Metformin suppresses proliferation and differentiation induced by BMP9 via AMPK signaling in human fetal lung fibroblast-1

Abstract

Adenosine monophosphosphate-activated protein kinase (AMPK) and its activator metformin were found to be involved in the regulation of fibroblast activation and pulmonary fibrosis. However, the regulatory mechanism has been undetermined. Recently, AMPK has been reported to exert its effect through inhibiting bone morphogenetic protein (BMP) pathway. In this study, human fetal lung fibroblast (HFL-1) cells were treated with metformin or specific AMPKα1 mutants, including constitutively activated mutant (AMPK-CA) and dominant negative mutant (AMPK-DN), combined with BMP9, and then the absorbance of these cells was measured by cell counting kit (CCK)-8 assay. The colony number of HFL-1 cells stimulated by metformin with or without BMP9 was examined by colony formation assay. The protein expressions of differentiated markers (α-smooth muscle actin, collagen I and collagen III) and the key molecules of BMP9 signaling, including activin receptor-like kinase (ALK) one and phosphorylated small mother against decapentaplegic (p-Smad)1/5, were also evaluated by western blot. Data revealed that BMP9 induced the proliferation and differentiation of HFL-1 cells which was suppressed by metformin or AMPK-CA. Meanwhile, the effect of metformin on BMP9-induced activation was counteracted by AMPK-DN. In addition, we found that the expressions of ALK1 and p-Smad1/5 induced by BMP9 were attenuated by metformin and AMPK-CA, whereas the inhibitory responses of metformin to the increased ALK1 and p-Smad1/5 were reduced by AMPK-DN. Accordingly, these results suggested that metformin mitigated BMP9-induced proliferation and differentiation of HFL-1 cells, which was achieved partly through the activation of AMPK and inhibition of ALK1/Smad1/5 signaling.

Keywords: AMPK; BMP9; differentiation; lung fibroblast; metformin; proliferation.

FIGURE 1

Metformin inhibited BMP9-induced proliferation and differentiation of HFL-1 cells. (A): HFL-1 cells were treated with different concentrations of metformin (0, 1, 5 and 10 mM) combined with BMP9 (50 ng/ml) for 24, 48 and 72 h. The proliferative ability was detected using a CCK-8 assay and represented as the relative absorbance (n = 3). (B,C): HFL-1 cells were treated with different concentrations of metformin (0, 1, 5 and 10 mM) combined with BMP9 (50 ng/ml) for 10 days. The ability of colony formation was evaluated using colony formation assay (n = 3). (D,E): HFL-1 cells were treated with different concentrations of metformin (0, 1, 5 and 10 mM) combined with BMP9 (10 ng/ml) for 48 h. The expression levels of fibroblast differentiation marker proteins (ɑ-SMA, Collagen I, Collagen III) were examined by western blot (n = 3). Cells dealt with only culture medium were served as a control group. Data are presented as mean ± SD. ^* p < 0.05 and ^** p < 0.01 versus control group; ^# p < 0.05 and ^## p < 0.01 versus only BMP9 stimulated group; ^& p < 0.05 and ^&& p < 0.01 versus the group exposed to 1 mM of metformin with BMP9; ^ΔP < 0.05 and ^ΔΔP < 0.01 versus the group exposed to 5 mM of metformin with BMP9.

FIGURE 2

Role of metformin in the expression of BMP9 downstream signalling molecules and AMPK. (A,B): Western blot was used to detect the expression of p-AMPK, ALK1 and p-Smad1/5 in HFL-1 cells with different concentrations of metformin (0, 1, 5, 10 mM) and 10 ng/ml BMP9 for 0.5 h (n = 3). (C,D): Western blot was applied to test the expression of p-AMPK, ALK1 and p-Smad1/5 in HFL-1 cells with 10 mM metformin for different times (2, 4, 8 and 24 h) and 10 ng/ml BMP9 for 0.5 h. Cells dealt with culture medium were served as control group (n = 3). Data are presented as mean ± SD. ^** p < 0.01 versus control group; ^## p < 0.01 versus only BMP9 stimulated group; ^& p < 0.05 and ^&& p < 0.01 versus 1 mM metformin for 24 h or 10 mM metformin for 2 h with BMP9 stimulated group; ^ΔP < 0.05 and ^ΔΔP < 0.01 versus 5 mM metformin for 24 h or 10 mM metformin for 4 h with BMP9 stimulated group; ^ɸɸP < 0.01 versus 10 mM metformin for 8 h with BMP9 treated group.

FIGURE 3

Effect of AMPK mutants on BMP9-induced proliferation and differentiation of HFL-1 cells. (A) CCK-8 assay was used to detect the effect of AMPK-CA on the proliferation of HFL-1 cells induced by BMP9 (n = 3). (B) CCK-8 assay was used to examine the role of AMPK-DN in the inhibitory effect of metformin on BMP9-induced proliferation of HFL-1 cells (n = 3). (C,D): The roles of AMPK-CA in the protein expression of fibroblast differentiation markers (ɑ-SMA, Collagen I and Collagen III) were detected by western blot (n = 3). (E,F): The effects of AMPK-DN on the protein expression of ɑ-SMA, Collagen I and Collagen III were tested by western blot (n = 3). Data are presented as mean ± SD. ^** p < 0.01 versus only GFP-AD group, ^# p < 0.05 and ^## p < 0.01 versus GFP-AD with BMP9 group, ^&& p < 0.01 versus GFP-AD with BMP9 and metformin group.

FIGURE 4

Impact of AMPK Mutants on the expression of BMP9 downstream signal molecules in HFL-1 cells. (A,B): Western blot was employed to examine the effect of AMPK-CA on the expression of ALK1 and p-Smad1/5 (n = 3). (C,D): Western blot was applied to detect the effect of AMPK-DN on the expression of ALK1 and p-Smad1/5 (n = 3). Data are presented as mean ± SD. ^* p < 0.05 and ^** p < 0.01 versus only GFP-AD group; ^## p < 0.01 versus GFP-AD with BMP9 group; ^& p < 0.05 and ^&& p < 0.01 versus GFP-AD with BMP9 and metformin group.