[Show abstract][Hide abstract] ABSTRACT: Wnt/β-catenin signaling via the β-catenin/TCF complex plays crucial roles in tissue homeostasis. Wnt stimulated β-catenin/TCF complex accumulation in the nucleus regulates cell survival, proliferation, and differentiation through the transcription of target genes. Compared with those in G1, LRP6 receptor activation and cytosolic β-catenin are both up-regulated in G2 cells. However, accumulation of the Wnt pathway negative regulator, AXIN2, also occurs in this phase. Therefore, it is unclear whether Wnt signaling is active in G2 phase cells. Here, we established a bimolecular fluorescence complementation (BiFC) biosensor system for the direct visualization of β-catenin/TCF interaction in living cells. Using the BiFC biosensor and co-immunoprecipitation experiments, we demonstrated the nucleus-localized β-catenin/TCF complex increases during the S and G2 phases, and declines in the next G1 phase. Accordingly, a subset of Wnt target genes was transcribed by the β-catenin/TCF complex during both S and G2 phases. In contrast, transient inhibition of this complex disturbed both cell survival and G2/M progression. Our results suggest that in S-G2 phase cells, Wnt/β-catenin signaling is highly active and functions to ensure cell survival and cell-cycle progression.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is an intracellular degradation process to clear up aggregated proteins or aged and damaged organelles. The Beclin1-Vps34-Atg14L complex is essential for autophagosome formation. However, how the complex formation is regulated is unclear. Here, we show that Dapper1 (Dpr1) acts as a critical regulator of the Beclin1-Vps34-Atg14L complex to promote autophagy. Dpr1 ablation in the central nervous system results in motor coordination defect and accumulation of p62 and ubiquitinated proteins. Dpr1 increases autophagosome formation as indicated by elevated puncta formation of LC3, Atg14L and DFCP1 (Double FYVE-containing protein 1). Conversely, loss of Dpr1 impairs LC3 lipidation and causes p62/SQSTM1 accumulation. Dpr1 directly interacts with Beclin1 and Atg14L and enhances the Beclin1-Vps34 interaction and Vps34 activity. Together, our findings suggest that Dpr1 enhances the Atg14L-Beclin1-Vps34 complex formation to drive autophagy.Cell Research advance online publication 1 July 2014; doi:10.1038/cr.2014.84.
[Show abstract][Hide abstract] ABSTRACT: Development of animal embryos before zygotic genome activation at the midblastula transition (MBT) is essentially supported by egg-derived maternal products. Nodal proteins are crucial signals for mesoderm and endoderm induction after the MBT. It remains unclear which maternal factors activate zygotic expression of nodal genes in the ventrolateral blastodermal margin of the zebrafish blastulas. In this study, we show that loss of maternal Eomesodermin a (Eomesa), a T-box transcription factor, impairs zygotic expression of the nodal genes ndr1 and ndr2 as well as mesodermal and endodermal markers, indicating an involvement in mesendoderm induction. Maternal Eomesa is also required for timely zygotic expression of the transcription factor gene mxtx2, a regulator of nodal gene expression. Eomesa directly binds to the Eomes-binding sites in the promoter or enhancer of ndr1, ndr2, and mxtx2 to activate their transcription. Furthermore, human and mouse Nodal genes are also regulated by Eomes. Transfection of zebrafish eomesa into murine embryonic stem cells promotes mesendodermal differentiation with constant higher levels of endogenous Nodal expression, suggesting a conserved function of Eomes. Taken together, our findings reveal a conserved role of maternal T-box transcription factors in regulating nodal gene expression and mesendoderm induction in vertebrate embryos.
Journal of Molecular Cell Biology 06/2014; · 8.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During the early vertebrate body plan formation, convergent extension (CE) of dorsal mesoderm and neurectoderm is coordinated by the evolutionarily conserved non-canonical Wnt/PCP signaling. Dishevelled (Dvl), a key mediator of Wnt/PCP signaling, is essential for the medial-lateral polarity formation in the cells undergoing convergent extension movements. NEDD4L, a highly conserved HECT type E3 ligase, has been reported to regulate the stability of multiple substrates including Dvl2. Here we demonstrate that NEDD4L is required for the cellular polarity formation and convergent extension in the early Xenopus embryos. Depletion of NEDD4L in early Xenopus embryos results in the loss of mediolateral polarity of the convergent-extending mesoderm cells and the shortened body axis, resembling those defects caused by the disruption of non-canonical Wnt signaling. Depletion of xNEDD4L also blocks the elongation of the animal explants in response to endogenous mesoderm inducing signals and partially compromises the expression of Brachyury. Importantly, reducing Dvl2 expression can largely rescue the cellular polarity and convergent extension defects in NEDD4L-depleted embryos and explants. Together with the data that NEDD4L reduces Dvl2 protein expression in the frog embryos, our findings suggest that regulation of Dvl protein levels by NEDD4L is essential for convergent extension during early Xenopus embryogenesis.
[Show abstract][Hide abstract] ABSTRACT: Transforming growth factor-β (TGF-β) signaling regulates diverse cellular processes, including cell proliferation, differentiation, apoptosis, cell plasticity, and migration. TGF-β signaling can be mediated by Smad proteins or other signaling proteins such as MAP kinases and Akt. TGF-β signaling is tightly regulated at different levels along the pathways to ensure its proper physiological functions in different cells and tissues. Deregulation of TGF-β signaling has been associated with various kinds of diseases, such as cancer and tissue fibrosis. This paper focuses on our recent work on regulation of TGF-β signaling.
[Show abstract][Hide abstract] ABSTRACT: The zinc finger transcription factor Smad-interacting protein-1 (Sip1; Zeb2, Zfhx1b) plays an important role during vertebrate embryogenesis in various tissues and differentiating cell types, and during tumorigenesis. Previous biochemical analysis suggests that interactions with several partner proteins, including TGFβ family receptor-activated Smads, regulate the activities of Sip1 in the nucleus both as a DNA-binding transcriptional repressor and activator. Using a peptide aptamer approach we mapped in Sip1 its Smad-binding domain (SBD), initially defined as a segment of 51 amino acids, to a shorter stretch of 14 amino acids within this SBD. Modelling suggests that this short SBD stretch is part of an extended α-helix that may fit the binding to a hydrophobic corridor within the MH2 domain of activated Smads. Four amino acids (two polar Q residues and two non-polar V residues) that form the tandem repeat (QxVx)2 in this 14-residue stretch were found to be crucial for binding to both TGFβ/Nodal/Activin-Smads and BMP-Smads. A full-length Sip1 with collective mutation of these Q and V residues (to A) no longer binds to Smads, while it retains its binding activity to its cognate bipartite target DNA sequence. This missense mutant Sip1(AxAx)2 provides a new molecular tool to identify SBD (in)dependent target genes in Sip1-controlled TGFβ and/or BMP (de)regulated cellular, developmental and pathological processes.
PLoS ONE 10/2013; 8(10):e76733. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The DNA damage checkpoint is tightly controlled. After its activation, the checkpoint machinery is inactivated once lesions are repaired, or undergoes adaptation if the DNA damage is unable to be repaired. Protein acetylation has been shown to play an important role in DNA damage checkpoint activation. However, the role of acetylation in checkpoint inactivation is unclear. Here we show that the histone deacetylase Rpd3-mediated deacetylation of Rad53 plays an important role in checkpoint adaptation. Deletion of Rpd3 or inhibition of its activity impairs adaptation. RPD3 deletion also leads to a higher acetylation level and enhanced kinase activity of Rad53. Substitution of two major acetylation sites of Rad53 with arginine reduces its activity, and further suppresses the adaptation defect of rpd3Δ cells, indicating that Rpd3 facilitates adaptation by preventing Rad53 over-activation. Similar to its role in adaptation, deletion of RPD3 or inhibiting its activity also suppressed checkpoint recovery. Altogether, our findings reveal an important role of Rpd3 in promoting checkpoint adaptation via deacetylation and inhibition of Rad53.
Molecular and Cellular Biology 08/2013; · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In hematopoietic system, hematopoietic stem cells (HSCs) are characterized by their self-renewal and multilineage differentiation capability. The abnormal behavior of HSCs causes emergence and progression of leukemia and other hematopoietic malignancies. Transforming growth factor β (TGF-β) is a multipotent factor which impacts the diverse fates of HSCs, including proliferation, self-renewal, differentiation, apoptosis, and mobilization from the niche. TGF-β signaling is finely controlled, and its deregulation is associated to leukemia development. In recent years, using genetically modified mice and hematopoietic stem cell transplantation system, the in vivo functions of TGF-β signaling in hematopoietic system have been greatly appreciated. This review summarizes the recent development on the role of TGF-β signaling in HSC fate decision and leukemia development.
[Show abstract][Hide abstract] ABSTRACT: Smad2/3-mediated transforming growth factor β signalling and the Ras-Raf-Mek-Erk cascade have important roles in stem cell and development and tissue homeostasis. However, it remains unknown whether Raf kinases directly crosstalk with Smad2/3 signalling and how this would regulate embryonic development. Here we show that Araf antagonizes mesendoderm induction and patterning activity of Nodal/Smad2 signals in vertebrate embryos by directly inhibiting Smad2 signalling. Knockdown of araf in zebrafish embryos leads to an increase of activated Smad2 with a decrease in linker phosphorylation; consequently, the embryos have excess mesendoderm precursors and are dorsalized. Mechanistically, Araf physically binds to and phosphorylates Smad2 in the linker region with S253 being indispensable in a Mek/Erk-independent manner, thereby attenuating Smad2 signalling by accelerating degradation of activated Smad2. Our findings open avenues for investigating the potential significance of Raf regulation of transforming growth factor β signalling in versatile biological and pathological processes in the future.
[Show abstract][Hide abstract] ABSTRACT: Wnt/β-catenin signaling plays key roles in embryonic development and tissue homeostasis. Dapper3/Dact3, one of the three members of the Dapper gene family, is transcriptionally repressed in colorectal cancer and may function as a negative regulator of Wnt/β-catenin signaling. To investigate its physiological functions, we generated a mouse strain harboring conditional null alleles of Dapper3 (Dapper3flox/flox), and homozygous Dapper3 deficient (Dapper3-/-) mice were produced after crossing with EIIa-cre transgenic mice. We found that Dapper3 is not essential for mouse embryogenesis, postnatal survival and reproduction. However, adult Dapper3-/- mice exhibited a mild reduction in body weight compared with their wild-type littermates, suggesting a functional role of Dapper3 in postnatal growth. To investigate the role of Dapper3 in renal fibrosis, we employed the unilateral ureteral obstruction (UUO) model. Dapper3 mRNA expression was upregulated in kidney after UUO. Loss of the Dapper3 gene enhanced myofibroblast activation and extracellular matrix overproduction in the obstructed kidney. Moreover, this aggravated fibrotic phenotype was accompanied with accumulation of Dishevelled2 and β-catenin proteins and activation of Wnt targeted fibrotic genes. In primary renal tubular cells, Dapper3 inhibits Wnt induced epithelial-to-mesenchymal transition. Consistently, Dapper3 interacted with and downregulated Dishevelled2 protein, and attenuated the Wnt-responsive Topflash reporter expression. These findings together suggest that Dapper3 antagonizes the fibrotic actions of Wnt signaling in kidney.
Journal of Biological Chemistry 04/2013; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BMP4 maintains self-renewal of mouse embryonic stem cells (ESCs) in collaboration with LIF. Here we report the identification of a novel key BMP target gene - cochlin (Coch) in mouse ESCs. Coch can be significantly upregulated by BMP4 specifically in ESCs but not in somatic differentiated cells, and this upregulation is dependent on the BMP signaling mediators Smad1/5 and Smad4. Overexpression of Coch can partially substitute BMP4 to promote self-renewal of mouse ESCs together with LIF, whereas knockdown of Coch impairs self-renewal marker gene expression even in the presence of both BMP4 and LIF. Further studies showed that COCH could mimic BMP4 in repressing neural differentiation of mouse ESCs upon LIF withdrawal and the inhibitory effect of BMP4 on neural differentiation is compromised by Coch knockdown. Taken together, our data suggest that COCH is a part of the downstream target network of BMP signaling and serves as another important effector to fine-tune mouse ESC fates.
Journal of Biological Chemistry 01/2013; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transforming growth factor β (TGF-β) is a potent antiproliferative factor in multiple types of cells. Deregulation of TGF-β signaling is associated with the development of many cancers, including leukemia, though the molecular mechanisms are largely unclear. Here, we show that Casitas B-lineage lymphoma (c-Cbl), a known proto-oncogene encoding an ubiquitin E3 ligase, promotes TGF-β signaling by neddylating and stabilizing the type II receptor (TβRII). Knockout of c-Cbl decreases the TβRII protein level and desensitizes hematopoietic stem or progenitor cells to TGF-β stimulation, while c-Cbl overexpression stabilizes TβRII and sensitizes leukemia cells to TGF-β. c-Cbl conjugates neural precursor cell-expressed, developmentally downregulated 8 (NEDD8), a ubiquitin-like protein, to TβRII at Lys556 and Lys567. Neddylation of TβRII promotes its endocytosis to EEA1-positive early endosomes while preventing its endocytosis to caveolin-positive compartments, therefore inhibiting TβRII ubiquitination and degradation. We have also identified a neddylation-activity-defective c-Cbl mutation from leukemia patients, implying a link between aberrant TβRII neddylation and leukemia development.
[Show abstract][Hide abstract] ABSTRACT: Sustained activation of X-box binding protein 1 (XBP1) results in endothelial cell (EC) apoptosis and atherosclerosis development. The present study provides evidence that XBP1 mRNA splicing triggered an autophagic response in ECs by inducing autophagic vesicle formation and markers of autophagy Beclin 1 and microtubule-associated protein 1 light chain 3 beta (LC3βII). Endostatin activated autophagic gene expression through XBP1 mRNA splicing in an inositol-requiring enzyme 1 alpha (IRE1α)-dependent manner. Knockdown of XBP1 or IRE1α by shRNA in ECs ablated endostatin-induced autophagosome formation. Importantly, data from arterial vessels from XBP1 EC conditional knockout (XBP1eko) mice demonstrated that XBP1 deficiency in ECs reduced the basal level of LC3β expression and ablated response to endostatin. Chromatin immuno-precipitation assays further revealed that the spliced XBP1 isoform bound directly to the Beclin 1 promoter at region -537~ -755nt. Beclin 1 deficiency in ECs abolished the XBP1-induced autophagy response, while spliced XBP1 did not induce transcriptional activation of a truncated Beclin 1 promoter. These results suggest that XBP1 mRNA splicing triggers an autophagic signal pathway through transcriptional regulation of Beclin 1.
Journal of Biological Chemistry 11/2012; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apoptosis is an essential cellular process in multiple diseases and a major pathway for neuronal death in neurodegeneration. The detailed signaling events/pathways leading to apoptosis, especially in neurons, require further elucidation. Here we identify a β-amyloid precursor protein (APP)-interacting protein, designated as appoptosin, whose levels are upregulated in brain samples from Alzheimer's disease and infarct patients, and in rodent stroke models, as well as in neurons treated with β-amyloid (Aβ) and glutamate. We further demonstrate that appoptosin induces reactive oxygen species release and intrinsic caspase-dependent apoptosis. The physiological function of appoptosin is to transport/exchange glycine/5-amino-levulinic acid across the mitochondrial membrane for heme synthesis. Downregulation of appoptosin prevents cell death and caspase activation caused by glutamate or Aβ insults. APP modulates appoptosin-mediated apoptosis through interaction with appoptosin. Our study identifies appoptosin as a crucial player in apoptosis and a novel pro-apoptotic protein involved in neuronal cell death, providing a possible new therapeutic target for neurodegenerative disorders.
Journal of Neuroscience 10/2012; 32(44):15565-15576. · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Embryonic stem (ES) cells, which can self-renew and can differentiate to various cell types, have great potential applications in regenerative medicine. BMP family members are evolutionarily conserved factors that play critical roles in embryogenesis and in adult tissue homeostasis in multicellular organisms, and their malfunction can lead to various human diseases. Consistent with its importance in early embryogenesis, BMP signaling has been established as a key determinant that directs a wide range of cell fate choices in both mouse and human embryonic stem cells, from self-renewal maintenance to multiple differentiation processes. Remarkably, BMPs exert their diverse functions via integrating with signal inputs from other extrinsic signals and intrinsic factors, including transcription factors and epigenetic regulators. Here we summarize the current understanding of BMP signaling in embryonic stem cell fate determination, and discuss the delicate cooperation between BMP signaling and its partners in these processes. The principles learned from these studies would pave a road for the potential medical applications of embryonic stem cells.
Experimental Cell Research 10/2012; · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein that interacts with C kinase 1 (PICK1) is a critical mediator of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking in neural synapses. However, its ubiquitous expression suggests that it may have other non-neural functions. Here we show that PICK1 antagonizes transforming growth factor beta (TGF-β) signaling by targeting TGF-β type I receptor (TβRI) for degradation. Biochemical analyses reveal that PICK1 directly interacts with the C-terminus of TβRI via its PDZ domain and acts as a scaffold protein to enhance the interaction between TβRI and caveolin-1, leading to enhanced lipid raft/caveolae localization. Therefore, PICK1 increases caveolin-mediated endocytosis, ubiquitination and degradation of TβRI. Moreover, a negative correlation between PICK1 expression and TβRI or phospho-Smad2 levels is observed in human breast tumors, indicating that PICK1 may participate in breast cancer development through inhibition of TGF-β signaling. Our findings reveal a non-neural function of PICK1 as an important negative regulator of TGF-β signaling.
Cell Research 06/2012; 22(10):1467-78. · 11.98 Impact Factor