Parallel Activation of Src and Hif1α Increases Localized Glycolytic 1 ATP Generation for Re-assembly of Endothelial Adherens 2 Junctions

S.K. , A.M.R. , A.K. , D.E. L. and J.R. The manuscript draft written by and J.R. reviewed the paper and feedback and revisions. Competing Interest Statement: The declare no competing interests. Abstract 34 Endothelial adherens junctions (AJs) are critical for the regulation of vascular 35 barrier integrity and undergo dis-assembly during inflammatory injury, thus causing 36 vascular leakiness. AJ re-assembly is thus necessary for restoration of the endothelial 37 barrier following the initial injury. Here we examine the metabolic underpinnings that drive 38 restoration of vascular integrity. In response to inflammatory stimuli, the glycolysis 39 regulatory enzyme PFKFB3 is activated, resulting in a rapid and sustained increase of 40 intracellular glycolytic ATP, especially in the proximity of AJs at the plasma membrane. 41 We engineered a novel chemo-genetic construct (RapT) which allowed for precise 42 temporal control of PFKFB3 recruitment to the plasma membrane. Activation of RapT by 43 rapamycin during the barrier restoration phase increased regional ATP and accelerated 44 AJ re-assembly. Mechanistically, we observed that PFKFB3 is activated through two 45 modes. Src-mediated post-translational phosphorylation rapidly increases PFKFB3 46 activity. Using another chemo-genetic approach to temporally control Src activity, we 47 found that Src activates PFKFB3 by binding to and phosphorylating it at residues Y175, 48 Y334, and Y363. Tyrosine-phospho-deficient mutants of PFKFB3 at these residues block 49 the glycolytic activation upon inflammatory stimuli. In parallel, elevated reactive oxygen 50 species generated during inflammatory stimulation create pockets of regional hypoxia and 51 allow for increased Hif1α -mediated transcription of PFKFB3, leading to sustained 52 glycolytic activation. Moreover, inhibition of PFKFB3 delays AJ reassembly and restoration 53 of vascular integrity both in vitro and in vivo. In conclusion, we show that while 54 inflammatory activation acutely compromises the endothelial barrier, inflammatory 55 signaling also concomitantly generates a metabolic milieu in anticipation of the 56 subsequent re-assembly of AJs and restoration of the vascular barrier. Here, we demonstrate that ECs respond to inflammatory stimuli by increasing glycolysis via PFKFB3 activation through two modes: a newly identified rapid post- translational mechanism of Src-mediated tyrosine phosphorylation, and a more sustained mechanism of Hif1α -mediated PFKFB3 transcription. Using RapR-Src, an engineered construct that allows for precise temporal regulation of Src, we show that Src binds, phosphorylates, and activates PFKFB3. Spatial control of PFKFB3 localization using a novel chemo-genetic engineered construct, RapT, shows that increased PFKFB3 activity at the membrane can accelerate restoration of the endothelial barrier. PFKFB3 inhibition during the resolution phase delayed the recovery of barrier function both in vivo and in vitro, thus highlighting the barrier restoration role for Src during the recovery period. We therefore posit that while inflammatory activation acutely compromises the EC barrier, inflammatory signaling concomitantly induces a metabolic shift towards increased glycolysis which promotes the restoration of the endothelial barrier and the resolution of tissue edema as well as inflammation.