-
[show abstract]
[hide abstract]
ABSTRACT: Epithelial to mesenchymal transition (EMT) and pulmonary fibrogenesis require epithelial integrin α3β1-mediated cross-talk
between TGFβ1 and Wnt signaling pathways. One hallmark of this cross-talk is pY654-β-catenin accumulation, but whether pY654-β-catenin
is a biomarker of fibrogenesis or functionally important is unknown. To clarify further the role of β-catenin in fibrosis,
we explored pY654-β-catenin generation and function. α3β1 was required for TGFβ1-mediated activation of Src family kinases,
and Src inhibition blocked both pY654 and EMT in primary alveolar epithelial cells (AECs). TGFβ1 stimulated β-catenin/Lef1-dependent
promoter activity comparably in immortalized AECs stably expressing WT β-catenin as well as Y654E or Y654F β-catenin point
mutants. But EMT was abrogated in the Tyr to Phe mutant. pY654-β-catenin was sensitive to the axin β-catenin turnover pathway
as inhibition of tankyrase 1 led to high AEC axin levels, loss of pY654-β-catenin, and inhibition of EMT ex vivo. Mice given a tankyrase inhibitor (50 mg/kg orally) daily for 7 days beginning 10 days after intratracheal bleomycin had
improved survival over controls. Treated mice developed raised axin levels in the lung that abrogated pY654-β-catenin and
attenuated lung Snail1, Twist1, α-smooth muscle actin, and type I collagen accumulation. Total β-catenin levels were unaltered.
These findings identify Src kinase(s) as a mediator of TGFβ1-induced pY654-β-catenin, provide evidence that pY654-β-catenin
levels are a critical determinant of EMT and fibrogenesis, and suggest regulation of axin levels as a novel therapeutic approach
to fibrotic disorders.
Journal of Biological Chemistry 02/2012; 287(7):5164-5172. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Epithelial to mesenchymal transition (EMT) and pulmonary fibrogenesis require epithelial integrin α3β1-mediated cross-talk between TGFβ1 and Wnt signaling pathways. One hallmark of this cross-talk is pY654-β-catenin accumulation, but whether pY654-β-catenin is a biomarker of fibrogenesis or functionally important is unknown. To clarify further the role of β-catenin in fibrosis, we explored pY654-β-catenin generation and function. α3β1 was required for TGFβ1-mediated activation of Src family kinases, and Src inhibition blocked both pY654 and EMT in primary alveolar epithelial cells (AECs). TGFβ1 stimulated β-catenin/Lef1-dependent promoter activity comparably in immortalized AECs stably expressing WT β-catenin as well as Y654E or Y654F β-catenin point mutants. But EMT was abrogated in the Tyr to Phe mutant. pY654-β-catenin was sensitive to the axin β-catenin turnover pathway as inhibition of tankyrase 1 led to high AEC axin levels, loss of pY654-β-catenin, and inhibition of EMT ex vivo. Mice given a tankyrase inhibitor (50 mg/kg orally) daily for 7 days beginning 10 days after intratracheal bleomycin had improved survival over controls. Treated mice developed raised axin levels in the lung that abrogated pY654-β-catenin and attenuated lung Snail1, Twist1, α-smooth muscle actin, and type I collagen accumulation. Total β-catenin levels were unaltered. These findings identify Src kinase(s) as a mediator of TGFβ1-induced pY654-β-catenin, provide evidence that pY654-β-catenin levels are a critical determinant of EMT and fibrogenesis, and suggest regulation of axin levels as a novel therapeutic approach to fibrotic disorders.
Journal of Biological Chemistry 12/2011; 287(7):5164-72. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Axin, a key modulator of the Wnt/beta-catenin pathway, acts as a scaffold protein in phosphorylating and degrading cytoplasmic beta-catenin. Canonical Wnt proteins appear to stabilize beta-catenin by inducing the interaction of LRP5/6 with Axin. This interaction requires the phosphorylation of the Ser or Thr residues in the PPPP(S/T)PX(T/S) motifs at the intracellular domain of LRP5/6. In this work, we identified a novel Axin-interacting protein, zinc-finger BED domain-containing 3 (Zbed3), by yeast two-hybrid screening. The interaction was confirmed in co-immunoprecipitation experiment in mammalian cells and in vitro pulldown assays. Moreover, we found Zbed3 also contains a PPPPSPT motif, which is crucial to its binding to Axin. The Ser and Thr residues in the motif appear to be also phosphorylated by glycogen synthase kinase 3beta (GSK3beta) and the CKI family kinases, as GSK3beta and CKIepsilon could enhance the interaction of Zbed3 with Axin. Mutation of the Ser (SA) or Thr (TA) residue to Ala in the motif markedly impaired its ability to interact with Axin. Expressing Zbed3, but not these mutants, led to inhibition of GSK3beta-mediated beta-catenin phosphorylation, cytoplasmic beta-catenin accumulation, and activation of lymphoid enhancer binding factor-1-dependent reporter gene transcription. Furthermore, knockdown of Zbed3 with RNA interference attenuated Wnt-induced beta-catenin accumulation, lymphoid enhancer binding factor-1-dependent luciferase reporter activity, and the Wnt target gene expression. These results together indicate that Zbed3 is a novel Axin-binding protein that is involved in Wnt/beta-catenin signaling modulation.
Journal of Biological Chemistry 02/2009; 284(11):6683-9. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) are coreceptors for Frizzled and transmit signals from the plasma membrane to the cytosol. However, the mechanism for LRP5/6 signal transmission remains undefined. Here, we identify cytoplasmic activation/proliferation-associated protein 2 (Caprin-2) as a LRP5/6-binding protein. Our data show that Caprin-2 stabilizes cytosolic beta-catenin and enhances lymphoid enhancer-binding factor 1/T cell factor-dependent reporter gene activity as well as the expression of Wnt target genes in mammalian cells. Morpholino-mediated knockdown of Caprin-2 in zebrafish embryos inhibits Wnt/beta-catenin signaling and results in a dorsalized phenotype. Moreover, Caprin-2 facilitates LRP5/6 phosphorylation by glycogen synthase kinase 3, and thus enhances the interaction between Axin and LRP5/6. Therefore, Caprin-2 promotes activation of the canonical Wnt signaling pathway by regulating LRP5/6 phosphorylation.
The Journal of Cell Biology 10/2008; 182(5):865-72. · 10.26 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In canonical Wnt signaling, Dishevelled (Dvl) is a critical cytoplasmic regulator that releases beta-catenin from degradation. Here, we find that Dvl and c-Jun form a complex with beta-catenin-T-cell factor 4 (TCF-4) on the promoter of Wnt target genes and regulate gene transcription. The complex forms via two interactions of nuclear Dvl with c-Jun and beta-catenin, respectively, both of which bind to TCF. Disrupting the interaction of Dvl with either c-Jun or beta-catenin suppresses canonical Wnt signaling-stimulated transcription, and the reduction of Dvl diminished beta-catenin-TCF-4 association on Wnt target gene promoters in vivo. Expression of a TCF-Dvl fusion protein largely rescued the c-Jun knockdown Wnt signaling deficiency in mammalian cells and zebrafish. Thus, we confirm that c-Jun functions in canonical Wnt signaling and show that c-Jun functions as a scaffold in the beta-catenin-TCFs transcription complex bridging Dvl to TCF. Our results reveal a mechanism by which nuclear Dvl cooperates with c-Jun to regulate gene transcription stimulated by the canonical Wnt signaling pathway.
The Journal of Cell Biology 04/2008; 180(6):1087-100. · 10.26 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Two transgenic mouse strains, in which the expression of human factor IX (hFIX) in the milk were different significantly, were bred, and the foreign gene integration as well as the content of hFIX in the milk were detected by PCR, Southern blot, FISH and ELISA, respectively. The results showed that approximately 50% offsprings were transgenic positive. Foreign gene integrated in mouse chromosomes was intact. The hFIX expression of each mouse in the same strain was different, the content of hFIX in the milk was (43.32 +/- 5.41) microgram/mL in FIX-33 transgenic strain and (1.16 +/- 0.45) microgram/mL in FIX-124 transgenic strain. Meanwhile, the hFIX gene expression between the two strains was different remarkably (P < 0.01). We conclude that the characteristics of inheritance and expression in the founder were able to be transferred to their offsprings stably.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 11/2002; 18(6):758-60.
-
[show abstract]
[hide abstract]
ABSTRACT: Fluorescence in situ hybridization (FISH) was used to detect the integration of hFIX on chromosomes of transgenic mice from F1 to F4 generation in two strains. For transgenic mice, 98%-100% of metaphases and 85%-94% of interphases showed hybridization signal. For negative control mice,100% of metaphases and 95%-96% of interphases showed no hybridization signal. The results demonstrated that FISH developed to detect the integration sites of hFIX was high efficient and specific. The integration sites of the transgenic mice analyzed were both single but different between the two strains. The integration chromosomes can be found in the transgenic mice from F1 to F4 generation and the integration sites were the same as each of the strains,which indicated that the transgene was stably integrated and transmitted to offspring.
Hereditas (Beijing) 06/2002; 24(3):232-6.
