Background Exposure to nicotine disrupts specific alveolar epithelial-mesenchymal paracrine signaling pathways, resulting in the transdifferentiation of pulmonary alveolar interstitial fibroblasts (AIFs) to myofibroblasts (MYFs). We have suggested that AIF-to-MYF transdifferention is a central phenomenon that contributes to the altered pulmonary development and function in infants born to mothers who smoke during pregnancy. Further, we have shown that modulation of specific parathyroid hormone-related protein (PTHrP)-driven signaling can almost completely prevent nicotine-induced AIF-to-MYF transdifferentiation. However, once this process has occurred, whether it can be reversed is not known.
Objectives To determine whether nicotine-induced AIF-to-MYF can be reversed by specifically targeting the PTHrP-mediated cAMP-dependent PKA signaling pathway.
Methods WI38 cells, a human embryonic pulmonary fibroblast cell line, cultured in MEM + 10%FBS at 37°C, were plated in 6-well plates, 60 mm, and 100 mm dishes, as needed. At near confluence, the cells were initially treated with nicotine (10-9-10-6 M) for 7 days and AIF-to-MYF was confirmed by determining the down-regulation of the key lipogenic marker, peroxisome proliferator activated receptor γ (PPARγ) and up-regulation of the key myogenic marker, α-smooth muscle actin (αSMA). Subsequently, the cells were treated with prostaglandin J2 (PGJ2) (10 μM), PTHrP (5 × 10-7 M), or dibutryl cAMP (10-4 M) for 7 days and the expression of PTHrP receptor, PPARγ, adipocyte differentiation related protein (ADRP), calponin, αSMA was determined at mRNA (RT-PCR) and protein levels (Western analysis and immunohistochemistry). Simultaneous studies to determine the functional status of these fibroblasts were performed by measuring triglyceride uptake.
Results Nicotine-induced AIF-to-MYF transdifferentiation was almost completely reversed by treatment with PTHrP, dibutryl cAMP, or PGJ2, as determined at the mRNA, protein, and functional assays.
Conclusions Using a specific molecular approach and targeting specific molecular intermediates in PTHrP-driven cAMP-mediated PKA signaling pathway, these data, for the first time, demonstrate the reversibility of nicotine-induced AIF-to-MYF transdifferentiation, suggesting not only the possibility of prevention but also the potential for reversal of nicotine-induced lung injury. This clearly has significant potential therapeutic implications for both in utero and postnatal nicotine-induced lung injury.
Supported by Philip Morris USA Inc. and Philip Morris International.
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