La piccola GTPasi Rap1 interposta tra la proteina mitocondriale Opa1 e l'inibizione dell'angiogenesi

OPA1 is a protein with pleiotropic functions ranging from the orchestration of mitochondrial fusion and cristae remodeling to transcriptional reprogramming 1, 2. Here we present two different mechanisms by which OPA1 exerts a transcriptional regulation activity in endothelial and breast cancer cells. Mitochondria are dynamic organelles that are now recognized as regulators of signal transduction able to impact on cellular genetic programs 3, 4. Increasing evidence support a fundamental role for mitochondrial shape in the orchestration of cellular transcriptional programs, but how cells sense and respond to changes in mitochondrial shape is unclear 5. We recently discovered that angiogenesis is transcriptionally modulated by the key mitochondrial fusion gene OPA1 through NFκB activation1. In particular, ablation of OPA1 in vivo and in vitro leads to developmental and tumor angiogenesis inhibition 1. A deep RNA sequencing analysis identified a signature for the Ras-proximate-1 RAP1, and its cyclic AMP (cAMP)-activated nucleotide exchange factor EPAC1 upon OPA1 deletion in Human Umbilical Vein Endothelial Cells (HUVECs). Previously, several studies had reported the essential role of Rap1 in developmental angiogenesis and vessel stabilization 6, 7. After birth, Rap1 is not essential, but it participates in the maintenance of vasculature and nitric oxide (NO) homeostasis 6, 8. Albeit EPAC1 is highly abundant and cover numerous functions in endothelial cells, its role in angiogenesis remained to be clarified 9. Likewise, EPAC1 was shown to be important for endothelial cells biology 10, 11. A handful of studies retrieved EPAC1 in mitochondria, and RAP1 in mitochondria associated membranes (MAMs) by proteomics, suggesting that they might be linked to mitochondria 12, 13. Whether the EPAC1/RAP1 axis could sense changes in mitochondria driven by OPA1 deletion was unknown. Our results show that EPAC1 and RAP1 localize in proximity to mitochondria and in MAMs, that are emerging as hubs for mitochondria-derived signals. Moreover, EPAC1 accumulates on mitochondria upon pharmacological activation and following OPA1 silencing. OPA1 silencing results in an increase in Ca2+ and in localized cAMP increase in proximity of mitochondria that in turn activated EPAC1 and therefore RAP1. Notably, Ca2+ chelation by BAPTA-AM treatment suppress the EPAC1/RAP1 activation elicited by OPA1 downregulation. Furthermore, the analysis of angiogenic parameters like migration and tubulogenesis revealed that the blockage of EPAC1 and RAP1 signaling could correct the defects caused by OPA1 downregulation. Thus, our work places EPAC1/RAP1 in the retrograde signaling pathway connecting mitochondria to angiogenesis and highlights the intricate network of signals and second messengers that can execute transcriptional changes when mitochondria are perturbed.