Fig 2: Expression of collagen, α-SMA and Smad3 in WI-38 lung fibroblasts after TGF-β stimulation with or without co-incubation of syndecan-4. (a,b) The co-incubation of recombinant syndecan-4 significantly decreased the mRNA expression of TGF-β-induced collagen and α-SMA upregulation at 24 h (n = 5–9 per each condition). (c,d) TGF-β-induced phosphorylation of Smad3 was attenuated by the co-incubation of recombinant syndecan-4 at 15 min. (c) were representatives of three separate experiments, and (d) showed intensity of p-Smad3 evaluated by densitometric analysis. The difference was compared using the ANOVA test and Fisher’s least significant difference test as a post hoc test. *, p < 0.05 vs. TGF-β (−) and syndecan-4 (−), †, p < 0.05 versus TGF-β (+) and syndecan-4 (−).

Fig 3: Effects of TIMP3 silencing on Smad2/3 phosphorylation in TGF-β1/2 signaling. A control RT-PCR of the knockdown effectiveness for TIMP3 was performed to ensure silencing of TIMP3 (A). The 7 × 104 93RS2 TIMP3-silenced cells were treated with TGF-β1 or TGF-β2 (both 10 ng/mL) for 1 h. Cell lysates were analyzed for P-Smad2 and P-Smad3 by ELISAs. Silencing of TIMP3 attenuated TGF-β2 but not TGF-β1-dependent phosphorylation of Smad2 (B) and of Smad3 (C) as well as proliferation of 93RS2 cells by 50% (D). Each bar represents the mean ± SEM of 3 independent experiments performed in duplicate. Dunnett’s test was used for statistical analysis; *p ≤ 0.05, **p < 0.01; ***p < 0.001; Ctrl, control; nc-siRNA, negative control siRNA; M, Marker; +, addition of substance; -, without addition of substance.

Fig 4: Scheme for the different modes of signaling by TGF-β1 and TGF-β2 under the influence of BG and TIMP3. (A) Normally binding of TGF-β1 (b1) to the TGF-β receptor complex results in phosphorylation of Smad3. However, TIMP3 counteracts MMP-mediated BG shedding, thus resulting in interference of membrane-bound and ligand-activated BG with the TGF-β receptor complex. This interaction attenuates TGF-β1-dependent Smad3 phosphorylation via TβRI. sBG reduces binding of TGF-β1 to the TGF-β receptor complex and thus counteracts TGF-β1 signaling. (B) In contrast, binding of TGF-β2 (b2) to BG enhances phosphorylation of TGF-β2-dependent Smad3 which is further increased when TIMP3 inhibits MMP-dependent BG shedding. sBG reduces binding of TGF-β2 to the TGF-β receptor complex and thus counteracts TGF-β2 signaling.

Fig 5: Expression of collagen, α-SMA and Smad3 in WI-38 lung fibroblasts after TGF-β stimulation with or without knockdown of syndecna-4. (a,b) TGF-β-induced type I collagen (COL1A1) and α-SMA upregulation significantly increased 24 h after knockdown of syndecan-4 at 24 h (n = 5–9 per each condition). (c,d) TGF-β-induced phosphorylation of Smad3 was increased by knockdown of syndecan-4 at 15 min. Figures (c) were representatives of three separate experiments, and figure (d) showed intensity of p-Smad3 evaluated by densitometric analysis. The difference was compared using the ANOVA and Fisher’s least significant difference test as a post hoc test. *, p < 0.05 vs. TGF-β (−), siControl (−) and siSyndecan-4 (−), †, p < 0.05 vs. TGF-β (+), siControl (−) and siSyndecan-4 (+). siControl: control siRNA, siSyndecan-4: siRNA for syndecan-4, t-Smad3: total Smad3, p-Smad3: phosphorylated Smad3.