Antifibrotic effects of noscapine through activation of prostaglandin E2 receptors and protein kinase A

Abstract

Myofibroblast differentiation is a key process in the pathogenesis of fibrotic disease. We have shown previously that differentiation of myofibroblasts is regulated by microtubule polymerization state. In this work, we examined the potential antifibrotic effects of the antitussive drug, noscapine, recently found to bind microtubules and affect microtubule dynamics. Noscapine inhibited TGF-β-induced differentiation of cultured human lung fibroblasts (HLFs). Therapeutic noscapine treatment resulted in a significant attenuation of pulmonary fibrosis in the bleomycin model of the disease. Noscapine did not affect gross microtubule content in HLFs, but inhibited TGF-β-induced stress fiber formation and activation of serum response factor without affecting Smad signaling. Furthermore, noscapine stimulated a rapid and profound activation of protein kinase A (PKA), which mediated the antifibrotic effect of noscapine in HLFs, as assessed with the PKA inhibitor, PKI. In contrast, noscapine did not activate PKA in human bronchial or alveolar epithelial cells. Finally, activation of PKA and the antifibrotic effect of noscapine in HLFs were blocked by the EP2 prostaglandin E2 receptor antagonist, PF-04418948, but not by the antagonists of EP4, prostaglandin D2, or prostacyclin receptors. Together, we demonstrate for the first time the antifibrotic effect of noscapine in vitro and in vivo, and we describe a novel mechanism of noscapine action through EP2 prostaglandin E2 receptor-mediated activation of PKA in pulmonary fibroblasts.

Keywords: Fibrosis; Microtubules; Myofibroblast; Noscapine; Prostaglandins; Protein Kinase A (PKA); SRF; Stress Fibers.

FIGURE 1.

Antifibrotic effect of noscapine in vitro and in vivo. A, inhibition of myofibroblast differentiation by noscapine in vitro. Quiescent HLF were treated with 1 ng/ml TGF-β1 in the presence of 100 μm noscapine for 48 h. Cell lysates were analyzed by Western blotting with the indicated antibodies. Shown are the representative images and the quantitative analysis of ECL from at least three independent experiments. B, effect of 100 μm noscapine or 1 mm H₂O₂ (48 h) on lactate dehydrogenase (LDH) release by HLF. C and D, attenuation of bleomycin-induced pulmonary fibrosis by noscapine. Mice were treated with 1 unit/kg bleomycin intratracheally. 7 days after bleomycin administration, 100 mg/kg noscapine or vehicle was delivered intraperitoneally daily for 14 more days. Animals were sacrificed, and lungs were removed. C, right lungs were processed for hydroxyproline assay. DMSO, dimethyl sulfoxide. D, left lungs were formalin-fixed, paraffin-embedded, sectioned, and processed for H&E or trichrome staining or for immunohistochemistry with collagen I, collagen III, or SM-α-actin (SMA) antibodies. The specificity of primary antibodies is demonstrated by the lack of staining in airway epithelial cells. No staining was observed in the absence of primary antibodies or with the use of normal IgG (data not shown). Representative images are shown. Error bars, S.E.; *, p < 0.05.

FIGURE 2.

Noscapine inhibits SRF activation without affecting Smad signaling in HLF. Effect of 100 μm noscapine (Nosc) on TGF-β1-induced MKL1 expression and accumulation in the nuclear fraction (A), on SRF-luciferase activity (B), on −125-bp SMA promoter activity (C), on Smad2 phosphorylation (D), on Smad2 nuclear accumulation (E), and on SBE-luciferase activity (F) is shown. Error bars, S.E.; *, p < 0.05.

FIGURE 3.

Noscapine has no effect on gross microtubule polymerization state but inhibits stress fiber formation in HLF. A, HLFs were treated with 100 μm noscapine or 10 μm Taxol for 24 h, followed by immunostaining with β-tubulin antibodies (green) and counterstaining for nuclei with DAPI (blue). Shown are the representative images at ×400 and ×1000 magnification. B, HLFs were treated with 100 μm noscapine or 10 μm Taxol for 24 h. Soluble and polymerized β-tubulin were in vitro fractionated and detected by Western blotting. C, HLFs were treated with 1 ng/ml TGF-β1 with or without 100 μm noscapine for 24 h. In vitro stress fiber preparations or total cell lysates were analyzed by Western blotting for β-actin or SMA as indicated.

FIGURE 4.

Activation of PKA mediates the regulation of myofibroblast differentiation by noscapine. A, quiescent HLFs were treated with 10 μm Taxol or 100 μm noscapine for the indicated times. Lysates were analyzed by Western blotting with antibodies against VASP or against PKA-substrate (PKAS) antibodies. B, HLFs, mouse lung fibroblasts (MLF), human bronchial epithelial cells (16HBE), or human alveolar epithelial cells (A549) were treated with 100 μm noscapine or 10 μm forskolin for the indicated times. Lysates were analyzed by Western blotting for VASP shift. C and D, HLF were transduced with Ad-PKI or control Ad-LacZ adenoviruses. C, cells were then treated with 100 μm noscapine for 30 min, and equal amounts of protein were assessed by Western blotting for VASP shift. D, cells were treated with 100 μm noscapine followed by 1 ng/ml TGF-β1 for 48 h. Equal amounts of cell lysates were analyzed by Western blotting for the desired proteins. Cell lysates were analyzed by Western blotting with the indicated antibodies. Shown are the representative images and the quantitative analysis of enhanced chemiluminescence from three independent experiments. Error bars, S.E.; *, p < 0.05.

FIGURE 5.

Activation of PKA and regulation of myofibroblast differentiation by noscapine are mediated by EP₂ receptors. A, HLFs were pretreated for 1 h with the EP/DP receptor antagonist, AH-6809 (10 μm), with the IP receptor antagonist, RO-1138452 (20 μm), with the COX-I and COX-II inhibitors, SC-560 (3 μm) and NS-398 (3 μm), or with the sigma opioid receptor antagonist, rimcazole (10 μm), followed by 100 μm noscapine for 10 min. Equal amounts of lysates were analyzed by Western blotting for VASP shift. B, HLFs were treated with EP₂ receptor antagonist, PF-04418948 (1 μm), with EP₄ receptor antagonist, L-161,982 (10 μm), or with DP receptor antagonist, BW A868C (10 μm) for 1 h, followed by stimulation with 100 μm noscapine for 10 min. Equal amounts of lysates were analyzed by Western blotting for VASP shift. C, HLFs were treated with EP₂ receptor antagonist PF-04418948 (1 μm) for 1 h, followed by stimulation with 1 ng/ml TGF-β with or without 100 μm noscapine for 48 h. Cell lysates were analyzed by Western blotting with the indicated antibodies. Shown are the representative images and the quantitative analysis of ECL from three independent experiments. Error bars, S.E.; *, p < 0.05.