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The roles of ribosomal protein S19 C-terminus in a shortened neutrophil lifespan through delta lactoferrin
Immunobiology 220 (2015) 1085–1092
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The roles of ribosomal protein S19 C-terminus in a shortened neutrophil lifespan through delta lactoferrin Hiroshi Nishiura ∗ , Koji Yamanegi, Mutsuki Kawabe, Nahoko Kato-Kogoe, Naoko Yamada, Keiji Nakasho Division of Functional Pathology, Department of Pathology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan
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Article history: Received 23 January 2015 Received in revised form 25 February 2015 Accepted 1 May 2015 Available online 11 May 2015 Keywords: C5a receptor Delta lactoferrin Neutrophils Regulator of G protein signaling 3 Ribosomal protein S19
a b s t r a c t Cell lifespan is partially regulated by a balance between survival signals via constitutively active G proteincoupled receptors (GPCRs) and death signals via death receptors. We have demonstrated that neutrophils produce a mimic ligand of G protein-coupled C5a receptor (C5aR), ribosomal protein S19 (RP S19) polymer. In contrast to an original ligand C5a, RP S19 polymer induces not only inhibition of the guanine nucleotide exchange factor activity but also initiation of the regulator of G protein signaling 3 (RGS3) promoter in a RP S19 C-terminus dependent manner. To examine an antagonistic effect of the RP S19 C-terminus on G proteins, His-S-tagged C5a or C5a/RP S19, in which an RP S19 C-terminus is bound to the C5a C-terminus, was incubated with neutrophils, and a transcription factor delta lactoferrin (␦Lf) was identified as a specific binding protein via pull-down experiments. The S-tagged C5a-induced agonistic effects on chemotaxis, cytoplasmic Ca2+ influx and p38 mitogen-activated protein kinase phosphorylation were not changed by Lf knockdown and ␦Lf overexpression in neutrophil-like or macrophage-like cells, which were differentiated into mature cells from human promyelocytic leukemia HL-60 cells by dimethyl sulfoxide and phorbol-12-myristate-13-acetate, respectively. While, the S-tagged C5a/RP S19-induced antagonistic or agonistic effects on mature HL-60 neutrophil-like or macrophage-like cells were reversed by Lf knockdown and ␦Lf overexpression, respectively. Moreover, RGS3 expression was increased in another HL-60 neutrophil-like cells under spontaneous apoptosis induced by an apoptotic inducer MnCl2 . The RGS3 expression in apoptotic neutrophil-like cells was delayed not only by Lf knockdown but also by neutralization of the RP S19 polymer or C5aR. The inhibitory extension from G protein of C5aR to G␣ subsets of constitutively active GPCRs along with the RP S19 polymer-induced translocation of ␦Lf from the cytoplasmic face of the plasma membrane to the nucleus seems to shorten the neutrophil cell lifespan. © 2015 Elsevier GmbH. All rights reserved.
Introduction We have demonstrated that cell lifespan is partially regulated by a balance between a weak extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated survival signal via constitutively active heterotrimeric guanine nucleotide-regulating (G␣␥) proteincoupled receptors (GPCRs) and a strong apoptotic death signal via
Abbreviations: C5aR, C5a receptor; ␦Lf, delta lactoferrin; GPCR, G proteincoupled receptor; NY-1, anti-RP S19 rabbit IgGs; PMX-53, C5aR antagonistic peptide; RGS3, regulator of G protein signaling 3; RP S19, ribosomal protein S19; sLf, secreted form of Lactoferrin; TRPM, transient receptor potential melastatin. ∗ Corresponding author at: Division of Functional Pathology, Department of Pathology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan. Tel.: +81 79 845 6432; fax: +81 79 845 6431. E-mail address: [email protected] (H. Nishiura). http://dx.doi.org/10.1016/j.imbio.2015.05.006 0171-2985/© 2015 Elsevier GmbH. All rights reserved.
death receptors (Milligan, 2003; Dragovich et al., 1998; Nishiura et al., 2009). Neutrophils under spontaneous apoptosis produce an alternative G␣i ␥ protein-coupled C5a receptor (C5aR) ligand, ribosomal protein S19 (RP S19) polymer, which is distinct from the natural ligand C5a and is cross-linked by apoptosis-related activation of tissue transglutaminases (TGs) (Sanghavi et al., 1998; Knepper-Nicolai et al., 1998; Nishiura et al., 1996; Horino et al., 1998; Nishimura et al., 2001). The C5a-induced G␥ subset-dependent activation begins with the exchange of GDP to GTP in the G␣i ␥ protein through a conformational change in C5aR. This activation is limited by a posttranslational modification of the regulator of G protein signaling 3 (RGS3), which increases the GTPase activity of G␣ in an autocrine manner, and/or C-terminal phosphorylation of C5aR (Hsu et al., 2014; Kehrl, 2004; Ishii and Kurachi, 2003). Therefore, the ligandinduced strong ERK1/2 signal via the C5aR on neutrophils through
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calcium release-activated channels is suggested to participate in the regulation of cell lifespan length (Perianayagam et al., 2004; Brazil et al., 2014). However, C5aR deficient mice sometimes show unreasonable neutrophil infiltration into acute inflammation lesions in an antigen-specific manner (Hopken et al., 1996, 1997). We recently found that the C5aR on macrophages bound with the RP S19 polymer mimic use a similar two-step binding mechanism of C5a to mediate a weak G␥-dependent p38 mitogen-activated protein kinase (p38MAPK) signal through transient receptor potential melastatin (TRPM) Ca2+ /Mg2+ channels without receptor internalization (Nishiura et al., 2010a,b, 2011). The C5aR-mediated ERK1/2 signal induced by C5a is exchanged to the p38MAPK signal by simultaneous treatment with low concentrations of the phosphoinositide 3-kinase inhibitor LY294002 and phospholipase C inhibitors U73122. Conversely, binding affinity of the RP S19 polymer to the C5aR on macrophages is lower than that on neutrophils (Nishiura et al., 2011). The RP S19 polymer does not induce a C5aR-mediated downstream signal but stimulates RGS3 expression via the C5aR on neutrophils (Nishiura et al., 1998, 2009, 2010a,b). Therefore, we suggested that RGS3 expression via de novo-synthesized C5aR on neutrophils participates in the shortening of the cell lifespan (Nishiura, 2013; Nishiura et al., 2013). To examine the mechanism of C5aR-mediated RGS3 expression, we identified a different C-terminus (IAGQVAAANKKH) of RP S19 from the C5a C-terminus (Nishiura et al., 2010a,b). C5a/RP S19 was prepared by connecting IAGQVAAANKKH to the C-terminus of C5a with a mutation at Gly73Asp, and we confirmed the different C5aRmediated outputs (Oda et al., 2008; Revollo et al., 2005). His-Stagged C5a or C5a/RP S19 was incubated here with neutrophils, and the transcription factor delta lactoferrin (␦Lf) was identified as a specific binding molecule to the RP S19 C-terminus in neutrophils (Breton et al., 2004). Materials and methods Materials Recombinant C5a, C5a/RP S19 and ␦Lf were prepared with the pET32a-Rosetta gami(B) Lys-S system. His-S-tagged proteins for binding assays and S-tagged proteins for biological assays were sometimes coupled to BrCN-activated Sepharose 4B beads (5 mg/mL) (GE, Little Chalfont, UK). Phosphorylated and unphosphorylated anti-p38MAPK rabbit IgGs were produced by Cell Signaling Technology (MA, USA). Anti-ANXA3 rabbit IgGs was purchased from Proteintech Group, Inc. (IL, USA). Anti-Lf and anti-C5aR rabbit IgGs, fluorescein isothiocyanate (FITC)-conjugated anti-C5aR mouse IgG and FITC- and horseradish peroxidase (HRP)-conjugated anti-rabbit goat IgGs were purchased from Santa Cruz Biotechnology (CA, USA). Cells Human promyelocytic leukemia HL-60 cells were obtained from the RIKEN BioResource Center (Tsukuba, Japan). Neutrophil-like cells and macrophage-like cells were differentiated from HL-60 cells into mature cells by dimethyl sulfoxide and phorbol-12myristate 13-acetate, respectively (Zinzar et al., 1989). To make another neutrophil-like cells under spontaneous apoptosis, HL60 cells were undergone into apoptosis by a caspase 12 activator MnCl2 (Nishiura et al., 2009; Sanghavi et al., 1998; Knepper-Nicolai et al., 1998; Oubrahim et al., 2002). Peripheral blood was collected from healthy volunteers who provided written consent according to a protocol approved by the Research Ethics Committee of the Japanese Red Cross, Kumamoto Hospital and Kumamoto University (No. 439) in accordance with the Helsinki Declaration of 1975,
as revised in 2002. Monocytes and neutrophils were isolated from the heparinized venous blood of at least three different healthy donors (Matsubara et al., 1991). Enriched neutrophils with satisfactory purity (>98%) were prepared using Ficoll-PaqueTM PLUS (GE). The cell identities were confirmed by fluorescence-activated cell forting (FACS) analysis using anti-CD16b antibodies (BD, Tokyo, Japan).
Vectors and HL-60 cells cDNAs corresponding to sLf and ␦Lf were a kind gift from Prof CT Teng at the National Institutes of Health (Liu et al., 2003). The sLf and ␦Lf cDNAs were inserted into a pCAGGS-IRES neomycin-resistant vector (Fukushima et al., 1997). shLf lentivirus (Santa Cruz Biotechnology) was used to knockdown Lf mRNA. Successfully transformed cells were sorted using a BD FACSAriaTM II Cell Sorter. Conversely, to generate HL-60 transformed cells containing pCIN-␦Lf cDNA, linearized plasmid DNA was transformed into HL-60 cells by electroporation using a Gene Pulsar II (Bio-Rad, Berkeley, CA, USA). The optimal clone was selected based on its growth rate.
Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting His-S-tagged C5a/RP S19 or His-S-tagged C5a (10−7 M) was incubated for 10 min at 22 ◦ C in 20 mL of Ca2+ buffer containing neutrophils (107 cells/mL). The proteins bound to the His-S-tagged proteins were cross-linked using dithiobis (2 mM) for 30 min at 22 ◦ C (Pierce, IL, USA). The membrane was segmented using 2% Triton X-100 and 4% CHAPS (Dojindo), and whole proteins were applied to a Hi-Trap Ni2+ chelating column (GE). The bound proteins were eluted using 10 mM dithiothreitol buffer. Neutrophils (1 × 107 cells/mL) in 10 mL of PBS were treated with 1 mM diisopropyl fluorophosphate and 2% Triton X-100 for 10 min at 37 ◦ C. After sucrose gradient centrifugation, the cytoplasmic proteins in Ca2+ buffer were incubated with S-tagged ␦Lf beads for 10 min at 22 ◦ C. The bound proteins were separated from the beads with 50 mM glycine, pH 3.0. The bound proteins were suspended in lysis buffer (200 g/125 L) (7 M urea, 2 M thiourea, 4% CHAPS, 2% Triton X-100, 18 mM dithiothreitol, 0.5% immobilized pH gradient buffer, and 0.1% bromothymol blue) and applied to an Immobiline DryStrip (7 cm/pH 3.0–10.0) for 24 h at 22 ◦ C. The proteins on the DryStrip gel were arranged along the immobilized pH gradients in an Ettan IPGphor II Isoelectric Focusing Unit (GE). Four-step programs were run at 50 A current and 20 ◦ C: 300 V for 30 min in the step and hold mode, 1000 V for 30 min in the gradient mode, 5000 V for 90 min in the gradient mode, and 5000 V for 4 h in the step and hold mode. The proteins on the DryStrip gel were reduced in equilibration buffer A (50 mM Tris–HCl buffer [pH 8.8] containing 9 M urea, 30% glycerol, 2% SDS, 0.25% dithiothreitol, and 0.1% bromothymol blue) and buffer B (50 mM Tris–HCl buffer [pH 8.8] containing 9 M urea, 30% glycerol, 1% SDS, 4.5% iodoacetamide, and 0.1% bromothymol blue) for 15 min at 22 ◦ C and were loaded onto 12% SDS-PAGE gels. Total proteins were detected using silver staining. The proteins were electrophoretically transferred to an Immobilon Transfer MembraneTM (Millipore, MA, USA) using a semi-dry electroblotter (Sartorius, Göttingen, BRD) for 90 min with a current of 15 V. Primary antibodies were incubated (200 ng/mL) for 1 h at 22 ◦ C, and HRP-conjugated anti-rabbit IgGs goat IgGs secondary antibody (20 ng/mL) was incubated for 30 min at 22 ◦ C. The enhanced chemiluminescence reaction was performed using the ECL Plus Western Blotting Detection SystemTM (GE).
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Time-of-flight mass spectrometry Proteins in the SDS polyacrylamide gel were excised and digested with trypsin (10 g/mL) (GE) in 30 L of 50 mM NH4 HCO3 (pH 7.8) overnight at 37 ◦ C. The protein fragments were washed in ZipTip® C18 and eluted in 0.1% trifluoroacetic acid containing 70% acetonitrile. Approximately 0.5 L of the protein fragments was covered with 0.5 L of 5 mg/mL ␣-cyano-4-hydroxy-cinnamic acid matrix solution on a MALDI plate and analyzed in a 4700 MALDI TOF/TOF mass spectrometer (ABI, Foster, MA, USA). Chemotaxis assay Cells (2 × 106 cells/mL) were prepared in Ca2+ buffer (HBSS containing 20 mM HEPES and 3% BSA, pH 7.4) for the chamber assay. The assay was performed for 90 min using a nucleopore filter with a pore size of 5 m for HL-60 cells. The total number of cells that migrated beyond the lower surface of the membrane was counted in five high-power microscopic fields. Calcium imaging Cells (2 × 106 cells/mL) were loaded with the calcium-sensitive dye Fura 2-AM (1 M) in Ca2+ buffer for 30 min at 37 ◦ C (Dojindo, Kumamoto, Japan). Samples were placed directly into the cell suspension in a cuvette after 5 min of baseline recording. Recordings were made with an F-2500 calcium imaging system (FL Solutions; Hitachi, Tokyo, Japan), which calculated the ratio of fluorescence signals obtained at 37 ◦ C with excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Statistical analysis Each result was confirmed by multiple experiments with a minimum of three samples. Statistical significance was calculated by non-parametric and parametric tests with two-way analysis of variance. The values are presented as the means ± SD. A Pvalue < 0.05 was considered to be statistically significant and is presented as P < 0.05 (*) or P < 0.01 (**). Results His-S-tagged C5a/RP S19 binds to neutrophil-specific proteins We suggested that the RP S19 C-terminus additionally binds neutrophil-specific proteins for closing an interactive space between the active C5aR and the GDP form of G␣i ␥ protein. To identify the binding partners, His-S-tagged C5a/RP S19 or His-S-tagged C5a was incubated with neutrophils. In addition to neutrophil proteins that bound the His-S-tagged C5a, the specific proteins that bound to His-S-tagged C5a/RP S19 were identified via pull-down experiments and assessed using 2D SDS-PAGE prior to preparing samples for TOF-MS. To confirm complexes of the RP S19 C-terminus with neutrophil-specific molecules on plasma membrane, we at least verified a presence of C5aR in the samples by Western blotting (data not shown). The specific protein spots corresponding to an apparent molecular weight (MW) of 75 kDa and an isoelectric point (pI) of 7.0–8.5 were specifically observed in silver stained 2D gels (Fig. 1). Six specific protein spots corresponding to MW of 74–78 kDa and pI of 7.0–8.5 were detected by anti-Lf rabbit IgGs (Fig. 1B upper). However, the N-terminal amino acid residues were not detected in ten trypsin-digested fragments of the secreted form of lactoferrin (sLf) (Supplemental Fig. 1). To confirm whether the RP S19 C-terminus bound to sLf (calculated MW, 78 kDa, calculated pI, 8.5 using the theoretical pI and MW computation tool of ExPASy) on the extracellular face of the
Fig. 1. His-S-tagged C5a/RP S19 binding protein. Proteins on the plasma membrane of neutrophils bound to either (A) His-S-tagged C5a or (B) His-S-tagged C5a/RP S19 were applied to a 2D-SDS gel and silver stained (n = 6). After transferring the proteins to a membrane, Western blotting was performed using anti-Lf rabbit IgGs.
plasma membrane or to ␦Lf (calculated MW, 74 kDa, calculated pI, 8.0) on the cytoplasmic face of the plasma membrane, protein spots in the cell fractions of the 2D gels and the transferred membranes were incubated with silver staining solution (Fig. 2 upper) and antiLf rabbit IgGs (Fig. 2 lower), respectively. The 78 kDa protein spots with a pI of 8.5 and 8.2 were mainly detected in the nuclear and the granular fractions, respectively. Conversely, the main 74 kDa protein spot with a pI of 7.0 was detected in the membrane fraction. These data indicate that the 78 kDa sLf is trafficked through the endoplasmic reticulum-Golgi secretion pathway, while the 74 kDa ␦Lf localizes on the cytoplasmic face of the plasma membrane. Moreover, we generated fresh preparations of BrCN-activated Sepharose 4B beads coupled with S-tagged C5a or S-tagged C5a/RP S19 without a His tag and incubated them with neutrophils or the membrane fractions of neutrophils and macrophages, respectively. In contrast to S-tagged C5a beads, S-tagged C5a/RP S19 beads were strongly bound to the 74 kDa ␦Lf, not only in neutrophils (Fig. 3A) but also in neutrophil membrane fractions but not macrophage membrane fractions (Fig. 3B). S-tagged C5a/RP S19 switches from an antagonist to an agonist of C5aR on HL-60 neutrophil-like cells upon ıLf knockdown To validate the antagonistic or agonistic effects of ␦Lf on the C5aR, we prepared Lf-knockdown HL-60 neutrophil-like cells or ␦Lf-overexpressing HL-60 macrophage-like cells and analyzed the relationship between the ratio of ␦Lf to sLf/C5aR/actin expression (␦Lf ratio) and relevant biological processes, such as chemotaxis, cytoplasmic Ca2+ influx, and p38MAPK signaling. The Lf ratio was calculated based on the actin expression density (= ␦Lf + sLf/C5aR/actin) in Western blots using ImageJ 1.46 (National Institutes of Health, MD, USA). The Lf ratio in HL-60 neutrophil-like cells decreased from 2.08 to 0.82 after transfection with Lf shRNA (Fig. 4A). The antagonistic effect of 10−8 M S-tagged C5a/RP S19 on the neutrophil C5aR was replaced by an agonistic effect, as reflected by chemotaxis, cytoplasmic Ca2+ influx, and p38MAPK phosphorylation upon Lf knockdown (Fig. 4B–D). Moreover, the switch of
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Fig. 2. ␦Lf localization. The proteins in neutrophil cell fractions were applied to 2D-SDS gels and silver stained (n = 3). After transferring the proteins to a membrane, Western blotting was performed using anti-Lf rabbit IgGs.
Fig. 3. The complex of S-tagged C5a/RP S19 with ␦Lf on the membrane fractions of neutrophils. S-tagged C5a and S-tagged C5a/RP S19 beads were prepared and incubated with neutrophils (A) or the membrane fractions of neutrophils or macrophages (B), respectively. After centrifugation, the proteins with (Ppt: pellet) and without (Sup: supernatant) S-tagged protein beads were applied to 2D-SDS gels and transferred to a membrane. The 74 kDa delta lactoferrin was detected by Western blotting using anti-Lf rabbit IgGs (n = 3).
Fig. 4. An exchange of antagonistic functions of S-tagged C5a/RP S19 to agonistic functions under ␦Lf knockdown in HL-60 neutrophil-like cells. (A) Lf expression levels in HL-60 neutrophil-like cells containing control or Lf shRNA were assessed via Western blot (n = 3). (B) The chemotactic potential of the control and Lf-knockdown HL-60 neutrophil-like cells against S-tagged C5a and S-tagged C5a/RP S19 was measured in a 48-well chemotaxis chamber (n = 6). (C) The cytoplasmic Ca2+ influx induced by S-tagged C5a or S-tagged C5a/RP S19 was measured in LF-knockdown HL-60 neutrophil-like cells (n = 4). (D) p38MAPK phosphorylation induced by S-tagged C5a or S-tagged C5a/RP S19 was assessed in the Lf-knockdown HL-60 neutrophil-like cells by Western blotting (n = 3).
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Fig. 5. An exchange of agonistic functions of S-tagged C5a/RP S19 to partially agonistic functions under ␦Lf overexpression in HL-60 macrophage-like cells. (A) Lf and C5aR expression levels in HL-60 macrophage-like cells containing control, Lf, and ␦Lf cDNAs were assessed via Western blot (n = 3). (B) C5aR expression on the Lf and ␦Lf HL-60 macrophage-like cells was detected by FACS (n = 3). (C) The chemotactic potential of mock, Lf, and ␦Lf HL-60 macrophage-like cells against S-tagged C5a and S-tagged C5a/RP S19 was measured in a 48-well chemotaxis chamber (n = 6). (D) Cytoplasmic Ca2+ influx induced by S-tagged C5a and S-tagged C5a/RP S19 was assessed in the mock, Lf, and ␦Lf HL-60 macrophage-like cells (n = 4). (E) p38MAPK phosphorylation induced by S-tagged C5a and S-tagged C5a/RP S19 in the presence or absence of 1 mM MgCl2 was assessed in mock, Lf, and ␦Lf-overexpressing HL-60 macrophage-like cells via Western blotting (n = 3).
S-tagged C5a/RP S19 from a C5aR antagonist to a C5aR agonist by Lf knockdown was also observed in human mast cell-1 (HMC-1) cells (Supplemental Fig. 2). In contrast to the ␦Lf ratio for the C5aR in macrophage-like cells differentiated from the mock HL-60 cells (1.55), the overexpression of sLf and ␦Lf shifted the ␦Lf ratios to 0.59 and 2.16, respectively (Fig. 5A). No significant difference in C5aR expression was noted between the sLf- and ␦Lf-overexpressing HL-60 macrophage-like cells (Fig. 5B). However, the effect on the biological processes of the mock HL-60 macrophage-like cells upon treatment with 10−8 M S-tagged C5a/RP S19 was inversely proportional to the ␦Lf ratio, as shown by chemotaxis, cytoplasmic Ca2+ influx, and p38MAPK phosphorylation (Fig. 5C–E). To further confirm the p38MAPK phosphorylation through TRPM Ca2+ /Mg2+ channels, HL-60 macrophage-like cells were stimulated with either S-tagged C5a or S-tagged C5a/RP S19 in a simultaneous presence of a Mg2+ channel blocker
1 mM MgCl2 and detected p38MAPK phosphorylation (Fig. 5E) (Nishiura et al., 2011). In contrast to the sLf-overexpressing HL60 macrophage-like cells, a significant decrement of the S-tagged C5a/RP S19-induced p38MAPK phosphorylation was detected in the ␦Lf-overexpressing HL-60 macrophage-like cells. Conversely, S-tagged C5a-induced biological responses were not reflected by the ␦Lf ratio. In addition, we observed similar biological effects in sLf and ␦Lf U937 macrophage-like cells upon treatment with 10−8 M S-tagged C5a/RP S19 (data not shown). The f-Met-Leu-Phe-induced chemotactic potential of HL-60 and U937 macrophage-like cells was not modified by ␦Lf overexpression (data not shown), as previously shown with CD14+ macrophages (Laemmli, 1970). Strangely, the sLf overexpression also endowed U937 macrophage-like cells with a high intensity of cytoplasmic Ca2+ influx. We hypothesized that neutrophil C5aR is specifically blocked by the complex of the RP S19 C-terminus with ␦Lf.
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Fig. 6. The ␦Lf-RGS3 pathway. (A) Lf in control and Lf-knockdown HL-60 cells was detected by Western blotting (n = 4). (B) RGS3 in control and Lf-knockdown HL-60 cells post-MnCl2 loading was detected by Western blotting (n = 4). (C) RGS3 in control and Lf-knockdown HL-60 cells post-MnCl2 loading in the presence of control PBS, anti-human RP S19 rabbit IgGs, or PMX-53 was detected by Western blotting (n = 4).
Postponement of RGS3 expression in HL-60 cells under spontaneous apoptosis upon ıLf knockdown To examine a contribution of ␦Lf on RGS3 production in neutrophils under spontaneous apoptosis, an effect of Lf knockdown on RGS3 expression was observed in HL-60 cells during apoptosis. We confirmed an almost 67% depletion of total sLf and ␦Lf protein expression in control HL-60 cells by Lf knockdown (Fig. 6A). In this experimental setting, the expression of RGS3 and its truncated variant RGS3T was delayed until 48 h post-MnCl2 loading by Lf knockdown (Fig. 6B) (Falk et al., 1980). To further confirm that the interaction between the RP S19 C-terminus and ␦Lf through RP S19 polymer-C5aR binding induces RGS3 expression, the RP S19 polymer was neutralized with anti-RP S19 rabbit IgGs (NY-1) or C5aR was blocked by a C5aR antagonistic peptide PMX-53 (Fig. 6C). The expression of RGS3 and RGS3T during apoptosis was blocked, even in control HL-60 cells. Discussion We have demonstrated that a conditional change to the apoptotic microenvironment sometimes enables transcription factor NF-Y to initiate the C5aR promoter and type II TGs with a crosslinkage of RP S19 in all cell types. After autocrine binding of the K38 LAKHK moiety of one RP S19 molecule in the RP S19 polymer to the C5aR N-terminus on neutrophils under spontaneous apoptosis, the L131 DR moiety of another RP S19 molecule in the RP S19 polymer geometrically enters into an active pocket of the C5aR on the extracellular face of the plasma membrane (Nishiura et al., 2010a,b). The dissociated G␥-mediated activation of p38MAPK induces ␦Lf phosphorylation and a translocation to the cytoplasmic face of the plasma membrane (Figs. 2C and 3B). RP S19 C-terminus (K143 KH) increases the chance for complex formation with ␦Lf, resulting in a complete interference with a further interaction of active C5aR with the GDP form of G␣i ␥ protein. ␦Lf next moves into the nucleus by a complicated translocation system and works as a transcription factor, initiating the RGS3 promoter to increase the GTPase activity of G␣ in neutrophils under spontaneous apoptosis (Figs. 2A and 6). Two-step inhibitory mechanisms of ␦Lf against G protein seem to launch programmed cell death. Moreover, the initiation of RGS3 promoter was also observed in apoptotic HMC-1 cells induced by MnCl2 (data not shown). Conversely, the de novosynthesized GPCRs on apoptotic cells and their ligands, such as ANXA1, Resolving, and others, are also reported to participate in shortening the cell lifespan (Dalli et al., 2013; El Kebir et al., 2012). In studying transcriptional mechanisms of ␦Lf, sLf and RGS3 during apoptosis, we found a NF-Y binding CCAAT moiety 17 nucleotides upstream of the ATG start codon in the sLf gene (NM 001199149.1), but not the RGS3 gene. Conversely, ␦Lf expres-
sion is commonly suppressed in cancer cell lines and lung cancer cells (Benaissa et al., 2005; Siebert and Huang, 1997). We at least found a ␦Lf transcript in apoptotic HL-60 cells during apoptosis induced by MnCl2 , which was relatively delayed compared to sLf expression (Supplemental Fig. 3). These data indicated an alternative promoter in exon 1. There is a GGAA moiety at 71 nucleotides downstream of the ATG start codon in the sLf gene, which binds to the Ets-1 transcription factor, indicating a P2 promoter in exon 1 for specific ␦Lf expression (Liu et al., 2003). Recruitment of histone deacetylase to the P2 promoter is regulated by the phosphorylation/SUMOylation cycling system of Ets-1 (Findlay et al., 2013). There is a complicated translocation system of ␦Lf from the cytoplasmic membrane to the nucleus via the phosphorylation/OGlcNAcation cycling system at S10 and/or the SUMOylation system (Mariller et al., 2012). In the present study, we observed not only the main protein spot of ␦Lf with a MW of 74 kDa and pI of 7.0 but also a minor protein spot with the same pI but with a higher MW in the membrane fraction (Fig. 2C). Similar protein spots to the latter of these were also observed in the granular fraction (Fig. 2B). Although we have ignored the weak C5aR-mediated p38MAPK signal that lacks biological outputs, an MAPK interacting molecule carrying the docking motif K580 VERLKQVLL and a p38MAPK-dependent phosphorylation motif GKDKS258 PKFQ are found in ␦Lf (AAA59511.1) (Eukaryotic Linear Motifs tool and NetPhosK 1.0 Server in the ExPASy Bioinformatics Resource Portal). Two motifs seem to turn in the same direction in the binding pocket for p38MAPK in the ␦Lf structure (MMDB ID: 34912) (Thomassen et al., 2005). In contrast, we found that a protein phosphatase 1 catalytic subunit interacting motif, G269 QKDLLFK, lies near the p38MAPK-dependent phosphorylation motif, GKDKS258 PKFQ, in the ␦Lf structure (Eukaryotic Linear Motifs tool in the ExPASy Bioinformatics Resource Portal). Moreover, we at least found that RGS3 expression in apoptotic HL-60 cells was delayed by the O-GlcNAcase inhibitor Thiamet G (Supplemental Fig. 4). We identified a SUMOylation motif PVS10 CIK13 RD near the phosphorylation/O-GlcNAcation cycling system at S10 in ␦Lf (SUMOplotTM Analysis Program in the ExPASy Bioinformatics Resource Portal). We suggested that the translocation system for ␦Lf to the cytoplasmic face of the plasma membrane used the phosphorylation/dephosphorylation cycling system at S258 , while translocation to the nucleus was mediated by the phosphorylation/O-GlcNAcation cycling system at S10 and the SUMOylation system at K13 (Liu et al., 2000). To examine other roles of ␦Lf, in silico analyses of the same promoter sequence, AGCACTTTGG, were performed, and we found expression of the ubiquitin-conjugating enzyme E2 E1 in the cancerous mammary gland MDA-MB-231 cells (Hoedt et al., 2014), suggesting the participation of ␦Lf in the maintenance of cell homeostasis. We identified the same promoter against ␦Lf at −468 bp upstream of the ATG start codon in the RGS3 gene
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(NG 029512.1) in this study. RGS3 expression was suppressed by Lf knockdown (Fig. 6). In contrast to RGS14, in the D/R12 subfamily, we at least confirmed the transcription of the B/R4 subfamily members RGS1, RGS3, RGS16, and RGS18 was increased in HL-60 cells at 24 h post-MnCl2 loading (Supplemental Fig. 5) (Bansal et al., 2007). In particular, RGS3 and RGS16 expression were delayed by Lf knockdown. When a participation of other chemotactic GiPCRs in cell lifespan, the ANAX1 N-terminus was reported to function as an antagonist in neutrophils via the fMet-Leu-Phe (fMLP) receptor (FPR) at the apoptotic microenvironment (Parente and Solito, 2004). In contrast to FPR and C5aR, we confirmed that the 60 kDa leukotriene B4 (LTB4) receptor (BLT1), which couples with the Gi protein but not BLT2, is synthesized de novo during MnCl2 -induced apoptosis (Supplemental Fig. 6) (Ihara et al., 2007). There is a CCAAT moiety at 143 nucleotides upstream of the ATG start codon in the BLT1 gene (NM 181657.3). We at least confirmed that rescuing HL-60 cells from MnCl2 -induced apoptosis with a combined administration of LTB4, fMLP and C5a was more efficient than that of a single administration of LTB4. It is likely that other de novosynthesized chemotactic Gi PCRs on apoptotic cells participate in shortening the cell lifespan. We have suggested a secretion of RP S19 polymer crosslinked by TGs not only from apoptotic cells in synovial tissues of rheumatoid arthritis and vascular lesions of atherosclerosis but also from neutrophils in acute inflammatory lesions or a production of RP S19 polymer cross-linked by an activated factor XIII in serum (Nishiura et al. 1996, 2010a,b, 2013; Shi et al., 2005). From this paper, we strongly speculated the roles of RP S19 polymer in cell clearance before undergoing to secondary necrosis of neutrophils by macrophages. This mechanism might protect host from tumorgenesis of anti-neutrophil cytoplasmic antibodyrelated vasculitis, such as Microscopic poly-vasculitis, Allergic granulomatosis-vasculitis and Wegener’s granulomatosis (KoseljKajtna et al., 2002). To clearly show a participation of RP S19 polymer in above diseases, we at least measure a concentration of RP S19 polymer in the patient serums by a sandwich enzymelinked immunosorbent assay system with anti-RP S19 and anti-C5a rabbit IgGs (Nishiura et al., 2010a,b). Conclusions Neutrophil lifespan is partially regulated by a balance between survival signals through constitutively active GPCRs and death signals through death receptors. Neutrophils using programmed cell death release RP S19 polymer. RP S19 C-terminus makes the complex with ␦Lf on the cytoplasmic face of the plasma membrane. ␦Lf moves into the nucleus and initiates the RGS3 promoter. The twostep inhibitory mechanism of ␦Lf from G␣i␥ protein of C5aR to G␣ subsets of constitutively active GPCRs shortens the neutrophil cell lifespan. Conflict of interest The authors declare no conflicts of interest. Acknowledgments Norie Araki (Department of Tumor Genetics and Biology, Faculty of Life Science, Kumamoto University Graduate School, 1-1-1 Honjo, Chuou-ku, Kumamoto 860-8556, Japan) helped to analyze the TOF-MS experiment. Grant numbers and sources of support: the Ministry of Education, Culture, Sports, Science, and Technology [26462863].
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The roles of ribosomal protein S19 C-terminus in a shortened neutrophil lifespan through delta lactoferrin Hiroshi Nishiura ∗ , Koji Yamanegi, Mutsuki Kawabe, Nahoko Kato-Kogoe, Naoko Yamada, Keiji Nakasho Division of Functional Pathology, Department of Pathology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan
a r t i c l e
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Article history: Received 23 January 2015 Received in revised form 25 February 2015 Accepted 1 May 2015 Available online 11 May 2015 Keywords: C5a receptor Delta lactoferrin Neutrophils Regulator of G protein signaling 3 Ribosomal protein S19
a b s t r a c t Cell lifespan is partially regulated by a balance between survival signals via constitutively active G proteincoupled receptors (GPCRs) and death signals via death receptors. We have demonstrated that neutrophils produce a mimic ligand of G protein-coupled C5a receptor (C5aR), ribosomal protein S19 (RP S19) polymer. In contrast to an original ligand C5a, RP S19 polymer induces not only inhibition of the guanine nucleotide exchange factor activity but also initiation of the regulator of G protein signaling 3 (RGS3) promoter in a RP S19 C-terminus dependent manner. To examine an antagonistic effect of the RP S19 C-terminus on G proteins, His-S-tagged C5a or C5a/RP S19, in which an RP S19 C-terminus is bound to the C5a C-terminus, was incubated with neutrophils, and a transcription factor delta lactoferrin (␦Lf) was identified as a specific binding protein via pull-down experiments. The S-tagged C5a-induced agonistic effects on chemotaxis, cytoplasmic Ca2+ influx and p38 mitogen-activated protein kinase phosphorylation were not changed by Lf knockdown and ␦Lf overexpression in neutrophil-like or macrophage-like cells, which were differentiated into mature cells from human promyelocytic leukemia HL-60 cells by dimethyl sulfoxide and phorbol-12-myristate-13-acetate, respectively. While, the S-tagged C5a/RP S19-induced antagonistic or agonistic effects on mature HL-60 neutrophil-like or macrophage-like cells were reversed by Lf knockdown and ␦Lf overexpression, respectively. Moreover, RGS3 expression was increased in another HL-60 neutrophil-like cells under spontaneous apoptosis induced by an apoptotic inducer MnCl2 . The RGS3 expression in apoptotic neutrophil-like cells was delayed not only by Lf knockdown but also by neutralization of the RP S19 polymer or C5aR. The inhibitory extension from G protein of C5aR to G␣ subsets of constitutively active GPCRs along with the RP S19 polymer-induced translocation of ␦Lf from the cytoplasmic face of the plasma membrane to the nucleus seems to shorten the neutrophil cell lifespan. © 2015 Elsevier GmbH. All rights reserved.
Introduction We have demonstrated that cell lifespan is partially regulated by a balance between a weak extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated survival signal via constitutively active heterotrimeric guanine nucleotide-regulating (G␣␥) proteincoupled receptors (GPCRs) and a strong apoptotic death signal via
Abbreviations: C5aR, C5a receptor; ␦Lf, delta lactoferrin; GPCR, G proteincoupled receptor; NY-1, anti-RP S19 rabbit IgGs; PMX-53, C5aR antagonistic peptide; RGS3, regulator of G protein signaling 3; RP S19, ribosomal protein S19; sLf, secreted form of Lactoferrin; TRPM, transient receptor potential melastatin. ∗ Corresponding author at: Division of Functional Pathology, Department of Pathology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan. Tel.: +81 79 845 6432; fax: +81 79 845 6431. E-mail address: [email protected] (H. Nishiura). http://dx.doi.org/10.1016/j.imbio.2015.05.006 0171-2985/© 2015 Elsevier GmbH. All rights reserved.
death receptors (Milligan, 2003; Dragovich et al., 1998; Nishiura et al., 2009). Neutrophils under spontaneous apoptosis produce an alternative G␣i ␥ protein-coupled C5a receptor (C5aR) ligand, ribosomal protein S19 (RP S19) polymer, which is distinct from the natural ligand C5a and is cross-linked by apoptosis-related activation of tissue transglutaminases (TGs) (Sanghavi et al., 1998; Knepper-Nicolai et al., 1998; Nishiura et al., 1996; Horino et al., 1998; Nishimura et al., 2001). The C5a-induced G␥ subset-dependent activation begins with the exchange of GDP to GTP in the G␣i ␥ protein through a conformational change in C5aR. This activation is limited by a posttranslational modification of the regulator of G protein signaling 3 (RGS3), which increases the GTPase activity of G␣ in an autocrine manner, and/or C-terminal phosphorylation of C5aR (Hsu et al., 2014; Kehrl, 2004; Ishii and Kurachi, 2003). Therefore, the ligandinduced strong ERK1/2 signal via the C5aR on neutrophils through
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calcium release-activated channels is suggested to participate in the regulation of cell lifespan length (Perianayagam et al., 2004; Brazil et al., 2014). However, C5aR deficient mice sometimes show unreasonable neutrophil infiltration into acute inflammation lesions in an antigen-specific manner (Hopken et al., 1996, 1997). We recently found that the C5aR on macrophages bound with the RP S19 polymer mimic use a similar two-step binding mechanism of C5a to mediate a weak G␥-dependent p38 mitogen-activated protein kinase (p38MAPK) signal through transient receptor potential melastatin (TRPM) Ca2+ /Mg2+ channels without receptor internalization (Nishiura et al., 2010a,b, 2011). The C5aR-mediated ERK1/2 signal induced by C5a is exchanged to the p38MAPK signal by simultaneous treatment with low concentrations of the phosphoinositide 3-kinase inhibitor LY294002 and phospholipase C inhibitors U73122. Conversely, binding affinity of the RP S19 polymer to the C5aR on macrophages is lower than that on neutrophils (Nishiura et al., 2011). The RP S19 polymer does not induce a C5aR-mediated downstream signal but stimulates RGS3 expression via the C5aR on neutrophils (Nishiura et al., 1998, 2009, 2010a,b). Therefore, we suggested that RGS3 expression via de novo-synthesized C5aR on neutrophils participates in the shortening of the cell lifespan (Nishiura, 2013; Nishiura et al., 2013). To examine the mechanism of C5aR-mediated RGS3 expression, we identified a different C-terminus (IAGQVAAANKKH) of RP S19 from the C5a C-terminus (Nishiura et al., 2010a,b). C5a/RP S19 was prepared by connecting IAGQVAAANKKH to the C-terminus of C5a with a mutation at Gly73Asp, and we confirmed the different C5aRmediated outputs (Oda et al., 2008; Revollo et al., 2005). His-Stagged C5a or C5a/RP S19 was incubated here with neutrophils, and the transcription factor delta lactoferrin (␦Lf) was identified as a specific binding molecule to the RP S19 C-terminus in neutrophils (Breton et al., 2004). Materials and methods Materials Recombinant C5a, C5a/RP S19 and ␦Lf were prepared with the pET32a-Rosetta gami(B) Lys-S system. His-S-tagged proteins for binding assays and S-tagged proteins for biological assays were sometimes coupled to BrCN-activated Sepharose 4B beads (5 mg/mL) (GE, Little Chalfont, UK). Phosphorylated and unphosphorylated anti-p38MAPK rabbit IgGs were produced by Cell Signaling Technology (MA, USA). Anti-ANXA3 rabbit IgGs was purchased from Proteintech Group, Inc. (IL, USA). Anti-Lf and anti-C5aR rabbit IgGs, fluorescein isothiocyanate (FITC)-conjugated anti-C5aR mouse IgG and FITC- and horseradish peroxidase (HRP)-conjugated anti-rabbit goat IgGs were purchased from Santa Cruz Biotechnology (CA, USA). Cells Human promyelocytic leukemia HL-60 cells were obtained from the RIKEN BioResource Center (Tsukuba, Japan). Neutrophil-like cells and macrophage-like cells were differentiated from HL-60 cells into mature cells by dimethyl sulfoxide and phorbol-12myristate 13-acetate, respectively (Zinzar et al., 1989). To make another neutrophil-like cells under spontaneous apoptosis, HL60 cells were undergone into apoptosis by a caspase 12 activator MnCl2 (Nishiura et al., 2009; Sanghavi et al., 1998; Knepper-Nicolai et al., 1998; Oubrahim et al., 2002). Peripheral blood was collected from healthy volunteers who provided written consent according to a protocol approved by the Research Ethics Committee of the Japanese Red Cross, Kumamoto Hospital and Kumamoto University (No. 439) in accordance with the Helsinki Declaration of 1975,
as revised in 2002. Monocytes and neutrophils were isolated from the heparinized venous blood of at least three different healthy donors (Matsubara et al., 1991). Enriched neutrophils with satisfactory purity (>98%) were prepared using Ficoll-PaqueTM PLUS (GE). The cell identities were confirmed by fluorescence-activated cell forting (FACS) analysis using anti-CD16b antibodies (BD, Tokyo, Japan).
Vectors and HL-60 cells cDNAs corresponding to sLf and ␦Lf were a kind gift from Prof CT Teng at the National Institutes of Health (Liu et al., 2003). The sLf and ␦Lf cDNAs were inserted into a pCAGGS-IRES neomycin-resistant vector (Fukushima et al., 1997). shLf lentivirus (Santa Cruz Biotechnology) was used to knockdown Lf mRNA. Successfully transformed cells were sorted using a BD FACSAriaTM II Cell Sorter. Conversely, to generate HL-60 transformed cells containing pCIN-␦Lf cDNA, linearized plasmid DNA was transformed into HL-60 cells by electroporation using a Gene Pulsar II (Bio-Rad, Berkeley, CA, USA). The optimal clone was selected based on its growth rate.
Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting His-S-tagged C5a/RP S19 or His-S-tagged C5a (10−7 M) was incubated for 10 min at 22 ◦ C in 20 mL of Ca2+ buffer containing neutrophils (107 cells/mL). The proteins bound to the His-S-tagged proteins were cross-linked using dithiobis (2 mM) for 30 min at 22 ◦ C (Pierce, IL, USA). The membrane was segmented using 2% Triton X-100 and 4% CHAPS (Dojindo), and whole proteins were applied to a Hi-Trap Ni2+ chelating column (GE). The bound proteins were eluted using 10 mM dithiothreitol buffer. Neutrophils (1 × 107 cells/mL) in 10 mL of PBS were treated with 1 mM diisopropyl fluorophosphate and 2% Triton X-100 for 10 min at 37 ◦ C. After sucrose gradient centrifugation, the cytoplasmic proteins in Ca2+ buffer were incubated with S-tagged ␦Lf beads for 10 min at 22 ◦ C. The bound proteins were separated from the beads with 50 mM glycine, pH 3.0. The bound proteins were suspended in lysis buffer (200 g/125 L) (7 M urea, 2 M thiourea, 4% CHAPS, 2% Triton X-100, 18 mM dithiothreitol, 0.5% immobilized pH gradient buffer, and 0.1% bromothymol blue) and applied to an Immobiline DryStrip (7 cm/pH 3.0–10.0) for 24 h at 22 ◦ C. The proteins on the DryStrip gel were arranged along the immobilized pH gradients in an Ettan IPGphor II Isoelectric Focusing Unit (GE). Four-step programs were run at 50 A current and 20 ◦ C: 300 V for 30 min in the step and hold mode, 1000 V for 30 min in the gradient mode, 5000 V for 90 min in the gradient mode, and 5000 V for 4 h in the step and hold mode. The proteins on the DryStrip gel were reduced in equilibration buffer A (50 mM Tris–HCl buffer [pH 8.8] containing 9 M urea, 30% glycerol, 2% SDS, 0.25% dithiothreitol, and 0.1% bromothymol blue) and buffer B (50 mM Tris–HCl buffer [pH 8.8] containing 9 M urea, 30% glycerol, 1% SDS, 4.5% iodoacetamide, and 0.1% bromothymol blue) for 15 min at 22 ◦ C and were loaded onto 12% SDS-PAGE gels. Total proteins were detected using silver staining. The proteins were electrophoretically transferred to an Immobilon Transfer MembraneTM (Millipore, MA, USA) using a semi-dry electroblotter (Sartorius, Göttingen, BRD) for 90 min with a current of 15 V. Primary antibodies were incubated (200 ng/mL) for 1 h at 22 ◦ C, and HRP-conjugated anti-rabbit IgGs goat IgGs secondary antibody (20 ng/mL) was incubated for 30 min at 22 ◦ C. The enhanced chemiluminescence reaction was performed using the ECL Plus Western Blotting Detection SystemTM (GE).
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Time-of-flight mass spectrometry Proteins in the SDS polyacrylamide gel were excised and digested with trypsin (10 g/mL) (GE) in 30 L of 50 mM NH4 HCO3 (pH 7.8) overnight at 37 ◦ C. The protein fragments were washed in ZipTip® C18 and eluted in 0.1% trifluoroacetic acid containing 70% acetonitrile. Approximately 0.5 L of the protein fragments was covered with 0.5 L of 5 mg/mL ␣-cyano-4-hydroxy-cinnamic acid matrix solution on a MALDI plate and analyzed in a 4700 MALDI TOF/TOF mass spectrometer (ABI, Foster, MA, USA). Chemotaxis assay Cells (2 × 106 cells/mL) were prepared in Ca2+ buffer (HBSS containing 20 mM HEPES and 3% BSA, pH 7.4) for the chamber assay. The assay was performed for 90 min using a nucleopore filter with a pore size of 5 m for HL-60 cells. The total number of cells that migrated beyond the lower surface of the membrane was counted in five high-power microscopic fields. Calcium imaging Cells (2 × 106 cells/mL) were loaded with the calcium-sensitive dye Fura 2-AM (1 M) in Ca2+ buffer for 30 min at 37 ◦ C (Dojindo, Kumamoto, Japan). Samples were placed directly into the cell suspension in a cuvette after 5 min of baseline recording. Recordings were made with an F-2500 calcium imaging system (FL Solutions; Hitachi, Tokyo, Japan), which calculated the ratio of fluorescence signals obtained at 37 ◦ C with excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Statistical analysis Each result was confirmed by multiple experiments with a minimum of three samples. Statistical significance was calculated by non-parametric and parametric tests with two-way analysis of variance. The values are presented as the means ± SD. A Pvalue < 0.05 was considered to be statistically significant and is presented as P < 0.05 (*) or P < 0.01 (**). Results His-S-tagged C5a/RP S19 binds to neutrophil-specific proteins We suggested that the RP S19 C-terminus additionally binds neutrophil-specific proteins for closing an interactive space between the active C5aR and the GDP form of G␣i ␥ protein. To identify the binding partners, His-S-tagged C5a/RP S19 or His-S-tagged C5a was incubated with neutrophils. In addition to neutrophil proteins that bound the His-S-tagged C5a, the specific proteins that bound to His-S-tagged C5a/RP S19 were identified via pull-down experiments and assessed using 2D SDS-PAGE prior to preparing samples for TOF-MS. To confirm complexes of the RP S19 C-terminus with neutrophil-specific molecules on plasma membrane, we at least verified a presence of C5aR in the samples by Western blotting (data not shown). The specific protein spots corresponding to an apparent molecular weight (MW) of 75 kDa and an isoelectric point (pI) of 7.0–8.5 were specifically observed in silver stained 2D gels (Fig. 1). Six specific protein spots corresponding to MW of 74–78 kDa and pI of 7.0–8.5 were detected by anti-Lf rabbit IgGs (Fig. 1B upper). However, the N-terminal amino acid residues were not detected in ten trypsin-digested fragments of the secreted form of lactoferrin (sLf) (Supplemental Fig. 1). To confirm whether the RP S19 C-terminus bound to sLf (calculated MW, 78 kDa, calculated pI, 8.5 using the theoretical pI and MW computation tool of ExPASy) on the extracellular face of the
Fig. 1. His-S-tagged C5a/RP S19 binding protein. Proteins on the plasma membrane of neutrophils bound to either (A) His-S-tagged C5a or (B) His-S-tagged C5a/RP S19 were applied to a 2D-SDS gel and silver stained (n = 6). After transferring the proteins to a membrane, Western blotting was performed using anti-Lf rabbit IgGs.
plasma membrane or to ␦Lf (calculated MW, 74 kDa, calculated pI, 8.0) on the cytoplasmic face of the plasma membrane, protein spots in the cell fractions of the 2D gels and the transferred membranes were incubated with silver staining solution (Fig. 2 upper) and antiLf rabbit IgGs (Fig. 2 lower), respectively. The 78 kDa protein spots with a pI of 8.5 and 8.2 were mainly detected in the nuclear and the granular fractions, respectively. Conversely, the main 74 kDa protein spot with a pI of 7.0 was detected in the membrane fraction. These data indicate that the 78 kDa sLf is trafficked through the endoplasmic reticulum-Golgi secretion pathway, while the 74 kDa ␦Lf localizes on the cytoplasmic face of the plasma membrane. Moreover, we generated fresh preparations of BrCN-activated Sepharose 4B beads coupled with S-tagged C5a or S-tagged C5a/RP S19 without a His tag and incubated them with neutrophils or the membrane fractions of neutrophils and macrophages, respectively. In contrast to S-tagged C5a beads, S-tagged C5a/RP S19 beads were strongly bound to the 74 kDa ␦Lf, not only in neutrophils (Fig. 3A) but also in neutrophil membrane fractions but not macrophage membrane fractions (Fig. 3B). S-tagged C5a/RP S19 switches from an antagonist to an agonist of C5aR on HL-60 neutrophil-like cells upon ıLf knockdown To validate the antagonistic or agonistic effects of ␦Lf on the C5aR, we prepared Lf-knockdown HL-60 neutrophil-like cells or ␦Lf-overexpressing HL-60 macrophage-like cells and analyzed the relationship between the ratio of ␦Lf to sLf/C5aR/actin expression (␦Lf ratio) and relevant biological processes, such as chemotaxis, cytoplasmic Ca2+ influx, and p38MAPK signaling. The Lf ratio was calculated based on the actin expression density (= ␦Lf + sLf/C5aR/actin) in Western blots using ImageJ 1.46 (National Institutes of Health, MD, USA). The Lf ratio in HL-60 neutrophil-like cells decreased from 2.08 to 0.82 after transfection with Lf shRNA (Fig. 4A). The antagonistic effect of 10−8 M S-tagged C5a/RP S19 on the neutrophil C5aR was replaced by an agonistic effect, as reflected by chemotaxis, cytoplasmic Ca2+ influx, and p38MAPK phosphorylation upon Lf knockdown (Fig. 4B–D). Moreover, the switch of
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Fig. 2. ␦Lf localization. The proteins in neutrophil cell fractions were applied to 2D-SDS gels and silver stained (n = 3). After transferring the proteins to a membrane, Western blotting was performed using anti-Lf rabbit IgGs.
Fig. 3. The complex of S-tagged C5a/RP S19 with ␦Lf on the membrane fractions of neutrophils. S-tagged C5a and S-tagged C5a/RP S19 beads were prepared and incubated with neutrophils (A) or the membrane fractions of neutrophils or macrophages (B), respectively. After centrifugation, the proteins with (Ppt: pellet) and without (Sup: supernatant) S-tagged protein beads were applied to 2D-SDS gels and transferred to a membrane. The 74 kDa delta lactoferrin was detected by Western blotting using anti-Lf rabbit IgGs (n = 3).
Fig. 4. An exchange of antagonistic functions of S-tagged C5a/RP S19 to agonistic functions under ␦Lf knockdown in HL-60 neutrophil-like cells. (A) Lf expression levels in HL-60 neutrophil-like cells containing control or Lf shRNA were assessed via Western blot (n = 3). (B) The chemotactic potential of the control and Lf-knockdown HL-60 neutrophil-like cells against S-tagged C5a and S-tagged C5a/RP S19 was measured in a 48-well chemotaxis chamber (n = 6). (C) The cytoplasmic Ca2+ influx induced by S-tagged C5a or S-tagged C5a/RP S19 was measured in LF-knockdown HL-60 neutrophil-like cells (n = 4). (D) p38MAPK phosphorylation induced by S-tagged C5a or S-tagged C5a/RP S19 was assessed in the Lf-knockdown HL-60 neutrophil-like cells by Western blotting (n = 3).
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Fig. 5. An exchange of agonistic functions of S-tagged C5a/RP S19 to partially agonistic functions under ␦Lf overexpression in HL-60 macrophage-like cells. (A) Lf and C5aR expression levels in HL-60 macrophage-like cells containing control, Lf, and ␦Lf cDNAs were assessed via Western blot (n = 3). (B) C5aR expression on the Lf and ␦Lf HL-60 macrophage-like cells was detected by FACS (n = 3). (C) The chemotactic potential of mock, Lf, and ␦Lf HL-60 macrophage-like cells against S-tagged C5a and S-tagged C5a/RP S19 was measured in a 48-well chemotaxis chamber (n = 6). (D) Cytoplasmic Ca2+ influx induced by S-tagged C5a and S-tagged C5a/RP S19 was assessed in the mock, Lf, and ␦Lf HL-60 macrophage-like cells (n = 4). (E) p38MAPK phosphorylation induced by S-tagged C5a and S-tagged C5a/RP S19 in the presence or absence of 1 mM MgCl2 was assessed in mock, Lf, and ␦Lf-overexpressing HL-60 macrophage-like cells via Western blotting (n = 3).
S-tagged C5a/RP S19 from a C5aR antagonist to a C5aR agonist by Lf knockdown was also observed in human mast cell-1 (HMC-1) cells (Supplemental Fig. 2). In contrast to the ␦Lf ratio for the C5aR in macrophage-like cells differentiated from the mock HL-60 cells (1.55), the overexpression of sLf and ␦Lf shifted the ␦Lf ratios to 0.59 and 2.16, respectively (Fig. 5A). No significant difference in C5aR expression was noted between the sLf- and ␦Lf-overexpressing HL-60 macrophage-like cells (Fig. 5B). However, the effect on the biological processes of the mock HL-60 macrophage-like cells upon treatment with 10−8 M S-tagged C5a/RP S19 was inversely proportional to the ␦Lf ratio, as shown by chemotaxis, cytoplasmic Ca2+ influx, and p38MAPK phosphorylation (Fig. 5C–E). To further confirm the p38MAPK phosphorylation through TRPM Ca2+ /Mg2+ channels, HL-60 macrophage-like cells were stimulated with either S-tagged C5a or S-tagged C5a/RP S19 in a simultaneous presence of a Mg2+ channel blocker
1 mM MgCl2 and detected p38MAPK phosphorylation (Fig. 5E) (Nishiura et al., 2011). In contrast to the sLf-overexpressing HL60 macrophage-like cells, a significant decrement of the S-tagged C5a/RP S19-induced p38MAPK phosphorylation was detected in the ␦Lf-overexpressing HL-60 macrophage-like cells. Conversely, S-tagged C5a-induced biological responses were not reflected by the ␦Lf ratio. In addition, we observed similar biological effects in sLf and ␦Lf U937 macrophage-like cells upon treatment with 10−8 M S-tagged C5a/RP S19 (data not shown). The f-Met-Leu-Phe-induced chemotactic potential of HL-60 and U937 macrophage-like cells was not modified by ␦Lf overexpression (data not shown), as previously shown with CD14+ macrophages (Laemmli, 1970). Strangely, the sLf overexpression also endowed U937 macrophage-like cells with a high intensity of cytoplasmic Ca2+ influx. We hypothesized that neutrophil C5aR is specifically blocked by the complex of the RP S19 C-terminus with ␦Lf.
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Fig. 6. The ␦Lf-RGS3 pathway. (A) Lf in control and Lf-knockdown HL-60 cells was detected by Western blotting (n = 4). (B) RGS3 in control and Lf-knockdown HL-60 cells post-MnCl2 loading was detected by Western blotting (n = 4). (C) RGS3 in control and Lf-knockdown HL-60 cells post-MnCl2 loading in the presence of control PBS, anti-human RP S19 rabbit IgGs, or PMX-53 was detected by Western blotting (n = 4).
Postponement of RGS3 expression in HL-60 cells under spontaneous apoptosis upon ıLf knockdown To examine a contribution of ␦Lf on RGS3 production in neutrophils under spontaneous apoptosis, an effect of Lf knockdown on RGS3 expression was observed in HL-60 cells during apoptosis. We confirmed an almost 67% depletion of total sLf and ␦Lf protein expression in control HL-60 cells by Lf knockdown (Fig. 6A). In this experimental setting, the expression of RGS3 and its truncated variant RGS3T was delayed until 48 h post-MnCl2 loading by Lf knockdown (Fig. 6B) (Falk et al., 1980). To further confirm that the interaction between the RP S19 C-terminus and ␦Lf through RP S19 polymer-C5aR binding induces RGS3 expression, the RP S19 polymer was neutralized with anti-RP S19 rabbit IgGs (NY-1) or C5aR was blocked by a C5aR antagonistic peptide PMX-53 (Fig. 6C). The expression of RGS3 and RGS3T during apoptosis was blocked, even in control HL-60 cells. Discussion We have demonstrated that a conditional change to the apoptotic microenvironment sometimes enables transcription factor NF-Y to initiate the C5aR promoter and type II TGs with a crosslinkage of RP S19 in all cell types. After autocrine binding of the K38 LAKHK moiety of one RP S19 molecule in the RP S19 polymer to the C5aR N-terminus on neutrophils under spontaneous apoptosis, the L131 DR moiety of another RP S19 molecule in the RP S19 polymer geometrically enters into an active pocket of the C5aR on the extracellular face of the plasma membrane (Nishiura et al., 2010a,b). The dissociated G␥-mediated activation of p38MAPK induces ␦Lf phosphorylation and a translocation to the cytoplasmic face of the plasma membrane (Figs. 2C and 3B). RP S19 C-terminus (K143 KH) increases the chance for complex formation with ␦Lf, resulting in a complete interference with a further interaction of active C5aR with the GDP form of G␣i ␥ protein. ␦Lf next moves into the nucleus by a complicated translocation system and works as a transcription factor, initiating the RGS3 promoter to increase the GTPase activity of G␣ in neutrophils under spontaneous apoptosis (Figs. 2A and 6). Two-step inhibitory mechanisms of ␦Lf against G protein seem to launch programmed cell death. Moreover, the initiation of RGS3 promoter was also observed in apoptotic HMC-1 cells induced by MnCl2 (data not shown). Conversely, the de novosynthesized GPCRs on apoptotic cells and their ligands, such as ANXA1, Resolving, and others, are also reported to participate in shortening the cell lifespan (Dalli et al., 2013; El Kebir et al., 2012). In studying transcriptional mechanisms of ␦Lf, sLf and RGS3 during apoptosis, we found a NF-Y binding CCAAT moiety 17 nucleotides upstream of the ATG start codon in the sLf gene (NM 001199149.1), but not the RGS3 gene. Conversely, ␦Lf expres-
sion is commonly suppressed in cancer cell lines and lung cancer cells (Benaissa et al., 2005; Siebert and Huang, 1997). We at least found a ␦Lf transcript in apoptotic HL-60 cells during apoptosis induced by MnCl2 , which was relatively delayed compared to sLf expression (Supplemental Fig. 3). These data indicated an alternative promoter in exon 1. There is a GGAA moiety at 71 nucleotides downstream of the ATG start codon in the sLf gene, which binds to the Ets-1 transcription factor, indicating a P2 promoter in exon 1 for specific ␦Lf expression (Liu et al., 2003). Recruitment of histone deacetylase to the P2 promoter is regulated by the phosphorylation/SUMOylation cycling system of Ets-1 (Findlay et al., 2013). There is a complicated translocation system of ␦Lf from the cytoplasmic membrane to the nucleus via the phosphorylation/OGlcNAcation cycling system at S10 and/or the SUMOylation system (Mariller et al., 2012). In the present study, we observed not only the main protein spot of ␦Lf with a MW of 74 kDa and pI of 7.0 but also a minor protein spot with the same pI but with a higher MW in the membrane fraction (Fig. 2C). Similar protein spots to the latter of these were also observed in the granular fraction (Fig. 2B). Although we have ignored the weak C5aR-mediated p38MAPK signal that lacks biological outputs, an MAPK interacting molecule carrying the docking motif K580 VERLKQVLL and a p38MAPK-dependent phosphorylation motif GKDKS258 PKFQ are found in ␦Lf (AAA59511.1) (Eukaryotic Linear Motifs tool and NetPhosK 1.0 Server in the ExPASy Bioinformatics Resource Portal). Two motifs seem to turn in the same direction in the binding pocket for p38MAPK in the ␦Lf structure (MMDB ID: 34912) (Thomassen et al., 2005). In contrast, we found that a protein phosphatase 1 catalytic subunit interacting motif, G269 QKDLLFK, lies near the p38MAPK-dependent phosphorylation motif, GKDKS258 PKFQ, in the ␦Lf structure (Eukaryotic Linear Motifs tool in the ExPASy Bioinformatics Resource Portal). Moreover, we at least found that RGS3 expression in apoptotic HL-60 cells was delayed by the O-GlcNAcase inhibitor Thiamet G (Supplemental Fig. 4). We identified a SUMOylation motif PVS10 CIK13 RD near the phosphorylation/O-GlcNAcation cycling system at S10 in ␦Lf (SUMOplotTM Analysis Program in the ExPASy Bioinformatics Resource Portal). We suggested that the translocation system for ␦Lf to the cytoplasmic face of the plasma membrane used the phosphorylation/dephosphorylation cycling system at S258 , while translocation to the nucleus was mediated by the phosphorylation/O-GlcNAcation cycling system at S10 and the SUMOylation system at K13 (Liu et al., 2000). To examine other roles of ␦Lf, in silico analyses of the same promoter sequence, AGCACTTTGG, were performed, and we found expression of the ubiquitin-conjugating enzyme E2 E1 in the cancerous mammary gland MDA-MB-231 cells (Hoedt et al., 2014), suggesting the participation of ␦Lf in the maintenance of cell homeostasis. We identified the same promoter against ␦Lf at −468 bp upstream of the ATG start codon in the RGS3 gene
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(NG 029512.1) in this study. RGS3 expression was suppressed by Lf knockdown (Fig. 6). In contrast to RGS14, in the D/R12 subfamily, we at least confirmed the transcription of the B/R4 subfamily members RGS1, RGS3, RGS16, and RGS18 was increased in HL-60 cells at 24 h post-MnCl2 loading (Supplemental Fig. 5) (Bansal et al., 2007). In particular, RGS3 and RGS16 expression were delayed by Lf knockdown. When a participation of other chemotactic GiPCRs in cell lifespan, the ANAX1 N-terminus was reported to function as an antagonist in neutrophils via the fMet-Leu-Phe (fMLP) receptor (FPR) at the apoptotic microenvironment (Parente and Solito, 2004). In contrast to FPR and C5aR, we confirmed that the 60 kDa leukotriene B4 (LTB4) receptor (BLT1), which couples with the Gi protein but not BLT2, is synthesized de novo during MnCl2 -induced apoptosis (Supplemental Fig. 6) (Ihara et al., 2007). There is a CCAAT moiety at 143 nucleotides upstream of the ATG start codon in the BLT1 gene (NM 181657.3). We at least confirmed that rescuing HL-60 cells from MnCl2 -induced apoptosis with a combined administration of LTB4, fMLP and C5a was more efficient than that of a single administration of LTB4. It is likely that other de novosynthesized chemotactic Gi PCRs on apoptotic cells participate in shortening the cell lifespan. We have suggested a secretion of RP S19 polymer crosslinked by TGs not only from apoptotic cells in synovial tissues of rheumatoid arthritis and vascular lesions of atherosclerosis but also from neutrophils in acute inflammatory lesions or a production of RP S19 polymer cross-linked by an activated factor XIII in serum (Nishiura et al. 1996, 2010a,b, 2013; Shi et al., 2005). From this paper, we strongly speculated the roles of RP S19 polymer in cell clearance before undergoing to secondary necrosis of neutrophils by macrophages. This mechanism might protect host from tumorgenesis of anti-neutrophil cytoplasmic antibodyrelated vasculitis, such as Microscopic poly-vasculitis, Allergic granulomatosis-vasculitis and Wegener’s granulomatosis (KoseljKajtna et al., 2002). To clearly show a participation of RP S19 polymer in above diseases, we at least measure a concentration of RP S19 polymer in the patient serums by a sandwich enzymelinked immunosorbent assay system with anti-RP S19 and anti-C5a rabbit IgGs (Nishiura et al., 2010a,b). Conclusions Neutrophil lifespan is partially regulated by a balance between survival signals through constitutively active GPCRs and death signals through death receptors. Neutrophils using programmed cell death release RP S19 polymer. RP S19 C-terminus makes the complex with ␦Lf on the cytoplasmic face of the plasma membrane. ␦Lf moves into the nucleus and initiates the RGS3 promoter. The twostep inhibitory mechanism of ␦Lf from G␣i␥ protein of C5aR to G␣ subsets of constitutively active GPCRs shortens the neutrophil cell lifespan. Conflict of interest The authors declare no conflicts of interest. Acknowledgments Norie Araki (Department of Tumor Genetics and Biology, Faculty of Life Science, Kumamoto University Graduate School, 1-1-1 Honjo, Chuou-ku, Kumamoto 860-8556, Japan) helped to analyze the TOF-MS experiment. Grant numbers and sources of support: the Ministry of Education, Culture, Sports, Science, and Technology [26462863].
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