Cytohesin-1 Regulates the Arf6-Phospholipase D Signaling Axis in Human Neutrophils: Impact on Superoxide Anion Production and Secretion

Abstract

Polymorphonuclear neutrophil (PMN) stimulation with fMLP stimulates small G proteins such as ADP-ribosylation factors (Arfs) Arf1 and Arf6, leading to phospholipase D (PLD) activation and functions such as degranulation and the oxidative burst. However, the molecular links between fMLF receptors and PLD remain unclear. PMNs express cytohesin-1, an Arf-guanine exchange factor that activates Arfs, and its expression is strongly induced during the acquisition of the neutrophilic phenotype by neutrophil-like cells. The role of cytohesin-1 in the activation of the fMLF-Arf-PLD signaling axis, and the accomplishment of superoxide anion production, and degranulation was investigated in PMNs using the selective inhibitor of cytohesin, Sec 7 inhibitor H3 (secinH3). Cytohesin-1 inhibition with secinH3 leads to Arf6 but not Arf1 inhibition, demonstrating the specificity for Arf6, and fMLF-mediated activation of PLD and of the oxidative burst as well. We observed a decrease in fMLF-mediated protein secretion and expression of cell surface markers corresponding to primary (CD63/myeloperoxidase), secondary (CD66/lactoferrin), and tertiary (matrix metalloproteinase-9) granules in PMNs incubated with secinH3. Similarly, silencing cytohesin-1 or Arf6 in PLB-985 cells negatively affected fMLF-induced activation of PLD, superoxide production, and expression of granule markers on the cell surface. In contrast, stable overexpression of cytohesin-1 in PLB-985 cells enhanced fMLF-induced activation of Arf6, PLD, and NADPH oxidase. The results of this study provide evidence for an involvement of cytohesin-1 in the regulation of the functional responses of human PMNs and link these events, in part at least, to the activation of Arf6.

The GTPase-activating protein ARAP3 regulates chemotaxis and adhesion-dependent processes in neutrophils

“…PI3Ks and Rho, Rap, and Arf family small GTPases are involved in regulating the phagocyte oxidase. 6,9,30 We found no difference in ROS production by neutrophils from tamoxifen-or mock-induced Arap3 fl/fl ERT2Cre ϩ mice or controls after stimulation with the soluble agonist fMLF ( Figure 2A-B and supplemental Figure 1A for controls), nor with the nonphysiologic, soluble stimulus phorbol 12-myristate 13-acetate (data not shown). This indicated that the machinery required for ROS production was intact in neutrophils lacking ARAP3.…”

“…Rho family small GTPases are involved in regulating the neutrophil NADPH oxidase and chemotaxis, 6,7 whereas Arf family members regulate phagocytosis, ROS production, and chemotaxis. 8,9 ARAP3 was identified from porcine neutrophils in a screen for PtdIns(3,4,5)P 3 -binding proteins. In vitro, ARAP3 is a PtdIns(3,4,5)P 3 -dependent Arf6 GAP and a Rap-activated RhoA GAP.…”

Phagocytic NADPH Oxidase Links ARNO-Arf6 Signaling Pathway in Glucose-Stimulated Insulin Secretion from the Pancreatic �-Cell

“…[14,15]. An elegantly described study in human neutrophils, demonstrated the regulation of Arf6-PLD signaling in superoxide production by cytohesin-1, another family member of ARNO [23]. As a logical connection to all the evidences, we tested the effect of secinH3 on the translocation of p47 phox to the membrane.…”

“…Furthermore, in the context of our current investigation, activation of Nox2 has been linked to Arf6-PLD signaling axis in polymorphonuclear leukocytes [23]. Therefore, we next determined if glucose-induced phosphorylation and membrane association of p47 phox , a member of the cytosolic core of Nox2 [24] are regulated by a signaling step involving activation of Arf6 by ARNO.…”

Molecular structure of phospholipase D and regulatory mechanisms of its activity in plant and animal cells

“…ARF1 activates PLD1 in vitro by increasing the catalytic constant without changes in the K m value [80]. The replacement of GDP by GTP in ARF6 is important for activation of PLD [114], and this explains the stimulatory effect of guanine nucleotide exchange factors on the activity of PLD1 in vivo [115,116]. The N-terminus of PLD2 (amino acids 1-308) is essential for modification of this enzyme activity through ARF.…”