Upregulation of COX-2 and PGE Induced by TNF-α Mediated Through TNFR1/MitoROS/PKCα/P38 MAPK, JNK1/2/FoxO1 Cascade in Human Cardiac Fibroblasts.

Purpose: Tumor necrosis factor-α (TNF-α) has been shown to exert as a pathogenic factor in cardiac fibrosis and heart failure which were associated with the up-regulation of cyclooxygenase (COX)-2/prostaglandin E (PGE) axis. However, whether TNF-α-induced COX-2/PGE upregulation mediated through ROS-dependent cascade remains elusive in human cardiac fibroblasts (HCFs). This study aims to address the underlying mechanisms of TNF-α-induced COX-2/PGE expression.

Methods: Here, we used TNF receptor neutralizing antibody (TNFR nAb), pharmacologic inhibitors, and siRNAs to dissect the involvement of signaling components examined by Western blot and ELISA in TNF-α-mediated responses in HCFs. MitoSOX Red was used to measure mitoROS generation. Isolation of subcellular fractions was performed to determine membrane translocation of PKCα. Promoter luciferase assay and chromatin immunoprecipitation (ChIP) assay were used to determine the role of transcription factor.

Results: We found that TNF-α time- and concentration-dependently upregulated COX-2 protein and mRNA expression as well as PGE synthesis which was attenuated by TNFR1 nAb, the inhibitor of mitochondrial ROS scavenger (MitoTEMPO), protein kinase C [(PKC)α, Gö6976], p38 MAPK [p38 inhibitor VIII, (p38i VIII)], JNK1/2 (SP600125), or forkhead box protein O1 [(FoxO1), AS1842856], and transfection with their respective siRNAs in HCFs. TNF-α-stimulated PKCα phosphorylation was inhibited by TNFR1 nAb, MitoTEMPO, or Gö6976. TNF-α stimulated phosphorylation of p38 MAPK and JNK1/2 was attenuated by TNFR1 nAb, MitoTEMPO, Gö6976, and their inhibitors p38i VIII and SP600125. Moreover, TNF-α-triggered FoxO1 phosphorylation was abolished by AS1842856, TNFR1 nAb, and its upstream inhibitors MitoTEMPO, Gö6976, p38i VIII, and SP600125. Phosphorylation of FoxO1 could enhance its interaction with the COX-2 promoter element revealed by ChIP assay, which was attenuated by AS1842856.

Conclusion: Our results suggested that TNF-α-induced COX-2/PGE upregulation is mediated through TNFR1-dependent MitoROS/PKCα/p38 MAPK and JNK1/2 cascade to activate FoxO1 binding with the COX-2 promoter in HCFs.