Intracellular tPA–PAI-1 interaction determines VLDL assembly in hepatocytes

Apolipoprotein B (apoB)–lipoproteins initiate and promote atherosclerotic cardiovascular disease. Plasma tissue plasminogen activator (tPA) activity is negatively associated with atherogenic apoB-lipoprotein cholesterol levels in humans, but the mechanisms are unknown. We found that tPA, partially through the lysine-binding site on its Kringle 2 domain, binds to the N terminus of apoB, blocking the interaction between apoB and microsomal triglyceride transfer protein (MTP) in hepatocytes, thereby reducing very-low-density lipoprotein (VLDL) assembly and plasma apoB-lipoprotein cholesterol levels. Plasminogen activator inhibitor 1 (PAI-1) sequesters tPA away from apoB and increases VLDL assembly. Humans with PAI-1 deficiency have smaller VLDL particles and lower plasma levels of apoB-lipoprotein cholesterol. These results suggest a mechanism that fine-tunes VLDL assembly by intracellular interactions among tPA, PAI-1, and apoB in hepatocytes. Description Editor’s summary Apolipoprotein B (apoB) is a protein that carries lipids and cholesterol. Lipoproteins composed of apoB and its lipid cargo play a key role in the pathogenesis of atherosclerosis. Tissue plasminogen activator (tPA), an anticoagulant protein that can be used to break down a blood clot causing a heart attack or stroke, inversely correlates with the amount of cholesterol-loaded apoB lipoproteins, but it was not clear why this inverse correlation exists. Dai et al. discovered that in both mouse and human cells, tPA produced by the liver binds directly to apoB, preventing it from being loaded with lipids and assembled into lipoproteins. Conversely, an inhibitor of tPA has the opposite effect, binding up free tPA and preventing its interaction with apoB. —Yevgeniya Nusinovich Intracellular tissue plasminogen activator in the endoplasmic reticulum of hepatocytes lowers circulating atherogenic cholesterol levels. INTRODUCTION Tissue plasminogen activator (tPA) is a serine protease that initiates fibrinolysis to remove excessive blood clots and restore blood flow. Intravenous infusion of recombinant tPA is approved as a thrombolytic therapy in thrombotic cardiovascular diseases, including ischemic stroke and myocardial infarction. Low plasma tPA activity is associated with a higher risk of atherosclerotic cardiovascular disease and atherogenic apolipoprotein B (apoB)–lipoprotein cholesterol levels in humans. However, whether low tPA elevates apoB-lipoprotein cholesterol is unknown. RATIONALE Given the central role of hepatocytes in apoB-lipoprotein production and recent studies showing that hepatocytes synthesize tPA, we sought to identify possible links between tPA and apoB-lipoprotein assembly and secretion from hepatocytes. RESULTS To investigate the role of hepatocyte tPA in apoB-lipoprotein metabolism, tPA expression was silenced or deleted in the hepatocytes of mice. These manipulations resulted in higher plasma apoB and cholesterol levels, independent of any changes in hepatic low-density lipoprotein receptor (LDLR) or apolipoprotein E (apoE) expression or Apob mRNA level. The higher plasma cholesterol in these mice was distributed in the very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) fractions. In human primary hepatocytes, silencing tPA increased the secretion of newly synthesized [3H]-labeled apoB despite no change in Apob mRNA. Thus, hepatocyte-tPA deficiency increases the secretion of apoB-lipoproteins. Lipidation is a key factor determining the fate of intrahepatic apoB. Poorly lipidated apoB undergoes intracellular degradation, whereas fully lipidated apoB is efficiently secreted as VLDL particles with larger size and lower density. Silencing hepatocyte tPA in Ldlr−/− mice led to larger VLDL particles in the plasma, with more triglyceride per VLDL particle, indicating increased intrahepatic apoB lipidation. apoB is lipidated in the endoplasmic reticulum (ER) by microsomal triglyceride transfer protein (MTP), which incorporates neutral lipids onto nascent apoB. Transfecting human primary hepatocytes with a plasmid encoding tPA led to lower secretion of newly synthesized [3H]-labeled apoB. Proximity ligation, confocal imaging, and immunoprecipitation assays revealed that tPA interacts with apoB in the hepatocyte ER. In addition, recombinant tPA interacts with solid-phase immobilized LDL, inhibits MTP-apoB interaction, and reduces neutral lipid transfer to apoB. Moreover, the serine protease inactive tPA (S513A) also interacts with solid-phase immobilized LDL, reducing both apoB secretion by human primary hepatocytes and MTP-mediated lipid transfer activity to the same degree as wild-type tPA, which indicates that this action of tPA is independent of its protease activity. The tPA-LDL interaction is inhibited by antibodies against the Kringle 2 (K2) domain of tPA, the MTP-interacting regions at the N terminus of apoB, and the lysine analog tranexamic acid. Further, deleting the K2 domain or mutating the lysine-binding site in the K2 domain of tPA abrogates the effects of tPA on limiting apoB secretion. These data indicate that tPA, partially through the lysine-binding site on its K2 domain, binds to the N terminus of apoB, blocking the interaction between apoB and MTP in hepatocytes. This process reduces VLDL assembly and plasma apoB-lipoprotein cholesterol levels. Plasminogen activator inhibitor 1 (PAI-1) is a rapidly acting serine protease inhibitor (serpin) of tPA. Upon lipid loading of hepatocytes, PAI-1 forms a complex with tPA and sequesters tPA away from apoB, which allows apoB to be lipidated and facilitates VLDL assembly and secretion. Consistent with these findings, humans with PAI-1 deficiency have smaller VLDL particles and lower plasma levels of apoB-lipoprotein cholesterol. CONCLUSION The findings in this study suggest a mechanism that fine-tunes VLDL assembly through intracellular interactions among tPA, PAI-1, and apoB in hepatocytes, thereby affecting the plasma levels of atherogenic apoB-lipoproteins. Knowledge of this mechanism of hepatic lipoprotein regulation may suggest therapeutic strategies for lowering atherogenic apoB-lipoproteins and cardiovascular risk. Intracellular tPA–PAI-1 interaction in the ER of hepatocytes determines apoB lipidation, VLDL assembly, and secretion. In the basal state, tPA interacts with apoB and inhibits MTP-apoB interaction in the ER of hepatocytes, limiting MTP-mediated apoB lipidation, VLDL assembly, and secretion. When hepatocytes are loaded with lipid, PAI-1 sequesters tPA away from apoB by forming a complex with tPA, allowing the lipidation of apoB and the assembly and secretion of VLDL. [Figure created with Biorender]