In a family of 11 isoforms, protein kinase C-epsilon (PKC-?) has unique binding and regulatory properties that cannot be compensated for by other PKCs. It is involved in neurological, vascular, and wound healing as evidenced from studies in the global PKC-? knockout mouse. Notably, the global KO has underlying immune defects that prevent homozygous breeding and PKC-? KO mice succumb to infections cleared by their wild type counterparts. As virtually every disease has an immune/inflammatory component, the results from in vivo studies with the KO mouse must be interpreted in the context of the unknown effects of the impaired immune system. Thus, there is an unmet need for a ?cleaner? mouse in which to study the role of PKC-? in disease, one in which PKC-? can be deleted in a tissue specific manner. That is, a PKC-?loxP/loxP (flox?d) mouse. This application will generate a PKC-? floxed mouse, which, when crossed to a tissue-selective Cre, will specifically delete PKC-? in the cells of choice. The significance is that PKC-? flox?d mice will have an intact immune system that, when crossed to a Cre of choice, will produce offspring lacking PKC-? only in the tissue of interest.? The PKC-? flox?d mouse can be considered a gateway strain, providing a tool for other investigators to study PKC-? in their model of choice independently of the immune defects documented for the global knockout. We will cross them to reporter mice expressing lox-STOP-lox ZsGreen and LysM-Cre. The resulting mouse will have PKC-? selectively deleted in macrophages (M) and neutrophils, which will also express the ZsGreen reporter. Our preliminary data indicate that M from PKC-???? mice accumulate more, and larger, lipid droplets and produce more TNF-?, but less ResolvinD1, in response to lipid feeding or immune complexes. We hypothesize that PKC-? expression in M will slow atherosclerosis development. In vivo, we will examine the role of M PKC-? in the AAV8-PCSK9 model of atherosclerosis, using PKC-ef/fLysM-Cre+/ mice. AAV8- PCSK9 is a gain of function virus expressing a mutant that produces hypercholesterolemia and atherosclerosis in mice. The mutant PCSK9 gene is expressed in humans with hypercholesterolemia, bringing translational relevance to the model. Aortic root plaques from WT and PKC-?f/fLysM-Cre+/ mice will be scored for metrics of plaque stability applied to the human disease. Imaging will be used to quantify the number and localization of plaque M, their polarization state (immunofluorescence for markers of M1, M2, and Mox) and lipid content (Oil Red O staining). Additionally, serum levels of cytokines and lipid mediators will be quantified. In vitro studies will identify specific steps in foam cell formation in which PKC-? acts and will define the signature of cytokines and lipid mediators produced by WT and PKC-?f/fLysM-Cre+/ M in response to immune complexes and apoptotic cells. In conclusion, we have identified PKC-? as a novel player in regulating lipid retention and inflammation in an atherogenic environment. These studies will identify atherosclerosis-relevant pathways that are regulated by macrophage PKC-?, pathways that can be examined in detail in a future R01.
Protein kinase C-epsilon has unique properties that endow it with non-redundant functions; its? dysregulation correlates with a variety of human diseases. Notably, it is expressed in hematopoetic stem cells and PKC-? null mice succumb to infections cleared by their wild type counterparts, suggesting that the immune system of PKC-? null mice is compromised. This R03 will generate PKC-? flox?d mice with an intact immune system that can be crossed to Cre mice for tissue-specific deletion of PKC-? for studies on the role of PKC-? in atherosclerosis (our studies) or other diseases (e.g., cancer, infection, tissue fibrosis).
