- Email: [email protected]
Protein kinase C epsilon: a new target to control inflammation and immune-mediated disorders
The International Journal of Biochemistry & Cell Biology 36 (2004) 183–188
Molecules in focus
Protein kinase C epsilon: a new target to control inflammation and immune-mediated disorders Ezra Aksoy, Michel Goldman∗ , Fabienne Willems Laboratory of Experimental Immunology, Université Libre de Bruxelles, B-1070 Brussels, Belgium Received 23 December 2002; received in revised form 16 May 2003; accepted 16 May 2003
Abstract Recent advances in understanding the molecular basis for mammalian host immune responses to microbial invasion suggest that the first line of defense against microbes is the recognition of pathogen-associated molecular patterns by a set of germline-encoded receptors: the Toll-like receptors (TLRs). TLRs have been identified as being part of a large family of pathogen-recognition receptors that play a decisive role in the induction of both innate and adaptive immunity. Indeed, activation of T lymphocytes depends on their interaction with dendritic cells previously stimulated by TLR agonists such as bacterial lipopolysaccharide (LPS), a TLR-4 ligand. A novel PKC epsilon (ε) was recently found to be a critical component of TLR-4 signaling pathway and thereby to play a key role in macrophage and dendritic cell (DC) activation in response to LPS. Thus, controlling the kinase activity of PKC ε might represent an efficient strategy to prevent or treat certain inflammatory disorders of microbial origin. © 2003 Elsevier Ltd. All rights reserved. Keywords: Protein kinase C epsilon; Innate immunity; IL-12; TH 1
1. Introduction Protein kinase C (PKC) is a phospholipid-dependent serine/threonine kinase family, consisting of at least 11 isoforms that exhibit related homologies in their structures. The PKC isoforms can be classified into three main subfamilies based on their homology and cofactor requirements for activation: (1) conventional Abbreviations: ERK, Extracellular receptor-activated kinase; IP-10, IFN-␥ inducible protein 10; IRF, Interferon response factor; ISG15, IRF-stimulated gene 15; JNK, c Jun-N-terminal kinase; PGN, Peptidoglycan; TAK, Transforming growth factor--activated kinase; TAB, TAK binding protein; TIR, Toll-IL-1 receptor region; TICAM-1, TIR-containing adaptor molecule ∗ Corresponding author. Fax: +32-2-555-44-99. E-mail address: [email protected] (M. Goldman).
(cPKCs) ␣, I, II and ␥ that require negatively charged phosholipids, diacylglycerol (DAG) or phorbol ester and calcium; (2) novel (nPKCs) ␦, ε, , /L (mouse/human) that do not require calcium; and (3) atypical (aPKCs) / (mouse/human) and that only require negatively charged phosholipids, and the PKC D/ (murine/human) that form a distinct class and are more related to calmodulin-dependent kinase (Mellor & Parker, 1998). Differences between the PKC isoforms for their substrate specificity suggest that a particular PKC isoform within a single cell may regulate specific physiological responses. The mammalian Toll-like receptor (TLR) recognition system for pathogens is the first line of anti-microbial defense (Medzhitov, 2001; Takeda & Akira, 2003). The TLRs recognize molecular patterns
1357-2725/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1357-2725(03)00210-3
184
E. Aksoy et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 183–188
that are characteristic of particular classes of microorganism, such as lipopolysaccharide (LPS; Gram-negative bacteria), double-stranded RNA (viruses) and mannan (fungi). LPS, a major integral component from the outer membrane of gram negative bacteria, is a prototypical pathogen-associated molecular pattern (PAMP) which was demonstrated to induce dendritic cell (DC) or macrophage activation through TLR-4 triggering (Kaisho & Akira, 2001). TLR-4-mediated signaling events has two major consequences in DC: (1) production of several cytokines including interleukin (IL)-12 which promotes the differentiation of CD4+ T lymphocytes into TH 1 inflammatory effector cells, and (2) upregulation of cell surface molecules including major histocompatibility complex (MHC) and co-stimulatory molecules (Fig. 2) (Trinchieri, 2003). Multiple signaling molecules are initiated upon LPS binding to TLR-4, predominantly leading to nuclear factor (NF)-B and mitogen-activated protein (MAP) kinases activation. PKC isoforms actively participate in several TLR signaling pathways. LPS inducible IRAK activation/degradation has been demonstrated to require a functional TLR-4 that can be inhibited by a protein kinase C inhibitor, calphostin (Hu, Jacinto, McCall, & Li, 2002). There is evidence that LPS stimulation of macrophages or DCs results in PKC activation (Aksoy, Amraoui, Goriely, Goldman, & Willems, 2002; Castrillo et al., 2001; Shapira et al., 1997). Furthermore, macrophages from the C3H/HeJ mouse strain which carries a point mutation in the intro-cytoplasmic domain of TLR-4 receptor was found to exhibit deficient translocation of novel and conventional PKC in response to LPS (Shinji, Akagawa, & Yoshida, 1994). Herein, we review the structure and regulation of PKC isoforms and further discuss the mechanisms by which PKC ε participates in inflammatory responses.
2. Structure and regulation of PKC The classification of PKC isoforms, based on their enzymatic properties, is best understood by comparing the structural homologies of these isoforms at the protein-sequence level. Protein-sequence alignments between PKC isoforms reveal the existence
of homologous domains conserved between family members (Newton, 1997). The structure of PKC isoforms includes conserved domains referred as C1–C4 (Fig. 1). Variable regions (V1–V5), whose functions still under study, interrupt the conserved domains. Two cysteine-rich sequences, which are found at the N-terminus of C1 regions of cPKCs and nPKCs, are necessary for DAG and phorbol ester binding. The C2 region in cPKCs is important for Ca2+ binding and is absent in nPKCs and aPKCs. Activation and translocation of the PKC isoforms to specific subcellular sites is achieved by the binding of activated PKC to distinct anchoring proteins. Receptor for activated C kinases (RACKs) are an example of these anchoring proteins. Binding of RACK to PKC isoforms is selective and there may be more than one RACK for each isoform. For cPKC, the PKC–RACK interaction occurs via C2-domain whereas the C2-like region is involved in the interaction of nPKCs. Mochly-Rosen and Gordon demonstrated that peptide fragments corresponding to C2 domain (mapped to the amino acids 186–198, 209–216) specifically inhibit activation-induced translocation of cPKCs but not the C2-less nPKCs (Mochly-Rosen & Gordon, 1998). Furthermore, they found that the binding of the nPKC to its RACKs occurs via the V1 domain that is homologous to the C2 domain of cPKC. Peptides containing the V1 domain of novel PKC ε or PKC ␦ (amino acids 1–142) selectively inhibit their translocation and thus their activation (Mochly-Rosen & Gordon, 1998). The RACK binding site on PKC ε has been further mapped to amino acids 14–21 and peptides corresponding to this region selectively inhibit PKC ε translocation. The C-terminal regions, C3–V5, are recognized in all PKC isoforms and represent the catalytic domain (Fig. 1). The C3 region contains the ATP-binding sequence and only PKC has a slightly different ATP-binding site. It is noteworthy to point out that ATP-binding site inhibitors are the most potent drugs developed to inhibit PKC function. The C4 region contains the pseudo-substrate-binding site, which is important for the enzyme’s interaction with its substrates. Synthetic peptide analogues, which correspond to the pseudo-substrate site, are demonstrated to act as potent inhibitors of PKC isoforms (Mochly-Rosen & Gordon, 1998).
E. Aksoy et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 183–188
185
Fig. 1. Schematic representation of the primary structures of protein kinase C isoforms. The PKC structure can be divided into an N-terminal (N) regulatory domain and C-terminal (C) catalytic domain. Indicated are the pseudo-substrate (red), the C1 domain which binds diacylgycerol and phorbol esters (containing the cysteine-rich motif), the C2 domain which binds to acidic lipids and for cPKCs, Ca2+ binding (orange), and the C3 and C4 domains that comprise the ATP- and substrate-binding regions of the kinase (blue). Peptide inhibitors of PKC translocation target the C2 region of cPKC and V1 region of nPKC. Members of the each isoform subfamily are listed on the left. N, amino terminus; C, carboxyl terminus. The figure is a modification from Mochly-Rosen and Gordon (1998).
3. Biological function of PKC in the immune system TLR signal transduction network is an active area of investigation. The cytoplasmic domains of the TLRs and members of the interleukin-1 receptor (IL-1R) family contain a conserved signal-transduction module—the Toll-interleukin 1 receptor (TIR) domainthrough which TIR-containing adaptor molecules recruit the putative serine/threonine kinase, IL-1Rassociated kinase (IRAK) (Fig. 2). Subsequently, tumor-necrosis factor receptor (TNFR)-associated factor 6 (TRAF-6) is activated, triggering the NF-B and mitogen-activated protein kinase (MAPK) pathways, which leads to secretion of pro-inflammatory cytokines (O’Neill, 2002; Takeda, Kaisho, & Akira,
2003). Current evidence indicates that the TLR pathways differ from one another and elicit distinct biological responses. For example, stimulation of DCs by Escherichia coli LPS results in the bioactive IL-12 p70 and interferon (IFN)-stimulated genes expression through TLR-4 whereas stimulation through TLR-2 with Staphylococcus aureus peptidoglycan (PGN) results only in the release of the IL-12 p40 (Fig. 2) (Re & Strominger, 2001; Nau, Schlesinger, Richmond, & Young, 2003). The diversity of the adapter molecules involved in executing distinct TLR signals may account for the nature of the inflammatory responses to different PAMPs. Signaling downstream from certain TLRs, such as TLR-2, appears to be completely dependent on MyD88 (Fig. 2) (Yamamoto et al., 2002). Nevertheless, NF-B and MAPKs can still be activated
186
E. Aksoy et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 183–188
Fig. 2. A simplified scheme of signaling pathways induced by ligand stimulation of the representative Toll-like receptors. TLR-4 and TLR-2 require the co-receptor, CD14 and a secreted protein, MD-2 to transmit LPS or PGN signals. TIRAP and MyD88 are recruited to both TLR-4 and TLR-2 by TIR domain interactions (dashed-lined region). IRAK is recruited by its DD (grey region), transduces signals that are mediated by MyD88. IRAK associates with TRAF-6 in the cytoplasm. Both TIRAP and MyD88 cooperate to regulate NF-B and MAP kinases activation: TRAF-6 forms a complex with TAB1-TAB2-TAK1 upon which TAK1 mediates downstream NF-B, p38, ERK and JNK activation. The core TLR-2/-4 responses result in the production of IL-12 p40, TNF and IL-1. TIRAP exclusively regulates phenotypic maturation of DC: increase of CD40, CD80 and CD86 surface expression. IL-12 p70 is produced only in response to TLR-4 signaling. TIRAP/MyD88-independent pathway downstream of TLR-4 activates IRF-3 through TICAM-1 which in turn induces INF-. Secreted IFN- then binds to type I IFN ␣/ receptor and instigates an autocrine/paracrine loop leading to the production of a set of secondary genes, such as IP-10, ISG15 and many of which are involved in antiviral and/or antimicrobial responses (on the right hand side).
by TLR-4 but not TLR-2 in DCs derived from MyD88 −/− mice, supporting the existence of an alternative, MyD88-independent pathway (Akira, Hoshino, & Kaisho, 2000). More recent attention has been focused on the role of type I interferon beta (IFN- as the key cytokine that link the innate and adaptive immune system (Taniguchi, Ogasawara, Takaoka, & Tanaka, 2001; Taniguchi & Takaoka, 2002). A recently identified TIR-containing adaptor molecule (TICAM-1), can activate type I IFN- gene promoter directly through interferon response factor (IRF)-3 upon TLR-4 triggering and may be responsible for the fundamental differences observed in cellular responses to LPS and PGN (Fig. 2) (Doyle et al., 2002;
Oshiumi, Matsumoto, Funami, Akazawa, & Seya, 2003). Recent evidence suggests that type I IFN- synthesis upon LPS can contribute to endotoxic shock. IFN- −/− but not STAT1 −/− mice were found resistant to high dose of LPS treatment (Karaghiosoff et al., 2003). These data suggest that IFN- may be an essential effector in LPS-induced lethality. The use of gene knockout mice models reveals fascinating new findings about PKC ε function and role in the regulation of the innate immune responses to pathogens. Initially, Bosca et al. established that novel PKC ε −/− mice display a major defect in clearing both gram negative and gram positive bacterial infections (Castrillo et al., 2001). Macrophages from
E. Aksoy et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 183–188
PKC ε −/− mice have a severely attenuated response to LPS marked by a significant reduction in cytokine production. In vitro experiments using inhibitors of PKC isoforms further confirmed the non-redundant role of PKC ε in the early phases of the innate immune responses. Recently, we demonstrated that PKC ε activation in human monocyte-derived DCs is essential for IL-12 synthesis in response to LPS (Aksoy et al., 2002). Specifically, inhibition of PKC ε activity by translocation inhibitory peptides diminishes LPS-induced cytokine production without altering the upregulation of costimulatory molecules. Recent evidence suggests that PKC ε may be also critical for cytokine gene expression in response to PGN (unpublished observations). Furthermore, PKC activity was found to be involved in the generation of TH 1 response since DC activation in presence of a PKC inhibitor, bisindolylmaleimide, diminishes their capacity to induce IFN-␥ production by T cells (Aksoy et al., 2002). In vivo, administration of bisindolylmaleimide to rats during autoantigen stimulation was shown to prevent the development of T cell-mediated autoimmune diseases in the Lewis rat model of experimental allergic encephalitis and the Lewis adjuvant arthritis model (Zhou et al., 1999). These observations are very reminiscent of the findings in MyD88 −/− mice that are deficient in the TH 1 response generation and are highly resistant to endotoxic shock (Schnare et al., 2001). It would be interesting to examine if PKC ε −/− mice are also resistant to endotoxic shock. Furthermore, the role of PKC ε in the generation of TH 1 responses in vivo is awaiting to be examined. The NF-B family of transcription factors have multiple roles in regulating events associated with DC maturation and macrophage activation. Recent in vivo studies proved that c-Rel and NF-B1/p50 are essential for LPS-induced IL-12 production (Ouaaz, Arron, Zheng, Choi, & Beg, 2002). There is evidence that PKC ε is a downstream target of LPS signals that leads to NF-B and MAPKs activation. PKC ε −/− macrophages display a severe defect in NF-B activation kinetics upon LPS challenge (Castrillo et al., 2001). Moreover, pharmacological inhibitors of PKC significantly impair LPS-mediated NF-B (Aksoy et al., 2002; Leitges et al., 2001; Sanz, Diaz-Meco, Nakano, & Moscat, 2000). On this basis, one can elaborate a scenario in which novel PKC ε plays an important role in Toll-like recep-
187
tor 4 (TLR-4) signaling that drives IL-12-dependent TH 1 responses of the host immune system. However, further studies are needed to elucidate how PKC ε is integrated in the TLR-4 signaling pathway.
4. Potential medical and pharmaceutical applications Although responsiveness to LPS is of primary importance in the innate resistance to infectious agents, exaggeration of LPS responses can be detrimental. Indeed, the overproduction of cytokines may result in severe inflammation and eventually endotoxic shock. Besides, excessive IL-12 production can result in organ specific autoimmune disease and block antiparasitic immunity by inhibiting effective TH 2 responses (Gately et al., 1998; Falcone & Sarvetnick, 1999). Preliminary data suggests that inhibition of PKC ε activity could represent a novel therapeutic approach for certain T cell-mediated inflammatory diseases as previously suggested by the beneficial effects of bisindolylmaleimide in rats. Although the efficacy of introducing peptides or antisense oligonucleotides to cells in vivo represents a present challenge, potential therapeutic value of PKC isoform-selective inhibitors represents a great appeal to both fundamental research and pharmaceutical industry. Peptide antagonists of PKC ε or antisense oligonucleotides which specifically block PKC ε function within the intracellular communication networks represent promising tools for the development of new immuno-intervention strategies. References Akira, S., Hoshino, K., & Kaisho, T. (2000). The role of Toll-like receptors and MyD88 in innate immune responses. Journal Endotoxin Research, 6, 383–387 (JID-9433350). Aksoy, E., Amraoui, Z., Goriely, S., Goldman, M., & Willems, F. (2002). Critical role of protein kinase C epsilon for lipopolysaccharide-induced IL-12 synthesis in monocytederived dendritic cells. European Journal of Immunology, 32, 3040–3049. Castrillo, A., Pennington, D. J., Otto, F., Parker, P. J., Owen, M. J., & Lisardo, B. (2001). Protein kinase C epsilon is required for macrophage activation and defense and defense against bacterial infection. The Journal of Experimental Medicine, 194, 1231–1242. Doyle, S., Vaidya, S., O’Connell, R., Dadgostar, H., Dempsey, P., Wu, T., Rao, G., Sun, R., Haberland, M., Modlin, R., & Cheng,
188
E. Aksoy et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 183–188
G. (2002). IRF3 mediates a TLR-3/TLR-4-specific antiviral gene program. Immunity, 17, 251–263. Falcone, M., & Sarvetnick, N. (1999). Cytokines that regulate autoimmune responses. Current Opinion in Immunology, 11, 670–676. Gately, M. K., Renzetti, L. M., Magram, J., Stern, A. S., Adorini, L., Gubler, U., & Presky, D. H. (1998). The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses. Annual Review of Immunology, 16, 495– 521. Hu, J., Jacinto, R., McCall, C., & Li, L. (2002). Regulation of IL-1 receptor-associated kinases by lipopolysaccharide. Journal of Immunology, 168, 3910–3914. Kaisho, T., & Akira, S. (2001). Dendritic-cell function in Toll-like receptor- and MyD88-knockout mice. Trends in Immunology, 22, 78–83. Karaghiosoff, M., Steinborn, R., Kovarik, P., Kriegshauser, G., Baccarini, M., Donabauer, B., Reichart, U., Kolbe, T., Bogdan, C., Leanderson, T., Levy, D., Decker, T., & Muller, M. (2003). Central role for type I interferons and Tyk2 in lipopolysaccharide-induced endotoxin shock. Nature Immunology, 4, 471–477. Leitges, M., Sanz, L., Martin, P., Duran, A., Braun, U., Garcia, J. F., Camacho, F., Diaz-Meco, M. T., Rennert, P. D., & Moscat, J. (2001). Targeted disruption of the zetaPKC gene results in the impairment of the NF-kappaB pathway. Molecular Cell, 8, 771–780. Medzhitov, R. (2001). Tolls. Current Biology, 11, R763. Mellor, H., & Parker, P. J. (1998). The extended protein kinase C superfamily. Biochemistry Journal, 332, 281–292. Mochly-Rosen, D., & Gordon, A. S. (1998). Anchoring proteins for protein kinase C: a means for isozyme selectivity. FASEB Journal, 12, 35–42. Nau, G. J., Schlesinger, A., Richmond, J. F., & Young, R. A. (2003). Cumulative Toll-like receptor activation in human macrophages treated with whole bacteria. Journal of Immunology, 170, 5203–5209. Newton, A. C. (1997). Regulation of protein kinase C. Current Opinion in Cell Biology, 9, 161–167. O’Neill, L. A. (2002). Wanted: a molecular basis for specificity in Toll-like receptor signal transduction. Molecular Cell, 10, 969–971. Oshiumi, H., Matsumoto, M., Funami, K., Akazawa, T., & Seya, T. (2003). TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction. Nature Immunology, 4, 161–167.
Ouaaz, F., Arron, J., Zheng, Y., Choi, Y., & Beg, A. A. (2002). Dendritic cell development and survival require distinct NF-kappaB subunits. Immunity, 16, 257–270 (JID-9432918). Re, F., & Strominger, J. L. (2001). Toll-like receptor 2 (TLR-2) and TLR-4 differentially activate human dendritic cells. The Journal of Biological Chemistry, 276, 37692–37699. Sanz, L., Diaz-Meco, M. T., Nakano, H., & Moscat, J. (2000). The atypical PKC-interacting protein p62 channels NF-kappaB activation by the IL-1-TRAF6 pathway. EMBO Journal, 19, 1576–1586. Schnare, M., Barton, G. M., Holt, A. C., Takeda, K., Akira, S., & Medzhitov, R. (2001). Toll-like receptors control activation of adaptive immune responses. Nature Immunology, 2, 947–950. Shapira, L., Sylvia, V. L., Halabi, A., Soskolne, W. A., Van Dyke, T. E., Dean, D. D., Boyan, B. D., & Schwartz, Z. (1997). Bacterial lipopolysaccharide induces early and late activation of protein kinase C in inflammatory macrophages by selective activation of PKC-epsilon. Biochemical and Biophysical Research Communications, 240, 629–634. Shinji, H., Akagawa, K. S., & Yoshida, T. (1994). LPS induces selective translocation of protein kinase C-beta in LPS-responsive mouse macrophages, but not in LPS-non-responsive mouse macrophages. Journal of Immunology, 153, 5760–5771. Takeda, K., & Akira, S. (2003). Toll receptors and pathogen resistance. Cell Microbiology, 5, 143–153. Takeda, K., Kaisho, T., & Akira, S. (2003). Toll-like receptors. Annual Review of Immunology, 21, 335–376. Taniguchi, T., & Takaoka, A. (2002). The interferon-alpha/beta system in antiviral responses: a multimodal machinery of gene regulation by the IRF family of transcription factors. Current Opinion in Immunology, 14, 111–116. Taniguchi, T., Ogasawara, K., Takaoka, A., & Tanaka, N. (2001). IRF family of transcription factors as regulators of host defense. Annual Review of Immunology, 19, 623–655. Trinchieri, G. (2003). Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nature Review: Immunology, 3, 133–146. Yamamoto, M., Sato, S., Hemmi, H., Sanjo, H., Uematsu, S., Kaisho, T., Hoshino, K., Takeuchi, O., Kobayashi, M., Fujita, T., Takeda, K., & Akira, S. (2002). Essential role for TIRAP in activation of the signalling cascade shared by TLR-2 and TLR-4. Nature, 420, 324–329. Zhou, T., Song, L., Yang, P., Wang, Z., Lui, D., & Jope, R. S. (1999). Bisindolylmaleimide VIII facilitates Fas-mediated apoptosis and inhibits T cell-mediated autoimmune diseases. Nature Medicine, 5, 42–48.
Molecules in focus
Protein kinase C epsilon: a new target to control inflammation and immune-mediated disorders Ezra Aksoy, Michel Goldman∗ , Fabienne Willems Laboratory of Experimental Immunology, Université Libre de Bruxelles, B-1070 Brussels, Belgium Received 23 December 2002; received in revised form 16 May 2003; accepted 16 May 2003
Abstract Recent advances in understanding the molecular basis for mammalian host immune responses to microbial invasion suggest that the first line of defense against microbes is the recognition of pathogen-associated molecular patterns by a set of germline-encoded receptors: the Toll-like receptors (TLRs). TLRs have been identified as being part of a large family of pathogen-recognition receptors that play a decisive role in the induction of both innate and adaptive immunity. Indeed, activation of T lymphocytes depends on their interaction with dendritic cells previously stimulated by TLR agonists such as bacterial lipopolysaccharide (LPS), a TLR-4 ligand. A novel PKC epsilon (ε) was recently found to be a critical component of TLR-4 signaling pathway and thereby to play a key role in macrophage and dendritic cell (DC) activation in response to LPS. Thus, controlling the kinase activity of PKC ε might represent an efficient strategy to prevent or treat certain inflammatory disorders of microbial origin. © 2003 Elsevier Ltd. All rights reserved. Keywords: Protein kinase C epsilon; Innate immunity; IL-12; TH 1
1. Introduction Protein kinase C (PKC) is a phospholipid-dependent serine/threonine kinase family, consisting of at least 11 isoforms that exhibit related homologies in their structures. The PKC isoforms can be classified into three main subfamilies based on their homology and cofactor requirements for activation: (1) conventional Abbreviations: ERK, Extracellular receptor-activated kinase; IP-10, IFN-␥ inducible protein 10; IRF, Interferon response factor; ISG15, IRF-stimulated gene 15; JNK, c Jun-N-terminal kinase; PGN, Peptidoglycan; TAK, Transforming growth factor--activated kinase; TAB, TAK binding protein; TIR, Toll-IL-1 receptor region; TICAM-1, TIR-containing adaptor molecule ∗ Corresponding author. Fax: +32-2-555-44-99. E-mail address: [email protected] (M. Goldman).
(cPKCs) ␣, I, II and ␥ that require negatively charged phosholipids, diacylglycerol (DAG) or phorbol ester and calcium; (2) novel (nPKCs) ␦, ε, , /L (mouse/human) that do not require calcium; and (3) atypical (aPKCs) / (mouse/human) and that only require negatively charged phosholipids, and the PKC D/ (murine/human) that form a distinct class and are more related to calmodulin-dependent kinase (Mellor & Parker, 1998). Differences between the PKC isoforms for their substrate specificity suggest that a particular PKC isoform within a single cell may regulate specific physiological responses. The mammalian Toll-like receptor (TLR) recognition system for pathogens is the first line of anti-microbial defense (Medzhitov, 2001; Takeda & Akira, 2003). The TLRs recognize molecular patterns
1357-2725/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1357-2725(03)00210-3
184
E. Aksoy et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 183–188
that are characteristic of particular classes of microorganism, such as lipopolysaccharide (LPS; Gram-negative bacteria), double-stranded RNA (viruses) and mannan (fungi). LPS, a major integral component from the outer membrane of gram negative bacteria, is a prototypical pathogen-associated molecular pattern (PAMP) which was demonstrated to induce dendritic cell (DC) or macrophage activation through TLR-4 triggering (Kaisho & Akira, 2001). TLR-4-mediated signaling events has two major consequences in DC: (1) production of several cytokines including interleukin (IL)-12 which promotes the differentiation of CD4+ T lymphocytes into TH 1 inflammatory effector cells, and (2) upregulation of cell surface molecules including major histocompatibility complex (MHC) and co-stimulatory molecules (Fig. 2) (Trinchieri, 2003). Multiple signaling molecules are initiated upon LPS binding to TLR-4, predominantly leading to nuclear factor (NF)-B and mitogen-activated protein (MAP) kinases activation. PKC isoforms actively participate in several TLR signaling pathways. LPS inducible IRAK activation/degradation has been demonstrated to require a functional TLR-4 that can be inhibited by a protein kinase C inhibitor, calphostin (Hu, Jacinto, McCall, & Li, 2002). There is evidence that LPS stimulation of macrophages or DCs results in PKC activation (Aksoy, Amraoui, Goriely, Goldman, & Willems, 2002; Castrillo et al., 2001; Shapira et al., 1997). Furthermore, macrophages from the C3H/HeJ mouse strain which carries a point mutation in the intro-cytoplasmic domain of TLR-4 receptor was found to exhibit deficient translocation of novel and conventional PKC in response to LPS (Shinji, Akagawa, & Yoshida, 1994). Herein, we review the structure and regulation of PKC isoforms and further discuss the mechanisms by which PKC ε participates in inflammatory responses.
2. Structure and regulation of PKC The classification of PKC isoforms, based on their enzymatic properties, is best understood by comparing the structural homologies of these isoforms at the protein-sequence level. Protein-sequence alignments between PKC isoforms reveal the existence
of homologous domains conserved between family members (Newton, 1997). The structure of PKC isoforms includes conserved domains referred as C1–C4 (Fig. 1). Variable regions (V1–V5), whose functions still under study, interrupt the conserved domains. Two cysteine-rich sequences, which are found at the N-terminus of C1 regions of cPKCs and nPKCs, are necessary for DAG and phorbol ester binding. The C2 region in cPKCs is important for Ca2+ binding and is absent in nPKCs and aPKCs. Activation and translocation of the PKC isoforms to specific subcellular sites is achieved by the binding of activated PKC to distinct anchoring proteins. Receptor for activated C kinases (RACKs) are an example of these anchoring proteins. Binding of RACK to PKC isoforms is selective and there may be more than one RACK for each isoform. For cPKC, the PKC–RACK interaction occurs via C2-domain whereas the C2-like region is involved in the interaction of nPKCs. Mochly-Rosen and Gordon demonstrated that peptide fragments corresponding to C2 domain (mapped to the amino acids 186–198, 209–216) specifically inhibit activation-induced translocation of cPKCs but not the C2-less nPKCs (Mochly-Rosen & Gordon, 1998). Furthermore, they found that the binding of the nPKC to its RACKs occurs via the V1 domain that is homologous to the C2 domain of cPKC. Peptides containing the V1 domain of novel PKC ε or PKC ␦ (amino acids 1–142) selectively inhibit their translocation and thus their activation (Mochly-Rosen & Gordon, 1998). The RACK binding site on PKC ε has been further mapped to amino acids 14–21 and peptides corresponding to this region selectively inhibit PKC ε translocation. The C-terminal regions, C3–V5, are recognized in all PKC isoforms and represent the catalytic domain (Fig. 1). The C3 region contains the ATP-binding sequence and only PKC has a slightly different ATP-binding site. It is noteworthy to point out that ATP-binding site inhibitors are the most potent drugs developed to inhibit PKC function. The C4 region contains the pseudo-substrate-binding site, which is important for the enzyme’s interaction with its substrates. Synthetic peptide analogues, which correspond to the pseudo-substrate site, are demonstrated to act as potent inhibitors of PKC isoforms (Mochly-Rosen & Gordon, 1998).
E. Aksoy et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 183–188
185
Fig. 1. Schematic representation of the primary structures of protein kinase C isoforms. The PKC structure can be divided into an N-terminal (N) regulatory domain and C-terminal (C) catalytic domain. Indicated are the pseudo-substrate (red), the C1 domain which binds diacylgycerol and phorbol esters (containing the cysteine-rich motif), the C2 domain which binds to acidic lipids and for cPKCs, Ca2+ binding (orange), and the C3 and C4 domains that comprise the ATP- and substrate-binding regions of the kinase (blue). Peptide inhibitors of PKC translocation target the C2 region of cPKC and V1 region of nPKC. Members of the each isoform subfamily are listed on the left. N, amino terminus; C, carboxyl terminus. The figure is a modification from Mochly-Rosen and Gordon (1998).
3. Biological function of PKC in the immune system TLR signal transduction network is an active area of investigation. The cytoplasmic domains of the TLRs and members of the interleukin-1 receptor (IL-1R) family contain a conserved signal-transduction module—the Toll-interleukin 1 receptor (TIR) domainthrough which TIR-containing adaptor molecules recruit the putative serine/threonine kinase, IL-1Rassociated kinase (IRAK) (Fig. 2). Subsequently, tumor-necrosis factor receptor (TNFR)-associated factor 6 (TRAF-6) is activated, triggering the NF-B and mitogen-activated protein kinase (MAPK) pathways, which leads to secretion of pro-inflammatory cytokines (O’Neill, 2002; Takeda, Kaisho, & Akira,
2003). Current evidence indicates that the TLR pathways differ from one another and elicit distinct biological responses. For example, stimulation of DCs by Escherichia coli LPS results in the bioactive IL-12 p70 and interferon (IFN)-stimulated genes expression through TLR-4 whereas stimulation through TLR-2 with Staphylococcus aureus peptidoglycan (PGN) results only in the release of the IL-12 p40 (Fig. 2) (Re & Strominger, 2001; Nau, Schlesinger, Richmond, & Young, 2003). The diversity of the adapter molecules involved in executing distinct TLR signals may account for the nature of the inflammatory responses to different PAMPs. Signaling downstream from certain TLRs, such as TLR-2, appears to be completely dependent on MyD88 (Fig. 2) (Yamamoto et al., 2002). Nevertheless, NF-B and MAPKs can still be activated
186
E. Aksoy et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 183–188
Fig. 2. A simplified scheme of signaling pathways induced by ligand stimulation of the representative Toll-like receptors. TLR-4 and TLR-2 require the co-receptor, CD14 and a secreted protein, MD-2 to transmit LPS or PGN signals. TIRAP and MyD88 are recruited to both TLR-4 and TLR-2 by TIR domain interactions (dashed-lined region). IRAK is recruited by its DD (grey region), transduces signals that are mediated by MyD88. IRAK associates with TRAF-6 in the cytoplasm. Both TIRAP and MyD88 cooperate to regulate NF-B and MAP kinases activation: TRAF-6 forms a complex with TAB1-TAB2-TAK1 upon which TAK1 mediates downstream NF-B, p38, ERK and JNK activation. The core TLR-2/-4 responses result in the production of IL-12 p40, TNF and IL-1. TIRAP exclusively regulates phenotypic maturation of DC: increase of CD40, CD80 and CD86 surface expression. IL-12 p70 is produced only in response to TLR-4 signaling. TIRAP/MyD88-independent pathway downstream of TLR-4 activates IRF-3 through TICAM-1 which in turn induces INF-. Secreted IFN- then binds to type I IFN ␣/ receptor and instigates an autocrine/paracrine loop leading to the production of a set of secondary genes, such as IP-10, ISG15 and many of which are involved in antiviral and/or antimicrobial responses (on the right hand side).
by TLR-4 but not TLR-2 in DCs derived from MyD88 −/− mice, supporting the existence of an alternative, MyD88-independent pathway (Akira, Hoshino, & Kaisho, 2000). More recent attention has been focused on the role of type I interferon beta (IFN- as the key cytokine that link the innate and adaptive immune system (Taniguchi, Ogasawara, Takaoka, & Tanaka, 2001; Taniguchi & Takaoka, 2002). A recently identified TIR-containing adaptor molecule (TICAM-1), can activate type I IFN- gene promoter directly through interferon response factor (IRF)-3 upon TLR-4 triggering and may be responsible for the fundamental differences observed in cellular responses to LPS and PGN (Fig. 2) (Doyle et al., 2002;
Oshiumi, Matsumoto, Funami, Akazawa, & Seya, 2003). Recent evidence suggests that type I IFN- synthesis upon LPS can contribute to endotoxic shock. IFN- −/− but not STAT1 −/− mice were found resistant to high dose of LPS treatment (Karaghiosoff et al., 2003). These data suggest that IFN- may be an essential effector in LPS-induced lethality. The use of gene knockout mice models reveals fascinating new findings about PKC ε function and role in the regulation of the innate immune responses to pathogens. Initially, Bosca et al. established that novel PKC ε −/− mice display a major defect in clearing both gram negative and gram positive bacterial infections (Castrillo et al., 2001). Macrophages from
E. Aksoy et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 183–188
PKC ε −/− mice have a severely attenuated response to LPS marked by a significant reduction in cytokine production. In vitro experiments using inhibitors of PKC isoforms further confirmed the non-redundant role of PKC ε in the early phases of the innate immune responses. Recently, we demonstrated that PKC ε activation in human monocyte-derived DCs is essential for IL-12 synthesis in response to LPS (Aksoy et al., 2002). Specifically, inhibition of PKC ε activity by translocation inhibitory peptides diminishes LPS-induced cytokine production without altering the upregulation of costimulatory molecules. Recent evidence suggests that PKC ε may be also critical for cytokine gene expression in response to PGN (unpublished observations). Furthermore, PKC activity was found to be involved in the generation of TH 1 response since DC activation in presence of a PKC inhibitor, bisindolylmaleimide, diminishes their capacity to induce IFN-␥ production by T cells (Aksoy et al., 2002). In vivo, administration of bisindolylmaleimide to rats during autoantigen stimulation was shown to prevent the development of T cell-mediated autoimmune diseases in the Lewis rat model of experimental allergic encephalitis and the Lewis adjuvant arthritis model (Zhou et al., 1999). These observations are very reminiscent of the findings in MyD88 −/− mice that are deficient in the TH 1 response generation and are highly resistant to endotoxic shock (Schnare et al., 2001). It would be interesting to examine if PKC ε −/− mice are also resistant to endotoxic shock. Furthermore, the role of PKC ε in the generation of TH 1 responses in vivo is awaiting to be examined. The NF-B family of transcription factors have multiple roles in regulating events associated with DC maturation and macrophage activation. Recent in vivo studies proved that c-Rel and NF-B1/p50 are essential for LPS-induced IL-12 production (Ouaaz, Arron, Zheng, Choi, & Beg, 2002). There is evidence that PKC ε is a downstream target of LPS signals that leads to NF-B and MAPKs activation. PKC ε −/− macrophages display a severe defect in NF-B activation kinetics upon LPS challenge (Castrillo et al., 2001). Moreover, pharmacological inhibitors of PKC significantly impair LPS-mediated NF-B (Aksoy et al., 2002; Leitges et al., 2001; Sanz, Diaz-Meco, Nakano, & Moscat, 2000). On this basis, one can elaborate a scenario in which novel PKC ε plays an important role in Toll-like recep-
187
tor 4 (TLR-4) signaling that drives IL-12-dependent TH 1 responses of the host immune system. However, further studies are needed to elucidate how PKC ε is integrated in the TLR-4 signaling pathway.
4. Potential medical and pharmaceutical applications Although responsiveness to LPS is of primary importance in the innate resistance to infectious agents, exaggeration of LPS responses can be detrimental. Indeed, the overproduction of cytokines may result in severe inflammation and eventually endotoxic shock. Besides, excessive IL-12 production can result in organ specific autoimmune disease and block antiparasitic immunity by inhibiting effective TH 2 responses (Gately et al., 1998; Falcone & Sarvetnick, 1999). Preliminary data suggests that inhibition of PKC ε activity could represent a novel therapeutic approach for certain T cell-mediated inflammatory diseases as previously suggested by the beneficial effects of bisindolylmaleimide in rats. Although the efficacy of introducing peptides or antisense oligonucleotides to cells in vivo represents a present challenge, potential therapeutic value of PKC isoform-selective inhibitors represents a great appeal to both fundamental research and pharmaceutical industry. Peptide antagonists of PKC ε or antisense oligonucleotides which specifically block PKC ε function within the intracellular communication networks represent promising tools for the development of new immuno-intervention strategies. References Akira, S., Hoshino, K., & Kaisho, T. (2000). The role of Toll-like receptors and MyD88 in innate immune responses. Journal Endotoxin Research, 6, 383–387 (JID-9433350). Aksoy, E., Amraoui, Z., Goriely, S., Goldman, M., & Willems, F. (2002). Critical role of protein kinase C epsilon for lipopolysaccharide-induced IL-12 synthesis in monocytederived dendritic cells. European Journal of Immunology, 32, 3040–3049. Castrillo, A., Pennington, D. J., Otto, F., Parker, P. J., Owen, M. J., & Lisardo, B. (2001). Protein kinase C epsilon is required for macrophage activation and defense and defense against bacterial infection. The Journal of Experimental Medicine, 194, 1231–1242. Doyle, S., Vaidya, S., O’Connell, R., Dadgostar, H., Dempsey, P., Wu, T., Rao, G., Sun, R., Haberland, M., Modlin, R., & Cheng,
188
E. Aksoy et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 183–188
G. (2002). IRF3 mediates a TLR-3/TLR-4-specific antiviral gene program. Immunity, 17, 251–263. Falcone, M., & Sarvetnick, N. (1999). Cytokines that regulate autoimmune responses. Current Opinion in Immunology, 11, 670–676. Gately, M. K., Renzetti, L. M., Magram, J., Stern, A. S., Adorini, L., Gubler, U., & Presky, D. H. (1998). The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses. Annual Review of Immunology, 16, 495– 521. Hu, J., Jacinto, R., McCall, C., & Li, L. (2002). Regulation of IL-1 receptor-associated kinases by lipopolysaccharide. Journal of Immunology, 168, 3910–3914. Kaisho, T., & Akira, S. (2001). Dendritic-cell function in Toll-like receptor- and MyD88-knockout mice. Trends in Immunology, 22, 78–83. Karaghiosoff, M., Steinborn, R., Kovarik, P., Kriegshauser, G., Baccarini, M., Donabauer, B., Reichart, U., Kolbe, T., Bogdan, C., Leanderson, T., Levy, D., Decker, T., & Muller, M. (2003). Central role for type I interferons and Tyk2 in lipopolysaccharide-induced endotoxin shock. Nature Immunology, 4, 471–477. Leitges, M., Sanz, L., Martin, P., Duran, A., Braun, U., Garcia, J. F., Camacho, F., Diaz-Meco, M. T., Rennert, P. D., & Moscat, J. (2001). Targeted disruption of the zetaPKC gene results in the impairment of the NF-kappaB pathway. Molecular Cell, 8, 771–780. Medzhitov, R. (2001). Tolls. Current Biology, 11, R763. Mellor, H., & Parker, P. J. (1998). The extended protein kinase C superfamily. Biochemistry Journal, 332, 281–292. Mochly-Rosen, D., & Gordon, A. S. (1998). Anchoring proteins for protein kinase C: a means for isozyme selectivity. FASEB Journal, 12, 35–42. Nau, G. J., Schlesinger, A., Richmond, J. F., & Young, R. A. (2003). Cumulative Toll-like receptor activation in human macrophages treated with whole bacteria. Journal of Immunology, 170, 5203–5209. Newton, A. C. (1997). Regulation of protein kinase C. Current Opinion in Cell Biology, 9, 161–167. O’Neill, L. A. (2002). Wanted: a molecular basis for specificity in Toll-like receptor signal transduction. Molecular Cell, 10, 969–971. Oshiumi, H., Matsumoto, M., Funami, K., Akazawa, T., & Seya, T. (2003). TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction. Nature Immunology, 4, 161–167.
Ouaaz, F., Arron, J., Zheng, Y., Choi, Y., & Beg, A. A. (2002). Dendritic cell development and survival require distinct NF-kappaB subunits. Immunity, 16, 257–270 (JID-9432918). Re, F., & Strominger, J. L. (2001). Toll-like receptor 2 (TLR-2) and TLR-4 differentially activate human dendritic cells. The Journal of Biological Chemistry, 276, 37692–37699. Sanz, L., Diaz-Meco, M. T., Nakano, H., & Moscat, J. (2000). The atypical PKC-interacting protein p62 channels NF-kappaB activation by the IL-1-TRAF6 pathway. EMBO Journal, 19, 1576–1586. Schnare, M., Barton, G. M., Holt, A. C., Takeda, K., Akira, S., & Medzhitov, R. (2001). Toll-like receptors control activation of adaptive immune responses. Nature Immunology, 2, 947–950. Shapira, L., Sylvia, V. L., Halabi, A., Soskolne, W. A., Van Dyke, T. E., Dean, D. D., Boyan, B. D., & Schwartz, Z. (1997). Bacterial lipopolysaccharide induces early and late activation of protein kinase C in inflammatory macrophages by selective activation of PKC-epsilon. Biochemical and Biophysical Research Communications, 240, 629–634. Shinji, H., Akagawa, K. S., & Yoshida, T. (1994). LPS induces selective translocation of protein kinase C-beta in LPS-responsive mouse macrophages, but not in LPS-non-responsive mouse macrophages. Journal of Immunology, 153, 5760–5771. Takeda, K., & Akira, S. (2003). Toll receptors and pathogen resistance. Cell Microbiology, 5, 143–153. Takeda, K., Kaisho, T., & Akira, S. (2003). Toll-like receptors. Annual Review of Immunology, 21, 335–376. Taniguchi, T., & Takaoka, A. (2002). The interferon-alpha/beta system in antiviral responses: a multimodal machinery of gene regulation by the IRF family of transcription factors. Current Opinion in Immunology, 14, 111–116. Taniguchi, T., Ogasawara, K., Takaoka, A., & Tanaka, N. (2001). IRF family of transcription factors as regulators of host defense. Annual Review of Immunology, 19, 623–655. Trinchieri, G. (2003). Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nature Review: Immunology, 3, 133–146. Yamamoto, M., Sato, S., Hemmi, H., Sanjo, H., Uematsu, S., Kaisho, T., Hoshino, K., Takeuchi, O., Kobayashi, M., Fujita, T., Takeda, K., & Akira, S. (2002). Essential role for TIRAP in activation of the signalling cascade shared by TLR-2 and TLR-4. Nature, 420, 324–329. Zhou, T., Song, L., Yang, P., Wang, Z., Lui, D., & Jope, R. S. (1999). Bisindolylmaleimide VIII facilitates Fas-mediated apoptosis and inhibits T cell-mediated autoimmune diseases. Nature Medicine, 5, 42–48.