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  • 1
    Keywords: TRANSPLANTATION ; C-JUN ; cell-cell interaction ; cell proliferation ; GM-CSF ; organotypic culture ; cell differentiation ; dermal fibroblasts ; epidermal keratinocytes ; epithelial cell culture ; dermal equivalent ; paracrine factors ; collagen matrix ; IL-1 ; KGF ; JunB
    Abstract: This chapter focuses on the role of dermal fibroblasts in epidermal keratinocyte proliferation and differentiation and proves the usefulness of an organotypic in vitro skin equivalent model to study mechanisms of tissue regeneration. Examples are given how these can be used to study the growth of keratinocytes after transplantation in vivo, alone or with a collagen matrix. This leads on to the development of in vitro organotypic models to generate skin equivalents composed of an organized epidermis on matrix-embedded dermal fibroblasts. Protocols are provided for both the in vivo transplantation models and the combined stromal/epithelial in vitro organotypic models, along with several variations of the methods. Characterization of the proliferation and differentiation of the cultures is described with an extensive discussion of recent results on the factors controlling tissue regeneration, with emphasis on the role of c-jun and junB gene products in the production of KGF and GM-CSF by fibroblasts in response to epithelium-derived IL1, and the effect of these cytokines on the proliferation and differentiation of epidermal keratinocytes.
    Type of Publication: Book chapter
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  • 2
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    Keywords: RECEPTOR ; EXPRESSION ; GROWTH ; GROWTH-FACTOR ; proliferation ; Germany ; IN-VIVO ; NETWORKS ; SYSTEM ; GENE ; GENE-EXPRESSION ; HYBRIDIZATION ; transcription ; DIFFERENTIATION ; TISSUE ; COMPLEX ; COMPLEXES ; TRANSCRIPTION FACTOR ; AP-1 ; KERATINOCYTES ; SKIN ; C-JUN ; fibroblasts ; cytokines ; antibodies ; antibody ; MOUSE ; IN-SITU ; gene expression ; REPAIR ; SIGNALING PATHWAY ; HUMAN KERATINOCYTES ; Jun ; gene expression profiling ; expression profiling ; COLONY-STIMULATING FACTOR ; FACTOR GENE-EXPRESSION ; FUNCTIONAL EXPRESSION ; in situ hybridization ; keratinocyte ; regulation ; mesenchyme ; SDF-1 ; CHEMOKINE RECEPTORS ; INFLAMMATORY CYTOKINES ; CELL-DERIVED FACTOR ; CXCL12 ; GROWTH-ASSOCIATED MOLECULE ; HB-GAM ; LARGE INDUCTION ; pleiotrophin
    Abstract: In skin, fibroblasts of the connective tissue play a decisive role in epidermal homeostasis and repair by contributing to the regulation of keratinocyte proliferation and differentiation. The AP-1 transcription factor subunit JUN plays a crucial role in this mesenchymal-epithelial interplay by regulating the expression of two critical paracrine-acting cytokines, keratinocyte growth factor (KGF) and granulocyte-macrophage colony-stimulating factor (GMCSF). We have performed gene expression profiling of wildtype and Jun(-/-) mouse embryonic fibroblasts to identify additional players involved in this complex network, and have found pleiotrophin (PTN) and the stromal cell-derived factor 1 (SDF-1) as novel JUN-regulated factors. Both cytokines are expressed by dermal fibroblasts in vivo, as shown by semi-quantitative RT-PCR and in situ hybridization on murine skin sections. Using a heterologous feeder layer co-culture system, we demonstrated that PTN and SDF-1 exert a mitogenic effect on primary human keratinocytes. Moreover, SDF-1-induced keratinocyte proliferation could be specifically inhibited by neutralizing antibodies against SDF-1 or its receptor, CXCR4. Consistent with its role in promoting keratinocyte growth, PTN was upregulated during cutaneous wound healing in vivo. Interestingly, co-cultivation with keratinocytes stimulated PTN expression but repressed SDF-1 production in fibroblasts, demonstrating the complexity of the paracrine regulatory cytokine networks that control skin homeostasis and regeneration
    Type of Publication: Journal article published
    PubMed ID: 15840658
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  • 4
    Keywords: RECEPTOR ; CELLS ; GROWTH ; GROWTH-FACTOR ; proliferation ; FACTOR RECEPTOR ; Germany ; IN-VIVO ; MICE ; ACTIVATION ; KERATINOCYTES ; SKIN ; fibroblasts ; knockout ; MOUSE ; PLASMA ; MEMBRANE ; HUMAN KERATINOCYTES ; PLASMA-MEMBRANE ; EPIDERMAL DIFFERENTIATION ; PHENOTYPE ; DEGRADATION ; RECEPTORS ; epidermis ; protease ; ABSENCE ; FACTOR RECEPTORS ; RE ; keratinocyte ; cathepsins ; HAIR FOLLICLE ; HYPERPLASIA ; plasma membrane ; INTERNALIZATION ; MOUSE KERATINOCYTES ; CYSTEINE PROTEASE ; DESQUAMATION ; HAIR FOLLICLE MORPHOGENESIS ; lysosomes ; PROTEOLYSIS
    Abstract: Mice deficient for cathepsin L (CTSL) show epidermal hyperplasia due to a hyperproliferation of basal keratinocytes. Here we show that the critical function of CTSL in the skin is keratinocyte specific. This is revealed by transgenic re-expression of CTSL in the keratinocytes of ctsl(-/-) mice, resulting in a rescue of the ctsl(-/-) skin phenotype. Cultivation of primary mouse keratinocytes with fibroblast- and keratinocyte-conditioned media, as well as heterologous organotypic co-cultures of mouse fibroblasts and human keratinocytes, showed that the altered keratinocyte proliferation is caused primarily by CTSL-deficiency in keratinocytes. In the absence of EGF, wild type and CTSL-knockout keratinocytes proliferate with the same rates, while in presence of EGF, ctsl(-/-) keratinocytes showed enhanced proliferation compared with controls. Internalization and degradation of radioactively labeled EGF was identical in both ctsl(-/-) and ctsl(+/+) keratinocytes. However, ctsl(-/-) keratinocytes recycled more EGF to the cell surface, where it is bound to the EGF-receptor, which is also more abundant in ctsl(-/-) cells. We conclude that the hyperproliferation of keratinocytes in CTSL-knockout mice is caused by an enhanced recycling of growth factors and growth factor receptors from the endosomes to the keratinocyte plasma membrane, which result in sustained growth stimulation
    Type of Publication: Journal article published
    PubMed ID: 16079282
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  • 5
    Keywords: RECEPTOR ; APOPTOSIS ; EXPRESSION ; IN-VITRO ; proliferation ; CELL ; Germany ; INHIBITION ; VITRO ; SYSTEM ; DEATH ; PROTEIN ; PROTEINS ; DRUG ; DIFFERENTIATION ; EPITHELIA ; ACTIVATION ; MARKER ; CONTRAST ; SKIN ; treatment ; ALPHA ; culture ; MATURATION ; CELL-DEATH ; ADHESION ; CELL-ADHESION ; RECEPTORS ; PROGRAMMED CELL-DEATH ; BARRIER FUNCTION ; TERMINAL DIFFERENTIATION ; AUTOIMMUNITY ; FUNCTIONAL-CHARACTERIZATION ; cell adhesion ; DRUGS ; ORGANOTYPIC COCULTURE ; cholinergic ; DARIERS-DISEASE ; KERATINOCYTE ADHESION ; NICOTINIC ACETYLCHOLINE-RECEPTOR ; PEMPHIGUS-VULGARIS
    Abstract: The aim of this study was to analyze the influence of cholinergic and anticholinergic drugs on epidermal physiology using organotypic cocultures (OTCs). Blocking of all acetylcholine receptors (AChRs) by combined treatment with mecamylamine and atropine or treatment with strychnine (blocking alpha 9nAChR) for 7-14 days resulted in a complete inhibition of epidermal differentiation and proliferation. Blockage of nicotinic (n) AChR with mecamylamine led to a less pronounced delay in epidermal differentiation and proliferation than blockage of muscarinic ( m) AChR with atropine, evidenced by reduced epithelial thickness and expression of terminal differentiation markers like cytokeratin 2e or filaggrin. In OTCs treated with atropine, mecamylamine, or strychnine, we could demonstrate intracellular lipid accumulation in the lower epidermal layers, indicating a severely disturbed epidermal barrier. In addition, we observed prominent acantholysis in the basal and lower suprabasal layers in mecamylamine-, atropine-, and strychnine-treated cultures, accompanied by a decreased expression of cell adhesion proteins. This globally reduced cell adhesion led to cell death via intrinsic activation of apoptosis. In contrast, stimulation of nAChR and mAChR with cholinergic drugs resulted in a significantly thickened epithelium, accompanied by an improved epithelial maturation. In summary, we show that epidermal AChR are crucially involved in the regulation of epidermal homeostasis
    Type of Publication: Journal article published
    PubMed ID: 16810300
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  • 6
    Keywords: RECEPTOR ; APOPTOSIS ; CELLS ; EXPRESSION ; GROWTH ; GROWTH-FACTOR ; IN-VITRO ; proliferation ; CELL ; CELL-PROLIFERATION ; FACTOR RECEPTOR ; Germany ; human ; MODEL ; MODELS ; VITRO ; cell line ; DIFFERENTIATION ; EPITHELIA ; TISSUE ; MICE ; RELEASE ; MECHANISM ; CARCINOGENESIS ; KERATINOCYTES ; SKIN ; fibroblasts ; culture ; cytokines ; EQUIVALENT ; NUDE-MICE ; CELL-LINE ; LINE ; MARKERS ; SURFACE ; differentiation markers ; EPIDERMAL DIFFERENTIATION ; organotypic cocultures ; EPITHELIAL-CELLS ; RECEPTORS ; ORGANIZATION ; nude mice ; HaCaT cell ; TRANSPLANTS ; growth factors ; BASEMENT-MEMBRANE FORMATION ; CULTURED HUMAN KERATINOCYTES ; epithelial-mesenchymal interaction ; FACTOR GENE-EXPRESSION ; GM-CSF ; HaCaT ; HUMAN DERMAL FIBROBLASTS ; HUMAN SKIN KERATINOCYTES ; KERATINOCYTE GROWTH-FACTOR ; LINE HACAT ; organotypic culture ; skin equivalent ; STIMULATING FACTOR
    Abstract: The human keratinocyte cell line HaCaT expresses essentially all epidermal differentiation markers but exhibits deficiencies in tissue organization as surface transplants in nude mice and even more so in organotypic co-cultures with fibroblasts. Whereas tissue differentiation by normal keratinocytes (NEKs) is regulated by stromal interactions, this mechanism is impaired in HaCaT cells. This regulatory process is initiated by interleukin-1 (IL-1) release in keratinocytes, which induces expression of keratinocyte growth factor (KGF/FGF-7) and granulocyte macrophage-colony stimulating factor (GM-CSF) in fibroblasts. Production and release of IL-1 is very low and, consequently, expression of the fibroblast-derived growth factors KGF/FGF-7 and GM-CSF is absent in HaCaT-fibroblast co-cultures. However, addition of KGF and GMCSF, respectively, is inefficient to improve stratification and differentiation by HaCaT cells due to the low expression of their cognate receptors. More importantly, expression and release of the autocrine keratinocyte growth factor TGF-alpha is dramatically decreased in HaCaT cells. Addition of TGF-alpha or EGF stimulated HaCaT cell proliferation but, even more effectively, suppressed apoptosis, thus facilitating the formation of a regularly stratified epithelium. Furthermore, TGF-alpha enhanced the expression of the receptors for KGF and GM-CSF so that addition of these growth factors, or of their inducer IL-1, further improved epidermal tissue differentiation leading to in vitro skin equivalents comparable with cultures of NEKs. Thus, supplementing TGF-alpha normalized epidermal tissue regeneration by immortal HaCaT keratinocytes and their interaction with stromal cells so that regular skin equivalents are produced as standardized in vitro models
    Type of Publication: Journal article published
    PubMed ID: 12771184
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  • 7
    Keywords: APOPTOSIS ; EXPRESSION ; GROWTH ; IN-VITRO ; SURVIVAL ; human ; VITRO ; DEATH ; GENE-EXPRESSION ; DIFFERENTIATION ; KERATINOCYTES ; SKIN ; FIBROBLAST-GROWTH-FACTOR ; EXTRACELLULAR-MATRIX ; HUMAN KERATINOCYTES ; INTEGRIN ; FAILURE ; epidermis ; TERMINAL DIFFERENTIATION ; BASEMENT-MEMBRANE FORMATION ; CULTURED HUMAN KERATINOCYTES ; HUMAN SKIN ; HEPARAN-SULFATE PROTEOGLYCANS ; BIOLOGICAL-ACTIVITY ; RECEPTOR-BINDING ; ORGANOTYPIC SKIN CULTURE ; PROTEIN CORE
    Abstract: Heparin-binding growth factors are crucial for the formation of human epidermis, but little is known about the role of heparan sulfate proteoglycans in this process. Here we investigated the role of the heparan sulfate proteoglycan, perlecan, in the formation of human epidermis, by utilizing in vitro engineered human skin. By disrupting perlecan expression either in the dermis or the epidermis, we found that epidermally derived perlecan is essential for epidermal formation. Perlecan-deficient keratinocytes formed a strikingly thin and poorly organized epidermis because of premature apoptosis and failure to complete their stratification program. Exogenous perlecan fully restored epidermal formation. Perlecan deposition in the basement membrane zone correlated with formation of multilayered epidermis. Perlecan deficiency, however, had no effect on the lining and deposition of major basement membrane components as was evident by a continuous linear staining of laminin and collagen IV. Similarly, perlecan deficiency did not affect the distribution of beta 1 integrin. Addition of the perlecan ligand, fibroblast growth factor 7, protected perlecan-deficient keratinocytes from cell death and improved the thickness of the epidermis. Taken together, our results revealed novel roles for perlecan in epidermal formation. Perlecan regulates both the survival and terminal differentiation steps of keratinocytes. Our results suggested a model whereby perlecan regulates these processes via controlling the bioavailability of perlecan-binding soluble factors involved in epidermal morphogenesis
    Type of Publication: Journal article published
    PubMed ID: 16269412
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    Keywords: RECEPTOR ; EXPRESSION ; Germany ; MODEL ; SYSTEM ; DISEASE ; DISEASES ; LONG-TERM ; DIFFERENTIATION ; EPITHELIA ; CONTRAST ; SKIN ; BIOLOGY ; ALPHA ; culture ; smoking ; PATHOGENESIS ; MORPHOLOGY ; RECEPTORS ; epidermis ; physiology ; LAYER ; LIFE ; analysis ; EPITHELIUM ; pharmacology ; UNIT ; NICOTINE ; coculture ; acetylcholine ; acne inversa ; epidermis equivalent ; EXTRANEURONAL CHOLINERGIC SYSTEM ; hidradenitis ; HIDRADENITIS-SUPPURATIVA
    Abstract: In recent years, the physiological role of non-neuronal acetylcholine (ACh) and its receptors (AChR) in epidermal physiology has been under intense investigation. However, little is known about the role of the non-neuronal cholinergic system in inflammatory skin diseases. We chose the clinically nicotine-dependent skin disease hidradenitis suppurativa (HS) as model to study the influence of long term nicotine ingestion on epidermal morphology and AChR expression. HS is a chronic inflammatory, disabling disease of unknown pathogenesis emerging from the pilosebaceous unit of the intertriginous areas. In order to correlate our findings to specific nicotine effects, we used the organotypical coculture system (OTC) and raised artificial epidermis in the presence of nicotine. After 12 days in culture control OTC showed a mature epithelium, while nicotine treated OTCs were significantly thicker. Using immunofluorescence analysis, nicotine treated OTCs produced significantly stronger immunoreactivity (IR) for the alpha 3, M-3 and M-5 AChR antisera than control. In contrast, the alpha 7 nAChR antiserum showed a slightly reduced IR in the granular layer and the alpha 9 nAChR IR retracted to the lower suprabasal layers. In HS epidermis we found the strongest IR for all AChR around the follicular infundibulum while in the sinus epithelia it was only weak. In contrast to the nicotine treated OTC, the alpha 7 nAChR IR in the hyperplastic HS epidermis was clearly extended to all living layers. Altogether we provide first hints for a causative role of the non-neuronal cholinergic system in the pathogenesis of HS by promoting infundibular epithelial hyperplasia and thus follicular plugging. (c) 2007 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17363005
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