A role for Sarcolemmal Membrane‐Associated Protein (SLMAP) in mediating autophagy in endothelial cells through an AMPK‐dependent mechanism

Abstract

ObjectiveSarcolemmal membrane associated protein (SLMAP) belongs to the superfamily of tail‐anchored membrane proteins and is a component of endoplasmic reticulum (ER)/sarcoplasmic reticulum as well as mitochondria, cell surface membranes and the nuclear envelope. It has been reported to be involved in various physiological functions mainly in, for example, the regulation of ion channels, membrane fusion and vesicle transport. Previously, we have shown that the up‐regulation of SLMAP protein in diabetic db/db and Tally Ho mice was linked to endothelial dysfunction; however, until now no mechanistic evidence has been provided as to how an up‐regulation of SLMAP disrupts endothelial function in type 2 diabetes mellitus. The primary objective for the current study was to elucidate the cellular pathway(s) whereby an overexpression of SLMAP in human umbilical vein endothelial cells (HUVECs) results in endothelial dysfunction.Materials and MethodsHUVECs were cultured in M199 medium (Sigma Aldrich, USA) supplemented with 15% FBS, heparin (0.1% of 100mg/ml), ECGS (0.01% of 100 mg/ml), 100 μg/mL penicillin, and 100 U/mL streptomycin, at 37°C in a 5% CO2—95% air‐humidified incubator. The 41kDa isoform of SLMAP was overexpressed in HUVECs using SLMAP Lentiviral particles designed for humans (Accession number: AF304450.1; Genecopoeia), and SLMAP was partially knocked using small hairpin RNA (shRNA) lentiviral particles for humans (Santa Cruz Biotechnology, Catalogue number: sc‐78464‐V). The procedures were performed according to the manufacturer's instructions using polybrene at a concentration of 5μg/ml. After overexpression, the HUVECs were treated with, or without, the AMP‐activated protein kinase activator (AMPK), AICAR, 500μM, for 24 h. Protein markers of apoptosis, oxidative stress and autophagy were evaluated by Western Blotting, FACS, DHE staining and immunofluorescence techniques respectively.ResultsThe ER chaperone protein, GRP78; the unfolded protein response (UPR) signaling proteins, such as IRE‐1α, PERK, XBP1s, cleaved caspase‐3, p‐JNK, Bcl‐XL; and markers of autophagy, such as LC3B‐II, Beclin‐1 were found to be significantly elevated (p<0.05), while protein levels of p‐AMPKα (Thr172) and SERCA2 were significantly decreased (p<0.05) in HUVECs overexpressed with SLMAP. Interestingly, treatment with the AMPK activator, AICAR, significantly (p<0.05) reversed all of the changes in HUVECs overexpressed with SLMAP. Moreover, the partial knock down of SLMAP significantly reduced IRE‐1α, PERK, XBP1s, p‐JNK, Bcl‐XL, LC3B‐II, Beclin‐1 proteins level, and increased p‐AMPKα (Thr172) and SERCA2 proteins level in SLMAP shRNA‐transfected HUVECs.ConclusionCollectively, this study has demonstrated for the first time that changes in the expression of SLMAP protein (41kDa) play an important role in ER stress and autophagy in endothelial cells via an AMPK‐dependent mechanism.Support or Funding InformationThis abstract is part of the project funded by NPRP: 5‐149‐3‐040