ABSTRACT
Extracellular adenosine triphosphate (eATP) released by damaged cells, and its purinergic receptors, comprise a crucial signaling network after injury. Purinergic receptor P2X7 (P2RX7), a major driver of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and IL-1ß processing, has been shown to play a role in liver injury in murine diet- and chemically-induced liver injury models. It is unclear, however, whether P2RX7 plays a role in non-alcoholic steatohepatitis (NASH) and which cell type is the main target of P2RX7 pharmacological inhibition. Here, we report that P2RX7 is expressed by infiltrating monocytes and resident Kupffer cells in livers from NASH-affected individuals. Using primary isolated human cells, we demonstrate that P2RX7 expression in CD14+ monocytes and Kupffer cells primarily mediates IL-1ß release. In addition, we show that pharmacological inhibition of P2RX7 in monocytes and Kupffer cells, blocks IL-1ß release, reducing hepatocyte caspase 3/7 activity, IL-1ß-mediated CCL2 and CCL5 chemokine gene expression and secretion, and hepatic stellate cell (HSC) procollagen secretion. Consequently, in a chemically-induced nonhuman primate model of liver fibrosis, treatment with a P2RX7 inhibitor improved histological characteristics of NASH, protecting from liver inflammation and fibrosis. Taken together, these findings underscore the critical role of P2RX7 in the pathogenesis of NASH and implicate P2RX7 as a promising therapeutic target for the management of this disease.
Subject(s)
Inflammation/prevention & control , Liver Cirrhosis/drug therapy , Purinergic P2X Receptor Antagonists/therapeutic use , Receptors, Purinergic P2X7/metabolism , Animals , Caspase 3/metabolism , Cells, Cultured , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Hepatic Stellate Cells/cytology , Hepatic Stellate Cells/drug effects , Hepatic Stellate Cells/metabolism , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Inflammation/pathology , Interleukin-1beta/metabolism , Kupffer Cells/cytology , Kupffer Cells/drug effects , Kupffer Cells/metabolism , Lipopolysaccharides/pharmacology , Liver/metabolism , Liver/pathology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/pathology , Macaca fascicularis , Male , Monocytes/cytology , Monocytes/drug effects , Monocytes/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Procollagen/metabolism , Purinergic P2X Receptor Antagonists/pharmacology , Receptors, Purinergic P2X7/chemistry , Receptors, Purinergic P2X7/geneticsABSTRACT
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a soluble protein that directs membrane-bound receptors to lysosomes for degradation. In the most studied example of this, PCSK9 binding leads to the degradation of low density lipoprotein receptor (LDLR), significantly affecting circulating LDL-C levels. The mechanism mediating this degradation, however, is not completely understood. We show here that LDLR facilitates PCSK9 interactions with amyloid precursor like protein 2 (APLP2) at neutral pH leading to PCSK9 internalization, although direct binding between PCSK9 and LDLR is not required. Moreover, binding to APLP2 or LDLR is independently sufficient for PCSK9 endocytosis in hepatocytes, while LDL can compete with APLP2 for PCSK9 binding to indirectly mediate PCSK9 endocytosis. Finally, we show that APLP2 and LDLR are also required for the degradation of another PCSK9 target, APOER2, necessitating a general role for LDLR and APLP2 in PCSK9 function. Together, these findings provide evidence that PCSK9 has at least two endocytic epitopes that are utilized by a variety of internalization mechanisms and clarifies how PCSK9 may direct proteins to lysosomes.
