Ulcerative colitis (UC) and Crohn's disease (CD) are two forms of chronic inflammatory bowel disease. CD4 T cells play a central role in the pathogenesis of both diseases. Smoking affects both UC and ...CD but with opposite effects, ameliorating UC and worsening CD. We hypothesized that the severity of gut inflammation could be modulated through T cell nicotinic acetylcholine receptors (nAChRs) and that the exact clinical outcome would depend on the repertoire of nAChRs on CD4 T cells mediating each form of colitis. We measured clinical and immunologic outcomes of treating BALB/c mice with oxazolone- and trinitrobenzene sulfonic acid (TNBS)-induced colitides by nicotine. Nicotine attenuated oxazolone colitis, which was associated with an increased percentage of colonic regulatory T cells and a reduction of Th17 cells. TCR stimulation of naive CD4(+)CD62L(+) T cells in the presence of nicotine upregulated expression of Foxp3. In marked contrast, nicotine worsened TNBS colitis, and this was associated with increased Th17 cells among colonic CD4 T cells. Nicotine upregulated IL-10 and inhibited IL-17 production, which could be abolished by exogenous IL-12 that also abolished the nicotine-dependent upregulation of regulatory T cells. The dichotomous action of nicotine resulted from the up- and downregulation of anti-inflammatory α7 nAChR on colonic CD4 T cells induced by cytokines characteristic of the inflammatory milieu in oxazolone (IL-4) and TNBS (IL-12) colitis, respectively. These findings help explain the dichotomous effect of smoking in patients with UC and CD, and they underscore the potential for nicotinergic drugs in regulating colonic inflammation.
Ulcerative colitis (UC) and Crohn's disease (CD) are two forms of chronic inflammatory bowel disease. CD4 T cells play a central role in the pathogenesis of both diseases. Smoking affects both UC and ...CD but with opposite effects, ameliorating UC and worsening CD. We hypothesized that the severity of gut inflammation could be modulated through T cell nicotinic acetylcholine receptors (nAChRs) and that the exact clinical outcome would depend on the repertoire of nAChRs on CD4 T cells mediating each form of colitis. We measured clinical and immunologic outcomes of treating BALB/c mice with oxazolone- and trinitrobenzene sulfonic acid (TNBS)-induced colitides by nicotine. Nicotine attenuated oxazolone colitis, which was associated with an increased percentage of colonic regulatory T cells and a reduction of Th17 cells. TCR stimulation of naive CD4+CD62L+ T cells in the presence of nicotine upregulated expression of Foxp3. In marked contrast, nicotine worsened TNBS colitis, and this was associated with increased Th17 cells among colonic CD4 T cells. Nicotine upregulated IL-10 and inhibited IL-17 production, which could be abolished by exogenous IL-12 that also abolished the nicotine-dependent upregulation of regulatory T cells. The dichotomous action of nicotine resulted from the up- and downregulation of anti-inflammatory alpha 7 nAChR on colonic CD4 T cells induced by cytokines characteristic of the inflammatory milieu in oxazolone (IL-4) and TNBS (IL-12) colitis, respectively. These findings help explain the dichotomous effect of smoking in patients with UC and CD, and they underscore the potential for nicotinergic drugs in regulating colonic inflammation.
A loss of epidermal cohesion in pemphigus vulgaris (PV) results from autoantibody action on keratinocytes (KCs) activating the signaling kinases and executioner caspases that damage KCs, causing ...their shrinkage, detachment from neighboring cells, and rounding up (apoptolysis). In this study, we found that PV antibody binding leads to activation of epidermal growth factor receptor kinase, Src, p38 MAPK, and JNK in KCs with time pattern variations from patient to patient. Both extrinsic and intrinsic apoptotic pathways were also activated. Although Fas ligand neutralizing antibody could inhibit the former pathway, the mechanism of activation of the latter remained unknown. PV antibodies increased cytochrome c release, suggesting damage to mitochondria. The immunoblotting experiments revealed penetration of PVIgG into the subcellular mitochondrial fraction. The antimitochondrial antibodies from different PV patients recognized distinct combinations of antigens with apparent molecular sizes of 25, 30, 35, 57, 60, and 100 kDa. Antimitochondrial antibodies were pathogenic because their absorption abolished the ability of PVIgG to cause keratinocyte detachment both in vitro and in vivo. The downstream signaling of antimitochondrial antibodies involved JNK and late p38 MAPK activation, whereas the signaling of anti-desmoglein 3 (Dsg3) antibody involved JNK and biphasic p38 MAPK activation. Using KCs grown from Dsg3⁻/⁻ mice, we determined that Dsg3 did not serve as a surrogate antigen allowing antimitochondrial antibodies to enter KCs. The PVIgG-induced activation of epidermal growth factor receptor and Src was affected neither in Dsg3⁻/⁻ KCs nor due to absorption of antimitochondrial antibodies. These results demonstrated that apoptolysis in PV is a complex process initiated by at least three classes of autoantibodies directed against desmosomal, mitochondrial, and other keratinocyte self-antigens. These autoantibodies synergize with the proapoptotic serum and tissue factors to trigger both extrinsic and intrinsic pathways of cell death and break the epidermal cohesion, leading to blisters. Further elucidation of the primary signaling events downstream of PV autoantigens will be crucial for the development of a more successful therapy for PV patients.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A novel paradigm of keratinocyte (KC) regulation via nicotinic acetylcholine receptors (nAChR) has been discovered in studies of SLURP (secreted mammalian Ly-6/urokinase-type plasminogen activator ...receptor-related protein)-1 in Mal de Meleda. We cloned human SLURP-1 and produced recombinant protein and the monoclonal antibody 336H12-1A3 that visualized native SLURP-1. SLURP-1 ligated the conventional ligand-binding site of KC nAChR, showing a higher affinity to the 3Hnicotine-, compared with the 3Hepibatidine-sensitive nAChR. SLURP-1 significantly (p<0.05) increased the activities of caspases 3 and 8, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling-positive cells. The pro-apoptotic activity of SLURP-1 exceeded that of tumor necrosis factor-α, suggesting the involvement of separate pathways. In a series of real-time PCR and in-cell western experiments, SLURP-1 significantly (p<0.05) upregulated expression of transglutaminase type I cytokeratin 10, p21, and caspase-3. In the presence of the agonist carbachol, the effects of SLURP-1 on gene expression were augmented, which is in keeping with the notion that SLURP-1 acts as an allosteric agonist at the KC nAChR. Thus, the changes in the cell state induced by SLURP-1 could result from nAChR-mediated effects on the KC gene expression. These results suggest that the biological role of SLURP-1 in the epidermis is to provide fine tuning of the physiologic regulation of KC functions through the cholinergic pathways.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The use of tobacco products is associated with an increased incidence of periodontal disease, poor response to periodontal therapy, and a high risk for developing head and neck cancer. Nicotine and ...tobacco-derived nitrosamines have been shown to exhibit their pathobiologic effects due in part to activation of the nicotinic acetylcholine (ACh) receptors (nAChRs), mainly α7 nAChR, expressed by oral keratinocytes (KCs). This study was designed to gain mechanistic insight into α7-mediated morbidity of tobacco products in the oral cavity. We investigated the signaling pathways downstream of α7 nAChR in monolayers of oral KCs exposed for 24 h to aged and diluted sidestream cigarette smoke (ADSS) or an equivalent concentration of pure nicotine. By both real-time polymerase chain reaction (PCR) and In-cell Western, the KCs stimulated with ADSS or nicotine showed multifold increases of STAT-3. These effects could be completely blocked or significantly (P<0.05) diminished if the cells were pretreated with the α7 antagonist α-bungarotoxin (αBTX) or transfected with anti-α7 small interfering RNA (siRNA-α7). The use of pathway inhibitors revealed that signaling through the Ras/Raf-1/MEK1/ERK steps mediated α7-dependent up-regulation of STAT-3. Targeted mutation of the α7 gene prevented ERK1/2 activation by nicotine. Using the gel mobility shift assay, we demonstrated that an increased protein binding activity of STAT-3 caused by ADSS or pure nicotine was mediated by janus-activated kinase (JAK)-2. Activation of JAK-2/STAT-3 pathway could be prevented by αBTX or siRNA-α7. Thus, nuclear transactivation of STAT-3 in KCs exposed to tobacco products is mediated via intracellular signaling downstream from α7, which proceeds via two complementary pathways. The Ras/Raf-1/MEK1/ERK cascade culminates in up-regulated expression of the gene encoding STAT-3, whereas recruitment and activation of tyrosine kinase JAK-2 phosphorylates it. Elucidation of this novel mechanism of nicotine-dependent nuclear transactivation of STAT-3 identifies oral α7 nAChR as a promising molecular target to prevent, reverse, or retard tobacco-related periodontal disease and progression of head and neck cancer by receptor inhibitors.--Arredondo, J., Chernyavsky, A. I., Jolkovsky, D. L., Pinkerton, K. E., Grando, S. A. Receptor-mediated tobacco toxicity: cooperation of the Ras/Raf-1/MEK1/ERK and JAK-2/STAT-3 pathways downstream of α7 nicotinic receptor in oral keratinocytes.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
This study reveals a function of endogenous galectin-3, an animal lectin recognizing β-galactosides, in regulating dendritic cell motility both in vitro and in vivo, which to our knowledge is ...unreported. First, galectin-3-deficient (gal3−/-) bone marrow-derived dendritic cells exhibited defective chemotaxis compared to gal3+/+ cells. Second, cutaneous dendritic cells in gal3−/- mice displayed reduced migration to draining lymph nodes upon hapten stimulation compared to gal3+/+ mice. Moreover, gal3−/- mice were impaired in the development of contact hypersensitivity relative to gal3+/+ mice in response to a hapten, a process in which dendritic cell trafficking to lymph nodes is critical. In addition, defective signaling was detected in gal3−/- cells upon chemokine receptor activation. By immunofluorescence microscopy, we observed that galectin-3 is localized in membrane ruffles and lamellipodia in stimulated dendritic cells and macrophages. Furthermore, galectin-3 was enriched in lipid raft domains under these conditions. Finally, we determined that ruffles on gal3−/- cells contained structures with lower complexity compared to gal3+/+ cells. In view of the participation of membrane ruffles in signal transduction and cell motility, we conclude that galectin-3 regulates cell migration by functioning at these structures.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
: Understanding the acantholytic pathways leading to blistering in pemphigus vulgaris (PV) is a key to development of novel treatments. A novel paradigm of keratinocyte damage in PV, termed ...apoptolysis, links the suprabasal acantholytic and cell death pathways to basal cell shrinkage rendering a ‘tombstone’ appearance to PV lesions. In contrast to apoptolysis, the classic keratinocyte apoptosis mediating toxic epidermal necrolysis causes death and subsequent sloughing of the entire epidermis. Apoptolysis includes five consecutive steps. (1) Binding of autoantibodies to PV antigens. (2) Activation of EGF receptor, Src, mTOR, p38 MAPK and other signalling elements downstream of ligated antigens, elevation of intracellular calcium and launching of the cell death cascades. (3) Basal cell shrinkage due to: (i) collapse and retraction of the tonofilaments cleaved by executioner caspases; and (ii) dissociation of interdesmosomal adhesion complexes caused by phosphorylation of adhesion molecules. (4) Massive cleavage of cellular proteins by activated cell death enzymes leading to cell collapse, and tearing off desmosomes from the cell membrane stimulating secondary autoantibody production. (5) Rounding up and death of acantholytic cells. Thus, the structural damage (acantholysis) and death (apoptosis) of keratinocytes are mediated by the same cell death enzymes. Appreciation of the unifying concept of apoptolysis have several important implications: (i) linking together a number of seemingly unrelated events surrounding acantholysis; (ii) opening new avenues of investigation into the pathomechanism of pemphigus; and (iii) creating new approaches to the treatment of pemphigus based on blocking the signalling pathways and enzymatic processes that lead to blistering.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Pemphigus vulgaris (PV) is a potentially lethal mucocutaneous blistering disease characterized by cell-cell detachment within the stratified epithelium (acantholysis) caused by IgG autoantibodies. ...Intravenous immunoglobulin (IVIg) therapy effectively treats PV, but the mechanism is not fully understood. To further understand acantholysis and the efficacy of IVIg, we measured effects of IgG fractions from PV patients on keratinocyte death processes. Using IgGs from representative PV patients who improved with IVIg, we identified apoptotic and oncotic signaling pathways in
in vitro
and
in vivo
PV models. We identified two groups of PV patients, each producing autoantibodies activating predominantly either apoptotic or oncotic cell death pathway. Experimental treatments with caspase 3 or calpain inhibitors demonstrated that PV IgGs induced acantholysis through both pathways. Upstream, the apoptotic signaling involved activation of caspases 8 and 3 and up-regulation of Fas ligand mRNA, whereas calpain-mediated cell death depended on elevated intracellular free Ca
2+. IVIg reduced PV IgG-mediated acantholysis and cell death and up-regulated the caspase inhibitor FLIP and the calpain inhibitor calpastatin. These results indicate that in different PV patients, IgG-induced acantholysis proceeds predominantly via distinct, yet complementary, pathways of programmed cell death differentially mediated by apoptosis and oncosis effectors, with IVIg protecting target cells by up-regulating endogenous caspase and calpain inhibitors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Corneal epithelial erosion is one of the most common problems in clinical ophthalmology. Despite significant progress in understanding how the cornea heals, clinically available pharmacological ...therapies that can promote repair and prevent visual complications remain limited. We have recently demonstrated that the acetylcholine (ACh) axis of corneal epithelium plays an important role in regulation and coordination of distinct activities of corneal epithelial cells (CEC) mediating re-epithelialization, but mechanisms remained unclear. We hypothesized that the grounds for synergistic effects of corneal ACh receptors lie within the signaling pathways linking different receptors to specific elements of the CEC pro-epithelialization activities. In this study, we sought to elucidate the molecular mechanisms of cooperation of corneal muscarinic and nicotinic ACh receptors (mAChRs and nAChRs) in upregulation of E-cadherin expression. The roles of individual corneal mAChRs and nAChRs subtypes were investigated by in-cell western assay of the ACh-treated CEC, in which different ACh receptor genes were silenced by receptor-specific shRNAs. Functional inactivation of M3, but not M4, mAChR subtype, or α3 or α7, but not α9, nAChR subunit significantly inhibited E-cadherin expression. To gain a mechanistic insight, we blocked the key steps of the downstream signaling pathways. Results demonstrated that cholinergic agonists can upregulate E-cadherin expression by activating M3 mAChR, and α3β2 and α7 nAChRs via the common signaling cascade Ca2+–CaMKII–PKC–Ras–Raf–MEK–ERK. Activation of α7 nAChR can launch the Ras–Raf–MEK–ERK cascade both indirectly, through the Ca2+–CaMKII–PKC step, and directly, perhaps, due to its direct interaction with Ras. Although the biological significance of such redundancy remained to be elucidated, results of the present study point to a new direction to pharmacologically accelerate corneal re-epithelialization, and should have salient clinical implication.
•Signaling linking the M3 muscarinic, and the α3-, and α7-made nicotinic receptors upregulates E-cadherin gene expression.•Cholinergic agonists can upregulate E-cadherin gene expression by activating the common signaling cascade Ca2+-CaMKII-PKC-Ras-Raf-MEK-ERK.•Activation of α7 nicotinic receptor launches Ras-Raf-MEK-ERK cascade both indirectly, through Ca2+-CaMKII-PKC step, and directly through Ras.•Cholinergic drugs activating the Ca2+-CaMKII-PKC-Ras-Raf-MEK-ERK pathway may facilitate re-epithelialization of corneal wounds.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
It is well established that auto/paracrine acetylcholine (ACh) is essential for wound epithelialization, and that the mechanisms include regulation of keratinocyte motility and adhesion via nicotinic ...ACh receptors (nAChRs). Keratinocyte nAChRs can be also activated by non‐canonical ligands, such as secreted mammalian Ly‐6/urokinase‐type plasminogen activator receptor‐related protein (SLURP)‐1 and ‐2. In this study, we determined effects of recombinant (r)SLURP‐1 and‐2 on migration of human epidermal and oral keratinocytes under agarose and epithelialization of cutaneous and oral mucosal excisional wounds in mice, and also identified nAChRs mediating SLURP signals. Both in vitro and in vivo, rSLURP‐1 decreased and SLURP‐2 increased epithelialization rate. The mixture of both peptides accelerated epithelialization even further, indicating that their simultaneous signaling renders an additive physiologic response. The specificity of rSLURP actions was illustrated by similar effects on cutaneous and oral wounds, which feature distinct responses to injury, and also by abrogation of rSLURP effects with neutralizing antibodies. rSLURP‐1 acted predominantly via the α7 nAChR‐coupled up‐regulation of the sedentary integrins α2 and α3, whereas SLURP‐2—through α3, and α9 nAChRs up‐regulating migratory integrins α5 and αV. The biologic effects of rSLURPs required the presence of endogenous ACh, indicating that auto/paracrine SLURPs provide for a fine tuning of the physiologic regulation of crawling locomotion via the keratinocyte ACh axis. Since nAChRs have been shown to regulate SLURP production, cholinergic regulation of keratinocyte migration appears to be mediated by a reciprocally arranged network. The cholinergic peptides, therefore, may become prototype drugs for the treatment of wounds that fail to heal.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
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