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1.
Neuroendocrinology ; 112(5): 446-456, 2022.
Article in English | MEDLINE | ID: mdl-34120115

ABSTRACT

INTRODUCTION: Targeting the α7 nicotinic acetylcholine receptor (α7nAChR) has recently been suggested as a potential new treatment for fibrotic skin diseases. Here, we performed a genetic and pharmacologic approach to clarify the role of this receptor in the bleomycin (BLM) mouse model of skin fibrosis using α7nAChR KO mice. METHODS: We analyzed the expression of extracellular matrix (ECM) components in murine skin using quantitative RT-PCR, pepsin digestion/SDS-PAGE of proteins and performed hydroxyproline assays as well as histological/immunohistochemical staining of skin sections. To identity the target cells of the α7nAChR agonist PHA-543613, we used murine dermal fibroblasts (MDF). We tested their response to the profibrotic cytokine transforming growth factor-ß1 (TGF-ß1) and utilized gene silencing to elucidate the role of the α7nAChR. RESULTS: We confirmed our previous findings on C3H/HeJ mice and detected a suppressive effect of PHA-543613 on BLM-induced skin fibrosis in the mouse strain C57BL/6J. This antifibrotic effect of PHA-543613 was abrogated in α7nAChR-KO mice. Interestingly, α7nAChR-KO animals exhibited a basal profibrotic signature by higher RNA expression of ECM genes and hydroxyproline content than WT mice. In WT MDF, PHA-543613 suppressed ECM gene expression induced by TGF-ß1. Gene silencing of α7nAChR by small interfering RNA neutralized the effects of PHA-543613 on TGF-ß1-mediated ECM gene expression. CONCLUSION: In summary, we have identified the α7nAChR as the essential mediator of the antifibrotic effect of PHA-543613. MDF are directly targeted by PHA-543613 to suppress collagen synthesis. Our findings emphasize therapeutic exploitation of α7nAChR receptor agonists in fibrotic skin diseases.


Subject(s)
Skin Diseases , alpha7 Nicotinic Acetylcholine Receptor , Animals , Bleomycin/metabolism , Bleomycin/toxicity , Bridged Bicyclo Compounds, Heterocyclic , Disease Models, Animal , Fibrosis , Hydroxyproline , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Quinuclidines , Transforming Growth Factor beta1/metabolism , alpha7 Nicotinic Acetylcholine Receptor/agonists , alpha7 Nicotinic Acetylcholine Receptor/genetics , alpha7 Nicotinic Acetylcholine Receptor/metabolism
2.
Exp Dermatol ; 29(9): 924-931, 2020 09.
Article in English | MEDLINE | ID: mdl-32780438

ABSTRACT

Nicotinic acetylcholine receptors (nAChRs) are members of the superfamily of neurotransmitter-gated ion channels. The natural ligand for nAChRs is the endogenous neurotransmitter acetylcholine. Among the nAChRs is the α7nAChR. It is not only expressed by neural tissues but also in the skin. A number of different resident cutaneous cell types including epidermal keratinocytes, sebocytes and dermal fibroblasts express functional α7nAChR. Moreover, cells of the immune system such as lymphocytes, macrophages and monocytes, playing an important role in skin homeostasis, also express α7nAChR. Translational research focusing on the exploitation of the α7nAChR in dermatology has revealed that this neuroendocrine receptor could be promising target for the treatment of inflammatory skin diseases. For example, α7nAChR agonists can counteract transforming growth factor-ß1-mediated responses in dermal fibroblasts, key effector cells in scleroderma. In accordance with this α7nAChR, agonists are effective in both inflammation and non-inflammation-driven models of experimentally induced skin fibrosis. Moreover, α7nAChR agonists can modulate expression of proinflammatory cytokines in epidermal keratinocytes that are crucially involved in the pathogenesis of psoriasis and other inflammatory skin diseases. Finally, the capability of α7nAChR agonists to suppress ultraviolet light A/B-induced responses, for example production of proinflammatory cytokines and oxidative stress, the latter crucially involved in dermal photoageing, points to a potential of such agents in the prevention of extrinsic skin ageing. Therefore, emphasis on translational research targeting the α7nAChR in skin may lead to the development of new treatment and prevention modalities against fibrosclerotic skin diseases, psoriasis vulgaris, atopic dermatitis, acne, photodermatoses and extrinsic skin ageing.


Subject(s)
Molecular Targeted Therapy , Skin Diseases/drug therapy , Skin/metabolism , alpha7 Nicotinic Acetylcholine Receptor/agonists , alpha7 Nicotinic Acetylcholine Receptor/metabolism , Animals , Humans
3.
Acta Derm Venereol ; 100(10): adv00157, 2020 May 28.
Article in English | MEDLINE | ID: mdl-32449780

ABSTRACT

Although recent therapeutic developments raise hope, melanoma remains a devastating disease with a need for new treatment targets. In other tumours prohormone convertases have been shown to be pro-tumourigenic as they are involved in processing preforms of matrix-metalloproteinases, growth factors and adhesion molecules. The aim of this study was to look for new treatment options for melanoma, by investigating the role of the prohormone convertase Paired basic Amino acid-Cleaving Enzyme 4 (PACE4/PCSK6) in melanoma cell lines and human melanoma tissue. PACE4-transfected A375 melanoma cells displayed significantly increased proliferation, MMP-2 production, gelatinase activity and migratory capacity in vitro compared with sham-transfected cells. In vivo, elevated PACE4 expression resulted in significantly increased tumour growth on immunodeficient mice. In the majority of 45 human primary melanomas and melanoma metastases ex vivo PACE4 immunoreactivity was detectable, while it was absent in in situ melanomas. These results indicate PACE4 as a regulator of melanoma cell aggressiveness.


Subject(s)
Melanoma/enzymology , Proprotein Convertases/metabolism , Serine Endopeptidases/metabolism , Skin Neoplasms/enzymology , Animals , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Melanoma/drug therapy , Melanoma/genetics , Melanoma/pathology , Mice, Hairless , Mice, SCID , Molecular Targeted Therapy , Neoplasm Invasiveness , Proprotein Convertases/antagonists & inhibitors , Proprotein Convertases/genetics , Serine Endopeptidases/genetics , Serine Proteinase Inhibitors/therapeutic use , Signal Transduction , Skin Neoplasms/drug therapy , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Tumor Burden
4.
J Invest Dermatol ; 140(12): 2371-2379, 2020 12.
Article in English | MEDLINE | ID: mdl-32335129

ABSTRACT

Targeting neuroendocrine receptors can be considered as another interesting approach to treating fibrotic disorders. Previously, we could demonstrate that tropisetron, a classical serotonin receptor blocker, can modulate collagen synthesis and acts in vitro through the α7 nicotinic acetylcholine receptor (α7nAchR). Here, we used a pharmacologic approach with specific α7nAchR agonists to validate this hypothesis. PHA-543613, an α7nAchR-specific agonist, not only prevented but also reversed established skin fibrosis of mice injected with bleomycin. Interestingly, agonistic stimulation of α7nAchR also attenuated experimental skin fibrosis in the non-inflammation driven adenovirus coding for TGFß receptor Iact mouse model, indicating fibroblast-mediated and not only anti-inflammatory effects of such agents. The fibroblast-mediated effects were confirmed in vitro using human dermal fibroblasts, in which the α7nAchR-specific agonists strongly reduced the impact of TGFß1-mediated expression on collagen and myofibroblast marker expression. These actions were linked to modulation of the redox-sensitive transcription factor JunB and impairment of the mitochondrial respiratory system. Our results indicate that pharmacologic stimulation of the α7nAchR could be a promising target for treatment of patients with skin fibrotic diseases. Moreover, our results suggest a mechanistic axis of collagen synthesis regulation through the mitochondrial respiratory system.


Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Quinuclidines/pharmacology , Scleroderma, Systemic/drug therapy , Skin/pathology , alpha7 Nicotinic Acetylcholine Receptor/agonists , Adenoviridae/genetics , Animals , Bleomycin/toxicity , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Cells, Cultured , Collagen/metabolism , Disease Models, Animal , Drug Evaluation, Preclinical , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibrosis , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Humans , Male , Mice , Primary Cell Culture , Quinuclidines/therapeutic use , Receptor, Transforming Growth Factor-beta Type I/genetics , Scleroderma, Systemic/chemically induced , Scleroderma, Systemic/genetics , Scleroderma, Systemic/pathology , Skin/cytology , Skin/drug effects , alpha7 Nicotinic Acetylcholine Receptor/metabolism
6.
J Pineal Res ; 67(4): e12610, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31532834

ABSTRACT

Melanogenesis is a key parameter of differentiation in melanocytes and melanoma cells; therefore, search for factors regulating this pathway are strongly desired. Herein, we investigated the effects of melatonin, a ubiquitous physiological mediator that is found throughout animals and plants. In mammals, the pineal gland secretes this indoleamine into the blood circulation to exert an extensive repertoire of biological activities. Our in vitro assessment indicates an oncostatic capacity of melatonin in time-dependent manner (24, 48, 72 hours) in highly pigmented MNT-1 melanoma cells. The similar pattern of regulation regarding cell viability was observed in amelanotic Sk-Mel-28 cells. Subsequently, MNT-1 cells were tested for the first time for evaluation of melanin/melatonin interaction. Thus primary, electron paramagnetic resonance (EPR) spectroscopy demonstrated that melatonin reduced melanin content. Artificially induced disturbances of melanogenesis by selected inhibitors (N-phenylthiourea or kojic acid) were slightly antagonized by melatonin. Additionally, analysis using transmission electron microscopy has shown that melatonin, particularly at higher dose of 10-3  mol/L, triggered the appearance of premelanosomes (stage I-II of melanosome) and MNT-1 cells synthesize de novo endogenous melatonin shown by LC-MS. In conclusion, these studies show a melanogenic-like function of melatonin suggesting it as an advantageous agent for treatment of pigmentary disorders.


Subject(s)
Melanins/biosynthesis , Melanoma/metabolism , Melatonin/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Humans , Melanoma/drug therapy , Melanoma/pathology , Pigmentation Disorders/drug therapy , Pigmentation Disorders/metabolism , Pigmentation Disorders/pathology
7.
Exp Dermatol ; 28(3): 276-282, 2019 03.
Article in English | MEDLINE | ID: mdl-30653770

ABSTRACT

Tropisetron is a serotonin receptor (5-HT-R)-modulating agent and approved as an antiemetic for patients undergoing chemotherapy. In the gut, it acts via specific serotonin receptors, 5-HT3 -R, to elicit its beneficial effects against nausea. We investigated whether tropisetron can affect inflammatory cell responses of human primary epidermal keratinocytes (NHK) which are key cells in the regulation of skin homoeostasis. Tropisetron significantly and dose-dependently suppressed tumor necrosis factor (TNF)-α-mediated mRNA expression and protein secretion of interleukin (IL)-6 and IL-8 in these cells. This effect of tropisetron was independent of p65/NF-κB as shown by various NF-κB signal transduction read-outs. Importantly, the anti-inflammatory tropisetron effect on NHK was neither mediated by 5-HT3 -R nor 5-HT4 -R since these receptors were absent in NHK. In contrast, NHK expressed α7 nicotinic acetylcholine receptors (α7nAchR) which previously were found to bind tropisetron. The α7nAchR antagonist α-bungarotoxin neutralized, whereas AR-R17779, a specific α7nAchR agonist, mimicked the suppressive effect of tropisetron on TNF-α-mediated IL-6 and IL-8 expression in NHK. Our findings suggest that tropisetron and probably other α7nAchR-activating agents could be useful for the future therapy of inflammatory skin diseases.


Subject(s)
Keratinocytes/metabolism , Tropisetron/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , alpha7 Nicotinic Acetylcholine Receptor/metabolism , Anti-Inflammatory Agents/pharmacology , Homeostasis , Humans , Inflammation , Interleukin-6/metabolism , Interleukin-8/metabolism , Keratinocytes/drug effects , Phosphorylation , Protein Binding , Psoriasis/metabolism , Serotonin 5-HT3 Receptor Antagonists/pharmacology , Signal Transduction , Skin/metabolism , Transcription Factor RelA/metabolism
8.
Int J Mol Sci ; 19(12)2018 Nov 28.
Article in English | MEDLINE | ID: mdl-30487387

ABSTRACT

Melatonin (Mel) is the major biologically active molecule secreted by the pineal gland. Mel and its metabolites, 6-hydroxymelatonin (6(OH)Mel) and 5-methoxytryptamine (5-MT), possess a variety of functions, including the scavenging of free radicals and the induction of protective or reparative mechanisms in the cell. Their amphiphilic character allows them to cross cellular membranes and reach subcellular organelles, including the mitochondria. Herein, the action of Mel, 6(OH)Mel, and 5-MT in human MNT-1 melanoma cells against ultraviolet B (UVB) radiation was investigated. The dose of 50 mJ/cm² caused a significant reduction of cell viability up to 48%, while investigated compounds counteracted this deleterious effect. UVB exposure increased catalase activity and led to a simultaneous Ca++ influx (16%), while tested compounds prevented these disturbances. Additional analysis focused on mitochondrial respiration performed in isolated mitochondria from the liver of BALB/cJ mice where Mel, 6(OH)Mel, and 5-MT significantly enhanced the oxidative phosphorylation at the dose of 10-6 M with lower effects seen at 10-9 or 10-4 M. In conclusion, Mel, 6(OH)Mel and 5-MT protect MNT-1 cells, which express melatonin receptors (MT1 and MT2) against UVB-induced oxidative stress and mitochondrial dysfunction, including the uncoupling of oxidative phosphorylation.


Subject(s)
Melanoma/metabolism , Melatonin/metabolism , Melatonin/pharmacology , 5-Methoxytryptamine/pharmacology , Animals , Calcium/metabolism , Catalase/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/radiation effects , Humans , Liver/drug effects , Liver/metabolism , Liver/radiation effects , Melatonin/analogs & derivatives , Mice, Inbred BALB C , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/radiation effects , Oxidative Phosphorylation/drug effects , Oxidative Phosphorylation/radiation effects , Oxidative Stress/drug effects , Oxidative Stress/radiation effects , Ultraviolet Rays
9.
Exp Dermatol ; 26(1): 73-81, 2017 01.
Article in English | MEDLINE | ID: mdl-27576129

ABSTRACT

Although there is increasing evidence that oxidative stress is involved in collagen synthesis and myofibroblast activation, the NADPH oxidase (Nox) system is incompletely investigated in the context of human dermal fibroblasts (HDFs) and skin fibrosis. Using the pan-Nox inhibitor diphenyleneiodonium (DPI) as an initial tool, we show that gene expression of collagen type I, α-smooth muscle actin (α-SMA) and fibronectin 1 is suppressed in HDFs. Detailed expression analysis of all Nox isoforms and adaptors revealed expression of RNA and protein expression of Nox4, p22phox and Poldip2 but neither Nox1 nor Nox2. Nox4 could be immunolocalized to the endoplasmic reticulum. Importantly, TGF-ß1 had a dose- and time-dependent upregulating effect on NADH activity and Nox4 gene expression in HDFs. Genetic silencing of Nox4 as demonstrated by siRNA in HDFs as well as in murine fibroblasts established from Nox4 knockout mice confirmed that TGF-ß1 -mediated collagen type I gene, α-SMA and fibronectin 1 gene expressions were Nox4-dependent. This TGF-ß1 effect was mediated by Smad3 as shown by in silico promoter analysis, pharmacological inhibition and gene silencing of Smad3. The relevance of these findings is highlighted in the bleomycin-induced scleroderma mouse model. DPI treatment attenuated skin fibrosis and myofibroblast activation. Moreover, Nox4 knockdown by siRNA reduced skin collagen synthesis, α-SMA and fibronectin 1 expression in vivo. Finally, analyses of HDFs from patients with systemic sclerosis confirmed the expression of Nox4 and its adaptors, whereas Nox1 and Nox2 were not detectable. Our findings indicate that Nox4 targeting is a promising future treatment for fibrotic skin diseases.


Subject(s)
Fibroblasts/enzymology , NADPH Oxidase 4/genetics , Scleroderma, Systemic/enzymology , Skin/enzymology , Skin/pathology , Actins/genetics , Adult , Animals , Cells, Cultured , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Computer Simulation , Cytokines/genetics , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Female , Fibronectins , Fibrosis , Gene Expression/drug effects , Gene Expression Profiling , Gene Silencing , Humans , Infant, Newborn , Isoenzymes/genetics , Male , Mice , Middle Aged , Multienzyme Complexes/metabolism , Myofibroblasts , NADH, NADPH Oxidoreductases/metabolism , NADPH Oxidase 1/genetics , NADPH Oxidase 2/genetics , NADPH Oxidase 4/metabolism , Onium Compounds/pharmacology , Primary Cell Culture , RNA, Messenger/metabolism , Scleroderma, Systemic/chemically induced , Scleroderma, Systemic/pathology , Transforming Growth Factor beta/pharmacology , Young Adult
11.
Exp Dermatol ; 25(11): 887-894, 2016 11.
Article in English | MEDLINE | ID: mdl-27307019

ABSTRACT

Mouse models resembling systemic sclerosis can be chemically induced by application of bleomycin or hypochloric acid (HOCl). To date, little is known about inflammatory cells and their potential role in scleroderma (Scl)-related fibrosis. Therefore, we compared both Scl models to define the early immune cell subsets in relation to fibrosis-related parameters. Both agents induced a significant increase in dermal thickness and collagen deposition after 4 weeks, as hallmarks of Scl. However, clinical skin thickness, densely packed, sirius red-stained collagen bundles and collagen cross-links were more pronounced in HOCl-induced Scl. In parallel, there was a significant upregulation of procollagen α1(I), α-SMA and TGF-ß transcripts in HOCl animals, whereas IL-1ß and MMP-13 mRNA levels were significantly increased in bleomycin-treated mice. Flow cytometric analysis of the Scl skin demonstrated an early cellular infiltrate containing mainly CD19+ B cells, CD4+ T cells, CD11c+ DC and CD11b+ myeloid cells, the latter ones being significantly more prominent after HOCl injection. Subanalysis revealed that Scl mice exhibited a significant increase of inflammatory myeloid CD11b+  Ly6Clow-high  CD64low-high cells (HOCl>bleomycin). In particular, in the HOCl model, activated dermal macrophages (CCR2low  MHCIIhigh ) and monocyte-derived DC (CCR2high MHCIIhigh ) predominated over less activated CD11b+ myeloid cells. In conclusion, the two models differ in certain aspects of the murine and human scleroderma but in the HOCl model, myeloid CD11b+  MHCIIhigh cells correlate with some fibrosis-related parameters. Therefore, analysis of both models is suggested to cover a comprehensive profile of Scl symptoms but with focus on the HOCl model when the role of early myeloid immune cells will be evaluated.


Subject(s)
Disease Models, Animal , Myeloid Cells/immunology , Scleroderma, Systemic/immunology , Animals , Bleomycin , Female , Fibrosis , Hypochlorous Acid , Mice, Inbred C57BL , Scleroderma, Systemic/chemically induced , Scleroderma, Systemic/metabolism , Scleroderma, Systemic/pathology , Skin/metabolism , Skin/pathology
12.
Exp Dermatol ; 23(6): 431-3, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24698097

ABSTRACT

The melanocortin-1 receptor (MC1 ) binds α-melanocyte-stimulating hormone (α-MSH) with high affinity and has a physiological role in cutaneous melanin pigmentation. Previously, we reported that human dermal fibroblasts also express functional MC1 . α-MSH suppressed transforming growth factor-ß1 - and bleomycin (BLM)-induced collagen synthesis in vitro and in vivo. Using MC1 signalling-deficient C57BL/6J-Mc1r(e/e) mice, we tested as to whether MC1 has a regulatory role on dermal collagen synthesis in the BLM model of scleroderma. Notably, mice with a C57BL/6J genetic background were previously shown to be BLM-non-susceptible. Interestingly, treatment of C57BL/6J-Mc1r(e/e) but not of C57BL/6J-wild-type mice with BLM increased cutaneous collagen type I content at RNA and protein level along with development of skin fibrosis. Cutaneous levels of connective tissue growth factor and monocyte chemotactic protein-1 were also increased in BLM-treated C57BL/6J-Mc1r(e/e) mice. Primary dermal fibroblasts from C57BL/6J-wt mice further expressed MC1 , suggesting that these cells are directly targeted by melanocortins to affect collagen production of the skin. Our findings support the concept that MC1 has an endogenous regulatory function in collagen synthesis and controls the extent of fibrotic stress responses of the skin.


Subject(s)
Bleomycin/adverse effects , Collagen/metabolism , Melanocortins/physiology , Receptor, Melanocortin, Type 1/deficiency , Scleroderma, Systemic/metabolism , Scleroderma, Systemic/physiopathology , Signal Transduction/physiology , Skin/pathology , Animals , Chemokine CCL2/metabolism , Connective Tissue Growth Factor/metabolism , Disease Models, Animal , Fibrosis/chemically induced , Fibrosis/pathology , Gene Deletion , Genetic Predisposition to Disease/genetics , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mutation/genetics , Receptor, Melanocortin, Type 1/genetics , Receptor, Melanocortin, Type 1/physiology , Scleroderma, Systemic/genetics , Skin/metabolism
13.
J Invest Dermatol ; 134(1): 168-175, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23884247

ABSTRACT

Prohormone convertases (PCs) are endoproteases that process many substrates in addition to hormone precursors. Although overexpression of PCs is linked to carcinogenesis in some solid tumors, the role of subtilisin-kexin isoenzyme-1 (SKI-1) in this context is unknown. We show that SKI-1 is constitutively expressed in human pigment cells with higher SKI activity in seven out of eight melanoma cell lines compared with normal melanocytes. SKI-1 immunoreactivity is also detectable in tumor cells of melanoma metastases. Moreover, tissue samples of the latter display higher SKI-1 mRNA levels and activity than normal skin. From various stimuli tested, 12-O-tetradecanoylphorbol-13-acetate and tunicamycin affected SKI-1 expression. Importantly, SKI-1 inhibition by the cell-permeable enzyme inhibitor decanoyl-RRLL-chloromethylketone (dec-RRLL-CMK) not only suppressed proliferation and metabolic activity of melanoma cells in vitro but also reduced tumor growth of melanoma cells injected intracutaneously into immunodeficient mice. Mechanistic studies revealed that dec-RRLL-CMK induces classical apoptosis of melanoma cells in vitro and affects expression of several SKI-1 target genes including activating transcription factor 6 (ATF6). However, ATF6 gene silencing does not result in apoptosis of melanoma cells, suggesting that dec-RRLL-CMK induces cell death in an ATF6-independent manner. Our findings encourage further studies on SKI-1 as a potential target for melanoma therapy.


Subject(s)
Melanoma/metabolism , Melanoma/secondary , Proprotein Convertases/antagonists & inhibitors , Proprotein Convertases/metabolism , Serine Endopeptidases/metabolism , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Activating Transcription Factor 6/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/physiology , Cell Line, Tumor , Endoplasmic Reticulum Chaperone BiP , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Heat-Shock Proteins/genetics , Humans , Melanoma/drug therapy , Mice , Mice, Inbred NOD , Mice, Mutant Strains , Mice, SCID , Proprotein Convertases/genetics , Serine Endopeptidases/genetics , Skin Neoplasms/drug therapy , Xenograft Model Antitumor Assays
14.
J Invest Dermatol ; 134(2): 526-537, 2014 Feb.
Article in English | MEDLINE | ID: mdl-23921952

ABSTRACT

Proper activation of macrophages (Mφ) in the inflammatory phase of acute wound healing is essential for physiological tissue repair. However, there is a strong indication that robust Mφ inflammatory responses may be causal for the fibrotic response always accompanying adult wound healing. Using a complementary approach of in vitro and in vivo studies, we here addressed the question of whether mesenchymal stem cells (MSCs)-due to their anti-inflammatory properties-would control Mφ activation and tissue fibrosis in a murine model of full-thickness skin wounds. We have shown that the tumor necrosis factor-α (TNF-α)-stimulated protein 6 (TSG-6) released from MSCs in co-culture with activated Mφ or following injection into wound margins suppressed the release of TNF-α from activated Mφ and concomitantly induced a switch from a high to an anti-fibrotic low transforming growth factor-ß1 (TGF-ß1)/TGF-ß3 ratio. This study provides insight into what we believe to be a previously undescribed multifaceted role of MSC-released TSG-6 in wound healing. MSC-released TSG-6 was identified to improve wound healing by limiting Mφ activation, inflammation, and fibrosis. TSG-6 and MSC-based therapies may thus qualify as promising strategies to enhance tissue repair and to prevent excessive tissue fibrosis.


Subject(s)
Cell Adhesion Molecules/metabolism , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/metabolism , Skin/injuries , Wound Healing/physiology , Animals , Cell Adhesion Molecules/genetics , Cells, Cultured , Coculture Techniques , Disease Models, Animal , Fibrosis/metabolism , Fibrosis/pathology , Fibrosis/prevention & control , Humans , Injections, Intradermal , Macrophages/pathology , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred C57BL , Skin/pathology
15.
Arthritis Rheum ; 65(3): 792-804, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23440693

ABSTRACT

OBJECTIVE: There is increasing evidence that serotonin (5-hydroxytryptamine [5-HT]) and distinct 5-HT receptors are involved in the pathogenesis of systemic sclerosis. The aim of this study was to test the hypothesis that tropisetron, a routinely used antiemetic agent previously characterized as a 5-HT(3/4) receptor-modulating agent, can directly affect collagen synthesis in vitro and attenuate experimentally induced fibrosis in vivo. METHODS: Functional in vitro studies were performed using human dermal fibroblasts (HDFs). Signal transduction studies included immunofluorescence analysis, Western immunoblotting, promoter reporter assays, cAMP/Ca(2+) measurements, and use of pharmacologic activators and inhibitors. Gene silencing was performed using small interfering RNA. Putative receptors of tropisetron were detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. The murine model of bleomycin-induced scleroderma was used to assess the antifibrogenic and antifibrotic effects of tropisetron in vivo. Collagen expression in vitro, ex vivo, and in situ was determined by real-time RT-PCR analysis, Western immunoblotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunohistochemical analysis. RESULTS: Tropisetron suppressed collagen synthesis induced by transforming growth factor ß1 (TGFß1). This effect was independent of 5-HT(3/4) receptor but was mediated via α7 nicotinic acetylcholine receptor (α7nAChR). Suppression of TGFß1-induced collagen synthesis occurred via an unknown molecular mechanism not involving modulation of the Smad, cAMP, Akt, c-Jun, or MAPK pathway. In vivo, tropisetron not only prevented skin fibrosis but also reduced the collagen content in established dermal fibrosis induced by bleomycin. CONCLUSION: Tropisetron directly reduces collagen synthesis in HDFs via an α7nAChR-dependent mechanism. The antifibrogenic and antifibrotic effects of this agent observed in a mouse model of bleomycin- induced scleroderma indicate the future potential of tropisetron in the treatment of fibrotic diseases such as scleroderma.


Subject(s)
Collagen/biosynthesis , Indoles/pharmacology , Receptors, Nicotinic/metabolism , Scleroderma, Systemic/drug therapy , 3T3 Cells , Adult , Aged , Animals , Antibiotics, Antineoplastic/toxicity , Bleomycin/toxicity , Dermis/drug effects , Dermis/metabolism , Dermis/pathology , Disease Models, Animal , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , Fibrosis/drug therapy , Fibrosis/metabolism , Fibrosis/pathology , Humans , Mice , Middle Aged , Scleroderma, Systemic/metabolism , Scleroderma, Systemic/pathology , Serotonin Antagonists/pharmacology , Signal Transduction/drug effects , Transforming Growth Factor beta1/metabolism , Tropisetron , alpha7 Nicotinic Acetylcholine Receptor
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