PECAM-1-Dependent Regulation of Heme Oxygenase-1 via the Antioxidant Nrf2 Pathway in the Endothelium

Abstract

Heme oxygenase-1 (HO-1) is the inducible isoform of the first and rate-limiting enzyme of heme degradation and has major anti-inflammatory and immunomodulatory functions. Overexpression of HO-1 in endothelial cells (EC) provides major protection against graft rejection in experimental animal transplantation models. Platelet EC adhesion molecule (PECAM)-1 is a multifunctional cell adhesion receptor that belongs to the immunoglobulin-like (Ig) superfamily and is highly expressed on EC. In addition to its role in modulating leukocyte transmigration through the endothelial monolayer, PECAM-1 has more recently been shown to be a major regulator of intracellular signaling. To investigate whether PECAM-1 might be involved in endothelial regulation of HO-1, we performed a knockdown in human umbilical vein EC (HUVEC) with an adenoviral-based vector approach encoding specific PECAM-1 shRNA sequences. Knockdown of PECAM-1 caused a marked up-regulation of HO-1 expression, but not that of two other inducible genes, Bcl-2 and cyclooxygenase-2, as determined by real time PCR and immunofluorescence studies. PECAM-1 deficiency in HUVEC led to increased intracellular levels of reactive oxygen species (ROS) and to nuclear translocation of the transcription factor Nrf2, which is a key regulator of the cellular antioxidant response. In addition, overexpression of PECAM-1 in two stably transfected cell lines (Chinese hamster ovary cells and L-cells) led to a marked down-regulation of HO-1 gene expression. Similarly, reporter gene activity of a HO-1 promoter gene construct with a specific Nrf2 target sequence was down-regulated in PECAM-1 overexpressing cells. In conclusion, these findings show a regulatory link of the signaling receptor PECAM-1 to HO-1 gene expression in the endothelium and may afford novel therapeutic approaches in organ transplantation.