Our results reveal ADGRL4/ELTD1 as a regulator of endothelial metabolism and KIT expression and identify new relationships with the Notch pathway. The finding that ADGRL4/ELTD1 down-regulates ACLY and SLC25A1 expression is interesting as both encode critical enzymes in cellular metabolism [13
]. Interestingly, ADGRL4/ELTD1 overexpression in HUVECs did not downregulate ACLY
, potentially because they are already maximally suppressed by endogenous levels of ADGRL4/ELTD1. Although ADGRL4/ELTD1 did not affect fatty acid or cholesterol synthesis or acetylation reactions, metabolomics showed that ADGRL4/ELTD1 has an effect on metabolites in multiple metabolic pathways, the most significant being pyrimidine metabolism. Through its conversion of citrate to acetyl-coA, cytoplasmic ACLY links glucose metabolism with both fatty acid and cholesterol synthesis pathways [14
] whilst SLC25A1 traffics citrate from the citric acid cycle out of the mitochondria into the cytoplasm [16
]. This citrate provides the acetyl units for lipogenesis and is catalysed by ACLY, producing acetyl-CoA which feeds fatty acid synthesis or cholesterol synthesis, or regulates glycolytic histone modification [18
]. Endothelial cells depend on glycolysis for ATP generation [20
] and use beta oxidation to generate carbon for endothelial DNA synthesis rather than using fatty acid oxidation as an alternative source of energy [21
]. VEGFA, the initiating ligand for endothelial cell sprouting, increases endothelial glycolysis [20
] and induces ADGRL4/ELTD1 expression [1
]. T rans-differentiation of fibroblasts into induced endothelial cells requires a glycolytic switch involving increased expression of SLC25A1 and ACLY as well as the intracellular accumulation of citrate, suggesting that both are important in the development and maintenance of the endothelial cell metabolic phenotype [22
] . Furthermore, complete ablation of ACLY induces the endothelial to mesenchymal transition (EMT), while increasing acetyl-coA suppresses EMT [23
]. These findings suggest that ADGRL4/ELTD1’s regulation of ACLY and SLC25A1 expression may help maintain endothelial homeostasis and metabolism. It remains unclear why ADGRL4/ELTD1 silencing upregulates ACLY, but downstream acetylation and/or lipid droplet formation are unaffected. This is most likely due to tight regulation of glycolysis in endothelial cells due to the integral role of glycolysis in endothelial cell function [21
ADGRL4/ELTD1 silencing induced the accumulation of cis-aconitate (a citric acid cycle intermediary), UDP-glucoronate (a sugar metabolism intermediary), fructose 2,6 diphosphate (a potent stimulator of glycolysis [25
]), uridine 5-diphosphate (a glycogen metabolism intermediary), aspartic acid (an amino acid synthesised from oxaloacetate and a citric acid cycle intermediary) and uridine (a pyrimidine analog). ADGRL4/ELTD1′s suppression of the above metabolites suggests a suppressive effect on glycolysis and metabolites related to the TCA cycle. Interestingly, ACLY expression positively regulates glycolysis (with ACLY silencing leading to a decrease in glycolysis) [19
], as does fructose 2,6 diphosphate [25
]. ADGRL4/ELTD1′s repressive effect on both fructose 2,6 diphosphate and ACLY suggests a possible role for ADGRL4/ELTD1 in regulating glycolysis.
In the LC-MS experiments, the majority of metabolites were suppressed with the most suppressed being phosphocreatine (a high energy phosphate metabolite), N-acetylaspartyglutamate (a metabolite derived from glutamate), deoxyadenosine triphosphate (a nucleotide involved in DNA synthesis), and sedoheptulose 1-phosphate (a pentose phosphate pathway intermediary). Pathway analysis showed significant enrichment of metabolites associated with pyrimidine metabolism, amino acid metabolism pathways (alanine, aspartate, and glutamine metabolism; cysteine and methionine metabolism; taurine metabolism; and arginine and proline metabolism) and amino and nucleotide sugar metabolism. The metabolomics experiments were limited by the low number of significant FDR-adjusted p values, despite substantial FC effects. This is most likely related to the relatively low number of biological replicates (five). Future studies will use larger numbers of replicates to investigate whether metabolite changes are dependent on the expression of induced/repressed transcripts downstream of ADGRL4/ELTD1 expression.
ADGRL4/ELTD1 silencing upregulating KIT mRNA and surface protein is interesting as KIT has an important role in haematopoiesis and endothelial development. In haematopoiesis, KIT is expressed by early haematopoietic progenitor cells (HPC) (regulating haematopoietic stem cell (HSC) renewal as well lineage differentiation [26
]) and within the haemogenic endothelial niche by haemogenic endothelial cells (HECs) [27
]. Interestingly, ADGRL4/ELTD1 is part of a larger transcriptional programme regulating the differentiation of HECs to HSCs [28
]. Together these results suggest new ways in which ADGRL4/ELTD1 regulates the endothelial phenotype.
Our findings reveal new relationships between ADGRL4/ELTD1 and the Notch pathway (JAG1
). The findings regarding DLL4 confirm the previously documented relationship between ADGRL4/ELTD1 and DLL4 [1
]. DLL4-Notch (which is repressed by ADGRL4/ELTD1 [1
]) decreases endothelial glycolysis and mitochondrial respiration and increases endothelial exogenous fatty acid uptake [29
]. By contrast, VEGFA (which induces ADGRL4/ELTD1 [1
]) increases glycolysis [20
]. ADGRL4/ELTD1′s hypothesised regulatory effect on glycolysis is thus likely to be nuanced and complex. KIT signalling is known to induce fatty acid metabolism through the transcriptional coactivator PPARGC1A, which upregulates fatty acid synthesis pathway genes [30
In summary, this work establishes ADGRL4/ELTD1 as a regulator of endothelial metabolism through its suppression of ACLY and SLC25A1 and its effect on various metabolites. It further extends ADGRL4/ELTD1′s relationship with the Notch signalling pathway and shows a new relationship with KIT. New insights into ADGRL4/ELTD1′s function within endothelial biology, and new experimental targets within the endothelial tumour microenvironment are clinically relevant findings that could be investigated as a means to better treat cancer.