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. Author manuscript; available in PMC: 2012 Jul 19.
Published in final edited form as: Sci Signal. 2012 Feb 7;5(210):ra12. doi: 10.1126/scisignal.2002448

PTEN Activates the Actin Depolymerization Factor Cofilin-1 During PGE2-Mediated Inhibition of Fungal Phagocytosis

CH Serezani 1, S Kane 1, AI Medeiros 2, A M Cornett 1, SH Kim 3, MM Marques 4, SP Lee 4, C Lewis 1, E Bourdonnay 1, MN Ballinger 1, E S White 1, M Peters-Golden 1,*
PMCID: PMC3400468  NIHMSID: NIHMS385040  PMID: 22317922

Abstract

Macrophage ingestion of Candida albicans requires recognition by multiple receptors and activation of diverse signaling programs. Synthesis of the lipid mediator prostaglandin E2 (PGE2) and generation of cyclic adenosine monophosphate (cAMP) also accompany this process. Here we characterized the mechanisms underlying PGE2-mediated inhibition of phagocytosis and of F-actin polymerization in response to ingestion of C. albicans. PGE2 suppressed macrophage phagocytosis and F-actin content through E-series prostanoid 2 and 4 receptors, cAMP, and activation of types I and II protein kinase A, but not guanine nucleotide exchange protein activated by cAMP. Dephosphorylation and activation of the actin depolymerizing factor cofilin-1 was necessary for these inhibitory effects of PGE2. Unexpectedly, cofilin-1 activation by PGE2 was mediated by the protein phosphatase activity of PTEN (phosphatase and tensin homolog deleted on chromosome 10), with which it directly associated. Because PGE2 overproduction accompanies many immunosuppressed states, the PTEN-dependent pathway described here may contribute to impaired antifungal defenses.

INTRODUCTION

Invasive Candida albicans infections are a serious clinical threat in patients who are immunosuppressed or undergo major surgical procedures. Mortality associated with disseminated candidiasis can be as high as 30–40%, despite the availability of new antifungal drugs (1). Host defense against candidiasis relies mainly on the ingestion and elimination of C. albicans by cells of the innate immune system, especially neutrophils, monocytes, and macrophages. In the lungs, C. albicans is a normal colonizer in humans and animals, but becomes a serious threat for immunocompromised patients (2, 3). Alveolar macrophages are the principal resident phagocytic cells protecting the alveolar compartment from microbial invasion (4). The antimicrobial effector functions of alveolar macrophages are modulated by pro- and-anti-inflammatory signals that act in an autocrine and paracrine manner (4). Recognition of C. albicans by alveolar macrophages is mediated by pathogen recognition receptors such as Toll like receptors and the C-type lectin mannose and dectin-1 receptors which recognize mannan and beta-glucan, respectively (5-8). Engagement of pathogen recognition receptors triggers fungal ingestion and killing; these processes require cytoskeletal rearrangement, including F-actin polymerization, an event orchestrated by actin depolymerization factors such as cofilin-1 (9).

Macrophage responses to pathogens also include generation of soluble inflammatory mediators such as cytokines and lipid mediators including prostaglandin E2 (PGE2) (10, 11). PGE2 is derived from the cyclooxygenase (COX) metabolism of arachidonic acid (12). PGE2 has the potential to exert important effects on innate immunity, as it is produced in abundance in the context of inflammation and infection (13). Moreover, PGE2 production has been reported to be increased in various conditions associated with increased susceptibility to infection, including protein-calorie malnutrition (14), cancer (15), infancy (16), aging (17), cystic fibrosis (18), cigarette smoke exposure (19), bone marrow transplantation (13), and HIV infection (20). The effects of PGE2 follow the ligation of four distinct cell membrane-associated G protein-coupled E-series prostanoid (EP) receptors termed EP1 to EP4 (21). In alveolar macrophages, EP2 and EP4 primarily mediate the effects of PGE2 through stimulatory G protein (GαS), increasing the activity of adenylyl cyclases which generate cyclic adenosine monophosphate (cAMP) (21). cAMP is a second messenger that influences numerous cellular functions through the activation of two downstream effector molecules, protein kinase A (PKA) and the guanine nucleotide exchange proteins directly activated by cAMP (Epac1 and Epac2) (21). We have shown that these two cAMP effectors differentially contribute to the inhibition of various functions of alveolar macrophages: Whereas Epacs inhibit FcR-mediated phagocytosis, PKA inhibit bacterial killing and cytokine secretion (22). FcR-mediated phagocytosis is inhibited by PTEN (phosphatase and tensin homolog deleted on chromosome 10) (23). Indeed, we have identified an essential role for PTEN in mediating the inhibitory effects of PGE2 on FcR-mediated phagocytosis in alveolar macrophages (23). PTEN is a protein and lipid phosphatase that can dephosphorylate PIP3, thereby inhibiting Akt activation (24), and our previous results implicated the lipid phosphatase activity of PTEN in the inhibitory actions of PGE2 on FcR-mediated phagocytosis (23).

It remains to be determined if PGE2 impairs host defense against C. albicans. In addition, the specific role of cAMP, its effectors, and PTEN in inhibiting phagocytosis of this fungus or any other unopsonized pathogen is unknown. Thus, we sought to investigate if PGE2 production during C. albicans infection inhibits phagocytosis in alveolar macrophages and to identify the operative molecular mechanisms. In this report we demonstrate that PGE2-cAMP-PKA-PTEN activates cofilin-1, preventing F-actin polymerization and therefore yeast ingestion. Unexpectedly, cofilin-1 dephosphorylation and activation in our model was mediated by the protein phosphatase activity of PTEN.

RESULTS

The PGE2-cAMP axis dampens C. albicans phagocytosis

To investigate the relevance of the PGE2-cAMP axis as a potential modulator of phagocytosis of C. albicans by alveolar macrophages, we initially determined if these cells produce PGE2 and subsequently cAMP when challenged with yeast. C. albicans induced a ~2.5-fold increase in PGE2 synthesis above that observed in uninfected cells (Fig. 1A). In parallel, cAMP was also significantly increased upon C. albicans challenge (Fig. 1B). Next, we sought to investigate if PGE2 generated during yeast challenge influenced the extent of ingestion. Treatment of alveolar macrophages with the COX inhibitor aspirin increased yeast ingestion by ~40% when compared to untreated control (Fig. 1C). Using two different approaches, we confirmed that PGE2 is the endogenous COX metabolite responsible for suppression of phagocytosis whose synthesis was inhibited by aspirin. First, pretreatment with selective antagonists to the PGE2 receptors EP2 and EP4 both enhanced C. albicans phagocytosis (Fig. 1C). Second, exogenous PGE2 as well as relatively selective EP2 and EP4 agonists decreased C. albicans ingestion (Fig. 1D). Both antagonist and agonist data suggest a greater role for ligation of EP2 than EP4 in mediating PGE2 inhibition of yeast phagocytosis (Fig. 1C and D). Because EP2 and EP4 receptors are both coupled to Gαs protein, and because C. albicans infection results in increased cAMP production, we speculated that the inhibitory effect of PGE2 was due to enhanced cAMP concentrations generated by activation of adenylyl cyclases. To confirm this, cells were pretreated with the adenylyl cyclase inhibitor SQ22536, then challenged with C. albicans. Indeed, inhibition of adenylyl cyclases enhanced fungal phagocytosis by 50% when compared to untreated cells (Fig. 1C). Together, these data show that cAMP generated by PGE2 through ligation of EP2 and to a lesser extent EP4 dampened ingestion of C. albicans.

Fig. 1. EP2 or EP4 and cAMP mediate PGE2 inhibition of C. albicans phagocytosis.

Fig. 1

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(A) PGE2 production in alveolar macrophages challenged with C. albicans (10:1). (B) Intracellular cAMP concentration in alveolar macrophages challenged with C. albicans (10:1). In (A) and (B), data are presented as fold increase relative to the unchallenged control. (C) Alveolar macrophages were pretreated with aspirin (ASP), the EP2 antagonist AH6809, the EP4 antagonist ONO-AE3-208, or the adenylyl cyclase inhibitor SQ22536 prior to infection with FITCC. albicans for 90 min, and phagocytosis was measured by fluorometry. (D) Alveolar macrophages were pretreated for 5 min with the EP2 agonist butaprost free acid, EP4 agonist ONO-AE1-329, or PGE2 prior to infection with FITCC. albicans and phagocytosis was measured by fluorometry. In (C) and (D), data are presented as % of control with the control value representing untreated and infected alveolar macrophages. Data are mean ± SEM from at least 3 separate experiments. * p < 0.05 compared to untreated control, represented by the dashed line.

PKA types I and II mediate inhibition of C. albicans phagocytosis through A-kinase anchoring proteins

cAMP actions in alveolar macrophages are due to activation of two effectors, PKA and Epac-1 (21). In alveolar macrophages, Epac-1 activation decreases FcR-mediated phagocytosis, whereas PKA is involved in modulating bacterial killing and cytokine release (22). To investigate the role of these two cAMP effectors in the inhibitory effects on C. albicans ingestion, we used cell membrane-permeable analogs specific for activation of PKA (6-bnz-cAMP) or Epac-1 (8-pCPT-2-O-Me-cAMP). The PKA agonist, but not the Epac-1 agonist, decreased phagocytosis (Fig. 2A). To confirm that endogenous PKA activation restrains phagocytosis, cells were treated with the PKA inhibitor KT5720 or with the cell-permeable PKA inhibitory peptide PKI, and both inhibitors potentiated alveolar macrophage phagocytosis (Fig. 2B). These data show that, in contrast to FcR-mediated phagocytosis (22), inhibition of C. albicans phagocytosis by PGE2-cAMP is mediated by PKA rather than Epac-1.

Fig. 2. PKA-AKAP interactions inhibit C. albicans phagocytosis.

Fig. 2

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(A) Alveolar macrophages were pretreated with the PKA-specific cAMP analog 6-Bnz-cAMP or the Epac-specific cAMP analog 8-pCPT-2-O-Me-cAMP, followed by incubation with FITCC. albicans, after which phagocytosis was determined by fluorometry. (B) Alveolar macrophages were incubated with the PKA inhibitors KT5720 or PKI peptide before the addition of FITCC. albicans, after which phagocytosis was determined by fluorometry. (C) Cells were pretreated with regulatory subunit-selective cAMP analogs to inhibit PKA type I (2-Cl-8-MA-cAMP) or PKA type II (6-MBC-cAMP), or with the AKAP-PKA-II-specific disruptor peptide Ht31 or the AKAP-PKA-I-specific disruptor peptide RIAD. Cells were then challenged with FITCC. albicans followed by fluorometric determination of phagocytosis. The data are means ± SEM values of 3 separate experiments, each performed in triplicate. * p < 0.05 compared to FITCC. albicans alone (control); # p < 0.05 compared to PKA-II antagonist or Ht31.

PKA consists of two catalytic subunits, Cα and Cβ, as well as two cAMP-binding regulatory subunits, RI and RII (25). To investigate the ability of PKA isoenzymes to modulate phagocytosis of C. albicans, we employed isoenzyme-selective inhibitors of PKA-I (Rp-8-Cl-cAMPs) or PKA-II (Rp-8-Piperidino-cAMPs) as described (26). Both PKA-I and PKA-II restrained C. albicans ingestion, but the role of PKA-II appeared to be quantitatively greater (Fig. 2C). By binding to the regulatory subunits of PKA, A-kinase anchoring proteins (AKAPs) target PKA to specific microdomains and thereby enhance proximity to particular substrates (27). To determine whether AKAPs are involved in the PKA-dependent inhibition of phagocytosis, we disrupted PKA RI-AKAP interactions with the RIAD peptide (28) and PKA RII-AKAP interactions with the Ht31 peptide (29, 30). Both Ht31 and RIAD enhanced fungal phagocytosis, with the action of the former exceeding that of the latter. These data suggest that both type I PKA and especially type II PKA participate in inhibiting the ingestion of C. albicans in a manner that depends on AKAP anchoring.

PKA activation decreases polymerization of F-actin

F-actin nucleation is a key step involved in the cytoskeletal remodeling necessary for phagocytic target ingestion (31). Thus, we hypothesized that PGE2-PKA inhibition of phagocytosis of yeast is associated with decreased polymerized F-actin and increased monomeric G-actin. We performed confocal fluorescence microscopy to determine the amounts of F- and G-actin, as indicated by binding of phalloidin-FITC which binds to F-actin and DNase-Alexa-555 which binds to G-actin. Alveolar macrophages were pretreated with PGE2, the COX inhibitor indomethacin, or the adenylyl cyclase inhibitor SQ22536, followed by C. albicans for 15 min. Untreated phagocytosing cells exhibited similar amounts of both F- and G-actin (as evidenced by similar intensity of green and red fluorescence) (Fig. 3A). In this experimental condition, F-actin accumulated at the periphery of the cell, whereas G-actin was more centrally localized near the nucleus. When alveolar macrophages were pretreated with PGE2 prior to challenge with yeast, G-actin abundance increased throughout the cell whereas F-actin abundance was diminished (Fig. 3B). In contrast, COX inhibition with indomethacin and AC inhibition with both substantially increased the peripheral accumulation of F-actin (Fig. 3, C and D). To confirm the effect of PGE2 on F-actin polymerization as visualized by microscopy as well as to determine which cAMP effector was responsible, we utilized an in vitro assay in which FITC-phalloidin bound to F-actin was solubilized and the fluorescence was quantified by fluorometric analysis. C. albicans challenge itself enhanced the amount of F-actin above that in resting cells, and this was decreased below the amount in resting cells by both PGE2 and PKA agonists, but not an Epac agonist (Fig. 3E). In addition, both COX and AC inhibition enhanced C. albicans-induced F-actin amounts (Fig. 3E). We further investigated the relative roles of PKA RI and RII in F-actin accumulation during C. albicans phagocytosis using isoenzyme-selective agonists and antagonists. Activation of both PKA RI and RII inhibited C. albicans-induced F-actin formation, but as was the case for effects on phagocytosis (Fig. 2C), PKA RII had a greater effect than PKA RI (Fig. 3F). Together, these data show that activation of type II PKA and to a lesser extent type I PKA in response to endogenous PGE2 generated during C. albicans ingestion acts as a phagocytic brake by disrupting F-actin polymerization.

Fig. 3. PGE2 and PKA prevent F-actin polymerization.

Fig. 3

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(A to D) Alveolar macrophages were pretreated for 20 min with medium (A), PGE2 (B), the COX inhibitor indomethacin (C), or the adenylyl cyclase inhibitor SQ22536 (D) before the addition of C. albicans (10:1). Cells were stained for F-actin with Alexa435-Phalloidin (green) or for G-actin with Alexa555-DNase (red), and imaged with confocal microscopy. (E) Alveolar macrophages were pretreated with PGE2, cAMP analogs selective for PKA or Epac, indomethacin, or SQ22536 and challenged with C. albicans. Actin polymerization was determined by fluorometry. (F) Cells were pretreated with regulatory subunit-selective cAMP analogs which activate PKA type I (2-Cl-8-MA-cAMP) or type II (6-MBC-cAMP) or inhibit PKA type I (Rp-Cl-8-MA-cAMP, 10 μM) or type II (Rp-8-PIP-cAMP, 10 μM) and challenged with C. albicans. Actin polymerizarion was determined by fluorometry. The data are means ± SEM values of 3 separate experiments, each performed in triplicate. * p < 0.05 compared to uninfected control; # p < 0.05 compared to yeast alone.

Cofilin-1 mediates PGE2-PKA inhibition of F-actin polymerization

Actin depolymerizing factors prevent F-actin assembly by both depolymerizing F-actin and inhibiting the polymerization of monomeric G-actin (32). Thus, we speculated that PGE2-PKA enhances activation of actin depolymerizing factors. Initially, we determined the abundance of the actin depolymerizing factors cofilin-1, destrin and cofilin-2 in alveolar macrophages by immunoblotting. Although cofilin-1 and destrin were readily detectable, cofilin-2 was barely detectable (fig. S1). To determine the ability of PGE2 to activate cofilin-1 (as measured by a decrease in its phosphorylation) and thereby decrease F-actin assembly, we investigated the amount of phosphorylated cofilin-1 (red fluorescence) and F-actin (green fluorescence) in alveolar macrophages infected with C. albicans in the absence or the presence of PGE2, the selective PKA agonist 6-Bnz-cAMP, the COX inhibitor indomethacin, or the adenylyl cyclase inhibitor SQ22536 (Fig. 4, A to F). Compared to untreated phagocytosing cells (Fig. 4A), both PGE2 (Fig. 4B) and PKA agonists (Fig. 4C) decreased phosphorylation of cofilin-1 and formation of F-actin in parallel; the selective Epac agonist (Fig. 4D) had no effect. Conversely, inhibitors of COX (Fig. 4E) and adenylyl cyclases (Fig. 4F) increased cofilin-1 phosphorylation and F-actin abundance. These events were also investigated by immunoblotting. Alveolar macrophages challenged with C. albicans showed increased phosphorylation of cofilin-1 as compared to resting cells. As observed by microscopy, PGE2 or PKA agonists, but not the Epac agonist, decreased cofilin-1 phosphorylation, consistent with an increase in its activity (Fig. 4G). siRNA directed against cofilin-1 siRNA (Fig. 4H) increased the baseline extent of ingestion of C. albicans (Fig. 4F) and baseline abundance of F-actin (Fig. 4H) compared to control siRNA-treated alveolar macrophages, showing that cofilin-1 inhibition is required for optimal yeast ingestion. The inhibitory effect of PGE2 on both ingestion (Fig. 4I) and F-actin formation (Fig. 4J) was blunted in cofilin-1 siRNA-treated cells. Taken together, these data show that cofilin-1 is necessary for the inhibitory effects of PGE2 and PKA on F-actin polymerization and phagocytosis in response to C. albicans.

Fig. 4. PGE2 and PKA target cofilin-1 to inhibit actin polymerization and C. albicans phagocytosis.

Fig. 4

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(A to F) Alveolar macrophages were pretreated for 20 min with medium (A) or with PGE2 (B), PKA agonist (C), Epac agonist (D), the COX inhibitor indomethacin (E) and the adenylyl cyclase inhibitor SQ22536 (F), challenged with C. albicans, and imaged by confocal microscopy using anti-phosphorylated-cofilin-1 (red) and Alexa 435-Phalloidin (green). (G) Alveolar macrophages were incubated with or without PGE2, PKA agonist, or Epac agonist, followed by C. albicans infection, and subjected to immunoblot for phosphorylated cofilin-1 and actin (left). Relative cofilin-1 phosphorylation was determined by densitometry (right). (H) Cofilin-1 mRNA abundance quantified by real time RT-PCR in alveolar macrophages pretreated with pooled siRNAs directed against cofilin-1 or control siRNA. (I) Alveolar macrophages pretreated as in (C), incubated with or without PGE2, followed by determination of FITCC. albicans phagocytosis. (J) Alveolar macrophages were pretreated with siRNAs as in (C) challenged with FITCC. albicans as described in (A). F-actin polymerization was determined. Data are means ± SEM values of 3 separate experiments. * p<0.05 compared to control siRNA; # p<0.05 compared to infected control siRNA; p<0.05 compared to PGE2 alone.

PTEN activation is required for the effects of PGE2 on C. albicans ingestion

Phosphorylation of cofilin-1 is controlled by the opposing actions of Lim domain kinase (LIMK) and the phosphatase slingshot-1 (SSH1) (32, 33). We next sought to identify the molecular mechanisms by which PGE2 activates cofilin-1. Initially, we utilized immunoblotting to determine if PGE2 increases the activation of SSH1. Although C. albicans challenge increased SSH1 phosphorylation, which results in its inhibition (34), PGE2 and PKA agonists failed to attenuate this process, implying that SSH1 is not a target for PGE2 and PKA (Fig. 5A). We also observed a modest but consistent inhibition of LIMK-1 activation by PGE2 and PKA in alveolar macrophages challenged with yeast (Fig. 5A). Because modulation of neither SSH-1 nor LIMK-1 adequately accounted for the ability of PGE2 to decrease cofilin-1 phosphorylation, we considered other phosphatases, particularly PTEN because we previously reported that activation of the phosphatase PTEN is required for the inhibitory effects of PGE2 on FcR-mediated ingestion (23). PGE2 enhanced PTEN activation, as evidenced by its dephosphorylation on Ser380 (35) (Fig. 5B). The consequence of PGE2-mediated activation of PTEN during yeast ingestion was demonstrated by the decreased phosphorylation of Akt in PGE2-treated cells (Fig. 5B).

Fig. 5. PTEN targets cofilin-1 during PGE2-mediated inhibition of C. albicans ingestion.

Fig. 5

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(A) Alveolar macrophages were preincubated with PGE2 or PKA agonist, infected with C. albicans, and immunoblotted for phosphorylated SSH1 (Ser978), phosphorylated LIMK-1 (Thr508) and actin. (B) Alveolar macrophages treated as in (A) were immunoblotted for phosphorylated PTEN (Ser380), phosphorylated Akt (Ser473) and total PTEN (left). Relative PTEN and Akt phosphorylation determined by densitometry (right). (C) Alveolar macrophages were pretreated as in (A) and cofilin (left) or PTEN (right) were immunoprecipitated. Immunoprecipitates were immunoblotted with antibodies against the indicated proteins. (D) Lysates were subjected to SDS-PAGE and the membranes were incubated with buffer alone (control), active PTEN, or alkaline phosphatase and probed for phosphorylated or total cofilin-1. R1 – R4 represent data employing cells from four separate rats, with R1-2 and R3-4 being processed together. (E) Alveolar macrophages were transfected with adenoviral constructs consisting of empty vector alone or constitutively active PTEN, dominant negative PTEN (C124S), or the PTEN mutant lacking lipid phosphatase activity (G129E), and immunoblotted for the indicated proteins (left). Densitometry of phosphorylated cofilin-1 in alveolar macrophages (right). Data shown are from a single experiment representative of 3 independent experiments. *p < 0.05 compared to untreated control; # p < 0.05 compared to PGE2.

PGE2 utilizes PTEN to induce activation of cofilin-1

The data presented above established that the lipid phosphatase activity of PTEN was activated during PGE2 inhibition of yeast phagocytosis. To investigate if PTEN is involved in cofilin-1 activation, we employed both biochemical and molecular approaches. We sought PTEN and cofilin-1 could coimmunoprecipitate each other in untreated alveolar macrophages, and this interaction was not altered by yeast challenge but was enhanced by PGE2 treatment (Fig. 5C). Moreover, the PTEN in these complexes in PGE2-treated cells was catalytically active, as evidenced by decreased phosphorylation of both serine and tyrosine residues (Fig. 5C). We did not observe any association between PTEN and other actin depolymerizing factors (destrin and cofilin-2), or between PTEN and the cofilin-1 upstream kinase LIMK 1/2 (Fig. 5C). Together, these results demonstrate that PGE2 promotes the formation of a complex between PTEN and cofilin-1.

We next determined if PTEN could directly dephosphorylating cofilin-1 in vitro by employing an in-blot PTEN activity assay. Lysate proteins from untreated alveolar macrophages were transferred to membranes, which were then incubated with buffer alone, recombinant PTEN, or the positive control alkaline phosphatase, and then probed for phosphorylated cofilin-1. Membranes treated with buffer only yielded a prominent band for phosphorylated cofilin-1, which is indicative of an inactive species (Fig. 5D). Treatment of membranes with either recombinant PTEN or alkaline phosphatase resulted in the virtual disappearance of phosphorylated cofilin-1, consistent with its activation. When these membranes were stripped and reprobed for total cofilin-1, we observed no difference among the treatment groups, which excludes a potential protease activity of PTEN (Fig. 5D). We next investigated whether PTEN could dephosphorylate cofilin-1 in intact cells, and if so, whether this was attributable to the protein or the lipid phosphatase activity of PTEN. We transfected primary alveolar macrophages with adenoviral constructs encoding either constitutively active PTEN, a PTEN mutant lacking only lipid phosphatase activity (G129E), a dominant negative PTEN lacking both lipid and protein phosphatase activities (C124S), or empty vector alone. As compared to empty vector, transfection with active PTEN decreased phosphorylation of cofilin-1 whereas the dominant negative PTEN had no effect on cofilin-1 activation (Fig 5E). The PTEN mutant lacking lipid phosphatase activity decreased phosphorylation of cofilin-1, implicating the protein phosphatase active site of PTEN in dephosphorylating cofilin-1 (Fig. 5E). Taken together, these data indicate that PTEN can associate with and dephosphorylate cofilin-1 in a manner dependent on its protein phosphatase activity, and this association is enhanced by PGE2 and PKA.

Finally, we determined if PTEN participates in the PGE2-induced inhibition of C. albicans ingestion and actin polymerization and its concomitant activation of cofilin-1 by siRNA-mediated silencing of PTEN (Fig. 6A). PTEN knockdown resulted in enhanced C. albicans ingestion (Fig. 6B), implicating this phosphatase in restraining basal phagocytosis by decreasing F-actin abundance (Fig. 6C). PTEN silencing also abolished PGE2-mediated inhibition of C. albicans phagocytosis and F-actin formation (Fig. 6B and C). These findings were also confirmed with a pharmacologic inhibitor of PTEN, which enhanced baseline yeast ingestion and abolished the inhibitory effects of PGE2 (fig. S2). Additionally, PTEN silencing increased basal phosphorylation of cofilin-1 and also blunted the ability of PGE2 to induce dephosphorylation of cofilin-1 (Fig. 6D). We confirmed these findings by employing two individual siRNA constructs from the pool used in Fig 6. These constructs, designated siRNA 1 and 2, reduced total PTEN abundance by ~60-70% as compared with a control siRNA derived from the scrambled sequence of siRNA 1 (fig. S3). As was true for the pooled siRNA, transfection of alveolar macrophages with these individual PTEN siRNAs also abrogated the inhibition of phagocytosis (fig. S3A) and the activation of cofilin-1 caused by PGE2 (fig. S3B). Taken together, our results uncover a previously unappreciated role for PTEN as an inhibitor of yeast ingestion by virtue of its activation of cofilin-1 and subsequent inhibition of F-actin assembly. Furthermore, these actions of PTEN are essential in mediating the inhibitory effects of PGE2 on actin polymerization and C. albicans ingestion.

Fig. 6. PTEN is required for PGE2-mediated activation of cofilin-1 and inhibition of actin polymerization and yeast phagocytosis.

Fig. 6

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(A) Pten mRNA abundance in alveolar macrophages treated with pooled Pten siRNA or control siRNA (left). Quantification of PTEN protein abundance in alveolar macrophages treated as in (A) as determined by immunoblotting. Inset: PTEN protein abundance. (B) Alveolar macrophages were pretreated with siRNAs as in (A), then with PGE2. Phagocytosis of FITCC. albicans was assessed. Data are presented relative to the untreated control siRNA value. (C) Alveolar macrophages were treated as in (B) and actin polymerization was determined fluorometrically. (D) Alveolar macrophages silenced as in (A) were treated with PGE2, followed by C. albicans. Lysates were immunoblotted for phosphorylated cofilin-1 (Ser3) and actin (left). Relative cofilin-1 phosphorylation was determined by densitometry (right). Data represent mean ± SEM from at least 3 independent experiments. (E) Model of PTEN- and cofilin-1 activation by PGE2 and PKA during C. albicans phagocytosis. During yeast ingestion, PGE2, through EP2 and EP4 receptors (EP2/4) and adenylyl cyclase-generated cAMP, activates PKA [bound through RI or RII (RI/RII) to an unknown AKAP] and PTEN. PTEN dephosphorylates cofilin-1, enhancing G-actin abundance and lowering F-actin. Dashed arrow indicates that the phosphatase responsible for PTEN dephosphorylation in this pathway is unknown.

DISCUSSION

C. albicans is a fungal pathogen that infects mucosal surfaces, including those of the respiratory tract, and can disseminate systemically in immunocompromised hosts (1). Many states of immunosuppression are associated with overproduction of PGE2 (21), making the influence of this lipid mediator on innate immune responses to C. albicans clinically relevant. Here we provide evidence that PGE2 produced during C. albicans infection inhibits fungal phagocytosis by impairing key steps involved in F-actin formation and therefore pathogen ingestion. Ligation of both EP2 and EP4 contributes to the inhibitory effect of PGE2 on C. albicans ingestion. cAMP and its downstream effector PKA – but not Epac-1 – mediate these inhibitory effects on fungal phagocytosis in an AKAP-dependent manner. PGE2 and PKA decrease F-actin formation by enhancing cofilin-1 activation by promoting the formation of a complex between active PTEN and cofilin-1. Subsequent dephosphorylation and activation of cofilin-1 is mediated by the protein phosphatase activity of PTEN. Because pathogen recognition receptors that are ligated by C. albicans are also ligated by other fungi, it is likely that our findings extend to other pathogens that cause lung infection. A scheme illustrating this pathway is depicted in Fig. 6E.

We chose to study the effect of PGE2 on C. albicans phagocytosis because this is the most abundant prostanoid produced by alveolar macrophages during fungal infection (36). In addition, COX inhibitors, which are widely utilized clinically as anti-inflammatory agents, inhibit PGE2 synthesis and may offer new therapeutic opportunities for enhancing host responses to fungal infection. Although both EP2 and EP4 are coupled to Gαs and therefore increase cAMP formation, we have previously shown that only EP2, but not EP4, mediates the suppressive effect of PGE2 on FcR-mediated phagocytosis (37). In contrast, our current results indicate that both of these EP receptors participate in the inhibition of C. albicans phagocytosis by PGE2. Because EP2 can elicit higher concentrations of cAMP in alveolar macrophages than EP4 (37), it is possible that the amount of cAMP required to inhibit C. albicans phagocytosis is lower than that required to inhibit FcR-mediated phagocytosis. We further investigated the importance of the downstream cAMP effectors PKA and Epac-1 in mediating cAMP-induced inhibition of fungal ingestion. The divergent roles of the two cAMP effectors in mediating inhibition of fungal ingestion (predominantly PKA) or FcR-mediated ingestion (predominantly Epac-1) remain to be explained. Both PKA-I and -II contributed to inhibition of F-actin formation and subsequent C. albicans ingestion, and interactions with AKAPs appeared to be involved in the case of both isoenzymes. Our previous work (38) has indicated that rat alveolar macrophages express various AKAPs, each of which mediate specific functions. Defining the specific AKAP(s) involved in inhibition of fungal ingestion will require additional investigation.

cAMP can both inhibit (39) and promote (40) F-actin polymerization during FcR-mediated phagocytosis in human neutrophils. In macrophages, cofilin-1 activation prevents FcR-mediated phagocytosis and its associated respiratory burst (41). Kamanova et al. (42) have shown that the adenylyl cyclase toxin from Bordetellae as well as a cAMP analog increases cofilin-1 activity and thereby results in unproductive actin ruffling and impaired phagocytosis of complement receptor-mediated phagocytosis in macrophages. However, the importance of the cAMP pathway and of cofilin-1 in modulating F-actin polymerization during phagocytosis of unopsonized targets by macrophages is poorly understood. Here, we show that PGE2 and PKA activate cofilin-1 during macrophage infection. PKA regulates cofilin-1 activation in different experimental systems. PKA inhibits cofilin-1 activity during luteinizing hormone receptor activation in preovulatory granulosa cells (43), and it activates LIMK-1 in fibroblasts (44). The precise role of LIMK-1 inhibition by PKA in modulating F-actin and phagocytosis thus remains to be determined.

Although cofilin-1 dephosphorylation and activation is thought to be mediated mainly by the phosphatase SSH1 (34), other phosphatases can also be involved. To date, the list of phosphatases capable of cofilin-1 dephosphorylation includes PP1 and PP2A, PP2B, and chronophin (45). Unexpectedly, we found that PTEN, when activated by PGE2 and PKA, was responsible for cofilin-1 dephosphorylation and activation. Although the genetic deletion of PTEN has been reported by others to both increase (46) and decrease (47) phagocytosis of unopsonized targets, our data suggest that PTEN acts as a brake on C. albicans phagocytosis in alveolar macrophages. The enhancement of C. albicans ingestion caused by PTEN silencing may reflect not only the action of PTEN to activate cofilin-1, but its ability to inhibit PI3K activation, which is also essential for ingestion.

We have previously shown in a model of FcR phagocytosis that PGE2 induces PTEN activation through tyrosine dephosphorylation mediated by the phosphatase SHP-1 (23). However, because our current results in the C. albicans ingestion model demonstrate that PGE2 dephosphorylated PTEN on both tyrosine and serine residues, we speculate that the responsible phosphatase exhibits dual specificity for tyrosine as well as serine and threonine residues. Candidates include mitogen-activated protein kinase phosphatases, myotubularins, and phosphatase of regenerating liver family members (48, 49). Taken together, our data indicate that PGE2 and cAMP suppress the antimicrobial effector functions of alveolar macrophages through the recruitment and activation of a network of phosphatases. Beyond its role as a lipid phosphatase, PTEN is also a protein phosphatase that can dephosphorylate itself (50), focal adhesion kinase (51), the platelet derived growth factor receptor (52), CREB (53), and Rho-associated kinase (54). Our present data suggest that the protein phosphatase activity of PTEN also dephosphorylates and activates cofilin-1, which in turn decreases F-actin assembly and C. albicans phagocytosis. PTEN has been previously reported to inhibit cofilin-1 activity by decreasing SSH-1 effects in insulin-stimulated Vero cells (55). We provide evidence that PTEN physically associates with cofilin-1 and directly dephosphorylates it in vitro and in vivo. Thus, our findings characterize a regulatory network involved in PGE2 inhibitory effects on non-opsonized target ingestion and actin assembly in alveolar macrophages and further identify a previously unrecognized role of PTEN in activating a master regulator of F-actin assembly, cofilin-1.

Pharmacological agents that inhibit COX activity have been used for many years to identify the role of prostanoids in immune responses. PGE2 participates in many aspects of the inflammatory response, and its inhibition by NSAIDs is beneficial in the treatment of inflammatory diseases. Here, we elucidate a mechanism by which PGE2 and cAMP inhibit F-actin formation, an event necessary for not only phagocytosis, but for other aspects of leukocyte activation including cell migration and bacterial killing. By inhibiting PGE2 synthesis and interrupting downstream cellular events, NSAIDs may exert potential immunostimulatory effects.

MATERIAL AND METHODS

Animals and reagents

Pathogen-free female Wistar rats weighing 125-150 g (Charles River Laboratories) were utilized as a source for alveolar macrophages and were treated according to National Institutes of Health guidelines for the use of experimental animals with the approval of the University of Michigan Committee for the Use and Care of Animals.

RPMI 1640 culture medium and antibiotic/antimycotic solution and FBS were purchased from Gibco-Invitrogen. Escherichia coli (055:B5) SDS was from Sigma-Aldrich. The PKA inhibitors KT5720 and myristoylated PKI peptide (14-22) and the AC inhibitor SQ22536 were purchased from Enzo Life Sciences. The PKA-specific cAMP analog N6-benzoyladenosine-3',5'-cAMP (6-Bnz-cAMP), Epac-specific cAMP analog 8-4-chlorophenylthio)-2’-O-methyladenosine-3',5'-cyclic monophosphate (8-pCPT-2-O-Me-cAMP), PKA RI-selective agonist 2-Cl-8-MA-cAMP (2-chloro-8-methylaminoadenosine-3′, 5′-cyclic monophosphate), PKA-RII-selective agonist N6-mono- t butylcarbamoyladenosine- 3', 5'- cyclic monophosphate (6-MBC-cAMP), PKA RI-selective antagonist 8-chloroadenosine- 3', 5'- cyclic monophosphorothioate, Rp-isomer (Rp-8-Cl-cAMPS), and PKA RII-selective antagonist 8-piperidinoadenosine- 3', 5'- cyclic monophosphorothioate, Rp- isomer (Rp-8-PIP-cAMPS) were purchased from Biolog Life Science Institute (Howard, CA). The selective EP2 receptor agonist butaprost free acid, the EP2 receptor antagonist AH6809, and PGE2 were purchased from Cayman Chemicals. EP4 receptor agonist (ONO-AE1-329) and EP4 receptor antagonist (ONO-AE3-208) were generous gifts from Ono Pharmaceutical Co., Ltd. The type II PKA AKAP disuptor peptide Ht31 was obtained from Promega. The type I PKA AKAP disruptor peptide RIAD was purchased from Anaspec. Required dilutions of all compounds were prepared immediately before use, and equivalent quantities of vehicle were added to the appropriate controls. Experimental compounds showed no adverse effects on cell viability as determined by lactate dehydrogenase release.

Cell isolation and culture

Rat alveolar macrophages were obtained by lung lavage as described (56). Cells were cultured overnight in RPMI containing 10% fetal bovine serum (FBS) and were washed twice the next day with warm medium to remove nonadherent cells.

Measurement of PGE2 in the supernatant of alveolar macrophage cultures

The concentrations of PGE2 in the supernatants from alveolar macrophages (5 × 105) stimulated with C. albicans for 30 min were determined using enzyme immunoassay kits (Cayman Chemicals) as described previously (57).

Measurement of intracellular cAMP

Alveolar macrophages were cultured overnight in 6-well plates in RPMI 1640 plus 10% FBS at a concentration of 3 × 106 cells/well. Medium was then changed to serum-free medium and cells were incubated with C. albicans for 30 min. Culture supernatants were aspirated and the cells were lysed by incubation for 20 min with 0.1 M HCl (22°C), followed by disruption using a cell scraper. Intracellular cAMP concentrations were determined by ELISA according to the manufacturer (Enzo Life Sciences).

Fluorometric assay of alveolar macrophage phagocytosis

The ability of alveolar macrophages to phagocytose C. albicans was assessed using a previously published protocol for determining the ingestion of fluorescent, FITC-labeled C. albicans (FITCC. albicans) (37). Briefly, heat-killed C. albicans were labeled with FITC, as previously described (9). In our previous work, phagocytosis of both live and heat-killed yeast by alveolar macrophages were regulated in a similar manner (9). 4 × 105 rat alveolar macrophages were seeded in replicates of 5 in 96-well tissue culture plates with opaque sides and optically clear bottoms (Costar, Corning Life Sciences). On the following day, alveolar macrophages were infected with C. albicans using a multiplicity of infection (MOI) of 10:1 for 90 min to allow phagocytosis to occur. Trypan blue (250 μg/ml; Molecular Probes) was added for 10 min to quench the fluorescence of extracellular yeast, and fluorescence was determined using a Spectramax Gemini EM fluorometer 485 excitation/535 emission (Molecular Devices). The phagocytic index was calculated, as previously described, in relative fluorescence units (37).

Fluorometric assay for quantification of actin polymerization

4 × 105 rat alveolar macrophages were seeded in replicates of 5 in 96-well tissue culture plates with opaque sides and optically clear bottoms (Costar, Corning Life Sciences). On the following day, alveolar macrophages were infected with heat-killed C. albicans using a MOI of 10:1 for 30 min. Cells were fixed with 3% paraformaldehyde for 20 min, followed by cell blocking with 2% BSA in PBS for 1 hour at room temperature. Alveolar macrophages were stained with FITC-phalloidin (1 μM) to stain F-actin, according to the manufacturer's protocol (Molecular Probes). Cells were washed three times in PBS and fluorescence was determined using a Spectramax Gemini EM fluorometer at settings of 485 nm for excitation and 535 nm for emission (Molecular Devices).

RNA interference

RNA interference was performed according to a protocol provided by Dharmacon and as previously described (9, 38, 58). Alveolar macrophages were transfected using DharmaFECT 1 reagent with 30 nM of specific ON-TARGET SMARTpool siRNAs (or their scrambled controls) against cofilin-1 and PTEN. The cofilin-1 siRNA pool included the following constructs: (construct 1 GUGUCAUCAAGGUGUUCAA; construct 2 CUAACUGCUACGAGGAGGU; construct 3 CAGACCUGCUCUUGGGUGU; construct 4 CCAGAAGAAGUGAAGAAAC). The PTEN siRNA pool included the following constructs: (construct 1 GUAUAGAGCGUGCGGAUAA; construct 2 GCUAAGUGAAGACGACAAU; construct 3 AAGACAAGGCCAACCGAUA; construct 4 AGAGGAUGGAUUCGACUU). After 48 hours of transfection, alveolar macrophages were harvested for mRNA, protein, and fluorometric phagocytosis or F-actin assays. In a separate set of experiments, individual PTEN siRNA constructs 1 and 2 and a control siRNA consisting of the scrambled sequence of siRNA 1 were transfected into alveolar macrophages for 48 hours as above.

RNA isolation and semiquantitative real time RT-PCR

RNA from cultured cells was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions and real time RT-PCR was performed as described (59). Cofilin-1 and PTEN mRNA were normalized to GAPDH, and the respective untreated control was set to 100%.

Confocal microscopy

2 × 105 alveolar macrophages were plated in 4-well chamber slides (Nunc), incubated with or without 10:1 C. albicans for 15 min, and then washed with PBS. Slides were fixed in 4% paraformaldehyde in PBS for 30 min at room temperatureand permeabilized with 0.5% Triton X-100 in PBS for 3 min. Cells were then blocked with 2% BSA in PBS and incubated with anti-phospho-cofilin-1 antibody (Ser3, 1:200) in blocking buffer for 1 hour at room temperature. Slides were washed in PBS and incubated with Alexa 594-conjugated secondary antibody (1:200) in blocking buffer for 1 hour. Cells were also stained with phalloidin-Alexa 455 (1 μM) or DNAse I-Alexa 594 (1 μM) to stain F- or G-actin, respectively, according to instructions of the manufacturer (Molecular Probes). Slides were mounted in Prolong Gold mounting media with 4,6 diamidino-2-phenylindole (DAPI) (Molecular Probes). Cells were imaged on a Zeiss LSM 510 confocal microscope with an inverted Axiovert 100 M microscope stand using a C-apochromat 40/1.2 W corr. The 488-nm line from an Ar laser and the 543- and 633-nm lines from 2 He/Ne lasers were used for excitation. The images were analyzed using LS 2.5 image analysis software (Zeiss). Photographs were obtained only of alveolar macrophages containing bound or internalized C. albicans. Confocal images were taken with identical settings to allow comparison of staining. Single confocal sections of the cells were captured in multitrack. Each set of frames from a given treatment condition depicts a representative alveolar macrophage selected from 20 analyzed cells in each of at least 3 experiments.

Western blotting

2 × 106 alveolar macrophages were plated in 6-well tissue culture dishes and were pretreated with or without the PKA agonist, Epac-1 agonist, or PGE2 for 5 min, followed by the addition of 10:1 C. albicans for 15 min. Immunoblot analysis was performed as previously described (60) using the following primary antibodies: phosphorylated cofilin-1 (Ser3; 1:1000), phosphorylated LIMK1 (Thr508; 1:1000), phosphorylated LIMK1/2 (Thr505 and Thr508; 1:1000), phosphorylated PTEN (Ser380) and total PTEN (both 1:1000), phosphorylated SSH1 (Ser978; 1:500), phosphorylated tyrosine residues (pY 20; 1:1000), phosphorylated Akt (Ser473, 1:1000), destrin-1 (1:500), cofilin-2 (1:500), GAPDH (1:5000) and β-actin (1:10,000). Densitometric analysis was as described (9, 38, 58) previously .

In-blot PTEN activity assay

PTEN dephosphorylation of cofilin-1 was examined by an in-blot phosphatase assay as described (61). Briefly, histidine-tagged PTEN (His6-PTEN) was generated by inserting full-length PTEN cDNA into the pQE30 vector (Qiagen, Valencia, CA). The protein was purified using Ni-NTA beads (Qiagen) under denaturing conditions and then renatured by sequential dilution and concentration in renaturation buffer (PBS [pH 7.0] containing 2 mm MgCl2, 0.5 mm phenylmethanesulfonyl fluoride, 0.005% Tween 20, 10 mm DTT, and protease inhibitor mixture). Purity (>90%) was confirmed by SDS-PAGE and Coomassie blue staining. 3×106 rat alveolar macrophages were plated overnight and cells were lysed with RIPA buffer for western blot. Equal amounts of proteins were subjected to 10% SDS-PAGE and electrotransferred to nitrocellulose. Blots were incubated with 20 μg/ml recombinant His6-PTEN or 500 U/mL alkaline phosphatase in 50 mm HEPES buffer (pH 7.0) containing 10 mm MgCl2, 10 mm DTT at 30°C for 1 hour. Phosphorylated and total cofilin-1 or LIMK-1/2 were detected by immunoblot as mentioned above.

Immunoprecipitation

For PTEN or cofilin-1 immunoprecipitation, alveolar macrophages were pretreated with or without 1 μM PGE2 for 5 min, followed by C. albicans infection for 15 min. Alveolar macrophages were lysed with RIPA buffer and precleared with protein A-Sepharose for 30 min and incubated overnight at 4°C with anti-PTEN (1:80), or cofilin-1 (1:80). Protein A-Sepharose was added and incubated for 3 hours with rotation at 4°C, and immunoprecipitates were isolated and subjected to electrophoresis as described above. Membranes were probed with the antibodies as described above.

Adenoviral constructs and infection

Adenovirus containing cDNA constructs encoding constitutively active wild-type PTEN, dominant negative PTEN (C124S), the lipid phosphatase activity mutant PTEN (G129E), and the empty viral vector (control) containing no cDNA insert were prepared as previously described (62, 63). Adenoviruses were amplified in HEK-293 cells and purified by ultracentrifugation on a CsCl density gradient as described (64). 2 × 106 alveolar macrophages were seeded in 6-well plates in DMEM containing 10% FBS. Virus was added to the medium at a multiplicity of infection of 500. After 72 hours, the cells were harvested and the cell lysates subjected to immunoblotting.

Statistical analysis

Graphs represent the mean ± SEM from 3 to 6 independent experiments. The means from different treatments were compared by ANOVA. When significant differences were identified, individual comparisons were subsequently made with the Bonferroni t test for unpaired values. When two groups were compared, we performed paired Student's t-test. Statistical significance was set at a p value less than 0.05.

Supplementary Material

Supplementary data (figures)

Acknowledgments

We thank members of the Peters-Golden laboratory and Ana Paula Moreira for their thoughtful input. Funding: This work was supported by NIH grants HL058897 (to M. Peters-Golden); K99HL103777 (to C.H. Serezani) and HL085083 (E. White); by FAPESP (to M. Morato-Marques); and by CNPq-Brazil, 201061/2007-4 to Alexandra Medeiros.

Footnotes

Author contributions: C.H.S. designed the research, performed experiments, analyzed data, and wrote the paper; A.I.M., S.K., S-H.K, M.M.M., S-P. L., C.L., A.C., M.N.B., C.L., E.B., E.W performed experiments and analyzed data; and M.P.-G. designed research, supervised the work, analyzed data, and wrote the paper.

Competing interests: The authors declare that they have no competing interests.

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