Abstract 710: A Novel Mechanism by Which CPI-17 Is Down-regulated in Cultured Vascular Smooth Muscle Cells and Tissues by TNFα and in C57BL/6 Mice by Lipopolysaccharide
Objectives: Sepsis is characterized by a severe inflammatory response to infection, and its complications, including hypotension, can be fatal. It has long believed that hypotension in sepsis occur as a result of failure of the vascular smooth muscle cells (VSMC) to constrict. However, the molecular mechanism that links inflammation and decreases in vasoconstriction is largely unknown.
Approaches and Results: CPI-17, a key regulator in vasoconstriction, was markedly decreased in mesentery arteries (MA) in mice injected with lipopolysaccharide (LPS). Incubation of cultured MA or VSMC with TNFα had similar effects on CPI-17 as LPS injection in mice. To identify transcriptional factors that respond to TNFα to suppress CPI-17 expression, we cloned a 1 kb murine CPI-17 promoter (-1,015 to +2 bp). Promoter deletion analysis revealed that a CPI-17 promoter region (-115 to -50 bp) was critical for TNFα to inhibit CPI-17 promoter activity. Bioinformatics analysis revealed this region contained 3 GC boxes that are putative binding sites for transcription factors SP1 and KLF4. Mutation of the 2nd and 3rd GC boxes, but not 1st GC box, abolished TNFα-mediated inhibitory effect on CPI-17 promoter activity. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) illustrated that SP1 and KLF4 bound to the 2nd and 3rd GC boxes, but not 1st GC box. Interestingly, TNFα had no effect on SP1 expression but suppressed SP1 binding to the CPI-17 promoter. In contrast, TNFα increased KLF4 expression and KLF4 binding to the CPI-17 promoter. Down-regulation of either SP1 or KLF4 by siRNA abolished inhibition of CPI-17 promoter activity by TNFα but the effects were opposite: SP1 siRNA decreased CPI-17 promoter activity whereas KLF4 siRNA increased CPI-17 promoter activity. Moreover, KLF4 up-regulation, but not SP1, was also found in MA in mice injected with LPS. KLF4 up-regulation by LPS preceded CPI-17 down-regulation and correlated with a decrease in vasoconstriction and hypotension.
Conclusions: These studies reveal a novel mechanism by which CPI-17 is down-regulated by LPS/TNFα through SP1 and KLF4 in vascular wall and identify SP1, KLF4, and CPI-17 as new potential therapeutic targets for treatment of hypotension in patients with sepsis.
Author Disclosures: G. Zhao: None. S. Liu: None. W. Su: None. M.C. Gong: None. Z. Guo: None.
This research has received full or partial funding support from the American Heart Association.
- © 2015 by American Heart Association, Inc.