Smads transmit signals from transmembrane ser/thr kinase receptors to the nucleus. We now identify SARA (for Smad anchor for receptor activation), a FYVE domain protein that interacts directly with Smad2 and Smad3. SARA functions to recruit Smad2 to the TGFbeta receptor by controlling the subcellular localization of Smad2 and by interacting with the TGFbeta receptor complex. Phosphorylation of Smad2 induces dissociation from SARA with concomitant formation of Smad2/Smad4 complexes and nuclear translocation. Furthermore, mutations in SARA that cause mislocalization of Smad2 inhibit TGFbeta-dependent transcriptional responses, indicating that the regulation of Smad localization is important for TGFbeta signaling. These results thus define SARA as a component of the TGFbeta pathway that brings the Smad substrate to the receptor.

No abstract

Functional involvement of the Notch pathway in osteoblastic differentiation has been previously investigated using the truncated intracellular domain, which mimics Notch signaling by interacting with the DNAbinding protein CBF-1. However, it is unclear whether Notch ligands Delta1 and Jagged1 also induce an identical cellular response in osteoblastic differentiation. We have shown that both Delta1 and Jagged1 were expressed concomitantly with Notch1 in maturating osteoblastic cells during bone regeneration and that overexpressed and immobilized recombinant Delta1 and Jagged1 alone did not alter the differentiated state of MC3T3-E1 and C2C12 cells. However, they augmented bone morphogenetic protein-2 (BMP2)-induced alkaline phosphatase activity and the expression of several differentiation markers, except for osteocalcin, and ultimately enhanced calcified nodule and in vivo ectopic bone formation of MC3T3-E1. In addition, both ligands transmitted signal through the CBF-1-dependent pathway and stimulated the expression of HES-1, a direct target of Notch pathway. To test the necessity of Notch signaling in BMP2-induced differentiation, Notch signaling was inhibited by the dominant negative extracellular domain of Notch1, specific inhibitor, or small interference RNA. These treatments decreased alkaline phosphatase activity as well as the expression of other differentiation markers and inhibited the promoter activity of Id-1, a target gene of the BMP pathway. These results indicate the functional redundancy between Delta1 and Jagged1 in osteoblastic differentiation whereby Delta1/Jagged1-activated Notch1 enhances BMP2-induced differentiation through the identical signaling pathway. Furthermore, our data also suggest that functional Notch signaling is essential not only for BMP2-induced osteoblast differentiation but also for BMP signaling itself.

No abstract

No abstract

Abstract-Cardiac fibrosis results from proliferation of interstitial fibroblasts and concomitant increased biosynthesis of extracellular matrix (ECM) components and is often complicated by cardiac hypertrophy. This study was conducted to investigate whether norepinephrine (NE) potentiates transforming growth factor-␤ (TGF-␤)-induced cardiac fibrosis. The expression of the cardiac ECM proteins, plasminogen activator inhibitor-1 (PAI-1), fibronectin, and collagen type I, was examined by Western blotting using extracts from neonatal rat primary cardiac fibroblasts. In cardiac fibroblasts, treatment with a combination of NE and TGF-␤1 increased cell proliferation and ECM expression. Luciferase assays were conducted to clarify the effect of NE on TGF-␤ signaling. TGF-␤1 (1 ng/mL) increased the specific signaling activity 2-fold, whereas the combination of NE (10 mol/L) and TGF-␤1 (1 ng/mL) resulted in an approximate 10-fold increase in specific signaling activity. We confirmed that treatment with NE markedly enhances TGF-␤-induced phosphorylation of activating transcription factor 2 (ATF-2). These results indicated that NE has a synergistic effect on TGF-␤ signaling. To determine whether this activation by NE was mediated by the TGF-␤1 receptor, we used a dominant negative vector of the TGF-␤1 type II receptor, and the synergistic effects were inhibited. Furthermore, this synergistic effect was attenuated by a specific inhibitor of p38, SB203680. These data indicate that NE enhances cardiac fibrosis through TGF-␤1 post-receptor signaling, predominantly via the p38 MAP kinase pathway.

Human T-cell leukemia virus type I (HTLV-I)Tax is a potent transcriptional regulator that can activate or repress specific cellular genes and that has been proposed to contribute to leukemogenesis in adult T-cell leukemia. Previously, HTLV-Iinfected T-cell clones were found to be resistant to growth inhibition by transforming growth factor (TGF)-␤. Here it is shown that Tax can perturb Smad-dependent TGF-␤ signaling even though no direct interaction of Tax and Smad proteins could be detected. Importantly, a mutant Tax of CREB-binding protein (CBP)/p300 binding site, could not repress the Smad transactivation function, suggesting that the CBP/p300 binding domain of Tax is essential for the suppression of Smad function. Because both Tax and Smad are known to interact with CBP/p300 for the potentiation of their transcriptional activities, the effect of CBP/p300 on suppression of Smad-mediated transactivation by Tax was examined. Overexpression of CBP/p300 reversed Tax IntroductionHuman T-cell leukemia virus type I (HTLV-I) is an etiologic agent of an acute malignancy of CD4 ϩ T lymphocytes called adult T-cell leukemia (ATL). 1,2 The virus-encoded regulatory protein, Tax, is critical for HTLV-I replication and is thought to contribute to ATL development. Several experimental observations indicate that Tax mediates the oncogenic activity of HTLV-I. For example, Tax immortalizes primary human T lymphocytes and transforms rodent fibroblasts in vitro. [3][4][5] In addition, transgenic mice expressing Tax develop mesenchymal tumors or large granular lymphocytic leukemia in vivo. 6,7 The exact mechanism through which Tax exerts its oncogenic potential is still unknown. Tax was originally identified as a transcriptional activator for viral gene expression and then was shown to activate the expression of a number of cellular genes, many of which either encode proteins involved in the regulation of cellular proliferation (ie, interleukin [IL]-2, 8 IL-2 receptor ␣ chain, 8,9 and proliferating cell nuclear antigen), 10 or are protooncogenes (c-fos, 11,12 c-jun, 12 and c-myc). 13 In contrast to its transcriptional activation, Tax can also repress the expression of ␤ polymerase, an enzyme important for DNA repair, and Bax, an accelerator of apoptosis. 14,15 Furthermore, Tax alters the activity of a number of cell cycle regulators, including cyclin D, 16,17 the mitotic checkpoint regulator MAD1, 18 the cyclin-dependent kinases (Cdk) Cdk4 and Cdk6, 19 the Cdk inhibitors p15INK4b, p16INK4a, and p18INK4b, 20-22 the tumor suppressor p53, and the p53-related proteins p73 and p51. [23][24][25][26] Thus, it is likely that Tax dysregulates the cell cycle through many different mechanisms, leading to the eventual immortalization and transformation of the infected cells.Proliferation and differentiation of cells are tightly regulated by a delicate balance of growth factors and growth-inhibitory factors. Transforming growth factor (TGF)-␤ is one of the best-characterized members of growth-inhibitory factors. TGF-␤ can inhibit the growth of...

To investigate the molecular mechanism underlying the differentiation of osteoblasts and chondroblasts, we established a clonal cell lines, RD-C6, from Runx2-deficient mouse embryos. RD-C6 cells expressed almost undetectable levels of phenotypes related to osteoblast and chondroblast differentiation at basal culture condition, whereas treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2) or transduction of BMP-2 by adenovirus effectively induced this cell line to express mRNA related to the differentiation of osteoblasts and chondroblasts including alkaline phosphatase, osteocalcin, and osterix. Transduction of Runx2 also induced the expression of these mRNA in RD-C6 cells. BMP-2 transduction increased expression levels of mRNA for Msx2 and Dlx5, but Runx2 transduction induced no significant increases in expression levels of these mRNA. Microarray analysis using RD-C6 cells with or without rhBMP-2 treatment demonstrated that BMP-2 upregulated 66 genes including 13 transcription-related molecules such as Id1, Id2, Id4, Hey1, Smad6, Smad7, and Msx2. To confirm bone and cartilage formation ability of RD-C6 cells, we transplanted RD-C6 cells into the peritoneal cavity of athymic mice using diffusion chambers with rhBMP-2. RD-C6 cells generated unmineralized cartilage but not bone. These results indicate that BMP-2 induces Runx2-deficient cells to express markers related to osteoblast and chondroblast differentiation using a Runx2-independent pathway, but it failed to induce these cells to differentiate into bone-forming osteoblasts and mature chondrocytes.