Introduction: Receptor interacting kinase 3 (RIP3/ RIPK3) plays a decisive role in necroptosis. The upregulated levels of RIP3 found in atherosclerosis and aneurysm is likely to skew the cellular injury response from apoptosis toward necroptosis. Necroptosis of vascular smooth muscle cells (VSMCs) may contribute to disease progression directly by weakening the fibrosis cap/aortic wall of atherosclerotic plaques/aneurysmal aorta as well as indirectly by amplifying inflammation. Although RIP3 ablation protects against these diseases conditions, our knowledge regarding Rip3 gene regulation is nearly blank.
Objective: to define the signaling and transcriptional mechanisms underlying Rip3 regulation in vascular smooth muscle cells
Results: We treated mouse VSMCs with various cytokines and observed TNFα, LPS and Poly (I:C) increased RIP3 expression. To study the mechanism by which inflammation might upregulate RIP3, we analyzed the proximal and distal (Fig 1A) regions of Rip3 gene and identified a highly conserved GAS-motif in a distal region. In chromatin immunoprecipitation assay, binding of STAT3 to the GAS-motif was decreased by ~60% upon PKCδ gene deletion but increased by 6.2 fold upon PMA treatment which activates PKC-signaling (Fig 1B). Further, RIP3 upregulation in mouse VSMCs was lost when PKCδ was ablated; conversely, activation of PKCδ signaling could not induce RIP3 when STAT3 was knocked down (Fig 1C). Together, our data suggest PKCδ dependent phosphorylation of STAT3 at S727 causes its binding to the distal enhancer and may regulate RIP3 expression in a cytokine-inducible manner. Our study also warrants investigation into STAT3-inhibition to prevent vascular diseases.