Mi-Yeon Kim

Bone morphogenetic protein 4 stimulates neuronal differentiation of neuronal stem cells through the ERK pathway

Bone morphogenic protein 4 (BMP4), a member of the TGF-β superfamily, induced neural differentiation of neural stem cells (NSCs) grown in a medium containing basic fibroblast growth factor (bFGF). The Ras protein level and the activities of the downstream ERKs were increased by transfection of BMP4 or treatment with recombinant BMP4. The effects of BMP4, including activation of the Ras-ERK pathway and induction of the neuron marker β-tubulin type III (Tuj1), were blocked by co-treatment of the BMP4 antagonist, noggin. The roles of the Ras-ERK pathway in neuronal differentiation by BMP4 were revealed by measuring the effect of the ERK pathway inhibition by dominant negative Ras or PD98059, the MEK specific inhibitor. BMP4 is a transcriptional target of Wnt/β-catenin signaling, and both the mRNA and protein levels of BMP4 were increased by treatment of valproic acid (VPA), a chemical inhibitor of glycogen synthase kinase 3β (GSK3β) activating the Wnt/β-catenin pathway. The BMP4-mimicking effects of VPA, activation of the Ras-ERK pathway and induction of Tuj1, also were blocked by noggin. These results indicate the potential therapeutic usage of VPA as a replacement for BMP4.

CXXC5 is a transcriptional activator of Flk-1 and mediates bone morphogenic protein-induced endothelial cell differentiation and vessel formation

CXXC5 is a member of a small subset of proteins containing CXXC‐type zinc‐finger domain. Here, we show that CXXC5 is a transcription factor activating Flk‐1, a receptor for vascular endothelial growth factor. CXXC5 and Flk‐1 were accmulated in nucli and membrane of mouse embryonic stem cells (mESCs), respectively, during their endothelial differentiation. CXXC5 overexpression induced Flk‐1 transcription in both endothelium‐differentiated mESCs and human umbilical vein endothelial cells (HUVECs). In vitro DNA binding assay showed direct interaction of CXXC5 on the Flk‐1 promoter region, and mutation on its DNA‐binding motif abolished transcriptional activity. We showed that bone morphorgeneic protein 4 (BMP4) induced CXXC5 transcription in the cells, and inhibitors of BMP signaling suppressed the CXXC5 induction and the consequent Flk‐1 induction by BMP4 treatment. CXXC5 knockdown resulted in suppression of BMP4‐induced stress fiber formation (56.8±1.3% decrease, P<0.05) and migration (54.6±1.9% decrease, P<0.05) in HUVECs. The in vivo roles of CXXC5 in BMP‐signaling‐specific vascular development and angiogenesis were shown by specific defect of caudal vein plex vessel formation (57.9±11.8% decrease, P<0.05) in cxxc5 morpholino‐injected zebrafish embryos and by supression of BMP4‐induced angigogensis in subcutaneously injected Matrigel plugs in CXXC5‐/‐ mice. Overall, CXXC5 is a transcriptional activator for Flk‐1, mediating BMP signaling for differentiation and migration of endothelial cell and vessel formation.—Kim, H.‐Y., Yang, D.‐H., Shin, S.‐W., Kim, M.‐Y., Yoon, J.‐H., Kim, S., Park, H.‐C., Kang, D. W., Min, D., Hur, M.‐W., Choi, K‐Y. CXXC5 is a transcriptional activator of Flk‐1 and mediates bone morphogenic protein‐induced endothelial cell differentiation and vessel formation. FASEB J. 28, 615–626 (2014). www.fasebj.org

Regulation of Notch1 signaling by the APP intracellular domain facilitates degradation of the Notch1 intracellular domain and RBP-Jk

The Notch1 receptor is a crucial controller of cell fate decisions, and is also a key regulator of cell growth and differentiation in a variety of contexts. In this study, we have demonstrated that the APP intracellular domain (AICD) attenuates Notch1 signaling by accelerated degradation of the Notch1 intracellular domain (Notch1-IC) and RBP-Jk, through different degradation pathways. AICD suppresses Notch1 transcriptional activity by the dissociation of the Notch1-IC–RBP-Jk complex after processing by γ-secretase. Notch1-IC is capable of forming a trimeric complex with Fbw7 and AICD, and AICD enhances the protein degradation of Notch1-IC through an Fbw7-dependent proteasomal pathway. AICD downregulates the levels of RBP-Jk protein through the lysosomal pathway. AICD-mediated degradation is involved in the preferential degradation of non-phosphorylated RBP-Jk. Collectively, our results demonstrate that AICD functions as a negative regulator in Notch1 signaling through the promotion of Notch1-IC and RBP-Jk protein degradation.

Small-molecule binding of the axin RGS domain promotes β-catenin and Ras degradation.

Both the Wnt/β-catenin and Ras pathways are aberrantly activated in most human colorectal cancers (CRCs) and interact cooperatively in tumor promotion. Inhibition of these signaling may therefore be an ideal strategy for treating CRC. We identified KY1220, a compound that destabilizes both β-catenin and Ras, via targeting the Wnt/β-catenin pathway, and synthesized its derivative KYA1797K. KYA1797K bound directly to the regulators of G-protein signaling domain of axin, initiating β-catenin and Ras degradation through enhancement of the β-catenin destruction complex activating GSK3β. KYA1797K effectively suppressed the growth of CRCs harboring APC and KRAS mutations, as shown by various in vitro studies and by in vivo studies using xenograft and transgenic mouse models of tumors induced by APC and KRAS mutations. Destabilization of both β-catenin and Ras via targeting axin is a potential therapeutic strategy for treatment of CRC and other type cancers activated Wnt/β-catenin and Ras pathways.

Dual Regulation of Notch1 Signaling Pathway by Adaptor Protein Fe65

Background: The importance of Notch and Fe65 function during development has been well recognized. Results: Adaptor protein Fe65 attenuates Notch1 signaling via the accelerated degradation of the membrane-tethered Notch1. Conclusion: Dual regulation of Notch1 signaling pathway by adaptor protein Fe65. Significance: The findings of this study may begin to shed some light onto what may be a signal cross-talk mechanism of Notch1 signaling and the Fe65 adaptor protein. Notch1 receptor functions as a critical controller of cell fate decisions and also as a key regulator of cell growth, differentiation, and proliferation in invertebrates and vertebrates. In this study, we have demonstrated that the adaptor protein Fe65 attenuates Notch1 signaling via the accelerated degradation of the membrane-tethered Notch1 in the cytoplasm. Fe65 also suppresses Notch1 transcriptional activity via the dissociation of the Notch1-IC-recombining binding protein suppressor of hairless (RBP)-Jk complex within the nucleus. Fe65 is capable of forming a trimeric complex with Itch and membrane-tethered Notch1, and Fe65 enhances the protein degradation of membrane-tethered Notch1 via an Itch-dependent proteasomal pathway. Collectively, our results demonstrate that Fe65 carries out different functions depending on its location in the regulation of Notch1 signaling.

Polygonum aviculare L. and its active compounds, quercitrin hydrate, caffeic acid, and rutin, activate the Wnt/β‐catenin pathway and induce cutaneous wound healing

Polygonum aviculare L. is a member of the Polygonaceae family of plants, which has been known for its antioxidant and anti‐obesity effects. However, the wound healing function of P. aviculare extract has not been assessed. In this study, we identified a novel property of P. aviculare extract as a Wnt/β‐catenin pathway activator based on a screen of 350 plant extracts using HEK293‐TOP cells retaining the Wnt/β‐catenin signaling reporter gene. P. aviculare extract accelerated the migration of HaCaT keratinocytes without showing significant cytotoxicity. Moreover, P. aviculare extract efficiently re‐epithelized wounds generated on mice. Additionally, ingredients of P. aviculare extract, such as quercitrin hydrate, caffeic acid, and rutin, also accelerated the motility of HaCaT keratinocytes with the activation of Wnt/β‐catenin signaling. Therefore, based on our findings, P. aviculare extract and its active ingredients could be potential therapeutic agents for wound healing. Copyright

Presenilin-2 regulates the degradation of RBP-Jk protein through p38 mitogen-activated protein kinase

Transcriptional regulation performs a central role in Notch1 signaling by recombining binding protein Suppressor of Hairless (RBP-Jk) – a signaling pathway that is widely involved in determination of cell fate. Our earlier work demonstrated the possible regulation of the Notch1–RBP-Jk pathway through protein degradation of RBP-Jk; however, the potential regulator for the degradation of RBP-Jk remains to be determined. Here, we report that the expression of endogenous and exogenous RBP-Jk was increased significantly in cells treated with proteasome- and lysosome-specific inhibitors. The effects of these inhibitors on RBP-Jk occurred in a dose- and time-dependent manner. The level of RBP-Jk protein was higher in presenilin-2 (PS2)-knockout cells than in presenilin-1 (PS1)-knockout cells. Furthermore, the level of RBP-Jk was decreased by expression of PS2 in PS1 and PS2 double-knockout cells. We also found that PS1-knockout cells treated with a specific inhibitor of p38 mitogen-activated protein kinase ∂ (MAPK) had significantly increased levels of RBP-Jk. p38 MAPK phosphorylates RBP-Jk at Thr339 by physical binding, which subsequently induces the degradation and ubiquitylation of the RBP-Jk protein. Collectively, our results indicate that PS2 modulates the degradation of RBP-Jk through phosphorylation by p38 MAPK.

CXXC5 plays a role as a transcription activator for myelin genes on oligodendrocyte differentiation

Myelination in corpus callosum plays important role for normal brain functions by transferring neurological information between various brain regions. However, the factors controlling expression of myelin genes in myelination are poorly understood. Here, CXXC5, a recently identified protein with CXXC‐type zinc finger DNA binding motif, was characterized as a transcriptional activator of major myelin genes. We identified expression of CXXC5 expression was increased by Wnt/β‐catenin signaling. CXXC5 specifically expressed in the white matter induced expression of myelin genes through the direct binding of CXXC DNA‐binding motif of CXXC5 on the MBP promoter. During the differentiation of neural stem cells (NSCs) of CXXC5−/− mice, the expressions of myelin genes were simultaneously reduced. The CXXC5−/− mice exhibited severely reduction of myelin genes expression in corpus callosum as well as abnormalities in myelin structure. The disrupted structural integrity of myelin in the CXXC5−/− mice resulted in reduced electrical conduction amplitudes at corpus callosum. These findings indicate that the regulation of myelin genes expression by CXXC5 is important for forming myelin structure involved with axonal electrical signal transfer in the corpus callosum. GLIA 2016;64:350–362

Isolation and Maintenance of Cortical Neural Progenitor Cells In Vitro

Neural progenitor cells (NPCs) or neural stem cells are important tools for investigating central nervous system (CNS) development. NPCs can be used in therapeutic strategies and for characterizing differentiation mechanisms. Here, we describe methods for isolating and culturing embryonic NPCs.

Electrophoretron: a new method for enhancing resolution in electrokinetic separations.

Two capillaries, each of which have different surface preparations on their inside walls, are joined together to form a closed loop, and electrodes are placed inside the two capillaries. When the loop is filled with liquid and a potential difference is applied between the two electrodes, a circulating flow of liquid is established inside the loop because the resistance to flow is unequal in going from one electrode to another in a clockwise versus a counterclockwise direction. Consequently, a sample injected into this device, which we call an electrophoretron, repeatedly circulates between the two electrodes and the capillary separation column becomes effectively one of unlimited length. On each cycle the separation between analytes with different mobilities increases, thus enhancing resolution of analytes having nearly the same mobilities. The operation of a prototype electrophoretron is demonstrated.