Fig 1.
Lipo-PGE1 markedly reduces collagen expression and production in HDFs.
(A) HDFs were harvested after Lipo-PGE1 treatment (2.5–40 ng/mL) for 24 h. Total RNA was extracted and cDNA was synthesized for real-time RT-PCR. Bars indicate mean ± SD of three independent experiments, each with triplicate samples. *P<0.05 (B) Effects of Lipo-PGE1 on expression of type I collagen protein in HDFs. Cells were harvested after 36 h and collagen expression was analysed by Western blot using anti-type I collagen antibody. (C) HDFs were harvested after Lipo-PGE1 treatment (5 ng/mL) for the indicated duration. Total collagen was detected by the Sircol assay, which was performed in triplicate. Data are mean ± SD. *P<0.01 vs. control at the indicated time point.
Fig 2.
Lipo-PGE1 inhibited collagen gene expression and production induced by TGF- β.
(A) HDFs were pretreated with 10 ng/mL TGF-β for 2 h, before addition of 5 ng/mL Lipo-PGE1. After 24 h, total RNA was extracted and real-time RT-PCR was performed. Bars indicate mean ± SD of three independent experiments, each with triplicate samples. *P<0.05 vs. control, §P<0.05 vs. TGF-β-treated group. (B) The total collagen concentration in conditioned medium was determined by the Sircol assay after 48 h of culture. Data are mean ± SD. *P<0.05 vs. control, §P<0.05 vs. TGF-β-treated group. (C) Effects of Lipo-PGE1 on TGF-β-induced type I collagen expression. HDF cells were pretreated with TGF- β (10 ng/ml) for 2 h, and then incubated with 5 ng/mL Lipo-PGE1 for 36 h. Type I collagen production was analyzed by Western blot analysis. (D) Lipo-PGE1 inhibited TGF-β-induced phosphorylation of Smad2. HDF cells were treated with Lipo-PGE1, and then challenged with TGF- β (10 ng/ml). After 36 hours, whole cell lysates were probed with antibodies against Smad2, phospho-Smad2 in Western blots. The band intensities were quantitated and data are mean ± SD. *P<0.01 vs. control, §P<0.01 vs. TGF-β-treated group.
Fig 3.
Activation of the ERK pathway is involved in Lipo-PGE1-induced collagen inhibition.
(A) HDFs were pretreated with or without a specific inhibitor of ERK1/2 (PD98059) for 1 h and then incubated with 5 ng/mL Lipo-PGE1. Total RNA was extracted and cDNA was synthesized for real-time RT-PCR. Data are mean ± SD of three independent experiments. *P<0.05 vs. control; §P<0.05 vs. Lipo-PGE1-treated group. (B) Cells were pretreated with or without a specific inhibitor of ERK1/2 (PD98059) for 1 h and then treated with 5 ng/mL Lipo-PGE1. Total collagen was determined by the Sircol assay. A representative of three independently performed experiments is shown. Bars, mean ± SE. *P<0.05 vs. control, §P<0.05 vs. Lipo-PGE1-treated group. (C) Cells were treated with 5 ng/mL Lipo-PGE1 for 1 min, 5 min, 10 min, 30 min and 60 min. After cell lysis, the level of ERK1/2 phosphorylation was determined by Western blot analysis. The levels of total ERK were used to confirm equal loading of the cell lysates.
Fig 4.
Effects of Ets-1 on Lipo-PGE1-induced collagen inhibition in HDFs.
(A) HDFs were pretreated with or without a specific inhibitor of ERK1/2 (PD98059) for 1 h and then treated with 5 ng/mL Lipo-PGE1. Ets-1 protein was confirmed by Ets-1-specific western blot analysis. (B) HDFs were treated with Lipo-PGE1 and transfected with siRNA targeting Ets-1 or a negative control sequence. After stabilization, cells were collected and Ets-1-targeting siRNA transfection was confirmed by Ets-1—specific western blot analysis. (C) Collagen production after Ets-1-targeting siRNA transfection was detected by the Sircol assay. Data are mean ± SD of three independent experiments. *P<0.05 vs. control, §P<0.05 vs. non-treated cells transfected with negative control siRNA, #P<0.05 vs. Lipo-PGE1-treated cells transfected with negative control siRNA.
Fig 5.
Scheme representing the suppression of collagen production in HDFs after Lipo-PGE1 treatment.
Lipo-PGE1 strongly inhibits collagen synthesis in HDFs. Furthermore, an ERK- and Ets-1-dependent pathway is involved in Lipo-PGE1-inhibited collagen production. Lipo-PGE1 can be modulated to down-regulate collagen synthesis in human HDFs and may provide a therapeutic approach to inhibit fibrosis and its roles in the pathogenesis of skin disorders.