MAPK and JAK-STAT Signaling Pathways are Involved in the Oxidative Stress – Induced Decrease in Expression of Surfactant Protein Genes

Abstract

Oxidative stress is generated by reactive oxygen species (ROS) including hydrogen peroxide (H₂O₂), hydroxyl radical (•OH ) and superoxide anion (O^2––), which are produced as by-products of cellular metabolism. An imbalance in cellular redox status is a potent pathogenic factor that contributes to various chronic inflammatory diseases. In this study, we demonstrate that H₂O₂decreases surfactant protein A, B and ABCA3 mRNA level, and increases SP-D mRNA level in human pulmonary lung epithelial cells. The decreased mRNA level of SP-A and SP-B were significant with a maximum inhibition of 79 and 87%, respectively by 150 µM H₂O₂after 24 hrs of incubation. In addition, ABCA3 mRNA level was decreased with a maximum inhibition of 55% by 150 µM H₂O₂after 12 hrs of incubation. In contrast, 150 µM H₂O₂caused the SP-D mRNA level to increase to 200% of control after 8 hrs of incubation. The H₂O₂-induced gene repression or activation of SP-A, SP-B, SP-D and ABCA3 was blocked by pretreatment with the antioxidants N-acetyl-L-cysteine (NAC) and catalase. Furthermore, the inhibition of SP-A and SP-B was associated with reduced thyroid transcription factor -1 (TTF-1) DNA binding activity, and this reduced TTF-1 binding activity may be due to decreased TTF-1 protein expression level. The analyses of signal transduction pathways that may play a role in the regulation of gene expression by H₂O₂using several specific inhibitors showed that U0126, an inhibitor of ERK1/2 upstream kinase MEK1/2, blocked both H₂O₂-induced inhibition of SP-A and SP-B gene expression, whereas SB203580, an inhibitor of p38 MAPK, partially blocked H₂O₂-mediated inhibition of SP-A gene expression but not SP-B expression. In contrast, AG-490, a specific inhibitor of JAK-STAT pathway, blocked H₂O₂-mediated inhibition of SP-B gene expression but not SP-A expression. Immunoblot analyses using specific phosphor-antibodies demonstrated that ERK1/2, p38 MAPK and STAT3 are phosphorylated by oxidative stress suggesting that H₂O₂-induced inhibition of SP-A and SP-B gene expression is associated with MAPK and JAKSTAT signaling pathway. These data, therefore, suggest that H₂O₂affects SP-A and SP-B gene regulation by reducing TTF-1 DNA binding activity via MAPKs or STAT signaling pathways.