Journal: The Journal of Cell Biology

Article Title: BMP promotes motility and represses growth of smooth muscle cells by activation of tandem Wnt pathways

doi: 10.1083/jcb.201008060

Figure Lengend Snippet: BMP-2 activates βC via pAkt-dependent inhibition of GSK3β and is independent of Smad signaling. (A and B) βC activation in 10 ng/ml BMP-2–stimulated hPASMCs transfected with a nonfunctional pAkt (ΔAkt) in A and a Smad 1 construct (ΔSmad 1) in B was measured by immunoblot analysis and compared with values in cells transfected with vector alone. The pGSK3β activation in A was measured relative to total GSK3β. Bars represent means ± SEM from n = 3. ***, P < 0.0001 versus time 0 in A was determined using one-way ANOVA with Dunnett’s. ***, P < 0.0001 in B were determined using the unpaired t test.

Article Snippet: Phosphorylation of the substrate was detected by Western immunoblotting with anti-GSK3β Ser 21/9 antibody (Cell Signaling Technology; ).

Techniques: Inhibition, Activation Assay, Transfection, Construct, Western Blot, Plasmid Preparation

Journal: The Journal of Cell Biology

Article Title: BMP promotes motility and represses growth of smooth muscle cells by activation of tandem Wnt pathways

doi: 10.1083/jcb.201008060

Figure Lengend Snippet: Activation of integrin-linked kinase 1 (ILK-1) is required for BMP-2–mediated hPASMC motility. (A) ILK-1 kinase assay was performed as described in Materials and methods with GSK3β as the substrate on cells incubated in the presence (right) or absence (left) of 5 µg/ml CS-1. (B and C) Motility in hPASMCs exposed to CS-1 (B) or transfected with either scrambled (SC) or ILK-1–specific siRNA (C) and exposed to 10 ng/ml BMP-2 was measured using the Boyden assay. (D) Levels of active RhoA and Rac1 were measured as described in Materials and methods. Densitometry values are shown relative to total RhoA and Rac1 in whole cell lysates, which were run in different gels. Bars represent means ± SEM from n = 3. In A, *, P < 0.01; and ***, P < 0.0001 were determined using one-way ANOVA with Dunnett’s. In B and C, **, P < 0.001; and ***, P < 0.001 versus baseline; and ## , P < 0.001; and ### , P < 0.0001 versus respective scrambled siRNA control using one-way ANOVA with Bonferroni’s. In D, *, P < 0.01; and ***, P < 0.0001 versus time 0 were determined using an unpaired t test. CON, control.

Article Snippet: Phosphorylation of the substrate was detected by Western immunoblotting with anti-GSK3β Ser 21/9 antibody (Cell Signaling Technology; ).

Techniques: Activation Assay, Kinase Assay, Incubation, Transfection, Boyden Assay, Control

Journal: The Journal of Cell Biology

Article Title: BMP promotes motility and represses growth of smooth muscle cells by activation of tandem Wnt pathways

doi: 10.1083/jcb.201008060

Figure Lengend Snippet: Presence of ΔDEP Dvl promotes VSMC growth response to PDGF-BB by increasing βC levels. (A) Levels of active βC (left) and pGSK3β (middle) in hPASMCs transfected with either WT or ΔDEP Dvl and stimulated with 10 ng/ml BMP-2 were visualized by Western immunoblotting and quantified by densitometry. Levels of active βC were normalized to α-tubulin, and levels of pGSK3β were normalized to total GSK3β in whole cell lysates. Co-IP of pGSK3β and WT and ΔDEP (right) was carried under BMP-2 stimulation. Levels of pGSK3β were normalized relative to total GSK3β in whole cell lysates analyzed in a separate gel. (B) Cell count studies were performed after addition of PDGF-BB, BMP-2, or both for 72 h. (C) Cell count studies were performed in hPASMCs cotransfected with WT or ΔDEP Dvl and either scrambled or two independent βC siRNAs after addition of PDGF-BB, BMP-2, or both for 72 h. **, P < 0.001; and ***, P < 0.0001 as indicated in A were determined by one-way ANOVA with Dunnett’s. Bars represent mean ± SEM from n = 3. *, P < 0.01; **, P < 0.001; and ***, P < 0.0001 versus baseline; # , P < 0.01; and ## , P < 0.001 versus scrambled counterpart as indicated in B and C using one-way ANOVA with Bonferroni’s. CON, control; IP, immunoprecipitation. IB, immunoblot.

Article Snippet: Phosphorylation of the substrate was detected by Western immunoblotting with anti-GSK3β Ser 21/9 antibody (Cell Signaling Technology; ).

Techniques: Transfection, Western Blot, Co-Immunoprecipitation Assay, Cell Counting, Control, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: BMP promotes motility and represses growth of smooth muscle cells by activation of tandem Wnt pathways

doi: 10.1083/jcb.201008060

Figure Lengend Snippet: Proposed models for BMP regulation of hPASMC motility and proliferation by recruitment of Wnt–βC and Wnt–PCP signaling. (A) BMP-2 triggers βC accumulation via pAkt-mediated GSK3β inhibition followed by production and release of FN to the extracellular space (1). By binding to α4β1-integrin, FN activates ILK-1 and induces formation of a complex between ILK-1 and Dvl leading to RhoA and Rac1 activation (2) and simultaneous suppression of βC activation (3). (B) The inability to form or maintain a complex between ILK-1 and Dvl may facilitate hPASMC proliferation in response to growth factors such as PDGF-BB by enhancing βC signaling through ILK-1–mediated GSK3β inhibition. APC, adenomatous polyposis coli.

Article Snippet: Phosphorylation of the substrate was detected by Western immunoblotting with anti-GSK3β Ser 21/9 antibody (Cell Signaling Technology; ).

Techniques: Inhibition, Binding Assay, Activation Assay