Transforming growth factor (TGF)-beta 1 internalization: modulation by ligand interaction with TGF-beta receptors types I and II and a mechanism that is distinct from clathrin-mediated endocytosis

Transforming growth factor-β (TGF-β) internalization was studied by monitoring the uptake of125I-TGF-β1 in Mv1Lu cells, which endogenously express TGF-β receptors types I (RI), II (RII), and III (RIII), and 293 cells transfected with RI and RII. At 37 °C internalization occurred rapidly, within 10 min of ligand addition. Internalization was optimal in 293 cells expressing both RI and RII. Internalization was prevented by phenylarsine oxide, a nonspecific inhibitor of receptor internalization, but was not affected by reagents that interfere with clathrin-mediated endocytosis such as monodansylcadaverine, K44A dynamin, and inhibitors of endosomal acidification. Electron microscopic examination of Mv1Lu cells treated with 125I- TGF-β1 at 37 °C indicated that internalization occurred via a noncoated vesicular mechanism. Internalization was prevented by prebinding cells with TGF-β1 at 4 °C for 2 h prior to switching the cells to 37 °C. This was attributed to a loss of receptor binding, as indicated by a rapid decrease in the amount of TGF-β1 bound to the cell surface at 37 °C and by a reduction in the labeling intensities of RI and RII in125I-TGF-β1-cross-linking experiments. Mv1Lu or 293 (RI+RII) cells, prebound with TGF-β1 at 4 °C and subsequently stripped of ligand by an acid wash, nevertheless initiated a signaling response upon transfer to 37 °C, suggesting that prebinding promotes formation of stable RI·RII complexes that can signal independently of ligand

Adapting the Scar-in-a-Jar to Skin Fibrosis and Screening Traditional and Contemporary Anti-Fibrotic Therapies

Skin fibrosis still constitutes an unmet clinical need. Although pharmacological strategies are at the forefront of scientific and technological research and innovation, their clinical translation is hindered by the poor predictive capacity of the currently available in vitro fibrosis models. Indeed, customarily utilised in vitro scarring models are conducted in a low extracellular matrix milieu, which constitutes an oxymoron for the in-hand pathophysiology. Herein, we coupled macromolecular crowding (enhances and accelerates extracellular matrix deposition) with transforming growth factor beta 1 (TGF beta 1; induces trans-differentiation of fibroblasts to myofibroblasts) in human dermal fibroblast cultures to develop a skin fibrosis in vitro model and to screen a range of anti-fibrotic families (corticosteroids, inhibitors of histone deacetylases, inhibitors of collagen crosslinking, inhibitors of TGF beta 1 and pleiotropic inhibitors of fibrotic activation). Data obtained demonstrated that macromolecular crowding combined with TGF beta 1 significantly enhanced collagen deposition and myofibroblast transformation. Among the anti-fibrotic compounds assessed, trichostatin A (inhibitors of histone deacetylases); serelaxin and pirfenidone (pleiotropic inhibitors of fibrotic activation); and soluble TGF beta receptor trap (inhibitor of TGF beta signalling) resulted in the highest decrease of collagen type I deposition (even higher than triamcinolone acetonide, the gold standard in clinical practice). This study further advocates the potential of macromolecular crowding in the development of in vitro pathophysiology models.Peer reviewe

Predominant intracellular localization of the type I transforming growth factor-beta receptor and increased nuclear accumulation after growth arrest

NRC publication: Ye

Down-regulation of transforming growth factor-beta receptors: cooperativity between the types I, II, and III receptors and modulation at the cell surface

UI - 99458858NRC publication: Ye

Real-time monitoring of the interactions of transforming growth factor-beta (TGF-beta) isoforms with latency-associated protein and the ectodomains of the TGF-beta type II and III receptors reveals different kinetic models and stoichiometries of binding

Mature transforming growth factor-\u3b2 (TGF-\u3b2) is proteolytically derived from the C terminus of a precursor protein. Latency-associated protein (LAP), the N-terminal remnant of the TGF-\u3b2 precursor, is able to bind and neutralize TGF-\u3b2. Mature TGF-\u3b2 exerts its activity by binding and complexing members of two subfamilies of receptors, the type I and II receptors. In addition to these signaling receptors, TGF-\u3b2 can also interact with an accessory receptor termed the type III receptor. Using a surface plasmon resonance-based biosensor (BIAcore), we determined the mechanisms of interaction of four binding proteins (LAP, the type II and III receptor ectodomains (EDs), and a type II receptor ED/Fc chimera) with three TGF-\u3b2 isoforms, and we quantified their related kinetic parameters. Using global fitting based on a numerical integration data analysis method, we demonstrated that LAP and the type II receptor/Fc chimera interacted with the TGF-\u3b2 isoforms with a 1:1 stoichiometry. In contrast, the type II ED interactions with TGF-\u3b2 were best fit by a kinetic model assuming the presence of two independent binding sites on the ligand molecule. We also showed that the type III ED bound two TGF-\u3b2 molecules. Further experiments revealed that LAP was able to block the interactions of TGF-\u3b2 with the two EDs, but that the two EDs did not compete or cooperate with each other. Together, these results strongly support the existence of a cell-surface complex consisting of one type III receptor, two TGF-\u3b2 molecules, and four type II receptors, prior to the recruitment of the type I receptor for signal transduction. Additionally, our results indicate that the apparent dissociation rate constants are more predictive of the neutralizing potency of these TGF-\u3b2-binding proteins (LAP, the type II and III receptor EDs, and the type II receptor/Fc chimera) than the apparent equilibrium constants.NRC publication: Ye

Identification of a functional site on the type I TGF-b receptor by mutational analysis of its ectodomain

NRC publication: Ye

Analysis of the contribution of receptor subdomains to the cooperative binding and internalization of transforming growth factor-\u3b2 (TGF-\u3b2) type I and type II receptors

The mechanistic basis underlying the striking cooperativity observed for the assembly of TGF-\u3b2 family ligand/receptor complexes is not well understood. We report here an investigation in which we used a novel ligand sequestration assay, in combination with immunofluorescent light microscopy and flow cytometry analyses, to examine and quantify cooperative assembly of TGF-\u3b2 ligand/receptor complexes on the cell surface, as well as ligand/receptor complex internalization. We analyzed the roles played by the ecto/transmembrane (ecto/TM) domains and endodomains of RI and RII TGF-\u3b2 receptors in these processes by transfecting 293 or HeLa cells with different combinations of receptor mutants. We found that the ecto/TM domains of RII and RI cooperated together to promote the formation of cell surface receptor/ligand complexes. Furthermore, in agreement with the recently determined structure of the TGF-\u3b23/RII ectodomain/RI ectodomain complex [J. Groppe, C.S. Hinck, P. Samavarchi-Tehrani, C. Zubieta, J.P. Schuermann, A.B. Taylor, P.M. Schwarz, J.L. Wrana, A.P. Hinck, Cooperative assembly of TGF-beta superfamily signaling complexes is mediated by two disparate mechanisms and distinct modes of receptor binding, Mol. Cell 29 (2008) 157\u2013168], we observed that the N-terminus of the RII ectodomain was required for full assembly. With respect to endodomains, we found that the RI endodomain enhanced cooperative complex assembly at the cell surface, whereas both the RI and RII endodomains enhanced internalization. Finally, we observed that ligand/receptor internalization, but not complex assembly at the cell surface, was partly raft-dependent. In light of these results, currently proposed mechanisms of cooperative ligand/receptor assembly are discussed.Peer reviewed: YesNRC publication: Ye

Dual drug delivery collagen vehicles for modulation of skin fibrosis in vitro

Single molecule drug delivery systems have failed to yield functional therapeutic outcomes, triggering investigations into multi-molecular drug delivery vehicles. In the context of skin fibrosis, although multi-drug systems have been assessed, no system has assessed molecular combinations that directly and specifically reduce cell proliferation, collagen synthesis and transforming growth factor β1 (TGFβ1) expression. Herein, a core-shell collagen type I hydrogel system was developed for the dual delivery of a TGFβ trap, a soluble recombinant protein that inhibits TGFβ signalling, and Trichostatin A (TSA), a small molecule inhibitor of histone deacetylases. The antifibrotic potential of the dual delivery system was assessed in an in vitro skin fibrosis model induced by macromolecular crowding (MMC) and TGFβ1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography analyses revealed that ∼50% of the TGFβ trap and ∼30% of the TSA were released from the core and shell compartments, respectively, of the hydrogel system after 10 d (longest time point assessed) in culture. As a direct consequence of this slow release, the core (TGFβ trap)/shell (TSA) hydrogel system induced significantly (p < 0.05) lower than the control group (MMC and TGFβ1) collagen type I deposition (assessed via SDS-PAGE and immunocytochemistry), α smooth muscle actin (αSMA) expression (assessed via immunocytochemistry) and cellular proliferation (assessed via DNA quantification) and viability (assessed via calcein AM and ethidium homodimer-I staining) after 10 d in culture. On the other hand, direct TSA-TGFβ supplementation induced the lowest (p < 0.05) collagen type I deposition, αSMA expression and cellular proliferation and viability after 10 d in culture. Our results illustrate the potential of core-shell collagen hydrogel systems for sustained delivery of antifibrotic molecules.publishedVersionPeer reviewe