A tale of two proteins: differential roles and regulation of Smad2 and Smad3 in TGF-beta signaling

Transforming growth factor-beta (TGF-beta) is an important growth inhibitor of epithelial cells, and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. Smad2 and Smad3 are direct mediators of TGF-beta signaling, however little is known about the selective activation of Smad2 versus Smad3. The Smad2 and Smad3 knockout mouse phenotypes and studies comparing Smad2 and Smad3 activation of TGF-beta target genes, suggest that Smad2 and Smad3 have distinct roles in TGF-beta signaling. The observation that TGF-beta inhibits proliferation of Smad3-null mammary gland epithelial cells, whereas Smad3 deficient fibroblasts are only partially growth inhibited, suggests that Smad3 has a different role in epithelial cells and fibroblasts. Herein, the current understanding of Smad2 and Smad3-mediated TGF-beta signaling and their relative roles are discussed, in addition to potential mechanisms for the selective activation of Smad2 versus Smad3. Since alterations in the TGF-beta signaling pathway play an important role in promoting tumorigenesis and cancer progression, methods for therapeutic targeting of the TGF-beta signaling pathway are being pursued. Determining how Smad2 or Smad3 differentially regulate the TGF-beta response may translate into developing more effective strategies for cancer therapy.     

Mitochondrial localization of Smad5 in a human chondrogenic cell line

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily and regulate the formation of cartilage and bone tissues as well as other key events during development. TGF-beta superfamily signaling is mediated intracellularly by Smad proteins, some of which can translocate into the cell nucleus and influence gene expression. Although much progress has been made in understanding how TGF-beta superfamily signaling regulates expression of target genes, little formal proof has been presented regarding the intracellular distribution of the Smad proteins before their entry into the nucleus. In the literature, non-nuclear Smad proteins are generally referred to as cytoplasmic. Using confocal microscopy, we here show for the first time that immunofluorescent labeling of Smad5, one of the Smad proteins associated with BMP signaling, colocalizes with the mitochondrion-specific probe MitoTracker, demonstrating a mitochondrial distribution of Smad5 in non-stimulated chondroprogenitor cells.     

Dynamic control of TGF-beta signaling and its links to the cytoskeleton

Transforming growth factor beta (TGF-beta) regulates cellular behavior in embryonic and adult tissues. TGF-beta binding to serine/threonine kinase receptors on the plasma membrane activates Smad molecules and additional signaling proteins that coordinately regulate gene expression or cytoplasmic processes such as cytoskeletal dynamics. In turn, the activity and duration of the Smad pathway seems to be regulated by cytoskeletal components, which facilitate the shuttling process that segregates Smad proteins in the cytoplasm and nucleus. We discuss mechanisms and models that aim at explaining the coordination between several components of the signaling network downstream of the TGF-beta signal.     

Axin is a scaffold protein in TGF-beta signaling that promotes degradation of Smad7 by Arkadia

TGF-beta signaling involves a wide array of signaling molecules and multiple controlling events. Scaffold proteins create a functional proximity of signaling molecules and control the specificity of signal transduction. While many components involved in the TGF-beta pathway have been elucidated, little is known about how those components are coordinated by scaffold proteins. Here, we show that Axin activates TGF-beta signaling by forming a multimeric complex consisting of Smad7 and ubiquitin E3 ligase Arkadia. Axin depends on Arkadia to facilitate TGF-beta signaling, as their small interfering RNAs reciprocally abolished the stimulatory effect on TGF-beta signaling. Specific knockdown of Axin or Arkadia revealed that Axin and Arkadia cooperate with each other in promoting Smad7 ubiquitination. Pulse-chase experiments further illustrated that Axin significantly decreased the half-life of Smad7. Axin also induces nuclear export of Smad7. Interestingly, Axin associates with Arkadia and Smad7 independently of TGF-beta signal, in contrast to its transient association with inactive Smad3. However, coexpression of Wnt-1 reduced Smad7 ubiquitination by downregulating Axin levels, underscoring the importance of Axin as an intrinsic regulator in TGF-beta signaling.     

Endoglin differentially modulates antagonistic transforming growth factor-beta1 and BMP-7 signaling

Transforming growth factor-beta1 (TGF-beta1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-beta type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-beta/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-beta1 signaling and the role of endoglin for both pathways in endoglin-deficient L(6)E(9) cells. In this myoblastic cell line, TGF-beta1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-beta1) are two examples. TGF-beta1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-beta1-induced collagen I expression and (CAGA)(12)-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-beta1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway.     

Hypoxia inhibition of adipocytogenesis in human bone marrow stromal cells requires transforming growth factor-beta/Smad3 signaling

Although hypoxia and transforming growth factor-beta (TGF-beta) inhibit differentiation of adipocytes from preadipocytes and bone marrow-derived cells in several species, the relationship between hypoxia and TGF-beta signaling in adipocytogenesis is unknown. In this study, we evaluated the mechanisms of inhibition of adipocyte differentiation by hypoxia and TGF-beta in human and murine marrow stromal cells (MSCs) and the role of TGF-beta/Smad signaling in the inhibition of adipocytogenesis by hypoxia. Both hypoxia-mimetic deferoxamine mesylate (DFO) and TGF-beta1 inhibited adipocyte differentiation (1.0% versus the control at 15 microm DFO and 1.4% versus the control at 1 ng/ml TGF-beta1) and adipocyte gene expression (peroxisome proliferator-activated receptor-gamma2 and lipoprotein lipase) in human MSCs after 21 days of treatment. Hypoxia (2% O(2)) and DFO (but not TGF-beta1) increased hypoxia-inducible factor-1alpha as shown by Western blotting. Macroarrays and Western and Northern blot analyses showed that hypoxia activated the TGF-beta/Smad signaling pathway and that both hypoxia and TGF-beta1 modulated adipocyte differentiation pathways such as the insulin-, peroxisome proliferator-activated receptor-gamma-, phosphatidylinositol 3-kinase-, and MAPK-associated signaling pathways. Studies with mouse marrow stromal cell lines derived from Smad3(+/+) or Smad3(-/-) mice revealed that the TGF-beta type I receptor (ALK-5) and its intracellular signaling molecule Smad3 were necessary for the inhibition of adipocyte differentiation by both TGF-beta and hypoxia-mimetic DFO. Thus, the TGF-beta/Smad signaling pathway is required for hypoxia-mediated inhibition of adipocyte differentiation in MSCs.     

Smad7 antagonizes transforming growth factor beta signaling in the nucleus by interfering with functional Smad-DNA complex formation

Smad7 plays an essential role in the negative-feedback regulation of transforming growth factor beta (TGF-beta) signaling by inhibiting TGF-beta signaling at the receptor level. It can interfere with binding to type I receptors and thus activation of receptor-regulated Smads or recruit the E3 ubiquitin ligase Smurf to receptors and thus target them for degradation. Here, we report that Smad7 is predominantly localized in the nucleus of Hep3B cells. The targeted expression of Smad7 in the nucleus conferred superior inhibitory activity on TGF-beta signaling, as determined by reporter assay in mammalian cells and by its effect on zebrafish embryogenesis. Furthermore, Smad7 repressed Smad3/4-, Smad2/4-, and Smad1/4-enhanced reporter gene expression, indicating that Smad7 can function independently of type I receptors. An oligonucleotide precipitation assay revealed that Smad7 can specifically bind to the Smad-responsive element via its MH2 domain, and DNA-binding activity was further confirmed in vivo with the promoter of PAI-1, a TGF-beta target gene, by chromatin immunoprecipitation. Finally, we provide evidence that Smad7 disrupts the formation of the TGF-beta-induced functional Smad-DNA complex. Our findings suggest that Smad7 inhibits TGF-beta signaling in the nucleus by a novel mechanism.