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1 Tufts University School of Dental Medicine, One Kneeland Street, Boston, MA 02111, USA; and
2 Department of Immunology, The Forsyth Institute, Boston, MA, USA;
* corresponding author, paloma.valverde{at}tufts.edu
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ABSTRACT |
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 TOP ABSTRACT (I) INTRODUCTION(II) INVOLVEMENT OF BACTERIAL...(III) CELLULAR AND MOLECULAR...(IV) INHIBITION OF RANK...(V) TOPOLOGY AND EXPRESSION...(VI) FUNCTIONAL CHARACTERIZATION...(VII) BLOCKADE OF Kv1.3...(VIII) CONCLUSIONSREFERENCES |
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, interferon-; IP3, inositol (1,4,5)-triphosphate; (K)ir, inward rectifier; JNK, c-Jun N-terminal kinase; I(K), intermediate conductance; LPS, lipopolysaccharide; L, ligand; MCSF, macrophage colony-stimulating factor; MHC, major histocompatibility complex; NFAT, nuclear factor of activated T-cells; RANK, receptor activator of nuclear factor-
B; TCM, central memory T-cells; TEM, effector memory T-cells; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; OPG, osteoprotegerin; Omp29, 29-kDa outer membrane protein; PKC, protein kinase C; PLC, phospholipase C; RT-PCR, reverse-transcriptase polymerase chain-reaction; S(K), small conductance; TCR, T-cell receptor; and (K)v, voltage-gated.
KEY WORDS: periodontal disease • potassium channel-blockers • Kv1.3 • IKCa1 • RANKL
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(I) INTRODUCTION |
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TOPABSTRACT(I) INTRODUCTION(II) INVOLVEMENT OF BACTERIAL...(III) CELLULAR AND MOLECULAR...(IV) INHIBITION OF RANK...(V) TOPOLOGY AND EXPRESSION...(VI) FUNCTIONAL CHARACTERIZATION...(VII) BLOCKADE OF Kv1.3...(VIII) CONCLUSIONSREFERENCES |
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The primary ecological niche of Actinobacillus actinomycetemcomitans, a Gram-negative anaerobic micro-organism associated with aggressive periodontitis, is a potassium (K+)-rich environment such as dental plaque and gingival fluid (Tatevossian and Gould, 1976). In patients suffering from severe periodontitis, the gingival fluid K+ concentration has been reported to be high (Bang et al., 1973). This has been suggested to be due to higher numbers of degenerating epithelial cells, connective tissue, and blood cells liberating their intracellular contents, and hence contributing to increasing the K+ concentration of the exudates (Bang et al., 1973). The increased K+ concentration in periodontal lesion sites, together with moderate levels of oxygen tension, appears to constitute conditions favorable for the growth of Actinobacillus actinomycetemcomitans (Ohta et al., 2001), since high concentrations of extracellular K+ are required for respiration to occur in rapidly growing bacteria.
Under physiological conditions, the efflux of K+ to the extracellular media occurs selectively through membrane proteins called K+ channels. Two K+ channels, termed Kv1.3 and IKCa1, are engaged in myriad functions in cells commonly present in inflammatory lesions of periodontal disease, and in osteoclasts. To facilitate understanding of the involvement of Kv1.3 and IKCa1 in inflammatory bone resorption, we first summarize some of the mechanisms by which macrophages and chronically activated/memory T- and B-cells work in concert with bone cells to trigger bone resorption in periodontal disease and other osteopenic disorders. We then describe the different structural features and the roles of Kv1.3 and IKCa1 in T-cells, B-cells, macrophages, and osteoclasts in the context of periodontal disease progression. Finally, we describe the most exciting potential for the use of selective blockers for Kv1.3 and IKCa1 to ameliorate periodontal disease and other inflammatory/autoimmune disorders with or without associated bone resorption.
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(II) INVOLVEMENT OF BACTERIAL FACTORS AND IMMUNE CELLS IN PERIODONTAL DISEASE PROGRESSION |
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TOPABSTRACT(I) INTRODUCTION(II) INVOLVEMENT OF BACTERIAL...(III) CELLULAR AND MOLECULAR...(IV) INHIBITION OF RANK...(V) TOPOLOGY AND EXPRESSION...(VI) FUNCTIONAL CHARACTERIZATION...(VII) BLOCKADE OF Kv1.3...(VIII) CONCLUSIONSREFERENCES |
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(Baker, 2000). Among the bacterial factors are such moieties as lipopolysaccharide (LPS; Wilson, 1995), toxins (Tsai et al., 1979; Sugai et al., 1998; Henderson et al., 2003), and enzymes (Rifkin et al., 1993; Curtis et al., 2001). In diseased periodontal tissues, intervening between the infection and the targets of the disease (bone, connective tissue) is a dense mononuclear inflammatory infiltrate (Page and Schroeder, 1976). Inflammatory lesions of periodontal disease contain all the cellular components, including abundant CD45RO+ activated/memory T-cells (Gemmell et al., 1992; Yamazaki et al., 1993; Taubman et al., 1994; Taubman and Kawai, 2001), activated/memory B-cells (Gemmell and Seymour, 1991; Yamazaki et al., 1993), and dendritic cells (DC) (Cutler and Jotwani, 2004) necessary to control immunologically interactive networks.
A potential regulatory role for T-cells in periodontal disease was initially suggested when an altered CD4+ to CD8+ T-cell ratio and autologous mixed-lymphocyte reactions were found in patients with aggressive periodontitis (Taubman et al., 1982; Kinane et al., 1989; Taubman and Kawai, 2001). The evidence that IgG antibody to A. actinomycetemcomitans and to an antigenic outer membrane protein, 29 kDa (Omp29; Wilson, 1991), and to other protein antigens (Taubman et al., 1982) was elevated in patients’ sera indicated that T-lymphocytes were involved in the immunological recognition of this organism, by reason of requirement of T-cells for the IgM-IgG switch through CD40-CD40L signaling (Grewal and Flavell, 1998). Furthermore, activated/memory T-cells play a key role in the pathogenesis of periodontal disease through their potential to produce a variety of cytokines (Fujihashi et al., 1996; Takeichi et al., 2000) that can increase osteoclastic activity and bone resorption (Taubman and Kawai, 2001; Theill et al., 2002; Teng, 2003).
Periodontal antibody-producing B-cells have been suggested to be initially protective for the host (Hou et al., 2000), since B-cell deficiency predisposes mice to endodontic infections. However, depletion of B-cells in the humanized NOD/SCID periodontal disease mouse model does not significantly affect A. actinomycetemcomitans-induced alveolar bone resorption (Teng et al., 1999). In agreement with this report, an immunodeficient state in SCID mice, characterized by the lack of B- and T-cells, exhibited a decreased periodontal bone resorption compared with that of the immune-competent host when orally infected by Porphyromonas gingivalis (Baker et al., 1999a,b). In such an animal model, it was the T-cells, not the B-cells, which were primarily involved in mediating alveolar bone resorption in vivo (Baker et al., 1999a,b).
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(III) CELLULAR AND MOLECULAR COMPONENTS OF NORMAL AND PATHOLOGICAL BONE REMODELING |
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TOPABSTRACT(I) INTRODUCTION(II) INVOLVEMENT OF BACTERIAL...(III) CELLULAR AND MOLECULAR...(IV) INHIBITION OF RANK...(V) TOPOLOGY AND EXPRESSION...(VI) FUNCTIONAL CHARACTERIZATION...(VII) BLOCKADE OF Kv1.3...(VIII) CONCLUSIONSREFERENCES |
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B ligand (RANKL), its cellular receptor, RANK, and the decoy receptor, osteoprotegerin (OPG), have been identified as constituting the key molecular regulation system for bone remodeling (Fig. 1
) (Akatsu et al., 1998; Lacey et al., 1998; Yasuda et al., 1998; Kong et al., 1999a). RANKL is a member of the tumor necrosis factor (TNF) family of ligands that is expressed at its highest levels in osteoblasts and stromal cells. In the presence of the permissive factor macrophage colony-stimulating factor (MCSF), RANKL is both necessary and sufficient for the differentiation of osteoclast precursors into mature activated osteoclasts. RANKL also has capacity to inhibit or induce osteoclast apoptosis (Lacey et al., 1998; Yasuda et al., 1998; Bharti et al., 2004; Wu et al., 2005). The effects of RANKL are counteracted by OPG, which is secreted by various tissues and prevents the binding of RANKL to its receptor RANK on osteoclasts, thereby suppressing osteoclastogenesis (Fig. 1) (Akatsu et al., 1998).
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, TNF
, IL-11, IL-17) that induce RANKL expression in osteoblasts and bone marrow stromal cells (Taubman and Kawai, 2001; Theill et al., 2002) (Fig. 1). Chronically activated T-cells can also interfere with RANK signaling and therefore with osteoclast functions through the production of other factors, including IFN-, IL-4, and IL-10 (Fig. 1) (Taubman and Kawai, 2001; Theill et al., 2002). IFN-, with a second signal from LPS, can also induce the production of IL-1, TNF-, or IL-6 by macrophages (Fig. 1), which in turn can increase RANKL expression in osteoblasts and bone marrow stromal cells, or increase osteoclastogenesis by direct binding on the osteoclasts (Taubman and Kawai, 2001; Theill et al., 2002). Within the last couple of years, additional factors produced by activated/memory T-cells, such as IL-3 or TNF-related apoptosis-inducing ligand (TRAIL) (Khapli et al., 2003; Zauli et al., 2004), have been shown to inhibit RANKL-mediated osteoclastogenesis. In addition, cytokines normally present at high levels in inflammatory sites, such as IL-7, have been reported to increase inflammatory bone resorption by RANKL-dependent and -independent mechanisms (Toraldo et al., 2003). Thus, elevated levels of IL-7 can increase the production of RANKL and TNF- by T-cells as well as the proliferation of osteoclast progenitor cells (Toraldo et al., 2003).
Activated B-cells do not express OPG, but produce RANKL and other cytokines (Yun et al., 1998), and affect osteoclastogenesis in a positive or negative fashion, depending on whether they are stimulated in the presence of Th2- or Th1-like cytokines, respectively (Choi et al., 2001; Choi and Kim, 2003; Fig. 1). Furthermore, the B220+ cell subset of the B-lymphoid lineage can serve as osteoclast precursor cells (Manabe et al., 2001), as previously described for certain populations of mononuclear cells from bone marrow and peripheral blood (Flanagan and Massay, 2003; Hayashi et al., 2003; Horowitz and Lorenzo, 2004; Susa et al., 2004).
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(IV) INHIBITION OF RANK ACTIVATION AS A THERAPEUTIC APPROACH TO DECREASE BONE RESORPTION |
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TOPABSTRACT(I) INTRODUCTION(II) INVOLVEMENT OF BACTERIAL...(III) CELLULAR AND MOLECULAR...(IV) INHIBITION OF RANK...(V) TOPOLOGY AND EXPRESSION...(VI) FUNCTIONAL CHARACTERIZATION...(VII) BLOCKADE OF Kv1.3...(VIII) CONCLUSIONSREFERENCES |
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Despite the fact that inhibition of RANKL-mediated activation of RANK ameliorates systemic and local bone resorption, it does not decrease inflammation (Kong et al., 1999b; Teng et al., 2000; Schett et al., 2003), suggesting that even abundant local pro-inflammatory cytokines do not contribute to the destruction of bone in a major fashion in the absence of a functional RANK-RANKL system.
Development of therapeutic approaches to decrease inflammation and also to down-regulate the ratio of RANKL-to-OPG expression of chronically activated T-cells has hitherto been unexplored. The combined approaches constitute promising areas for intervention in periodontal disease pathogenesis and other inflammatory/autoimmune bone-resorptive diseases, such as rheumatoid arthritis. These novel approaches to therapeutic intervention in these diseases involve the use of K+ channel-blockers.
Several reports have demonstrated that Kv1.3 and IKCa1 K+ channels play crucial roles in T-cell activation, inflammation, progression of autoimmune diseases, and of other immunological disorders (Cahalan et al., 2001; Wulff et al., 2003a; Chandy et al., 2004; Vicente et al., 2004). Our preliminary results suggest that mononuclear inflammatory infiltrates isolated from patients with chronic periodontitis express Kv1.3 and IKCa1 at the mRNA level (Fig. 2). Furthermore, Kv1.3 expression appears to be up-regulated in the chronic periodontitis patients examined, while the expression of IKCa1 does not change significantly with respect to that of healthy gingiva (Fig. 2). This pattern of K+ channel expression correlates with an increased RANKL expression and a decreased OPG expression in these periodontal disease patients, as previously described by others (Mogi et al., 2004) (Fig. 2). In the following sections of this review, we will describe the structural and functional features of Kv1.3 and IKCa1, to lead to a better understanding of the putative functional link among these K+ channels, the RANKL/OPG system, and periodontal disease progression. Thereafter, we will summarize recent advances in the use of Kv1.3 and IKCa1 K+ channel-blockers to ameliorate several types of disorders, including periodontal disease.
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(V) TOPOLOGY AND EXPRESSION DISTRIBUTION OF Kv1.3 AND IKCa1 K+ CHANNELS |
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TOPABSTRACT(I) INTRODUCTION(II) INVOLVEMENT OF BACTERIAL...(III) CELLULAR AND MOLECULAR...(IV) INHIBITION OF RANK...(V) TOPOLOGY AND EXPRESSION...(VI) FUNCTIONAL CHARACTERIZATION...(VII) BLOCKADE OF Kv1.3...(VIII) CONCLUSIONSREFERENCES |
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K+ channels have been demonstrated to be involved in a wide variety of functions, including regulation of membrane potential, signal transduction, insulin secretion, hormone release, regulation of vascular tone, cell volume, and immune response (Hille, 2001). In this review, we will mostly focus on the voltage-gated K+ channel Kv1.3 (also referred to as KCNA3; http://www.iuphar-db.org/iuphar-ic/KV1x.html), and the intermediate-conductance Ca2+-activated IKCa1 channels (also known as KCNN4 or KCa3.1; http://www.iuphar-db.org/iuphar-ic/KCa.html), since they can be used as targets to control the progression of several immunological disorders (Cahalan et al., 2001; Jensen et al., 2001; Wulff et al., 2003a; Chandy et al., 2004), including periodontal disease (Valverde et al., 2004).
(A) Kv1.3 and IKCa1 Topology
Functional Kv1.3 and IKCa1 are tetramers. Hydropathy plots and indirect structural-functional analyses have led to the generally accepted topology for this class of K+ channels. Briefly, the amino- and carboxyl-terminal ends of the tetramer are located on the cytoplasmic side of the membrane, and each subunit is constituted by 6 transmembrane domains, referred to as S1 to S6 (Wulff et al., 2003a; Gulbis and Doyle, 2004; Yu and Catterall, 2004). The S5 and S6 transmembrane domains and the interconnecting loop or pore-forming loop form the conduction pathway of the channels, also known as the ‘pore’. The pore-forming loop contains the signature motif ‘GYG’, responsible for the selectivity of Kv1.3 and IKCa1 and other K+ channels for the conduction of K+ ions (Yu and Catterall, 2004). Activation of Kv1.3 channels in response to membrane depolarization occurs through a conformational change that involves movement of the S4 transmembrane segment (voltage sensor) and opening of the pore (Wulff et al., 2003a). IKCa1 channels open in response to an increase in cytosolic Ca2+ concentration, the Ca2+ sensor being calmodulin, which is bound to the carboxyl-terminal end of IKCa1 (Wulff et al., 2003a). Importantly, crystallography of the bacterial homologue (Jiang et al., 2003; Gulbis and Doyle, 2004) suggests that the generally accepted topology for this class of K+ channels may need to be revised.
(B) Expression Distribution
Kv1.3 has been shown to be expressed in lymphocytes, macrophages, osteoclasts, platelets, microglia, fat cells, oligo-dendrocytes, fibroblasts, olfactory bulb, and brain (Arkett et al., 1994; Komarova et al., 2001; Vicente et al., 2003; Wulff et al., 2003a). IKCa1 expression has been detected in lymphocytes, erythrocytes, vascular and bladder smooth muscles, endothelium, Paneth cells, colon, prostate, placenta, liver, pancreas, melanoma, and fibroblasts (Wulff et al., 2003a). A Ca2+-activated K+ channel current, resembling that of IKCa1, has also been reported in osteoclasts (Komarova et al., 2001, 2003).
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(VI) FUNCTIONAL CHARACTERIZATION OF Kv1.3 AND IKCa1 IN DIFFERENT CELL TYPES |
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TOPABSTRACT(I) INTRODUCTION(II) INVOLVEMENT OF BACTERIAL...(III) CELLULAR AND MOLECULAR...(IV) INHIBITION OF RANK...(V) TOPOLOGY AND EXPRESSION...(VI) FUNCTIONAL CHARACTERIZATION...(VII) BLOCKADE OF Kv1.3...(VIII) CONCLUSIONSREFERENCES |
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). Some of these K+ channel-blockers have been isolated from natural sources, such as scorpion venom, sea anemone venom, or tree extracts (Table), while others have been synthesized in the laboratories of academic or pharmaceutical companies (Table). Two of these synthetic compounds, TRAM-34 and ICA-17043, have been proven to be specific for IKCa1, while none of the blockers shown in the Table is fully selective for Kv1.3. However, due to the restricted expression of functionally tetrameric Kv1.3 channels, some Kv1.3 blockers (i.e., kaliotoxin, correolide, margatoxin) have been used in animal models for several diseases and did not exhibit major side-effects. This will be described in more detail in section VII.
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). Interaction of T-cells with APC leads to the formation of synapses at the T-cell/APC contact site. Synapse formation is associated with rapid clustering of T-cell receptor (TCR) molecules binding to peptide/MHC complexes on APC and activation of phospholipase C (PLC), which leads to the activation of protein kinase C (PKC) and the generation of inositol (1,4,5)-triphosphate (IP3) (Krogsgaard et al., 2003). IP3 then induces the release of Ca2+ from intracellular stores, which, in turn, leads to the opening of Ca2+ release-activated Ca2+ (CRAC) channels and results in a sustained increase in cytosolic Ca2+ (Krogsgaard et al., 2003; Randriamampita and Trautmann, 2004). K+ efflux through Kv1.3 and IKCa1 K+ channels prevents the membrane depolarization that would result from the sole Ca2+ influx through CRAC channels and would limit further Ca2+ influx (Cahalan et al., 2001; Wulff et al., 2003a). Briefly, membrane hyperpolarization results from the opening of Kv1.3 in response to membrane depolarization, followed by the opening of IKCa1 as a consequence of elevated concentrations of cytosolic Ca2+. Simultaneous activation of CD28 by the co-stimulatory molecule CD80 or CD86 further amplifies the resulting Ca2+ signaling (Fig. 3). The sustained increase in the cytosolic Ca2+ concentration activates the phosphatase calcineurin, which then dephosphorylates the cytoplasmic transcription factor NFAT (nuclear factor of activated T-cells), enabling it to translocate into the nucleus to bind to NFAT-response elements of several genes, including the T-cell growth factor interleukin-2 (IL-2) (Crabtree, 1999). In a separate pathway, activation of PKC leads to activation of the Ras and JNK (c-Jun N-terminal kinase) pathways (Baier, 2003), which results in the assembly of Fos/Jun transcription factors and binding to the promoter of several genes. Coordinated activity of Ca2+- and PKC-dependent signaling pathways culminates in cytokine production and T-cell proliferation (Fig. 3). As previously shown (Fig. 1), some of the cytokines produced by activated T-cells mediate pro-inflammatory or anti-inflammatory effects, while others induce or inhibit osteoclastogenesis and bone resorption (Figs. 1, 3). In addition, some cytokines of the TNF-related family (i.e., FasL, CD40L, and TRAIL) are also up-regulated upon T-cell activation and mediate a wide variety of functions, including activation-induced cell death (AICD) of T-cells (Roberts et al., 2003), B-cell co-stimulation, DC activation and differentiation (Bishop and Hostager, 2003; O’Sullivan and Thomas, 2003), and inhibition of osteoclastogenesis (Zauli et al., 2004).
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In the absence of classic immunological stimulatory signals, and when Kv1.3 is open, elevated extracellular K+ levels have been shown to activate T-cell ß1 integrin moieties to induce integrin-mediated adhesion and migration of human T-cells (Levite et al., 2000).
In the first few hours after mitogenic stimulation, specifically when Ca2+ elevation is necessary (Crabtree, 1999), K+ channel-blockers for Kv1.3 and IKCa1 have been demonstrated to inhibit Ca2+ influx and T-cell proliferation (Cahalan et al., 2001; Chandy et al., 2001; Fanger et al., 2001), as well as the production of a variety of cytokines, including IL2, TNF, IFN-, RANKL, and OPG (Beeton et al., 2001a; Valverde et al., 2004) in human or rat T-cell clones. K+ channel-blockers for Kv1.3 have also been shown to inhibit T-cell ß1 integrin-mediated adhesion and migration of human T-cells (Levite et al., 2000).
(B) Involvement of Kv1.3 and IKCa1 in Naïve and Activated/Memory B-cell Functions
Kv1.3 and IKCa1 have been reported to play a role in B-cell activation and B-cell mitogenesis, as described for the T-cell lineage, although the expression patterns of the channels may be different, depending upon the protocol of stimulation (Sutro et al., 1989; Amigorena et al., 1990; Brent et al., 1990; Partiseti et al., 1992, 1993). Most importantly, there is a switch in K+ channel expression during differentiation of human B-cells from naïve to memory cells (Wulff et al., 2004). Thus, naïve and early-memory B-cells (CD27+IgD+) express low levels of both Kv1.3 and IKCa1 in their quiescent state, but upon activation, IKCa1 expression is up-regulated (about 45-fold), whereas Kv1.3 expression does not change significantly. In contrast, class-switch late-memory B-cells (CD27+IgD–), which are a major source of pathogenic IgG autoantibodies in several autoimmune diseases (Atkinson and Eisenbarth, 2001; O’Connor et al., 2001; Dorner and Burmester, 2003), express very high levels of Kv1.3 in their quiescent or activated state (Wulff et al., 2004).
(C) Roles of K+ Channels in Macrophages and Osteoclasts
Several K+ channels, including Kv1.3, Kv1.5, and Kir2.1, have been shown to be expressed in primary cultures of bone-marrow-derived macrophages (Vicente et al., 2003). In this cell model, MCSF-dependent proliferation induced Kv1.3 and Kir2.1 expression. The authors of this study suggested that macrophages may require both channels to maintain sufficiently negative potential to open Ca2+ channels and thus initiate mitotic calcineurin-dependent Ca2+ signaling pathways. In the same study, LPS-induced activation (that blocks macrophage proliferation) differentially regulated the expression of Kv1.3 and Kir2.1 by TNF--dependent and -independent mechanisms. Thus, while Kv1.3 was further induced, Kir2.1 was down-regulated (Vicente et al., 2003). The authors suggested that, by inducing Kv1.3 and repressing Kir2.1, macrophages reduce the Ca2+-driving force and the intracellular K+ concentration while increasing extracellular K+ concentration. In the same study, margatoxin or a derivative of ShK toxin inhibited cell growth and expression of inducible nitric-oxide synthase expression, suggesting that Kv channels (mostly Kv1.3) may play crucial roles in macrophage proliferation and activation.
When actively resorbing bone, mature osteoclasts adhere to the bone surface via integrin receptors and transport protons (H+) through vacuolar ATPases across the ruffled membrane, thus acidifying the resorption lacunae (Teitelbaum, 2000). Transport of H+ alone would cause hyperpolarization of the membrane potential, which would ultimately prevent further H+ influx. To avoid this, ion transport through chloride and K+ channels, including Kv1.3, Kir2.1, and Ca2+-activated K+ channels, has been hypothesized to play a role by compensating for charge accumulation arising from the electrogenic transport of H+ in actively bone-resorbing osteoclasts (Komarova et al., 2001). During osteoclastic bone resorption, crystal hydroxyapatite is dissolved into free Ca2+ ions, leading to the increase in extracellular Ca2+ concentration in the resorbed area. High extracellular Ca2+ concentration then triggers an increase in cytosolic Ca2+ concentration. As a result, resorbing activity gradually decreases, and osteoclasts turn into non-resorbing/motile cells, which migrate and settle on a new surface area of the bone. Basal intracellular Ca2+ concentration is therefore higher in rat motile non-resorbing osteoclasts than in resorbing osteoclasts (Kajiya et al., 2003), and exposure to high K+ increases intracellular Ca2+ concentration in resorbing osteoclasts but reduces it in motile osteoclasts. As a consequence, high K+ inhibits the motility of non-resorbing osteoclasts and reduces pit formation in resorbing osteoclasts (Kajiya et al., 2003). Interestingly, Kir2.1 K+ current dominates in motile osteoclasts, whereas Kv1.3 appears to be more important in rounded resorbing osteoclasts (Arkett et al., 1992; Komarova et al., 2001), although their putative role in differentially regulating intracellular Ca2+ concentration has not been reported. In addition to Kir2.1 or Kv1.3, Ca2+-activated K+ currents have been suggested to be involved in the regulation of osteoclast movement and spreading on bone substrate (Espinosa et al., 2002). In those studies, treatment with charybdotoxin or apamin (which blocks SK channels) decreased the ability of osteoclasts to move and spread on bone substrate and to resorb bone in vitro (Espinosa et al., 2002), raising the possibility that several types of Ca2+-activated K+ channels and Kv channels may have been involved in the effects observed.
Studies performed with osteoclasts isolated from bone or the RAW264.7 osteoclast precursor cell line have shown that, upon RANK activation by RANKL, there is an elevation of cytosolic Ca2+ concentration (Komarova et al., 2003) that activates a Ca2+-activated K+ current resembling that of IKCa1 (Komarova et al., 2003), as well as the NFAT-calcineurin signaling pathway (Takayanagi et al., 2002; Hirotani et al., 2004). Although other signaling pathways and transcription factors are also activated upon RANK engagement (Lee and Kim, 2003; Kwan et al., 2004), the increase in cytosolic Ca2+ has been reported to promote osteoclast survival (Komarova et al., 2003), and the activation of NFAT/calcineurin signaling pathway has been demonstrated to play a crucial role in osteoclast differentiation (Takayanagi et al., 2002; Hirotani et al., 2004) and the inhibition of osteoclast apoptosis (Igarashi et al., 2004). The putative relationship between osteoclast survival, differentiation, or apoptosis and the K+ currents activated or inhibited upon RANK activation has not yet been established.
Since bone-marrow-derived macrophages express a variety of functional K+ channels and can be induced to differentiate into osteoclasts (Hayashi et al., 2003; Horowitz and Lorenzo, 2004), this cell model may be useful for the study of K+ channel involvement in osteoclast differentiation and bone resorption induced by different combinations of osteoclastogenic factors. Alternatively, a simpler and more economical cell system has been described to generate human primary osteoclasts from peripheral blood (Susa et al., 2004). This cell system may be the most appropriate for comparison of the putative effects of different K+ channel-blockers to regulate human bone resorption in individuals with normal and pathological bone remodeling.
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(VII) BLOCKADE OF Kv1.3 OR IKCa1 TO AMELIORATE AUTOIMMUNE DISEASES, PERIODONTAL DISEASE, AND OTHER IMMUNOLOGICAL DISORDERS |
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TOPABSTRACT(I) INTRODUCTION(II) INVOLVEMENT OF BACTERIAL...(III) CELLULAR AND MOLECULAR...(IV) INHIBITION OF RANK...(V) TOPOLOGY AND EXPRESSION...(VI) FUNCTIONAL CHARACTERIZATION...(VII) BLOCKADE OF Kv1.3...(VIII) CONCLUSIONSREFERENCES |
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TEM and/or CD27+IgD– B-cells have been reported to be implicated in the pathogenesis of several autoimmune diseases, such as multiple sclerosis (Iglesias et al., 2001; O’Connor et al., 2001), type-1 diabetes (Atkinson and Eisenbarth, 2001; Viglietta et al., 2002), and rheumatoid arthritis (Dorner and Burmester, 2003), through their ability to migrate to sites of inflammation and contribute to the inflammatory process through secretion of cytokines. In multiple sclerosis, the majority of autoreactive memory cells exhibit a high expression of functional Kv1.3 and very low expression of functional IKCa1 (Beeton et al., 2001a,b; Wulff et al., 2003b). As a consequence, Kv1.3 blockers, as opposed to IKCa1 blockers, have been shown to be effective in suppressing cytokine production and proliferation of these autoreactive memory cells, thus ameliorating the clinical signs of multiple sclerosis in a rat model of the disease (Beeton et al., 2001a, b). A Kv1.3-based therapy for the treatment of human multiple sclerosis and other autoimmune disorders would be expected to be safer than generalized immunomodulators, since naïve and early-memory lymphocytes (CD27+IgD+, TCM) would not be affected, leaving the bulk of the immune response unaltered.
CD27+IgD+ and/or TCM has been suggested to be probably involved in immune-mediated acute rejection of transplanted organs and acute graft-vs.-host disease (Chandy et al., 2004; Wulff et al., 2004). Because of the expression pattern of Kv1.3 and IKCa1 before and after activation, these cells are initially sensitive to Kv1.3 channel-blockers and then become sensitive to IKCa1 channel-blockers (Chandy et al., 2004; Wulff et al., 2004). Thus, initial combination therapy with Kv1.3 and IKCa1 channel-blockers, followed by IKCa1 channel blockade alone, could potentially be used to control the progression of these disorders (Chandy et al., 2004; Wulff et al., 2004). This K+ channel-blocker-based therapy would be expected to decrease the incidence of side-effects (i.e., gingival overgrowth, osteoporosis, and osteopenia) normally seen in renal-transplanted patients subjected to long-term cyclosporine A or nifedipine therapies (Cayco et al., 2000; Khoori et al., 2003). It is important to keep in mind, however, that high concentrations of nifedipine (in the µM but not in the nM range) can also block Kv1.3 in vitro (Chandy et al., 2004). Therefore, before Kv1.3 and IKCa1 blockers are used in transplanted patients, they should be formally evaluated, in appropriate animal models, for their pharmacokinetic profile, toxicity, and selectivity. A good animal model for these studies is the mini-swine, which has physiological parameters very similar to those of humans, including the fact that Kv1.3 regulates the membrane potential of peripheral blood T-cells (Koo et al., 1997, 1999). Indeed, the mini-swine model has been previously used to validate the concept that Kv1.3 is a target for immunomodulation. In that model, systemic administration with margatoxin inhibited a delayed-type hypersensitivity reaction as well as an antibody response to an allogeneic challenge (Koo et al., 1997). In this study, treatments with margatoxin did not result in overt toxicity, except for some animals that exhibited hypersalivation or appetite loss, and others that showed some transient hyperactivity when margatoxin was used at a very high dose. In a different study, correolide or its derivatives exhibited immunosuppressive properties in mini-swine when administered systemically, and caused only some reversible intestinal distress at the time of the dosage, or a small degree of thymic atrophy when administered at high doses (Koo et al., 1999). Therefore, the mini-swine model is probably the most useful for validation of the use of Kv1.3 and IKCa1 blockers in managing the rejection of transplanted organs and graft-vs.-host disease.
(B) Use of K+ Channel-blockers for Kv1.3 or IKCa1 to Decrease Periodontal Disease Progression and Other Bone-resorptive Disorders
Rat A. actinomycetemcomitans Omp29-specific memory T-cell clones (G23 or G21) have been previously described to induce experimental alveolar bone resoption in vivo (Kawai et al., 2000) and to express RANKL and OPG upon activation with APC and Omp29 in vitro or in vivo (Valverde et al., 2004). These T-cell clones express low levels of Kv1.3 (Valverde et al., 2004) and IKCa1 mRNA (Valverde et al., unpublished observations) in their quiescent state, and do not express either Kv1.1 or Kv1.5 (Valverde et al., unpublished observations). Activation with APC and specific antigen leads to an average of four-fold up-regulation in Kv1.3 mRNA and protein levels (Valverde et al., 2004), whereas IKCa1 mRNA levels are not significantly up-regulated (Valverde et al., unpublished observations). Since RANKL is expressed by T-cells following TCR stimulation (Kong et al., 1999b; Theill et al., 2002), and its transcriptional up-regulation is partly Ca2+-dependent (Kong et al., 1999b; Wang et al., 2002), down-regulation of Ca2+ signaling in T-lymphocytes would be expected to decrease RANKL expression and RANKL-mediated osteoclastogenesis and bone resorption induced by activated/memory T-cells. In agreement with this notion, the immunosuppressant cyclosporine (Kong et al., 1999b; Wang et al., 2002), as well as the inhibitor of intracellular calcium mobilization TMB-8 (Wang et al., 2002), were reported to decrease RANKL expression by T-cells in vitro. Similarly, we have found that kaliotoxin or charybdotoxin treatments decreased RANKL expression as well as the ratio of RANKL to OPG in rat Omp29-specific memory T-cells in vitro (Valverde et al., 2004; Valverde et al., unpublished observations). In contrast, clotrimazole did not affect the ratio of RANKL to OPG in experiments performed with this T-cell clone (Valverde et al., unpublished observations). Since clotrimazole mostly targets IKCa1, and both kaliotoxin and charybdotoxin can block Kv1.3 (Table), we believe that Kv1.3 plays a more important role than IKCa1 in regulating RANKL-dependent osteoclastogenesis and bone resorption induced by Omp29-specific T-cell clones.
Despite the ability of cyclosporine A, FK-506, or some K+ channel-blockers to decrease the RANKL-mediated bone resorption triggered by activated T-cells in vitro (Kong et al., 1999b; Wang et al., 2002; Valverde et al., 2004), their systemic administration in vivo may lead to unwanted side-effects, unless the drug target shows a restricted distribution. Thus, cyclosporine A or FK-506 targets the ubiquitously distributed calcineurin, which limits their therapeutic use to ameliorate bone resorption, as suggested by the high incidence of post-transplant osteoporosis and osteopenia in transplanted patients receiving systemic administration of these drugs (Epstein, 1996; Cayco et al., 2000). These side-effects might be partly mediated through the up-regulation in the ratio of RANKL-to-OPG expression in osteoblasts, as recently described for osteoblast cell cultures treated with cyclosporine A or FK-506 in vitro (Hofbauer et al., 2001). In contrast to the in vivo activation of bone resorption, these immunosuppressants decrease bone resorption in vitro by decreasing RANKL-induced osteoclast differentiation and increasing osteoclast apoptosis (Igarashi et al., 2004). Another important side-effect in renal-transplanted patients on cyclosporine therapy is gingival overgrowth (Boltchi et al., 1999; Khoori et al., 2003). This might be associated with decreased expression of OPG in gingival fibroblasts, as described for vascular smooth-muscle cells treated with cyclosporine A in culture (Hofbauer et al., 2001). Similar side-effects, due to the ubiquitous distribution of the drug target, have been described when transplanted patients are subjected to a calcium-channel-blocker-based therapy (i.e., nifedipine) (Cayco et al., 2000; Khoori et al., 2003).
Unlike calcineurin or calcium channels, functional Kv1.3 and IKCa1 appear to be expressed in a more restricted group of cells, and their selective blockade would be expected to down-regulate the Ca2+-driven transcriptional up-regulation of RANKL in memory T-cells with a lower incidence of side-effects. To test this hypothesis, we performed adoptive transfer of activated Omp29-specific G23 T-cells into naïve rat recipients, to cause experimental alveolar bone resorption. Systemic administration of kaliotoxin or charybdotoxin reduced experimental alveolar bone resorption without obvious side-effects in this rat periodontal disease model, whereas clotrimazole did not mediate any significant effects (Valverde et al., 2004; Valverde et al., unpublished observations). Furthermore, CD3+ T-cells infiltrating the gingival tissue expressed a lower RANKL-to-OPG ratio in kaliotoxin- or charybdotoxin-treated than in saline- or clotrimazole-treated rats (Valverde et al., 2004; Valverde et al., unpublished observations). Thus, Kv1.3 appears to play a more important role than IKCa1 in regulating T-cell-mediated and RANKL-dependent alveolar bone resorption in our rat experimental system.
Gating of Kv1.3 has been suggested to regulate the ability of elevated extracellular K+ concentration to affect T-cell ß1 integrin functions (Levite et al., 2000). Thus, opening of the channel leads to function, whereas its blockade prevents it (Levite et al., 2000). These Kv1.3 functions may be particularly relevant in inflammatory diseases with associated cell destruction that leads to an increase of the extracellular K+ concentration, such as in severe cases of periodontal disease. Thus, elevated K+ concentrations in the gingival fluid would not only be expected to favor the growth and metabolism of A. actinomycetemcomitans (Ohta et al., 2001), but also might activate T-cell integrin functions required for the recruitment of T-cells from blood circulation to inflamed gingival tissue. However, we found that gingival CD3+ T-cells from saline-treated or channel-blocker-treated animals expressed comparable levels of GAPDH mRNA at 2 days and 10 days after the T-cell transfer in our rat animal studies. Therefore, a similar number of CD3+ T-cells appear to have migrated and to have been retained within the gingival tissue in the different experimental animal groups. These results suggested that blockade of Kv1.3 and/or IKCa1 lacked effects on the migration and probably the adhesion of memory T-cells to inflamed gingival tissue prior to triggering alveolar bone resorption. The lack of effects of kaliotoxin in the migration and adhesion of pathogenic memory T-cells in a rat model for multiple sclerosis was reported prior to our study (Beeton et al., 2001b). Taken together, these animal studies appear to be in contrast to the role of Kv1.3 in regulating T-cell adhesion and migration functions (Levite et al., 2000). The discrepancy between the in vitro and in vivo data may be due to species differences (rat vs. human) and/or may be difficult to detect in vivo, requiring examination of tissue samples at earlier or later time points than those examined in the in vivo studies reported so far.
Although systemic administration of K+ channel-blockers in rat or mini-swine animal models appears to have very few side-effects at the doses reported (Koo et al., 1997, 1999; Beeton et al., 2001a,b; Valverde et al., 2004), higher doses than those used so far in animals may lead to more side-effects in humans, unless the blockers are completely selective for Kv1.3 or IKCa1. For example, clotrimazole has been reported to ameliorate some of the symptoms associated with rheumatoid arthritis in humans (Panayi, 1995; Jensen et al., 2001) but unfortunately causes side-effects, including a significant rise in plasma cortisol and a fall in white count, partly associated with the inhibition of cytochrome P450 enzymes. Systemic administration of clotrimazole did not inhibit alveolar bone resorption in our animal model (Valverde et al., unpublished observations), although clotrimazole treatment decreased resorption pit surface in soluble RANKL-treated MOCP-5 osteoclast cells in vitro (Valverde et al., unpublished observations). However, the inhibition of the resorption pit surface in vitro could have been the result of the inhibitory effect of clotrimazole on cytochrome P450 and/or blockade of IKCa1, since it has been previously described that inhibition of cytochrome P450 by clotrimazole can decrease bone resorption area (Choo and Chole, 1999).
Despite the encouraging but limited results with animal studies, the use of selective blockers for Kv1.3 and IKCa1 to decrease inflammatory bone resorption in humans requires a great deal of additional research before becoming a reality. Thus, it would be important to compare the effects of these blockers upon local or systemic administration in different animal models of bone resorption. Furthermore, the phenotype of the memory B- and T-cells implicated in periodontal disease progression and/or inflammatory bone resorption in humans needs to be further characterized, if the most appropriate K+-channel-based therapy is to be designed. Taken together, this line of investigation may help us not only to develop a new therapeutic approach to stop the progression of bone-resorptive disorders, but also to learn more about the cell and molecular components responsible for these diseases.
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(VIII) CONCLUSIONS |
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TOPABSTRACT(I) INTRODUCTION(II) INVOLVEMENT OF BACTERIAL...(III) CELLULAR AND MOLECULAR...(IV) INHIBITION OF RANK...(V) TOPOLOGY AND EXPRESSION...(VI) FUNCTIONAL CHARACTERIZATION...(VII) BLOCKADE OF Kv1.3...(VIII) CONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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Received June 8, 2004; Accepted January 11, 2005
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REFERENCES |
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TOPABSTRACT(I) INTRODUCTION(II) INVOLVEMENT OF BACTERIAL...(III) CELLULAR AND MOLECULAR...(IV) INHIBITION OF RANK...(V) TOPOLOGY AND EXPRESSION...(VI) FUNCTIONAL CHARACTERIZATION...(VII) BLOCKADE OF Kv1.3...(VIII) CONCLUSIONSREFERENCES |
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