Purinergic A2b Receptor Activation by Extracellular Cues Affects Positioning of the Centrosome and Nucleus and Causes Reduced Cell Migration

Abstract

The tight, relative positioning of the nucleus and centrosome in mammalian cells is important for the regulation of cell migration. Under pathophysiological conditions, the purinergic A2b receptor can regulate cell motility, but the underlying mechanism remains unknown. Expression of A2b, normally low, is increased in tissues experiencing adverse physiological conditions, including hypoxia and inflammation. ATP is released from such cells. We investigated whether extracellular cues can regulate centrosome-nucleus positioning and cell migration. We discovered that hypoxia as well as extracellular ATP cause a reversible increase in the distance between the centrosome and nucleus and reduced cell motility. We uncovered the underlying pathway: both treatments act through the A2b receptor and specifically activate the Epac1/RapGef3 pathway. We show that cells lacking A2b do not respond in this manner to hypoxia or ATP but transfection of A2b restores this response, that Epac1 is critically involved, and that Rap1B is important for the relative positioning of the centrosome and nucleus. Our results represent, to our knowledge, the first report demonstrating that pathophysiological conditions can impact the distance between the centrosome and nucleus. Furthermore, we identify the A2b receptor as a central player in this process.

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development

“…While AKAP9 is required for integrin-mediated adhesion and barrier function downstream of EPAC1, the scaffolding protein is not essential for EPAC-induced Rap activation, reorganization of cortical actin, or basal barrier properties (931). In relation to the function of EPAC1 in MT dynamics, a recent study shows that although EPAC1 activity is not required for maintaining the relative positioning of the centrosome and nucleus under normal conditions, EPAC1/ Rap1B signaling is essential for hypoxia/ATP-induced centrosome-nuclear separation and cell migration downstream of the purinergic A2b receptor in human retinal epithelial pigment (RPE) cells (794). The centrosome is the main MTorganizing center and promotes de novo assembly of MTs (77).…”

Ouabain affects cell migration via Na,K-ATPase-p130cas and via nucleus-centrosome association

“…We therefore analyzed if modulation of total p130cas using siRNA and of p130cas Tyr phosphorylation by ouabain affects cell motility. To measure this, we performed monolayer cell scratch damage assays, commonly used for monitoring cell migration [ 22 ], that we had employed previously [ 19 ]. RPE monolayers were scratched, cells were allowed to migrate into the gap in the presence or absence of ouabain, and the gap was measured after the scratch was made (0 hrs) and after 36 hrs.…”

“…While we were performing the above experiments which demonstrate that ouabain inhibits cell migration via p130cas, we were engaged in another line of investigation that showed that nucleus-centrosome separation can be induced by ATP resulting in inhibition of cell migration [ 19 ]. This suggested the possibility that ouabain’s effect on cell migration could involve nucleus-centrosome separation.…”

“…Second, we observed separation of nucleus and centrosome upon nanomolar ouabain treatment of cells. We had previously shown using a system of ATP and hypoxia that such separation causes a block in cell migration [ 19 ]. RNAi and kinase inhibitors suggested that ERK is critically involved in this pathway.…”

Hexose‐6‐phosphate dehydrogenase controls cancer cell proliferation and migration through pleiotropic effects on the unfolded‐protein response, calcium homeostasis, and redox balance

“…Upon H6PD silencing, we observed a loose appearance of the centrosome, as visualized through α‐tubulin immunofluorescence staining. Microtubule structure is indispensable for cell movement (40), and changes in the centrosome have been reported to cause defects in cell migration (41, 42). More detailed studies on the structure and composition of the microtubular network after H6PD down‐regulation will provide a clearer picture.…”