.

Li2ZrOx/carbon double-coated Li[Li0.2Ni0.16Mn0.56Co0.08]O2 cathodes for improved electrochemical performance

The electrochemical properties of the Li[Li0.2Ni0.16Mn0.56Co0.08]O2 cathode material were studied after surface modification with a Li2ZrOx/carbon double coating layer. Li2ZrOx, a stable oxide with ionic conductivity, suppresses the unwanted reaction between the cathode and the electrolyte. On the other hand, carbon layer, with high electronic conductivity, compensates for the low conductivity of the oxide cathode. The rate capability and cyclic performance of the Li[Li0.2Ni0.16Mn0.56Co0.08]O2 cathode were improved due to the surface modification. Moreover, the thermal stability of the cathode was also enhanced as the double coating layer protected the cathode surface from the electrolyte. The results show that the Li2ZrOx/carbon double coating is an effective approach to obtain enhanced electrochemical properties of the Li[Li0.2Ni0.16Mn0.56Co0.08]O2 cathode.

Isobavachalcone from Angelica keiskei Inhibits Adipogenesis and Prevents Lipid Accumulation

We isolated isobavachalcone (IBC) from Angelica keiskei (AK) as an anti-obesity component. IBC dose-dependently inhibited 3T3-L1 adipocyte differentiation by down-regulating adipogenic factors. At the mitotic clonal expansion stage (MCE), IBC caused cell cycle arrest in G0/G1 with decreased expression of cell cycle-regulating proteins. IBC also inhibited autophagic flux by inducing intracellular accumulation of LC3B and SQSTM1/p62 proteins while decreasing expression levels of regulating factors for autophagy initiation. In parallel with the inhibition of adipocyte differentiation, IBC decreased intrahepatic fat deposits and rescued the liver steatosis in high fat cholesterol diet-fed zebrafish. In this study, we found that IBC isolated from AK suppresses mitotic clonal expansion and autophagy flux of adipocytes and also shows anti-obesity activity in a high cholesterol-diet zebrafish model by decreasing intrahepatic fat deposits. These results suggest that IBC could be a leading pharmacological compound for the development of anti-obesity drugs.