Effect of tunicamycin on the synthesis, processing, and secretion of pro-opiomelanocortin peptides in mouse pituitary cells

Pro-opiomelanocortin (POMC) is glycosylated and proteolytically cleaved to produce a number of smaller peptide hormones including adrenocorticotropic hormone (ACTH) and endorphin in mammalian pituitary and the mouse pituitary cell line AtT-20/D16v. When glycosylation of POMC is inhibited in AtT-20 cells with the drug tunicamycin, a 26,000-dalton protein appears in place of the glycosylated 29,000- and 32,000-dalton forms of POMC. The 26,000-dalton form found in tunicamycin-treated cells has the same [35S]methionine tryptic peptides as 29,000- and 32,000-dalton POMC, indicating that the decrease in apparent mass is most likely due to loss of carbohydrate and not to changes in the peptide backbone. The 4,500-dalton form of alpha(1-39)ACTH and the 3,000- and 11,000-dalton forms of endorphin are all present in tunicamycin-treated cells. The glycosylated form of alpha(1-39)ACTH, however, is missing and the glycosylated ACTH intermediates are replaced by unglycosylated ACTH intermediates. Pulse-chase studies demonstrate that the 26,000-dalton unglycosylated POMC is the precursor of the smaller ACTH and endorphin molecules in tunicamycin-treated cells. Furthermore, all of the forms of ACTH and endorphin found in tunicamycin-treated cells are secreted. Thus, it appears that glycosylation is not an essential step for correct cleavage or secretion of POMC or its products.     

The role of FcgammaR signaling in the K/B x N serum transfer model of arthritis

Spontaneous arthritis in the KRN transgenic mouse (K/BxN) model is due to the autoreactivity of the transgenic TCR and subsequent induction of autoantibodies directed against glucose-6-phosphate isomerase. These autoantibodies transfer clinically apparent arthritis into most recipient mouse strains and systemic catabolism of the transferred Abs attenuates paw swelling. Although mice deficient in the common gamma-chain of the FcgammaR did not show clinical synovitis after receiving K/BxN sera, erosive lesions in the bone still developed. Further analysis demonstrated that FcgammaRII(-/-) mice manifested accelerated arthritis whereas the FcgammaRIII(-/-) mice had a more slowly progressing arthritis. Paw swelling required FcgammaR expression by bone marrow-derived cells and mast cells substantially contributed to the acute phase of paw swelling. In the K/BxN serum transfer model of arthritis, there is a clinically apparent acute phase, which is modulated by FcgammaRII and FcgammaRIII, and a subacute component, which results in bone erosion, even in the absence of FcgammaR signaling.     

Distribution of pro-opiomelanocortin and its peptide end products in the brain and hypophysis of the aquatic toad, Xenopus laevis

Using in situ hybridization with a pro-opiomelanocortin (POMC)-mRNA probe and immunocytochemistry with antisera to POMC and to various POMC-derived peptides, it is shown that melanotrope cells in the pars intermedia of the hypophysis of the South African aquatic toad Xenopus laevis contain POMC, α-melanophore-stimulating hormone (α-MSH), γ-MSH, acetylated and non-acetylated endorphins and adrenocorticotropic hormone (ACTH). With the exception of γ-MSH, these peptides are also found in the corticotrope cells in the rostral pars distalis. In the Xenopus brain, neuronal cell bodies in the ventral hypothalamic nucleus express POMC, α-MSH, γ-MSH, non-acetylated endorphins and ACTH, neurones in the anterior preoptic area reveal POMC, α-MSH, γ-MSH and non-acetylated endorphin, neurones in the suprachiasmatic nucleus contain α-MSH, non-acetylated endorphin and ACTH and neurones in the posterior tubercle show α-MSH, non-acetylated endorphin and ACTH immunoreactivities. In the locus coeruleus POMC and ACTH coexist, whereas α-MSH and non-acetylated endorphin occur together in the nucleus accumbens, the striatum and the nucleus of the paraventricular organ. Finally, α-MSH alone is present in the olfactory bulb, the medial septum, the medial and lateral parts of the amygdala, the ventromedial and posterior thalamic nuclei, the optic tectum and the anteroventral tegmental nucleus, and non-acetylated endorphin alone appears in the epiphysis. It is suggested that neurones that form POMC-derived peptides may play a direct or indirect role in the control of POMC-producing hypophyseal cells and/or in the physiological processes these endocrine cells regulate. This idea is supported by the fact that the suprachiasmatic nucleus and the locus coeruleus, both involved in melanotrope cell control, show POMC and POMC-peptide expression. A possible involvement in melanotrope and/or corticotrope control of the anterior preoptic and ventral hypothalamic nuclei, which both express POMC and various POMC-derived peptides, deserves future attention.     

Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis

BACKGROUND: It has previously been demonstrated that high levels of gene expression in skeletal muscles can be achieved after direct in vivo electrotransfer of naked plasmid DNA. The purpose of this study is to examine the potential of in vivo electroporation of plasmid DNA encoding human IL-1Ra for the prevention of murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were injected in gastrocnemius muscles with plasmid DNA followed by in vivo electroporation. To uncover the optimum conditions of gene transfer, various electric field strengths and different amounts of plasmid DNA were applied. Calf muscles around the injected areas were investigated with histological methods for damage to muscle tissue. The levels of human IL-1Ra expression in the injected area and also in the serum were determined with ELISA for human IL-1Ra. Based on these data, the effects of electrotransfer of plasmid DNA were tested using the murine CIA model. DBA/1 mice were immunized with bovine collagen type II at the base of the tail. On day 21, mice were given a booster injection with the same antigen. Mice were divided into two groups on day 26. One group of mice received plasmid containing the IL-1Ra cDNA sequence, while control mice were given plasmid lacking the IL-1Ra coding sequence. The incidence of arthritis was evaluated by macroscopic analysis, histological analysis, and the levels of inflammatory cytokines. RESULTS: IL-1Ra expression increased as a function of the electrical field strength and the amount of DNA. 200 V/cm (eight pulses; 20 ms per pulse; 1 Hz) and 15 microg of plasmid DNA per mouse were found to be optimum for gene transfer. After in vivo electroporation, gene expression in both muscle and serum increased gradually, reaching a peak value on day 10. Significant levels of human IL-1Ra expression were maintained for 20 days. Macroscopic analysis showed that the onset of CIA was significantly inhibited by direct electrotransfer of plasmid DNA encoding human IL-1Ra. Histological analysis of knee joints showed that the incidence of arthritis in knee joints was also prevented. The levels of mouse IL-1beta and IL-12 in paws were significantly lower in the group treated with IL-1Ra than those in the control group. CONCLUSIONS: These results demonstrate that direct electrotransfer of plasmid containing the human IL-1Ra cDNA sequence to skeletal muscle can reduce the incidence of CIA in mice.     

Blockade of endothelin-1 release contributes to the anti-angiogenic effect by pro-opiomelanocortin overexpression in endothelial cells

Pro-opiomelanocortin (POMC) is the precursor of several neuropeptides, such as corticotropin (ACTH), alpha-melanocyte-stimulating hormone (MSH), and the endogenous opioid, beta-endorphin (EP). ACTH-dependent Cushing's syndrome is characterized by ACTH overproduction and is associated with an increased risk of cardiovascular disease. Endothelial dysfunction has been recognized as an early marker of cardiovascular disease. However, the mechanism underlying endothelial dysfunction by ACTH overexpression in Cushing's patients remains elusive. Endothelial cells, the primary cells producing endothelin (ET)-1, are both the source and target of POMC-derived peptides. In the present study, we generated adenovirus vectors (Ad) encoding POMC (Ad-POMC) and green fluorescent protein (GFP; Ad-GFP) to investigate whether POMC gene transfer altered the ET-1 homeostasis and angiogenic functions in human EA.hy926 endothelial cells. Via adenovirus gene delivery, the POMC-transduced EA.hy926 cells released significantly elevated ACTH and beta-EP levels (P < 0.001). In addition, POMC gene delivery significantly decreased the ET-1 release (P < 0.001) without affecting the ET-1 messenger RNA (mRNA) level. Despite no effect on the secretion of matrix metalloproteinases (MMPs) and cell proliferation, POMC gene delivery significantly inhibited the migration (P < 0.01) and tube-forming capability (P < 0.01) of endothelial cells. Moreover, the POMC-induced inhibition of tube formation could be partially reversed by adding exogenous ET-1 (P < 0.05). In summary, the attenuated ET-1 release and angiogenic processes by POMC overexpression may contribute to endothelial dysfunction, thereby providing a link between Cushing's syndrome and cardiovascular diseases.     

Small interfering RNA targeting CD81 ameliorated arthritis in rats

CD81 belongs to a family of cell-surface protein (tetraspanin) known as one of the up-regulated elements in rheumatoid arthritis synoviocytes. In this study, the therapeutic effect of small interfering RNA targeting CD81 (siCD81) was examined by in vivo electroporation method. Treatment with siCD81 significantly ameliorated paw swelling of collagen-induced arthritic (CIA) rats. In histological examination, hypertrophy of synovium, bone erosion, and degeneration of articular cartilage were milder in rats treated with siCD81 than in the control group and the non-specific siRNA group. Expression of synoviolin, a rheumatoid regulator, was suppressed by siCD81. Thus, therapeutic intervention by targeting CD81 may be used in the treatment of rheumatoid arthritis.     

Effects of the Sri Lankan medicinal plant, Salacia reticulata, in rheumatoid arthritis

Salacia reticulata is a native plant of Sri Lanka. In the traditional medicine of Sri Lanka and India, Salacia reticulata bark is considered orally effective in the treatment of rheumatism, gonorrhea, skin disease and diabetes. We have investigated, both in vivo and in vitro, whether the leaf of Salacia reticulata (SRL) can ameliorate collagen antibody-induced arthritis (CAIA) in mice as the rheumatoid arthritis (RA) model. The mice were fed a lard containing chow diet (AIN-93G) or the same diet containing 1% (w/w) SRL powder. All mice were bred for 23 days. On day 7 or 14 after LPS injection, mice were killed, and tissue and blood samples were collected. Histological analysis was performed, and serum levels of inflammatory mediators and the mRNA levels of inflammation-related genes and osteoclast-related genes were measured. SRL treatment ameliorated the rapid initial paw swelling, inflammatory cells infiltration, skeletal tissues damage, osteoclast activation and the mRNA levels for osteoclast-related genes compared with the CAIA mice. However, the serum and mRNA levels of inflammatory mediators did not differ between the CAIA mice and the SRL-treated mice. SRL might reduce the inflammatory cells induction and skeletal tissue degradation by CAIA by the regulating osteoclastogenesis.     

Gene expression and immune response kinetics using electroporation-mediated DNA delivery to muscle

BACKGROUND: Injection of DNA encoding exogenic proteins into muscle tissue combined with electroporation often results in a transient increase of the encoded protein concentration in the muscle and the blood. The reduction is normally due to an immune response against the exogenic protein but other factors may also be involved. How various electroporation parameters affect the concentration kinetics of syngenic and exogenic proteins is studied in relation to immune response and muscle damage after electroporation-mediated DNA transfer to muscle. METHODS: Electroporation was applied to mouse quadriceps and rat tibialis anterior muscles after injection of DNA encoding either secreted alkaline phosphatase (SEAP), beta-galactosidase (beta-gal), luciferase or a mouse IgG molecule. Protein concentrations in blood or muscle and antibody responses were measured for a period up to 3 months. Tissue inflammation and muscle cell damage were studied on muscle cross-sections and assessed by measuring the concentrations of creatine phosphokinase (CPK) in blood. RESULTS: Mice with the highest SEAP concentration in blood at day 7 also had the highest rate of decrease afterwards, the strongest antibody responses against SEAP and the highest acute levels of CPK in blood. DNA-transfected muscle fibers were significantly reduced in number from days 7 to 14. Mononuclear cells surrounded the reporter gene expressing muscle fibers, thus indicating a cellular immune response. When using DNA encoding a syngenic protein the protein concentration in blood was relatively stabile over a 3-month period, but showed different kinetics for various electroporation parameters. CONCLUSIONS: Our findings suggest that the optimal electroporation parameters for DNA vaccination may be different from the optimal parameters for long-term expression of genes encoding syngenic proteins.     

Biosynthesis and processing of pro-opiomelanocortin-derived peptides during fetal pituitary development

We have investigated the molecular weight forms of pro-opiomelanocortin (POMC)-derived peptides present in rat pituitaries during fetal and early postnatal development (embryonic Day 14 to 3 day neonate). At all early ages examined, the major immunoreactive form of corticotropin (ACTH) was POMC. Only during late fetal and early postnatal stages did progressively larger amounts of 4.5K ACTH, a major POMC processing end product, appear. This form was found almost exclusively in isolated anterior lobes. In contrast, 3.5K size endorphin(s), another POMC derivative, were present in whole glands even at early stages (Day 14), and were the major POMC derivative(s) found in isolated intermediate-posterior lobes of older fetuses. Despite the early appearance of 3.5K endorphin(s), α-MSH did not appear until Day 19 and was detected only in isolated intermediate-posterior lobes. We have also cultured dispersed fetal pituitary cells in the presence of radioactive amino acids. After immunoprecipitation using affinity-purified antisera, followed by fractionation of the radiolabeled products, we found that POMC biosynthesis does occur in cultures of Day 14 embryonic pituitary cells, and that the major POMC-derived end product produced is 3.5K size endorphin(s). These findings demonstrate that POMC is synthesized at least by Day 14 of rat pituitary development and that lobe-specific processing characteristic of the corresponding adult lobe is apparent at the earliest stages that the lobes can be separated. The presence of 3.5K-sized endorphins at early ages is consistent with the possibility that POMC synthesis first occurs in the intermediate lobe. The noncoordinate appearance of α-MSH, 1–39 ACTH, and endorphins implies that the activities of certain cleavage enzymes and acetylation enzymes responsible for lobe-specific post-translational POMC processing may be expressed at different times during development.     

活体电穿孔法基因导入技术

活体电穿孔法(invivoelectroporation)可将外源基因有效导入靶组织或器官,导入效率较高,并且可在多种组织器官上应用。近年来活体电穿孔法用于转基因研究的报道不断增多,在基因治疗方面的优势也日趋显著,是一种很好的活体基因导入方法 。     

The feasibility of non-viral gene transfer to the diaphragm in vivo

Gene transfer using electroporation is an essential method for the study of developmental biology, especially to understand the internal control of degeneration and apoptosis of the muscle cells that occurs earlier and quicker than the usual degeneration process occurring by aging. Such experimental studies may have a role in developing new strategies for treating patients suffering from inherited primary myopathies such as Duchenne muscular dystrophy (DMD). The present study was designed to evaluate the feasibility of electroporation mediated transfer of reporter genes to the diaphragm in vivo. This is the first report of gene transfer of naked plasmid DNA into the diaphragm muscle in vivo using electroporation. Our results showed that in vivo gene transfer of naked plasmid DNA into the diaphragm muscle using electroporation is feasible.     

Gene therapy with an improved doxycycline-regulated plasmid encoding a tumour necrosis factor-alpha inhibitor in experimental arthritis

Inhibition of tumour necrosis factor (TNF)-alpha with biological molecules has proven an effective treatment for rheumatoid arthritis, achieving a 20% improvement in American College of Rheumatology score in up to 65% of patients. The main drawback to these and many other biological treatments has been their expense, which has precluded their widespread application. Biological molecules could alternatively be delivered by gene therapy as the encoding DNA. We have developed novel plasmid vectors termed pGTLMIK and pGTTMIK, from which luciferase and a dimeric TNF receptor II (dTNFR) are respectively expressed in a doxycycline (Dox)-regulated manner. Regulated expression of luciferase from the self-contained plasmid pGTLMIK was examined in vitro in a variety of cell lines and in vivo following intramuscular delivery with electroporation in DBA/1 mice. Dox-regulated expression of luciferase from pGTLMIK of approximately 1,000-fold was demonstrated in vitro, and efficient regulation was observed in vivo. The vector pGTTMIK encoding dTNFR was delivered by the same route with and without administration of Dox to mice with collagen-induced arthritis. When pGTTMIK was delivered after the onset of arthritis, progression of the disease in terms of both paw thickness and clinical score was inhibited when Dox was also administered. Vectors with similar regulation characteristics may be suitable for clinical application.     

Porphyromonas gingivalis oral infection exacerbates the development and severity of collagen-induced arthritis

Introduction

Clinical studies suggest a direct influence of periodontal disease (PD) on serum inflammatory markers and disease assessment of patients with established rheumatoid arthritis (RA). However, the influence of PD on arthritis development remains unclear. This investigation was undertaken to determine the contribution of chronic PD to immune activation and development of joint inflammation using the collagen-induced arthritis (CIA) model.

Methods

DBA1/J mice orally infected with Porphyromonas gingivalis were administered with collagen II (CII) emulsified in complete Freund’s adjuvant (CFA) or incomplete Freund’s adjuvant (IFA) to induce arthritis. Arthritis development was assessed by visual scoring of paw swelling, caliper measurement of the paws, mRNA expression, paw micro-computed tomography (micro-CT) analysis, histology, and tartrate resistant acid phosphatase for osteoclast detection (TRAP)-positive immunohistochemistry. Serum and reactivated splenocytes were evaluated for cytokine expression.

Results

Mice induced for PD and/or arthritis developed periodontal disease, shown by decreased alveolar bone and alteration of mRNA expression in gingival tissues and submandibular lymph nodes compared to vehicle. P. gingivalis oral infection increased paw swelling and osteoclast numbers in mice immunized with CFA/CII. Arthritis incidence and severity were increased by P. gingivalis in mice that received IFA/CII immunizations. Increased synovitis, bone erosions, and osteoclast numbers in the paws were observed following IFA/CII immunizations in mice infected with P gingivalis. Furthermore, cytokine analysis showed a trend toward increased serum Th17/Th1 ratios when P. gingivalis infection was present in mice receiving either CFA/CII or IFA/CII immunizations. Significant cytokine increases induced by P. gingivalis oral infection were mostly associated to Th17-related cytokines of reactivated splenic cells, including IL-1β, IL-6, and IL-22 in the CFA/CII group and IL-1β, tumor necrosis factor-α, transforming growth factor-β, IL-6 and IL-23 in the IFA/CII group.

Conclusions

Chronic P. gingivalis oral infection prior to arthritis induction increases the immune system activation favoring Th17 cell responses, and ultimately accelerating arthritis development. These results suggest that chronic oral infection may influence RA development mainly through activation of Th17-related pathways.     

Reexamination of the difference in susceptibility to adjuvant-induced arthritis among LEW/Crj, Slc/Wistar/ST and Slc/SD rats.

The present investigations were performed to assess the differences among rat colonies commonly used for neurophysiological research regarding the development of complete Freund's adjuvant (CFA)-induced arthritis. Inflammatory signs including edema in the paw fluctuated remarkably among individual Wistar (Slc/Wistar/ST) and Sprague-Dawley (Slc/SD) rats, while the inflammatory signs of Lewis (LEW/Crj) rats appeared earlier and was severer and more consistent than Slc/Wistar/ST and Slc/SD rats. Edema in the hind paw developed in 100% of LEW/Crj rats with the lowest dose of CFA (0.6 mg/rat) used as compared with 64% of Slc/Wistar/ST (CFA 1 mg/rat) and 38% of Slc/SD rats (CFA 1.2 mg/rat). Retardation of weight gain was observed in Slc/Wistar/ST and Slc/SD rats in contrast to a severe weight decrease in inflamed LEW/Crj rats after the development of arthritis.     

The M20 IL-1 inhibitor prevents onset of adjuvant arthritis

Cytokines, specifically IL-1 and TNF, have been implicated as important mediators of joint destruction in rheumatoid arthritis (RA). Elevated levels of IL-1 in the joint fluid of patients with RA have been reported, as well as the presence of IL-1 inhibitory activity. We have reported the characterization of an inhibitor derived from a myelomonocytic cell line cloned in our laboratory which is specific for IL-1. This IL-1 inhibitor is protein in nature which specifically inhibits activityin vitro andin vivo. Previous studies showed that the inhibitor reduced acute inflammatory reactions associated with IL-1 (fever, leukocytosis, local foot pad swelling, lymph node enlargement and acute phase reactants). Thus it was of interest to study whether the M20 IL-1 inhibitor could modify adjuvant-induced chronic inflammation in rats, which is often used as a model for human RA. Administration of complete Freund's adjuvant (CFA) into Lewis rats, resulted in a severe adjuvant arthritis (AA) which reached peak severity after 14 days. Daily administration of IL-1 inhibitor, beginning after injection of CFA, abolished the appearance of AA. The parameters investigated were: joint swelling (the increase in diameter of joints), peri-articular erythema, limping of the rats and histological examination. The effect of the M20 IL-1 inhibitor was shown to be dose dependent and the IL-1 inhibitor alone had no adverse effects. These results indicate that the M20 IL-1 inhibitor may have a role in the treatment of AA and may be used to reduce pathological processes in joint inflammation.     

Estrogenic effect on swelling and monocytic receptor expression in an arthritic temporomandibular joint model

Clinical presentation of temporomandibular joint (TMJ) disorders are more common in women and changes in the female hormone estrogen affect the level of swelling, pro-inflammatory cytokine release and pain in animal models of TMJ arthritis. Estrogen also modulates the expression of the CD16 receptor in vitro. This alters pro-inflammatory cytokine release in monocytes/macrophages when auto-antigens and arthritic factors bind the CD16 receptor. This study investigated the effects of various levels of estrogen on the intensity of inflammation and CD16 expression in a TMJ arthritic animal model. The experiments included rats that were intact or ovariectomized (OVX), eliminating the major source of estrogen output. A portion of the OVX animals had estrogen replaced with 17-beta estradiol (E2) using Alzet pumps. In OVX animals E2 levels were administered for 10 days to create an artificial estrus cycle or to simulate pregnancy. Following E2 treatment the rats were given an intra-articular TMJ injection of saline or complete Freund's adjuvant (CFA). CFA injection significantly increased TMJ swelling, stress induced chromodacryorrhea and attenuated food intake, thus indicating the adjuvant induced TMJ pain/inflammation. Removing endogenous E2 through OVX reduced CFA induced TMJ inflammation, whereas CFA increased the number of TMJ monocytes expressing the CD14 receptor equally in all groups irrespective of plasma E2 levels. Paradoxically, higher levels of E2 reduced the number of TNF-alpha positive, CD16+ and double labeled CD14+/CD16+ cells. The findings indicate that reduced plasma E2 levels attenuated CFA induced TMJ inflammation, whereas increasing E2 levels enhanced TMJ swelling in a dose dependent manner. Estrogenic group differences in CFA induced swelling were independent of TMJ CD14+, CD14+/CD16+ or CD16+ cell numbers suggesting E2 action on the CFA immune response primarily excluded CD16 receptor action.     

牛肾上腺髓质22肽削弱完全弗氏佐剂引起的早期痛觉过敏

本研究旨在探讨感觉神经元特异性受体(sensory neuron-specific receptor,SNSR)的内源性激动剂牛肾上腺髓质22肽(bovine adrenal medulla22,BAM22)对完全弗氏佐剂(complete Freund’s adjuvant,CFA)引起的早期炎性痛的影响。在大鼠后足皮下注射CFA形成炎性痛模型,通过撤足反射、脚肿测定和免疫组织化学等方法观察鞘内给予BAM22对炎性痛的影响。结果显示:在撤足反射实验中,BAM22能剂量依赖地延长CFA炎性鼠撤足反射潜伏期基础阈值,增强抗伤害作用,并降低脚肿程度。10nmol BAM22作用48h后,撤足反射潜伏期恢复至正常的83.2%,脚肿仅增加60.0%;24h时延长撤足反射潜伏期达最大可能作用的33.5%,并持续至少1h。在免疫组织化学实验中,BAM22能显著降低CFA引起的L3-L5脊髓背角神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)阳性细胞和降钙素基因相关肽(calcitonin gene-related peptide,CGRP)样免疫活性物质的表达,与生理盐水组相...