Abstract
Under physiological and pathological conditions, elastin is degraded to produce elastin-derived peptides (EDPs). EDPs are detected in the healthy human brain, and its concentration significantly increases after ischemic stroke. Both elastin and EDPs contains replications of the soluble VGVAPG hexapeptide, which has a broad range of biological activities. Effects of VGVAPG action are mainly mediated by elastin-binding protein (EBP), which is alternatively spliced, enzymatically inactive form of the GLB1 gene. This study was conducted to elucidate the activation and role of the N-methyl-D-aspartate receptor (NMDAR) in elastin-derived VGVAPG peptide-dependent calcium homeostasis in mouse cortical astrocytes in vitro. Cells were exposed to 10 nM VGVAPG peptide and co-treated with MK-801, nifedipine, verapamil, or Src kinase inhibitor I. After cell stimulation, we measured Ca2+ level, ROS production, and mRNA expression. Moreover, the Glb1 and NMDAR subunits (GluN1, GluN2A, and GluN2B) siRNA gene knockdown were applied. We found the VGVAPG peptide causes Ca2+ influx through the NMDA receptor in mouse astrocytes in vitro. Silencing of the Glb1, GluN1, GluN2A, and GluN2B gene prevented VGVAPG peptide-induced increase in Ca2+. Nifedipine does not completely reduce VGVAPG peptide-activated ROS production, whereas MK-801, verapamil, and Src inhibitor reduce VGVAPG peptide-activated Ca2+ influx and ROS production. These data suggest the role of Src kinase signal transduction from EBP to NMDAR. Moreover, the VGVAPG peptide affects the expression of NMDA receptor subunits.
Highlights
Elastin is an insoluble, amorphous, hydrophobic protein with widespread presence in the walls of blood vessels as well as in the microvasculature of the normal human brain[1]
We found that the VGVAPG peptide increases reactive oxygen species (ROS) production in an elastin-binding protein (EBP)-dependent manner both in mouse astrocytes in vitro[32] and a human neuroblastoma (SH-SY5Y) cell line[33]
The data we obtained suggest that the activation of EBP results in opening of the N-methyl-Daspartate receptor (NMDAR) and other types of calcium channels are less often blocked by nifedipine and verapamil
Summary
Amorphous, hydrophobic protein with widespread presence in the walls of blood vessels as well as in the microvasculature of the normal human brain[1]. Studies to date have described that κ-elastin increases Ca2+ influx in human monocytes and fibroblasts, as well as in smooth muscle cells from pig aorta[23]. It has been described that EDPs induce ROS production in murine monocytes and human fibroblasts[29,30,31] Such data suggest that the effects of EDPs are cell and/or tissue dependent. We found that the VGVAPG peptide increases ROS production in an EBP-dependent manner both in mouse astrocytes in vitro[32] and a human neuroblastoma (SH-SY5Y) cell line[33]. EBP-mediated cell proliferation has been described to involve the activation of Gi proteins with the opening of calcium L-type channels and resultant increase in Ca2+ levels in porcine smooth muscle cells[36]
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