Piclamilast inhibits the pro-apoptotic and anti-proliferative responses of A549 cells exposed to H₂O₂ via mechanisms involving AP-1 activation

Abstract

Aims. Reactive oxygen species (ROS) are involved in the pathogenesis of many inflammatory diseases such as chronic obstructive pulmonary disease (COPD). They can alter the expression of genes involved in cellular damage by activating transcription factors, including the NF-κB and the activator protein 1 (AP-1). Phosphodiesterase type 4 (PDE4) inhibitors have anti-inflammatory and antioxidant effects, as described in in vivo and in vitro COPD models. This study analysed the effects of piclamilast, a selective PDE4 inhibitor, on modulating the global gene expression profile in A549 cells exposed to H₂O₂. Main methods. Changes in gene expression were analysed using high-density Affymetrix microarrays and validated by RT-PCR. Cell proliferation was studied using BrdU incorporation. Apoptosis was assessed by flow cytometry using annexin V-fluorescein isothiocyanate. C-Jun phosphorylation and AP-1 activation were determined by ELISA and luciferase assay, respectively. Key findings. Our results indicate that H₂O₂ modified the expression of several genes related to apoptosis, cell cycle control and cell signalling, including IL8, FAS, HIG2, CXCL2, CDKN25 and JUNB. Piclamilast pre-treatment significantly inhibited the changes in 23 genes via mechanisms involving AP-1 activation and c-Jun phosphorylation at Ser63. Functional experiments confirmed our results, suggesting new targets related to the antioxidant properties of PDE4 inhibitors. Significance. This is the first study to demonstrate antioxidant effects of a selective PDE4 inhibitor at the global gene expression level, and the results support the importance of AP-1 as a key regulator of the expression of genes involved in the inflammatory response of epithelial cells to oxidative damage.

Keywords::

• oxidative stress
• apoptosis
• cAMP
• microarrays
• AP-1

Acknowledgements

This work was supported by grants SAF2005 - 00669/SAF2008 - 03113 (JC), PI10/02294 (MM) and CIBERES (CB06/06/0027) from the Ministry of Science and Innovation and Health Institute ‘Carlos III’ of Spain and by research grants from the Regional Government (GV2007/287 and AP073/10, from Generalitat Valenciana) SAF2009-08913 (EJM) and Prometeo/2008/045 (EJM).

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This paper was first published online on Early Online on 14 March 2012.