IL-13 induces YY1 through the AKT pathway in lung fibroblasts

Abstract

A key feature of lung fibrosis is the accumulation of myofibroblasts. Interleukin 13 (IL-13) is a pro-fibrotic mediator that directly and indirectly influences the activation of myofibroblasts. Transforming growth factor beta (TGF-β) promotes the differentiation of fibroblasts into myofibroblasts, and can be regulated by IL-13. However, IL-13's downstream signaling pathways are not completely understood. We previously reported that the transcription factor Yin Yang 1 (YY1) is upregulated in fibroblasts treated with TGF-β and in the lungs of mice and patients with pulmonary fibrosis. Moreover, YY1 directly regulates collagen and alpha smooth muscle actin (α-SMA) expression in fibroblasts. However, it is not known if IL-13 regulates fibroblast activation through YY1 expression. We hypothesize that IL-13 up-regulates YY1 expression through regulation of AKT activation, leading to fibroblast activation. In this study we found that YY1 was upregulated by IL-13 in lung fibroblasts in a dose- and time-dependent manner, resulting in increased α-SMA. Conversely, knockdown of YY1 blocked IL-13-induced α-SMA expression in fibroblasts. Furthermore, AKT phosphorylation was increased in fibroblasts treated with IL-13, and AKT overexpression upregulated YY1, whereas blockade of AKT phosphorylation suppressed the induction of YY1 by IL-13 in vitro. In vivo YY1 was upregulated in fibrotic lungs from CC10-IL-13 transgenic mice compared to that from wild-type littermates, which was associated with increased AKT phosphorylation. Taken together, these findings demonstrate that IL-13 is a potent stimulator and activator of fibroblasts, at least in part, through AKT-mediated YY1 activation.

Fig 1. IL-13 induces YY1, collagen and α-SMA expression in lung fibroblasts.

After 24 h serum starvation, lung fibroblasts (MRC5) were treated with 30 ng/ml of IL-13 in serum-free DMEM. (A) Expression of α-SMA, p-AKT and YY1 was determined by Western blot in MRC5 treated with IL-13 (30 ng/ml) for 0–12 h. Whole cell extracts were subjected to 8–16% SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-YY1 and anti-α-SMA antibodies. (B) MRC5 cells were treated with IL-13 (10, 20, 30 ng/ml) for 12 h, and then the level of YY1 was determined by Western blot. (C) The image of western blot was scanned three times with ImageJ, and the density of YY1 was analyzed after normalization into β-actin. The statistic of scanned bands was performed by T-test. ** indicated P<0.01. (D and E) MRC5 cells were treated with IL-13 (30 ng/ml) for 24h, and immunostaining was performed to determine α-SMA (green), collagen I (green), YY1 (red), and DAPI (blue). All figures are at original magnification of 63×. Supplemental data S1 Fig. showed low magnification of Fig. 1D and 1E. (F) YY1-luciferase plasmid was transiently transfected into MRC5 cells by electroporation overnight followed by treatment with PBS or IL-13 (30 ng/ml) for 24 h. Relative light units (RLU) were measured with a luminometer. The fold change was calculated after normalization to control. The data was presented with standard errors derived from at least three independent experiments, each performed in triplicate. ** indicated P<0.01. (G) Fibroblasts were incubated in complete medium (MEM 10% FBS) for 24 h. On the second day the complete medium was replaced by medium with 0.1% FBS. Different doses of IL-13 (0, 3, 10 and 30 ng/ml) were added into the cells for 24 h. The cells were harvested, and collagen I in fibroblasts was measured by western blot in at least three independent experiments. (H) Densitometry of Western blot from Fig. 1G was analyzed by ImageJ. ** indicated P≤0.01.

Fig 2. IL-13 induces α-SMA expression through YY1.

(A) The α-SMA luciferase reporter (α-SMA-Luc, 5 μg) along with a 2.5 μg of control vector or 2.5 μg of YY1 was transiently transfected by electroporation into MRC-5 cells. At 24 h after transfection, IL-13 (30 ng/ml) or PBS were used to treat the MRC-5 cells for 24 h. These cells were lyzed with luciferase lysis buffer. Relative light units (RLU) were measured with a luminometer. The data are presented with standard errors derived from at least three independent experiments, mean ± SEM. **P < 0.01. (B) LL97A cells were infected with μl of control lentivirus vector shRNA (1x10⁵) or 10μl of lentivirus vector YY1 shRNA (1x10⁵) for 2 days. Lentiviral particles were produced as described in our previous paper [23]. Puromycin (4 μg/ml) was then used to select the transduced cells, which were stimulated by IL-13 (30 ng/ml) for 24 h. The selected LL97A cells were immunostained with anti-α-SMA (green) and anti-YY1 (red) antibodies. Representative pictures are presented at the original magnification shown a ruler on image. Representative examples are from three independent experiments. (C) The levels of YY1 and α-SMA in LL97A cells transduced and stimulated with IL-13 (30 ng/ml) were determined by western blot using corresponding antibodies. (D) Densitometry of western blot from 2C is measured by ImageJ. ** indicated p≤0.01.

Fig 3. Fibroblast specific protein 1 (FSP1), α-SMA, YY1, and collagen were increased in CC10-IL-13 transgenic mice.

(A) Lung tissues were stained with Hematoxylin and eoxin and Masson trichrome. The histological features and collagen were compared in between wild type (WT) control mice and CC10-IL-13 transgenic mice. All figures are at original magnification with a ruler (upper panel) and with a ruler (lower panel) on the images. (B) Immunohistochemical staining was used to localize the sites of FSP1, α-SMA, and YY1 in lungs of CC10-IL-13 transgenic mice and WT l mice. Collagen stains blue in these panels. All figures are at original magnification: 10 × (left panel) and 63 × (right panel). YY1, FSP1, and α-SMA expression are increased in lung tissue from IL-13 transgenic mice (n = 4). (C) Lung tissues from IL-13 transgenic (IL-13tg) and wild-type mice were stained by immunofluorescence for YY1 (Red), FSP1 (Green) and DAPI (blue). The magnification is 63x. Low magnification 10x showed in supplemental data S1 Fig.

Fig 4. IL-13-induced YY1 expression is regulated by AKT pathway.

MRC-5 cells were transfected with AKT and pCDNA1 control plasmids with electroporation. (A). At 24 h after transfection, the cells were starved for 24 h. IL-13 (30 ng/ml) was added to the cells for 12 h, The cells were lyzed, and the levels of YY1, AKT, p-AKT and β-actin were determined by Western blot. (B). YY1 and β-actin expression from Fig. 4A were scanned and were conducted a densitometric analysis with Image J. YY1 expression was normalized to β-actin. Scanned data were analyzed by T-test. ** indicate p value <0.01. (C) After MRC-5 cells were starved for 24 h without serum, cells were pre-treated with or without wortmannin (10 μM) for 1 h, and then treated with or without IL-13 (30 ng/ml) for 12 h in serum-free DMEM. Whole cell extracts were subjected to Western blot analysis for determining the levels of YY1, α-SMA, p-AKT, collagen and β-actin. (D). the mRNA expression of YY1 was detected by quantitative PCR. GAPDH mRNA expression was used as an internal control. The data are presented with standard errors derived from at least three independent experiments, each performed in triplicate; n = 3 and **p < 0.01.

Fig 5. YY1 and p-AKT were up-regulated in the lungs of IL-13 transgenic mice.

(A) Lungs from IL-13 transgenic mice and wild type (WT) mice were perfused with PBS, fixed with 10% formaldehyde, and embedded in paraffin. Immunofluorescent staining was conducted with anti-YY1 (green) and anti-p-AKT (red). DAPI is blue. The pictures are presented at the original magnification showed as ruler on the image. Representative examples are from three independent experiments. In a big magnification image, yellow arrows indicate YY1 and p-AKT overexpressed in the same area. (B) A portion of lung was homogenized, and Western blot was performed to determine the levels of YY1 and p-AKT in both IL-13 transgenic and wild-type mice.