Jungmook Lyu

Cleavage of the Wnt Receptor Ryk Regulates Neuronal Differentiation during Cortical Neurogenesis

Ryk is a transmembrane receptor tyrosine kinase (RTK). It functions as a receptor of Wnt proteins required for cell-fate determination, axon guidance, and neurite outgrowth in different organisms; however, the molecular mechanism of Ryk signaling is unknown. Here, we show that Ryk is cleaved, permitting the intracellular C-terminal fragment of Ryk to translocate to the nucleus in response to Wnt3 stimulation. We also show that the cleaved intracellular domain of Ryk is required for Wnt3-induced neuronal differentiation in vitro and in vivo. These results demonstrate an unexpected mechanism of signal transduction for Ryk as a Wnt receptor, in which the intracellular domain itself functions as the transducing molecule to bring extracellular signals from the cell surface into the nucleus, to regulate neural progenitor cell differentiation.

Klf4 Interacts Directly with Oct4 and Sox2 to Promote Reprogramming

Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by ectopic expression of specific sets of transcription factors. Oct4, Sox2, and Klf4, factors that share many target genes in embryonic stem (ES) cells, are critical components in various reprogramming protocols. Nevertheless, it remains unclear whether these factors function together or separately in reprogramming. Here we show that Klf4 interacts directly with Oct4 and Sox2 when expressed at levels sufficient to induce iPS cells. Endogenous Klf4 also interacts with Oct4 and Sox2 in iPS cells and in mouse ES cells. The Klf4 C terminus, which contains three tandem zinc fingers, is critical for this interaction and is required for activation of the target gene Nanog. In addition, Klf4 and Oct4 co‐occupy the Nanog promoter. A dominant negative mutant of Klf4 can compete with wild‐type Klf4 to form defective Oct4/Sox2/Klf4 complexes and strongly inhibit reprogramming. In the absence of Klf4 overexpression, interaction of endogenous Klf4 with Oct4/Sox2 is also required for reprogramming. This study supports the idea that direct interactions between Klf4, Oct4, and Sox2 are critical for somatic cell reprogramming. STEM CELLS 2009;27:2969–2978

Wnt-7a Up-regulates Matrix Metalloproteinase-12 Expression and Promotes Cell Proliferation in Corneal Epithelial Cells during Wound Healing

Corneal wound repair involves the rapid coverage of a denuded area by residual epithelial cells. During wound healing, there are different cell behaviors in different regions of the epithelium: cell proliferation in the peripheral epithelium and cell migration in the central epithelium. We found that Wnt-7a was rapidly induced in the wounded cornea, promoted the proliferation of corneal epithelial cells, and enhanced wound closure. Matrix metalloproteinase-12 (MMP-12) was detected in the peripheral epithelium, where cell proliferation was enhanced, but was diminished in the migrating central epithelium. Wnt-7a induced the accumulation of β-catenin and the activation of Rac and β-catenin, and Rac synergistically induced the transcription of MMP-12. Blocking the function of MMP-12 delayed wound closure induced by Wnt-7a. Our results also suggest that, in addition to the β-catenin pathway, Wnt-7a might induce a β-catenin-independent pathway. By regulating the proliferation of corneal epithelial cells, Wnt-7a and MMP-12 appear to contribute to corneal wound healing.

Wnt signaling enhances FGF2-triggered lens fiber cell differentiation

Wnt signaling is implicated in many developmental processes, including cell fate changes. Several members of the Wnt family, as well as other molecules involved in Wnt signaling, including Frizzled receptors, LDL-related protein co-receptors, members of the Dishevelled and Dickkopf families, are known to be expressed in the lens during embryonic or postembryonic development. However, the function of Wnt signaling in lens fiber differentiation remains unknown. Here, we show that GSK-3β kinase is inactivated and thatβ -catenin accumulates during the early stages of lens fiber cell differentiation. In an explant culture system, Wnt conditioned medium (CM) induced the accumulation of β-crystallin, a marker of fiber cell differentiation, without changing cell shape. In contrast, epithelial cells stimulated with Wnt after priming with FGF elongated, accumulatedβ -crystallin, aquaporin-0, p57kip2, and altered their expression of cadherins. Treatment with lithium, which stabilizes β-catenin, induced the accumulation of β-crystallin, but explants treated with lithium after FGF priming did not elongate as they did after Wnt application. These results show that Wnts promote the morphological aspects of fiber cell differentiation in a process that requires FGF signaling, but is independent ofβ -catenin. Wnt signaling may play an important role in lens epithelial-to-fiber differentiation.

The Wnt receptor Ryk controls specification of GABAergic neurons versus oligodendrocytes during telencephalon development

GABAergic neurons and oligodendrocytes originate from progenitors within the ventral telencephalon. However, the molecular mechanisms that control neuron-glial cell-fate segregation, especially how extrinsic factors regulate cell-fate changes, are poorly understood. We have discovered that the Wnt receptor Ryk promotes GABAergic neuron production while repressing oligodendrocyte formation in the ventral telencephalon. We demonstrate that Ryk controls the cell-fate switch by negatively regulating expression of the intrinsic oligodendrogenic factor Olig2 while inducing expression of the interneuron fate determinant Dlx2. In addition, we demonstrate that Ryk is required for GABAergic neuron induction and oligodendrogenesis inhibition caused by Wnt3a stimulation. Furthermore, we showed that the cleaved intracellular domain of Ryk is sufficient to regulate the cell-fate switch by regulating the expression of intrinsic cell-fate determinants. These results identify Ryk as a multi-functional receptor that is able to transduce extrinsic cues into progenitor cells, promote GABAergic neuron formation, and inhibit oligodendrogenesis during ventral embryonic brain development.

Cdc37 Regulates Ryk Signaling by Stabilizing the Cleaved Ryk Intracellular Domain

Ryk is a Wnt receptor that plays an important role in neurogenesis, neurite outgrowth, and axon guidance. We have reported that the Ryk receptor is cleaved by γ-secretase and that its intracellular domain (ICD) translocates to the nucleus upon Wnt stimulation. Cleavage of Ryk and its ICD is important for the function of Ryk in neurogenesis. However, the question of how the Ryk ICD is stabilized and translocated into the nucleus remains unanswered. Here, we show that the Ryk ICD undergoes ubiquitination and proteasomal degradation. We have identified Cdc37, a subunit of the molecular chaperone Hsp90 complex, as a Ryk ICD-interacting protein that inhibits proteasomal degradation of the Ryk ICD. Overexpression of Cdc37 increases Ryk ICD levels and promotes its nuclear localization, whereas Cdc37 knockdown reduces Ryk ICD stability. Furthermore, we have discovered that the Cdc37-Ryk ICD complex is disrupted during neural differentiation of embryonic stem cells, resulting in Ryk ICD degradation. These results suggest that Cdc37 plays an essential role in regulating Ryk ICD stability and therefore in Ryk-mediated signal transduction.

Smek promotes histone deacetylation to suppress transcription of Wnt target gene brachyury in pluripotent embryonic stem cells

In embryonic stem cells (ESCs), Wnt-responsive development-related genes are silenced to maintain pluripotency and their expression is activated during differentiation. Acetylation of histones by histone acetyltransferases stimulates transcription, whereas deacetylation of histones by HDACs is correlated with transcriptional repression. Although Wnt-mediated gene transcription has been intimately linked to the acetylation or deacetylation of histones, how Wnt signaling regulates this type of histone modification is poorly understood. Here, we report that Smek, a regulatory subunit of protein phosphatase 4 (PP4) complex, plays an important role in histone deacetylation and silencing of the Wnt-responsive gene, brachyury, in ESCs. Smek mediates recruitment of PP4c and HDAC1 to the Tcf/Lef binding site of the brachyury promoter and inhibits brachyury expression in ESCs. Activation of Wnt signaling during differentiation causes disruption of the Smek/PP4c/HDAC1 complex, resulting in an increase in histones H3 and H4 acetylation at the brachyury gene locus. These results suggest that the Smek-containing PP4 complex represses transcription of Wnt-responsive development-related genes through histone deacetylation, and that this complex is essential for ESC pluripotency maintenance.

Expression of Wnt and MMP in epithelial cells during corneal wound healing

Purpose: To study the expression of Wnt during corneal wound healing and to understand the signaling mechanism involved. Methods: Rat cornea was demarcated on the central area by 4-mm trepine, and the epithelium within this area was removed by scalpel. The epithelium was scraped and isolated to extract RNA. To determine the proliferation of corneal epithelial cells, corneoscleral rims from human donors were treated with dispase II for 15 minutes, and epithelial cells were isolated. Cells were plated on a 3T3 feeder cells layer. The proliferation of corneal epithelial cells was evaluated by colony-forming efficiency. Results: Wnt 5b and 7a were rapidly induced in wounded cornea, and Wnt 7a promoted proliferation of corneal epithelial cells. In addition, matrix metalloproteinase (MMP)-12 was expressed in wounded rat corneal epithelium. Transcription of MMP-12 was responsive to Wnt/β-catenin signaling. Function blockade of MMP-12 delayed Wnt 7a-induced cell proliferation. Conclusion: These results indicate that Wnt proteins and MMP-12 regulate the proliferation of corneal epithelial cells and that Wnt signaling contributes to the resurfacing of defective areas during corneal wound healing.

Protein Phosphatase 4 and Smek Complex Negatively Regulate Par3 and Promote Neuronal Differentiation of Neural Stem/Progenitor Cells

Neural progenitor cells (NPCs) are multipotent cells that can self-renew and differentiate into neurons and glial cells. However, mechanisms that control their fate decisions are poorly understood. Here, we show that Smek1, a regulatory subunit of the serine/threonine protein phosphatase PP4, promotes neuronal differentiation and suppresses the proliferative capacity of NPCs. We identify the cell polarity protein Par3, a negative regulator of neuronal differentiation, as a Smek1 substrate and demonstrate that Smek1 suppresses its activity. We also show that Smek1, which is predominantly nuclear in NPCs, is excluded from the nucleus during mitosis, allowing it to interact with cortical/cytoplasmic Par3 and mediate its dephosphorylation by the catalytic subunit PP4c. These results identify the PP4/Smek1 complex as a key regulator of neurogenesis.

Over-expression of translationally controlled tumor protein in lens epithelial cells seems to be associated with cataract development

Inhibition of Na,K-ATPase causes opacification of the lens through abnormal increases in sodium and calcium levels, disturbed osmotic equilibrium, activation of proteolytic enzymes and cell damage. We previously identified Translationally Controlled Tumor Protein (TCTP) as a cytoplasmic repressor of Na,K-ATPase and confirmed that systemic hypertension is induced in transgenic mice over-expressing TCTP through inhibition of vascular Na,K-ATPase and increased intracellular calcium mobilization. In the current study, we confirmed the role of TCTP in causing intracellular calcium mobilization by inhibiting Na,K-ATPase in a human lens epithelial cell line and further showed that some of the TCTP-transgenic mice develop cataracts with an incidence rate of 7.38% compared to 1.47% in controls. We demonstrated that TCTP acts as a cataractogenic factor through the repression of Na,K-ATPase activity and calcium mobilization in lens epithelial cells.