Abstract
Lysophosphatidylcholine (LPC) may accumulate in the heart to cause fibrotic events, which is mediated through fibroblast activation and collagen accumulation. Here, we evaluated the mechanisms underlying LPC-mediated collagen induction via mitochondrial events in human cardiac fibroblasts (HCFs), coupling application of the pharmacologic cyclooxygenase-2 (COX-2) inhibitor, celecoxib, and genetic mutations in FOXO1 on the fibrosis pathway. In HCFs, LPC caused prostaglandin E2 (PGE2)/PGE2 receptor 4 (EP4)-dependent collagen induction via activation of transcriptional activity of forkhead box protein O1 (FoxO1) on COX-2 gene expression. These responses were mediated through LPC-induced generation of mitochondrial reactive oxygen species (mitoROS), as confirmed by ex vivo studies, which indicated that LPC increased COX-2 expression and oxidative stress. LPC-induced mitoROS mediated the activation of protein kinase C (PKC)α, which interacted with and phosphorylated dynamin-related protein 1 (Drp1) at Ser616, thereby increasing Drp1-mediated mitochondrial fission and mitochondrial depolarization. Furthermore, inhibition of PKCα and Drp1 reduced FoxO1-mediated phosphorylation at Ser256 and nuclear accumulation, which suppressed COX-2/PGE2 expression and collagen production. Moreover, pretreatment with celecoxib or COX-2 siRNA suppressed WT FoxO1; mutated Ser256-to-Asp256 FoxO1-enhanced collagen induction, which was reversed by addition of PGE2 Our results demonstrate that LPC-induced generation of mitoROS regulates PKCα-mediated Drp1-dependent mitochondrial fission and COX-2 expression via a PKCα/Drp1/FoxO1 cascade, leading to PGE2/EP4-mediated collagen induction. These findings provide new insights about the role of LPC in the pathway of fibrotic injury in HCFs.
Highlights
Lysophosphatidylcholine (LPC) may accumulate in the heart to cause fibrotic events, which is mediated through fibroblast activation and collagen accumulation
The phosphorylation of dynamin-related protein 1 (Drp1) Ser616 was inhibited after pretreatment with MitoTEMPO, mdivi-1, or Gö 6976, but not with SP600125 (Fig. 5F, G). These results suggested that LPC induced mitochondrial reactive oxygen species (mitoROS)-dependent protein kinase C (PKC) -Drp1 activation and, contributed to mitochondrial depolarization that was associated with mitochondrial fission in human cardiac fibroblasts (HCFs)
The transcriptional activity of p-forkhead box protein O1 (FoxO1) Ser256 was attenuated by these inhibitors (Fig. 6D). These results suggested that LPC-enhanced FoxO1 transcriptional activity on COX-2 expression is mediated via a mitoROS-PKC Drp1-JNK1/2-dependent cascade in HCFs
Summary
Lysophosphatidylcholine (LPC) may accumulate in the heart to cause fibrotic events, which is mediated through fibroblast activation and collagen accumulation. In HCFs, LPC caused prostaglandin E2 (PGE2)/PGE2 receptor 4 (EP4)-dependent collagen induction via activation of transcriptional activity of forkhead box protein O1 (FoxO1) on COX-2 gene expression. These responses were mediated through LPC-induced generation of mitochondrial reactive oxygen species (mitoROS), as confirmed by ex vivo studies, which indicated that LPC increased COX-2 expression and oxidative stress. We investigated to Abbreviations: CF, cardiac fibroblast; COX-2, cyclooxygenase-2; DMEM/F-12, DMEM/nutrient mixture F-12; Drp, dynamin-related protein 1; ECM, extracellular matrix; EP, prostaglandin E2 receptor; EtOH, ethanol; FoxO1, forkhead box protein O1; HCFs, human cardiac fibroblasts; IL-6, interleukin-6; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-. Tetraethylbenzimidazolocarbocyanine iodide; LPC, lysophosphatidylcholine; mitoROS, mitochondrial reactive oxygen species; PG, prostaglandin; PGE2, prostaglandin E2; quantitative PCR; ROS, reactive oxygen
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