No abstract

Epithelial-mesenchymal transition (EMT

TAK1 (transforming growth factor-␤-activated kinase-1), a MAP3K with considerable sequence similarity to Raf-1 and MEKK-1, has been identified as a transforming growth factor-␤/bone morphogenetic protein (BMP)-activated cytosolic component of the MAPK pathways. In this investigation, the molecular interactions between TAK1 and Smad proteins were characterized as well as their influence on BMP-mediated mesenchymal cell differentiation along the osteogenic/chondrogenic pathway. In co-immunoprecipitations we found an interaction of TAK1 with all Smads tested, R-Smads Smads1-5, the co-Smad Smad4, and the inhibitory Smads (I-Smad6 and I-Smad7). Smad interaction with TAK1 takes place through their MH2 domain. This interaction is dependent on the presence of an active kinase domain in TAK1. TAK1 dramatically interferes with RSmad transactivation in reporter assays and affects subcellular distribution of Smad proteins. Activated TAK1 also interferes with BMP-dependent osteogenic development in murine mesenchymal progenitor cells (C3H10T 1 ⁄2). A potential TAK1-mediated apoptosis process could be excluded for these cells. Both synergistic and interfering influences of TAK1 on BMP-mediated Smad-signaling have been reported previously. We suggest that TAK1 is a factor that is involved in the finetuning of BMP effects during osteogenic development.

simvastatin, as an intervention therapy, reverses the pulmonary vascular effects of cigarette smoke, including pulmonary hypertension, and prevents smoke-induced emphysema, but does not prevent small airway remodeling. This is the first demonstration that an intervention can reverse a COPD-associated cigarette smoke-induced anatomic abnormality. The study also shows the importance of examining all three anatomic lung compartments when assessing the effects of a potential drug intervention in patients with COPD.

Smad2 and Smad3 are intracellular mediators of transforming growth factor β (TGFβ) signaling that share various biochemical properties, but data emerging from functional analyses in several cell types indicate that these two Smad proteins may convey distinct cellular responses. Therefore, we have investigated the individual roles of Smad2 and Smad3 in mediating the cytostatic and proapoptotic effects of TGFβ as well as their function in epithelial-to-mesenchymal transition. For this purpose, we transiently depleted mouse mammary epithelial cells (Nme) of Smad2 and/or Smad3 mainly by a strategy relying on RNaseH-induced degradation of mRNA. The effect of such depletion on hallmark events of TGFβ-driven epithelial-to-mesenchymal transition was analyzed, including dissolution of epithelial junctions, formation of stress fibers and focal adhesions, activation of metalloproteinases, and transcriptional regulation of acknowledged target genes. Furthermore, we investigated the effect of Smad2 and Smad3 knockdown on the TGFβ-regulated transcriptome by microarray analysis. Our results identify Smad3 as a key factor to trigger TGFβ-regulated events and ascribe tumor suppressor as well as oncogenic activities to this protein. (Mol Cancer Res 2009;7(8):1342-53)

Smad3 is an essential component in the intracellular signaling of transforming growth factor-b (TGFb), which is a potent inhibitor of tumor cell proliferation. BRCA2 is a tumor suppressor involved in early onset of breast, ovarian and prostate cancer. Both Smad3 and BRCA2 possess transcription activation domains. Here, we show that Smad3 and BRCA2 interact functionally and physically. We found that BRCA2 forms a complex with Smad3 in vitro and in vivo, and that both MH1 and MH2 domains of Smad3 contribute to the interaction. TGFb1 stimulates interaction of endogenous Smad3 and BRCA2 in non-transfected cells. BRCA2 co-activates Smad3-dependent transcriptional activation of luciferase reporter and expression of plasminogen activator inhibitor-1 (PAI-1). Smad3 increases the transcriptional activity of BRCA2 fused to the DNA-binding domain (DBD) of Gal4, and reciprocally, BRCA2 co-activates DBD-Gal4-Smad3. Thus, our results show that BRCA2 and Smad3 form a complex and synergize in regulation of transcription.

Cigarette smoke-induced lung disease presents a morphologic contradiction in that the small airways become fibrotic but the parenchyma becomes emphysematous over time. To examine the mechanisms behind these phenomena, we exposed mice to cigarette smoke for up to 6 months and isolated small airways from histologic sections by laser capture microdissection. We then removed residual airway tissue and vessels, and collected the remaining parenchymal tissue. Gene expression of 13 fibrogenic growth/signaling factors (particularly TGF-beta-related genes), matrix proteins, or enzymes involved in matrix production was examined by real-time RT-PCR. Combining present and previously published data from our laboratory, in the airways over the long term there was a sustained and marked increase in expression of almost all of these genes. By contrast, in the parenchyma, expression of most genes was elevated at 2 and 24 hours after initial exposure, and all were elevated at 1 month; but by 6 months, when emphysema was present, most genes (9/13) were either at control values or down-regulated below control. At 3 months, several genes that were considerably elevated at 1 month were back to control levels, suggesting that loss of the parenchymal response precedes the development of emphysema. We conclude that with smoke exposure the airways demonstrate an ongoing profibrotic/proelastogenic response and the parenchyma a generally anti-fibrotic/anti-elastogenic response, but one that develops only with long-term exposure to smoke. These observations support the idea that the parenchyma largely fails to repair smoke-induced matrix damage, but this phenomenon is a relatively late event.

Chronic obstructive lung disease (COPD) is characterized by matrix deposition in the small airways but matrix loss from the parenchyma, phenomena which must depend on the ability of local fibroblasts to produce matrix after smoke exposure. To investigate this idea, we exposed C57Bl/6 mice once to cigarette smoke or to air (control) and prepared primary cultures of lung fibroblasts by microdissecting large airways (trachea, LAF), medium size airways (major bronchi, MAF) and parenchyma (PF). Control PF showed the lowest rate of wound closure and wound closure was depressed in all lines by a single in vivo smoke exposure. Gene expression of matrix proteins differed considerably among the sites; decorin, which may sequester TGFβ, was markedly higher in PF. PF showed higher intrinsic ratios of pSmad2/Smad2. Smoke caused much greater increases in secreted and matrix deposited collagens 1 and 3 in PF than in LAF or MAF. Expression of Thy-1, a gene that suppresses myofibroblast differentiation, was increased by smoke in PF. We conclude that there is considerable regional heterogeneity in murine lung fibroblasts in terms of matrix production, either basally or after in vivo smoke exposure; that PF have lower ability to repair wounds and higher intrinsic TGFβ signaling; and that a single exposure to smoke produces lasting changes in the pattern of matrix production and wound repair, changes that may be mediated in part by smoke-induced release of TGFβ. However, PF still retain the ability to repair by producing new matrix after a single in vivo smoke exposure.