Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/threonine kinase receptors.

Smad proteins are signal transducers for the members of the transforming growth factor-beta (TGF-beta) superfamily. Here we show that, in the absence TGF-beta stimulation, Smads exist as monomers in vivo. Smad2 and Smad3 form homo-oligomers upon phosphorylation by the constitutively active TGF-beta type I receptor, and this oligomerization does not require Smad4. Major portions of Smad4, Smad6 and Smad7 are also present as monomers in vivo. Analysis using a cross-linking reagent suggested that the Smad2 oligomer induced by receptor activation is a trimer. Studies by gel chromatography demonstrated that the Smad2-Smad4 heteromer is not larger than the Smad2 homomer. Moreover, overexpression of Smad4 prevented Smad2 from forming a homo-oligomer. These findings suggest that Smad2 may form a homotrimer, or heterotrimers with Smad4, which are probably composed of two and one, or one and two molecules of Smad2 and Smad4, respectively, depending on the amount of each protein. Gel-mobility shift assay revealed that the Smad3 homomer and Smad3-Smad4 heteromer constitute DNA-binding complexes. Transition of the Smad proteins from monomers to oligomers is thus a critical event in the signal transduction of the TGF-beta superfamily members.     

Transforming growth factor-beta 1 inhibition of macrophage activation is mediated via Smad3

Activated macrophages are critical cellular participants in inflammatory disease states. Transforming growth factor (TGF)-beta1 is a growth factor with pleiotropic effects including inhibition of immune cell activation. Although the pathway of gene activation by TGF-beta1 via Smad proteins has recently been elucidated, suppression of gene expression by TGF-beta1 remains poorly understood. We found that of Smad1-Smad7, Smad3 alone was able to inhibit expression of markers of macrophage activation (inducible nitric-oxide synthase and matrix metalloproteinase-12) following lipopolysaccharide treatment in gene reporter assays. Transient and constitutive overexpression of a dominant negative Smad3 opposed the inhibitory effect of TGF-beta1. Domain swapping experiments suggest that both the Smad MH-1 and MH-2 domains are required for inhibition. Mutation of a critical amino acid residue required for DNA binding in the MH-1 of Smad3 (R74A) resulted in the loss of inhibition. Transient overexpression of p300, an interactor of the Smad MH-2 domain, partially alleviated the inhibition by TGF-beta1/Smad3, suggesting that inhibition of gene expression may be due to increased competition for limiting amounts of this coactivator. Our results have implications for the understanding of gene suppression by TGF-beta1 and for the regulation of activated macrophages by TGF-beta1.     

Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGF-beta-induced fibronectin in mesangial cells

Activation of the transforming growth factor-beta (TGF-beta) system has been implicated in the pathological changes of diabetic nephropathy such as renal hypertrophy and accumulation of extracellular matrix. Streptozotocin-induced diabetic mice were used to examine whether the Smad pathway, which transduces the TGF-beta signal, is activated in the diabetic kidney, employing Southwestern histochemistry with labeled Smad-binding element (SBE) oligonucleotides and immunoblotting of nuclear protein extracts for Smad3. Mouse mesangial cells were used to study the role of Smads in mediating the effects of high glucose and TGF-beta on fibronectin expression, using transient transfections of Smad expression vectors and TGF-beta-responsive reporter assays. By Southwestern histochemistry, the binding of nuclear proteins to labeled SBE increased in both glomeruli and tubules at 1, 3, and 6 weeks of diabetes. Likewise, immunoblotting demonstrated that nuclear accumulation of Smad3 was increased in the kidney of diabetic mice. Both increases were prevented by insulin treatment. In mesangial cells, high glucose potentiated the effect of low-dose TGF-beta1 (0.2ng/ml) on the following TGF-beta-responsive constructs: 3TP-Lux (containing AP-1 sites and PAI-1 promoter), SBE4-Luc (containing four tandem repeats of SBE sequence), and the fibronectin promoter. Additionally, Smad3 overexpression increased fibronectin promoter activity, an effect that was enhanced by high ambient glucose or treatment with TGF-beta1 (2ng/ml). The TGF-beta-stimulated activity of the fibronectin promoter was prevented by transfection with either a dominant-negative Smad3 or the inhibitory Smad7. We conclude that hyperglycemia activates the intrarenal TGF-beta/Smad signaling pathway, which then promotes mesangial matrix gene expression in diabetic nephropathy.     

TGF-beta1-mediated fibroblast-myofibroblast terminal differentiation-the role of Smad proteins

It is now clear that resident myofibroblasts play a central role in the mediation of tissue fibrosis. The aim of the work outlined in this study is to increase our understanding of the mechanisms which drive the phenotypic and functional changes associated with the differentiation process. We have used an in vitro model of transforming growth factor-beta1 (TGF-beta1)-induced pulmonary fibroblast-myofibroblast differentiation to examine the role of the TGF-beta1 Smad protein signaling intermediates, in alterations of fibroblast phenotype and function associated with terminal differentiation. TGF-beta1 induced marked alteration in cell phenotype, such that cells resembled "epithelioid-postmitotic fibroblasts." This was associated with marked reorganization of the actin cytoskeleton and upregulation of alphaSMA gene expression. TGF-beta1 stimulation also induced alphaSMA protein expression with increased incorporation of alphaSMA into stress fibers. Following stimulation with TGF-beta1, subsequent addition of serum-free medium did not reverse TGF-beta1-induced morphological change, suggesting that TGF-beta1 induced a relatively stable alteration in fibroblast cell phenotype. Functionally, these phenotypic changes were associated with induction of type I, type III, and type IV collagen gene expression and an increase in the concentrations of the respective collagens in the cell culture supernatant. The role of Smad proteins in terminal differentiation of fibroblasts was examined by transfection of cells, with expression vectors for the TGFbeta1 receptor-regulated Smads (R-Smads) or the co-Smad, Smad 4. Transfection with Smad2 but not Smad3 resulted in TGF-beta1 independent alteration in fibroblast cell phenotype, up-regulation of alphaSMA mRNA and reorganization of the actin cytoskeleton. Transfection with Smad4 also induced alteration in cell phenotype, although this was not as pronounced as the effect of overexpression of Smad2. Overexpression of the Smad2, Smad3, or Smad4 proteins was associated with increased production of all collagen types. The study suggests that the phenotypic and functional changes associated with TGF-beta1-induced fibroblast terminal differentiation are differentially regulated by Smad proteins.