Abstract
Macroautophagy is a fundamental and evolutionarily conserved catabolic process that eradicates damaged and aging macromolecules and organelles in eukaryotic cells. Decorin, an archetypical small leucine-rich proteoglycan, initiates a protracted autophagic program downstream of VEGF receptor 2 (VEGFR2) signaling that requires paternally expressed gene 3 (PEG3). We have discovered that PEG3 is an upstream transcriptional regulator of transcription factor EB (TFEB), a master transcription factor of lysosomal biogenesis, for decorin-evoked endothelial cell autophagy. We found a functional requirement of PEG3 for TFEB transcriptional induction and nuclear translocation in human umbilical vein endothelial and PAER2 cells. Mechanistically, inhibiting VEGFR2 or AMP-activated protein kinase (AMPK), a major decorin-activated energy sensor kinase, prevented decorin-evoked TFEB induction and nuclear localization. In conclusion, our findings indicate a non-canonical (nutrient- and energy-independent) mechanism underlying the pro-autophagic bioactivity of decorin via PEG3 and TFEB.
Highlights
Macroautophagy is a fundamental and evolutionarily conserved catabolic process that eradicates damaged and aging macromolecules and organelles in eukaryotic cells
We have discovered that paternally expressed gene 3 (PEG3) is an upstream transcriptional regulator of transcription factor EB (TFEB), a master transcription factor of lysosomal biogenesis, for decorin-evoked endothelial cell autophagy
As decorin suppresses mTOR activity and initiates prolonged autophagic responses, we evaluated the existence of a mechanistic link between PEG3 and TFEB for endothelial cell autophagy
Summary
To evaluate a potential mechanistic link between PEG3 and TFEB, we conducted time course experiments in both human umbilical vein endothelial cells (HUVECs) and porcine aortic endothelial cells overexpressing VEGFR2 (PAER2). The profile obtained for Tfeb protein (Fig. 2B) paralleled that obtained for the mRNA (Fig. 2A) These data reinforce the concept that Peg is necessary and sufficient for driving Tfeb expression and further substantiate the role of Peg in autophagic progression. Generating HA-tagged truncation fragments (Fig. 2G) of either the SCAN domain (HA-SCAN) or the zinc fingers (HA-ZF) prevented nuclear translocation of these fragments following decorin stimulation in comparison with full-length HAtagged PEG3 [60] We expressed these fragments in our PAER2PEG3 cells and assayed Tfeb expression to determine whether they acted in a dominant negative fashion. AMPK (Fig. 3B) or VEGFR2 (Fig. 3C) significantly prevented decorin-evoked TFEB expression These results were extended at the protein level insofar as Compound C (Fig. 3D) or SU5416 (Fig. 3E) precluded an increase in TFEB following decorin stimulation. We conclude that decorin requires the VEGFR2– AMPK signaling axis for proficient nuclear translocation of TFEB protein and proper induction of TFEB
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