Abstract
Recently, we found that 5,8-dihydroxy-4′,7-dimethoxyflavone (DDF) upregulated the expression of heme oxygenase (HO)-1 via p38 mitogen-activated protein kinase/nuclear factor-erythroid factor 2-related factor 2 (MAPK/Nrf2) pathway in human cardiac fibroblasts (HCFs). However, the alternative processes by which DDF induces the upregulation of HO-1 expression are unknown. Activation of epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), and protein kinase C (PKC)α may initiate specificity protein (Sp)1 activity, which has been reported to induce expression of antioxidant molecules. Thus, we explored whether these components are engaged in DDF-induced HO-1 upregulation in HCFs. Western blotting, promoter-reporter analyses, and real-time polymerase chain reactions were adopted to measure HO-1 and vascular cell adhesion molecule (VCAM)-1 expressions in HCFs. Respective small interfering (si)RNAs and pharmacological inhibitors were employed to investigate the signaling components engaged in DDF-induced HO-1 upregulation. The chromatin immunoprecipitation assay was conducted to detect the binding interaction of Sp1 and antioxidant response elements (ARE) on the promoter of HO-1. An adhesion assay of THP-1 monocyte was undertaken to examine the functional effect of HO-1 on tumor necrosis factor (TNF)-α-induced VCAM-1 expression. DDF stimulated the EGFR/PKCα/PI3K/Akt pathway leading to activation of Sp1 in HCFs. The roles of these protein kinases in HO-1 induction were ensured by transfection with their respective siRNAs. Chromatin immunoprecipitation assays revealed the interaction between Sp1 and the binding site of proximal ARE on the HO-1 promoter, which was abolished by glutathione, AG1478, Gö6976, LY294002, or mithramycin A. HO-1 expression enhanced by DDF abolished the monocyte adherence to HCFs and VCAM-1 expression induced by TNF-α. Pretreatment with an inhibitor of HO-1: zinc protoporphyrin IX reversed these inhibitory effects of HO-1. We concluded that DDF-induced HO-1 expression was mediated via an EGFR/PKCα/PI3K/Akt-dependent Sp1 pathway and attenuated the responses of inflammation in HCFs.
Highlights
Fibroblasts are crucial components in the pathogenesis of hearts induced by chemical signals or mechanical forces
Our recent report has shown that reactive oxygen species (ROS) generation via GSH depletion mediates upregulated expression of heme oxygenase (HO)-1 induced by DDF in human cardiac fibroblasts (HCFs) [12]
We demonstrated that the tumor necrosis factor (TNF)-α-mediated adhesion of THP-1 monocytes associated with expression of vascular cell adhesion molecule (VCAM)-1 protein could be protected by increased protein expression of HO-1 generated by DDF, which was rescued by Zinc protoporphyrin (ZnPP) IX, one inhibitor of HO-1
Summary
Fibroblasts are crucial components in the pathogenesis of hearts (e.g., cardiac inflammation) induced by chemical signals or mechanical forces. Proinflammatory cytokines and mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and sphingosine 1-phosphate, promote the activation and proliferation of fibroblasts [1–3]. TNF-α overexpression-induced fibrotic cardiomyopathy and interactions between mast cells and fibroblasts are needed for the progress of cardiac fibrosis [4]. Turner et al [5] suggested that TNF-α increases the levels of IL-1β and IL-6 in cardiac fibroblasts. Our recent report indicated that TNF-α and sphingosine 1-phosphate induce cyclooxygenase-2/prostaglandin E2 upregulation in human cardiac fibroblasts (HCFs) [3, 6]. Those findings imply that TNF-α is a crucial factor in cardiovascular disorders
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
