Latent TGF-β Activation Is a Hallmark of the Tenascin Family

Abstract

Transforming growth factor-β (TGF-β) isoforms are secreted as inactive complexes formed through non-covalent interactions between bioactive TGF-β entities and their N-terminal pro-domains called latency-associated peptides (LAP). Extracellular activation of latent TGF-β within this complex is a crucial step in the regulation of TGF-β activity for tissue homeostasis and immune cell function. We previously showed that the matrix glycoprotein Tenascin-X (TN-X) interacted with the small latent TGF-β complex and triggered the activation of the latent cytokine into a bioactive TGF-β. This activation most likely occurs through a conformational change within the latent TGF-β complex and requires the C-terminal fibrinogen-like (FBG) domain of the glycoprotein. As the FBG-like domain is highly conserved among the Tenascin family members, we hypothesized that Tenascin-C (TN-C), Tenascin-R (TN-R) and Tenascin-W (TN-W) might share with TN-X the ability to regulate TGF-β bioavailability through their C-terminal domain. Here, we demonstrate that purified recombinant full-length Tenascins associate with the small latent TGF-β complex through their FBG-like domains. This association promotes activation of the latent cytokine and subsequent TGF-β cell responses in mammary epithelial cells, such as cytostasis and epithelial-to-mesenchymal transition (EMT). Considering the pleiotropic role of TGF-β in numerous physiological and pathological contexts, our data indicate a novel common function for the Tenascin family in the regulation of tissue homeostasis under healthy and pathological conditions.

Keywords: immune cell modulation; latent TGF-β activation; tenascins; tissue homeostasis; transforming growth factor (TGF)-β; tumor microenvironment.

Figure 1

Sequence alignment and homology degree between the Tenascin family FBG-like domains. (A) Multiple sequence alignment analysis of the FBG-like domains of human TN-X, -C, -W and -R (FBG-X, -C, -W, -R) performed with CLUSTALW software. Red (*) labelled residues are identical between the four FBG-like domains, green labelled (:) residues are highly conserved and blue labelled (.) residues are poorly conserved. The four conserved cysteine residues are highlighted in yellow. (B) Percentages of identity and similarity amongst the amino-acid sequences of the FBG-like domains of the human Tenascins performed with paired comparisons using CLUSTALW software.

Figure 2

Recombinant full-length Tenascins and their respective FBG-like domains co-purify with latent TGF-β1. (A) Schematic representation of the recombinant proteins used in this study (dark grey). Structural modules of Tenascins are depicted in the inset. (B, D) SDS-PAGE analysis of the purified recombinant proteins and stained with Coomassie blue. MM, molecular mass markers. (B) Purified full-length TN-X, TN-C, TN-W and the TN-X^ΔEΔF fragment (1 μg each) were loaded on 6% acrylamide gels under reducing conditions. (D) Purified recombinant FBG-like domains of the four human Tenascins and the recombinant CUB1CUB2 (C1C2) domain (2 μg) were resolved on 15% acrylamide gels under reducing conditions. (C, E) Western blot analysis indicating the level of human mature TGF-β1 and LAP(β1) pro-domain associated with equimolar quantity of purified recombinant full-length TN or TN-X^ΔEΔF fragment (5 pmol each) (C) and recombinant Tenascin FBG-like domains or CUB1CUB2 (C1C2) protein (20 pmol) (E).

Figure 3

The recombinant FBG-like domains of Tenascins interact with TGF-β1. (A–D) Interactions of TGF-β1 with FBG-like domains analyzed by SPR. Increasing concentrations of TGF-β1 were injected in single-cycle mode over immobilized FBG-X (A), FBG-W (B), FBG-C (C) and FBG-R (D). The experimental curves are shown in red and the best fits with the heterogeneous ligand model in black. The concentrations of TGF-β1 were as follows: (A) 15.6 – 31.2 – 62.5 – 125 – 250 nM; (B) 7.8 – 15.6 – 31.25 – 62.5 – 125 nM; (C) 20.2 – 40.5 – 81 – 162 – 324 nM; (D) 9.4 – 18.8 – 37.5 – 75 – 150 nM. (E, F) Homology models showing the predicted docking between the FBG-like domain of TN-C (E) or TN-W (F) and the pro-TGF-β1. The dimeric LAP(β1) pro-domain is shown in green, the TGF-β1 dimer in orange and the FBG-like domains in purple. ‘Straitjacket’ and arm domains of the LAP(β1) dimer are indicated.

Figure 4

Full-length Tenascins and their respective FBG-like domains stimulate TGF-β/Smad intracellular signaling pathway in epithelial cells. (A) Western Blot analysis showing phosphorylated Smad2 (P-Smad2), total Smad2/3 and GAPDH levels in NMuMG cells cultured for 3h onto control non-coated (N-C) dishes or dishes coated with equimolar quantity (22,2 pmol/cm²) of full-length Tenascins (TN) or central TN-X fragment (TN-X^ΔEΔF), or stimulated with soluble active TGF-β1 (2 ng/mL). (B) Fold changes of P-Smad2 to total Smad2 levels or to GAPDH levels. Graph shows means ± SD of n = 3 independent experiments. (C) Western Blot analysis showing P-Smad2, total Smad2/3 and Actin levels in NMuMG cultured for 3h onto equimolar quantity of FBG-like domains (333 pmol/cm²) or stimulated with soluble active TGF-β1 (2 ng/mL). (D) Fold changes of P-Smad2 to total Smad2 levels or to reference protein levels. Graph shows means ± SD of n = 3 independent experiments. (E) Western-blot analysis showing P-Smad2, Smad2/3 and GAPDH levels in NMuMG cells stimulated for 1h in the presence of active TGF-β1 (2 ng/mL), stimulated or not (PBS) with soluble FBG-like domains (300 nM) or MOCK-CM. “MOCK-CM” referred to the conditioned media of MOCK-transfected HEK293 EBNA cells, submitted to Nickel-affinity chromatography and eluted from the column as for the FBG-like domains. (F) Immunoblotting analysis showing P-Smad2 and GAPDH levels in NMuMG cells stimulated for 1h in the presence of active TGF-β1 (2 ng/mL), stimulated or not (PBS) with soluble CUB1CUB2 protein fragment (C1C2), bovine serum Albumin (BSA) or FBG-like domains (300 nM each). (G) Relative Luciferase activity of NMuMG cells transiently transfected with the Smad-responsive (CAGA)₁₂-Luc reporter construct and treated for 16h with soluble FBG-like domains (600 nM), MOCK-CM or TGF-β1 (2 ng/mL). Graph shows one representative result from 3 independent experiments.

Figure 5

The FBG-like domain of the Tenascin family members activates the latent TGF-β in epithelial cells. (A) Western-blot analysis showing P-Smad2, total Smad2/3 and GAPDH levels in NMuMG cultured for 3h onto Non-Coated (N-C) dishes, dishes coated with 22,2 pmol/cm² full-length Tenascins (TN) or stimulated with 2 ng/mL TGF-β1, in presence of anti-pan-TGF-β antibody or isotype-matched control IgG (10 μg/mL). (B) Western Blot analysis showing P-Smad2, total Smad2/3 and GAPDH levels in NMuMG cells cultured for 3h in presence of 333 pmol/cm² of FBG-like domains as described in (A). (C) SDS-PAGE analysis of purified recombinant FBG-like domains produced in mammalian cells (FBG) and E. coli (FBG*) resolved on 15% acrylamide gels under reducing conditions (2 µg each). MM, molecular mass markers. (D) Western-Blot analysis of the levels of mature TGF-β1 and LAP(β1) pro-domain associated with 20 pmol of FBG-like domains produced in mammalian cells (FBG) and E coli (FBG*). (E) Western-blot analysis showing P-Smad2, Smad2/3 and GAPDH levels in NMuMG cells stimulated for 1h with active TGF-β1 (2 ng/mL), soluble purified FBG-like domains produced in E Coli (720 nM) or vehicle (PBS).

Figure 6

The FBG-like domains of Tenascins trigger EMT and cytostasis in epithelial cells. (A) F-actin direct fluorescence (red) and E-Cadherin indirect immunofluorescence (green) performed on NMuMG cells cultured for 72h onto N-C dishes, dishes coated with 666 pmol/cm² FBG-like domains or stimulated with soluble TGF-β1 (5 ng/mL). Cell nuclei were counterstained with DAPI (blue). Bars, 15 µm. (B) Percentage of cell viability in NMuMG cells cultured for 48h onto N-C dishes, dishes coated with 333, 666 or 999 pmol/cm² FBG-like domains, or stimulated with 5 ng/mL TGF-β1. Error bars are means ± SD from 3 independent experiments. *p < 0.05 compared to N-C condition. ****p < 0.0001 compared to N-C condition. ns, not significant. (C) Percentage of cell viability in NMuMG cells cultured for 48h onto N-C dishes, dishes coated with 666 pmol/cm² FBG-like domains (FBG-X and FBG-C) or CUB1CUB2 protein fragment (C1C2), or stimulated with 5 ng/mL TGF-β1, in the presence of anti-pan-TGF-β antibody or isotype-matched control IgG (5 μg/mL). Error bars are means ± SD from 3 independent experiments. **, *** and **** respectively correspond to p < 0.01, p < 0.001 and p < 0.0001 compared to N-C condition. ^# and ^### respectively correspond to p < 0.05 and p < 0.001 versus their control IgG-treated counterpart. ns, not significant.