Fibrillin-1 regulates the bioavailability of TGFbeta1

Abstract

We have discovered that fibrillin-1, which forms extracellular microfibrils, can regulate the bioavailability of transforming growth factor (TGF) beta1, a powerful cytokine that modulates cell survival and phenotype. Altered TGFbeta signaling is a major contributor to the pathology of Marfan syndrome (MFS) and related diseases. In the presence of cell layer extracellular matrix, a fibrillin-1 sequence encoded by exons 44-49 releases endogenous TGFbeta1, thereby stimulating TGFbeta receptor-mediated Smad2 signaling. This altered TGFbeta1 bioavailability does not require intact cells, proteolysis, or the altered expression of TGFbeta1 or its receptors. Mass spectrometry revealed that a fibrillin-1 fragment containing the TGFbeta1-releasing sequence specifically associates with full-length fibrillin-1 in cell layers. Solid-phase and BIAcore binding studies showed that this fragment interacts strongly and specifically with N-terminal fibrillin-1, thereby inhibiting the association of C-terminal latent TGFbeta-binding protein 1 (a component of the large latent complex [LLC]) with N-terminal fibrillin-1. By releasing LLC from microfibrils, the fibrillin-1 sequence encoded by exons 44-49 can contribute to MFS and related diseases.

Figure 1.

Human recombinant fibrillin-1 fragments spanning full-length fibrillin-1. Fibrillin-1 fragments were cloned into pCEP-His, expressed in 293-EBNA cells, and purified as previously described (Cain et al., 2005, 2006; Marson et al., 2005). The asterisk denotes the six cbEGF-like domains in fragments PF10 and PF11 that regulate TGFβ.

Figure 2.

PF10 and PF11 stimulate Smad2 phosphorylation. 0.15 μM of fibrillin-1 recombinant fragments PF1, PF2, PF8, PF9, PF10, PF11, PF12, PF13, PF14, and PF15 were tested for their ability to stimulate Smad2 phosphorylation. The negative control (Con) contained no added proteins. 4 nM of human recombinant TGFβ1, which stimulates Smad2 signaling, was a positive control. 0.15 μM of human plasma fibronectin (FN) was an additional control. Only PF10 and PF11 stimulated Smad2 signaling. This experiment was repeated three times with similar results.

Figure 3.

PF10 stimulates Smad2 phosphorylation through TGFβRs and activates TGFβ1. (A) When TGFβRII was blocked using 15 μg/ml of a neutralizing antibody (RII), Smad2 phosphorylation caused by PF10 stimulation was ablated (***, P < 0.0001 by t test in comparison with the PF10 control). A control antibody that has no inhibitory effect on the TGFβ pathway (IgG) confirmed that ablation of PF10 stimulation was caused by the specific TGFβRII antibody. (B) PF10 stimulation of Smad2 signaling was ablated when TGFβRI was blocked using a TGFβRI kinase chemical inhibitor (RI; ***, P < 0.0001 by t test in comparison with the PF10 control). (A and B) Control samples (Con) contained no added proteins. The TGFβRII-inhibiting antibody (A) and TGFβRI kinase inhibitor (B) also blocked TGFβ1-activated Smad2 signaling (***, P < 0.0001 in comparison with the TGFβ1 control). (C) When active endogenous TGFβ1 was blocked using 15 μg/ml of a neutralizing antibody (β1), there was a marked reduction in PF10-induced Smad2 signaling (***, P < 0.0001 by t test in comparison with the PF10 control). In the presence of anti-TGFβ1 antibody, controls with added TGFβ1 showed reduced activation of Smad2 (***, P < 0.0001 in comparison with the TGFβ1 control). A further control contained no added proteins. A control antibody that has no inhibitory effect on the TGFβ pathway (IgG) confirmed that the effects were caused by the specific TGFβ1 antibody. (A–C) Quantitative analysis was performed by densitometry with data normalized against β-actin. Data are represented as the mean of three repeated experiments. Error bars represent the SD of the three experiments. (D) Endogenous TGFβ1 activity produced by stimulating cells for 90 min with PF10 and PF11 at a concentration of 1 μM was assayed using the TGFβ1 EMax Immunoassay kit. The control that contained no added proteins and the fibronectin (FN) control showed no increase in active TGFβ1. (E) PF10 stimulated active and total TGFβ1 levels in cell layers and lysed cell layers. ELISA assays revealed that when 1.5 μM PF10 was incubated in fresh serum-free medium with intact cell layers (90 min), high levels of total and active TGFβ1 activation occurred with a small but statistically significant (*, P < 0.05 by protected t test) decrease in active TGFβ relative to total TGFβ. When PF10 was incubated with lysed cell layers (PF10 (L)), levels of total and active TGFβ were statistically similar, and, in both cases, 83% of levels using intact cell layers (***, P < 0.0001; active and total TGFβ in PF10 lysed cells compared with PF10 in unlysed cells; two-way ANOVA followed by a posthoc Tukey's test). The control that contains no added proteins and was subjected to cell lysis (Con (L)) shows a small but statistical increase in both active and total TGFβ when compared with the unlysed control (**, P < 0.001 by two-way ANOVA followed by a posthoc Tukey's test). Thus, PF10 releases TGFβ mainly from the cell layer. (D and E) All experiments were performed in triplicate and on the same microtitre plate (D, R² 0.9989; E, R² 0.9988). Error bars represent the SD of a single experiment that was undertaken in triplicate. The experiment was repeated at least three times with similar results.

Figure 4.

Efficacies of different fibrillin-1 ligands in stimulating Smad2 phosphorylation. (A) The ability of PF10 and PF11 to stimulate Smad2 phosphorylation was compared at equal concentrations (0.15 μM). PF10 consistently generated a stronger signal than PF11. The control well (Con) contains no added proteins. (B) Time course of PF10-induced Smad2 phosphorylation. 0.15 μM PF10 induced an increase in Smad2 phosphorylation within 10 min. (C) SDS-PAGE analysis of 0.15 μM PF10 after treatment with 0.2 mg/ml porcine pancreatic elastase, a potent protease that degrades fibrillin-1 (Kielty et al., 1994), showing the presence of degraded fragments. Lane M is a molecular marker lane; lane (i) is PF10; lane (ii) is PF10 after elastase treatment. Blots (iii) show the effects of PF10 with or without elastase treatment. After elastase, PF10 exhibited an enhanced ability to stimulate Smad2 signaling. The control, which contained no added proteins, and 0.2 mg/ml of a further elastase-only control (El) did not induce Smad2 phosphorylation. The addition of 4 nM elastase to TGFβ1 did not increase Smad2 phosphorylation. (D) 0.15 μM of purified full-length fibrillin-1 molecules (FBN-1) stimulated Smad2 phosphorylation but weakly compared with 0.15 μM PF10. The control contains no added proteins. (E) TGFβ signaling activity was barely detectable after supplementing cultures with 0.15 μM microfibrils (MF) purified from bovine ciliary zonules. 0.15 μM of the PF10 control stimulated Smad2 phosphorylation as expected. The control contains no added proteins (Con). (A–E) Quantitative analysis was performed by densitometry with data normalized against β-actin. Data are represented as the mean of three repeated experiments. Error bars represent the SD of the three experiments. ***, P < 0.0001 by t test in comparison with the PF10 control.

Figure 5.

Quantification of active TGFβ1 after treatment with PF10, PF11, and TSP-1. (A) ELISA assays revealed that PF10, PF11, and fibrillin-1 molecules showed dose-dependent increases (0.0625–2 μM) in active TGFβ1 when HDF cells were stimulated for 90 min. A plot of the concentration of active TGFβ1 (picomolar) against the concentration of protein (micromolar) is shown with a regression line for each protein. The table below shows the B value and the 95% confidence interval (CI) for each protein. The slope of the regression line for PF10 is greater than that for PF11, although it is not statistically significant. PF10 shows an increase in active TGFβ1 when compared with intact fibrillin-1 molecules (R² 0.9993). The control contained no added proteins and showed no increase in active TGFβ1. All experiments were performed in triplicate and on the same microtitre plate (R² 0.9993). (B) Supplementation with 15 nM PF10 induced 1.1 pM more active TGFβ1 than 15 nM TSP-1 (***, P < 0.0001 by t test in comparison with TSP-1; R² 0.9989). The control, which contained medium only, and the fibronectin (FN) control both showed no increase in active TGFβ1. All experiments were performed in triplicate and on the same microtitre plate (R² 0.9989). Error bars represent the SD of a single experiment that was undertaken in triplicate. (A and B) The experiment was repeated at least three times with similar results.

Figure 6.

PF10-mediated increase in active TGFβ1 requires cell layers. Conditioned HDF medium that had been preincubated with HDF for 15 min, 60 min, and 24 h was stimulated with 1.5 μM PF10. In the absence of cells, PF10 induced only very low but statistically significant levels of active TGFβ1 in the 15- (**, P < 0.001 by t test) and 60-min (***, P < 0.0001 by t test) incubations compared with the controls. The conditioned medium control contains no PF10 and shows no active TGFβ1. The positive control contains 1.5 μM PF10 incubated in the presence of cells for 90 min and shows high levels of active TGFβ1. All experiments were performed in triplicate and on the same microtitre plate (R² 0.9985). Error bars represent the SD of a single experiment that was undertaken in triplicate. The experiment was repeated at least three times with similar results.

Figure 7.

Regulation of TGFβ1 by PF10 does not require cell surface receptors. (A) 20 μg/ml of integrin function–blocking antibodies to αv (17E6) and β1 (mAb 13) had no significant inhibitory effect on the PF10-mediated stimulation of Smad2 signaling by 0.15 μM of fibrillin-1 fragment PF10. The α5-blocking antibody (mAb 16) induced a small but significant (*, P < 0.05 by t test) increase in Smad2 signaling. The control (Con), which contained no added proteins, and antibody controls (β1, αv, and α5 antibodies) showed no Smad2 phosphorylation. Active TGFβ1 was used as a positive control. (B) 0.15 μM PF10 was able to induce significant stimulation of Smad2 signaling (***, P < 0.0001 by t test in comparison with the control) in a syndecan-4–null mouse embryonic fibroblast culture, indicating that absence of the syndecan-4 receptor does not block PF10-mediated Smad2 signaling. Wild-type (wt) fibroblasts were used as a positive control (*, P < 0.05 by t test; PF10 in comparison with the control). A negative control contains no added proteins. 4 nM TGFβ1 was an additional control for Smad2 signaling. (A and B) Quantitative analysis was performed by densitometry with data normalized against β-actin. Data are represented as the mean of three repeated experiments. Error bars represent the SD of the three experiments.

Figure 8.

PF10 interacts with the fibrillin-1 N-terminal region (PF1) and inhibits PF1 interaction with LTBP-1. (A) Solid-phase binding assays of 0–200 nM of soluble biotinylated PF10 to 200 nM of immobilized fibrillin-1 fragments showed that PF10 interacts specifically with the N-terminal region of fibrillin-1 (PF1) with moderately strong affinity (K_D = 90 ± 14 nM). Mutant PF1^V449I had increased affinity (K_D = 52 ± 13 nM), but mutant PF1^R62C bound very poorly. Nonspecific binding to BSA is shown. Results are presented as the mean ± SEM (error bars) of triplicate values. (B) Preincubation of 0.15 μM PF10 and PF1 for 15 min at 20°C caused a reduction in Smad2 signaling compared with the PF10-only control (these data were normalized against corresponding β-actin; ***, P < 0.0001 by t test). Preincubation of PF10 with mutant PF1^V449I also reduced Smad2 signaling (***, P < 0.0001 by t test in comparison with the PF10 control). However, there was no difference in Smad2 signaling after preincubation of PF10 and mutant PF1^R62C compared with the wild-type PF10 control experiment. The negative control (Con) contains no added proteins. Quantitative analysis was performed by densitometry with data normalized against β-actin. Data are represented as the mean of three repeated experiments. Error bars represent the SD of the three experiments (P < 0.05 by t test) in comparison with the PF10 control.

Figure 9.

PF10 inhibits the binding of PF1 and CT LTBP-1, and PF10 does not activate TGFβ1 in UMR-106 cells. (A) BIAcore analysis of the interaction of C-terminal LTBP-1 with the fibrillin-1 N-terminal fragment PF1 as well as inhibition by PF10. Fibrillin-1 protein fragments PF1 (i) or PF10 (ii) were injected over LTBP-1 immobilized using amine coupling on a CM5 sensor chip. Both sensorgrams show analyte concentrations ranging from 0 to 150 nM, and duplicate concentrations were included in every run. One representative experiment is shown in each case. Only PF1 interacted with LTBP-1. Response difference is the difference between experimental and control flow cells in response units. Time is shown in seconds. Inhibition of the maximum response of 50 nM PF1 to LTBP-1 is shown in panel iii. Increasing concentrations of PF10 (0–30 μM) was incubated with PF1 before addition to immobilized LTBP-1. PF10 inhibited PF1 binding to LTBP-1 (IC₅₀ = 2.42 ± 0.5 μM). (B) Densitometry analysis of Smad2 phosphorylation by UMR-106 cells revealed that treatment with PF10 failed to induce Smad2 signaling when compared with the control. The addition of active TGFβ1 was a positive control. No added protein was a negative control (Con). Error bars represent SD.

Figure 10.

Model of how PF10 regulates TGFβ bioavailability. Secreted LLC becomes associated with deposited fibrillin microfibrils (LTBP-1, a component of LLC, is shown in red). The PF10 fragment (orange), which is released by proteolysis, binds microfibrillar fibrillin-1 within the insoluble cell layer, interacting specifically with the fibrillin-1 N-terminal region (PF1; blue). PF10 binds assembled microfibrils at or adjacent to the beads where this N-terminal region localizes (Reinhardt et al., 1996; Baldock et al., 2001). PF10 inhibits the PF1 interaction with LTBP-1 (and thus with LLC), leading to the release of LLC and an increase in active TGFβ. Microfibril beads (gray ovals) and interbead regions (lines between ovals) are indicated.