All trans retinoic acid recruits Smad3 to drive TGF beta signaling in lung fibroblasts : new insights for the management of bronchopulmonary dysplasia with retinoids
There is currently much interest in using retinoids such as all trans retinoic acid (ATRA), or retinoid analogs such as vitamin A (retinol), in the management of premature neonates at risk for the development of bronchopulmonary dysplasia (BPD). Current clinical evidence is contradictory, with some reports suggesting benefits, while other reports suggest no impact, or a deleterious effect of retinoid administration. In contrast, retinoids generally perform very well in animal models of BPD, and serve to stimulate lung development, particularly alveolarization. In this study, we attempted to evaluate the impact of retinoids on the activity of the transforming growth factor (TGF) beta signaling pathway, a pathway that has been credited with high importance in driving lung development and alveolarization.We employed NIH/3T3 mouse fibroblasts to study the impact of ATRA on TGF beta signaling. While the NIH/3T3 cell line differs in some important aspects to that of lung fibroblasts, it is reasonable to assume that NIH/3T3 have a similar biological behavior to that of airway fibroblasts in tissue. We observed that ATRA had a dramatic effect on TGF beta signaling in NIH/3T3 cells. Pre-treatment of NIH/3T3 cells with ATRA caused a strong increase in TGF beta signaling, as assessed by phosphorylation of Smad3, and by a luciferase-based promoter-reporter assay. Further studies revealed that this effect was caused by the ability of ATRA to strongly increase Smad3 expression levels in NIH/3T3 cells. Using plasmid-mediated overexpression of the smad3 gene, we confirmed these observations, and showed that increased Smad3 levels in NIH/3T3 cells can indeed make the NIH/3T3 cells more responsive to TGF beta. To further support these data, we also down-regulated smad3 gene expression using siRNA directed against Smad3, and demonstrated that by down-regulating smad3 expression, the pro-TGF beta signaling effect of ATRA was lost. We also demonstrated that the degree of change in smad3 gene expression caused by ATRA stimulation is sufficient to cause a significant change in the TGF beta signaling pathway.Together, these data demonstrate that ATRA administration can upregulate TGF beta signaling in NIH/3T3 cells. This may have important consequences for lung development, since TGF beta drives multiple fibroblast functions such as extracellular matrix (ECM) production, as well as ECM reshaping in developing organs. Future studies should address the cross-talk of TGF beta and retinoid signaling on other lung cell types, primary cells, and in animal models of BPD, in order to complete the picture of how retinoids may influence lung organogenesis. With a better understanding about the impact of retinoids on lung fibroblasts function and lung development one might be able to optimize the administration of retinoids in patients with BPD and thus improve clinical outcome.
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