Bone morphogenetic protein (BMP)-4 and BMP-7 regulate differentially transforming growth factor (TGF)-beta1 in normal human lung fibroblasts (NHLF)

Abstract

Background: Airway remodelling is thought to be under the control of a complex group of molecules belonging to the transforming growth factor (TGF)-superfamily. The bone morphogenetic proteins (BMPs) belong to this family and have been shown to regulate fibrosis in kidney and liver diseases. However, the role of BMPs in lung remodelling remains unclear. BMPs may regulate tissue remodelling in asthma by controlling TGF-beta-induced profibrotic functions in lung fibroblasts.

Methods: Cell cultures were exposed to TGF-beta1 alone or in the presence of BMP-4 or BMP-7; control cultures were exposed to medium only. Cell proliferation was assessed by quantification of the incorporation of [3H]-thymidine. The expression of the mRNA encoding collagen type I and IV, tenascin C and fibronectin in normal human lung fibroblasts (NHLF) was determined by real-time quantitative PCR and the main results were confirmed by ELISA. Cell differentiation was determined by the analysis of the expression of alpha-smooth muscle actin (alpha-SMA) by western blot and immunohistochemistry. The effect on matrix metalloproteinase (MMP) activity was assessed by zymography.

Results: We have demonstrated TGF-beta1 induced upregulation of mRNAs encoding the extracellular matrix proteins, tenascin C, fibronectin and collagen type I and IV when compared to unstimulated NHLF, and confirmed these results at the protein level. BMP-4, but not BMP-7, reduced TGF-beta1-induced extracellular matrix protein production. TGF-beta1 induced an increase in the activity of the pro-form of MMP-2 which was inhibited by BMP-7 but not BMP-4. Both BMP-4 and BMP-7 downregulated TGF-beta1-induced MMP-13 release compared to untreated and TGF-beta1-treated cells. TGF-beta1 also induced a myofibroblast-like transformation which was partially inhibited by BMP-7 but not BMP-4.

Conclusions: Our study suggests that some regulatory properties of BMP-7 may be tissue or cell type specific and unveil a potential regulatory role for BMP-4 in the regulation of lung fibroblast function.

Figure 1

Effect of TGF-β superfamily members on BMP type I and type II receptor transcript levels. NHLF were stimulated with 5 ng/ml TGF-β1 or 100 ng/ml BMP-4 or BMP-7 for 24 h. Cells were harvested, RNA extracted and reverse transcribed, and a real-time quantitative PCR for ALK-2 (A), ALK-3 (B), ALK-6 (C), and BMPRII (D) was performed. Results are expressed as the ratio of each transcript relative to the geometric mean of mRNA expression of the housekeeping genes UBC, SDHA, and RPL13a. Data are mean ± SD of five independent experiments. *, p < 0.05, as compared to unstimulated cells.

Figure 2

Simultaneous incubation of NHLF with TGF-β1 and BMP-4 inhibits cell proliferation. [3H]thymidine incorporation in NHLF in response to tissue culture media with 2% FBS in the presence of 5 ng/ml TGF-β1 or 100 ng/ml BMP-4 or BMP-7 alone or with TGF-β1 in the presence of BMP-4 or BMP-7 for 36 h. [3H]thymidine was added for the last 6 h of incubation. Data are mean ± SD of five independent experiments. *, p < 0.05, as compared to unstimulated cells and †, p < 0.05, as compared to TGF-β1-stimulated cells.

Figure 3

TGF-β1-induced collagen expression in NHLF is downregulated by BMP-4. NHLF were stimulated with 5 ng/ml TGF-β1 or 100 ng/ml BMP-4 or BMP-7 alone, or with TGF-β1 in the presence of BMP-4 or BMP-7 for 24 h (A and B) or 72 h (C). Cells were harvested, RNA was extracted, reverse transcribed, and a real-time quantitative PCR for collagen type I alpha 1 chain (COL1a1, A) and collagen type IV alpha 1 chain (COL4a1, B) was performed. Results are expressed as the ratio of each transcript relative to the geometric mean of mRNA expression of the housekeeping genes UBC, SDHA, and RPL13a. Total soluble collagen release was quantified in the cell supernatants by Sircol assay (C). Data are mean ± SD of five independent experiments. *, p < 0.05, as compared to unstimulated cells and †, p < 0.05, as compared to TGF-β1-stimulated cells.

Figure 4

TGF-β1-induced ECM protein expression in NHLF is down-regulated by BMP-4. NHLF were stimulated with 5 ng/ml TGF-β1 or 100 ng/ml BMP-4 or BMP-7 alone or with TGF-β1 in the presence of BMP-4 or BMP-7 for 24 h (A and B) or 48 h (C and D). Cells were harvested, RNA was extracted, reverse transcribed, and a real-time quantitative PCR for tenascin C (A) and fibronectin (C) was performed. Results are expressed as the ratio of each transcript relative to the geometric mean of mRNA expression of the housekeeping genes UBC, SDHA, and RPL13a. Tenascin C and fibronectin protein were quantified in the cell supernatants by specific ELISAs (B and D, respectively). Data are mean ± SD of five independent experiments. *, p < 0.05, as compared to unstimulated cells and †, p < 0.05, as compared to TGF-β1-stimulated cells.

Figure 5

Effect of TGF-β superfamily members on MMP activity and expression level. NHLF were stimulated with 5 ng/ml TGF-β1 or 100 ng/ml BMP-4 or BMP-7 alone or with TGF-β1 in the presence of BMP-4 or BMP-7 for 72 h. Cell supernatants were collected to perform zymography (A and B) and ELISA (C). Representative gelatin zymograms and related graphic plot of the bands obtained in zymographs for the pro-forms of MMP-1 (A) and MMP-2 (B) were performed. Gelatinolytic activity of the pro- and active forms of MMP-1 (57/52 and 47/42 kDa) and pro- and active forms of MMP-2 (72 and 67 kDa) are indicated. MMP-13 release was quantified in the cell supernatants by specific ELISA (C). Data are mean ± SD of five independent experiments. *, p < 0.05, as compared to unstimulated cells and †, p < 0.05, as compared to TGF-β1-stimulated cells.

Figure 6

TGF-β1-induced myofibroblast like phenotype in NHLF is partially inhibited by BMP-7. NHLF were stimulated with 5 ng/ml TGF-β1 or 100 ng/ml BMP-4 or BMP-7 or with TGF-β1 in the presence of BMP-4 or BMP-7 for 72 h. Representative panel of α-SMA expression was obtained by immunohistochemistry (A) and western blot of cell lysates for α-SMA is shown in (B). Data are representative of five independent experiments.

Figure 7

TGF-β1-induced CTGF promoter and SBE-SEAP reporter activities are not modulated by the BMPs. (A) The CTGF promoter pCT-sb was transiently transfected into NHLF, cells were then treated with 5 ng/ml TGF-β1 or 100 ng/ml BMP-4 or BMP-7 or with TGF-β1 in the presence of BMP-4 or BMP-7 in FGM containing 0.2% FBS. All assays were performed with 150000 cells/well in 2 ml total volume in 6-well plates and luciferase activity was measured after 24 h induction in 50 μl cell pellet. (B) MFB-F11 cells stably transfected with SBE-SEAP were stimulated with 5 ng/ml TGF-β1 or 100 ng/ml BMP-4 or BMP-7 or with TGF-β1 in the presence of BMP-4 or BMP-7 in serum-free DMEM. All assays were performed with 40000 cells/well in 100 μl total volume in 96-well plates and SEAP activity was measured after 24 h induction in 10 μl supernatant. Data are mean ± SD of five independent experiments. *, p < 0.05, as compared with unstimulated cells.