Vitamin D deficiency exacerbates bleomycin-induced pulmonary fibrosis partially through aggravating TGF-+ACY-beta+ADs-/Smad2/3-mediated epithelial-mesenchymal transition

Abstract

Background: Our earlier report indicated that active vitamin D3 inhibited epithelial-mesenchymal transition (EMT) in bleomycin (BLM)-induced pulmonary fibrosis. The objective of this study was to further investigate whether vitamin D deficiency exacerbates BLM-induced pulmonary fibrosis. Methods: This study consists of two independent experiments. Experiment 1, male mice were fed with vitamin D deficient (VDD) fodder. Experiment 2, Cyp27b1 +ADw-sup+AD4APA-/sup+AD4-, Cyp27b1 +ADw-sup+AD4APA-/sup+AD4- and Cyp27b1 +ADw-sup+AD4-/-+ADw-/sup+AD4- mice were fed with standard diet. For pulmonary fibrosis, mice were intratracheally instilled with a single dose of BLM (1.5 mg/kg). Serum 25(OH) D level was measured. Pulmonary collagen deposition was assessed by Sirius red staining. EMT was measured and transforming growth factor-beta (TGF-+ACY-beta+ADs-)/Smad3 signaling was evaluated in the lungs of BLM-treated mice. Results: The relative weight of lungs was elevated in BLM-treated mice. Col1+ACY-alpha+ADs-1 and Col1+ACY-alpha+ADs-2, two collagen protein genes, were upregulated, and collagen deposition, as determined by Sirius red staining, was observed in the lungs of BLM-treated mice. E-cadherin, an epithelial marker, was downregulated. By contrast, vimentin and +ACY-alpha+ADs-SMA, two EMT markers, were upregulated in the lungs of BLM-treated mice. Pulmonary TGF-+ACY-beta+ADs-/Smad3 signaling was activated in BLM-induced lung fibrosis. Further analysis showed that feeding VDD diet, leading to vitamin D deficiency, aggravated elevation of BLM-induced relative lung weight. Moreover, feeding VDD diet aggravated BLM-induced TGF-+ACY-beta+ADs-/Smad3 activation and subsequent EMT in the lungs. In addition, feeding VDD diet exacerbated BLM-induced pulmonary fibrosis. Additional experiment showed that Cyp27b1 gene knockout, leading to active vitamin D3 deficiency, exacerbated BLM-induced pulmonary fibrosis. Moreover, Cyp27b1 gene knockout aggravated pulmonary TGF-+ACY-beta+ADs-/Smad2/3 activation and subsequent EMT in BLM-induced lung fibrosis. Conclusion: Vitamin D deficiency exacerbates BLM-induced pulmonary fibrosis partially through aggravating TGF-+ACY-beta+ADs-/Smad2/3-mediated EMT in the lungs.