TGF-β1 stimulates HDAC4 nucleus-to-cytoplasm translocation and NADPH oxidase 4-derived reactive oxygen species in normal human lung fibroblasts

Abstract

Myofibroblasts are important mediators of fibrogenesis; thus blocking fibroblast-to-myofibroblast differentiation (FMD) may be an effective strategy to treat pulmonary fibrosis (PF). Previously, we reported that histone deacetylase 4 (HDAC4) activity is necessary for transforming growth factor-β₁ (TGF-β₁)-induced human lung FMD. Here, we show that TGF-β₁ increases NADPH oxidase 4 (NOX4) mRNA and protein expression in normal human lung fibroblasts (NHLFs) and causes nuclear export of HDAC4. Application of the NOX family inhibitor diphenyleneiodonium chloride reduces TGF-β₁-induced HDAC4 nuclear export, expression of the myofibroblast marker α-smooth muscle actin (α-SMA), and α-SMA fiber formation. Inhibition of HDAC4 nucleus-to-cytoplasm translocation using leptomycin B (LMB) had little effect on α-SMA expression but blocked α-SMA fiber formation. A coimmunoprecipitation assay showed that HDAC4 associates with α-SMA. Moreover, LMB abolishes TGF-β₁-induced α-SMA fiber formation and cell contraction. Relevant to human pulmonary fibrosis, idiopathic PF specimens showed significantly higher NOX4 RNA expression and scant HDAC4 staining within nuclei of fibroblast foci myofibroblasts. Taken together, these results indicate that reactive oxygen species promote TGF-β₁-mediated myofibroblast differentiation and HDAC4 nuclear export. The physical association of HDAC4 with α-SMA suggests that HDAC4 has a role in regulating the α-SMA cytoskeleton arrangement.

Keywords: HDAC4; IPF; NOX4; myofibroblasts; reactive oxygen species; α-smooth muscle actin.

Fig. 1.

TGF-β₁ promotes nuclear export of HDAC4. NHLFs were treated with TGF-β₁ (1 ng/ml) for 12 h. A: immunostaining of HDAC4. Arrows indicate nuclei. DAPI, 4′,6′-diamidino-2-phenylindole. B: HDAC4 subcellular distribution in cytosolic and nuclear fractions was analyzed using immunoblot. C: densitometry measurements of triplicates for B. *P < 0.05 vs. control.

Fig. 2.

TGF-β₁ stimulates NOX4-derived ROS to augment α-SMA expression in NHLFs. A: NOX4 expression was analyzed using quantitative real-time PCR 9 h after TGF-β₁ treatment. B: NOX4 expression in the membrane fraction was analyzed using immunoblot. α-TU, α-tubulin. C: ROS were measured using the DCFH-DA method after TGF-β₁ treatment for 12 h with or without DPI (*P < 0.05 vs. control, **P < 0.05 vs. TGF-β₁ + DMSO). D: NHLFs were transfected with NOX4 siRNA or nontarget control siRNA (NC) for 48 h and then treated with TGF-β₁ for 12 h. ROS were measured using the DCFH-DA method (#P < 0.05 vs. control, ##P < 0.05 vs. NC + DMSO). E: NHLFs were pretreated with DPI for 1 h followed by treatment with TGF-β₁ for 12 h. α-SMA expression was assessed using immunoblot. F: immunoblot for type I human collagen 1 (h-Col1). Immunoblot results represent at least 2 independent assays. RT-PCR represents 3 independent assays.

Fig. 3.

Inhibition of ROS reduces HDAC4 nuclear export. A: NHLFs were transfected with GFP-HDAC4-expressing plasmids using electroporation and then treated with TGF-β₁ ± DPI for 24 h. Green fluorescent protein (GFP)-positive cells were counted for each treatment. The results represent the percentage of cells lacking nuclear GFP (*P < 0.05 vs. control, **P < 0.05 vs. TGF-β₁ + DMSO). B: immunostaining of endogenous HDAC4 in NHLFs. NHLFs were treated with DPI for 2 h before TGF-β₁ treatment for 12 h. C: immunoblot analysis of HDAC4 disulfide bond formation. The upper blot, “HDAC4 S=S,” represents BIAM HDAC4 (BIAM-labeled HDAC4 disulfide bonds). The lower blot, “Input,” indicates total HDAC4. D: immunoblot analysis showing HDAC4 distribution within cytoplasm and nuclear compartments. E: immunostaining of HDAC4 localization. NHLFs were pretreated with nontargeting control (NC) siRNA or NOX4 siRNA for 48 h and then treated with or without TGF-β₁ for 24 h. Results represent at least 2 independent assays. Arrows indicate nuclei. DM, DMSO.

Fig. 4.

HDAC4 associates with α-SMA, and inhibition of ROS reduces myofibroblast contractility. A: immunostaining of α-SMA fibers. NHLFs were pretreated with DMSO, TSA, or LMB for 2 h and then treated with TGF-β₁ for 24 h. B: immunostaining of α-SMA fibers. NHLFs were transfected with HDAC4 siRNA or nontarget control siRNA (NC) for 48 h and then treated with TGF-β₁ for 24 h. C: immunostaining of endogenous HDAC4 localization. NHLFs were pretreated with LMB for 2 h and then treated with TGF-β₁ for 12 h. D: immunoblot analysis of α-SMA protein expression in NHLFs treated with TGF-β₁, with or without LMB or TSA. E: coimmunoprecipitation (IP) of endogenous HDAC4 with α-SMA. NHLFs were stimulated with TGF-β₁ for 24 h. F: NHLF collagen gel contraction assay. The ratio of the area of gel to culture well was used to express the contractile ability (*P < 0.05 vs. control, **P < 0.05 vs. DMSO). G: colocalization of stress fibers (phalloidin staining) and α-SMA. Results represent at least 2 independent assays.

Fig. 5.

Expressions of NOX4 and HDAC4 in IPF lung biopsy specimens. A: total RNA was isolated from lung biopsy specimens of IPF patients [n = 10; 3 patients with forced vital capacity (FVC) of 50–80% predicted and 7 patients with FVC of <50% predicted] or controls [very mild chronic obstructive pulmonary disease; COPD; Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage 1; n = 11]. NOX 4 mRNA expression was measured using real-time PCR and normalized to 18S. *P < 0.05 vs. control. B: immunohistochemistry localizing HDAC4 in IPF lung biopsy specimens. Scale bars are for the panoramic view 100 μm (bottom left), middle magnification 20 μm (top left), and high magnification 10 μm (top right and bottom right).