Integrin b1 acetylation-induced transcriptomic reprograming of mouse embryonic endothelial cells and functions

Reciprocal regulation of integrin-dependent cell-matrix adhesions and cell-cell junctions is critical for controlling endothelial permeability and proliferation in cancer and inflammatory diseases, but remain poorly understood. Here, we investigated how acetylation of the distal NPKY-motif of b1-integrin influences endothelial cell physiology and barrier function. Expression of an acetylation-mimetic B1A-K794Q-GFP mutant induced transcriptomic reprograming of embryonic endothelial cells particularly in genes responsible for cell adhesion, proliferation, polarity and barrier function. B1A-K794Q-GFP induced a constitutive activation of MAPK signaling, an impairment of junctions, increased proliferation and an altered contact inhibition at confluency. Structural analysis of integrin b1 interaction with KINDLIN-2, biochemical pulldown assay and molecular dynamic analysis showed that acetylation of K794 increases KINDLIN-2 binding to the integrin b1 cytoplasmic tail. Accordingly, altering KINDLIN-2 levels modified Claudin-5 expression and endothelial barrier function. Revealing how integrin b1 acetylation regulates endothelial cell junctions offers new therapeutic possibilities for controlling vascular permeability. Overall design: To understand the influence of integrin b1 acetylation on gene transcription in endothelial cells, we have expressed a chimeric form of human integrin ?1 with an extracellular eGFP tag in mouse embryonic endothelial eEnd2 cells. The integrin b1 was used either as wildtype (WT) or carrying the acetyl-mimetic mutation (lysine-to-glutamin at Lys794 or K794Q). For expression of human integrin ?1, we used the previously (Vega, Kastberger et al. 2020) generated pCDNA3 plasmid with the CMV promoter replaced by a fragment of matrix attachment region of chicken lysozyme (MAR) and a small fragment of human actin ? promoter (SAP), and encoding for human integrin b1A containing an extracellular tagging after the K107 with the green fluorescence protein. Mouse embryonic endothelial cell line eEnd2 was transfected with this pcDNA-SAPMAR-ITGB1AK107-GFP encoding for human integrin ?1A (wildtype or K794Q mutant)-eGFP chimera, by using Neon nucleofector (Thermo Fisher Scientific) according to the manufacturer recommendation. Two populations of GFP positive cells (GFPlow and GFPhigh) were sorted by fluorescence-activated cell sorting (FACS) and sub-cultured until use. Sorted cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS) in humidified atmosphere with 5% CO2 at 37°C. Confluent cells in 10-cm dishes were lysed and the total mRNA were extracted with the RNA and protein extraction kit (Macherey-Nagel). Total RNA of three different cultures of each sorted cells were sequenced, aligned and the differential expression of genes analysed at the iGE3 Genomics Platform (University of Geneva, Geneva, Switzerland). Reference: Vega, M. E., et al. (2020). "Stimulation of Fibronectin Matrix Assembly by Lysine Acetylation." Cells 9(3): 655.