Abstract
With increasing worldwide rates of morbidity and mortality of pulmonary fibrosis, the development of effective therapeutics for this disease is of great interest. Secretoglobin (SCGB) 3A2, a novel cytokine-like molecule predominantly expressed in pulmonary airways epithelium, exhibits anti-inflammatory and growth factor activities. In the current study SCGB3A2 was found to inhibit TGFβ-induced differentiation of fibroblasts to myofibroblasts, a hallmark of the fibrogenic process, using pulmonary fibroblasts isolated from adult mice. This induction was through increased phosphorylation of STAT1 and expression of SMAD7 and decreased phosphorylation of SMAD2 and SMAD3. To demonstrate the effect of SCGB3A2 on the TGFβ signaling in vivo, a bleomycin-induced pulmonary fibrosis mouse model was used. Mice were administered bleomycin intratracheally followed by intravenous injection of recombinant SCGB3A2. Histological examination in conjunction with inflammatory cell counts in bronchoalveolar lavage fluids demonstrated that SCGB3A2 suppressed bleomycin-induced pulmonary fibrosis. Microarray analysis was carried out using RNAs from lungs of bleomycin-treated mice with or without SCGB3A2 and normal mice treated with SCGB3A2. The results demonstrated that SCGB3A2 affects TGFβ signaling and reduces the expression of genes involved in fibrosis. This study suggests the potential utility of SCGB3A2 for targeting TGFβ signaling in the treatment of pulmonary fibrosis.
Highlights
With increasing worldwide rates of morbidity and mortality of pulmonary fibrosis, the development of effective therapeutics for this disease is of great interest
Fibrosis arises from inflammation initiated by cell injury, and injured tissues are gradually replaced by collagen fibers that are produced from fibroblasts and accumulate as myofibroblasts
Effect of SCGB3A2 on Differentiation of Fibroblasts into Myofibroblasts—It was previously demonstrated that SCGB3A2 has at least two biological functions; an anti-inflammatory function was revealed using an ovalbumin-induced allergic airway inflammation model [24], and growth factor activity was studied using ex vivo fetal lung organ cultures and in vivo injection of SCGB3A2 to pregnant mice [25]
Summary
Isolation and Primary Culture of Lung Fibroblasts—Lung tissues from 7–9-week-old female mice were cut into small pieces, mounted on collagen type I-coated 60-mm plate (IWAKI, Shizuoka, Japan), and cultured for 7 days. C57BL/6N mice (7– 8 weeks old) were treated with PBS or SCGB3A2 by intravenous administration and were euthanized 12 h later. Immunohistochemical staining for SCGB3A2, pSMAD2, pSMAD3, and SMAD2/3 was performed in BLM-treated lungs using anti-mouse SCGB3A2 antibody (produced in our laboratory) [14], anti-pSMAD2(ser465/467) (Cell Signaling Technology), anti-pSMAD3 (Epitomics, Burlingame, CA), and anti-SMAD2/3 (BD Biosciences), respectively. Fibroblasts cultured on 8-well Lab Tek Chamber Glass Slide (Nalge Nunc International, Naperville, IL) with and without TGF and/or SCGB3A2 were incubated at room temperature in PBS containing 0.1% Triton X 100 for 15 min followed by fixing with 4% paraformaldehyde for 15 min at room temperature. Ten (group mice analysis) or 20 g (SCGB3A2 treatment) of purified total RNA were reverse-transcribed to label with Cy3 and Cy5 (GE Healthcare) using the FairPlay Microarray Labeling kit (Stratagene, La Jolla, CA) or SuperScriptTM Indirect cDNA Labeling Core kit (Invitrogen), respectively. Statistical analysis was performed one-way ANOVA followed by Bonferroni multiple comparison test
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