Abstract 1061: The Phospholipase cγ2 And Protein Kinase cα Signaling Axis Is A Key Regulator Of The Extracellular Digestion Of Lipoprotein Aggregates By Macrophages

Abstract

Atherosclerosis is characterized by cholesterol ester accumulation in macrophages, leading to foam cell formation. In atherosclerotic lesions, most lipoproteins are aggregates (agLDL) that are tightly crosslinked to the extracellular matrix, making digesting them by endocytosis or phagocytosis difficult. Thus, it has been unclear how the cholesteryl esters in the core of agLDL are hydrolyzed. Our laboratory discovered a novel mechanism macrophages use to digest agLDL and generate free cholesterol in an extracellular, acidic, hydrolytic compartment known as the lysosomal synapse. Macrophages form a tight seal around agLDL through actin polymerization and deliver lysosomal contents into this space in a process termed digestive exophagy. Vacuolar ATPase on the plasma membrane lowers the pH of the lysosomal synapse, enabling lysosomal acid lipase (LAL) activity. LAL generates free cholesterol, which accumulates in macrophages and leads to foam cell formation. Our laboratory has begun to characterize the signaling pathways that regulate digestive exophagy, having previously identified TLR4 activation of MyD88/Syk as critical. Here, our goal was to identify digestive exophagy regulators downstream of Syk. Syk activates Bruton’s tyrosine kinase (BTK) and phospholipase Cγ2 (PLCγ2). We tested the hypothesis that Syk phosphorylates and activates PLCγ2 during digestive exophagy. Using pharmacological treatment and genetic manipulation, we found that PLCγ2 and to a lesser extent BTK regulate digestive exophagy. We confirmed that inhibition of Syk or BTK leads to a loss of PLCγ2 activation. PLCγ2 cleaves PI(4,5)P 2 into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3 ). Soluble IP 3 then activates the release of Ca 2+ from the endoplasmic reticulum (ER) into the cytoplasm. We demonstrated that Ca 2+ release from the ER is upregulated by agLDL and plays a role in digestive exophagy. Both DAG and Ca 2+ activate protein kinase Cα (PKCα). We also found that PKCα regulates digestive exophagy. In conclusion, we identified new signaling mechanisms regulating digestive exophagy downstream of Syk. We hope that expanding our understanding of the mechanisms of digestive exophagy will be helpful in identifying therapeutics to slow atherosclerosis.