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Endorepellin Evokes Autophagy in Endothelial Cells*

  • Chiara Poluzzi
    Footnotes
    Affiliations
    Department of Pathology, Anatomy, and Cell Biology and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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  • Joshua Casulli
    Footnotes
    Affiliations
    Department of Pathology, Anatomy, and Cell Biology and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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  • Atul Goyal
    Affiliations
    Department of Pathology, Anatomy, and Cell Biology and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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  • Thomas J. Mercer
    Affiliations
    Department of Pathology, Anatomy, and Cell Biology and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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  • Thomas Neill
    Footnotes
    Affiliations
    Department of Pathology, Anatomy, and Cell Biology and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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  • Renato V. Iozzo
    Correspondence
    To whom correspondence should be addressed: Dept. of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, 1020 Locust St., Ste. 336 JAH, Philadelphia, PA 19107. Tel.: 215-503-2208; Fax: 215-923-7969
    Affiliations
    Department of Pathology, Anatomy, and Cell Biology and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants RO1 CA3948, RO1 CA47282, and RO1 CA164462 (to R. V. I.).
    1 Both authors contributed equally to this work.
    2 Supported by National Institutes of Health Training Grant T32 AA07463.
Open AccessPublished:April 15, 2014DOI:https://doi.org/10.1074/jbc.M114.556530
      Endorepellin, the C-terminal fragment of the heparan sulfate proteoglycan perlecan, possesses angiostatic activity via dual receptor antagonism, through concurrent binding to the α2β1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2). Here, we discovered that soluble endorepellin induced autophagy in endothelial cells by modulating the expression of Beclin 1, LC3, and p62, three established autophagic markers. Moreover, endorepellin evoked expression of the imprinted tumor suppressor gene Peg3 and its co-localization with Beclin 1 and LC3 in autophagosomes, suggesting a major role for this gene in endothelial cell autophagy. Mechanistically, endorepellin induced autophagy by down-regulating VEGFR2 via the two LG1/2 domains, whereas the C-terminal LG3 domain, the portion responsible for binding the α2β1 integrin, was ineffective. Endorepellin also induced transcriptional activity of the BECN1 promoter in endothelial cells, and the VEGFR2-specific tyrosine kinase inhibitor, SU5416, blocked this effect. Finally, we found a correlation between endorepellin-evoked inhibition of capillary morphogenesis and enhanced autophagy. Thus, we have identified a new role for this endogenous angiostatic fragment in inducing autophagy through a VEGFR2-dependent but α2β1 integrin-independent pathway. This novel mechanism specifically targets endothelial cells and could represent a promising new strategy to potentiate the angiostatic effect of endorepellin and perhaps other angiostatic matrix proteins.

      Introduction

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      The abbreviations used are:
      VEGFR2
      VEGF receptor 2
      LG
      laminin-like globular domain
      HUVEC
      human umbilical vein endothelial cells
      PAE
      porcine aortic endothelial cells
      DIC
      differential interference contrast microscopy
      mTOR
      mammalian target of rapamycin
      Atg
      autophagy-related
      LC3
      microtubule associated light chain 3
      qPCR
      quantitative PCR
      HBSS
      Hanks' balanced salt solution.
      (
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      ). This ultimately inhibits VEGFA-induced endothelial cell migration and angiogenesis.
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      ). Thus, we hypothesized that endorepellin could evoke autophagy via suppression of VEGFR2-dependent signaling by suppressing the canonical mTOR pathway. In this work, we demonstrate for the first time that endorepellin induces autophagy in endothelial cells through VEGFR2 but independently of the α2β1 integrin. We found that nanomolar concentrations of human recombinant endorepellin induced Beclin 1- and LC3-positive autophagosomes in nutrient-enriched conditions in both human and porcine endothelial cells. Moreover, p62 protein was dynamically modulated by endorepellin and co-localized with LC3 in autophagosomes. Thus, we have discovered a novel mechanism that specifically targets endothelial cells and could provide a promising strategy to potentiate the angiostatic effect of endorepellin and perhaps other proteolytically processed matrix proteins harboring angiostatic activity.

      DISCUSSION

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      Autophagy modulation as a potential therapeutic target for diverse diseases.
      ). In addition to eliminating protein aggregates and damaged organelles, autophagy promotes cellular senescence and cell surface antigen presentation. Moreover, autophagy protects against genome instability and prevents necrosis, thereby counteracting diseases such as cancer, neurodegeneration, cardiomyopathy, diabetes, liver disease, autoimmune diseases, and infections (
      • Mizushima N.
      • Komatsu M.
      Autophagy: renovation of cells and tissues.
      ). Autophagy induction in response to stress and starvation also has a crucial role in normal cells. For example, Atg5-deficient mice fail to live through the neonatal survival period, during which tissues show signs of amino acid depletion and metabolic insufficiency. These findings support a pro-survival role for autophagy in both normal tissues and in response to metabolic stress (
      • Rubinsztein D.C.
      • Codogno P.
      • Levine B.
      Autophagy modulation as a potential therapeutic target for diverse diseases.
      ).
      A working model summarizing our most recent findings concerning the mechanism of action for endorepellin-evoked endothelial cell autophagy and anti-angiogenesis is provided in Fig. 8. Accordingly, we propose that endorepellin engages VEGFR2 via its two proximal LG domains and acts as a partial agonist. This signaling emanated by VEGFR2 evokes the simultaneous formation of Peg3-, Beclin 1-, and LC3-positive complexes and suppression of the anti-autophagic PI3K/Akt/mTOR pathway (Fig. 9). Transcriptional induction of PEG3 is also required for downstream activation of BECN1 and MAPLC3A. Accumulated Peg3, Beclin 1, and lipidated LC3 promote autophagosome nucleation and maturation with concomitant dissolution of the actin cytoskeleton for angiogenic inhibition mediated by interaction of LG3 with the α2β1 integrin (Fig. 9).
      Figure thumbnail gr9
      FIGURE 9.Working model elucidating the mechanism in which endorepellin induces autophagy through the VEGFR2 α2β1 integrin-independent manner. Please, see the text for detailed information.
      We hypothesized that endorepellin could remove the repression of autophagy induced by mTOR, a downstream component of the PI3K pathway, through the binding to the VEGFR2 in endothelial cells and consequent inhibition of the PI3K/PDK1/AKT/mTOR pathway (
      • Goyal A.
      • Poluzzi C.
      • Willis C.D.
      • Smythies J.
      • Shellard A.
      • Neill T.
      • Iozzo R.V.
      Endorepellin affects angiogenesis by antagonizing diverse VEGFR2-evoked signaling pathways: transcriptional repression of HIF-1α and VEGFA and concurrent inhibition of NFAT1 activation.
      ). Using DIC microscopy, we found that in both endothelial cell types, endorepellin induced the formation of characteristic autophagosomes comparable with those evoked by rapamycin or nutrient deprivation. This observation was further corroborated by the finding that endorepellin induced expression of the Atg proteins Beclin 1, LC3, and p62 and promoted their co-localization and physical interaction in HUVEC. Importantly, endorepellin-evoked autophagy was analogous to traditional autophagic stimuli such as rapamycin and nutrient deprivation.
      The Atg proteins are required for activation of autophagy, formation of autophagosomes, sequestration of intracellular constituents, and targeting and fusion of autophagosomes to lysosomes where the contents are degraded and recycled (
      • Mizushima N.
      • Komatsu M.
      Autophagy: renovation of cells and tissues.
      ). Beclin 1 is part of the class III PI3K-Vps34 complex that is also required for autophagy. ATG8 (also known as LC3) is another ubiquitin-like protein that is cleaved and lipidated and becomes a component of the autophagosomal membrane. This membrane translocation event is commonly used to monitor autophagy (
      • Choi A.M.
      • Ryter S.W.
      • Levine B.
      Autophagy in human health and disease.
      ). Notably, endorepellin induced a re-distribution of Vps34 in the cytoplasm of HUVEC and a subsequent co-localization with Beclin 1, suggesting that inhibiting mTOR precedes and evokes the formation of Beclin 1/Vps34 pre-autophagic signaling complexes.
      We have recently discovered that Peg3, a decorin-induced gene (
      • Buraschi S.
      • Neill T.
      • Owens R.T.
      • Iniguez L.A.
      • Purkins G.
      • Vadigepalli R.
      • Evans B.
      • Schaefer L.
      • Peiper S.C.
      • Wang Z.X.
      • Iozzo R.V.
      Decorin protein core affects the global gene expression profile of the tumor microenvironment in a triple-negative orthotopic breast carcinoma xenograft model.
      ), is a master regulator of autophagy through the recruitment and transcriptional induction of Beclin 1 and LC3 (
      • Buraschi S.
      • Neill T.
      • Goyal A.
      • Poluzzi C.
      • Smythies J.
      • Owens R.T.
      • Schaefer L.
      • Torres A.
      • Iozzo R.V.
      Decorin causes autophagy in endothelial cells via Peg3.
      ). Peg3 was originally identified as a paternally imprinted gene and is a relatively large protein (∼180 kDa) that contains an N-terminal SCAN protein-protein interaction domain and 12 Kruppel-type zinc fingers (
      • Kuroiwa Y.
      • Kaneko-Ishino T.
      • Kagitani F.
      • Kohda T.
      • Li L.-L.
      • Tada M.
      • Suzuki R.
      • Yokoyama M.
      • Shiroishi T.
      • Wakana S.
      • Barton S.C.
      • Ishino F.
      • Surani M.A.
      Peg3 imprinted gene on proximal chromosome 7 encodes for a zinc finger protein.
      ,
      • Relaix F.
      • Weng X.
      • Marazzi G.
      • Yang E.
      • Copeland N.
      • Jenkins N.
      • Spence S.E.
      • Sassoon D.
      Pw1, a novel zinc finger gene implicated in the myogenic and neuronal lineages.
      ). Notably, Peg3−/− mice show growth retardation and impaired maternal nurturing (
      • Li L.-L.
      • Keverne E.B.
      • Aparicio S.A.
      • Ishino F.
      • Barton S.C.
      • Surani M.A.
      Regulation of maternal behavior and offspring growth by paternally expressed Peg3.
      ). Peg3 is involved in TNF-NFκB signaling (
      • Relaix F.
      • Wei X.J.
      • Wu X.
      • Sassoon D.A.
      Peg3/Pw1 is an imprinted gene involved in the TNF-NFκB signal transduction pathway.
      ) as well as p53-mediated apoptosis (
      • Relaix F.
      • Wei Xj
      • Li W.
      • Pan J.
      • Lin Y.
      • Bowtell D.D.
      • Sassoon D.A.
      • Wu X.
      Pw1/Peg3 is a potential cell death mediator and cooperates with Siah1a in p53-mediated apoptosis.
      ) and Wnt signaling (
      • Jiang X.
      • Yu Y.
      • Yang H.W.
      • Agar N.Y.
      • Frado L.
      • Johnson M.D.
      The imprinted gene PEG3 inhibits Wnt signaling and regulates glioma growth.
      ). We found transcriptional induction of PEG3 upon endorepellin treatment with a dynamic co-distribution of Peg3 with both Beclin 1 and LC3, supporting a role for this gene in endorepellin-induced autophagy. In accordance with the finding that Peg3 binds Beclin 1 following decorin stimulation in HUVEC (
      • Buraschi S.
      • Neill T.
      • Goyal A.
      • Poluzzi C.
      • Smythies J.
      • Owens R.T.
      • Schaefer L.
      • Torres A.
      • Iozzo R.V.
      Decorin causes autophagy in endothelial cells via Peg3.
      ), we found that endorepellin also promotes Peg3-Beclin 1 interaction. This finding suggests that a general feature of soluble matrix constituents with anti-angiogenic activities is to promote Peg3/Beclin 1 association consummate with autophagic induction. Moreover, the role of Peg3 in relaying endorepellin-evoked Atg gene expression was confirmed as silencing of Peg3 prevented BECN1 and MAPLC3A expression. These data support a role for Peg3 as a master autophagic regulator responsible for coordinating downstream responses from matrix signaling.
      We have discovered that endorepellin specifically targets endothelial cells via a dual receptor antagonism, acting as a molecular bridge, ligating both VEGFR2 and α2β1 integrin (
      • Goyal A.
      • Pal N.
      • Concannon M.
      • Paul M.
      • Doran M.
      • Poluzzi C.
      • Sekiguchi K.
      • Whitelock J.M.
      • Neill T.
      • Iozzo R.V.
      Endorepellin, the angiostatic module of perlecan, interacts with both the α2β1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2).
      ,
      • Goyal A.
      • Poluzzi C.
      • Willis C.D.
      • Smythies J.
      • Shellard A.
      • Neill T.
      • Iozzo R.V.
      Endorepellin affects angiogenesis by antagonizing diverse VEGFR2-evoked signaling pathways: transcriptional repression of HIF-1α and VEGFA and concurrent inhibition of NFAT1 activation.
      ). Following engagement, endorepellin seemingly attenuates VEGFR2 phosphorylation at Tyr1175, a key residue from which the phospholipase C-γ signaling cascade originates (
      • Olsson A.-K.
      • Dimberg A.
      • Kreuger J.
      • Claesson-Welsh L.
      VEGF receptor signalling-in control of vascular function.
      ). However, endorepellin requires positive VEGFR2-dependent signaling for sustained autophagic induction concurrent with PEG3, BECN1, and MAPLC3A transcriptional induction as SU5416 abrogated this response. Furthermore, a similar outcome was obtained by RNAi-mediated silencing of VEGFR2. Together, these results support the importance of the major pro-angiogenic VEGFR2 tyrosine kinase in endorepellin-induced autophagy.
      Recently, we have dissected the bioactivity of endorepellin and its two main fragments (LG1/2 and LG3) on VEGFR2 and α2β1 integrin (
      • Willis C.D.
      • Poluzzi C.
      • Mongiat M.
      • Iozzo R.V.
      Endorepellin laminin-like globular repeat 1/2 domains bind Ig3–5 of vascular endothelial growth factor (VEGF) receptor 2 and block pro-angiogenic signaling by VEGFA in endothelial cells.
      ), respectively. Together with the present results, our findings suggest a major role for VEGFR2 in binding endorepellin and inducing autophagy in an α2β1 integrin-independent manner. This assumption was confirmed by the discovery that, unlike LG3, LG1/2 promotes the formation of large autophagosomes and mediates transcriptional regulation of Atg genes comparable with those induced by endorepellin, mTOR inhibition, or nutrient deprivation. In contrast, LG3 signaling via α2β1 integrin results in autophagic gene suppression. Moreover, the necessity of VEGFR2 and dispensability of the α2β1 integrin are underscored by the finding that utilization of an α2β1 integrin-blocking mAb has no effect on autophagic induction. The N-terminal LG1/2 domains are inherently pro-autophagic and require VEGFR2. Concomitant with endorepellin-evoked autophagic induction, branching morphogenesis is also abrogated but with a concurrent increase in GFP-LC3 fluorescence. Thus, deciphering whether autophagy via Peg3 is required for suppressing capillary morphogenesis is an important goal of our future research.
      The intrinsic nature of endorepellin abrogating tumor angiogenesis and metabolism, by selectively targeting the tumor vasculature, has been verified in vivo (
      • Bix G.
      • Castello R.
      • Burrows M.
      • Zoeller J.J.
      • Weech M.
      • Iozzo R.A.
      • Cardi C.
      • Thakur M.L.
      • Barker C.A.
      • Camphausen K.
      • Iozzo R.V.
      Endorepellin in vivo: targeting the tumor vasculature and retarding cancer growth and metabolism.
      ). As endorepellin activates endothelial cell autophagy downstream of VEGFR2 signaling, it is plausible that autophagic elimination of critical organelles might inhibit neovascularization, thereby impeding tumorigenesis.
      Autophagic induction is emerging as a more common theme among matrix constituents. Endostatin, for example, is a well characterized angiogenesis inhibitor generated by C-terminal proteolysis of the heparan sulfate proteoglycan, collagen XVIII (
      • Seppinen L.
      • Pihlajaniemi T.
      The multiple functions of collagen XVIII in development and disease.
      ). In addition to apoptosis, endostatin also activates autophagy by Beclin 1 and β-catenin levels in HUVEC (
      • Nguyen T.M.
      • Subramanian I.V.
      • Xiao X.
      • Ghosh G.
      • Nguyen P.
      • Kelekar A.
      • Ramakrishnan S.
      Endostatin induces autophagy in endothelial cells by modulating Beclin 1 and β-catenin levels.
      ) and induces autophagic cell death in the EAhy926 human endothelial cell line (
      • Chau Y.-P.
      • Lin J.-Y.
      • Chen J.H.
      • Tai M.-H.
      Endostatin induces autophagic cell death in EAhy926 human endothelial cells.
      ). Moreover, Kringle 5, an antiangiogenic factor derived from human plasminogen, induces both autophagy and apoptotic cell death in HUVEC (
      • Nguygen T.M.
      • Subramanian I.V.
      • Kelekar A.
      • Ramakrishnan S.
      Kringle 5 of human plasminogen, an angiogenesis inhibitor, induces both autophagy and apoptotic death in endothelial cells.
      ). Thus, proteolytically processed fragments of larger parent molecules can evoke autophagy, suggesting the potential involvement of a common signaling pathway for angiostatic proteins (
      • Ramakrishnan S.
      • Nguygen T.M.
      • Subramanian I.V.
      • Kelekar A.
      Autophagy and angiogenesis inhibition.
      ). Moreover, autophagic regulation via matrix components might be a highly dynamic process. For example, CD47, the receptor for the anti-angiogenic effector thrombospondin 1 (
      • Murphy-Ullrich J.E.
      • Iozzo R.V.
      Thrombospondins in physiology and disease: new tricks for old dogs.
      ) and for regulating cardiovascular function and responses to stress (
      • Roberts D.D.
      • Miller T.W.
      • Rogers N.M.
      • Yao M.
      • Isenberg J.S.
      The matricellular protein thrombospondin-1 globally regulates cardiovascular function and responses to stress via CD47.
      ), suppresses autophagy (
      • Soto-Pantoja D.R.
      • Miller T.W.
      • Pendrak M.L.
      • DeGraff W.G.
      • Sullivan C.
      • Ridnour L.A.
      • Abu-Asab M.
      • Wink D.A.
      • Tsokos M.
      • Roberts D.D.
      CD47 deficiency confers cell and tissue radioprotection by activation of autophagy.
      ). Specifically, lack of CD47 permits autophagic activation in Jurkat T-cells and HUVEC (
      • Soto-Pantoja D.R.
      • Miller T.W.
      • Pendrak M.L.
      • DeGraff W.G.
      • Sullivan C.
      • Ridnour L.A.
      • Abu-Asab M.
      • Wink D.A.
      • Tsokos M.
      • Roberts D.D.
      CD47 deficiency confers cell and tissue radioprotection by activation of autophagy.
      ), which ultimately confers a radioprotective state for cells and tissues.
      In conclusion, we propose that endorepellin, the C-terminal cleavage product of perlecan, functions not only as an endogenous anti-angiogenic cue but also as a potent pro-autophagic effector for endothelial cells while residing in a nutrient-rich microenvironment. Endorepellin, by specifically binding VEGFR2, induces the formation of the Peg3-Vps34-Beclin 1 autophagic complexes via inhibition of the PI3K/Akt/mTOR pathway. Importantly, endorepellin-evoked autophagy requires the recently discovered master regulator Peg3 for initiation. Therefore, this autophagic response may be a novel target for enhancing the therapeutic efficacy of angiogenesis inhibitors.

      Acknowledgments

      We thank S. Ramakrishnan for providing the GFP-LC3 plasmid, L. Claesson-Welsh for providing the PAE-VEGFR2 cells, F. Demarchi for providing the BECN1 promoter-luciferase construct, and A. Shellard and A. Torres for valuable help in the initial stages of this work.

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