Pleiotropic Effects of Phosphatidylinositol 3‐Kinase in Monocyte Cell Regulation

Pleiotropic Effects of Phosphatidylinositol 3‐Kinase in Monocyte Cell Regulation Sanaaˆ Noubir,* Jimmy S. Lee,* and Neil E. Reiner*{ *Department of Medicine (Division of Infectious Diseases), University of British Columbia, Faculties of Medicine and Science, Vancouver Coastal Health Research Institute (VCHRI), Vancouver, British Columbia, Canada V5Z 3J5 {

Department of Microbiology and Immunology, University of British Columbia, Faculties of Medicine and Science, Vancouver Coastal Health Research Institute (VCHRI), Vancouver, British Columbia, Canada V5Z 3J5 I. Background .............................................................................. A. Classification and Structure of PI 3‐Kinases. ................................... B. Lipid Products as Downstream Mediators of PI 3‐Kinase Signaling ....... II. PI 3‐Kinase and Lipopolysaccharide ................................................. A. LPS Signaling Pathway and LPS Receptors .................................... B. LPS‐Induced Association and Coordinate Activation of p53/56lyn and PI 3‐Kinase ..................................................................... C. PI 3‐Kinase‐Dependent Activation of PKC‐z in LPS‐Treated Human Monocytes ................................................................. D. Bimodal Regulation of IRAK‐1 Dependent on CD14/TLR4, CR3, and PI 3‐Kinase ..................................................................... E. LPS‐Induced Adherence Is Regulated by Rho and PI 3‐Kinase ............ III. PI 3‐Kinase and 1a,25‐Dihydroxyvitamin D3 ....................................... A. Vitamin D3 Action and Signaling................................................. B. A VDR
PI 3‐Kinase Signaling Complex Regulates Myeloid Cell Differentiation................................................................. C. D3‐Induced Monocyte Antimycobacterial Activity Is Regulated by PI 3‐Kinase....................................................................... D. D3‐Induced Antimicrobial Activity Against Salmonella enterica Is PI 3‐Kinase Dependent ............................................................... IV. PI 3‐Kinase and Phagocytosis of Mycobacteria..................................... A. Phagocytosis of Mycobacteria: Receptors and Signaling...................... B. Cross‐Talk Between CD14 and CR3: Regulation by PI 3‐Kinase and Cytohesin‐1.....................................................................

Progress in Nucleic Acid Research and Molecular Biology, Vol. 81 DOI: 10.1016/S0079-6603(06)81002-0

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Copyright 2006, Elsevier Inc. All rights reserved. 0079-6603/06 $35.00

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C. Rescue of Phagosome Maturation by Vitamin D3 Is PI 3‐Kinase Dependent ............................................................... V. Stable Gene Silencing of Isoforms of PI 3‐Kinase and Perspectives............ A. Silencing of P110a Subunit of PI 3‐Kinase: Differential Involvement in Monocyte Adherence.................................................................. B. Perspectives.......................................................................... References ...............................................................................

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Mononuclear phagocytes are important regulators and effectors of both the innate and acquired immune responses, and they are also prominently involved in inflammation. Consequently, regulation of monocyte function is an intense area of research interest and recent studies have highlighted an important role for phosphoinositides in monocyte cell regulation. For nearly two decades now, phosphoinositides have been recognized to function as second messengers in signal transduction pathways initiated through cell‐ surface receptors. Class I phosphatidylinositol 3‐kinase (PI 3‐kinase or PI 3‐K) is a lipid kinase that catalyzes the transfer of the g‐phosphate group of ATP to the 30 ‐position of the inositol ring of phosphatidylinositol (PI) and its derivatives. The phosphoinositide (P’tide) metabolites produced as a result are known to be involved in regulating a multitude of cellular events such as mitogenic responses, differentiation, apoptosis, cytoskeletal organization, membrane traffic along the exocytic and endocytic pathways (1), and various other aspects of monocyte function (2–5). This chapter will focus on those regulatory events involving PI 3‐kinase that have been extensively studied in our laboratory and will emphasize the role of PI 3‐kinase in three main areas. The first is activation of PI 3‐kinase following lipopolysaccharide (LPS) stimulation, our efforts to identify downstream effectors, and a role for this activation in regulating changes such as monocyte adherence and endotoxin tolerance. The second area will focus on the role of PI 3‐kinase in vitamin D3‐induced monocyte maturation, the phagocyte oxidative burst, and antimicrobial activity during both infection with mycobacteria and Salmonella. Third, we will review aspects of the roles PI 3‐kinase has been shown to play in regulating phagosome biology. Finally, the last part of this chapter will address important advances in a new approach to stable gene silencing in human monocytic cell lines using lentiviral‐delivered small interfering RNA. The power of this strategy is illustrated by experiments that examined the specific role of the p110a subunit of PI 3‐kinase in vitamin D3 but not LPS‐induced monocyte adherence. To provide further perspective, several ongoing and promising experiments ultimately made possible by using stable and isoform‐ specific PI 3‐kinase gene silencing are discussed.

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I. Background A. Classification and Structure of PI 3‐Kinases The phosphoinositide 3‐kinases (PI 3‐K) constitute a family of at least eight different lipid kinases that phosphorylate the hydroxyl group of the inositol ring of phosphoinositides at the 30 position. A considerable amount of research has led to the conclusion that an apparently vast diversity of cellular functions are differentially regulated by distinct PI 3‐K family members and their corresponding isoforms (6–8). Based on their selective in vitro substrate specificity, the multiple PI 3‐Ks have been divided into three classes (6). In vitro, class I PI 3‐K isoforms phosphorylate phosphatidylinositol (PtdIns), PtdIns 4‐phosphate, and PtdIns 4,5‐bisphosphate. In intact cells, however, their preferred substrate appears to be PtdIns(4,5)P2. Class I PI 3‐K is further divided into subclasses IA and IB, which signal downstream of tyrosine kinases and heterotrimeric G‐protein–coupled receptors (GPCRs) respectively. All class I PI 3‐K members are able to bind to Ras, but the role of this interaction in physiological PI 3‐K signaling is not entirely clear. Mammalian class IA PI 3‐Ks are heterodimers consisting of a regulatory subunit (p85a, p85b, p55, or other splice variants) and a p110 (a, b, or
isoforms) catalytic subunit (9–11). Through their Src‐homology 2 domain‐ containing p85 subunits, class IA PI 3‐Ks bind to phosphorylated tyrosine residues that are generated by activated tyrosine kinases in receptors and various receptor‐associated adaptor proteins. The class IAp85 subunits prefer to bind to proteins containing the consensus phosphotyrosine motif Y(P)xxM in which x is any amino acid. Phosphotyrosine binding is thought to allow translocation of the cytosolic PI 3‐Ks to the plasma membrane, where their lipid substrates and Ras reside. Nonphosphotyrosine‐based recruitment mechanisms may also contribute to PI 3‐K activation (12, 13). Some membrane proteins may even show a constitutive association with class IA PI 3‐Ks, as reported for CD2 (14). All mammalian cell types investigated express at least one class IA PI 3‐K isoform, and stimulation of almost every receptor that induces tyrosine kinase activity also leads to class IA PI 3‐K activation (15–17).

B. Lipid Products as Downstream Mediators of PI 3‐Kinase Signaling Certain types of ligand receptor interactions may trigger a rapid rise of cellular PtdIns(3,4,5)P3, and with some delay, PtdIns(3,4,)P2, the latter generated by the action of phosphoinositide 5‐phosphatases. Several molecular targets for PtdIns(3,4,5)P3 and PtdIns(3,4,)P2 have been identified, which are translocated and activated on interaction with phosphoinositides. Two protein subdomains in particular, the FYVE domain (based on the first letters of four

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proteins containing this domain, Fab1, YOTB/ZK632.12, Vac1 and EEA‐1) and the pleckstrin homology (PH) domain, have been defined whose structures confer specificity of lipid interaction at the molecular level. FYVE domains selectively bind PtdIns3P, whereas a subgroup of PH domains shows specificity for PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3. PH domains that selectively bind PtdIns3P have also been identified (18). The potential for some PH domains to specifically interact with PtdIns(3,4)P2 and PtdIns(3,4,5)P3 correlates with in vivo data defining the same PH domain‐containing proteins as PI 3‐kinase effectors (19, 20). One of the most thoroughly studied PI 3‐K effectors is the protein Ser/Thr kinase Akt/PKB (also called RAC), which translocates to the plasma membrane by binding PtdIns(3,4)P2. It has been conclusively demonstrated that the activity of type 1 phosphoinositide‐dependent kinases (PDK), which phosphorylates and activates Akt/PKB on Thr308, is also specifically controlled through the binding of PtdIns(3,4,5)P3, or PtdIns(3,4)P2 to its PH domain (21–24). Furthermore, PDK1s have been recently shown to phosphorylate and activate protein kinases C (PKC) z and
directly (25, 26). Taken together, these data illustrate the complexity and fine tuning that has come to be recognized as characteristic of lipid‐mediated signaling.

II. PI 3‐Kinase and Lipopolysaccharide Lipopolysaccharide (LPS), or endotoxin, is the major component of the outer surface of Gram‐negative bacteria. LPS is an activator of both immune and inflammatory responses and has particularly potent effects on macrophages, monocytes, dendritic cells, endothelial cells, and others (27, 28). An important property of LPS is its ability to promote leukocyte adherence and the accumulation of leucocytes at inflammatory foci in vitro or in vivo (29, 30). Our first indication that the monocyte response to LPS was regulated by PI 3‐kinase derived from the finding that LPS treatment led to activation of this lipid kinase and this was required for the activation of monocyte PKC‐z as well as the tyrosine kinase p53/56lyn (4, 31, 32). Based on these findings, further efforts were focused on understanding the role of the PI 3‐kinase activation on monocyte cell regulation. Prior exposure of monocytes and macrophages to LPS induce a transient state of refractoriness to subsequent LPS restimulation, known as endotoxin tolerance (33, 34). The phenomenon of endotoxin tolerance has been widely investigated (2, 33–35), but to date, the molecular mechanisms of endotoxin tolerance remain to be resolved clearly. Recent research has identified distinct roles for PI 3‐kinase in regulating monocyte adherence as well as endotoxin tolerance (2, 36). To put these findings in proper context, it is useful to review briefly key aspects of what is known about LPS signaling and different receptors for LPS.

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A. LPS Signaling Pathway and LPS Receptors It has become clear that cellular responses to LPS involve specific cell‐ surface receptors leading to the activation of pathways containing both tyrosine and serine/threonine protein kinases (32, 37, 38). Proximal events involved in at least one dominant LPS signaling pathway are dependant on the cell‐surface molecule CD14 (39). The binding of LPS to mCD14 is enhanced by LPS‐ binding protein, a plasma protein (40). Unlike other more conventional receptors, CD14 is linked to the membrane via a glycosylphosphatidylinositol anchor, thereby lacking both transmembrane and cytoplasmic domains. The mechanisms, by which CD14 mediates stimulus–response coupling has been a central question in macrophages cell biology. Evidence has been presented to suggest that this may involve an auxiliary receptor subunit(s) that interacts with a ternary complex comprised of CD14, LPS, and LPS‐binding protein. It has been shown that CD14 uses TLR‐4 as a cell‐surface coreceptor that mediates signal transmission. Genetic and other evidence has established that LPS signals predominantly through CD14 and TLR‐4 (41, 42) and utilizes signaling elements in common with the interleukin‐1 receptor including MyD88, IRAK, and TRAF6 to elicit cellular responses (43). TIRAP, also known as MAL, an adaptor protein in the TLR signaling pathway, has also been identified and shown to function downstream of TLR‐4 (44, 45). The b2 integrin CD11/CD18 represents another putative LPS receptors. CD11b and CD11c, the alpha subunits of CR3 and CR4, respectively, are glycoproteins that associate noncovalently with the b2 integrin subunit, CD18. Although there has been some ambiguity as to whether CR3 and CR4 are bona fide LPS signaling receptors, recent findings show that expression of CD11b/CD18 or CD11c/CD18 in Chinese hamster ovary cells confers a phenotype of LPS responsiveness to these cells which are normally CD14 (46–49). Of interest, CD11b/CD18 mutants, deficient in cytoplasmic domains thereby rendering them incapable of functioning to internalize Gram‐negative bacteria, were still competent for LPS‐induced cellular activation (47). This has led to the suggestion that CR3 like CD14 may function to initiate signal transduction via an as yet unidentified coreceptor (47). To function as an LPS receptor, TLR4 must interact with a secreted protein, MD‐2 (50–52). Site‐directed mutagenesis has shown that one region of MD‐2 is capable of binding TLR4 and may mediate its correct targeting to the cell membrane, whereas another region, rich in basic and aromatic residues, most likely functions to bind LPS (53). Incubation of naive cells with LPS triggers activation of IRAK‐1 (IL‐1R associated serine/threonine‐specific protein kinase) and its association with MyD88, an adaptor protein linking IRAK‐1 to the intracellular domain of TLR‐4 (43, 54, 55). On stimulation, IRAK‐1 rapidly translocates to the active receptor complex and interacts via its

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N‐terminal death domain with MyD88 (56–58). IRAK‐1 then auto‐ and/or cross‐phosphorylates itself at the active receptor complex resulting in phosphorylation in the kinase domain. Subsequently, IRAK‐1 catalyzes multiple auto‐phosphorylations in its ProST region, a domain rich in proline, serine, and threonine residues. IRAK‐1 interacts with the downstream adapter TRAF6, which has polyubiquitinylated itself (59). TRAF6 then facilitates autophosphorylation of TAK1 (60, 61), which in turn phosphorylates I‐kB‐kinase (IKK) and MKK6 thus activating the NF‐kB pathway as well as p38 MAP kinase. Rapid activation of nuclear factor‐kB (NF‐kB) in both monocytic (62, 63) and endothelial cells (64) is required for the production of proinflammatory cytokines, including interleukin‐1 (IL‐1), interleukin‐6 (IL‐6), and tumor necrosis factor (TNF) (65, 66). Tyrosine phosphorylation and activation of ERK1, ERK2, p38 MAP kinase, and c‐Jun N‐terminal kinase also appear to be important for cell activation by LPS (67).

B. LPS‐Induced Association and Coordinate Activation of p53/56lyn and PI 3‐Kinase Increased tyrosine phosphorylation of monocyte proteins (68, 69) and activation of the src family tyrosine kinases p53/56lyn, p58hck, and p59fgr (37, 70, 71) have been reported to take place within minutes of exposure of macrophages to LPS. Moreover, we and others have shown that activation of serine‐threonine protein kinases, such as p42 and p44 mitogen‐activated protein kinases and PKC, also occurs following LPS stimulation (32, 38, 72, 73). Based on these findings and coupled with the knowledge that PI‐3 kinase has been shown to be a downstream effector of tyrosine kinases (17), we investigated whether LPS brings about the activation of PI 3‐kinase in human monocytes and whether this involves direct kinase–kinase interactions. 1. BACTERIAL LPS INDUCES CD14‐DEPENDENT ACTIVATION OF PI 3‐KINASE IN MONOCYTES To examine whether LPS activates PI 3‐kinase, inositol phospholipids were detected after extraction and separation of lipids by thin‐layer chromatography (TLC). Levels of Ptdlns 3,4,5‐trisphosphate (Ptdlns 3,4,5‐P3) were elevated within minutes of exposure of human monocytes to LPS (Fig. 1). Analysis of 32P‐labeled lipid extracts of U937 cells by HPLC confirmed that levels of Ptdlns 3,4,5‐P3 increased rapidly following LPS treatment (31). Moreover, anti‐PI 3‐kinase immunoprecipitates prepared from unlabeled monocytes and assayed in an in vitro phosphorylation assay using Ptdlns as substrate, showed higher enzymatic activity when these were prepared from lysates of LPS‐treated cells as compared with control cells (31). These results suggest that increased levels of Ptdlns 3,4,5‐P in LPS‐treated cells resulted from an

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FIG. 1. Ptdlns 3,4,5‐P3 levels in monocytes after LPS treatment. (A) Human peripheral blood monocytes were labeled with Pi for 2 hr before incubation with LPS (100 ng/ml) for different periods. Lipids were extracted and separated by TLC using a solvent system. (B) Spots corresponding to Ptdlns 3,4,5‐P3 (PIP) were identified by comigration with standard. The spots were cut from the plate and analyzed by liquid scintillation counting. Activity is expressed relative to control. The visible spots at the origin reflect residual, water‐soluble 32P‐labeled material in the samples, the amount of which is not relevant to the results. Adapted from the work of Herrera‐Velit et al. (31), with permission.

increase in the specific activity of PI 3‐kinase rather than decreased activity of phosphomonoesterases. PI 3‐kinase activity was elevated as early as 2 min after LPS exposure and was observed with LPS concentrations in a range of 10–100 pg/ml, which are similar to those required to induce monocytic functional responses (31). Furthermore, activation of PI 3‐kinase was shown to involve signaling through the monocyte cell‐surface molecule CD14, since pretreatment of cells with Abs to CD14 abrogated LPS‐induced increases in Ptdlns 3,4,5‐P3 (31). 2. LPS ACTIVATES P53/56 WITH

LYN

PI 3‐KINASE

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PROMOTES ITS ASSOCIATION

Activation of several src family tyrosine kinases, including p53/56lyn, p58fgr, and p59hck has been shown to occur in response to LPS, dependent on CD14. In the case of p53/56lyn, a physical association of lyn with CD14 is involved (37). It has also been observed that PI 3‐kinase may associate with and be

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activated by a variety of both receptor and nonreceptor tyrosine kinases (17, 74), including p53/56lyn in B cells (75). Taken together, these findings suggested the possibility that activation of monocyte PI 3‐kinase in response to LPS may involve its association with activated p53/56lyn. This hypothesis in fact proved to be correct. Anti‐lyn immunoprecipitates from LPS‐treated THP‐1 cells were examined and showed increased PI 3‐kinase activity (31). Maximal lyn‐ associated PI 3‐kinase activity was observed after 5–10 min of LPS treatment and was gone by 40 min. In addition, when parallel p53/56lyn immunoprecipitates were examined, kinetics of lyn activation was maximal at 10 min, consistent with its maximal association with PI 3‐kinase (31). Taken together, these results indicate that LPS brings about the coordinate activation of p53/56lyn and PI 3‐kinase and these enzymes become associated transiently for a period of time that corresponds to the duration of PI 3‐kinase activation. Further evidence to suggest that activation of PI 3‐kinase is dependent on its association with activated lyn was provided by the finding that inhibition of lyn with the tyrosine kinase inhibitor herbimycin abrogated activation of PI 3‐kinase (31).

C. PI 3‐Kinase‐Dependent Activation of PKC‐z in LPS‐Treated Human Monocytes 1. LPS INDUCES ACTIVATION

OF

PKC‐z

IN

MONOCYTES

In light of findings that LPS activated both PI 3‐kinase and PKC in monocytes, an important objective was to determine whether these events were related and the isoform of PKC involved. Resolution of detergent‐soluble lysates prepared from LPS‐treated human blood monocytes using Mono Q chromatography revealed two principal peaks of myelin basic protein kinase activity. Immunoblotting and immunoprecipitation with different isoform‐ specific anti‐PKC antibodies showed that the major and latest eluting peak was accounted for by PKC‐z (4). Consistent with its identity as PKC‐z, the LPS‐activated monocyte kinase did not depend on the presence of lipids, Ca2þ, or diacylglycerol for activity. In addition, the kinase phosphorylated peptide epsilon and myelin basic protein with equal efficiency, but phosphorylated kemptide and protamine sulfate poorly, features all consistent with PKC‐z. Translocation of PKC‐z from the cytosolic to the particulate membrane fraction on exposure of monocytes to LPS provided further evidence for activation of the kinase (4). 2. PI 3‐KINASE IS REQUIRED FOR LPS‐INDUCED ACTIVATION OF PKC‐z Previous work had suggested a role for PI 3‐kinase metabolites in the activation of PKC. In particular, the PI 3‐kinase metabolite PIP3 was known to be an activator of the PKC isoforms z, e, and
(26, 76, 77). Based on this information and the observation from this laboratory and others that PKC

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activity was increased in LPS‐treated monocytes (32, 38), we investigated the potential involvement of PI 3‐kinase in LPS‐induced activation of PKC‐z. Human monocytes were incubated with two structurally unrelated PI 3‐kinase inhibitors, wortmannin or LY294002, prior to the addition of LPS. Both inhibitors markedly attenuated activation of PKC‐z (4). Other results showed that activation of PKC‐z was abrogated in U937 cells transfected with a dominant negative mutant of the p85 subunit of PI 3‐kinase (Fig. 2). These findings are consistent with a model in which PI 3‐kinase is required for the activation of PKC‐z in LPS‐treated human moncytes. Further support for this argument was provided by experiments in which PKC‐z activity was observed to be enhanced in vitro by the addition of phosphatidylinositol (3,4,5)P3 (4).

FIG. 2. Attenuation of LPS‐induced activation of PKC‐z by a dominant negative mutant of the p85 subunit (Dp85) of PI 3‐kinase. Quiescent transfected U937 cells were treated either with LPS (1 mg/ml, 15 min) or medium alone and lysed in FPLC extraction buffer. Detergent lysates were fractionated by Mono Q chromatography. Aliquots (5 ml) of each fraction from cells transfected with Dp85 (A) or with wild‐type p85 (Wp85) (B) were assayed for MBP kinase activity in triplicate. The standard deviations of each data point were <20% of the mean. (C) Aliquots from LPS‐treated cells were analyzed by immunoblotting with anti‐PKC‐z antibodies. Adapted from the work of Herrera‐Velit et al. (4).

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D. Bimodal Regulation of IRAK‐1 Dependent on CD14/TLR4, CR3, and PI 3‐Kinase 1. IRAK‐1 DEGRADATION

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TOLERANCE

TO

LPS

Infection with Gram‐negative bacteria often results in the development of a sepsis syndrome in which uncontrolled production of proinflammatory cytokines results in shock with multiorgan failure (78, 79). To prevent the morbidity and mortality that can develop under these conditions, a variety of intrinsic control mechanisms have evolved to exert negative feedback regulation of cellular responses to LPS. For example, macrophages and other phagocytic cells incubated with LPS become unresponsive or tolerant to a second challenge with endotoxin (33). One level at which this type of control may be exerted involves attenuation of LPS signaling. IRAK‐1 plays a crucial role in facilitating formation of the multiprotein signaling complex or LPS signalosome. IRAK‐1 links the active receptor complex to the central adapter and coactivator protein TRAF6. Thus, IRAK‐1 functions as a switch or relay for the signalosome and is also capable of switching off or interrupting the signalosome through autoinduced removal by degradation. Recently, it has been shown that in response to prolonged LPS exposure, cellular levels of IRAK protein and correspondingly IRAK activity are markedly diminished, and this correlates tightly with LPS tolerance (80). Multiple lines of evidence including study of IRAK deficient mice, macrophages lacking IRAK, and over expression of dominant negative IRAK in THP‐1 cells, have established an essential role for IRAK in conferring a phenotype of tolerance to LPS (43, 81, 82). 2. INVOLVEMENT OF CD14/TLR4, CR3, IN IRAK‐1 DEGRADATION

AND

PI 3‐KINASE

Although it was known that IRAK undergoes rapid inactivation/degradation in response to LPS, providing negative feedback leading to LPS tolerance, mechanisms governing IRAK degradation were not fully understood. In previous study that had examined normal THP‐1 cells, which expressed little or no CD14, evidence for changes in IRAK abundance and activity were not evident until at least 1.5 hr after the addition of LPS (80). However, several lines of evidence suggested that more rapid degradation of IRAK in response to LPS was likely to occur in cells that were CD14þ (2). The prediction that rapid IRAK degradation was likely to involve CD14 proved to be correct. Based on a kinetic analysis comparing both CD14 and CD14þ cells, we found that the expression of CD14 confers a phenotype of rapid IRAK degradation in response to LPS, a property not shared by cells expressing low or undetectable levels of CD14 (2). Whereas CD14 cells were also capable of degrading IRAK, this occurred with distinctly delayed kinetics, providing evidence for bimodal regulation of IRAK expression.

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The discovery that IRAK degradation was in fact bimodal inferred that it was likely to be regulated by multiple mechanisms. This inference was confirmed with the use of blocking antibodies that established that only the rapid phase of IRAK degradation was TLR‐4‐ and CD14‐dependent (2). To address how the delayed phase was regulated, we considered other putative LPS receptors that might be involved. CR3 appeared to be a potential candidate as it had been shown to be capable of transducing LPS signals (47, 49). Blocking antibody experiments using THP‐1rsv cells (CD14/CR3þ) clearly showed that CR3 was required for late phase IRAK degradation. Similar blocking experiments using both THP‐1rsv (CD14) and THP‐1wt (CD14þ) cells showed that CR3 functioned independently of TLR‐4 in signaling delayed IRAK degradation (2). The finding that dual receptors appeared to be involved in invoking independent pathways of IRAK degradation raised the important question of how these pathways were regulated. In examining this question, an important lead was provided by the findings that PKC activity was required for LPS‐ induced phosphorylation and degradation of IRAK and the demonstration that PKC‐z associated with IRAK in LPS‐treated THP‐1 cells (83). These results, indicating a possible role for PKC‐z in a pathway regulating IRAK degradation, led us to investigate whether this may be regulated by PI 3‐kinase. As can be seen in Fig. 3, use of two distinct PI 3‐kinase inhibitors, LY294002 and wortmannin, showed that PI 3‐kinase was required for the early, but not the late phase of IRAK degradation. These findings suggested a model in which on exposure to LPS, PI 3‐kinase becomes activated downstream of CD14/TLR4 and signals through a pathway leading to activation of PKC‐z to bring about the rapid degradation of IRAK (Fig. 4). Investigation of other elements that may be involved in this pathway is presently underway. One potential candidate is phosphoinositide‐dependent kinase type‐1 because it is known to be activated by phosphatidylinositol 3,4,5‐trisphosphate and 3,4‐bisphosphate through its PH domain (84), and it has been shown to phosphorylate and activate PKC‐z directly (25, 26).

E. LPS‐Induced Adherence Is Regulated by Rho and PI 3‐Kinase 1. MONOCYTE ADHERENCE INDUCED CD14 AND LFA‐1

BY

LPS INVOLVES

Adherence of monocytes to endothelial cells is an essential requirement for the localization of these cells to sites of tissue inflammation (85–87). Several reports have shown that this process is dependent on the monocyte surface molecule lymphocyte function‐associated antigen‐1 (LFA‐1) (CD11a/ CD18; aLb2) [(87–89) and reviewed in (90, 91)]. Intercellular adhesion

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FIG. 3. PI 3‐kinase is involved in early but not late phase IRAK‐1 degradation. THP‐1wt (CD14þ) cells were preincubated either with 20 mM LY294002 (LY), 50 nM wortmannin (Wm), or medium alone for 20 min at room temperature and then treated with 500 ng/ml LPS for 30 min (A) or 2 hr (B). Cells were then washed, lysed, and equal amounts of proteins were analyzed by SDS‐PAGE and Western blotting with anti‐IRAK‐1 antibodies. Adapted from the work of Noubir et al. (2).

molecule‐1 (ICAM‐1) (CD54) has been identified as a high affinity counter‐ receptor for LFA‐1 (92), and interactions between ICAM‐1 and LFA‐1 mediate several important functions in the immune system in addition to adherence (90). The affinity of LFA‐1 under basal conditions for ICAM‐1 or its other ligands is low, and LFA‐1 must be activated to mediate stable adhesion (88, 89). Changes in the affinity of LFA‐1 for ligand are known to be regulated by a process of ‘‘inside‐out’’ signaling that converts LFA‐1 into an activated form capable of mediating increased adhesion (91, 93). We examined signaling events required for LPS‐induced monocyte adherence using a quantitative, microtiter adhesion assay, CD14 transfected THP1 cells, and immobilized sICAM‐1. Experiments that examined competitive inhibition of LPS‐induced adherence using mAbs to CD14, CD18, and CD11a provided direct evidence that LPS‐induced adherence to sICAM‐1 involved a CD14‐ mediated signal leading to activation of LFA‐1. These findings were consistent with previous data showing that antibody cross‐linking of cell surface CD14 induces LFA‐1 activation (94). LPS effects on LFA‐1 did not involve changes in the expression of CD18 or CD11a indicating that LPS‐induced adhesion was rather related to increased affinity of LFA‐1. Such a change in the properties of LFA‐1 is presumably mediated by a specific pathway of inside‐out signaling initiated through CD14.

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FIG. 4. Dual, independent pathways of IRAK‐1 degradation are initiated separately through CD14/TLR‐4 and CR3. Both CD14/TLR‐4 and CR3 have the capacity to initiate the innate immune responses to LPS through IRAK‐1–dependent pathways leading to activation of NF‐kB–dependent gene transcription. Negative feedback is provided by dual, independent pathways also triggered through CD14/TLR‐4 and CR3 leading to IRAK‐1 degradation and attenuation of the inflammatory response. LPS signaling through CD14 and TLR‐4 leads to rapid IRAK‐1 degradation, and this is dependent on PI 3‐kinase (PI3‐K). In parallel, a delayed wave of IRAK‐1 degradation initiated by CR3 occurs independently of both TLR‐4 and PI 3‐kinase. The latter pathway is likely to be of particular importance in cells that are CR3þ and express little or no CD14. Adapted from the work of Noubir et al. (2).

2. REGULATION BY RHO FOR CYTOHESIN

AND

PI 3‐KINASE

AND

REQUIREMENT

The signaling events that link cell stimulation to activation of LFA‐1 are incompletely understood. The requirement for PI 3‐kinase activity in a variety of leukocyte functions, together with its apparent role in the adhesion of platelets (95), lymphocytes (96), and neutrophils (97), made this enzyme an attractive candidate for mediating signaling through CD14 for monocyte adhesion. This hypothesis was supported by the fact that cytohesin‐1 contains a PH domain that binds the PI 3‐kinase metabolite, phosphatidylinositol 3,4, 5‐triphosphate (PtdIns‐3,4,5‐P3), leading to changes in properties of the protein (98). Cytohesin‐1 is a regulatory or adaptor protein that has been shown

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to interact with the cytoplasmic tail of CD18 (99). Based on these findings, a potential role for PI 3‐kinase in regulating the activity of LFA‐1 was suggested. Two different approaches provided evidence that PI 3‐kinase regulated LPS‐induced monocyte adherence. The first involved the use of wortmannin and LY294002 and the findings that both inhibitors abrogated LPS‐induced adherence. The second approach demonstrated complete abrogation of LPS‐ induced adherence to sICAM‐1 when a dominant negative mutant of PI 3‐kinase (Dp85) was expressed in U937 cells (36). It has been shown previously that incubation of monocytes with LPS activates PI 3‐kinase, leading to increased cellular levels of PtdIns‐3,4,5‐P3 (31). Thus, the most likely mechanism for the attenuation of adherence by either wortmannin, LY294002, or Dp85 is inhibition of the formation of PtdIns‐3,4,5‐P3. Based on previous reports that the small G‐protein Rho regulated PI 3‐kinase activation in different cell systems (100–102), the possibility that LPS‐induced adherence may be Rho‐regulated and mediated by PI 3‐kinase was investigated. A requirement for Rho in LPS‐induced adherence was suggested by studies that used Clostridium difficile toxin B, which specifically inhibits Rho family proteins (103, 104). Pretreatment of THP‐1wt (CD14þ) cells with toxin B attenuated LPS‐induced adherence to sICAM, whereas PMA‐induced adherence appeared to be mediated by a toxin B‐insensitive pathway (36). The findings that both Rho and PI 3‐kinase appeared to be essential for LPS‐induced adherence raised the question as to whether they act independently or they are positioned together in a single signaling pathway. Toxin B prevented activation of PI 3‐kinase in LPS‐stimulated THP‐1wt cells, suggesting that Rho regulates this LPS response in monocytes as well (36). This observation is consistent with previous reports showing involvement of Rho in PI 3‐kinase activation in other systems (100–102). Although we cannot completely eliminate the possibility of a direct, PI 3‐kinase–independent role for Rho in regulating monocyte adherence, the data suggest that LPS triggers Rho‐mediated activation of PI 3‐kinase, leading to downstream effects on LFA‐1 and monocyte adherence. An important question that arose from these observations was how PI 3‐kinase activation modulates the properties of LFA‐1. Recently, cytohesin‐1 was shown to interact with the cytoplasmic tail of CD18 (99). Cytohesin‐1 contains a domain homologous to the yeast Sec7 gene product and a PH domain. The Sec7 domain binds to and regulates LFA‐1 (99), and this process is positively regulated by the binding of the PI 3‐kinase metabolite PtdIns‐ 3,4,5‐P3 to the cytohesin‐1 PH domain (98). To directly address the role of cytohesin‐1 in LPS‐induced adherence, cytohesin‐1–specific antisense oligonucleotides were used. The finding that antisense treatment of THP‐1 cells, but not treatment with sense oligonucleotide, significantly attenuated LPS‐ induced adherence to ICAM‐1 provided compelling evidence to suggest that

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cytohesin‐1 plays an essential role in adherence induced by LPS (Fig. 5). Taken together, these results are consistent with a model (Fig. 6) in which LPS binding to CD14 switches on the small G‐protein Rho leading to activation of PI 3‐kinase. This is linked to increased monocyte adherence, dependent on changes in the adhesive properties of LFA‐1. The latter appears to involve the interaction of PtdIns‐3,4,5‐P3, cytohesin‐1, and LFA‐1.

FIG. 5. Cytohesin‐1 antisense S‐oligos inhibit LPS‐induced adherence to ICAM‐1. (A) THP‐ 1wt cells were incubated with various concentrations of S‐oligos for 2 hr at 37 C in 250 ml of RPMI 1640 containing 2.5% lipofectamine. The medium was then adjusted to 1 ml and supplemented with 10% FCS, and culture was continued for 18 hr. Cells were then washed and tested in the adhesion assay (36). (B) THP‐1wt cells were incubated with 5 mM fluoresceinated‐50 antisense S‐oligo using the same conditions as for unmodified S‐oligo. Cells were washed and analyzed by FACS (36). The data shown in (A) are the means  S.E. of values obtained in three independent experiments. Results in (B) represent one of two independent experiments with similar results. The tracing on the left in (B) represents cells incubated with medium alone (autofluorescence), and the tracing on the right corresponds to cells incubated with fluorescein‐labeled, sense S‐oligo. Adapted from the work of Hmama et al. (36).

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FIG. 6. LPS‐induced monocyte adherence to ICAM‐1. Monocyte adhesion to ICAM‐1 involves activated LFA‐1 and the adaptor molecule cytohesin‐1. The latter is believed to be dependent on PtdIns‐3,4,5‐P3 (PIP3). LPS binding to CD14 (A) engages the small G‐protein Rho (B) leading to activation of PI 3‐kinase (C). PtdIns‐3,4,5‐P3 (D) binds to the PH domain of cytohesin‐1, thereby modifying its interaction, through its Sec7 domain, with the cytoplasmic tail of CD18 (E). This leads to altered properties of LFA‐1 (F) and increased affinity for its counter receptor ICAM‐1. Adapted from the work of Hmama et al. (36).

III. PI 3‐Kinase and 1a,25‐Dihydroxyvitamin D3 1a,25‐dihydroxycholecalciferol (Vit D3 or D3) is the biologically active form of vitamin D that plays an important role in numerous cellular and physiological processes. In addition to its well established importance in plasma calcium homeostasis (105, 106) and bone remodeling (107, 108), Vit D3 also plays a functional role in the hematopoietic system. D3 modulates the expression of several genes in promonocytic cell lines, thereby regulating their differentiation (109). Moreover, other physiological processes, such as cellular adherence and host resistance to infection, have also been attributed to Vit D3 (110–114). Recent results from this laboratory showing that D3 activates PI 3‐kinase and PI 3‐kinase activity is required for the D3 induced myeloid cell differentiation as well as adherence and antimicrobial effector function are reviewed in the next section.

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A. Vitamin D3 Action and Signaling The pleiotropic effects of D3 are principally mediated through the intracellular vitamin D receptor (VDR). The VDR is a member of the superfamily of nuclear steroid, thyroid, and retinoic acid receptors. The classical mechanism of action of D3 involves genomic signaling in which hormone binds to the VDR, a ligand‐dependent transcription factor. The D3˙VDR complex then translocates to the nucleus, where it directly regulates transcription by binding to the vitamin D response element (VDRE) consensus sequence located upstream of many D3‐activated genes (115). This mode of signaling, referred to as ‘‘genomic action,’’ is exemplified for instance in D3‐treated immature myeloid cells, which express the Cdk inhibitor p21 after hormone treatment. p21 has been shown to regulate gene expression and promote myleoid cell differentiation (116) and is transcriptionally induced by D3 via a functional VDRE within the p21 promoter (116). When immature myeloid leukemia cells, such as HL60, U937, THP‐1, and M1, are incubated with D3, they differentiate into cells expressing functional properties and differentiation markers of monocytes and/or macrophages, including CD14 and CD11b/CD18 (117–120). CD14 is undetectable on the surface of monocytic precursors and increases dramatically during differentiation into monocytes (121–123). This provides an excellent model in which to study mechanisms of myeloid cell maturation regulated by D3. Induction of CD14 expression in response to D3 occurs at the level of gene transcription and requires new protein synthesis (123, 124). However, a curious paradox of this response is that no VDRE consensus sequence has been identified in the promoter regions of CD14 or CD11b, and this also applies to certain other D3‐inducible genes (123, 125). The region located at –128 to –70 bp upstream from the transcription start site of the CD14 promoter has been shown to be critical for CD14 expression in response to D3 (123). Rather than a VDRE, this region contains two GC boxes that bind the nonreceptor transcription factor Sp1, and this is believed to be essential for CD14 expression (123, 126). Likewise, the integrin CD11b also possess a sequence at bp –60 from the transcription start site that binds Sp1 in vitro and in vivo and is essential to the promoter activity (127). In various attempts to define the molecular mechanisms regulating this type of cellular responses to D3, efforts have been made to identify alternative signaling pathways to the classical mode of genomic action. Indeed, during the past decade, experimental evidence for ‘‘nongenomic’’ signaling has challenged the concept of exclusive VDR‐mediated genomic action. For example, D3 has been shown to stimulate the rapid formation of second messengers, including ceramides, cAMP, inositols, and Ca2þ, and to activate a variety of protein kinases including PKC, Raf, mitogen‐activated protein (MAP) kinase, and Src

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family kinases (125, 128–130). MAP kinase activity was also shown to be activated by D3 in keratinocytes, enterocytes, and in the promyelocytic cell lines HL60 and NB4 (130–132). The physiological complexity of nongenomic signaling itself and to what extent it acts cooperatively with VDR‐mediated genomic signaling is unclear. Moreover, it has not been clear whether D3 uses the classical VDR for nongenomic signaling or an alternative receptor is involved.

B. A VDR
PI 3‐Kinase Signaling Complex Regulates Myeloid Cell Differentiation 1. D3‐INDUCED CD14 AND CD11B EXPRESSION IS PI 3‐KINASE DEPENDENT In light of the important roles played by PI 3‐kinase in regulating the differentiation of a variety of cell types (133–135), the possibility that D3‐ induced monocyte differentiation involves PI 3‐kinase was investigated. Incubation of serum‐starved THP‐1 cells with 10 nM D3 brought about a significant increase in PI 3‐kinase activity, with a maximum response observed at 1 mM (3). Time course experiments using 100 nM D3 detected PI 3‐kinase activation as early as 5 min and a maximum level by 20 min (3). Subsequent experiments provided several lines of evidence to show that D3‐induced CD14 expression is PI 3‐kinase dependent including: (a) D3‐induced CD14 expression was abrogated in THP‐1 cells incubated with either wortmannin and LY294002 (3); (b) antisense targeting of p110a PI 3‐kinase attenuated D3‐induced CD14 expression (Fig. 7); and (c) expression of a dominant negative mutant of PI 3‐kinase (Dp85) in U937 cells also abrogated D3‐induced expression of CD14 (3). Similarly, CD11b induction in response to D3 was also attenuated by wortmannin and LY294002 in both THP‐1 cells and in human peripheral blood monocytes (3). Taken together, these results established that D3‐induced CD14 and CD11b expression are regulated by PI 3‐kinase. Of particular note, D3 induction of p21 expression in THP‐1 cells was found to be resistant to both wortmannin and LY294002 (data not shown) (3). 2. INDUCTION OF CD14 IN RESPONSE TO D3 INVOLVES THE ASSOCIATION OF PI 3‐KINASE WITH THE VDR Many responses to D3, such as induction of expression of osteopontin, osteocalcin, calbindin, and 24‐hydroxylase, are brought about by a mechanism involving binding of the VDR to a specific VDRE in the corresponding promoters (136). Since no VDRE has been identified within the CD14 gene promoter (123), this raised the question as to whether the VDR plays any role at all in regulating D3‐induced CD14 expression. Experiments using antisense S‐oligo specific to VDR mRNA showed attenuation of D3‐induced CD14 expression (3).

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FIG. 7. Antisense S‐oligo to the p110 subunit of PI 3‐kinase inhibits D3‐induced CD14 expression. THP‐1 cells were suspended in RPMI 1640 containing 2.5% Lipofectamine alone (Control), or the same solution containing 5 mM of either sense (S) or antisense (AS) S‐oligo to p110. Cells were then incubated on a rotary shaker for 4 hr at 37 C. The incubation was brought up to 10 ml of medium, and cells were cultured for an additional 18 hr at 37 C and 5% CO2. (A) THP‐1 cells were stimulated with 100 nM D3 for 20 min and assayed for PI 3‐kinase assay (3). The upper rectangle shows PIP spots obtained in one representative experiment. The graphical data shown below represent PI 3‐kinase activities (means  SEM of values obtained in three separate experiments) (3). (B) Aliquots of control or S‐oligo–treated cells (~1  106) were incubated in medium alone or with D3 (100 nM, 24 hr), washed with staining buffer, and then labeled with anti‐CD14 mAb or irrelevant mAb, followed by FITC‐conjugated secondary Abs. Flow cytometric analyses were performed, and MFI indices were determined (3). Results are expressed as histograms of fluorescence intensity. Histograms displaced to the right represent cells stained with anti‐CD14, and histograms on the left represent cells stained with irrelevant IgG2b. Bold, italicized values are means  SEM (n ¼ 3) of MFI indices (3). (C) Control or S‐oligo–treated cells were incubated with D3 (100 nM, 24 hr). Total RNA was extracted and RT‐ PCR was carried out for CD14 and b‐actin (3). Adapted from the work of Hmama et al. (3), with permission.

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RT‐PCR experiments showed that this was related to a marked reduction in CD14 mRNA accumulation in response to D3 (3). Taken together, these findings indicate that the VDR is essential for D3‐induced CD14 gene expression. Given the evidence that both PI 3‐kinase and the VDR were found to be essential for induction of CD14 in response to D3, it was important to know whether VDR was required for activation of PI 3‐kinase. In fact, antisense targeting of VDR almost completely abrogated PI 3‐kinase activation in response to D3 (Fig. 8).

FIG. 8. The VDR is required for D3‐induced PI 3‐kinase activation. (A) THP‐1 cells were treated overnight with 5 mM of either sense (S) or antisense (AS) S‐oligo to VDR (3). Cells were then stimulated for 20 min with 100 nM D3, and lysates were prepared and assayed for PI 3‐kinase activities (3). The upper rectangle shows PIP spots obtained in one representative experiment. The data shown below are PI 3‐kinase activities (averages of values obtained in two separate experiments) (3). (B) Cells were stimulated with D3 as in (A) and cell lysates were prepared and incubated overnight at 4  C either with 9A7 mAb (anti‐VDR) or with mAb specific for the p85 subunit of PI 3‐kinase. Immune complexes were adsorbed onto protein A and assayed for PI 3‐kinase activities. The upper rectangle shows PIP spots obtained in one representative experiment. The data shown below are PI 3‐kinase activities (average of two separate experiments). (C) Cell lysates prepared from either control cells or D3‐treated cells were immunoprecipitated with specific mAbs to either the VDR or the p85 subunit of PI 3‐kinase. Immunoprecipitates were then analyzed by SDS‐PAGE and immunoblotting with Abs to p85 (40). Immunoprecipitates with normal mouse IgG (MIgG) and normal rat IgG (RIgG) were used to control for the specificity of anti‐p85 and anti‐VDR Abs, respectively. Adapted from the work of Hmama et al. (3), with permission.

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The finding that the D3 receptor was required for both PI 3‐kinase activation and CD14 expression in response to D3 suggested the possibility that this might involve a signaling complex containing both the VDR and PI 3‐kinase. Our results in fact indicated that immunoprecipitates of the VDR prepared from cells activated with D3 contained PI 3‐kinase activity and the p85 subunit (Fig. 8). Of particular interest was the finding that the association of p85 with the VDR was only observed in cells treated with hormone (Fig. 8). These results demonstrated that D3 treatment induces the formation of a signaling complex containing the VDR and PI 3‐kinase resulting in activation of the lipid kinase, which is required for induction of CD14 expression. In summary, D3‐induced signaling in THP‐1 cells initiated through the VDR appears to involve at least two distinct pathways. One is a genomic pathway involving p21, which regulates aspects of monocyte differentiation independently of PI 3‐kinase. A second pathway is nongenomic and involves a signaling complex containing at least PI 3‐kinase and VDR, which regulates the induction of CD14 and CD11b and perhaps other targets.

C. D3‐Induced Monocyte Antimycobacterial Activity Is Regulated by PI 3‐Kinase Several lines of evidence have indicated that D3 regulates host resistance to Mycobacterium tuberculosis. D3 deficiency and VDR receptor polymorphisms have been linked to increased susceptibility to infection with M. tuberculosis and Mycobacterium leprae (110–112). In this regard, D3 production in vivo is promoted by exposure to ultraviolet light, providing a link between exposure to sunlight and antimycobacterial resistance (110, 113). In addition, D3 has been shown directly to activate mononuclear phagocyte antimycobacterial activity (113, 114). However, the molecular basis through which D3 regulates host resistance to M. tuberculosis has not been identified. To elucidate this further, we examined whether PI 3‐K is involved in regulating D3‐induced antimycobacterial activity and investigated the mechanistic basis for this effector function. 1. D3‐INDUCED MONOCYTE RESISTANCE IS REGULATED BY PI 3‐KINASE

TO

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To investigate the basis for the induction of monocyte antimycobacterial activity by D3, THP‐1 cells or human peripheral blood monocyte‐derived macrophages were infected with M. tuberculosis H37Rv and then incubated with hormone. As expected, this resulted in decreased recovery of mycobacterial colony‐forming units from D3‐treated cells compared with control cells. The potential role of PI 3‐K in this response was studied by examining the growth of M. tuberculosis in cells treated with D3 in the presence or absence of

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LY294002 or wortmannin as well as in cells where the p110a subunit of PI 3‐kinase was targeted by antisense. Treatment with either LY294002 or wortmannin inhibited PI 3‐K activity in M. tuberculosis‐infected, D3‐treated THP‐ 1 cells and also significantly abrogated D3‐induced antimycobacterial activity (137). Essentially identical effects were observed in antisense‐treated cells with reduced expression of the p110a subunit of PI 3‐Kinase (137). Taken together, these findings indicated that activation of PI 3‐K p85/p110a is required for the antimycobacterial action of D3. 2. PHAGOCYTE OXIDATIVE BURST IS REQUIRED FOR THE ANTIMYCOBACTERIAL ACTION OF D3 AND IS PI 3‐KINASE–DEPENDENT The phagocyte oxidative burst is a major antimicrobial effector mechanism. To assess the role of macrophage reactive oxygen intermediates (ROI) in the antimycobacterial action of D3, infected cells were incubated with hormone in the presence of either 4‐hydroxy‐TEMPO, PEG‐SOD, or PEG‐cat. These reagents act to eliminate ROI, either by scavenging (4‐hydroxy‐TEMPO) or enzymatically (PEG‐cat or PEG‐SOD), and each of them significantly attenuated the ability of D3 to induce resistance to M. tuberculosis in THP‐1 cells and monocyte‐derived macrophages (137). Consistent with these observations, M. tuberculosis‐infected macrophages secreted significant amounts of O2 when treated with D3. Of particular interest, this response was inhibited by LY294002 or wortmannin as well as by antisense targeting of p110a PI 3‐K (137). These results suggested an interaction between M. tuberculosis infection and D3 in triggering the phagocyte oxidative burst in human monocyte‐derived macrophages as well as in THP‐1 cells and that this process is regulated by PI 3‐kinase. 3. TRANSLOCATION OF PHAGOCYTE OXIDASE COMPONENTS TO THE PLASMA MEMBRANE Phagosome oxidase activity requires recruitment of the cytosolic components, p47phox and p67phox, to the membrane for assembly with flavocytochrome b558 (138, 139). The relative distribution of p47phox and p67phox in cytosolic and membrane fractions was examined. A marked translocation of both p47phox and p67phox to the membrane fraction was observed in M. tuberculosis‐infected cells treated with D3, whereas treatment of cells with either live or heat‐killed M. tuberculosis alone did not bring about detectable changes (138, 139). We have shown that redistribution of p47phox and p67phox to the membrane fraction in response to infection with M. tuberculosis and treatment with D3 was markedly reduced in cells pretreated with either of the PI 3‐K inhibitors, LY294002 or wortmannin (Fig. 9). Consistent with this result, PI 3‐kinase was shown to be required for triggering the NADPH

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FIG. 9. THP‐1 cell cytosol and membrane immunoblots probed with anti‐p47phox (upper panel) or anti‐p67phox (lower panel). In panels A and B, membrane and cytosolic fractions, respectively, were purified from resting cells (lane 1), cells differentiated with PMA (lane 2), differentiated cells coincubated with M. tuberculosis for 2 (lane 3) or 4 hr (lane 4), differentiated cells coincubated with M. tuberculosis for 4 hr followed by treatment with D3 (lane 5), differentiated cells coincubated with heat‐killed M. tuberculosis for 2 (lane 6) or 4 hr (lane 7), differentiated cells coincubated with heat‐killed M. tuberculosis for 4 hr followed by treatment with D3 (lane 8), and differentiated cells treated with D3 only (lane 9). In panels C and D, membrane fractions were purified from differentiated cells treated in the presence or absence of the PI 3‐K inhibitor, LY or Wm, respectively. Shown are PMA‐differentiated cells (lane 1), cells coincubated with M. tuberculosis for 4 hr (lane 2), D3‐treated cells (lane 3), cells coincubated with M. tuberculosis followed by D3 treatment (lane 4), PMA‐differentiated cells treated with PI 3‐K inhibitor (lane 5), cells treated with PI 3‐K inhibitor followed by coincubation with M. tuberculosis for 4 hr (lane 6), cells treated with PI 3‐K inhibitor followed by D3 treatment (lane 7), cells treated with PI 3‐K inhibitor followed by coincubation with M. tuberculosis for 4 hr and then D3 treatment (lane 8), and cells coincubated with M. tuberculosis followed by treatment with PI 3‐K inhibitor and then D3 (lane 9). Adapted from the work of Sly et al. (137).

oxidase in response to particulate stimuli through activating Fca and Fcg receptors in neutrophils (140). Similarly, based on effects of wortmannin, a role for PI 3‐kinase in regulating the production of superoxide by human neutrophils in response to soluble stimuli, such as fMLP (141) and PMA (142), has also been suggested. In summary, a key regulatory pathway of D3‐ induced host defense against tuberculosis has been identified and appears to represent another novel example of nongenomic signaling by vitamin D3. PI 3‐Kinase is required to trigger the oxidative burst in response to D3 in M. tuberculosis‐infected cells. However, activation of PI 3‐kinase alone is insufficient to bring about full oxidase activation, since treatment of THP‐1 cells with D3 alone activates PI 3‐kinase, but at best this elicits only modest

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superoxide production. Prior infection of cells with live M. tuberculosis was observed to prime cells for an enhanced D3‐induced oxidative burst (137). This suggests that live M. tuberculosis or some factor unique to the phagosome containing viable M. tuberculosis potentiates phagocytes to undergo a vigorous oxidative burst in response to D3.

D. D3‐Induced Antimicrobial Activity Against Salmonella enterica Is PI 3‐Kinase Dependent Salmonella enterica serovar Typhimurium causes self‐limiting gastroenteritis in immunocompetent adults and may also cause life‐threatening extraintestinal infections (143). The phagocyte oxidative burst is a key host defense mechanism against S. enterica (144–146) and Salmonella SodCI and SodCII isoenzymes have been implicated in virulence (144, 146). In light of our new understanding of how D3 induces macrophage resistance to M. tuberculosis, we examined the role of PI 3‐kinase in D3‐induced killing of S. enterica as well as the potential roles for SodCI and SodCII in resisting this response (147). Vitamin D3 treatment had a pronounced antimicrobial effect against wild‐ type S. enterica serovar Typhimurium in THP‐1 cells, reducing the number of viable bacteria by almost tenfold compared to the number of bacteria in untreated control cells. Incubation of monocytes with wortmannin or LY204002 prior to addition of D3, abrogated this effect indicating that it was PI 3‐K dependent (147). Vitamin D3‐induced killing of S. enterica was not dependent on nitric oxide (NO), as no nitrite was detectable in culture media by Griess assay. Furthemore, killing was not reversed by incubation of cells with NO inhibitor NG‐monomethyl‐L‐arginine (L‐MMA). Vitamin D3‐treated, Salmonella‐infected THP‐1 cells produced significantly more superoxide than did cells exposed to either infection alone or to D3 alone (147). Furthermore, the amount of superoxide produced by vitamin D3‐treated, Salmonella‐infected THP‐1 cells was reduced to background levels by pretreatment with either LY294002 or wortmannin (147). These results indicated that D3 induces bactericidal activity against intracellular S. enterica serovar Typhimurium in human monocyte‐like cells that are PI 3‐kinase dependent and suggest that the ulimate effector mechanism is the phagocyte oxidative burst. The latter argument is consistent with studies showing that control of S. enterica infection in the mouse model requires NADPH oxidase activity (144, 145, 148). Further direct support for this model was provided by study of SodC mutants, which we found to be more susceptible to the effect of D3 in THP‐1 cells than were wild‐type organisms. While each mutant strain (SodCI, SodCII, and the SodCI‐SodCII double knockout) grew normally in untreated THP‐1 cells, significant loss of viability was observed in D3‐treated cells. While this was observed for both individual SodCI and SodCII mutants, the effect was

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more pronounced in the SodCI‐SodCII double mutant, which was nearly completely eliminated from D3‐treated cells (147). This result provided evidence that both SodC enzymes contribute to bacterial resistance to oxidative damage in a nonredundant fashion.

IV. PI 3‐Kinase and Phagocytosis of Mycobacteria A. Phagocytosis of Mycobacteria: Receptors and Signaling Several receptors involved in the internalization of pathogenic mycobacteria by macrophages have been tentatively characterized, but not extensively studied. Binding of mycobacteria to macrophages is believed to occur in cholesterol‐rich domains of the host cell plasma membrane (149), and involvement of b2 integrins (complement receptor CR3 and CR4), together with other molecules including the mannose receptor, CR1 and CD14 (150), have been suggested. Other surface molecules, such as Toll‐like receptors, are also involved in mycobacterial interactions with phagocytic cells (151). In monocytes and immature macrophages, which do not express the mannose receptor (152), CR3 and CD14 have been shown to be potential gates for entry of mycobacteria (153, 154). However, whether these two receptors act individually or cooperatively is not known. Interest in signaling events that link cell stimulation to engagement of b2 integrins has been considerable in recent years (155, 156). The mycobacterial cell surface glycolipid lipoarabinomannan (LAM) is known to enhance phagocytosis of mycobacteria (157). It has also been shown that LAM‐regulated phagocytosis is dependent at least in part on CR3 (158). Studies by Kolanus et al. (99) led to the discovery that cytohesin‐1 acts as an adaptor protein and interacts with the cytoplasmic tail of CD18 leading to changes in the functional properties of b2 integrins. Cytohesin‐1 contains a PH domain that binds the PI 3‐ kinase metabolite, phosphatidylinositol 3,4,5‐trisphosphate (PtdIns‐3,4,5‐P3), leading to changes in the functional properties of the protein (98, 159). These findings have suggested a role for PI 3‐kinase and cytohesin in regulating the activity of b2 integrins (36, 160), including CR3. The receptors that cooperate to bring about optimal uptake of mycobacteria and how they are linked are important issues in innate immunity. In the next section, we have reviewed data from studies that focused on deciphering the mechanisms that lead to the internalization of Mycobacterium bovis bacillus Calmette‐Guerin (BCG), the role of vitamin D3 in regulating phagosome maturation as well as the important role of PI 3‐kinase in regulating these events.

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B. Cross‐Talk Between CD14 and CR3: Regulation by PI 3‐Kinase and Cytohesin‐1 1. ROLE OF CD14/CR3 OF TLR‐2

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We investigated the hypothesis that binding of mycobacteria to macrophages modulates the activity of cell‐surface receptors so as to enhance bacterial internalization. Using THP‐1 and CHO cells transfected to express individual receptors, evidence was obtained to show that CD14 played a major role in phagocytosis of M. bovis BCG and this required serum factors including lipopolysaccharide binding protein (LBP) (161). These results were consistent with a previous report showing that CD14 mediated uptake of mycobacteria by monocyte‐derived microglial cells (162). Whereas our results showed that some degree of mycobacterial internalization occurred through CD14 alone and CR3 by itself functioned poorly as a phagocytic receptor for BCG uptake was significantly potentiated in the presence of both CD14 and CR3 (161). Transfected CHO cells were also used to investigate the potential role of TLR‐2 in regulating CD14‐dependent phagocytosis. Exposure of CHO cells transfected with CD14 or TLR‐2 alone to opsonized BCG led to a modest degree of bacterial ingestion. In contrast, coexpression of both TLR‐2 and CD14 (CHO/CD14/TLR2) resulted in a four‐ to fivefold increase in the proportion of cells that ingested bacteria (161). 2. CD14‐DEPENDENT PHAGOCYTOSIS PI 3‐KINASE AND TLR‐2

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An important question raised by these findings was the mechanism of communication between CD14 and CR3. PI 3‐kinase has been demonstrated to have a direct role in regulating a range of leukocyte functions dependent on rearrangement of the cytoskeleton such as adhesion (163), phagocytosis (164, 165), and phagosome biogenesis (166–168). These properties, together with the knowledge that the activities of b2 integrin receptors can be modulated through PI 3‐kinase (36, 160), identified this lipid kinase as an attractive candidate for regulating cross‐talk between CD14 and CR3 in the context of the binding and uptake of mycobacteria. Incubation of THP‐1wt cells with serum‐opsonized BCG brought about a rapid and significant increase in PI 3‐kinase activity with a maximum response observed within 10 min (3.5‐fold increase) (161). A threefold increase in PI 3‐kinase activity was also observed in cells incubated with LAM in the presence of LBP. In contrast, THP‐1wt cells incubated with BCG in the absence of opsonization with either serum or LAM had minimal effects on PI 3‐kinase activity. PI 3‐kinase inhibitors were used to examine further the potential involvement of the enzyme in CD14‐ induced phagocytosis. Preincubation of THP‐1wt cells with either wortmannin

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or LY294002 inhibited phagocytosis in a dose‐dependent manner with maximum inhibition of 78% and 75%, respectively. Taken together, these findings suggested that PI 3‐kinase activation plays a central role in CD14‐dependent phagocytosis of BCG (53). Furthermore, whether PI 3‐kinase is involved in TLR‐2–dependent ingestion of mycobacteria was investigated by examining the intracellular distribution of the PI 3‐kinase product PtdIns‐3,4,5‐P3 in vivo. CHO cells transfected with both CD14 and TLR‐2 (CHO/CD14/TLR‐2) were infected with BCG conjugated to the fluorescent tracer rhodamine B isothiocyanate (RITC) and then incubated with mAb RC6F8 and fluorescein isothiocynate (FITC)‐conjugated secondary Ab. RC6F8 recognizes specifically PtdIns‐3,4,5‐P3, with minimal cross‐reactivity for either PtdIns‐ (4,5)‐diphosphate or for the two monophosphates PtdIns‐(3)‐monophosphate and PtdIns‐(4)‐monophosphate (169). Marked colocalization of PtdIns‐3,4, 5‐P3 with RITC‐BCG in CHO/CD14/TLR‐2 cells, but not in CHO/CR3 or CHO/CD14 cells, was observed showing the important role of TLR‐2 in the CD14‐induced activation of PI 3‐kinase (161). Finally, using the PI 3‐kinase inhibitor LY294002, increased avidity of CR3 induced by LAM was also shown to be dependent on PI 3‐kinase (161). 3. CR3‐MEDIATED PHAGOCYTOSIS BY CYTOHESIN‐1

OF

M. BOVIS BCG IS REGULATED

An important question raised by these findings was the molecular basis by which activation of CD14/TLR‐2 and PI 3‐kinase–dependent signaling led to modulation of the properties of CR3. Cytohesin‐1 is known to be a key regulator of leukocyte adhesion, and this is mediated by its interaction with the cytoplasmic tail of CD18 to bring about increased avidity of LFA‐1 (CD18/ CD11a) for ICAM‐1 (36, 99, 170). The interaction of cytohesin‐1 with b2 integrins is positively regulated by the binding of the PI 3‐kinase metabolite, PtdIns‐3,4,5‐P3, to the cytohesin‐1 PH domain (98, 171). These findings suggested the hypothesis that CD14/PI 3‐kinase–dependent enhancement of CR3 avidity brought about by mycobacterial cell wall fraction components may involve cytohesin‐1. To investigate this possibility, cytohesin‐1 expression was downregulated by incubation of THP‐1wt cells in the presence of antisense S‐oligos. Treatment of cells with antisense S‐oligo to cytohesin‐1 mRNA, but not with control sense S‐oligo, eliminated the expression of this protein concomitant with significant attenuation of serum‐dependent uptake of BCG (53% inhibition) (161). Direct association between cytohesin‐1 and CR3 was shown using flow cytometry based ‘‘pull down’’ experiments. Anticytohesin‐1 Ab was coupled to latex beads and used to pull down cytohesin‐1 and from lysates of activated cells. Beads were then stained with antibodies to either PtdIns‐3,4,5‐P3 or CR3 and analyzed flow cytometry. LAM stimulation of THP‐1wt cells increased

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the level of PtdIns‐3,4,5‐P3 associated with cytohesin‐1 (3.5‐fold increase) concomitant with increased recruitment of CR3 (6.5‐fold increase) (161). Pretreatment of cells with PI 3‐kinase inhibitor or CD14 blocking mAb before LAM abrogated the recruitment of both PtdIns‐3,4,5‐P3 and CR3 to cytohesin‐1. Taken together, these results suggested a direct role for a CD14/PI3‐kinase/ cytohesin‐1 signaling pathway resulting in enhanced CR3‐dependent phagocytosis of BCG. In summary, several lines of evidence support a role for cytohesin‐1 in CR3‐dependent phagocytosis of BCG: (i) the uptake of serum‐ but not LBP‐opsonized BCG was significantly attenuated in THP‐1wt cells with reduced cytohesin‐1, (ii) intracellular staining showed a CD14‐ and PI 3‐kinase–dependent colocalization of cytohesin‐1 with CR3 in LAM‐stimulated cells, and (iii) pull‐down experiments detected the physical association between cytohesin‐1 and both PtdIns‐3,4,5‐P3 and CR3 in LAM‐stimulated cells (161). These findings suggested a dynamic model regulating the uptake of mycobacteria by macrophages (Fig. 10). In this model, binding of BCG or LAM to CD14 triggers signaling through TLR‐2 leading to activation of PI 3‐kinase. This creates a point of bifurcation in which in one direction based on the effects of PI 3‐kinase on the cytoskeleton alone, suboptimal levels of

FIG. 10. Cooperativity between CD14 and CR3 mediates optimal uptake of BCG. Signaling through membrane‐bound CD14 initiated by BCG or LAM leads to TLR‐2–mediated activation of PI 3‐K. The latter is responsible for triggering two pathways for bacterial internalization: a unique CD14 pathway that can function in the absence of CR3 and a cytohesin‐1–regulated CR3‐ dependent pathway that together with CD14 results in optimal phagocytosis of BCG. Adapted from the work of Sendide et al. (161), with permission.

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CD14‐dependent bacterial ingestion can proceed independently of CR3. In the other direction, through the action of the PI 3‐kinase product PtdIns‐3,4,5‐P3, the molecular adaptor cytohesin‐1 is recruited to the membrane where it binds to the b‐chain of CR3 and converts it into an active receptor for BCG. Thus, CD14‐dependent activation of PI 3‐kinase through TLR‐2 leads to recruitment of a second receptor that acts cooperatively to bring about optimal bacterial internalization.

C. Rescue of Phagosome Maturation by Vitamin D3 Is PI 3‐Kinase Dependent To investigate processes regulating phagosome maturation, we established a technology to analyze phagosomes using flow cytometry (166). This involved labeling the plasma membrane of intact macrophages with the red‐fluorescent marker PKH26 after which the cells were allowed to ingest latex beads resulting in the generation of phagosomes surrounded by a red‐fluorescent membrane suitable for flow cytometry. Following cell disruption and partial purification of phagosomes, these vesicles were readily distinguished from both cell debris and free beads released from disrupted vacuoles. Using this system, we were able to show that phagosomes stained with specific mAbs showed progressive acquisition of both Rab7 and LAMP‐1 consistent with their expected movement along the endocytic pathway. Alternatively, macrophages were preloaded with the lysosomal tracer FITC‐dextran before membrane labeling with PKH and incubation with latex beads. Phagosome–lysosome fusion was then quantified on the basis of the colocalization of red and green signals [described in detailed (166)]. Using this system, we found that internalization of latex beads in the presence of lysates of M. tuberculosis, but not in the presence of lysates from the nonpathogenic organism Mycobacterium smegmatis markedly decreased phagosome acquisition of Rab7 and LAMP‐1 and vesicle fusion with FITC‐dextran–loaded lysosomes. Inhibition of phagolysosome fusion could be attributed, at least in part, to the mycobacterial cell wall glycolipid lipoarabinomannan. Further analysis showed complete rescue of phagosome maturation when cells were pretreated with Vit D3 before exposure to lipoarabinomannan. To examine the role of PI 3‐kinase in D3‐induced phagolysosome fusion, acquisition of FITC‐ DXT by phagosomes was studied in D3‐treated cells in the presence or absence of the PI 3‐kinase inhibitor LY‐294002. FITC‐DXT–loaded THP‐1 cells were treated with LY‐294002 for 30 min and washed before D3 stimulation. Twenty‐ four hours later, macrophages were labeled with PKH and exposed to latex beads in the presence or absence of LAM. The results indicated that in the absence of PI 3‐kinase inhibitor, the ability of LAM to inhibit phagolysosome fusion was completely prevented by D3. However, the ability of D3 to prevent phagosome maturation induced by LAM was nearly completely abrogated by

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LY‐294002, suggesting that D3 promotes phagolysosome fusion via a PI 3‐kinase signaling pathway (166).

V. Stable Gene Silencing of Isoforms of PI 3‐Kinase and Perspectives Genetic approaches to assigning function to individual class I PI 3‐kinase p110 isoforms have been limited since gene knockouts of p110a and p110b in mice were found to be embryonically lethal (172, 173). Consequently, it has not been possible to determine with precision the roles of these isoforms in immune cells. Recently, instead of gene knockouts, mutant p110
mice were created and have been a useful alternative (174). Thus far, however, no phenotypes in monocytic cells from either knockout or mutant p110
animals have been reported (174–176). Studying mononuclear phagocyte cell biology through genetic manipulation by nonviral transfection methods has been challenging due to the dual problems of low transfection efficiency and the difficulty in obtaining stable transfection. Methods involving cationic lipid and liposome‐mediated delivery of DNA or physical methods, such as electroporation, result in low transfection efficiency in monocytic cells, loss of viability, and the difficulty of obtaining stable transfection (177, 178). An approach that has achieved greater success in monocytic cell lines is viral‐mediated transduction. Although not all viruses can transduce monocytic cells efficiently, lentiviruses have been shown to do so at >90% efficiency (179–181). In what follows, we review our successful efforts to develop a strategy that allows for transduction of human monocytic cell lines using a lentiviral vector leading to stable silencing of a specific isoform of PI 3‐kinase through RNA interference (RNAi) (182).

A. Silencing of P110a Subunit of PI 3‐Kinase: Differential Involvement in Monocyte Adherence 1. STABLE SILENCING OF P110a PI 3‐KINASE SUBUNIT MONOCYTIC CELL LINES

IN

HUMAN

RNAi is a sequence‐specific posttranscriptional gene silencing mechanism initiated by the introduction of double‐stranded RNA (dsRNA) into target cells (183). RNAi is a natural regulatory mechanism that occurs in many organisms, including plants, Caenorhabditis elegans, Drosophila, and mammalian cells [reviewed in (183)]. The RNAi pathway begins by processing dsRNA into short (<30 bp) dsRNA duplexes called small interference RNA (siRNA) by a host RNase Dicer. The siRNA then becomes incorporated into a multicomponent nuclease complex called the RNA‐induced silencing complex (RISC).

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RISC then uses the siRNA sequence as a guide to recognize cognate mRNAs for degradation. To produce recombinant lentiviral vectors for transduction of monocytes, we used the packaging cell line HEK 293T transiently cotransfected by the vector plasmid (pHR‐U6‐shRNA), helper plasmid (pCMV
R8.2), and envelope plasmid (pMD.G). This strategy [for detailed protocol see (182)] segregates the trans‐acting sequences that encode for viral proteins (made by the helper plasmid) from the cis‐acting sequences (regions recognized by viral proteins on the vector plasmid) involved in the transfer of vector sequences encoding a short hairpin RNA (shRNA) [reviewed in (184)]. This approach results in the production of progeny virions that do not contain any genetic elements that code for viral proteins. Therefore, transduced cells are not capable of producing any viral proteins. The vector plasmid contains a U6 promoter, which drives transcription of the shRNA coding sequence of interest as well as a CMV‐driven enhanced green fluorescent protein (EGFP) reporter. These elements are flanked by long terminal repeats (LTRs) and contain cis‐acting sequences allowing only the vector RNA to be packaged. Following transfection of HEK 293T cells, the plasmids pCMVDR8.2 and pMD.G provide in trans the viral structural proteins required for the assembly of viral particles. Using this approach, we harvested viral particles from conditioned medium and used them for transduction of monocytes. Transduction efficiency was then quantitated by GFP expression. The shRNA expressed from the integrated vector elements then silenced the target gene via the RNAi pathway. We designed a shRNA sequence specific to the PI 3‐kinase p110a mRNA. THP‐1 and U‐937 cells were either mock transduced or transduced with viral vectors targeting either p110a, control shRNA, or with a U6 promoter control. Transduction of THP‐1 and U‐937 with the viral vectors targeting the p110a isoform resulted in nearly complete elimination of PI 3‐kinase p110a isoform expression (Fig. 11). In contrast, transduction of cells either with the lentiviral vector expressing the control shRNA sequence, the U6‐promoter control vector, or mock transduction did not affect p110a protein levels. This result was specific in that levels of other Class IA PI 3‐kinase catalytic subunits p110b and p110
, or the p85a regulatory subunits were not affected. The stability of p110a silencing was confirmed by Western blotting of cells that were stored in liquid nitrogen for greater than 1 year, and cells that had been in continuous culture for more than 6 weeks. 2. MONOCYTE ADHERENCE INDUCED IS DEPENDENT ON P110a

BY

D3, BUT

NOT

LPS

Previous results from this laboratory showed that LPS‐induced adherence of THP‐1 cells is dependent on PI 3‐kinase (3). However, use of pharmacological inhibitors did not permit determination of whether this involved class IA PI

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FIG. 11. Transduction of monocytic cell lines by lentiviral vectors is efficient and generates stable cell lines deficient in p110a. (A) Flow cytometry analysis of transduced (solid histogram) or mock transduced cells (clear histogram). Ten thousand cells were analyzed, and the GFP fluorescence intensity was measured on the FL1 channel. Approximately 97% of the THP‐1 and U‐937 cells were GFP‐positive. The mean fluorescence intensity (MFI) for mock‐infected cells was 8.5 for THP‐1 and 6.6 for U‐937. Transduced cells had MFI values of 38.3 for THP‐1 and 132.3 for U‐937. (B and C) Western blot analyses of class IA PI 3‐K p110 catalytic subunit isoforms (a, b, and
), and p85 regulatory subunit in THP‐1 cells and U‐937 cells. Actin was used as a loading control. Adapted from the work of Lee et al. (182).

3‐kinase and, if so, which p110 isoform (3). To address this question, LPS‐ induced adherence was examined in THP‐1 cells rendered p110a‐deficient using lentiviral transduction of shRNA. Consistent with previous findings that PMA‐induced monocyte adherence was resistant to PI 3‐kinase inhibitors (3), p110a‐deficient cells showed normal PMA‐induced adherence (182). Using PMA‐treated cells as a control, LPS‐induced adherence was determined as the percentage of PMA‐induced adherence. Whereas PI 3‐kinase inhibitor LY294002 significantly reduced LPS‐induced adherence in all transduced

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cells, silencing of p110a did not affect LPS‐induced adherence (182). Similar to LPS, LY294002 also inhibited D3‐induced adherence in all types of transduced cells. In contrast to LPS, however, monocyte adherence induced by D3 was found to be attenuated in p110a‐deficient cells (182). Moreover, this inhibitory effect was comparable to that observed in LY294002‐treated cells. Expression of the monocyte differentiation marker CD11b was also induced by D3 in a PI 3‐kinase–dependent manner, and gene silencing using shRNA showed that p110a was also required for this effect (Fig. 12). Taken together, these findings demonstrate that LPS and D3 use distinct isoforms of PI 3‐kinase to induce functional responses and lentiviral‐mediated delivery of shRNA is a powerful approach to study the role of specific PI 3‐kinase isoforms in monocytic cells.

FIG. 12. CD11b induction by D3 is attenuated in p110a‐deficient THP‐1 cells. THP‐1 cells were either incubated or not with LY294002 (25 mM) for 30 min, followed by 100 nM D3 for 72 hr at 37  C and 5% CO2. Cells were then washed and stained with anti‐CD11b RPE‐conjugated antibody or isotype matched RPE‐conjugated control antibody. After paraformaldehyde fixation, 10,000 cells were analyzed for RPE fluorescence. For transduced cells, the analysis was restricted to GFP‐positive cells. Data were collected after compensation of GFP and RPE fluorochromes on FL1 and FL2 channels. MFI index is the ratio of (MFI of D3‐treated samples stained with anti‐ CD11b antibody–MFI of isotype‐matched control antibody)/(MFI of anti‐CD11b antibody‐ stained, untreated samples–MFI isotype‐matched control antibody). Therefore, an MFI index of 1 indicates that there was no induction of CD11b by D3, or a complete inhibition by LY294002. All of the LY294002 plus D3‐treated samples were significantly different from control cells treated with D3 alone (post‐ANOVA Tukey test, p < 0.05 for all pairs), but not from HR‐p110a3 plus D3 alone (p > 0.05). Error bars indicate S.D., n ¼ 3. Adapted from the work of Lee et al. (182).

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B. Perspectives The ability of lentiviral vectors to transduce both dividing and nondividing cells and stable expression of the transgene make this a versatile strategy for gene silencing based on siRNA. Moreover, the finding that lentiviral‐ transduced cells expressing shRNA can be further manipulated by transfection with reporter plasmids, for instance luciferase, significantly expands the utility of this approach. One important application of this technique may be in gene therapy research, such as silencing genes in myeloid leukemia cells or other difficult to transfect cells to better understand their biology and to identify potential therapeutic targets. One area of investigation in our laboratory focuses on the construction of stable siRNA‐silenced mutants of other isoforms of PI 3‐kinase such as p110b and p110
and studying their role in monocyte cell regulation. Ongoing experiments also are taking advantage of discovery‐based approaches such as gene array analysis. In modern genomics, traditional gene‐ by‐gene approaches are not adequate to meet the demand of processing information generated from mapping the complex biology of the human genome. Microarray technology is rapidly defining broad patterns of gene regulation, insight into gene function, processes, and pathways. Substantial new knowledge is being generated about transcriptional regulation and biological control mechanisms. Using gene array technology coupled to specific siRNA silencing of individual PI 3‐kinase isoforms we expect will yield substantial insight into the multiplicity of cellular events and cell regulation that are under the specific controls of these PI 3‐kinase family members. Acknowledgments The authors thank the collaborators and trainees who contributed to the studies reviewed herein. This research was supported by Canadian Institutes of Health Research (CIHR) operating grant MOP‐8633.

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