Young-Eun Leem

Cdo Interacts with APPL1 and Activates AKT in Myoblast Differentiation

Cdo activates Akt via indirect interaction with APPL1 during myoblast differentiation, and this complex likely mediates some of the promyogenic effect of cell–cell interaction. The promyogenic function of Cdo involves a coordinated activation of p38MAPK and Akt via interaction with scaffold proteins, JLP and Bnip-2 for p38MAPK and APPL1 for Akt.

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.

TGF-β-activated Kinase 1 (TAK1) and Apoptosis Signal-regulating Kinase 1 (ASK1) Interact with the Promyogenic Receptor Cdo to Promote Myogenic Differentiation via Activation of p38MAPK Pathway

Background: p38MAPK plays an essential role in myoblast differentiation. Results: TAK1 and ASK1 interact with Cdo and JLP to promote myogenesis. Conclusion: TAK1 and ASK1 act as MAP3Ks to activate p38MAPK in Cdo-mediated myogenesis. Significance: This might be the first report to identify MAP3Ks in Cdo-mediated myogenesis. p38MAPK plays an essential role in the transition of myoblasts to differentiated myotubes through the activation of MyoD family transcription factors. A promyogenic cell surface molecule, Cdo, promotes myogenic differentiation mainly through activation of the p38MAPK pathway. Two MAP3Ks, TAK1 and ASK1, can activate p38MAPK via MKK6 in various cell systems. Moreover TAK1 has been shown to promote myogenic differentiation via p38MAPK activation. In this study, we hypothesized that TAK1 and ASK1 might function as MAP3Ks in Cdo-mediated p38MAPK activation during myoblast differentiation. Both ASK1 and TAK1 were expressed in myoblasts and interacted with the cytoplasmic tail of Cdo and a scaffold protein, JLP. The depletion of TAK1 or ASK1 in C2C12 cells decreased myoblast differentiation, whereas overexpression of TAK1 or ASK1 in C2C12 cells enhanced myotube formation. In agreement with this, overexpression of ASK1 or TAK1 resulted in enhanced p38MAPK activation, and their knockdown inhibited p38MAPK in C2C12 cells. Overexpression of TAK1 or ASK1 in Cdo−/− myoblasts and Cdo-depleted C2C12 cells restored p38MAPK activation as well as myotube formation. Furthermore, ASK1 and TAK1 compensated for each other in p38MAPK activation and myoblast differentiation. Taken together, these findings suggest that ASK1 and TAK1 function as MAP3Ks in Cdo-mediated p38MAPK activation to promote myogenic differentiation.

Cdo suppresses canonical Wnt signalling via interaction with Lrp6 thereby promoting neuronal differentiation

Canonical Wnt signalling regulates expansion of neural progenitors and functions as a dorsalizing signal in the developing forebrain. In contrast, the multifunctional co-receptor Cdo promotes neuronal differentiation and is important for the function of the ventralizing signal, Shh. Here we show that Cdo negatively regulates Wnt signalling during neurogenesis. Wnt signalling is enhanced in Cdo-deficient cells, leading to impaired neuronal differentiation. The ectodomains of Cdo and Lrp6 interact via the Ig2 repeat of Cdo and the LDLR repeats of Lrp6, and the Cdo Ig2 repeat is necessary for Cdo-dependent Wnt inhibition. Furthermore, the Cdo-deficient dorsal forebrain displays stronger Wnt signalling activity, increased cell proliferation and enhanced expression of the dorsal markers and Wnt targets, Pax6, Gli3, Axin2. Therefore, in addition to promoting ventral central nervous system cell fates with Shh, Cdo promotes neuronal differentiation by suppression of Wnt signalling and provides a direct link between two major dorsoventral morphogenetic signalling pathways.

Esco2 promotes neuronal differentiation by repressing Notch signaling.

Esco2 is an acetyltransferase that is required for the establishment of sister chromatid cohesion. Roberts-SC phocomelia (RBS) syndrome caused by the mutations of Esco2 gene, is an autosomal recessive development disorder characterized by growth retardation, limb reduction and craniofacial abnormalities including cleft lip and palate. Here, we show that Esco2 protein co-immunoprecipitates with Notch but not with CBF1. Esco2 represses the transactivational activity of Notch protein in an acetyltransferase-independent manner. Chromatin immunoprecipitation experiments suggest that Esco2 might regulate the activity of NICD-CBF1 via attenuating NICD binding to CBF1 on the promoter of Hes1, the downstream target gene of Notch. Furthermore, we demonstrate that the overexpression of Esco2 promotes the neuronal differentiation of P19 embryonic carcinoma cells and C17.2 neural progenitor cells and the knockdown of Esco2 by siRNA blocks the differentiation. The inhibitory effects of Notch protein on neuronal differentiation of P19 cells was suppressed by Esco2 overexpression. Taken together, our study suggests that Esco2 may play an important role in neurogenesis by attenuating Notch signaling to promote neuronal differentiation.

Inhibition of master transcription factors in pluripotent cells induces early stage differentiation

Significance Though the potential of stem cells to differentiate into diverse specialized cell types has given much hope to the field of regenerative medicine, low efficiency of commitment is still a major obstacle to practical application. We hypothesized that initial differentiation can be enhanced if the transcriptional activity of core stemness regulators is suppressed. By taking advantage of a sex determining region Y-box 2 (Sox2) interacting protein from heterologous origin, we proved that the inhibition of transcriptional activity of Sox2 resulted in the expression of early differentiation markers and appearance of early neuronal and cardiac progenitors. This strategy can possibly be applied to induce efficient differentiation of stem cells and provide a clue to understanding the mechanism of early differentiation. The potential for pluripotent cells to differentiate into diverse specialized cell types has given much hope to the field of regenerative medicine. Nevertheless, the low efficiency of cell commitment has been a major bottleneck in this field. Here we provide a strategy to enhance the efficiency of early differentiation of pluripotent cells. We hypothesized that the initial phase of differentiation can be enhanced if the transcriptional activity of master regulators of stemness is suppressed, blocking the formation of functional transcriptomes. However, an obstacle is the lack of an efficient strategy to block protein–protein interactions. In this work, we take advantage of the biochemical property of seventeen kilodalton protein (Skp), a bacterial molecular chaperone that binds directly to sex determining region Y-box 2 (Sox2). The small angle X-ray scattering analyses provided a low resolution model of the complex and suggested that the transactivation domain of Sox2 is probably wrapped in a cleft on Skp trimer. Upon the transduction of Skp into pluripotent cells, the transcriptional activity of Sox2 was inhibited and the expression of Sox2 and octamer-binding transcription factor 4 was reduced, which resulted in the expression of early differentiation markers and appearance of early neuronal and cardiac progenitors. These results suggest that the initial stage of differentiation can be accelerated by inhibiting master transcription factors of stemness. This strategy can possibly be applied to increase the efficiency of stem cell differentiation into various cell types and also provides a clue to understanding the mechanism of early differentiation.

PKN2 and Cdo interact to activate AKT and promote myoblast differentiation

Skeletal myogenesis is coordinated by multiple signaling pathways that control cell adhesion/migration, survival and differentiation accompanied by muscle-specific gene expression. A cell surface protein Cdo is involved in cell contact-mediated promyogenic signals through activation of p38MAPK and AKT. Protein kinase C-related kinase 2 (PKN2/PRK2) is implicated in regulation of various biological processes, including cell migration, adhesion and death. It has been shown to interact with and inhibit AKT thereby inducing cell death. This led us to investigate the role of PKN2 in skeletal myogenesis and the crosstalk between PKN2 and Cdo. Like Cdo, PKN2 was upregulated in C2C12 myoblasts during differentiation and decreased in cells with Cdo depletion caused by shRNA or cultured on integrin-independent substratum. This decline of PKN2 levels resulted in diminished AKT activation during myoblast differentiation. Consistently, PKN2 overexpression-enhanced C2C12 myoblast differentiation, whereas PKN2-depletion impaired it, without affecting cell survival. PKN2 formed complexes with Cdo, APPL1 and AKT via its C-terminal region and this interaction appeared to be important for induction of AKT activity as well as myoblast differentiation. Furthermore, PKN2-enhanced MyoD-responsive reporter activities by mediating the recruitment of BAF60c and MyoD to the myogenin promoter. Taken together, PKN2 has a critical role in cell adhesion-mediated AKT activation during myoblast differentiation.

The Shh coreceptor Cdo is required for differentiation of midbrain dopaminergic neurons

Sonic hedgehog (Shh) signaling is required for numerous developmental processes including specification of ventral cell types in the central nervous system such as midbrain dopaminergic (DA) neurons. The multifunctional coreceptor Cdo increases the signaling activity of Shh which is crucial for development of forebrain and neural tube. In this study, we investigated the role of Cdo in midbrain DA neurogenesis. Cdo and Shh signaling components are induced during neurogenesis of embryonic stem (ES) cells. Cdo(-/-) ES cells show reduced neuronal differentiation accompanied by increased cell death upon neuronal induction. In addition, Cdo(-/-) ES cells form fewer tyrosine hydroxylase (TH) and microtubule associated protein 2 (MAP2)-positive DA neurons correlating with the decreased expression of key regulators of DA neurogenesis, such as Shh, Neurogenin2, Mash1, Foxa2, Lmx1a, Nurr1 and Pitx3, relative to the Cdo(+/+) ES cells. Consistently, the Cdo(-/-) embryonic midbrain displays a reduction in expression of TH and Nurr1. Furthermore, activation of Shh signaling by treatment with Purmorphamine (Pur) restores the DA neurogenesis of Cdo(-/-) ES cells, suggesting that Cdo is required for the full Shh signaling activation to induce efficient DA neurogenesis.

CDO, an Hh-coreceptor, mediates lung cancer cell proliferation and tumorigenicity through Hedgehog signaling.

Hedgehog (Hh) signaling plays essential roles in various developmental processes, and its aberrant regulation results in genetic disorders or malignancies in various tissues. Hyperactivation of Hh signaling is associated with lung cancer development, and there have been extensive efforts to investigate how to control Hh signaling pathway and regulate cancer cell proliferation. In this study we investigated a role of CDO, an Hh co-receptor, in non-small cell lung cancer (NSCLC). Inhibition of Hh signaling by SANT-1 or siCDO in lung cancer cells reduced proliferation and tumorigenicity, along with the decrease in the expression of the Hh components. Histological analysis with NSCLC mouse tissue demonstrated that CDO was expressed in advanced grade of the cancer, and precisely co-localized with GLI1. These data suggest that CDO is required for proliferation and survival of lung cancer cells via Hh signaling.