Crucial role of fibroblasts in regulating epidermal morphogenesis

Ghalbzouri, Abdoelwaheb El; Lamme, Evert N.; Ponec, Maria

doi:10.1007/s00441-002-0621-0

Cited by 127 publications

(106 citation statements)

References 28 publications

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“…Immunohistochemistry-Human skin biopsies were processed for immunohistochemistry as described previously (13,26). Immunohistochemical staining was performed according to the avidin biotinylatedenzyme-complex method (Vector, Burlingame, CA) using polyclonal rabbit-anti-human cystatin M/E antibodies (12), polyclonal sheep-antihuman legumain antibodies (24), monoclonal mouse-anti-human CTSV antibodies (R&D Systems), and monoclonal rat-anti-human/ mouse CTSL antibodies (R&D Systems).…”

Section: Methodsmentioning

confidence: 99%

Cystatin M/E Is a High Affinity Inhibitor of Cathepsin V and Cathepsin L by a Reactive Site That Is Distinct from the Legumain-binding Site

Cheng

Hitomi

Vlijmen-Willems

et al. 2006

Journal of Biological Chemistry

100

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Cystatin M/E is a high affinity inhibitor of the asparaginyl endopeptidase legumain, and we have previously reported that both proteins are likely to be involved in the regulation of stratum corneum formation in skin. Although cystatin M/E contains a predicted binding site for papain-like cysteine proteases, no high affinity binding for any member of this family has been demonstrated so far. We report that human cathepsin V (CTSV) and human cathepsin L (CTSL) are strongly inhibited by human cystatin M/E. Kinetic studies show that K i values of cystatin M/E for the interaction with CTSV and CTSL are 0.47 and 1.78 nM, respectively. On the basis of the analogous sites in cystatin C, we used site-directed mutagenesis to identify the binding sites of these proteases in cystatin M/E. We found that the W135A mutant was rendered inactive against CTSV and CTSL but retained legumain-inhibiting activity. Conversely, the N64A mutant lost legumain-inhibiting activity but remained active against the papain-like cysteine proteases. We conclude that legumain and papain-like cysteine proteases are inhibited by two distinct non-overlapping sites. Using immunohistochemistry on normal human skin, we found that cystatin M/E co-localizes with CTSV and CTSL. In addition, we show that CTSL is the elusive enzyme that processes and activates epidermal transglutaminase 3. The identification of CTSV and CTSL as novel targets for cystatin M/E, their (co)-expression in the stratum granulosum of human skin, and the activity of CTSL toward transglutaminase 3 strongly imply an important role for these enzymes in the differentiation process of human epidermis.The cellular activity of a protease is the result of many regulatory mechanisms such as the concentration and compartmentalization of substrates, the enzyme itself, and its cognate inhibitors. Cystatins are the natural and specific inhibitors of endogenous mammalian lysosomal cysteine proteases and have shown important regulatory and protective functions in cells and tissues against proteolysis by cysteine proteases of host, bacterial, and viral origin (1-3). The inhibitory activity of cystatins is regulated by a reversible, tight-binding interaction between the protease inhibitor and its target protease (4). Disturbance of the normal balance between cysteine proteases and their inhibitors at a wrong time and location can lead to several pathological conditions such as chronic inflammatory reactions (5), tumor malignancy (6), and faulty differentiation processes in the epidermis and hair follicle (7). Little is known on the specific biological functions of cystatin family members. However, mutations in the genes encoding the cystatin family members cystatin B and C cause neurological phenotypes in humans (8, 9).Cystatin M/E is a 14-kDa secreted protein that shares only 35% homology with other human type 2 cystatins. Nevertheless, it has a similar overall structure including the two characteristic intrachain disulfide bridges (10, 11). Expression of cystatin M/E is found to be restricted to ...

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Section: Methodsmentioning

confidence: 99%

Cystatin M/E Is a High Affinity Inhibitor of Cathepsin V and Cathepsin L by a Reactive Site That Is Distinct from the Legumain-binding Site

Cheng

Hitomi

Vlijmen-Willems

et al. 2006

Journal of Biological Chemistry

100

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“…These HSEs have been shown to have fairly similar morphology, expression of differentiation markers, and lipid properties compared with native human skin. [12][13][14][15] However, usage of primary keratinocytes has several disadvantages, for example, limited availability of fresh skin, limited in vitro lifespan, and, in particular, the large donor-donor variation. 16 By using an established immortalized keratinocyte cell line that can be kept in culture for prolonged time, such limitations can be circumvented.…”

Section: Introductionmentioning

confidence: 99%

Barrier Properties of an N/TERT-Based Human Skin Equivalent

Drongelen

Danso²,

Mulder³

et al. 2014

Tissue Engineering Part A

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Human skin equivalents (HSEs) can be considered a valuable tool to study aspects of human skin, including the skin barrier, or to perform chemical or toxicological screenings. HSEs are three-dimensional skin models that are usually established using primary keratinocytes and closely mimic human skin. The use of primary keratinocytes has several drawbacks, including a limited in vitro life span and large donor-donor variation. This makes them less favorable for in vitro toxicity screenings. Usage of an established keratinocyte cell line circumvents these drawbacks and enables the generation of easy-to-generate and reproducible HSEs, which can be used for pharmacological and/or toxicological screenings. For such screenings, a proper barrier function is required. In this study, we investigated the barrier properties of HSEs established with the keratinocyte cell line N/TERT (N-HSEs). N-HSEs showed comparable tissue morphology and expression of several epidermal proteins compared with HSEs established with primary keratinocytes. Our results clearly demonstrate that N-HSEs not only contain several stratum corneum (SC) barrier properties similar to HSEs, including the presence of the long periodicity phase and a comparable SC permeability, but also show some differences in lipid composition. Nonetheless, the similarities in barrier properties makes N/TERT cells a promising alternative for primary keratinocytes to generate HSEs.

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“…Os fibroblastos exercem profundos efeitos na proliferação e na diferenciação dos queratinócitos (essenciais na reepitelização) e também na deposição de proteínas. Somente na presença dos fibroblastos é que a diferenciação epidermal e a deposição de laminina 5, colágeno tipo IV e VII, ocorrem e, por isso, tem um papel fundamental no processo cicatricial (EL GHALBZOURI et al, 2002), levando a entender que a utilização da calêndula no atual experimento contribuiu no processo cicatricial. Ciência Rural, v.38, n.6, set, 2008.…”

Section: Resultsunclassified