bioRxiv preprint doi: https://doi.org/10.1101/2020.06.15.152942; this version posted June 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Protein tyrosine phosphatase-PEST (PTP-PEST) mediates hypoxia- 2 induced endothelial autophagy and angiogenesis through AMPK 3 activation. 4 Shivam Chandel1, Amrutha Manikandan1, Nikunj Mehta1, Abel Arul Nathan1, Rakesh 5 Kumar Tiwari1, Samar Bhallabha Mohapatra1, Mahesh Chandran2, Abdul Jaleel2, 6 Narayanan Manoj1 and Madhulika Dixit1. 7 8 1. Department of Biotechnology, 9 Bhupat and Jyoti Mehta School of Biosciences, 10 Indian Institute of Technology Madras (IIT Madras), 11 Chennai, INDIA, 600036 12 2. Rajiv Gandhi Centre for Biotechnology (RGCB), 13 Thyacaud Post, Thiruvananthpuram, 14 Kerala, INDIA, 695014 15 16 Corresponding Author: 17 Madhulika Dixit 18 Department of Biotechnology, 19 Bhupat and Jyoti Mehta School of Biosciences, 20 Indian Institute of Technology Madras 21 Chennai, INDIA, 600036 22 23 Phone: +91-44-22574131 24 E-mail: [email protected] 25 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.15.152942; this version posted June 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 26 Abstract: 27 Global and endothelial loss of PTP-PEST is associated with impaired cardiovascular 28 development and embryonic lethality. Although hypoxia is implicated in vascular 29 morphogenesis and remodelling, its effect on PTP-PEST remains unexplored. Here we 30 report that hypoxia (1% oxygen)increases protein levels and catalytic activity of PTP- 31 PEST in primary endothelial cells. Immunoprecipitation followed by mass spectrometry 32 (LC/MS/MS) revealed that AMP-activated protein kinase alpha subunits (AMPK α1 and 33 α2) interact with PTP-PEST under normoxia but not in hypoxia. Co-immunoprecipitation 34 experiments confirmed this observation and determined that AMPK α subunits interact 35 with the catalytic domain of PTP-PEST. Knock-down of PTP-PEST abrogated hypoxia 36 mediated tyrosine dephosphorylation and activation of AMPK (Thr172 phosphorylation). 37 Absence of PTP-PEST also blocked hypoxia-induced autophagy (measured as LC3 38 degradation and puncta formation) which was rescued by AMPK activator, metformin 39 (500µM). Since endothelial autophagy is a pre-requisite for angiogenesis, knock-down 40 of PTP-PEST also attenuated endothelial cell migration and capillary tube formation 41 with autophagy inducer rapamycin (200nM) rescuing these effects. In conclusion, this 42 work identifies for the first time PTP-PEST as a regulator of hypoxia-induced AMPK 43 activation and endothelial autophagy to promote angiogenesis. 44 45 46 Key words: PTP-PEST, hypoxia, AMPK, autophagy, angiogenesis 47 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.15.152942; this version posted June 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 48 Introduction: 49 Physiological hypoxia is a potent agonist for embryonic development and post-natal 50 angiogenesis. Oxygen concentrations ranging from 1% to 5% are observed in the 51 uterine environment between embryonic days 3.5 to 14.5 (E3.5-E14.5) to facilitate 52 development of placenta. Similarly, in response to hypoxia (<2% oxygen), endocardial 53 and vascular endothelial cells mediate formation of foetal heart and vasculature 54 respectively in mice between embryonic days E7.5 to E15 [1;2]. Post-natal 55 angiogenesis as seen in female reproductive tract during menstrual cycle in humans, as 56 well as formation of collateral circulation to over-come coronary artery blocks also 57 depend on hypoxia-induced endothelial signalling. Other than the known classical 58 mediators of angiogenesis, multiple studies in rodents have reported increased activity 59 of cytosolic protein tyrosine phosphatases (PTPs) at the sites of post-natal 60 angiogenesis, including ischemic myocardium and skeletal muscles [3;4]. An increase 61 in the cytosolic PTP activity during hypoxia is also seen in the cerebral cortex of new 62 born piglets [5]. Paradoxically, others have shown that non-selective PTP inhibitor, 63 sodium orthovanadate, enhances VEGFR2 signalling and capillary morphogenesis 64 [3;6]. Although these studies allude to the involvement of different PTPs in hypoxia- 65 induced angiogenic signalling, barring the involvement of few, for instance VE-PTP (a 66 receptor PTP), the identity of hypoxia responsive cytosolic angiogenic PTPs remains 67 largely unknown. 68 69 Human genome encodes 38 classical PTPs which are specific for tyrosine residues [7]. 70 Among these, PTP-PEST is a ubiquitously expressed cytosolic PTP with an N-terminal 71 catalytic domain (1-300 amino acids) and a regulatory C-terminal PEST domain (301- 72 780 amino acids) containing four PEST motifs. The latter plays a crucial role in protein- 73 protein interaction allowing the enzyme to interact with its known substrates such as 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.15.152942; this version posted June 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 74 Cas, Paxillin, FAK and Pyk2 in addition to being a protein degradation signal [8-10]. 75 The catalytic domain harbours the conserved phosphatase ‘HC(X)5R’ motif surrounded 76 by the ‘WPD loop’ and the ‘Q loop’ on one side to assist in catalysis and by the ‘P-Tyr- 77 loop’on the other side to regulate substrate recognition and specificity [11]. Both global 78 and endothelial deficiency of PTP-PEST in mice leads to embryonic lethality between 79 E9.5-E10.5 days due to defective heart formation and impaired endothelial network in 80 the yolk sac [12;13]. It is worth noting that this stage in embryonic development 81 coincides with hypoxia-induced morphogenesis [1;2]. PTP-PEST appears to be crucial 82 for vascular development even in humans since a partial deletion of PTP-PEST is 83 associated with disrupted aortic arch development [14]. Intriguingly, in a recent study, 84 enhanced endothelial expression of PTP-PST was indeed observed in the vascularized 85 core of glioblastoma tumours with others demonstrating involvement of PTP-PEST in 86 integrin mediated endothelial cell adhesion and migration [13;15]. Based on these 87 observations which illustrate an essential role of PTP-PEST in vascular development 88 and given the fact that hypoxia is indispensable for cardiovascular development and 89 angiogenesis, in the present study we set out to determine the functional role of PTP- 90 PEST in hypoxia-induced endothelial responses. We assessed the effect of hypoxia on 91 the expression, activity and binding partners of PTP-PEST in primary human umbilical 92 vein derived endothelial cells (HUVECs). 93 94 Materials and methods: 95 Experimental procedure involving isolation of endothelial cells from umbilical cords was 96 approved by the IIT-Madras Institutional Ethics Committeeas per the Indian Council of 97 Medical Research (ICMR), Government of India guidelines. These guidelines are in 98 accordance with the declaration of Helsinki, which was revised in 2000. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.15.152942; this version posted June 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 99 Antibodies and Chemicals: Tissue culture grade plastic ware was from Tarsons 100 Products Pvt. Ltd., Kolkata, India. Antibodies against HIF-1α, phospho-AMPK, total 101 AMPK, LC3, phospho-ULK1, total ULK1, phospho-ACC, total ACC, PTP-PEST (AG10) 102 and anti-phospho-tyrosine (P-Tyr-102) antibody were obtained from Cell Signaling 103 Technology (CST), Boston, MA, USA. Dulbecco’s modified Eagle’s medium (DMEM) 104 and MCDB131 were purchased from HiMediaTM, Mumbai, India and fetal bovine serum 105 (FBS) was from Gibco, Thermo Fisher Scientific, Waltham, MA, USA. Unless specified 106 otherwise rest of the molecular biology and biochemistry reagents were from Sigma, St. 107 Louis, MO, USA. 108 Cell culture: HUVECs were isolated from freshly collected umbilical cords by 109 collagenase digestion. They were cultured in fibronectin coated T25 flasks until 110 monolayer was formed. MCDB131 medium with endothelial growth supplements was 111 used for culturing HUVECs. All the experiments were performed in passage one. For 112 cell lines HEK293, HeLa, Huh7 and HASMC, cells were cultured in DMEM with 10% 113 FBS. For hypoxia exposure, cells were incubated in hypoxia incubator (5% CO2, 1% O2 114 and 95% relative humidity) for specified duration. After degassing, the cell culture 115 media used for hypoxia experiments was pre-equilibrated to the hypoxic environment 116 prior to start of the experiment. 117 Immunoblotting: All the buffers used for washing and lysing of cells were degassed 118 before use. Cells were washed with 1X cold PBS and lysed in buffer containing 50mM 119 Tris-HCl (pH 7.5), 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% TritonX-100, 0.1% SDS, 120 1% sodium deoxycholate, 1mM PMSF and protease inhibitor cocktail from Sigma (St. 121 Louis, MO, USA) followed by sonication for 1 min (30% amplitude, 3 second on/off 122 cycle). 50 µg worth total protein was resolved through 8% SDS-PAGE and transferred 123 onto PVDF membrane. Blotted proteins were incubated with corresponding primary 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.15.152942; this version posted June 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 124 antibodies at 4°C overnight followed by incubation with horseradish peroxidase 125 conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA).