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To whom correspondence should be addressed: Division of Pulmonary, Critical Care, and Occupational Medicine, Rm. 100, EMRB, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242. Tel.: 319-335-7590; Fax: 319-335-6530
* This work was supported in part by a Veterans Administration Merit Review grant, National Institutes of Health Grants HL-60316 and ES-09607, Environmental Protection Agency Grant R826711 (to G. W. H.), National Institutes of Health Grant HL-03860 (to A. B. C.), and Grant RR00059 from the General Clinical Research Centers Program, NCRR, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Human alveolar macrophages have both lipopolysaccharide (LPS)-induced and constitutive phosphatidylinositol 3-kinase (PI3K) activity. We observed that blocking PI3K activity increased release of prostaglandin E2 after LPS exposure, and increasing PI3K activity (interleukin-13) decreased release of prostaglandin E2 after LPS exposure. This was not because of an effect of PI3K on phospholipase 2 activity. PI3K inhibition resulted in an increase in cyclooxygenase 2 (COX2) protein, mRNA, and mRNA stability. PI3K negatively regulated activation of the p38 pathway (p38, MKK3/6, and MAPKAP2), and an active p38 was necessary for COX2 production. The data suggest that PI3K inhibition of p38 modulates COX2 expression via destabilization of LPS-induced COX2 mRNA.
nuclear factor κB
extracellular signal-regulated kinase
c-Jun NH2-terminal kinase
enzyme-linked immunosorbent assay
mitogen-activated protein kinase kinase 3/6
mitogen-activated protein kinase activated protein kinase-2
Alveolar macrophages are major effector cells of the innate immune system. They play a central role in the response to Gram-negative bacteria in the lung. Endotoxin (LPS)1 is the principle activating component of the Gram-negative cell wall and is a major activator of these macrophages. After initial exposure to LPS, expression of prostaglandin endoperoxide H synthase 2 (COX2) and production of prostaglandin E2 (PGE2) appears at 12 to 24 h after stimulation (
). Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid and O2 to PGH2. It is the rate-limiting step in the metabolism of arachidonic acid to prostanoid products. Arachidonic acid is a 20-carbon unsaturated fatty acid that is hydrolyzed from membrane-bound phospholipids by the actions of phospholipases (PLA) (secretory PLA2 and cytosolic PLA2). Both COX1 and -2 catalyze the same step in the arachidonic acid pathway (a cyclooxygenase reaction in which arachidonic acid is converted to PGG2 and a peroxidase reaction in which PGG2 is reduced to PGH2) (
). It is the only cyclooxygenase present in platelets and has been linked to platelet production of thromboxane A2. In contrast, COX2 is induced by inflammatory mediators and has been linked to inflammation, fever, pain, and a number of cancers (
). Class 1A PI3Ks (found in alveolar macrophages) consist of a p85-kDa subunit protein (α and β) and a p110-kDa catalytic subunit (α, β, and δ) or a p101-kDa regulatory unit and a p110-kDa catalytic unit (γ). The p85 regulatory unit is activated via interaction of the SH2 domain with YXXM motifs of multiple receptors. The p101 regulatory unit is activated by γβ subunits of G proteins downstream of G protein-coupled receptors (
). Once activated PI3K catalyzes the transfer of ATP to thed-3 position of the inositol ring of membrane-localized phosphoinositides. This results in the production of a number of bioactive lipid species including PI3P, PI3,4P, and PI3,4,5P. Both PI3,4P and PI3,4,5P are absent in most unstimulated cells and increase dramatically following PI3K activation. The presence of PI3,4,5P results in the membrane recruitment of proteins containing pleckstrin homology domains. This includes PI3K-dependent kinase (PDK-1), which phosphorylates a number of biologically important substrates (Akt, protein kinase A, and multiple protein kinase C isoforms) (
). Activation of Akt is linked to NFκB translocation and transactivation, endothelial nitric oxide synthase activation, and inhibition of a number of substrates positively involved in apoptosis. The apoptosis-related factors that are inhibited by Akt include glycogen synthase kinase 3, forkhead transcription factors, Bad, and caspase 9 (
). Glycogen synthase kinase 3 inhibition results in increased signaling from a number of transcription factors, β catenin, nuclear factor of activated T-cells, CCAAT/enhancer binding protein, GATA 4, and some of the activator protein 1 proteins (
). Activation of PI3K is therefore linked to multiple biological effects. One possible role of PI3K activity is as a modulator of MAP kinase signaling. Akt, in some conditions, has been shown to negatively regulate c-Raf (part of the ERK pathway in some cells) (
The MAP kinases are a family of evolutionarily conserved enzymes that connect cell surface receptors to regulatory targets that include both cytoplasmic and nuclear proteins. The three major MAP kinase families are the ERK (1 and 2), p38 (α, β, γ, and δ), and the JNK (1, 2, and 3) (
In these studies, we found that inhibition of the PI3K pathway increased LPS-induced COX2 and PGE2. Lack of PI3K activity increased the stability of COX2 mRNA, and this increased stability correlated with increased PGE2 release. We found PI3K activity to be correlated inversely with LPS-induced p38 activity. Inhibition of PI3K resulted in increased p38 activity. These studies suggest that constitutive and LPS-induced PI3K activity in alveolar macrophages delays and decreases the production of COX2 and release of PGE2.
We have shown previously that LPS induces production of PGE2 in human alveolar macrophages by increasing amounts of COX2 protein and mRNA (
). In this study, we show that PI3K activity regulates expression of COX2 and release of PGE2 negatively. A significant effect of active PI3K is to decrease stability of COX2 mRNA. We further showed that the PI3K pathway suppressed p38 MAPK activity, and active p38 regulates stability of the COX2 mRNA positively. Of interest, we also found that human alveolar macrophages have high constitutive PI3K activity, as well as LPS-induced PI3K activity. The expression of COX2 did not increase after LPS stimulation until there was a decrease in PI3K activity to below baseline level. This occurred at late time points after LPS stimulation. These studies suggest that PI3K activity must decrease below a threshold level to permit expression of COX2.
Fig. 12 shows the role we feel PI3K plays in COX2 expression. The figure shows formation of the well described Toll-like receptor (TLR 4) signaling pathway after macrophage LPS exposure (
). Downstream of this complex, signaling intermediates activate NFκB, all three MAP kinases (this diagram focuses on p38 only), and PI3K. The exact mechanism of PI3K activation after LPS is not known at this time, but it is known that there are two major pathways downstream of TLR 4, MyD88-dependent and MyD88-independent (
). Future studies should determine the exact upstream activators of PI3K. PI3K, via Akt, is known to be a positive regulator of NFκB activity, suggesting the need for some PI3K activity in COX2 transcription (
). Our studies, however, focus on the negative regulation of p38 by PI3K and its consequence (decreased COX2 mRNA stability). The end effect of PI3K activity in LPS-treated alveolar macrophages is the decreased production of PGE2.
This study doesn’t exclude an effect on COX2 transcription by PI3K inhibition. In fact, we have shown that an active p38 is necessary for expression of both NFκB and AP-1 luciferase activity after LPS (NFκB) or phorbol myristate acetate (AP-1) (
). This effect of p38 is mediated by phosphorylation of TATA-binding protein, which permits this component of the basal transcription complex to bind to the TATA box and interact physically with NFκB and AP-1 proteins. However, we believe the major regulatory effect of PI3K on COX2 is at the message stability level. Mestre et al. (
) have shown in a recent study that there is significant redundancy in the pathways that lead to COX2 transcription in monocyte/macrophages. They found that mutations in the NFκB, NFIL-6, or CRE sites alone did not change COX2 promoter activity. They also showed that dominant negative MAP kinases (ERK, p38, or JNK) did not decrease COX2 promoter activity.
Several previous studies have suggested a role for p38 in the stabilization of some mRNAs. A study by Guan et al. (
) in 1998 links p38 activation to production of COX2. They showed that a dominant negative p38 upstream kinase would reduce expression of COX2. This study was followed in 2000 by a group of studies showing that p38 activity had a positive effect on COX2 mRNA stability (
) used a tetracycline-regulated reporter system to investigate the regulation of COX2 mRNA stability. They found that a chimeric transcript with the COX2 3′ region was stabilized by a constitutively active MKK6, as well as an active MAPKAP2. They localized the p38 effect to the first 123 nucleotides 3′ to the stop codon (
). These studies on regulation of COX2 mRNA are consistent with our observation that PI3K inhibition increased p38 activity and stability of COX2 mRNA. p38 activation alone is not enough to induce COX2 production. In our cells, PI3K inhibition without LPS induced an increase in p38 activity but did not increase either COX2 or PGE2. This suggests that alternative pathways are activated by LPS (ERK, JNK, NFκB) that are necessary for transcriptional activation of the COX2 gene.
A possible negative role for PI3K in MAP kinase signaling has been described. A study by Park et al. (
) have shown, in bovine aortic endothelial cells, that Akt phosphorylates and inactivates MEKK3 leading to a down-regulation of MKK3/6 and p38 activity. In Gratton's study, vascular endothelial growth factor-induced PI3K activity inhibits p38, protecting the cells from apoptosis. These studies, combined with our data, suggest that Akt may decrease activation of the stress kinases.
PI3K negative regulation of COX2 production is a possible explanation for the observation that IL-10 decreases COX2 (
) looked at intestinal epithelial cells and showed that a PI3K inhibitor blocked COX2 production by activated K-Ras. Finally, a study in colon carcinoma cells found that active Akt increased COX2 expression (
). The difference in findings between these studies and ours could be because of cell specificity (none of these cells are immune cells) or different stimuli. The only study that has shown PI3K inhibition increasing COX2 is a study by Weaver et al. (
). Using colonic epithelial cell lines, they showed that the inhibitor wortmannin increased COX2 protein, and the inhibitor LY294002 increased COX2 mRNA. Their data were limited to these observations. Our study utilizing human alveolar macrophages and a sepsis-relevant stimulus (LPS) is the first to show negative regulation of COX2 by PI3K activity in a primary human cell. In addition, it is the first study to show a link between PI3K and destabilization of COX2 mRNA.
In summary, the novel findings of this study include constitutive PI3K activity in primary macrophages (alveolar), PI3K-dependent suppression of p38 activity and COX2 mRNA stability, and increased LPS-induced COX2 and PGE2 production with PI3K inhibition. These observations suggest that there is both constitutive and inducible early negative regulation of COX2 activity. The late rise in COX2 activity coincides with an increase in p38 activity and a decline in PI3K activity.