Endocytic mechanism of internalization of dietary peptide lunasin into macrophages in inflammatory condition associated with cardiovascular disease

PLoS One. 2013 Sep 5;8(9):e72115. doi: 10.1371/journal.pone.0072115. eCollection 2013.

Abstract

Cardiovascular disease (CVD) is the leading cause of death in the United States. Diet influences risk factors associated with CVD and atherosclerosis, a major vascular disease that arises from inflammation. Lunasin, a peptide derived from plant foods such as soybeans, contains a unique Arg-Gly-Asp cell-adhesion motif and inhibits the pathways involved in the inflammatory cascade. The objective was to determine the mechanism by which lunasin is internalized into human THP-1 macrophages, investigate the expression of endocytic membrane proteins in inflammatory conditions and to identify the pathways involved. While lipopolysaccharide (10 nM), vitronectin (130 nM) and a combination of these two molecules enhanced lunasin uptake and increased basal αVβ3 integrin expression, lunasin reduced αVβ3 expression by 25.5, 26.8 and 49.2%, respectively. The pretreatment of cells with brefeldin A (71 µM), an inhibitor of protein trafficking, inhibited lunasin internalization by up to 99.8%. Lunasin increased caveolin-1 expression by up to 204.8%, but did not modulate clathrin. The pretreatment of macrophages with nystatin (54 µM), an inhibitor of caveolae-dependent endocytosis, reduced lunasin internalization. The presence of amantadine (1 mM) and amiloride (1 mM), inhibitors of clathrin-mediated endocytosis and macropinocytosis, abolished lunasin cell entry. Lunasin elicited a transient reduction in intracellular levels of Ca²⁺ in LPS-induced macrophages. The results suggest that internalization of lunasin into macrophages is amplified in inflammatory conditions and is primarily mediated by endocytic mechanisms that involve integrin signaling, clathrin-coated structures and macropinosomes. Lunasin may be responsible for attenuation of CVD risk factors by interacting with pathways involved in endocytosis and inflammation.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Brefeldin A / pharmacology
  • Calcium / metabolism
  • Cardiovascular Diseases / immunology*
  • Caveolin 1 / metabolism
  • Cell Line, Tumor
  • Cell Membrane / metabolism
  • Clathrin / metabolism
  • Clathrin-Coated Vesicles / metabolism
  • Endocytosis*
  • Humans
  • Integrin alphaVbeta3 / metabolism
  • Lipopolysaccharides / pharmacology
  • Macrophages / immunology
  • Macrophages / metabolism*
  • Protein Synthesis Inhibitors / pharmacology
  • Protein Transport / drug effects
  • Soybean Proteins / metabolism*
  • Soybean Proteins / pharmacology

Substances

  • Caveolin 1
  • Clathrin
  • GM2S-1 protein, Glycine max
  • Integrin alphaVbeta3
  • Lipopolysaccharides
  • Protein Synthesis Inhibitors
  • Soybean Proteins
  • Brefeldin A
  • Calcium

Grant support

This work is supported by U.S. Department of Agriculture grant number: CREES 698 AG 2010-34644-20970 EGdM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.