Mechanisms and redox effects of epi/pericellular protein disulfide isomerase

Abstract

Protein disulfide isomerase (PDI) is a thioredoxin family redox chaperone from the endoplasmic reticulum. Our group has previously shown that PDI associates with the NADPH oxidase complex and regulates its function, so that PDI has a role events such as vascular smooth muscle cell migration. Moreover, PDI can translocate towards the cell surface or peri-cellular compartment (epcPDI) and, in this location, it can promote changes in thiol redox state of several membrane or secreted proteins associated with the regulation of processes such as blood coagulation, platelet function, cell adhesion, proteolysis, immune response and viral infection. EpcPDI also promotes internalization of NO via transnitrosation. Mechanisms of cellular effects of epcPDI are yet unclear and poorly explored. Studies from our group indicate that in vivo epcPDI inhibition in rabbit iliac arteries post-injury induces decrease in extracellular reactive oxygen species generation and significant decrease in vascular caliber via constrictive By analogy with known PDI functions in the endoplasmic reticulum, it is possible that epcPDI promotes folding or oligo/multimerization of proteins at the cell surface or peri-cellular milieu. Studying the mechanisms of these processes in vivo brings about intrinsic technical limitations. The aim of our project is to develop, in cultured primary vascular smooth muscle cells, suitable models to understand which type of stimulus promote secretion/translocation of PDI towards the cell surface or pericellular milieu. Our preliminary data show that epcPDI is detectable by immunofluorescence. We will addresss multiple types of stimuli with the aim of modulating epcPDI expression, including growth factors, serum deprivation, calcium ionophore, endoplasmic reticulum stressors, cytokines (TNFalpha, TGFbeta) and hypoxia. EpcPDI will be characterized with respect to redox state, reductase activity and capacity to modulate (protein) thiol redox state at the cell surface. These results will be compared to those obtained in cells with low epcPDI amount, achieved either constitutively or via imuno-inhibition of epcPDI. An important question to be addressed focuses on possible routes of externalization of PDI, which will provide evidence for possible specific types of substrates. The in-depth understanding of mechanisms of epcPDI function will help understand the implications of the apparently central function of epcPDI in the pathophysiology of vascular remodeling.