Immunol Res (2013) 56:9–19 DOI 10.1007/s12026-012-8341-3
Immune regulation by phospholipase C-b isoforms Wenbin Xiao • Yuko Kawakami • Toshiaki Kawakami
Published online: 26 May 2012 Ó Springer Science+Business Media, LLC 2012
Abstract Rapid progress has recently been made regarding how phospholipase C (PLC)-b functions downstream of G protein-coupled receptors and how PLC-b functions in the nucleus. PLC-b has also been shown to interplay with tyrosine kinase-based signaling pathways, specifically to inhibit Stat5 activation by recruiting the protein-tyrosine phosphatase SHP-1. In this regard, a new multimolecular signaling platform, named SPS complex, has been identified. The SPS complex has important regulatory roles in tumorigenesis and immune cell activation. Furthermore, a growing body of work suggests that PLC-b also participates in the differentiation and activation of immune cells that control both the innate and adaptive immune systems. Keywords Phospholipase C G protein Stat5 SHP-1 SPS complex Immune cells
Introduction PLC is a family of enzymes that catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) [1]. These second messengers are
W. Xiao (&) Department of Pathology, University Hospital Case Medical Center, Case Western Reserve University, Cleveland, OH 44106, USA e-mail:
[email protected] Y. Kawakami T. Kawakami (&) Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA e-mail:
[email protected]
positive regulators of mitogenic signaling—IP3 induces calcium release from intracellular stores and DAG can initiate activation of Ras proteins via the RasGRP family of guanine nucleotide exchange factors. Ca2? and DAG also activate protein kinase C (PKC), among other targets [1, 2]. Molecular cloning has identified 13 mammalian PLC isozymes that can be classified into six subfamilies (PLC-b, PLC-c, PLC-d, PLC-e, PLC-f and PLC-g) on the basis of their size, amino acid sequence, domain structure, and activation mechanism [3]. The regulation of PLC-b and c isoforms has been studied in more depth than that of the other subfamilies. The four isozymes of PLC-b (b1*b4) show different tissue profiles in expression and G protein regulation. PLC-b1 and PLC-b3 are expressed in a wide range of tissues and cell types, whereas PLC-b2 and PLCb4 have been found only in hematopoietic and neuronal tissues, respectively. All four isoforms are activated by the Ga subunits of the Gq class. PLC-b2 and PLC-b3 can also be activated by bc subunits of the Gai/o family of G proteins and the small GTPases such as Rac and Cdc42. In addition, PLC-bs are GTPase-activating proteins (GAPs) for the Gaq proteins that activate them. G proteins cycle from the GDP-bound inactive to the GTP-bound active state (Fig. 1). GTP-bound G proteins activate the effectors such as PLC-b. G protein-coupled receptors (GPCRs) act by catalyzing the release of GDP and binding of GTP. GAPs accelerate the deactivation process, which would otherwise be very slow. In contrast to PLC-b isoforms, PLC-c isoforms are regulated by receptor and nonreceptor tyrosine kinases. PLC-e is regulated directly by small GTPases of the Ras and Rho families, as well as subunits of G proteins [4–6]. These studies suggest a complex interplay among different families and their regulatory network. This notion is further supported by recent studies showing that PLC-b3 interacts
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Fig. 1 G protein activation cycle. GTP-bound G protein is an active form while GDP-bound G protein is inactive. Thus, the signal output of the G protein is proportional to the balance of the rates of receptorcatalyzed GDP/GTP exchange (activation) and GAP-accelerated GTP hydrolysis (deactivation). PLC-b is not only an effector of the G protein, but also the GAP (indicated by dotted line)
with tyrosine kinase signaling network [7–9]. Here we review recent progresses in the research of signaling pathways of PLC-b and the role of PLC-b in the regulation of immune cells.
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Fig. 2 Structure of PLC-b isoforms and PLC-b3/Gaq complex. a PLC-b consists of an N-terminal PH domain, four EF hands, a catalytic TIM barrel, a C2 domain, and a carboxy-terminal (CT) domain. The CT domain is not necessary for Gaq binding. Three distinct regions of PLC-b3 that interact with Gq are indicated by red numerals. b PLC-b3/Gaq complex. PLC-b3 is depicted as a ribbon cartoon with domains colored as in (a). Activated Gaq is depicted as a green surface with nucleotide-dependent switches (Sw1–Sw3) in shades of red. The X/Y linker (orange) connects the two halves of the catalytic TIM barrel, and an ordered portion of the linker occludes the active site of the lipase highlighted by the Ca2? (yellow ball) cofactor. Taken from Waldo et al. [18] with permission (Color figure online)
Structures, localizations, and functions of PLC-b Members of the PLC-b subfamily share a well-conserved core architecture consisting of an N-terminal pleckstrin homology (PH) domain, four EF hands, split X ? Y catalytic domain, C2 domain, and C-terminal (CT) domain (Fig. 2a). The catalytic domain is the most conserved domain across all PLC isozymes both structurally and functionally, which recognize and hydrolyze phosphoinositides, with a substrate preference for PIP2 over phosphatidylinositol 5-phosphate and phosphatidylinositol. PH domain Binding targets of the PH domain vary. The PLC-b1, PLCb2, and PLC-b3 isozymes do not have a high-affinity PIP2or phosphatidylinositol 3,4,5-trisphosphate-binding site [10]. However, the PH domains of PLC-b1 and PLC-b2 isozymes bind tightly to membranes composed of a neutral lipid, phosphatidylcholine, and this binding is not affected by addition of PIP2 or phosphatidylserine [11]. The PH domains of PLC-b exhibit higher affinity to Gbc subunits, which is one of the major signals for PLC-b activation except for PLC-b4 [12–14]. Furthermore, membraneassociated small GTPases such as Rac and Cdc42
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specifically activate PLC-b2 and PLC-b3 through direct interaction with the PH domains [15–17]. EF hands and C2 domain Compared to the PH domain, little has been known about the function of the EF hands and C2 domain of PLC-b. Waldo et al. recently solved the structure of a complex of Gaq and the globular core of PLC-b3 and provided new insights into the function of the EF hands and C2 domains of PLC-bs [18]. The PLC-bs used in this study are composed of two pieces—the core and a long, C-terminal coiled-coil domain. The core is composed of a PH domain, four EF hands, a triosephosphate isomerase (TIM) barrel that includes the catalytic domain, and a C2 domain. PLCb3 binds to Gaq over a large contact area through three separate segments: a loop at the N terminus of the C2 domain, a loop off the EF hands, and an additional helixturn-helix extension at the C terminus of the C2 domain (Fig. 2b). The contact sites at both ends of the C2 domain are required for activation of PLC-b3 by Gaq; the loop off the EF hands mediates Gq GAP activity. When the helixturn-helix segment of the C2 domain from PLC-b3 was appended to the C terminus of PLC-c1, which is not
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regulated by Gaq, the resulting chimera was stimulated about twofold in response to Gq and a Gq-coupled receptor, suggesting that all of the mammalian PIP2-specific PLCs can be activated in fundamentally the same way. On the other hand, Waldo et al. noted that asparagine residue (N260) on a loop off one of the EF hands contacts Gaq in a way reminiscent of the contact of Ga subunits with RGS proteins, a class of G protein-activating proteins (GAPs). Mutation of this asparagine on PLC-b3 blocks Gq GAP activity [18]. Since there is no sequence similarity between the PLC-bs and RGS proteins, it seems that the asparagine is all that matters in the flexible loops on the surface of the effector [19]. C-terminal domain The most unique structural feature of PLC-b is a C-terminal long tail (residues 846–1234 in human PLC-b3). Interestingly, each PLC-b isoform has an alternative splicing variant with a truncated C-terminal region [3]. The CT domain can bind a Ga subunit, an activator of PLC-b enzymes. The structure of this domain from PLC-b has been solved, revealing a novel fold composed almost entirely of three long helices forming a coiled-coil [17, 20]. This structure contains elements involved in dimerization and an electrostatically positive surface proposed to be the major site of interaction with Gaq. In spite of the conserved overall structure, sequence alignments indicate that each C-terminal long tail has relatively low homology compared with other domains. This explains why each PLC-b isoform shows a different binding affinity to Ga subunits as well as Gbc subunits [21, 22]. Results from many reports have implicated that the CT domain is important for Gaq-induced PLC-b activation. Removal or mutation of the CT domain blocks stimulation of PLC-b by Gaq in cells and in vitro assays [23–25], and the isolated CT domain both displays some Gq GAP activity and inhibits PLC-b activation by Gq [26, 27]. However, it had been unclear how Gaq enhances PLC-b activity and how CT domain contributes to its regulation. Two recent studies shed new light on these mysteries [18, 28]. Waldo et al. showed that PLC-b core (PLC-b3 D887, lacking residues 887–1234) that includes a very small portion of CT domain (residues 846 to 886) still responds to Gaq and retains Gaq GAP activity. Moreover, the affinity of this truncated PLC-b3 for Gaq is similar to that of the full-length protein. Structurally, this small portion of CT domain consisting of residues 848–882 in human PLCb3 forms a helix-turn-helix motif (Ha1-Ha2) that docks with the effector-binding site of Ga. By analyzing the crystal structures of cephalopod PLC21, Lyon et al. identified a Ha20 helix that corresponds to the C-terminal end of the PLC-b3 Ha2 helix and interacts with a conserved
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surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLC-b3 significantly increased basal activity and diminish stimulation by Gaq. It was expected that Gaq-GTP binding to the Ha1-Ha2 motif displaced the Ha20 helix from the catalytic core, leading to enhanced PIP2 hydrolysis. In addition, PLC-b3 full length had tenfold higher basalspecific activity and 40-fold higher affinity to Gaq than PLC-b3 D892, confirming that more distal regions of the CT domain (residues 892–1234) strongly contribute to high-affinity Gaq binding and to basal and Gaq-stimulated activity. PLC-b3 is known to be phosphorylated by stimulation of GPCRs. Ser-1105 of PLC-b3 can be phosphorylated by PKC [29] or protein kinase A (PKA) [30]. Activation of PKC can result from stimulation of the GPCR-G proteinPLC-b pathway. PKC, but not PKA, mediates GPCRsinduced phosphorylation of PLC-b3 [31]. PKC-mediated phosphorylation of PLC-b3 diminishes the interaction of Gaq/11 with PLC-b3. This mechanism represents one of the several ways to terminate the activity of PLC-b3, whose activation by GPCR stimulation is typically rapid and transient. Other proposed mechanisms for PLC-b deactivation include the phosphorylation and internalization of activated GPCRs [32] and GTPase-mediated inactivation of Ga proteins [33–35]. All PLC-b isoforms have consensus sequences known as PDZ domain-binding motifs, which include an –X(S/T)X(V/L)-COOH sequence at the extreme C-terminal end [36]. PDZ domains exist in a large number of multifunctional proteins that mediate protein–protein interactions at highly specialized submembranous sites, such as the postsynaptic density and cellular junctions [37]. An early study showed that the PDZ-binding motif in PLC-b subfamily binds to Par3 and Par6, two PDZ proteins participating symmetric cell division [38]. More recently, Par3 was shown to bind the bradykinin receptor B2R and PLC-b1, and another PDZ domain-containing protein NHERF2 (Na?/H? exchanger regulatory factor 2) to bind the lysophosphatidic acid receptor LPA2 and PLC-b3 [39]. Therefore, these results suggest that each isoform of PLC-b is selectively coupled to GPCR via PDZ scaffold proteins [40]. PLC-b3 CT as an adaptor We recently uncovered a tumor suppressor function of PLC-b3 [8]. Mice deficient in PLC-b3 developed a variety of neoplastic diseases. Specifically, the majority of older mice developed a myeloproliferative neoplasm (MPN), a hematopoietic malignancy that is frequently caused by mutations that activate signaling molecules such as BCRABL, N-Ras, SHP-2, and JAK-2 [41, 42]. Interestingly, a
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minor proportion of MPN patients has reduced PLC-b3 expression regardless of Jak2 V617F status (our unpublished data). PLC-b3 also plays an important role in the pathogenesis of lymphoid leukemia and lymphoma, as PLC-b3-deficient mice developed lymphoma. Loss of PLC-b3 cooperates with deregulated c-Myc expression in a classic model of B lineage lymphoma [43]. Consistent with this, two of six human Burkitt’s lymphoma cell lines tested showed low levels of PLC-b3. Furthermore, a subset of human chronic lymphocytic leukemia samples showed low PLC-b3 expression. Deletion of regions of chromosome 11 including PLC-b3 locus has been reported in patients with acute lymphoblastic leukemia [44]. In an attempt to reveal the biochemical mechanism of the tumor suppressor activity of PLC-b3, we unexpectedly found that PLC-b3 functions as an adaptor rather than an enzyme. PLC-b3 can recruit Stat5 and SHP-1 to a multimolecular complex termed SPS complex, and within this
complex, SHP-1 dephosphorylates Stat5 and thus inhibits Stat5 activity (Fig. 3). Interestingly, this adaptor function resides in the CT domain. This growth-inhibitory activity of PLC-b3, functioning as a signaling scaffold by its C-terminal domain, is required to prevent hematopoietic neoplasia. More recently, we finely mapped the Stat5- and SHP-1binding sites within PLC-b3-CT (positions 809–1234) and found that three noncontiguous regions, designated a (positions 917–943), b (positions 983–1000), and c (positions 1032–1069), bound to Stat5 and two regions, designated c (positions 1032–1069) and d (positions 1182–1209) bound to SHP-1 [45]. Because region b peptide exhibited 80 % potency of PLC-b3-CT in inhibiting the growth of Ba/F3 cells, we tested even shorter peptides in this region. Among them, an 11-residue peptide (peptide b11; positions 988–998) exhibited a growth-inhibitory activity as strong as [80 % of that of PLC-b3-CT. Stat5 phosphorylation at
Fig. 3 Signaling Platform for SHP-1 activity. a SHP-1 is phosphorylated at Tyr564 by Lyn and at Tyr536 by Lyn and other kinase(s) including Jak2. Inactive SHP-1 (Top left) has a closed conformation with the catalytic site of the PTP domain capped by the N-SH2 domain [81]. The Tyr536 phosphorylation site of SHP-1 interacts with Stat5 most probably via the SH2 domain of the latter molecule (Bottom). The Tyr564 phosphorylation site of SHP-1
interacts with N-SH2 to render the enzyme active. N-terminal ends of SHP-1 and Stat5 proteins are shown by ‘‘N’’. b SHP-1 and Lyn interact with PLC-b3-CT [7, 8]. Tyr694 phosphorylated Stat5 is recruited to the PLC-b3-nucleated SPS complex containing Lyn and SHP-1, and dephosphorylated by an active SHP-1 enzyme that is phosphorylated at Tyr536 and Tyr564. Stat5 dimer is depicted as a monomer for simplicity
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Tyr694 was significantly reduced in peptide b11-expressing cells. The relevance of these in vitro studies was verified by reduced myeloid colony-forming activity of the peptide b11-expressing cells and by the failure of peptide b11expressing lin- bone marrow cells derived from PLCb3-/- mice to cause MPN development upon transplantation [45]. In search of additional components in the SPS complex, we found that Lyn is another component in the SPS complex [7]. Lyn is required to phosphorylate SHP-1 at both tyrosine residue 536 and 564. Phosphorylation of Tyr564 is critically important for SHP-1 phosphatase activity. On the other hand, phosphorylation of Tyr536 facilitates the interaction between SHP-1 and its substrate, that is Stat5. Therefore, Lyn-dependent phosphorylation on both Tyr536 and Tyr564 maximizes SHP-1 activity toward its substrate. In this process, PLC-b3 is indispensable for the organization of the multimolecular complex. Intranuclear signaling of PLC-bs Nuclear localization of PLC-bs has also been reported [46]. For example, PLC-b1 has been shown to be located in nuclear speckles, suggesting that PLC-b1 signaling occurs not only at or beneath the plasma membrane but also in the nucleus. Interestingly, the CT region of the protein is required for its nuclear localization. A recent report showed that PKC-mediated phosphorylation of PLC-b1 at Ser887 might regulate the level of PLC-b1 in the cytosol and nuclei [47]. The significance of nuclear PLC-b1 has been investigated by Cocco and his colleagues. Overexpression of nuclear PLC-b1 in a murine erythroleukemic cell line increased CD24 expression, and knocking down of PLC-b1 reduced it, suggesting that nuclear PLC-b1 constitutes a key step in erythroid differentiation in vitro [48]. This group further extended the study to hematological malignancies, focusing particularly on patients with myelodysplastic syndromes (MDS) at high risk of evolution into acute myeloid leukemia. Their results showed that monoallelic cryptic deletion of the PLC-b1 gene is associated with an increased risk of developing acute myeloid leukemia and a worse clinical outcome, as compared with patients having both alleles [49]. Moreover, the expression profile of PLC-b1a and PLC-b1b mRNAs, which are the two alternative splicing subtypes of PLC-b1, is altered in high-risk MDS, as compared with healthy donors. Interestingly, MDS cells always expressed higher levels of PLC-b1b mRNA as compared with PLC-b1a mRNA; this difference may reflect a specific role of PLC-b1b in MDS, given that the PLC-b1a splicing subtype demonstrates both nuclear and cytoplasmatic localization, while PLC-b1b splicing subtype is localized only in the nucleus [50]. An increased nuclear PLC-b1 expression is also correlated
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with an almost complete remission in patients with MDS after azacitidine treatment [51]. The results demonstrate not only that PLC-b1 promoter is hypermethylated in highrisk MDS patients, but also that the amount of PLC-b1 mRNA could predict the clinical response to azacitidine, therefore indicating a promising new therapeutic approach. However, the detailed molecular mechanisms remain unclear.
PLC-b in myeloid cells Macrophages Macrophages are innate immune cells with well-established roles not only in the primary response to pathogens, but also in tissue homeostasis, coordination of the adaptive immune response, inflammation, resolution, and repair [52]. Macrophages are heterogeneous populations due to different mechanisms governing their differentiation, tissue distribution, and responsiveness to stimuli. PLC-bs regulate both the function and differentiation of macrophages. Activated macrophages are classified into two groups: classically activated (M1) and alternatively activated (M2) macrophages. M1 and M2 macrophages differ in terms of receptors, cytokine and chemokine expression, and effector functions. M1 macrophages are anti-inflammatory, but M2 macrophages are pro-inflammatory, but immunomodulatory and angiogenic [53]. Toll-like receptor (TLR) 4 agonist LPS, together with adenosine A2A receptor (A2AR) agonists, downregulates expression of tumor necrosis factor a and interleukin-12, and upregulates expression of vascular endothelial growth factor (VEGF) and interleukin10, therefore switching macrophages from M1 to M2 phenotype [54]. Interestingly, this switching is accompanied by a rapid and specific posttranscriptional downregulation of PLC-b1 and PLC-b2 expression due to destabilization of their mRNAs. LPS also suppresses PLCb1 and -b2 expression in vivo in a MyD88-dependent manner. Further investigation revealed that expression of M2 markers such as VEGF is increased in macrophages treated with PLC-b inhibitors, or a small-interfering RNA specific to PLC-b2 but not PLC-b1, and in macrophages from PLC-b2-/- mice, arguing that suppression of PLC-b2 plays an important role in switching M1 macrophages into an M2 state. In line with this, the number of alveolar macrophages that express Ym-1 and arginase I, markers of M2 macrophages, is increased in PLC-b3-/- mice [7], suggesting that PLC-b3, similar to PLC-b2, might inhibit the polarization of macrophages into M2 phenotype. Atherosclerosis is an inflammatory disease that is associated with monocyte recruitment and subsequent differentiation into lipid-laden macrophages at sites of arterial
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lesions, leading to the development of atherosclerotic plaques [55]. PLC-b is a key member of signaling pathways initiated by GPCR ligands in macrophages. Wu’s group studied macrophages from mice lacking PLC-b2, PLC-b3, or both and found that PLC-b3 is the major functional PLC-b isoform in murine macrophages [56]. PLC-b3 deficiency did not affect macrophage migration, adhesion, or phagocytosis, suggesting a compensatory role of other PLC-b members. However, PLC-b3 deficiency rendered macrophages highly sensitive to multiple inducers of apoptosis. PLC-b3 appeared to regulate this sensitivity via PKC-dependent upregulation of Bcl-XL, an antiapoptotic member of the Bcl-2 family. The significance of PLC-b signaling in vivo was examined using the apoE-deficient mouse model of atherosclerosis. Mice lacking both PLC-b3 and apoE exhibited fewer total macrophages and increased macrophage apoptosis in atherosclerotic lesions, as well as reduced atherosclerotic lesion size when compared with mice lacking only apoE. These results demonstrate a novel role for PLC-b3 activity in promoting macrophage survival in atherosclerotic plaques and identify PLC-b3 as a potential target for the treatment of atherosclerosis. The role of PLC-b in atherosclerosis was further investigated in two mouse models with hyperlipidemia and genetically imposed hypercoagulability, that is, TMPro/Pro ApoE2/2 and FVLQ/Q ApoE2/2 mice [57]. In both models, hypercoagulability resulted in larger plaques, but vascular stenosis was not enhanced secondary to positive vascular remodeling. Plaque stability was increased in hypercoagulable mice with less necrotic cores, more extracellular matrix, more smooth muscle cells, and fewer macrophages. The reduced frequency of intraplaque macrophages in hypercoagulable mice is due to thrombin-induced inhibitory effect on monocyte transendothelial migration. In this context, thrombin/protease-activated receptor-1-mediated signaling in macrophages is blocked by PLC-b inhibitor U73122, suggesting that PLC-b might have a protective role against atherosclerosis under hypercoagulable status.
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response to the CC chemokine MIP-1a both in vitro and in vivo [59], suggesting that PLC-b rather has a negative regulatory role in neutrophil functions under certain circumstances. PLC-b might also control neutrophil production. A recent study investigated genetic variations that regulate neutrophil count by performing a genome-wide association study in Japanese subjects [60]. Two genetic loci were identified as significantly associated with neutrophil count (rs4794822 in PSMD3–CSF3 and rs2072910 in PLC-B4). These loci are perhaps specifically associated with neutrophil count as no association with the counts of the other subtypes of WBCs (lymphocytes, monocytes, eosinophils, and basophils) is present. The subjects who were homozygous for ‘‘neutrophil-increasing alleles’’ in both of the SNPs (T alleles for rs4794822 and rs2072910) had 1.17fold higher neutrophil count when compared with the subjects homozygous for ‘‘neutrophil-decreasing alleles’’ (C alleles for rs4794822 and rs2072910). Therefore, both PSMD3–CSF3 and PLC-b4 might regulate neutrophil production. Recently, we reported that aged PLC-b3-/- mice developed MPN with increased mature neutrophils. This is unique to PLC-b3-/- mice as PLC-b2-/- mice do not develop MPN. Aged PLC-b3-/- mice typically have increased numbers of hematopoietic stem cells (HSCs) and myeloid progenitors in the bone marrow and spleens. Furthermore, PLC-b3-/- HSCs and myeloid progenitors have an increased predisposition to differentiate into granulocytes and are hypersensitive to cytokines such as GM-CSF and IL-3, a hallmark of human MPNs. In vivo BrdU incorporation, in vitro cultures, and cell cycle analysis of HSCs demonstrate increased proliferation in PLC-b3-/- HSC-enriched populations. Apoptosis is less abundant in PLC-b3-/- HSCs. These abnormal HSCs can give rise to MPN upon transferring into lethally irradiated wt mice, which is largely Stat5 dependent. Therefore, PLC-b3 regulates neutrophil differentiation and production at the level of HSCs via Stat5 inhibition [8].
Neutrophils Mast cells A pioneer study from Wu’s group reported that chemokineinduced IP3 production and Ca2? signaling were reduced in PLC-b2-/- neutrophils [58] and were abrogated in PLCb2;PLC-b3 double knockout (dko) neutrophils, supporting the conclusion that PLC-b2 and PLC-b3 are the sole PLC isoforms that are activated by chemoattractants in mouse neutrophils [59]. Related to this, PKC phosphorylation and associated p47phox translocation and JNK phosphorylation were not detected in fMLP-stimulated dko neutrophils. Thus, the PLC-b pathway is essential in chemoattractantinduced neutrophil activation. On the other hand, the dko neutrophils showed enhanced chemotactic activities in
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Mast cells are the major effector cells in immediate hypersensitivity and chronic allergic reactions, and contribute to asthma and other allergic diseases. Cross-linking of the FceRI (high-affinity IgE receptors) on mast cells activates multiple signaling pathways that lead to degranulation, de novo synthesis of arachidonic acid metabolites, and production of various cytokines and chemokines [61]. IgE/antigen-mediated mast cell activation via FceRI can be markedly enhanced by the activation of other receptors expressed on mast cells, and these receptors may thus contribute to the allergic response in vivo. One such
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receptor family is the GPCRs [62]. For example, prostaglandin (PG) E2, adenosine, sphingosine 1-phosphate, MIP-1a (CCL3), and RANTES (CCL5) can augment antigen-mediated degranulation in mouse mast cells. This enhancement, which is the most robust by PGE2, and the ability of PGE2 to amplify antigen-induced calcium mobilization, was linked to a pertussis toxin-sensitive synergistic membrane translocation of PLC-c and PLC-b (i.e., b2 and b3) and tyrosine phosphorylation of PLC-c1 and PLC-c2 [63]. This ‘‘trans-synergistic’’ activation of PLC-b and PLC-c, in turn, enhanced the production of IP3, store-operated calcium entry, and activation of PKC-a and PKC-b. These responses were critical for the promotion of degranulation. Therefore, GPCR-linked PLC-b can activate mast cells synergistically with FceRI-linked PLC-c. In contrast, adenosine- and FceRI-mediated signals are integrated at PI3Kc, but not PLC [64]. Like murine macrophages [56], PLC-b3 appears to be the major functional isoform in mast cells as well [9]. In a passive cutaneous anaphylaxis model, intradermal injection of antigen induced rapid and robust increase in vascular permeability and skin edema in IgE-sensitized mice. This acute reaction is dependent on histamine and serotonin released from activated mast cells [65]. PLC-b3-/- and wt mice showed similar acute responses, which is consistent with the normal degranulation of PLC-b3-/- mast cells. However, late-phase anaphylactic reaction is cytokine (especially TNFa and IL-33)-dependent [66, 67]. PLCb3-/- mice have blunted FceRI-dependent late-phase anaphylactic responses. Consistent with this, FceRI stimulation of PLC-b3-/- mast cells exhibited substantially reduced cytokine production. In a mast cell-dependent airway inflammation model, PLC-b3-/- mice showed significantly reduced response. Therefore, PLC-b3 is indispensible for Ag ? IgE-induced cytokine production in mast cells and thus is important for mast cell-mediated late-phase anaphylaxis and airway inflammation. Mechanistically, reduced cytokine production in PLCb3-/- cells could be accounted for by increased activity of the negative regulator Lyn and reduced activities of the positive regulatory MAPKs. Furthermore, PLC-b3 constitutively interacts with Lyn and SHP-1, and these interactions were increased by FceRI stimulation. SHP-1 likely recognizes its substrates Lyn and MAPKs via the recently described kinase tyrosine-based inhibitory motif, KTIM [68, 69]. Consistent with PLC-b3/SHP-1-mediated repression of Lyn kinase activity by dephosphorylation of Lyn-Tyr396, FceRI-mediated phenotypes were similar in PLC-b3-/- and SHP-1-mutant mast cells. Interestingly, PLC-b3 was shown to bind to FceRI by immunoprecipitation and pull-down assay, suggesting that PLC-b3, along with Lyn and SHP-1, regulates early signaling events of mast cell activation. Parenthetically, tyrosine
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phosphorylation of PLC-c2 was not significantly affected by PLC-b3 deficiency, despite the expectation based on the study of Kuehn et al. [63]. Further, PLC-b2 deficiency did not affect the FceRI phenotype in BMMCs. Thus, PLC-b3 plays a unique role in FceRI signaling probably because of its ability to interact with Lyn and SHP-1. Platelets In an attempt to understand the role of PLC-b in platelets, Abrams’s group found PLC-b2/b3 dko mice were unable to form stable thrombi in a carotid injury model [70]. This could be explained by the fact that PLC-b2/b3 dko platelets failed to assemble filamentous actin, had defects in both secretion and mobilization of intracellular calcium, and were unable to form stable aggregates following low doses of agonists. Remarkably, dko platelets spread more slowly upon fibrinogen. These results suggest that both PLC-b2 and PLC-b3 play vital roles in platelet cytoskeletal dynamics.
PLC-b in lymphocytes T cells T cell receptor (TCR) recognition of peptide antigen presented by antigen-presenting cells via MHC class II or MHC class I molecules and subsequent T cell activation are essential initial processes of adaptive immunity. Following the antigen recognition by TCR, Lck, a Src family tyrosine kinase associated with coreceptors CD4 and CD8, is activated, which is one of the earliest activation events. Lck phosphorylates the immunoreceptor tyrosine-based activation motif (ITAMs) on the e, d, c, and f subunits of the TCR/CD3 complex, generating the sites for recruitment of ZAP-70, a Syk family tyrosine kinase. ZAP-70 phosphorylates tyrosine residues in the cytoplasmic portion of the transmembrane adaptor LAT. LAT acts as a scaffold for the assembly of signaling complexes. These ‘‘signalosomes’’ trigger several downstream pathways that eventually activate transcription factors, leading to gene transcription characteristic of activated T cells. In this activation scheme, PLC-c1 is critical for the generation of IP3 and DAG and Ca2? mobilization [71], but PLC-b subfamily, specifically, b2 and b3, appears to be nonessential ([72] and our unpublished data). Unlike this conventional TCR signaling, bacterial superantigens (SAg) can activate an alternative pathway that does require TCR but does not involve Lck, ZAP-70 or PLC-c1. Using a set of SAg derived from Staphylococcus and Streptococcus bacteria, Madrenas’ group showed that neither CD4 nor Lck is required for SAg-induced T cell
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activation [73]. PLC-b inhibitor U73122 abrogated SAginduced T cell activation. Furthermore, introducing a PLCb1-specific siRNA had similar effect, albeit to a lesser degree, demonstrating that PLC-b1 (and probably other PLC-b isoforms as well) is part of the alternative signaling pathway activated by SAg. Consistent with the view that PLC-b family is downstream of G proteins, a dominant negative Ga11 mutant, but not pertussis toxin (Gai inhibitor), inhibited SAg-induced T cell activation. Therefore, the triggering of this Ga11-PLC-b-dependent alternative pathway by SAg suggests that these toxins use a GPCR as a coreceptor on T cells. Chemokines acting through GPCRs play an essential role in the immune response, as lymphocyte traffic is a key element in immune surveillance. PLC-b has been studied in the regulation of chemokine-mediated T cell migration [72]. Abrams’s group demonstrated that loss of PLC-b2 and PLC-b3 significantly impaired T cell migration. T cell migration induced by stromal cellderived factor-1a (SDF-1a = CXCL12), the sole ligand of CXCR4, was inhibited by chelation of Ca2?. Ca2? influx induced by SDF-1a was undetectable in PLC-b2/b3 dko lymphocytes, suggesting that the migration defect is due to the impaired ability to increase intracellular Ca2?. This study, together with another study showing that human T cell migration through a chemokine receptor CXCR3 is sensitive to PLC inhibitor [74], demonstrates that, in contrast to neutrophils, phospholipid second messengers generated by PLC-b play a critical role in T lymphocyte chemotaxis. Upon SDF-1a binding to CXCR4, CXCR4 heterodimerizes with the TCR [75]. The CXCR4-TCR heterodimer stimulates increased intracellular Ca2? concentrations, prolonged ERK activation, gene transcription, and cytokine production. These responses occur via pathways that use several traditional TCR signaling molecules including ZAP-70 and SLP-76 as well as Gi-type G proteins [76]. Furthermore, in Jurkat T cells, SDF-1a signaling via the CXCR4-TCR heterodimer uses PLC-b3 to activate the RasERK pathway and increase intracellular Ca2? concentrations, whereas PLC-c1 is dispensable for these events. In contrast, PLC-c1, but not PLC-b3, is required for SDF-1amediated migration via a mechanism independent of LAT [77]. These results characterize new roles for PLC-b3 and PLC-c1 in T cells, and suggest that multiple PLCs may also be activated downstream of chemokine receptors to distinctly regulate migration versus other signaling functions. The lack of effects of PLC-b3 in SDF-1a-mediated migration of Jurkat T cells might be explained by the redundancy of other PLC-b members, probably PLC-b2. Human T cells express PLC-b and -c isoforms [78]. Interestingly, PLC-b2 expression is reduced in T cells from
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elderly people, suggesting that an impaired expression of PLC-b2 in aged T lymphocytes might explain the agerelated defect of PLC activity and contribute to immune suppression in this group of people [79]. B cells Antibody production by B cells is regulated by PLC-b subfamily. Mice lacking both PLC-b2 and PLC-b3 consistently produced larger amounts of antigen-specific antibodies composed of immunoglobulin k light chain (TI-Igk) than did wild-type mice when immunized with T cellindependent (TI) antigen hydroxylnitrophenyl (NP)-Ficoll. Enhancement in TI-Igk production appeared to be primarily dependent on PLC-b3 deficiency. PLC-b2 or PLCb3 deficiency did not affect the production of TI-Igj or of T cell-dependent antigen NP-chicken gamma globulin (NP-CCG)-specific antibodies composed of either k or j light chains. Together, these data suggest that the production of TI-Igk may be subjected to regulation by G proteinmediated signaling pathways [59]. A recent study showed that the Igk locus of B cell progenitors in IL-7-rich environment is epigenetically repressed by Stat5 tetramermediated recruitment of the histone methyltransferase Ezh2 [80]. Thus, if Stat5 is high in PLC-b3-/- (or dko) B cell progenitors as expected from our study [8], Igj production might be suppressed relative to Igk production. When these mice become aged, both the Th1- and Th2associated antibodies are increased in their serum, suggesting that PLC-b3 is a brake in antibody production (our unpublished data).
Conclusion It is clear now that PLC-bs are crucial players in immune regulation. However, much remains to be learned (Box 1). Although we have an improved understanding of the molecular details of enzymatic regulation of PLC-bs, several lines of evidence suggest that more complicated signaling mechanisms are to be uncovered. The newly identified function of PLC-b3 CT domain and its resulting SPS multimolecular signaling platform shed new insights into the complexity of PLC-b molecules. The interplay between GPCR-mediated signaling pathways and tyrosine kinase-based pathways converging at the levels of PLC-bs probably has more pathophysiological significance than previously thought. Therefore, elucidation of these mysteries will be important to help us understand the complexity of, and combat, immune-mediated diseases and malignancies.
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Box 1 Outstanding questions in the study of PLC-b in immunity Here we list specific immunologic questions that can be experimentally testable: How does TLR4 regulate PLC-b1 and –b2 expression? How do PLC-b2 and –b3 contribute to M2 macrophage conversion? Is M2 macrophage differentiation in the absence of PLC-b3 dependent on Stat5? Is skewed M2 macrophage differentiation in the absence of PLC-b3 dependent on Stat5? Does PLC-b3 regulated TLR-mediated DC activation? Is mast cell versus basophil differentiation regulated by PLC-b3? Is Stat5 activity under the control of SPS complex in signaling pathways other than those for the common b cytokines (e.g., IL-3, GM-CSF) and FceRI? Given that Stat5 activity is necessary for Th2 and Treg differentiation, does PLC-b3 regulate T cell differentiation? Is Igj versus Igk production regulated by Stat5-mediated epigenetic control downstream of PLC-b3?
Acknowledgments We thank Dr. Tohru Kozasa, University of Tokyo, for his critical reading of this manuscript. The study in the Kawakami laboratory is funded by the National Institutes of Health and the MPN Foundation. This study is publication No. 1515 from the La Jolla Institute for Allergy and Immunology.
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