Abstract 483: EGR1 Mediates Suppression of PKD1 Expression, an Action Antagonized by Pioglitazone in Differentiated Angiotensin II-Treated THP-1 Cells

Abstract

Aims: Perixosome proliferator-activated receptor γ (PPARγ) agonists have been shown to inhibit Angiotensin II-induced experimental abdominal aortic aneurysms. Since increased macrophage infiltration in the vascular wall is a hallmark for initiation of aneurysmal pathology, we set out to explore the effects of the PPARγ agonist pioglitazone on Angiotensin II-treated, phorbol-ester-differentiated monocytes, with the scope to identify molecular changes that may be responsible for the infiltrating phenotype of macrophages. Approach and Results: Using microarray-based expression profiling of phorbol-ester-stimulated THP-1 monocytic leukemia cells, we found that Angiotensin II (Ang II) or Ang II and pioglitazone were able to cause a number of aneurysm-related gene changes. Among those, polycystic kidney disease 1 (PKD1) was significantly up-regulated. Mutations that lead to changes in PKD1 expression are the main cause of autosomal dominant polycystic kidney disease, which is linked to increased incidence of aneurysms. Online-software analysis of the PKD1 proximal promoter revealed a putative Early Growth Response 1 (EGR1) binding site, which we confirmed by chromatin immuneprecipitation (ChIP) and quantitative real time (RT) PCR. Further analyses of publicly available ChIP-sequencing data (ENCODE project) revealed that this putative binding site in the PKD1 proximal promoter overlapped with a conserved EGR1-binding peak present in 5 other cell lines. Quantitative RT-PCR showed that EGR1 suppressed PKD1, while Ang II significantly up-regulated PKD1, an effect counteracted by pioglitazone. Conversely, in THP-1 cells that were lenti-virally transduced with EGR1 short-hairpin RNA, reduced EGR1 levels led to significant up-regulation of PKD1, especially after treatment with pioglitazone. Conclusion: PKD1 is an EGR1-target gene that is suppressed during monocytic differentiation at the time of EGR1 expression. Ang II treatment up-regulates the expression of PKD1 and pioglitazone mediates the opposite effect by displacement of EGR1 from their shared binding site in the PKD1 proximal promoter through a mechanism that requires further investigation.