ABSTRACT
BACKGROUND: Eosinophils contribute to the pathology of several types of disorders, in particular of allergic nature, and strategies to limit their actions are therefore warranted. OBJECTIVE: We sought to evaluate the possibility of targeting the acidic, lysosome-like eosinophil granules as a potential means of inducing eosinophil cell death. METHODS: To this end, we used monensin, an ionophoric drug that has previously been shown to permeabilize the secretory granules of mast cells, thereby inducing cell death. RESULTS: Our findings reveal that monensin induces cell death in human eosinophils, whereas neutrophils were less affected. Blockade of granule acidification reduced the effect of monensin on the eosinophils, demonstrating that granule acidity is an important factor in the mechanism of cell death. Furthermore, monensin caused an elevation of the granule pH, which was accompanied by a decrease of the cytosolic pH, hence indicating that monensin caused leakage of acidic contents from the granules into the cytosol. In agreement with a granule-targeting mechanism, transmission electron microscopy analysis revealed that monensin caused extensive morphological alterations of the eosinophil granules, as manifested by a marked loss of electron density. Eosinophil cell death in response to monensin was caspase-independent, but dependent on granzyme B, a pro-apoptotic serine protease known to be expressed by eosinophils. CONCLUSIONS: We conclude that monensin causes cell death of human eosinophils through a granule-mediated mechanism dependent on granzyme B.
Subject(s)
Eosinophils , Monensin , Humans , Monensin/pharmacology , Monensin/metabolism , Granzymes/metabolism , Granzymes/pharmacology , Secretory Vesicles/metabolism , Cytoplasmic GranulesABSTRACT
Mastocytosis is a KIT-related myeloproliferative disease characterised by abnormal expansion of neoplastic mast cells (MC) in the skin or virtually any other organ system. The cutaneous form of adult-onset mastocytosis is almost invariably combined with indolent systemic involvement for which curative therapy is yet not available. Here we evaluated a concept of depleting cutaneous MCs in mastocytosis lesions ex vivo by targeting their secretory granules. Skin biopsies from mastocytosis patients were incubated with or without mefloquine, an antimalarial drug known to penetrate into acidic organelles such as MC secretory granules. Mefloquine reduced the number of dermal MCs without affecting keratinocyte proliferation or epidermal gross morphology at drug concentrations up to 40 µM. Flow cytometric analysis of purified dermal MCs showed that mefloquine-induced cell death was mainly due to apoptosis and accompanied by caspase-3 activation. However, caspase inhibition provided only partial protection against mefloquine-induced cell death, indicating predominantly caspase-independent apoptosis. Further assessments revealed that mefloquine caused an elevation of granule pH and a corresponding decrease in cytosolic pH, suggesting drug-induced granule permeabilisation. Extensive damage to the MC secretory granules was confirmed by transmission electron microscopy analysis. Further, blockade of granule acidification or serine protease activity prior to mefloquine treatment protected MCs from apoptosis, indicating that granule acidity and granule-localised serine proteases play major roles in the execution of mefloquine-induced cell death. Altogether, these findings reveal that mefloquine induces selective apoptosis of MCs by targeting their secretory granules and suggest that the drug may potentially extend its range of medical applications.
Subject(s)
Mastocytosis, Cutaneous , Mastocytosis , Adult , Humans , Mast Cells/metabolism , Mefloquine/metabolism , Mastocytosis, Cutaneous/metabolism , Secretory Vesicles/metabolism , Secretory Vesicles/pathology , Apoptosis , Caspases/metabolismSubject(s)
Mast Cells , Monensin , Apoptosis , Homeostasis , Humans , Mast Cells/metabolism , Monensin/metabolism , Monensin/pharmacology , Secretory Vesicles/metabolismABSTRACT
BACKGROUND: Mast cells (MCs) have a profound impact on allergic asthma. Under such conditions, MCs undergo degranulation, resulting in the release of exceptionally large amounts of MC-restricted proteases. However, the role of these proteases in asthma is only partially understood. OBJECTIVES: We sought to test our hypothesis that MC proteases can influence the functionality of human lung fibroblasts (HLFs). METHODS: Primary HLFs were treated with MC chymase or tryptase, followed by assessment of parameters related to fibroblast function. RESULTS: HLFs underwent major morphologic changes in response to chymase, showing signs of cellular contraction, but were refractory to tryptase. However, no effects of chymase on HLF viability or proliferation were seen. Chymase, but not tryptase, had a major impact on the output of extracellular matrix-associated compounds from the HLFs, including degradation of fibronectin and collagen-1, and activation of pro-matrix metalloprotease 2. Further, chymase induced the release of various chemotactic factors from HLFs. In line with this, conditioned medium from chymase-treated HLFs showed chemotactic activity on neutrophils. Transcriptome analysis revealed that chymase induced a proinflammatory gene transcription profile in HLFs, whereas tryptase had minimal effects. CONCLUSIONS: Chymase, but not tryptase, has a major impact on the phenotype of primary airway fibroblasts by modifying their output of extracellular matrix components and by inducing a proinflammatory phenotype.
Subject(s)
Asthma/etiology , Chymases/toxicity , Fibroblasts/drug effects , Lung/drug effects , Mast Cells/enzymology , Apoptosis/drug effects , Cells, Cultured , Collagen Type I/metabolism , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Lung/metabolism , Lung/pathology , Mast Cells/physiology , Transcriptome , Tryptases/toxicityABSTRACT
Persistent and impaired inflammation impedes tissue healing and is a characteristic of chronic wounds. A better understanding of the mechanisms controlling wound inflammation is needed. In this study, we show that in human wound-edge keratinocytes, the expressions of microRNA (miR)-17, miR-18a, miR-19a, miR-19b, and miR-20a, which all belong to the miR-17â¼92 cluster, are upregulated during wound repair. However, their levels are lower in chronic ulcers than in acute wounds at the proliferative phase. Conditional knockout of miR-17â¼92 in keratinocytes as well as injection of miR-19a/b and miR-20a antisense inhibitors into wound edges enhanced inflammation and delayed wound closure in mice. In contrast, conditional overexpression of the miR-17â¼92 cluster or miR-19b alone in mice keratinocytes accelerated wound closure in vivo. Mechanistically, miR-19a/b and miR-20a decreased TLR3-mediated NF-κB activation by targeting SHCBP1 and SEMA7A, respectively, reducing the production of inflammatory chemokines and cytokines by keratinocytes. Thus, miR-19a/b and miR-20a being crucial regulators of wound inflammation, the lack thereof may contribute to sustained inflammation and impaired healing in chronic wounds. In line with this, we show that a combinatory treatment with miR-19b and miR-20a improved wound healing in a mouse model of type 2 diabetes.
Subject(s)
Diabetes Mellitus, Type 2/complications , Diabetic Foot/pathology , MicroRNAs/metabolism , Pressure Ulcer/pathology , Wound Healing/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Case-Control Studies , Cell Line , Cytokines/metabolism , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/immunology , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/immunology , Diabetes Mellitus, Type 2/pathology , Diabetic Foot/immunology , Disease Models, Animal , Female , Gene Expression Regulation , Gene Knockout Techniques , Healthy Volunteers , Humans , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Keratinocytes/immunology , Keratinocytes/metabolism , Keratinocytes/pathology , Male , Mice , Mice, Knockout , MicroRNAs/genetics , Middle Aged , Pressure Ulcer/immunology , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Streptozocin/administration & dosage , Wound Healing/immunologyABSTRACT
Tryptase is a tetrameric serine protease located within the secretory granules of mast cells. In the secretory granules, tryptase is stored in complex with negatively charged heparin proteoglycans and it is known that heparin is essential for stabilizing the enzymatic activity of tryptase. However, recent findings suggest that enzymatically active tryptase also can be found in the nucleus of murine mast cells, but it is not known how the enzmatic activity of tryptase is maintained in the nuclear milieu. Here we hypothesized that tryptase, as well as being stabilized by heparin, can be stabilized by DNA, the rationale being that the anionic charge of DNA could potentially substitute for that of heparin to execute this function. Indeed, we showed that double-stranded DNA preserved the enzymatic activity of human ß-tryptase with a similar efficiency as heparin. In contrast, single-stranded DNA did not have this capacity. We also demonstrated that DNA fragments down to 400 base pairs have tryptase-stabilizing effects equal to that of intact DNA. Further, we showed that DNA-stabilized tryptase was more efficient in degrading nuclear core histones than heparin-stabilized enzyme. Finally, we demonstrated that tryptase, similar to its nuclear localization in murine mast cells, is found within the nucleus of primary human skin mast cells. Altogether, these finding reveal a hitherto unknown mechanism for the stabilization of mast cell tryptase, and these findings can have an important impact on our understanding of how tryptase regulates nuclear events.
Subject(s)
DNA/chemistry , Mast Cells/enzymology , Tryptases/chemistry , Cells, Cultured , Enzyme Stability , Humans , Mast Cells/chemistry , Mast Cells/cytology , Skin/chemistry , Skin/cytology , Skin/enzymologyABSTRACT
Venous ulcers are the most common type of human chronic nonhealing wounds and are stalled in a constant and excessive inflammatory state. The molecular mechanisms underlying the chronic wound inflammation remain elusive. Moreover, little is known about the role of regulatory RNAs, such as microRNAs, in the pathogenesis of venous ulcers. We found that both microRNA (miR)-34a and miR-34c were upregulated in the wound-edge epidermal keratinocytes of venous ulcers compared with normal wounds or the skin. In keratinocytes, miR-34a and miR-34c promoted inflammatory chemokine and cytokine production. In wounds of wild-type mice, miR-34a-mimic treatment enhanced inflammation and delayed healing. To further explore how miR-34 functions, LGR4 was identified as a direct target mediating the proinflammatory function of miR-34a and miR-34c. Interestingly, impaired wound closure with enhanced inflammation was also observed in Lgr4 knockout mice. Mechanistically, the miR-34-LGR4 axis regulated GSK-3ß-induced p65 serine 468 phosphorylation, changing the activity of the NF-κB signaling pathway. Collectively, the miR-34-LGR4 axis was shown to regulate keratinocyte inflammatory response, the deregulation of which may play a pathological role in venous ulcers.
Subject(s)
MicroRNAs/metabolism , Receptors, G-Protein-Coupled/genetics , Varicose Ulcer/immunology , Wound Healing/genetics , Aged , Aged, 80 and over , Animals , Biopsy , Cell Movement/genetics , Cell Movement/immunology , Cell Proliferation/genetics , Disease Models, Animal , Female , Gene Expression Regulation/immunology , Glycogen Synthase Kinase 3 beta/metabolism , Healthy Volunteers , Humans , Keratinocytes , Male , Mice , Mice, Knockout , Middle Aged , Phosphorylation/genetics , Phosphorylation/immunology , Signal Transduction/genetics , Signal Transduction/immunology , Skin/immunology , Skin/pathology , Transcription Factor RelA/metabolism , Varicose Ulcer/pathology , Wound Healing/immunologyABSTRACT
An increasing number of studies reveal the importance of long noncoding RNAs (lncRNAs) in gene expression control underlying many physiological and pathological processes. However, their role in skin wound healing remains poorly understood. Our study focused on a skin-specific lncRNA, LOC105372576, whose expression was increased during physiological wound healing. In human nonhealing wounds, however, its level was significantly lower compared with normal wounds under reepithelialization. We characterized LOC105372576 as a nuclear-localized, RNAPII-transcribed, and polyadenylated lncRNA. In keratinocytes, its expression was induced by TGF-ß signaling. Knockdown of LOC105372576 and activation of its endogenous transcription, respectively, reduced and increased the motility of keratinocytes and reepithelialization of human ex vivo skin wounds. Therefore, LOC105372576 was termed "wound and keratinocyte migration-associated lncRNA 1" (WAKMAR1). Further study revealed that WAKMAR1 regulated a network of protein-coding genes important for cell migration, most of which were under the control of transcription factor E2F1. Mechanistically, WAKMAR1 enhanced E2F1 expression by interfering with E2F1 promoter methylation through the sequestration of DNA methyltransferases. Collectively, we have identified a lncRNA important for keratinocyte migration, whose deficiency may be involved in the pathogenesis of chronic wounds.
Subject(s)
Cell Movement , Keratinocytes/metabolism , RNA, Long Noncoding/biosynthesis , Signal Transduction , Skin/metabolism , Wound Healing , Wounds and Injuries/metabolism , Chronic Disease , E2F1 Transcription Factor/metabolism , Gene Expression Regulation , Humans , Keratinocytes/pathology , Skin/pathology , Transforming Growth Factor beta/metabolism , Wounds and Injuries/pathologyABSTRACT
Mastocytosis is a rare and multifaceted disease group characterized by mast cell accumulation in the skin and/or internal organs. In its most common form solitary or widespread, often itchy, red-brown skin lesions appear in childhood or during adulthood (cutaneous mastocytosis). The skin lesions are not always easy to recognize by medical professionals; hence, a correct diagnosis is often delayed. In children, the lesions tend to resolve before puberty, whereas most post-adolescent patients experience a chronic course combined with extra-cutaneous mast cell infiltration (systemic mastocytosis). Therefore, adult patients with cutaneous mastocytosis should be examined for signs of systemic involvement. This article describes the symptoms and signs in cutaneous mastocytosis, and provides guidelines based on international consensus documents. In addition, a newly updated classification of different forms of cutaneous mastocytosis is given.
Subject(s)
Mastocytosis, Cutaneous , Adult , Child , Disease Management , Humans , Mastocytosis, Cutaneous/classification , Mastocytosis, Cutaneous/diagnosis , Mastocytosis, Cutaneous/pathology , Mastocytosis, Cutaneous/therapy , Practice Guidelines as TopicABSTRACT
Direct microscopy of keratinised specimens is a standard screening procedure that assists clinicians to differentiate true superficial mycoses from non-fungal disorders of the skin, nail and hair. Most clinical dermatologists use bright-field microscopy when searching for dermatophyte fungi in clinical samples while laboratory-based mycologists increasingly favour fluorescence microscopy in order to optimise visualisation of fungal elements. This study compared the validity and speediness of fluorescence microscopy vs. conventional light microscopy when screening for fungi in 206 dermatological samples from dermatology outpatients. Both senior dermatologist and a less experienced investigator (medical student) attained high and comparable levels of specificity (91.7-93.8%), positive predictive value (77.1-81.4%) and negative predictive value (83.7-89.9%) using either method. Fluorostaining with Blankophor prior to fluorescence microscopy increased the sensitivity by 22 ± 1% as compared to light microscopy of unstained samples. For both investigators, the time required to identify fungal elements by the fluorescence-based technique was reduced by at least 50%, thus improving the performance of direct microscopy in the clinical setting.
Subject(s)
Arthrodermataceae/isolation & purification , Hair/microbiology , Microscopy, Fluorescence/methods , Mycology/methods , Nails/microbiology , Skin/microbiology , Tinea/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Arthrodermataceae/genetics , Arthrodermataceae/ultrastructure , Child , Child, Preschool , DNA, Fungal/analysis , Female , Humans , Male , Microscopy, Polarization , Middle Aged , Onychomycosis/diagnosis , Onychomycosis/microbiology , Outpatients , Predictive Value of Tests , Sensitivity and Specificity , Staining and Labeling , Tinea/microbiology , Tinea Capitis/diagnosis , Tinea Capitis/microbiology , Young AdultABSTRACT
Mast cells are known to have a detrimental impact on numerous types of inflammatory skin diseases such as contact dermatitis, atopic eczema and cutaneous mastocytosis. Regimens that dampen skin mast cell-mediated activities can thus offer an attractive therapeutic option under such circumstances. As mast cells are known to secrete a large array of potentially pathogenic compounds, both from preformed stores in secretory lysosomes (granules) and after de novo synthesis, mere inhibition of degranulation or interference with individual mast cell mediators may not be sufficient to provide an effective blockade of harmful mast cell activities. An alternative strategy may therefore be to locally reduce skin mast cell numbers. Here, we explored the possibility of using lysosomotropic agents for this purpose, appreciating the fact that mast cell granules contain bioactive compounds prone to trigger apoptosis if released into the cytosolic compartment. Based on this principle, we show that incubation of human skin punch biopsies with the lysosomotropic agents siramesine or Leu-Leu methyl ester preferably ablated the mast cell population, without causing any gross adverse effects on the skin morphology. Subsequent analysis revealed that mast cells treated with lysosomotropic agents predominantly underwent apoptotic rather than necrotic cell death. In summary, this study raises the possibility of using lysosomotropic agents as a novel approach to targeting deleterious mast cell populations in cutaneous mastocytosis and other skin disorders negatively influenced by mast cells.
Subject(s)
Lysosomes/drug effects , Mast Cells/drug effects , Skin/drug effects , Apoptosis/drug effects , Cell Count , Cell Proliferation/drug effects , Dipeptides/pharmacology , Fibroblasts/drug effects , Humans , Immunosuppressive Agents/pharmacology , In Vitro Techniques , Indoles/pharmacology , Keratinocytes/drug effects , Lysosomes/immunology , Lysosomes/pathology , Mast Cells/immunology , Mast Cells/pathology , Mastocytosis, Cutaneous/drug therapy , Mastocytosis, Cutaneous/immunology , Mastocytosis, Cutaneous/pathology , Skin/immunology , Skin/pathology , Spiro Compounds/pharmacologyABSTRACT
BACKGROUND: Many of the functions attributed to mast cells depend on the various pro-inflammatory mediators that are secreted upon mast cell activation. These include a panel of mast cell-specific proteases. In addition, recent studies have indicated that murine mast cells also express granzyme D, a protease previously thought to be confined to cytotoxic lymphocytes. Here, we address the human relevance of the latter findings by investigating whether human mast cells express granzyme H, the granzyme that may represent the functional counterpart to murine granzyme D. METHODS: Cord blood-derived mast cells, LAD2 cells and skin mast cells in situ were evaluated for their expression of granzymes using quantitative PCR, Western blot analysis and immunostaining. Mast cells were activated by either calcium ionophore stimulation or IgE receptor cross-linking. RESULTS: Cord blood-derived mast cells and LAD2 cells were shown to express granzyme H and B mRNA, while granzyme A, K and M expression was undetectable. Mast cell activation by either calcium ionophore or IgE receptor cross-linking caused down-regulated expression of granzyme H. In contrast, granzyme B expression was up-regulated by the same stimuli. Granzyme H expression was also confirmed at the protein level, as shown by both Western blot analysis and confocal microscopy. Further, we show that granzyme H is expressed by human skin mast cells in situ. CONCLUSIONS: The present findings implicate granzyme H as a novel protease expressed by human mast cells and support earlier findings obtained in natural killer cells suggesting that granzymes B and H are reciprocally regulated.
Subject(s)
Granzymes/biosynthesis , Mast Cells/enzymology , Cell Line , Granzymes/genetics , Granzymes/metabolism , Humans , Immunohistochemistry , Microscopy, Confocal , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Real-Time Polymerase Chain ReactionABSTRACT
Venous leg ulcers (VLUs) are one of the most prevalent types of chronic wounds. The aim of this study was to determine the safety and dose-response efficacy of the human synthetic peptide LL-37 in the treatment of hard-to-heal VLUs. This first-in-man trial included 34 participants with VLUs and comprised a 3-week, open-label, run-in period on placebo, followed by a 4-week randomized double-blind treatment phase with twice weekly applications of LL-37 (0.5, 1.6, or 3.2 mg/mL) or placebo, and a 4-week follow-up. The healing rate constants for 0.5 and 1.6 mg/mL of LL-37 were approximately six- and threefold higher than for placebo (p = 0.003 for 0.5 mg/mL and p = 0.088 for 1.6 mg/mL). Square-root transformed wound area data showed improved healing for the 0.5 and 1.6 mg/mL dose groups compared with pretreatment values (p < 0.001 and p = 0.011, respectively). Consistently, treatment with the two lower doses markedly decreased the mean ulcer area (68% for 0.5 mg/mL and 50% for 1.6 mg/mL groups). No difference in healing was observed between the groups receiving 3.2 mg/mL of LL-37 and placebo. There were no safety concerns regarding local or systemic adverse events. In conclusion, topical treatment with LL-37 for chronic leg ulcers was safe and well tolerated with the marked effect on healing predictors at the two lower doses warranting further investigations.
Subject(s)
Antimicrobial Cationic Peptides/therapeutic use , Varicose Ulcer/drug therapy , Aged , Aged, 80 and over , Double-Blind Method , Female , Humans , Male , Middle Aged , Treatment Outcome , Wound Healing , CathelicidinsABSTRACT
BACKGROUND: Ligand-stimulated epidermal growth factor receptor (EGFR/HER1) plays a fundamental role in skin biology as potent transducer of mitotic and anti-apoptotic stimuli in keratinocytes. In human epidermis, at least two additional EGFR family members--HER2 and HER3--are expressed but their biological functions in normal and diseased human skin remain obscure. OBJECTIVE: Here, we studied the expression and biological impact of HER3 in regenerating human epidermis formed from skin explants adhered to acellular dermis. METHODS: Neoepidermal HER3 expression was examined by quantitative real-time reverse transcriptase polymerase chain reaction, immunohistochemistry and Western blot analysis. The dynamic effect of HER3 receptor stimulation by recombinant heregulin (HRG)-beta1 was assessed by fluorescence imaging of re-epithelialization. RESULTS: In the neoepidermis, HER3 mRNA and protein were detected with activated receptors being immunolocalized at basal and low suprabasal levels. Exogenous HRG-beta1 at 10-20 ng/ml increased the outgrowth rate corresponding to approximately 30% the response of exogenous EGF. The growth-promoting effect of HRG-beta1 was associated with enhanced HER3 phosphorylation, keratinocyte proliferation and thickening of viable neoepidermis whereas blockade of ligand-binding to HER3 delayed the outgrowth process and inhibited both constitutive and ligand-induced HER3 phosphorylation. HER2 antagonism using an anti-dimerization antibody, pertuzumab, impeded the re-epithelialization rate. In addition, a selective HER2 kinase inhibitor, CP654577, downregulated phospho-HER3 expression suggesting that transactivation of kinase-deficient HER3 was accomplished through dimerization with HER2. CONCLUSION: The study emphasizes the central role of EGFR in epidermal renewal and demonstrates that HRG-activated HER3 contributes to the outgrowth process of epidermis in vitro.
Subject(s)
Epidermis/physiology , Receptor, ErbB-3/biosynthesis , Regeneration , Adult , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized , Culture Techniques , Epidermis/drug effects , Epidermis/metabolism , Female , Humans , Ligands , Middle Aged , Neuregulin-1/pharmacology , Protein Kinase Inhibitors/pharmacology , Receptor, ErbB-3/agonistsABSTRACT
In the skin, expression of several matrix metalloproteinases (MMPs) occurs in response to tissue injury, tumorigenesis, angiogenesis, apoptosis, and inflammation. The recently cloned MMP-21 has been implicated in skin development and various epithelial cancers. In this study, we found that it is also expressed by differentiated keratinocytes (KCs) in various benign skin disorders, in which it was not associated with KC apoptosis or proliferation, and in organotypic cultures. Furthermore, MMP-21 was induced in keratinocytes in association with increased calcium and presence of the differentiation marker filaggrin. In stably transfected A431 and HEK293 cell lines, MMP-21 increased invasion of cells but did not associate with increased apoptosis, proliferation, or epithelial-to-mesenchymal transition. Of various agents tested in HaCaT cell cultures, only retinoic acid (10(-6) M) and staurosporine (2.5 x 10(-8) M) upregulated MMP-21 mRNA and protein expression, whereas tumor promoters, hormones, or dexamethasone were without effect. Our results suggest that MMP-21 may be an important protease in the terminal differentiation of keratinocytes.
Subject(s)
Gene Expression Regulation, Enzymologic , Keratinocytes/metabolism , Matrix Metalloproteinases, Secreted/biosynthesis , Tretinoin/metabolism , Animals , Apoptosis , Cell Differentiation , Cell Line , Cell Line, Tumor , Cell Movement , Collagen/metabolism , Dexamethasone/pharmacology , Filaggrin Proteins , Humans , Rats , Staurosporine/pharmacology , Tretinoin/pharmacologyABSTRACT
The family of human epidermal growth factor receptors (EGFR, HER2-4) exerts key functions in normal and malignant epithelial cells. Both EGFR and HER2 are valuable targets for anti-cancer drugs by interfering with ligand binding, receptor dimerization, or tyrosine kinase activity. A similar therapeutic strategy has been advocated for chronic psoriasis since plaque lesions overexpress EGFR and its ligands. Our aim was to characterize EGFR/HER2 protein expression in skin cultures and to evaluate the effects of tyrosine kinase inhibitors on epidermal outgrowth, morphology, and EGFR activation. Human skin explants were established on cell-free dermis and cultured at the air-liquid interface. The impact of small-molecule HER inhibitors on outgrowth was assayed by fluorescence-based image analysis and histometry. Effects of a dual EGFR/HER2 kinase inhibitor, PKI166, on neoepidermis were studied by immunohistochemistry and Western blot. Receptor immunostaining showed in vivo-like distributions with highest EGFR intensity in the proliferative layers whereas HER2 was mainly expressed by suprabasal keratinocytes. Reepithelialization was associated with EGFR autophosphorylation irrespective of exogenous ligand stimulation. PKI166 inhibited neoepidermal EGFR activation, keratinocyte proliferation, and outgrowth from normal and psoriatic skin explants. The rate of epidermalization in presence of other HER inhibitors varied suggesting that drug specificity, potency, and reversibility determine the dynamic outcome. Overall, agents predominantly targeting EGFR kinase were more efficient inhibitors of epidermal regeneration than an HER2-selective drug. The study illustrates the usefulness of a dynamic skin model and emphasizes the potential of HER-directed approaches to control epidermal growth in hyperproliferative skin disorders.
Subject(s)
Dermis/physiology , Enzyme Inhibitors/pharmacology , Epidermis/physiology , ErbB Receptors/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Regeneration/physiology , Cell Proliferation/drug effects , Cells, Cultured , Dermis/metabolism , Dose-Response Relationship, Drug , Epidermal Growth Factor/pharmacology , Epidermis/drug effects , Epidermis/metabolism , ErbB Receptors/metabolism , Humans , Keratinocytes/drug effects , Keratinocytes/metabolism , Models, Biological , Organ Culture Techniques , Protein-Tyrosine Kinases/metabolism , Psoriasis/metabolism , Pyrimidines/pharmacology , Pyrroles/pharmacology , Receptor, ErbB-2/metabolism , Regeneration/drug effectsABSTRACT
The CCHCR1 gene (Coiled-Coil alpha-Helical Rod protein 1) within the major psoriasis susceptibility locus PSORS1 is a plausible candidate gene for the risk effect. We have previously generated transgenic mice overexpressing either the psoriasis-associated risk allele CCHCR1*WWCC or the normal allele of CCHCR1. All transgenic CCHCR1 mice appeared phenotypically normal, but exhibited altered expression of genes relevant to the pathogenesis of psoriasis, including upregulation of hyperproliferation markers keratins 6, 16 and 17. Here, we challenged the skin of CCHCR1 transgenic mice with wounding or 12-O-tetradecanoyl-13-acetate (TPA), treatments able to induce epidermal hyperplasia and proliferation that both are hallmarks of psoriasis. These experiments revealed that CCHCR1 regulates keratinocyte proliferation. Early wound healing on days 1 and 4 was delayed, and TPA-induced epidermal hyperproliferation was less pronounced in mice with the CCHCR1*WWCC risk allele than in mice with the normal allele or in wild-type animals. Finally, we demonstrated that overexpression of CCHCR1 affects basal keratinocyte proliferation in mice; CCHCR1*WWCC mice had less proliferating keratinocytes than the non-risk allele mice. Similarly, keratinocytes isolated from risk allele mice proliferated more slowly in culture than wild-type cells when measured by BrdU labeling and ELISA. Our data show that CCHCR1 may function as a negative regulator of keratinocyte proliferation. Thus, aberrant function of CCHCR1 may lead to abnormal keratinocyte proliferation which is a key feature of psoriatic epidermis.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Keratinocytes/cytology , Animals , Cell Movement/drug effects , Cell Proliferation , Hyperplasia/chemically induced , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, Transgenic , Psoriasis/chemically induced , Psoriasis/genetics , Psoriasis/metabolism , Tetradecanoylphorbol Acetate , Wound HealingABSTRACT
Drug screening procedures should preferably utilize experimental settings mimicking the in vivo situation. The aim of this study was to evaluate a skin explant model as a tool to identify topical agents with anti-proliferative properties in human epidermis. Re-epithelialization was initiated from a skin punch biopsy explanted onto de-epidermized dermis and cultured at the air-liquid interface in the presence of epidermal growth factor receptor inhibitor PKI166, tacrolimus or established topical anti-psoriatic drugs: betamethasone, calcipotriol, dithranol and tazarotene. Neo-epidermal extension was traced by fluorescence microscopy prior to histomorphometric analysis. PKI166 at 1 microM decreased the mean radial outgrowth rate (-19%), frequency of BrdU-positive (-37%) and laminin 5-positive (-45%) cells, indicating reduced proliferation and migration of neo-epidermal keratinocytes. However, the papillomatosis index and epithelial thickness were not significantly affected. Calcipotriol at 1 microM had a similar effect on the outgrowth rate (-15%) and fraction of laminin 5-stained keratinocytes (-40%). Furthermore, calcipotriol significantly reduced mean neo-epidermal thickness. Equimolar concentrations of the other test compounds had no apparent effect on histology or outgrowth parameters. This study exemplifies the versatility of combined dynamic and morphological analysis and emphasizes the potential of epidermal growth factor receptor-directed inhibition in hyperproliferative disorders of the epidermis.
Subject(s)
Cell Proliferation/drug effects , Dermatologic Agents/pharmacology , Epithelial Cells/drug effects , Skin/cytology , Apoptosis/drug effects , Cell Adhesion Molecules/metabolism , Cell Movement/drug effects , Drug Evaluation, Preclinical , Epidermal Cells , Epidermis/drug effects , Epithelial Cells/cytology , Humans , Immunohistochemistry , In Vitro Techniques , Keratinocytes/cytology , Keratinocytes/drug effects , Keratinocytes/metabolism , Keratinocytes/physiology , Microscopy, Fluorescence , Psoriasis , Skin/drug effects , KalininABSTRACT
BACKGROUND AND PURPOSE: Venous leg ulceration represents a global health problem affecting predominantly elderly women. Traditionally, functional problems in this group of patients have attracted modest attention from wound care providers and physiotherapists. The aim of the present study was to describe and quantify disease consequences in female leg ulcer patients as a background for future physiotherapy interventions, using the nomenclature of the WHO International Classification of Functioning, Disability and Health (ICF). METHOD: A prospective study was conducted in 34 women aged 60-85 years with current or previous venous leg ulcer as compared to 27 age-matched non-ulcer subjects. The outcome variables were pain, ankle range of motion, walking speed, walking endurance, self-perceived exertion, mobility, activities of daily living (ADL), physical activity, general health, life satisfaction and use of walking aids and community services. Established instruments were utilized and categorized within ICF domains to provide a conceptual framework and basis for physiotherapeutic research. RESULTS: Leg ulcer patients showed significantly reduced values of ankle range of motion, walking speed and endurance, self-perceived exertion, mobility, ADL and physical activity level as compared to control subjects. Patients suffering from active ulceration were more negatively affected, and more of them had pain than post-ulcer fellows. By contrast, general health and life satisfaction were similarly rated by the two study groups. CONCLUSIONS: Elderly females in our study with chronic leg ulcer of venous aetiology had significant mobility impairments, but the reasons and consequences of these impairments remain to be elucidated. The potential of preventive measures and physical rehabilitation to aid functioning and prospects of leg ulcer repair need to be investigated in future studies.
Subject(s)
Leg Ulcer/rehabilitation , Activities of Daily Living , Aged , Aged, 80 and over , Ankle Joint/physiopathology , Chronic Disease , Female , Health Status Indicators , Humans , Leg Ulcer/physiopathology , Middle Aged , Physical Therapy Modalities , Prospective Studies , Range of Motion, Articular , Recovery of FunctionABSTRACT
Psoriasis responds favourably to treatment with retinoids but the cellular pathways mediating these effects are poorly understood. Retinoids regulate keratinocyte proliferation and maturation via binding to nuclear retinoic acid receptors (mainly RARalpha and RARgamma) which form heterodimers with the 9-cis-RA receptor, RXRalpha. We have previously shown that mRNA expression of RARalpha and RXRalpha is down-regulated in psoriatic lesions as compared with non-lesional human skin. In the present study, we investigated the protein expression of RARalpha, RARgamma and RXRalpha in normal and psoriatic skin using indirect immunofluorescence analysis. Epidermal keratinocytes of normal and non-lesional psoriatic skin displayed similar nuclear localization of all three receptors; RARalpha was detected with decreasing intensity from basal to suprabasal layers, RARgamma showed the opposite trend, whereas RXRalpha was evenly expressed throughout the epidermis. In lesional psoriatic skin, however, all three receptor proteins showed a much higher staining intensity in the lower half of the epidermis; in particular, RARalpha immunoreactivity was low or even absent in the upper layers of epidermis. The results support the idea that psoriasis is associated with abnormal retinoid signalling in lesional epidermis.