LT biosynthesis in PMNs requires the dynamic construction of macromolecular complexes on the nuclear membrane. Although prior studies focused on the discrete step of 5-LO translocation to the nuclear envelope and the use of AA as a substrate for generating leukotrienes through the activation of cPLA2
(Nahas et al., 1996
), these studies did not address how LT synthesis is organized on the nuclear envelope. We report here that multistep assembly and organization of the LT synthetic complex in isolated murine PMNs involves processes that are both dependent on and independent of AA release. The flexibility of this process and response to different signals supports a role for the LT synthetic complex as the regulator of the qualitative interaction between 5-LO and FLAP, a relationship that is likely to be reflected in the output of LTA4
To elucidate whether assembly of the LT synthetic complex on the nuclear envelope of PMNs was altered by different signals, we exploited the concept of “priming,” in which a signal by a transmembrane cytokine receptor, such as the GM-CSF receptor, synergistically augments LTB4
synthesis in concert with a GPCR agonist such as C5a (DiPersio et al., 1988
). As expected, priming by GM-CSF increased LTB4
synthesis threefold to fourfold even though there was no difference in calcium flux compared with unprimed, stimulated cells.
The additional amplification of LTB4
synthesis imparted by GM-CSF priming has two effects. First, it amplifies the AA-driven assembly of the LT synthetic complex, regulating an increase in the percentage of nuclear membrane–associated 5-LO molecules interacting with FLAP. This is distinct from the effect seen by stimulation with C5a alone. GM-CSF also provides a signal that drives the closeness of the association of 5-LO and FLAP ( and and Table ). The mechanism by which GM-CSF exerts these two distinct activities may be related to the phosphorylation of cPLA2
and 5-LO. Priming by GM-CSF stimulates the MAPK pathway, which can phosphorylate cPLA2
(Geijsen et al., 2000
). This would augment the release of AA from membrane phospholipids. Phosphorylation of 5-LO can affect its intracellular trafficking; whether it directly affects the association of 5-LO and FLAP on the nuclear envelope, however, is not known. Both ERK and p38 MAPK inhibition alter the interaction of 5-LO and FLAP independent of AA (). However, whether this is secondary to phosphorylation of 5-LO or the posttranslational modification of a yet-to-be-determined complex member is unknown.
FIGURE 6: The multistep assembly of the LT synthetic complex. 1) Under resting conditions 5-LO resides in the cytoplasm and FLAP within the nuclear membrane. No LTs are made under this condition. 2) C5a-induced cell activation, followed by a rise in intracellular (more ...)
The ability of cells to qualitatively modify the association of 5-LO with FLAP in response to discrete signals has the potential to provide the flexibility to generate the appropriate amount of LTB4
in different biological settings. This can be illustrated by considering the diverse signaling responses of the GM-CSF receptor (Hercus et al., 2009
). At low (femtomole) levels of GM-CSF, the receptor is exclusively phosphorylated at βc Ser-585, engaging PI3 kinase and Akt pathways that do not lead to posttranslational modification of either cPLA2
or 5-LO, and are not known to prime PMNs for augmented LTB4
synthesis. This would potentially correspond to a setting of early or low-level tissue inflammation or injury, and the generation of LTB4
would be determined solely by the AA levels induced by C5a, fMLP, or chemokines. In settings of more severe inflammation or infection, higher levels of GM-CSF would be generated. This would lead to exclusive phosphorylation of βc Tyr-577 and extinction of Ser-585 phosphorylation. This engages the MAP kinase pathway, allowing for the phosphorylation of cPLA2
and increased release of free AA. In turn, this would lead to the incorporation of a higher percentage of nuclear membrane–associated 5-LO into LT synthetic complexes with increased closeness between 5-LO and FLAP. Phosphorylation of 5-LO would have an additional effect by increasing the closeness of its association with FLAP. The net effect of these changes would be the generation of more LTB4
and the recruitment of more PMNs and other LTB4
-responsive cells to the inflamed tissue. The levels of GM-CSF used in this study to prime PMNs are within this higher range.
It has been shown that ligands of scaffold proteins impart an allosteric effect on the scaffold protein's ability to recruit a second ligand (Good et al., 2011
). We found that AA has a direct effect on the interaction of the N- and C- termini of FLAP. Furthermore, in the presence of the cPLA2
Inh, primed and unprimed cells treated with C5a exhibited statistically decreased LTB4
production. We also found that in cells exposed to the cPLA2
Inh and C5a, τ1
values significantly decreased close to those observed in unstimulated cells. Similarly, cells that were incubated with the cPLA2
Inh, primed, and treated with C5a have significantly reduced τ1
values compared with cells primed and treated without the inhibitor. Taken together, these results imply that AA governs the closeness of the association of 5-LO and FLAP.
The use of AA as a means of modulating the LT synthetic complex yields increased flexibility in terms of regulating LT synthesis. Studies have shown that the regulation of the reacylation of free AA is a key step in LT synthesis in PMNs, especially under the conditions of priming (Zarini et al., 2006
). Therefore it will be important to understand the regulation of the transacylase enzymes.
In addition to the studies indicating that LTB4
dramatically amplified formyl peptide–mediated neutrophil chemotaxis and cytoskeletal rearrangement by acting on signaling pathways upstream of actin polymerization (Afonso et al., 2012
), C5a, fMLP, and IL-8 all stimulate PMNs to generate LTB4
, and all have LTB4
synthesis amplified by priming (Dahinden et al., 1988
; DiPersio et al., 1988
; Krump and Borgeat, 1994
; Afonso et al., 2012
). Therefore the observations that AA stimulates the assembly of the LT synthetic complex in response to C5a likely represent a general mechanism for transducing and amplifying signaling by chemoattractants.