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Open Access Publications from the University of California

Scholarly Works (3 results)

  • Article
  • Peer Reviewed
UC San Francisco Previously Published Works (2019)
Functional activation of the transforming growth factor-β (TGF-β) receptors (TGFBRs) is carefully regulated through integration of post-translational modifications, spatial regulation at the cellular level, and TGFBR availability at the cell surface. Although the bulk of TGFBRs resides inside the cells, AKT Ser/Thr kinase (AKT) activation in response to insulin or other growth factors rapidly induces transport of TGFBRs to the cell surface, thereby increasing the cell's responsiveness to TGF-β. We now demonstrate that TGF-β itself induces a rapid translocation of its own receptors to the cell surface and thus amplifies its own response. This mechanism of response amplification, which hitherto has not been reported for other cell-surface receptors, depended on AKT activation and TGF-β type I receptor kinase. In addition to an increase in cell-surface TGFBR levels, TGF-β treatment promoted TGFBR internalization, suggesting an overall amplification of TGFBR cycling. The TGF-β-induced increase in receptor presentation at the cell surface amplified TGF-β-induced SMAD family member (SMAD) activation and gene expression. Furthermore, bone morphogenetic protein 4 (BMP-4), which also induces AKT activation, increased TGFBR levels at the cell surface, leading to enhanced autocrine activation of TGF-β-responsive SMADs and gene expression, providing context for the activation of TGF-β signaling in response to BMP during development. In summary, our results indicate that TGF-β- and BMP-induced activation of low levels of cell surface-associated TGFBRs rapidly mobilizes additional TGFBRs from intracellular stores to the cell surface, increasing the abundance of cell-surface TGFBRs and cells' responsiveness to TGF-β signaling.
    Cover page: Transforming growth factor–β (TGF-β)–induced up-regulation of TGF-β receptors at the cell surface amplifies the TGF-β response
    • Thesis
    • Peer Reviewed
    UC San Francisco Electronic Theses and Dissertations (2017)

    The transforming growth factor (TGF)-beta family of proteins drive normal embryonic development, while dysregulation of TGF-beta signaling contributes to developmental disorders, cancer progression and dissemination, and fibrosis. Signaling by TGF-beta initiates upon ligand binding to transmembrane receptor proteins, which then promote gene expression changes through phosphorylation and activation of intracellular Smad proteins. Functional activation of the TGF-beta receptors is carefully regulated through integration of post-translational modifications, spatial regulation at the cellular level, and receptor availability at the cell surface.

    While the majority of TGF-beta receptors reside intracellularly, they can be rapidly mobilized to the cell surface in response to Akt activation, thereby increasing the cell’s responsiveness to TGF-beta. Because TGF-beta is known to induce phosphorylation of Akt, I investigated whether TGF-beta regulates translocation of its own receptors to the cell surface and thus amplifies its own response. Through selective biotinylation of cell surface proteins, I found that TGF-beta induced a rapid increase of type I and type II TGF-beta receptors at the plasma membrane. This receptor upregulation was inhibited by blocking Akt phosphorylation, or in the presence of TGF-beta type I receptor kinase inhibitors. Furthermore, attenuation of the Akt-mediated increase in cell surface receptor presentation decreased Smad activation and TGF-beta-induced gene expression responses. I also observed that while TGF-beta treatment caused an overall increase in cell surface TGF-beta receptors, it also induced an increased rate of receptor endocytosis, suggesting an overall amplification of TGF-beta receptor cycling. Together, these data outline a novel response amplification mechanism, through a ligand-induced, rapid mobilization of cell surface receptors from intracellular stores.

    Furthermore, BMP-4 caused cells to upregulate TGF-beta receptors, leading to subsequent autocrine activation of TGF-beta-responsive Smads and gene expression. Inhibition of Akt phosphorylation or inhibition of TGF-beta type I receptor kinase attenuated the autocrine TGF-beta signaling activation in response to BMP. The balance between BMP and TGF-beta signaling pathways is essential for proper spatial and temporal regulation of tissue specification during development, and has been observed biochemically, although the underlying mechanisms are not clear. This work provides context for activation of TGF-beta signaling by BMP, presenting a means by which cells could gain sensitivity to TGF-beta in the presence of BMP.

      Cover page: Ligand-induced upregulation of TGF-beta type I and type II receptors at the cell surface amplifies the TGF-beta response
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2017)
      TGF-β1 signaling is a critical driver of collagen accumulation and fibrotic disease but also a vital suppressor of inflammation and epithelial cell proliferation. The nature of this multifunctional cytokine has limited the development of global TGF-β1 signaling inhibitors as therapeutic agents. We conducted phenotypic screens for small molecules that inhibit TGF-β1-induced epithelial-mesenchymal transition without immediate TGF-β1 receptor (TβR) kinase inhibition. We identified trihydroxyphenolic compounds as potent blockers of TGF-β1 responses (IC50 ~50 nM), Snail1 expression, and collagen deposition in vivo in models of pulmonary fibrosis and collagen-dependent lung cancer metastasis. Remarkably, the functional effects of trihydroxyphenolics required the presence of active lysyl oxidase-like 2 (LOXL2), thereby limiting effects to fibroblasts or cancer cells, the major LOXL2 producers. Mechanistic studies revealed that trihydroxyphenolics induce auto-oxidation of a LOXL2/3-specific lysine (K731) in a time-dependent reaction that irreversibly inhibits LOXL2 and converts the trihydrophenolic to a previously undescribed metabolite that directly inhibits TβRI kinase. Combined inhibition of LOXL2 and TβRI activities by trihydrophenolics resulted in potent blockade of pathological collagen accumulation in vivo without the toxicities associated with global inhibitors. These findings elucidate a therapeutic approach to attenuate fibrosis and the disease-promoting effects of tissue stiffness by specifically targeting TβRI kinase in LOXL2-expressing cells.
        Cover page: Fibroblast-specific inhibition of TGF-β1 signaling attenuates lung and tumor fibrosis
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