RESUMEN
Given the role of macrophage-derived high mobility group box 1 (HMGB1) in chemotherapy-induced peripheral neuropathy (CIPN) caused by paclitaxel, we analyzed the role of HMGB1 and macrophages in the CIPN caused by bortezomib, a proteasome-inhibiting chemotherapeutic agent used for the treatment of multiple myeloma. Repeated administration of bortezomib caused CIPN accompanied by early-stage macrophage accumulation in the dorsal root ganglion. This CIPN was prevented by an anti-HMGB1-neutralizing antibody, thrombomodulin alfa capable of accelerating thrombin-dependent degradation of HMGB1, antagonists of the receptor for advanced glycation end-products (RAGE) and C-X-C motif chemokine receptor 4 (CXCR4), known as HMGB1-targeted membrane receptors, or macrophage depletion with liposomal clodronate, as reported in a CIPN model caused by paclitaxel. In macrophage-like RAW264.7 cells, bortezomib as well as MG132, a well-known proteasome inhibitor, caused HMGB1 release, an effect inhibited by caspase inhibitors but not inhibitors of NF-κB and p38 MAP kinase, known to mediate paclitaxel-induced HMGB1 release from macrophages. Bortezomib increased cleaved products of caspase-8 and caused nuclear fragmentation or condensation in macrophages. Repeated treatment with the caspase inhibitor prevented CIPN caused by bortezomib in mice. Our findings suggest that bortezomib causes caspase-dependent release of HMGB1 from macrophages, leading to the development of CIPN via activation of RAGE and CXCR4.
Asunto(s)
Antineoplásicos/efectos adversos , Bortezomib/efectos adversos , Proteína HMGB1/metabolismo , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Animales , Apoptosis , Modelos Animales de Enfermedad , Masculino , RatonesRESUMEN
BACKGROUND: Macrophage-derived high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) protein, plays a key role in the development of chemotherapy-induced peripheral neuropathy (CIPN) caused by paclitaxel in rodents. Endothelial thrombomodulin (TM) promotes thrombin-induced degradation of HMGB1, and TMα, a recombinant human soluble TM, abolishes peripheral HMGB1-induced allodynia in mice. We thus examined whether HMGB1, particularly derived from macrophages, contributes to oxaliplatin-induced neuropathy in mice and analyzed the anti-neuropathic activity of the TM/thrombin system. METHODS: CIPN models were created by the administration of oxaliplatin in mice and rats, and the nociceptive threshold was assessed by von Frey test or paw pressure test. Macrophage-like RAW264.7 cells were stimulated with oxaliplatin in vitro. Proteins were detected and/or quantified by Western blotting, immunostaining, or enzyme-linked immunosorbent assay. RESULTS: Intraperitoneal administration of an anti-HMGB1-neutralizing antibody (AB) at 1 mg/kg prevented the oxaliplatin-induced allodynia in mice and rats. Antagonists of Toll-like receptor (TLR) 4, receptor for advanced glycation end products (RAGE) and CXCR4 among the HMGB1-targeted pro-nociceptive receptors, also mimicked the anti-neuropathic activity of AB in mice. Macrophage accumulation in the sciatic nerve was observed in mice treated with paclitaxel, but not oxaliplatin, and neither macrophage depletion nor inhibitors of macrophage activation affected oxaliplatin-induced allodynia. Oxaliplatin was 10- to 100-fold less potent than paclitaxel in releasing HMGB1 from macrophage-like RAW264.7 cells. Like AB, TMα at 10 mg/kg prevented the oxaliplatin-induced allodynia in mice as well as rats, an effect abolished by argatroban at 10 mg/kg, a thrombin inhibitor. The anti-neuropathic activity of TMα in oxaliplatin-treated mice was suppressed by oral anticoagulants such as warfarin at 1 mg/kg, dabigatran at 75 mg/kg, and rivaroxaban at 10 mg/kg, but not antiplatelet agents such as aspirin at 50 mg/kg and clopidogrel at 10 mg/kg. Repeated administration of the anticoagulants gradually developed neuropathic allodynia and elevated plasma HMGB1 levels in mice treated with a subeffective dose of oxaliplatin. CONCLUSIONS: Our data thus suggests a causative role of HMGB1 derived from non-macrophage cells in oxaliplatin-induced peripheral neuropathy and a thrombin-dependent anti-neuropathic activity of exogenous TMα and, most probably, endogenous TM.
Asunto(s)
Anticoagulantes/administración & dosificación , Proteína HMGB1/metabolismo , Oxaliplatino/toxicidad , Enfermedades del Sistema Nervioso Periférico/prevención & control , Trombina/metabolismo , Trombomodulina/metabolismo , Animales , Anticoagulantes/efectos adversos , Antineoplásicos/toxicidad , Masculino , Ratones , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Células RAW 264.7 , Ratas , Ratas Wistar , RoedoresRESUMEN
We tested whether genetic deletion of Cav3.2 T-type Ca2+ channels abolishes hydrogen sulfide (H2S)-mediated pain signals in mice. In Cav3.2-expressing HEK293 cells, Na2S, an H2S donor, at 100 µM clearly increased Ba2+ currents, as assessed by whole-cell patch-clamp recordings. In wild-type C57BL/6 mice, intraplantar and intracolonic administration of Na2S evoked mechanical allodynia and visceral nociceptive behavior, respectively, which were abolished by TTA-A2, a T-type Ca2+ channel blocker. In Cav3.2-knockout mice of a C57BL/6 background, Na2S caused neither somatic allodynia nor colonic nociception. Our study thus provides definitive evidence for an essential role of Cav3.2 in H2S-dependent somatic and colonic pain.
Asunto(s)
Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/metabolismo , Sulfuro de Hidrógeno/farmacología , Dolor Nociceptivo/metabolismo , Transducción de Señal/efectos de los fármacos , Dolor Visceral/metabolismo , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Wistar , Canal Catiónico TRPA1/metabolismoRESUMEN
Bortezomib, a first-line agent for treatment of multiple myeloma, exhibits anticancer activity through proteasome inhibition. However, bortezomib-induced peripheral neuropathy (BIPN) is one of the most serious side effects. Since decreased proteasomal degradation of Cav3.2 T-type calcium channels in the primary afferents is involved in persistent pain, we investigated whether BIPN involves increased protein levels of Cav3.2 in mice. Six repeated i.p. administrations of bortezomib for 12 days developed persistent mechanical allodynia. Systemic administration of novel T-type calcium channel blockers, (2R/S)-6-prenylnaringenin and KTt-45, and of TTA-A2, the well-known blocker, reversed the BIPN. Ascorbic acid, known to block Cav3.2, but not Cav3.1 or 3.3, and silencing of Cav3.2 gene also suppressed BIPN. Protein levels of Cav3.2 in the dorsal root ganglion (DRG) at L4-L6 levels increased throughout days 1-21 after the onset of bortezomib treatment. Protein levels of USP5, a deubiquitinating enzyme that specifically inhibits proteasomal degradation of Cav3.2, increased in DRG on days 3-21, but not day 1, in bortezomib-treated mice. In DRG-derived ND7/23 cells, bortezomib increased protein levels of Cav3.2 and T-channel-dependent currents, as assessed by a patch-clamp method, but did not upregulate expression of Cav3.2 mRNA or USP5 protein. MG-132, another proteasome inhibitor, also increased Cav3.2 protein levels in the cultured cells. Given the previous evidence for USP5 induction following nociceptor excitation, our data suggest that BIPN involves the increased protein levels of Cav3.2 in nociceptors through inhibition of proteasomal degradation of Cav3.2 by bortezomib itself and then by USP5 that is upregulated probably in an activity-dependent manner.
Asunto(s)
Antineoplásicos/toxicidad , Bortezomib/toxicidad , Canales de Calcio Tipo T/biosíntesis , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/metabolismo , Inhibidores de Proteasoma/toxicidad , Animales , Canales de Calcio Tipo T/deficiencia , Canales de Calcio Tipo T/genética , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Técnicas de Silenciamiento del Gen/métodos , Masculino , Ratones , Enfermedades del Sistema Nervioso Periférico/genética , RatasRESUMEN
We examined the effect of repeated cold (RC) stress on cyclophosphamide (CPA)-induced cystitis/bladder pain in mice, in relation to macrophage activity. CPA, given i.p. at 400 mg/kg, caused bladder pain symptoms accompanying cystitis in both unstressed and RC-stressed mice, which were prevented by the macrophage inhibitor minocycline. A low dose, that is, 200 mg/kg, of CPA still produced bladder pain symptoms in unstressed but not RC-stressed mice. Lipopolysaccharide-induced cytokine production in peritoneal macrophages from RC-stressed mice was less than that from unstressed mice. Thus, RC stress appears to reduce CPA-induced bladder pain in mice, which may be associated with the decreased macrophage activity.
Asunto(s)
Frío , Ciclofosfamida/efectos adversos , Cistitis/inducido químicamente , Cistitis/inmunología , Activación de Macrófagos/efectos de los fármacos , Dolor/inducido químicamente , Estrés Fisiológico , Vejiga Urinaria/efectos de los fármacos , Animales , Citocinas/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Masculino , Ratones , Minociclina/farmacología , Dolor/inmunología , Vejiga Urinaria/inmunologíaRESUMEN
BACKGROUND: Protein production as secretory-form is a powerful tool in industrial enzyme production due to the simple purification procedure. Streptomyces lividans is a versatile host for secretory production of useful proteins. In order to expand the amount of secreted protein, signal peptide sequences, which encourage protein secretion from inside cell to extracellular environment, are one of the most significant factors. In this study, we focused on Streptomyces lividans as a host strain to secrete useful proteins, and screened for signal peptides from the biomass-degradation enzymes derived from Thermobifida fusca YX and S. lividans. RESULTS: Three candidate signal peptides were isolated and evaluated for their protein secretion ability using ß-glucosidase derived from T. fusca YX, which is a non-secreted protein, as a model protein. Using S. lividans xylanase C signal peptide, the amount of produced the ß-glucosidase reached 10 times as much as that when using Streptomyces cinnamoneus phospholipase D signal peptide, which was identified as a versatile signal peptide in our previous report. In addition, the introduction of the ß-glucosidase fused to xylanase C signal peptide using two kinds of plasmid, pUC702 and pTYM18, led to further protein secretion, and the maximal level of produced the ß-glucosidase increased up to 17 times (1.1 g/l) compared to using only pUC702 carrying the ß-glucosidase fused to S. cinnamoneus phospholipase D signal peptide. CONCLUSION: In the present study, we focused on signal peptide sequences derived from biomass degradation enzymes, which are usually secreted into the culture supernatant, and screened for signal peptides leading to effective protein secretion. Using the signal peptides, the hyper-protein secretion system was successfully demonstrated for the cytoplasmic ß-glucosidase.
Asunto(s)
Proteínas Bacterianas/metabolismo , Señales de Clasificación de Proteína/genética , Streptomyces lividans/enzimología , beta-Glucosidasa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , Datos de Secuencia Molecular , Transporte de Proteínas , Streptomyces lividans/genética , Streptomyces lividans/metabolismo , beta-Glucosidasa/genéticaRESUMEN
We screened for high-activity endoglucanase (EG) as a first step toward the creation of cellulose-assimilating Streptomyces lividans transformants. EGs derived from Thermobifida fusca YX, Tfu0901, and S. lividans, cellulase B (CelB), were successfully expressed. Genes encoding Tfu0901 or CelB were introduced into S. lividans using the integrative vector pTYM18 and the high-copy-number vector pUC702, and EG activity was detected in the supernatant of each transformant. To achieve coexpression of EG and transglutaminase, the transglutaminase gene was introduced into EG-secreting S. lividans using pUC702. S. lividans coexpressing EG and transglutaminase effectively assimilated phosphoric acid swollen cellulose. The yield of Streptomyces cinnamoneus transglutaminase in the culture supernatant was 7.2 mg/L, which was 18 times higher than that of the control strain. To demonstrate the versatility of our system, we also created an EG-producing S. lividans transformant capable of coexpressing endoxylanase. The EG-secreting S. lividans transformants constructed here can be used to produce other useful compounds through cellulose fermentation.
Asunto(s)
Carbono/metabolismo , Celulasa/metabolismo , Celulosa/metabolismo , Ingeniería Metabólica/métodos , Streptomyces lividans/enzimología , Streptomyces lividans/metabolismo , Biotecnología/métodos , Celulasa/genética , Expresión Génica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces lividans/genética , Streptomyces lividans/crecimiento & desarrollo , Transglutaminasas/genética , Transglutaminasas/metabolismoRESUMEN
Transglutaminase from Streptoverticillium cinnamoneum (StvcMTG) was produced using recombinant Streptomyces lividans. When grown on glycerol and xylose as sole carbon sources, S. lividans/StvcMTG produced 360 and 530 mg of StvcMTG per liter, respectively. With starch and xylan, the strain produced 230 and 400mg of StvcMTG per liter, respectively. Recombinant S. lividans/encP, which expresses phenylalanine ammonia lyase from Streptomyces maritimus, produced 160 mg/L of cinnamic acid from cellulose. These results show that S. lividans can assimilate various carbon sources and produce useful compounds in desirable quantities.
Asunto(s)
Carbono/metabolismo , Cinamatos/metabolismo , Mejoramiento Genético/métodos , Streptomyces/clasificación , Streptomyces/enzimología , Transglutaminasas/metabolismo , Especificidad de la Especie , Streptomyces/genéticaRESUMEN
Cinnamic acid production was demonstrated using Streptomyces as a host. A gene encoding phenylalanine ammonia lyase (PAL) from Streptomyces maritimus was introduced into Streptomyces lividans, and its expression was confirmed by Western blot analysis. After 4 days cultivation using glucose as carbon source, the maximal level of cinnamic acid reached 210 mg/L. When glycerol (30 g/L) was used as carbon source, the maximal level of produced cinnamic acid reached 450 mg/L. In addition, using raw starch, xylose or xylan as carbon source, the maximal level of cinnamic acid reached 460, 300, and 130 mg/L, respectively. We demonstrated that S. lividans has great potential to produce cinnamic acid as well as other aromatic compounds.