Smad family members are newly identified essential intracellular signalling components of the transforming growth factor‐β (TGF‐β) superfamily. Smad2 and Smad3 are structurally highly similar and mediate TGF‐β signals. Smad4 is distantly related to Smads 2 and 3, and forms a heteromeric complex with Smad2 after TGF‐β or activin stimulation. Here we show that Smad2 and Smad3 interacted with the kinase‐deficient TGF‐β type I receptor (TβR)‐I after it was phosphorylated by TβR‐II kinase. TGF‐β1 induced phosphorylation of Smad2 and Smad3 in Mv1Lu mink lung epithelial cells. Smad4 was found to be constitutively phosphorylated in Mv1Lu cells, the phosphorylation level remaining unchanged upon TGF‐β1 stimulation. Similar results were obtained using HSC4 cells, which are also growth‐inhibited by TGF‐β. Smads 2 and 3 interacted with Smad4 after TβR activation in transfected COS cells. In addition, we observed TβR‐activation‐dependent interaction between Smad2 and Smad3. Smads 2, 3 and 4 accumulated in the nucleus upon TGF‐β1 treatment in Mv1Lu cells, and showed a synergistic effect in a transcriptional reporter assay using the TGF‐β‐inducible plasminogen activator inhibitor‐1 promoter. Dominant‐negative Smad3 inhibited the transcriptional synergistic response by Smad2 and Smad4. These data suggest that TGF‐β induces heteromeric complexes of Smads 2, 3 and 4, and their concomitant translocation to the nucleus, which is required for efficient TGF‐β signal transduction.

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Members of the transforming growth factor-␤ (TGF-␤

Mouse embryonic stem-derived cells were recently shown to differentiate into endothelial and smooth muscle cells. In the present study, we investigated whether human umbilical vein endothelium-derived cells retain the potential to differentiate into smooth muscle cells. Examination of biochemical markers, including basic calponin, SM22␣, prostaglandin E synthase, von Willebrand factor, and PECAM-1, as well as cell contractility, showed that whereas endothelium-derived cells cultured with fibroblast growth factor can be characterized as endothelial cells, when deprived of fibroblast growth factor, a significant fraction differentiates into smooth muscle-like cells. Reapplication of fibroblast growth factor reversed this differentiation. Activin A was up-regulated in fibroblast growth factor-deprived, endothelium-derived cells; moreover, the inhibitory effects of exogenous follistatin and overexpressed Smad7 on smooth muscle-like differentiation confirmed that the differentiation was driven by activin A signaling. These findings indicate that when deprived of fibroblast growth factor, human umbilical vein endotheliumderived cells are capable of differentiating into smooth muscle-like cells through activin A-induced, Smad-dependent signaling, and that maintenance of the endothelial cell phenotype and differentiation into smooth muscle-like cells are reciprocally controlled by fibroblast growth factor-1 and activin A.

Internleukin-1 (IL-1) and IL-6 are the most potent proinflammatory cytokines being involved in inflammatory diseases such as periodontitis. The objective of this study was to examine the synergistic effects of IL-1β and IL-6 on gingival inflammation by targeting cultured human gingival fibroblasts (HGFs). HGFs were treated with IL-1β or IL-6/soluble IL-6R (sIL-6R), and total RNA and total cell lysate were collected to examine expression of gp130 known as a signal transducer of IL-6 using qRT-PCR and Western blotting. IL-1β-mediated IL-6 productivity in HGFs was examined using ELISA method. Likewise, after HGFs and THP-1 macrophages were treated with IL-1β, TNF-α and IL-6, sIL-6R productivity was examined. Next, HGFs were treated with IL-6/ sIL-6R after pretreatment of IL-1β, and the intracellular signals were examined using Western blotting. Finally, various mRNA/protein expressions in HGFs treated with IL-6/sIL-6R after pretreatment of IL-1β were examined using qRT-PCR and ELISA method. IL-1β increased significantly both gp130 and IL-6 expression in HGFs. IL-6 increased significantly sIL-6R production in THP-1 macrophages but not HGFs. Co-stimulation with IL-1β and IL-6/sIL-6R induced dramatically the phosphorylation of Stat3, ERK and JNK in HGFs. Interestingly, expression of various inflammation-related molecules such as MMP-1, MCP-1, IL-1ra, bFGF and VEGF were enhanced by co-stimulation with IL-1β and IL-6/sIL-6R in HGFs. Gingival inflammation is regulated by HGFs affected by both IL-1β and IL-6/sIL-6R synergistically through induction of gp130 expression, resulting in progression of periodontitis.