Signaling pathways regulating the extracellular digestion of lipoprotein aggregates by macrophages

The interaction between aggregated low-density lipoprotein (agLDL) and macrophages in arteries plays a major role in atherosclerosis. Macrophages 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. Our laboratory has identified TLR4 activation of MyD88/Syk as critical for digestive exophagy. Here we use pharmacological agents and siRNA knockdown to characterize signaling pathways downstream of Syk that are involved in digestive exophagy. Syk activates Bruton's tyrosine kinase (BTK) and phospholipase Cγ2 (PLCγ2). We show that PLCγ2 and to a lesser extent BTK regulate digestive exophagy. PLCγ2 cleaves PI(4,5)P₂ into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃). Soluble IP₃ activates release of Ca²⁺ from the endoplasmic reticulum (ER). We demonstrate that Ca²⁺ release from the ER is upregulated by agLDL and plays a key role in digestive exophagy. Both DAG and Ca²⁺ activate protein kinase Cα (PKCα). We find that PKCα is an important regulator of digestive exophagy. These results expand our understanding of the mechanisms of digestive exophagy, which could be useful in developing therapeutic interventions to slow development of atherosclerosis.