TGF-beta-induced interleukin-6 participates in transdifferentiation of human Tenon's fibroblasts to myofibroblasts

Abstract

Purpose: To gain a better understanding of the roles of interleukins (ILs) in subconjunctival fibrosis, we investigated their expression in transforming growth factor-beta1 (TGF-beta1)-stimulated Tenon's fibroblasts and examined their association with the transdifferentiation of fibroblasts to myofibroblasts.

Methods: After primary culture, fibroblasts derived from human Tenon's capsule were exposed to TGF-beta1. The expression of alpha-smooth muscle actin (alpha-SMA) protein was assessed by western immunoblots and immunofluorescence. The mRNA levels of various ILs were also evaluated by multiplex reverse transcription (RT)-PCR. Using the small interfering RNAs (siRNAs) specific for IL-6 and IL-11 and the promoter deletion assay, the contributions of IL-6 and IL-11 to TGF-beta1-induced induction of alpha-SMA were determined.

Results: In human Tenon's fibroblasts, TGF-beta1 stimulated the expression of alpha-SMA protein determined by western blot analysis and also increased the mRNA levels of IL-6 and IL-11 determined by multiplex RT-PCR. On the western immunoblots and immunofluorescence, the increased expression of alpha-SMA was attenuated only by the siRNAs specific for IL-6 but not by the siRNAs specific for IL-11. When the activator protein-1 binding sites of the IL-6 promoter region were deleted, the stimulation effects of TGF-beta1 decreased.

Conclusions: Our data show that autocrine IL-6 may participate in the TGF-beta1-induced transdifferentiation of human Tenon's fibroblasts to myofibroblasts, which is known to be an essential step for subconjunctival fibrosis.

Figure 1

Representative western blot bands for α-smooth muscle actin. A: Dose-response effects: fibroblasts were exposed to various concentrations of transforming growth factor (TGF)-β1 for 72 h. B: Time-response effects: fibroblasts were incubated with or without 10 ng/ml of TGF-β1 for up to 6 days. β-actin was used as an internal control. Densitimetric data represent the mean±SD of results from three independent experiments.

Figure 2

Multiplex reverse transcription PCR. Multiplex reverse transcription PCR sets for interleukins (ILs; A, B) and housekeeping genes (C). For each set of data, lane 1 represents the control group with no transforming growth factor (TGF)-β1 treatment and lane 2 represents the study group with 10 ng/ml TGF-β1 treatment for 72 h. Lane M contains positive markers. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-1RN, interleukin-1 receptor antagonist; RPL13a; SDHA, succinate dehydrogenase complex subunit A; β2M, β2-microblobulin. Two asterisks refer to a significant increase (p<0.001) in expression of IL-6 and IL-11 in the presence of TGF-β1.

Figure 3

RNA interference assay for IL-6 and IL-11. After administering small interfering RNAs targeting IL-6 (A) and IL-11 (B), fibroblasts were exposed to 10 ng/ml transforming growth factor (TGF)-β1 for 72 h. Then, the expression of α-smooth muscle actin (α-SMA) was evaluated using western blots. Densitimetric data represent the mean±SD of results from three independent experiments. The asterisks refer to a significant difference compared to TGF-β treatment only (p<0.001).

Figure 4

Immunofluorescent analysis of the expression of α-smooth muscle actin. A: No treatment control, B: 10 ng/ml transforming growth factor (TGF)-β1 for 72 h, C: IL-6-specific small interfering (si)RNA, D: IL-6-specific siRNA plus 10 ng/ml TGF-β1 for 72 h, E: IL-11-specific siRNA, and F: IL-11-specific siRNA plus 10 ng/ml TGF-β1 for 72 h. The secondary antibody was conjugated with fluoroscein isothiocyanate, resulting in green fluorescence. Total magnification was 400×.

Figure 5

Promoter deletion assay for interleukin-6 (IL-6). Three recombinant plasmids, in which different fragments of the activator protein-1 (AP-1)-binding site on the human IL-6 promoter were deleted, were used. After transfection with each plasmid, fibroblasts were incubated with 10 ng/ml transforming growth factor (TGF)-β1 for 72 h. The luciferase activity was then determined and expressed as the percent of the no-TGF-β1 treatment control. Data represent the mean±SD of results from three independent experiments. The asterisks refer to a significant difference (p<0.001) compared to the wild type.