Upregulation of COX-2 and PGE2 Induced by TNF-α Mediated Through TNFR1/MitoROS/PKCα/P38 MAPK, JNK1/2/FoxO1 Cascade in Human Cardiac Fibroblasts

Chuen-Mao Yang; Chien-Chung Yang; Li-Der Hsiao; Chia-Ying Yu; Hui-Ching Tseng; Chih-Kai Hsu; Jiro Hasegawa Situmorang

doi:10.2147/JIR.S313665

Upregulation of COX-2 and PGE₂ Induced by TNF-α Mediated Through TNFR1/MitoROS/PKCα/P38 MAPK, JNK1/2/FoxO1 Cascade in Human Cardiac Fibroblasts

J Inflamm Res. 2021 Jun 28:14:2807-2824. doi: 10.2147/JIR.S313665. eCollection 2021.

Authors

Chuen-Mao Yang^{1

2

3}, Chien-Chung Yang^{4

5}, Li-Der Hsiao¹, Chia-Ying Yu¹, Hui-Ching Tseng¹, Chih-Kai Hsu¹, Jiro Hasegawa Situmorang¹

Affiliations

¹ Department of Pharmacology, College of Medicine, China Medical University, Taichung, 40402, Taiwan.
² Ph.D. Program for Biotech Pharmaceutical Industry, China Medical University, Taichung, 40402, Taiwan.
³ Department of Post-Baccalaureate Veterinary Medicine, College of Medical and Health Science, Asia University, Wufeng, Taichung, 41354, Taiwan.
⁴ Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital at Tao-Yuan, Kwei-San, Tao-Yuan, 33302, Taiwan.
⁵ School of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, 33302, Taiwan.

Abstract

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 E₂ (PGE₂) axis. However, whether TNF-α-induced COX-2/PGE₂ 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/PGE₂ 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 PGE₂ 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/PGE₂ 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.

Keywords: COX-2; FoxO1; PGE2; PKC-α; TNF-α; mitochondrial ROS.

Grants and funding

This work was supported by the Ministry of Science and Technology, Taiwan [Grant numbers: MOST108-2320-B-039-061, MOST109-2320-B-039-061, MOST109-2813-C-039-029-B, and MOST108-2320-B-182-014]; China Medical University, Taiwan [Grant number: CMU109-MF-09]; Chang Gung Medical Research Foundation, Taiwan [Grant numbers: CMRPG5F0203, CMRPG5J0142, and CMRPG5J0143].