ABSTRACT
This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article is being retracted following correspondence from the Office of Accountability and Compliance at the University of Maryland, Baltimore. An internal investigation into this manuscript by the University of Maryland, Baltimore, found evidence that there are errors with the presentation of the standard deviations and statistical significance shown in Figure 6 which are not supported by the original data, and that these inaccuracies warrant retraction to correct the scientific record. Despite extensive efforts, the journal was unable to contact Dr. Ying-hua Yang and Dr. Hua Zhou with regard to this retraction.
ABSTRACT
The Editors-in-Chief have retracted this article [1] following an investigation by the University of Maryland. The institution found that in Figures 1B and 1D, the cell lines are different and all published histograms show SEMA4D mRNA level whereas Excel data have two histograms showing SEMA4D expression and two histograms showing VEGF expression. In Figure 2B, the metadata for one image shows different treatment conditions than those reported in the article. The published image labelled "VEGF + VEGFR-2 shRNA" has a metadata label of S4d-plexinB1 shRNA2". In Figure 2E, statistical significance was shown in the published figure for four comparisons, but upon recalculation, one comparison noted as significant was not. In Figure 6A, the lower left image is labelled "VEGF shRNA" in the published figure, but the metadata label is "S4DshRNA-HN121-20X". In Figure 6C, specifically, within columns 2-4, for each antibody used for immunocytochemistry, the three images have been swapped so that the original images do not match the shRNA labels in the figure (the labels for the two antibodies were correct). In Figure 7D, the first published image is labelled as "IgG" in the paper, but the metadata show a label of "Restore (V+S).tif". The third published image has a label of "anti-VEGF IgG", and the metadata show a label of "con sh.tif". Due to these errors, the Editors-in-Chief have found that the results are no longer reliable.
ABSTRACT
The Editors-in-Chief have retracted this article [1] following an investigation by the University of Maryland. The institution found that in Figure 1C, the graph showing PDGF-B does not match the original data for the 24-hour time point. The graph shows the value to be over 1000 pg/ml, but the original data have a value of 106.626. In Figure 1F, the data were entered manually to create the standard deviation bars. The data manually entered do not match the original data. When the standard deviations for the original data were calculated, the p values were no longer significant using a paired student t test. In Figure 2C, the original data do not match the published data. In Figure 4B, the images in the first lane and the fifth lane are from the same micrograph (i.e., the same set of conditions). However, the published figure claims that they are different conditions. The metadata in this figure also shows different cell lines than those noted in the article. The first and last images are labelled as "Du145 shAR3 anti AR3.jpg". The second image is labelled as "Du145 shAR8 anti AR8.jpg". The third image is labelled as "Cos1 mARs3 mS3-2 antibody-2.jpg." The fourth image is labelled as "R1 3634 bleed.jpg". Due to these errors, the Editors-in-Chief have found that the results are no longer reliable.
ABSTRACT
Figure 3c of this article originally contained standard deviation values which had not been calculated correctly. A single standard deviation value was used for all 5 time points for each condition.
ABSTRACT
The semaphorins and plexins comprise a family of cysteine-rich cell surface and secreted proteins originally shown to control nerve growth and the immune response, but that have recently been implicated in a wide variety of developmental and pathological processes that are influenced by cell adhesion and migration. Along those lines, our group and others have found that Semaphorin 4D (SEMA4D) plays an important role in angiogenesis by promoting chemotaxis of endothelial cells, which express its receptor, Plexin-B1. Indeed, some neoplasms produce SEMA4D along with other pro-angiogenic proteins for the purpose of enhancing blood vessel growth into a developing neoplasm. Here we describe the application of in vitro migration and tubulogenesis assays and the directed in vivo angiogenesis assay (DIVAA) in the measurement of the angiogenic potential of cell-derived and soluble SEMA4D.
Subject(s)
Antigens, CD/metabolism , Neovascularization, Physiologic , Semaphorins/metabolism , Signal Transduction , Culture Media, Serum-Free , Human Umbilical Vein Endothelial Cells , HumansABSTRACT
The semaphorins and the plexins are a family of large, cysteine-rich proteins originally identified as regulators of axon growth and lymphocyte activation that are now known to provide motility and positional information for a number of cell and tissue types. For example, our group and others have shown that some malignancies over express Semaphorin 4D (S4D), which acts through its receptor Plexin-B1 (PB1) on endothelial cells to attract blood vessels from the surrounding stroma for the purpose of supporting tumor growth. While plexins are the known functional receptors for the semaphorins, there is evidence that transmembrane semaphorins may transmit a signal themselves through their short cytoplasmic tail, a phenomenon known as 'reverse signaling.' We used computational methods based upon correlated evolution of sequences of interacting proteins, mutational analysis and in vitro and in vivo measurements of tumor aggressiveness to show that when bound to PB1, transmembrane S4D associates with the Rac GTPase exchange factor T lymphoma invasion and metastasis (Tiam) 1, which activates Rac and promotes proliferation, invasion and metastasis in oral squamous cell carcinoma (OSCC) cells. These results suggest that not only can S4D production by tumor cells affect the microenvironment, but engagement of this semaphorin at the cell surface activates a reverse signaling mechanism that influences tumor aggressiveness in OSCC.
Subject(s)
Antigens, CD/metabolism , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Guanine Nucleotide Exchange Factors/metabolism , Mouth Neoplasms/metabolism , Mouth Neoplasms/pathology , Semaphorins/metabolism , rac GTP-Binding Proteins/metabolism , Animals , Antigens, CD/chemistry , Biopsy , Carcinoma, Squamous Cell/mortality , Cell Line, Tumor , Cell Movement , Cell Proliferation , DNA-Binding Proteins , Disease Models, Animal , Gene Expression , Guanine Nucleotide Exchange Factors/chemistry , Humans , Mice , Mouth Neoplasms/mortality , Neoplasm Metastasis , Nuclear Proteins/metabolism , PDZ Domains , Prognosis , Protein Binding , Protein Interaction Domains and Motifs , Proteomics/methods , Semaphorins/chemistry , T-Lymphoma Invasion and Metastasis-inducing Protein 1 , Transcription Factors/metabolismABSTRACT
Bone density is controlled by interactions between osteoclasts, which resorb bone, and osteoblasts, which deposit it. The semaphorins and their receptors, the plexins, originally shown to function in the immune system and to provide chemotactic cues for axon guidance, are now known to play a role in this process as well. Emerging data have identified Semaphorin 4D (Sema4D) as a product of osteoclasts acting through its receptor Plexin-B1 on osteoblasts to inhibit their function, tipping the balance of bone homeostasis in favor of resorption. Breast cancers and other epithelial malignancies overexpress Sema4D, so we theorized that tumor cells could be exploiting this pathway to establish lytic skeletal metastases. Here, we use measurements of osteoblast and osteoclast differentiation and function in vitro and a mouse model of skeletal metastasis to demonstrate that both soluble Sema4D and protein produced by the breast cancer cell line MDA-MB-231 inhibits differentiation of MC3T3 cells, an osteoblast cell line, and their ability to form mineralized tissues, while Sema4D-mediated induction of IL-8 and LIX/CXCL5, the murine homologue of IL-8, increases osteoclast numbers and activity. We also observe a decrease in the number of bone metastases in mice injected with MDA-MB-231 cells when Sema4D is silenced by RNA interference. These results are significant because treatments directed at suppression of skeletal metastases in bone-homing malignancies usually work by arresting bone remodeling, potentially leading to skeletal fragility, a significant problem in patient management. Targeting Sema4D in these cancers would not affect bone remodeling and therefore could elicit an improved therapeutic result without the debilitating side effects.
Subject(s)
Antigens, CD/metabolism , Bone Neoplasms/metabolism , Breast Neoplasms/metabolism , Calcification, Physiologic , Neoplasm Proteins/metabolism , Semaphorins/metabolism , Animals , Antigens, CD/genetics , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Bone Neoplasms/secondary , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Heterografts , Humans , Interleukin-8/genetics , Interleukin-8/metabolism , Mice , Neoplasm Metastasis , Neoplasm Proteins/genetics , Neoplasm Transplantation , Semaphorins/geneticsABSTRACT
Semaphorin 4D (SEMA4D) is a member of a family of transmembrane and secreted proteins that have been shown to act through its receptor Plexin-B1 to regulate axon growth cone guidance, lymphocyte activation, and bone density. SEMA4D is also overexpressed by some malignancies and plays a role in tumor-induced angiogenesis similar to vascular endothelial growth factor (VEGF), a protein that has been targeted as part of some cancer therapies. In an attempt to examine the different effects on tumor growth and vascularity for these two pro-angiogenic factors, we previously noted that while inhibition of both VEGF and SEMA4D restricted tumor vascularity and size, vessels forming under conditions of VEGF blockade retained their association with pericytes while those arising in a background of SEMA4D/Plexin-B1 deficiency did not, an intriguing finding considering that alteration in pericyte association with endothelial cells is an emerging aspect of anti-angiogenic intervention in the treatment of cancer. Here we show through array analysis, immunoblots, migration and co-culture assays and VE-cadherin immunohistochemistry that SEMA4D production by head and neck carcinoma tumor cells induces expression of platelet-derived growth factor-B and angiopoietin-like protein 4 from endothelial cells in a Plexin-B1/Rho-dependent manner, thereby influencing proliferation and differentiation of pericytes and vascular permeability, whereas VEGF lacks these effects. These results partly explain the differences observed between SEMA4D and VEGF in pathological angiogenesis and suggest that targeting SEMA4D function along with VEGF could represent a novel anti-angiogenic therapeutic strategy for the treatment of solid tumors.
Subject(s)
Angiopoietins/biosynthesis , Antigens, CD/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Nerve Tissue Proteins/metabolism , Pericytes/metabolism , Proto-Oncogene Proteins c-sis/biosynthesis , Receptors, Cell Surface/metabolism , Semaphorins/metabolism , Signal Transduction , rhoA GTP-Binding Protein/metabolism , Angiopoietin-Like Protein 4 , Angiopoietins/genetics , Antigens, CD/genetics , Humans , Neoplasms/blood supply , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/therapy , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/therapy , Nerve Tissue Proteins/genetics , Proto-Oncogene Proteins c-sis/genetics , Receptors, Cell Surface/genetics , Semaphorins/genetics , rhoA GTP-Binding Protein/geneticsABSTRACT
Anabolic-androgenic steroids (AAS) are lipophilic hormones often taken in excessive quantities by athletes and bodybuilders to enhance performance and increase muscle mass. AAS exert well known toxic effects on specific cell and tissue types and organ systems. The attention that androgen abuse has received lately should be used as an opportunity to educate both athletes and the general population regarding their adverse effects. Among numerous commercially available steroid hormones, very few have been specifically tested for direct neurotoxicity. We evaluated the effects of supraphysiological doses of methandienone and 17-α-methyltestosterone on sympathetic-like neuron cells. Vitality and apoptotic effects were analyzed, and immunofluorescence staining and western blot performed. In this study, we demonstrate that exposure of supraphysiological doses of methandienone and 17-α-methyltestosterone are toxic to the neuron-like differentiated pheochromocytoma cell line PC12, as confirmed by toxicity on neurite networks responding to nerve growth factor and the modulation of the survival and apoptosis-related proteins ERK, caspase-3, poly (ADP-ribose) polymerase and heat-shock protein 90. We observe, in contrast to some previous reports but in accordance with others, expression of the androgen receptor (AR) in neuron-like cells, which when inhibited mitigated the toxic effects of AAS tested, suggesting that the AR could be binding these steroid hormones to induce genomic effects. We also note elevated transcription of neuritin in treated cells, a neurotropic factor likely expressed in an attempt to resist neurotoxicity. Taken together, these results demonstrate that supraphysiological exposure to the AAS methandienone and 17-α-methyltestosterone exert neurotoxic effects by an increase in the activity of the intrinsic apoptotic pathway and alterations in neurite networks.
ABSTRACT
Growth and metastasis of solid tumors requires induction of angiogenesis to ensure the delivery of oxygen, nutrients and growth factors to rapidly dividing transformed cells. Through either mutations, hypoxia generated by cytoreductive therapies, or when a malignancy outgrows its blood supply, tumor cells undergo a change from an avascular to a neovascular phenotype, a transition mediated by the hypoxia-inducible factor (HIF) family of transcriptional regulators. Vascular endothelial growth factor (VEGF) is one example of a gene whose transcription is stimulated by HIF. VEGF plays a crucial role in promoting tumor growth and survival by stimulating new blood vessel growth in response to such stresses as chemotherapy or radiotherapy-induced hypoxia, and it therefore has become a tempting target for neutralizing antibodies in the treatment of advanced neoplasms. Emerging evidence has shown that the semaphorins, proteins originally associated with control of axonal growth and immunity, are regulated by changes in oxygen tension as well and may play a role in tumor-induced angiogenesis. Through the use of RNA interference, in vitro and in vivo angiogenesis assays and tumor xenograft experiments, we demonstrate that expression of semaphorin 4D (SEMA4D), which is under the control of the HIF-family of transcription factors, cooperates with VEGF to promote tumor growth and vascularity in oral squamous cell carcinoma (OSCC). We use blocking antibodies to show that targeting SEMA4D function along with VEGF could represent a novel anti-angiogenic therapeutic strategy for the treatment of OSCC and other solid tumors.
Subject(s)
Antigens, CD/metabolism , Carcinoma, Squamous Cell/metabolism , Mouth Neoplasms/metabolism , Neoplasms, Experimental/metabolism , Neovascularization, Pathologic/metabolism , Semaphorins/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , Antigens, CD/biosynthesis , Carcinoma, Squamous Cell/blood supply , Carcinoma, Squamous Cell/pathology , Cell Proliferation , Cells, Cultured , HEK293 Cells , Humans , Mice , Mouth Neoplasms/blood supply , Mouth Neoplasms/pathology , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/pathology , Semaphorins/biosynthesisABSTRACT
The semaphorins and plexins comprise a family of cysteine-rich proteins implicated in control of nerve growth and development and regulation of the immune response. Our group and others have found that Semaphorin 4D (SEMA4D) and its receptor, Plexin-B1, play an important role in tumor-induced angiogenesis, with some neoplasms producing SEMA4D in a manner analogous to vascular endothelial growth factor (VEGF) in order to attract Plexin-B1-expressing endothelial cells into the tumor for the purpose of promoting growth and vascularity. While anti-VEGF strategies have been the focus of most angiogenesis inhibition research, such treatment can lead to upregulation of pro-angiogenic factors that can compensate for the loss of VEGF, eventually leading to failure of therapy. Here, we demonstrate that SEMA4D cooperates with VEGF to promote angiogenesis in malignancies and can perform the same function in a setting of VEGF blockade. We also show the potential value of inhibiting SEMA4D/Plexin-B1 signaling as a complementary mechanism to anti-VEGF treatment, particularly in VEGF inhibitor-resistant tumors, suggesting that this may represent a novel treatment for some cancers.
Subject(s)
Antigens, CD/metabolism , Neoplasms/blood supply , Neovascularization, Pathologic , Semaphorins/metabolism , Vascular Endothelial Growth Factor A/metabolism , Base Sequence , Cells, Cultured , DNA Primers , Disease Progression , Humans , Immunohistochemistry , Polymerase Chain ReactionABSTRACT
Perineural invasion (PNI) is a tropism of tumor cells for nerve bundles located in the surrounding stroma. It is a pathological feature observed in certain tumors, referred to as neurotropic malignancies, that severely limits the ability to establish local control of disease and results in pain, recurrent growth, and distant metastases. Despite the importance of PNI as a prognostic indicator, its biological mechanisms are poorly understood. The semaphorins and their receptors, the plexins, compose a family of proteins originally shown to be important in nerve cell adhesion, axon migration, and proper central nervous system development. Emerging evidence has demonstrated that these factors are expressed in tissues outside of the nervous system and represent a widespread signal transduction system that is involved in the regulation of motility and adhesion in different cell types. We believe that the plexins and semaphorins, which are strongly expressed in both axons and many carcinomas, play a role in PNI. In this study, we show that plexin-B1 is overexpressed in tissues and cell lines from neurotropic malignancies and is attracted to nerves that express its ligand, semaphorin 4D, in a Rho/Rho kinase-dependent manner. We also demonstrate that nerves are attracted to tumors through this same system of proteins, suggesting that both plexin-B1 and semaphorin 4D are important in the promotion of PNI.
Subject(s)
Antigens, CD/physiology , Nerve Tissue Proteins/physiology , Nervous System Neoplasms/pathology , Receptors, Cell Surface/physiology , Semaphorins/physiology , rhoA GTP-Binding Protein/metabolism , Animals , Antigens, CD/metabolism , Axons/physiology , Cell Line, Tumor , Cell Movement/physiology , Drug Synergism , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Neoplasm Invasiveness , Neoplasm Transplantation , Nerve Tissue Proteins/metabolism , Nervous System Neoplasms/metabolism , RNA, Small Interfering/pharmacology , Receptors, Cell Surface/metabolism , Semaphorins/metabolism , Signal Transduction , Transplantation, HeterologousABSTRACT
BACKGROUND: The semaphorins and their receptors, the plexins, are proteins related to c-Met and the scatter factors that have been implicated in an expanding signal transduction network involving co-receptors, RhoA and Ras activation and deactivation, and phosphorylation events. Our previous work has demonstrated that Semaphorin 4D (Sema4D) acts through its receptor, Plexin-B1, on endothelial cells to promote angiogenesis in a RhoA and Akt-dependent manner. Since NF-κB has been linked to promotion of angiogenesis and can be activated by Akt in some contexts, we wanted to examine NF-κB in Sema4D treated cells to determine if there was biological significance for the pro-angiogenic phenotype observed in endothelium. METHODS/PRINCIPAL FINDINGS: Using RNA interference techniques, gel shifts and NF-κB reporter assays, we demonstrated NF-κB translocation to the nucleus in Sema4D treated endothelial cells occurring downstream of Plexin-B1. This response was necessary for endothelial cell migration and capillary tube formation and protected endothelial cells against apoptosis as well, but had no effect on cell proliferation. We dissected Plexin-B1 signaling with chimeric receptor constructs and discovered that the ability to activate NF-κB was dependent upon Plexin-B1 acting through Rho and Akt, but did not involve its role as a Ras inhibitor. Indeed, inhibition of Rho by C3 toxin and Akt by LY294002 blocked Sema4D-mediated endothelial cell migration and tubulogenesis. We also observed that Sema4D treatment of endothelial cells induced production of the NF-κB downstream target IL-8, a response necessary for angiogenesis. Finally, we could show through co-immunofluorescence for p65 and CD31 that Sema4D produced by tumor xenografts in nude mice activated NF-κB in vessels of the tumor stroma. CONCLUSION/SIGNIFICANCE: These findings provide evidence that Sema4D/Plexin-B1-mediated NF-κB activation and IL-8 production is critical in the generation a pro-angiogenic phenotype in endothelial cells and suggests a new therapeutic target for the anti-angiogenic treatment of some cancers.
Subject(s)
Endothelial Cells/metabolism , Interleukin-8/metabolism , Neovascularization, Pathologic/metabolism , Nerve Tissue Proteins/metabolism , Receptors, Cell Surface/metabolism , Transcription Factor RelA/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/pharmacology , Apoptosis/drug effects , Capillaries/drug effects , Capillaries/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Transformation, Neoplastic , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/pathology , Gene Expression Regulation, Neoplastic/drug effects , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Mice , Nerve Tissue Proteins/genetics , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Cell Surface/genetics , Semaphorins/genetics , Semaphorins/pharmacology , Signal Transduction/drug effects , rhoA GTP-Binding Protein/metabolismABSTRACT
Autoimmune diseases, such as rheumatoid arthritis, frequently target one major tissue/organ despite the systemic nature of the immune response. This is particularly perplexing in the case of ubiquitously distributed antigens invoked in arthritis induction. We reasoned that selective targeting of the synovial joints in autoimmune arthritis might be due in part to the unique attributes of the joint vasculature. We examined this proposition using the adjuvant-induced arthritis model of human rheumatoid arthritis, and profiled the synovial vasculature using ex vivo and in vivo screening of a defined phage peptide-display library. We identified phage that preferentially homed to the inflamed joints. The corresponding synthetic peptides showed binding to the joint-derived endothelial cells, as well as specificity in inhibiting binding of the respective phage to the synovial vasculature. Intriguingly, the treatment of arthritic rats with one such peptide resulted in efficient inhibition of the progression of arthritis. The suppression of arthritis was attributable in part to the peptide-induced reduction of T-cell trafficking into the joints and the inhibition of angiogenesis. This peptide differed in sequence, in receptor binding specificity, and in angiogenesis/inflammation-related cell signaling from the previously characterized arginine-glycine-aspartic acid-containing peptide. Thus, our study reveals joint-homing peptides that can be further exploited for the selective delivery of antiarthritic agents into the inflamed joints to enhance their efficacy while reducing systemic toxicity, and also for examining intricacies of the pathogenesis of arthritis. This approach can be customized for application to other organ-specific autoimmune diseases as well.
Subject(s)
Arthritis, Experimental/immunology , Peptides/immunology , Synovial Membrane/immunology , Vasculitis/immunology , Amino Acid Sequence , Animals , Arthritis, Experimental/pathology , Arthritis, Experimental/prevention & control , Blotting, Western , Cell Adhesion/drug effects , Cell Adhesion/immunology , Cell Movement/drug effects , Cell Movement/immunology , Cells, Cultured , Endothelial Cells/immunology , Endothelial Cells/metabolism , Humans , Male , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Peptide Library , Peptides/genetics , Peptides/pharmacology , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Inbred Lew , Synovial Membrane/blood supply , Synovial Membrane/pathology , Vasculitis/prevention & controlABSTRACT
Phosphatidylinositol 4-phosphate 5-kinase (PI(4)P5K) is a type I lipid kinase that generates the lipid second messenger phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and functions downstream of RhoA in actin organization. It is known to play an essential role in neurite remodeling, yielding a phenotype identical to that seen in cells treated with Semaphorin 4D (Sema4D), a protein that regulates proliferation, adhesion and migration in many different cell types. Plexin-B1, the receptor for Sema4D, activates RhoA in order to generate a pro-angiogenic signal in endothelial cells. Therefore, we looked in human umbilical vein endothelial cells (HUVEC) to determine if Plexin-B1 exerted control over the cytoskeleton by regulation of PI(4)P5K activity. Here we demonstrate the Rho/Rho Kinase (ROK)-dependent generation of PI(4,5)P(2) upon treatment of HUVEC with Sema4D, as well as co-localization of PI(4)P5Kα with Plexin-B1. Formation of PI(4,5)P(2) was necessary for cytoskeletal polymerization, as expression of the phosphatase synaptojanin blocked this effect. We noted phosphorylation and activation of PLCγ and an increase in intracellular calcium upon treatment of HUVEC with Sema4D, responses that were necessary for a pro-angiogenic phenotype observed in vitro. Taken together, these results suggest that Plexin-B1 promotes angiogenesis in endothelial cells by signaling through PI(4)P5Kα and generating lipid second messengers.
Subject(s)
Antigens, CD/metabolism , Neovascularization, Physiologic/physiology , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Second Messenger Systems/physiology , Semaphorins/metabolism , rhoA GTP-Binding Protein/metabolism , Antigens, CD/genetics , Calcium/metabolism , Cytoskeleton/genetics , Cytoskeleton/metabolism , HEK293 Cells , Humans , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Phosphatidylinositol 4,5-Diphosphate/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Semaphorins/genetics , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/geneticsABSTRACT
OBJECTIVE: Rheumatoid arthritis (RA) is a debilitating autoimmune disease, and smoking is an important environmental factor in a subset of RA patients. A role of the cholinergic antiinflammatory pathway in autoimmune inflammation is increasingly being realized. Nicotine is a major component of cigarette smoke, and it stimulates the α7 nicotinic acetylcholine receptors. Therefore, defining the mechanisms underlying the immunomodulatory effects of nicotine on arthritis is of high relevance. The purpose of this study was to address this issue using the rat adjuvant-induced arthritis (AIA) model of human RA. METHODS: Lewis rats were immunized subcutaneously with heat-killed Mycobacterium tuberculosis H37Ra for disease induction. Rats were treated with nicotine intraperitoneally either before (pretreatment) or after (posttreatment) the onset of AIA. Control rats received the vehicle (buffer) in place of nicotine. The severity of arthritis was assessed and graded. The draining lymph node cells were tested for T cell proliferative and cytokine responses against the disease-related antigen mycobacterial heat-shock protein 65. The sera were tested for anti-cyclic citrullinated peptide (anti-CCP) antibodies and anti-mycobacterial Hsp65 antibodies. RESULTS: Nicotine pretreatment aggravated the arthritis, whereas nicotine posttreatment suppressed the disease. This altered severity of AIA directly correlated with the levels of the anti-CCP antibodies, of the Th1/Th17 cytokines, and of the corresponding dendritic cell-derived cytokines. The majority of these effects on cellular responses could be replicated in vitro. CONCLUSION: Nicotine-induced modulation of AIA involves specific alterations in the disease-related cellular and humoral immune responses in AIA. These results are of significance in advancing our understanding of the pathogenesis of RA.
Subject(s)
Antibodies, Anti-Idiotypic/metabolism , Arthritis, Experimental/metabolism , Autoimmune Diseases/metabolism , Bacterial Proteins/immunology , Chaperonin 60/immunology , Interleukin-17/metabolism , Nicotine/pharmacology , Peptides, Cyclic/immunology , Animals , Arthritis, Experimental/pathology , Autoimmune Diseases/pathology , Cell Proliferation/drug effects , Disease Models, Animal , Male , Rats , Rats, Inbred Lew , Severity of Illness Index , T-Lymphocytes/pathology , Time FactorsABSTRACT
Rheumatoid arthritis (RA) is one of the major autoimmune diseases of global prevalence. The use of the anti-inflammatory drugs for the treatment of RA is associated with severe adverse reactions and toxicity. This limitation has necessitated the search for novel therapeutic products. We report here a traditional Chinese medicine-based herbal formula, Huo luo xiao ling dan (HLXL), which has potent antiarthritic activity as validated in the rat adjuvant-induced arthritis (AA) model. HLXL (2.3 g/Kg) was fed to Lewis (RT.1(1)) rats daily by gavage beginning at the onset of arthritis and then continued through the observation period. HLXL inhibited the severity of ongoing AA. This suppression of arthritis was associated with significant alterations in the T cell proliferative and cytokine responses as well as the antibody response against the disease-related antigen, mycobacterial heat-shock protein 65 (Bhsp65). There was a reduction in the level of the proinflammatory cytokines IL-17 and IL-1ß but enhancement of the anti-inflammatory cytokine IL-10 level. In addition, there was inhibition of both the anti-Bhsp65 antibody response and the serum level of nitric oxide. Thus, HLXL is a promising CAM modality for further testing in RA patients.
ABSTRACT
OBJECTIVE: Angiopoietin-like protein 4 (Angptl4) is a secreted glycoprotein that has recently been implicated in the regulation of angiogenesis and metastasis. This study aimed to investigate the structural and cellular basis underlying the biological actions of Angptl4. METHODS AND RESULTS: Circulating Angptl4 was proteolytically cleaved into NH2-terminal coiled-coil domain (N-Angptl4) and COOH-terminal fibrinogen-like domain (C-Angptl4). Using amino acid sequencing analysis, we identified a major cleavage site between Lys(168) and Leu(169) and a minor cleavage site between Lys(170) and Met(171) in mouse Angptl4. C-Angptl4, but not N-Angptl4, potently inhibited both bFGF- and VEGF-induced cell proliferation, migration, and tubule formation in endothelial cells, and prevented neovascularization in mice. Treatment of C-Angptl4 with PNGase F (an N-glycosidase) ablated its N-linked glycosylation, and also significantly attenuated its antiangiogenic activities. C-Angptl4 blocked bFGF-induced activation of ERK1/2 MAP kinase, but had no obvious effect on Akt and P38 MAP kinase. Furthermore, C-Angptl4 abrogated bFGF-induced phosphorylation of Raf-1 and MEK1/2, whereas neither auto-phosphorylation of FGF receptor-1 nor activation of Ras was affected, suggesting that the blockage occurs at the level of Raf-1 activation. CONCLUSIONS: The carboxyl terminus of Angptl4 alone is sufficient to suppress angiogenesis, possibly through inhibiting the Raf/MEK/ERK1/2 MAP kinase pathway in endothelial cells.
Subject(s)
Blood Proteins/physiology , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , MAP Kinase Kinase Kinases/antagonists & inhibitors , Neovascularization, Physiologic/physiology , raf Kinases/antagonists & inhibitors , Amino Acid Sequence , Angiopoietin-Like Protein 4 , Angiopoietins , Animals , Cell Line , Cell Movement/drug effects , Cell Proliferation/drug effects , Endothelium, Vascular/drug effects , Endothelium, Vascular/pathology , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Fibroblast Growth Factor 2/pharmacology , Humans , MAP Kinase Kinase Kinases/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Nude , Molecular Sequence Data , Phosphorylation/drug effects , Signal Transduction , Vascular Endothelial Growth Factor A/pharmacology , raf Kinases/metabolismABSTRACT
In the present study, patch clamp experiments demonstrated the expression of multiple ionic currents, including a Ba2+-sensitive inward rectifier K+ current (IKir), a 4-aminopyridine- (4-AP) sensitive delayed rectifier K+ current (IKDR), and a nifedipine-sensitive, tetrodotoxin-resistant inward Na+ current (INa.TTXR) in the non-transformed rat gastric epithelial cell line RGM-1. RT-PCR revealed molecular identities of mRNAs for the functional ionic currents, including Kir1.2 for IKir, Kv1.1, Kv1.6, and Kv2.1 for IKDR, and Nav1.5 for INa.TTXR. Pharmacologic blockade of Kv and Nav, but not Kir, suppressed RGM-1 cell proliferation. To further elucidate which subtypes of the ion channels were involved in cell proliferation, RNA interference was employed to knockdown specific gene expression. Downregulation of Kv1.1 or Nav1.5 by RNA interference suppressed RGM-1 cell proliferation. To conclude, our study is the first to delineate the expression of ion channels and their functions as growth modulators in gastric epithelial cells.