PKCε-CREB-Nrf2 signalling induces HO-1 in the vascular endothelium and enhances resistance to inflammation and apoptosis

Aims Vascular injury leading to endothelial dysfunction is a characteristic feature of chronic renal disease, diabetes mellitus, and systemic inflammatory conditions, and predisposes to apoptosis and atherogenesis. Thus, endothelial dysfunction represents a potential therapeutic target for atherosclerosis prevention. The observation that activity of either protein kinase C epsilon (PKC epsilon) or haem oxygenase-1 (HO-1) enhances endothelial cell (EC) resistance to inflammation and apoptosis led us to test the hypothesis that HO-1 is a downstream target of PKC epsilon. Methods and results Expression of constitutively active PKC epsilon in human EC significantly increased HO-1 mRNA and protein, whereas conversely aortas or cardiac EC from PKC epsilon-deficient mice exhibited reduced HO-1 when compared with wild-type littermates. Angiotensin II activated PKC epsilon and induced HO-1 via a PKC epsilon-dependent pathway. PKC epsilon activation significantly attenuated TNF alpha-induced intercellular adhesion molecule-1, and increased resistance to serum starvation-induced apoptosis. These responses were reversed by the HO antagonist zinc protoporphyrin IX. Phosphokinase antibody array analysis identified CREB1((Ser133)) phosphorylation as a PKC epsilon signalling intermediary, and cAMP response element-binding protein 1 (CREB1) siRNA abrogated PKC epsilon-induced HO-1 up-regulation. Likewise, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was identified as a PKC epsilon target using nuclear translocation and DNA-binding assays, and Nrf2 siRNA prevented PKC epsilon-mediated HO-1 induction. Moreover, depletion of CREB1 inhibited PKC epsilon-induced Nrf2 DNA binding, suggestive of transcriptional co-operation between CREB1 and Nrf2. Conclusions PKC epsilon activity in the vascular endothelium regulates HO-1 via a pathway requiring CREB1 and Nrf2. Given the potent protective actions of HO-1, we propose that this mechanism is an important contributor to the emerging role of PKC epsilon in the maintenance of endothelial homeostasis and resistance to injury.