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.     

Pro-metastasis function of TGFbeta mediated by the Smad pathway

The transforming growth factor beta (TGFbeta) signaling pathway plays a vital role in the development and homeostasis of normal tissues. Abnormal function of this pathway contributes to the initiation and progression of cancer. Smad proteins are key signal transducers of the TGFbeta pathway and are essential for the growth suppression function of TGFbeta. Smads are bona fide tumor suppressors whose mutation, deletion, and silencing are associated with many types of human cancer. However, the involvement and functional mechanism of Smad proteins in cancer metastasis are poorly defined. Recent studies using genetically modified cancer cells and mouse tumor models have provided concrete evidence for a Smad-dependent mechanism for metastasis promotion by TGFbeta. Understanding the dual roles of Smad proteins in tumor initiation and progression has important implications for cancer therapeutics.     

Phospho-specific Smad3 signaling: Impact on breast oncogenesis

Members of the TGFβ superfamily are known to exert a myriad of physiologic and pathologic growth controlling influences on mammary development and oncogenesis. In epithelial cells, TGFβ signaling inhibits cell growth through cytostatic and pro-apoptotic activities but can also induce cancer cell EMT and, thus, has a dichotomous role in breast cancer biology. Mechanisms governing this switch are the subject of active investigation. Smad3 is a critical intracellular mediator of TGFβ signaling regulated through phosphorylation by the TGFβ receptor complex at the C terminus. Smad3 is also a substrate for several other kinases that phosphorylate additional sites within the Smad protein. This discovery has expanded the understanding of the significance and complexity of TGFβ signaling through Smads. This review highlights recent advances revealing the critical role of phospho-specific Smad3 in malignancy and illustrates the potential prognostic and therapeutic impact of Smad3 phospho-isoforms in breast cancer.     

TGFβ‐stimulated Smad1/5 phosphorylation requires the ALK5 L45 loop and mediates the pro‐migratory TGFβ switch

During the course of breast cancer progression, normally dormant tumour‐promoting effects of transforming growth factor β (TGFβ), including migration, invasion, and metastasis are unmasked. In an effort to identify mechanisms that regulate the pro‐migratory TGFβ ‘switch’ in mammary epithelial cells in vitro, we found that TGFβ stimulates the phosphorylation of Smad1 and Smad5, which are typically associated with bone morphogenetic protein signalling. Mechanistically, this phosphorylation event requires the kinase activity and, unexpectedly, the L45 loop motif of the type I TGFβ receptor, ALK5, as evidenced by studies using short hairpin RNA‐resistant ALK5 mutants in ALK5‐depleted cells and in vitro kinase assays. Functionally, Smad1/5 co‐depletion studies demonstrate that this phosphorylation event is essential to the initiation and promotion of TGFβ‐stimulated migration. Moreover, this phosphorylation event is preferentially detected in permissive environments such as those created by tumorigenic cells or oncogene activation. Taken together, our data provide evidence that TGFβ‐stimulated Smad1/5 phosphorylation, which occurs through a non‐canonical mechanism that challenges the notion of selective Smad phosphorylation by ALK5, mediates the pro‐migratory TGFβ switch in mammary epithelial cells.     

TGFbeta1 and TGFbeta3 are partially redundant effectors in brain vascular morphogenesis

Gene deletion experiments have shown that the three TGFβ isoforms regulate distinct developmental processes. Recent work by our group and others showed that the integrins αvβ6 and αvβ8 activate latent forms of TGFβ1 and TGFβ3. This raises the possibility that TGFβ1 and TGFβ3 act redundantly in developmental processes where both isoforms are expressed and activation is by integrins. To investigate this issue, we generated mice with defective integrin-mediated TGFβ1 activation (Tgfb1^RGE/RGE) that were also homozygous for a null mutation in the TGFβ3 gene. Tgfb1^RGE/RGE; Tgfb3^−/− mice have severely perturbed development of the brain vasculature that is highly similar to that in mice lacking αvβ8. Some Tgfb1^RGE/RGE; Tgfb3^+/− and Tgfb1^RGE/RGE; Tgfb3^+/+ mice have milder, background-dependent versions of the phenotype. In addition, we found that Tgfb3 gene status influences embryonic lethality due to TGFβ1 deficiency after limited backcrossing to the BALB/c background. Conversely, Tgfb1 gene status modifies the extent of palate fusion in Tgfb3^−/− mice after limited backcrossing to the ICR background. Our results are consistent with a functional connection between TGFβ1 and TGFβ3 during development based on a shared mechanism of activation.     

Creatine promotes cancer metastasis through activation of Smad2/3

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