3.Adjustment of the phosphorylation of 5-LOX 
3.1  Activation of 5-LOX by phosphorylation 
The 5-LO pathway was activated in RPM by 16:0/9al-PC in the presence of hBALF, however the amount of 5-LO products detected was half that observed when 16:0/9al-PC was added to RPM in HBSS (Table 1).
Increased production of 5-LO-derived eicosanoids was observed with 16:0/9al-PC (7.5–75 µM) pretreatment 3 minutes before the addition of zymosan compared to no lipid pretreatment (0 µM) or to pretreatment with POPC (7.5–75 µM) (Figure 2A). Pretreatment of RPM with POPC (7.5–150 µM) 3 min prior to zymosan addition did not alter the production of 5-LO-derived eicosanoids compared to no lipid pretreatment (0 µM)
(Figure 2A). After stimulation of RPM with 16:0/9al-PC phosphorylation of p38 MAPK was observed whereas activation of the ERK1/2 was not detected (Figure 4). This data suggests that phosphorylation of the p38 MAPK pathway mediates activation of the 5-LO pathway by16:0/9al-PC in RPM.
It was found that 16:0/9al-PC, as well as other oxidized phospholipids (Table 1), activates the 5-LO pathway in RPM (Figure 1). The first step in the leukotriene pathway is release of arachidonic acid from a glycerophospholipid via the action of cPLA2α and AA is transformed into LTA4, which is a precursor of LTB4 and LTC4, through the action of 5-LO/FLAP.10,12 A key step in both the cPLA2α and 5-LO enzymatic mechanism is the translocation of the enzyme to intracellular membranes. Elevation of intracellular calcium levels drives the translocation of both cPLA2α and 5-LO to intracellular membranes and is a key step in the enzymatic mechanisms of both enzymes.\cite{RN\cite{Feynman_1986}10} Therefore, the effect of 16:0/9al-PC on AA release and intracellular calcium levels was examined. In addition to calcium-mediated activation, 5-LO can be regulated through protein phosphorylation. Activation of the 5-LO pathway can occur by phosphorylation at Ser271 by a p38 MAPK dependent pathway or at Ser663 by an ERK2 dependent pathway.\cite{RN7,6} Depending on the cell type, the p38 MAPK or ERK pathway can stimulate 5-LO activity
under conditions that do not lead to increases in intracellular calcium levels and result in translocation to the nuclear membrane.\cite{RN9,8} In contrast, phosphorylation of Ser523 by PKA results in the inhibition 5-LO activity.\cite{RN18} Previous studies have demonstrated that oxidized phospholipids, specifically oxPAPC, have the ability to activate a number of protein kinase pathways.\cite{RN\ref{470973}\ref{535346}21, 20, 19} Additionally, it has been found that activation of p38 MAPK and ERK1/2 pathways occurs in mouse macrophages upon exposure to the membrane fatty acid oxidation products, 4-hydroxynonenal and acrolein, which results in 5-LO activation and production of leukotrienes.\cite{RN29,27,30} The effect of 16:0/9al-PC in RPM on the MAPK pathways involved in 5-LO activation was assessed using a specific p38 MAPK inhibitor (SB202190), a specific MEK/ERK inhibitor (U0126), and the non-specific JNK inhibitor (SP600125).\cite{RN32,31} It was found that both the p38
MAPK and MEK/ERK inhibitors significantly reduced 5-LO-derived eicosanoid production in RPM with 16:0/9al-PC (Figure 3). The JNK inhibitor did not have an effect on 5-LO production by in RPM treated with 16:0/9al-PC. \cite{RN5}
16:0/9al-PC plays a role in the regulation of 5-LO activity and therefore leukotriene production via the p38 MAPK pathway in RPM.\cite{RN5}
MAPK pathway activation enhances 5-LO activity and product formation in the presence of an additional stimulus, like zymosan,15 and with the evidence that intracellular calcium levels do not need to be elevated
for 5-LO activation to occur. \cite{RN9,8}
Oxidized phospholipids cause an enhancement of the 5-LO pathway coupled to a concomitant inhibition of the COX pathway. From these studies, the mechanism of the influence of oxidized phospholipids on the regulation of 5-LO activity and therefore leukotriene production occurs through the p38 MAPK pathway in RPM.
 
3.2  Inhibition of 5-LOX by phosphorylation 
5-LO eicosanoid production in RPM by 16:0/9al-PC after 1h was significantly reduced by
60% in the presence of a p38 MAPK inhibitor (SB202190) and by 37% with a MEK/ERK
inhibitor (U0126). However, the JNK inhibitor (SP600125) did not have a significant effect
on 5-LO eicosanoid production in RPM by 16:0/9al-PC (Figure 3A). While both the p38
MAPK and MEK/ERK inhibitors did result in reduced levels of 5-LO-derived eicosanoids in
the presence of 16:0/9al-PC, neither inhibitor completely returned the total 5-LO product
levels to basal levels. Additionally, the production of 5-LO-derived eicosanoids in RPM in
the presence of both 16:0/9al-PC and zymosan for 1h was significantly reduced by 65% in
the presence of the p38 MAPK inhibitor and by 71% with the MEK/ERK inhibitor (Figure
3B). Individually each of these inhibitors reduced the levels of 5-LO derived eicosanoids to
that observed in RPM stimulated with zymosan alone (Figure 3B). The presence of the JNK
inhibitor did not affect the production of 5-LO eicosanoids in RPM in the presence of both
16:0/9al-PC and zymosan (Figure 3B). This data suggested a possible role of p38 MAPK
and ERK1/2 in the activation of the 5-LO pathway by 16:0/9al-PC in RPM.\cite{RN5}
 
List the activators and inhibitors in a table
Inhibitors/Activators
Target Sites
Release of AA
Translocation of 5-LOX
Reference
Extra
p38 MAPK inhibitor
RPM
/
/
17 ,18
 
specific MEK/ERK inhibitor (U0126)
RPM
/
/
 
non-specific JNK inhibitor (SP600125)
RPM
/
/
 
 
 
 
 
 
 
 
 
 
 
 
 
 
4.   Application of controlling 5-LOX
ž   Oxidized phospholipids, which can activate the 5-LOX, formed by the reaction of surfactant phospholipids with act in a proinflammatory fashion in the lung and may play a role in exacerbating pulmonary inflammatory diseases, such as COPD and asthma.\cite{RN5}
ž   It could be that defects in 5LO phosphorylation could explain the muscle symptoms and/or elevation of muscle and liver enzymes associated with statin therapy. Moreover, it is plausible that in patients with 5LO phosphorylation deficits, statins may not decrease inflammation and, thus, may have fewer effects on atherosclerosis.\cite{RN44}
ž   There are also implications in medical fields other than atherosclerosis, because the increased production of LTs by COX2 and 5LO has been implicated with increased risks for colon cancer \cite{RN45}, Alzheimer’s disease\cite{RN46,48,47}, and asthma \cite{RN49}. Because statins may reduce the risk of colon cancer \cite{RN66} and the progression of Alzheimer’s disease \cite{RN67}, it is plausible that statins (and PIO) may have a role in preventing the membranous shift of 5LO and, hence, the production of LTs in various disease states.
 
 

\ref{312826}

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