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189 REVERSAL OF ALVEOLAR INTERSTITIAL FIBROBLAST TO MYOFIBROBLAST TRANSDIFFERENTIATION BY STIMULANTS OF PARATHYROID HORMONE-RELATED PROTEIN-MEDIATED CYCLIC ADENOSINE MONOPHOSPHATE-DEPENDENT PROTEIN KINASE A SIGNALING PATHWAY.
  1. R. Sakurai,
  2. Y. Wang,
  3. J. Santos,
  4. K. Huynh,
  5. J. S. Torday,
  6. V. K. Rehan
  1. Los Angeles BioMedical Research Institute, Los Angeles, CA

Abstract

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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