Epidermal morphogenesis during progressive <i>in vitro</i> 3<scp>D</scp> reconstruction at the air–liquid interface

Frankart, Aurélie; Malaisse, Jérémy; Vuyst, Évelyne De; Minner, Frédéric; Rouvroit, Catherine Lambert de; Poumay, Yves

doi:10.1111/exd.12020

Cited by 74 publications

(77 citation statements)

References 20 publications

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“…To this aim, a primary culture model of RHE on polycarbonate filter was used (21). RHE from different donors were grown for increasing times at an air-liquid interface in the presence or absence of 1 unit/ml hyaluronidase from the 3rd until the 11th day of reconstruction (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…5A). To determine whether the proliferation process was altered, a BrdU incorporation assay was performed between the 3rd and the 5th day of reconstruction, when the proliferation rate is highest in RHE (21). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We have previously shown, using concentration-response experiments, that 4MU at 0.5 mM is both effective in removing HA and relatively non-toxic in human keratinocytes cultured as monolayers (21). Subconfluent keratinocytes from different donors were thus grown in the presence or absence of 0.5 mM 4MU during 48 h. After treatment, the HA concentration decreased markedly (Fig.…”

Section: Mu Inhibits Cell Proliferation In Subconfluentmentioning

confidence: 90%

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Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation

Malaisse

Pendaries

Hontoir

et al. 2016

Journal of Biological Chemistry

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Hyaluronan (HA) is synthesized by three HA synthases (HAS1, HAS2, and HAS3) and secreted in the extracellular matrix. In human skin, large amounts of HA are found in the dermis. HA is also synthesized by keratinocytes in the epidermis, although its epidermal functions are not clearly identified yet. To investigate HA functions, we studied the effects of HA depletion on human keratinocyte physiology within in vitro reconstructed human epidermis. Inhibition of HA synthesis with 4-methylumbelliferone (4MU) did not modify the expression profile of the epidermal differentiation markers involucrin, keratin 10, and filaggrin during tissue reconstruction. In contrast, when keratinocytes were incubated with 4MU, cell proliferation was decreased. In an attempt to rescue the proliferation function, HA samples of various mean molecular masses were added to keratinocyte cultures treated with 4MU. These samples were unable to rescue the initial proliferation rate. Furthermore, treatments with HA-specific hyaluronidase, although removing almost all HA from keratinocyte cultures, did not alter the differentiation or proliferation processes. The differences between 4MU and hyaluronidase effects did not result from differences in intracellular HA, sulfated glycosaminoglycan concentration, apoptosis, or levels of HA receptors, all of which remained unchanged. Similarly, knockdown of UDP-glucose 6-dehydrogenase (UGDH) using lentiviral shRNA effectively decreased HA production but did not affect proliferation rate. Overall, these data suggest that HA levels in the human epidermis are not directly correlated with keratinocyte proliferation and differentiation and that incubation of cells with 4MU cannot equate with HA removal. Hyaluronan (HA),2 a large polysaccharide of the extracellular space, is composed of repetitive D-glucuronic acid and D-Nacetylglucosamine dimer units. Although it belongs to the glycosaminoglycan (GAG) family, HA is not synthesized in the Golgi network; the molecule is assembled at the inner face of the plasma membrane, and the newly formed polymer is extruded into the extracellular matrix as it lengthens by addition of the dimer units. This mechanism allows the formation of huge HA molecules with masses ranging from 10 5 to 10 7 Da and lengths ranging from 2 to 25 m (1).HA synthesis is performed by three different glycosyltransferases called hyaluronan synthases (HAS) located at the plasma membrane. These three enzymes, HAS1, HAS2, and HAS3, differ from each other with respect to temporal expression pattern during development, specific activity, and size of HA polymers generated (2, 3). The degradation of HA is carried out in somatic tissues by HYAL1 and HYAL2, two enzymes belonging to the hyaluronidase family (4).HA has been identified in most tissues of the organism. Half of the total amount of HA is localized in skin (5). The skin is a large organ making up the first barrier against physical, biological, and chemical damages to the body. In the dermis, the main cell type is represented by fibroblasts, which...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Mu Inhibits Cell Proliferation In Subconfluentmentioning

confidence: 90%

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Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation

Malaisse

Pendaries

Hontoir

et al. 2016

Journal of Biological Chemistry

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“…A valuable tool for unravelling these mechanisms are 3D skin models that mimic key features of the skin barrier function in vitro (Frankart et al, 2012) and which can be used for further dissection of normal and mutant channel proteins using both patient-derived keratinocytes and normal cells that have been genetically manipulated. These in vitro disease models can also be used to optimise therapies for these skin conditions, such as topical barrier creams containing small molecule inhibitors specific for the channel protein of interest.…”

Section: Perspectivesmentioning

confidence: 99%

Defective channels lead to an impaired skin barrier

Blaydon¹,

Kelsell²

2014

Journal of Cell Science

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Channels are integral membrane proteins that form a pore, allowing the passive movement of ions or molecules across a membrane (along a gradient), either between compartments within a cell, between intracellular and extracellular environments or between adjacent cells. The ability of cells to communicate with one another and with their environment is a crucial part of the normal physiology of a tissue that allows it to carry out its function. Cell communication is particularly important during keratinocyte differentiation and formation of the skin barrier. Keratinocytes in the skin epidermis undergo a programme of apoptosis-driven terminal differentiation, whereby proliferating keratinocytes in the basal (deepest) layer of the epidermis stop proliferating, exit the basal layer and move up through the spinous and granular layers of the epidermis to form the stratum corneum, the external barrier. Genes encoding different families of channel proteins have been found to harbour mutations linked to a variety of rare inherited monogenic skin diseases. In this Commentary, we discuss how human genetic findings in aquaporin (AQP) and transient receptor potential (TRP) channels reveal different mechanisms by which these channel proteins function to ensure the proper formation and maintenance of the skin barrier.

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“…Here, we have investigated HA synthesis in normal human keratinocytes using two culture models of epidermal differentiation induced by cell density, i.e., confluent keratinocyte monolayers and reconstructed human epidermis (RHE), both cultured without addition of molecules influencing the differentiation processes (Minner et al, 2010;Frankart et al, 2012), as well as in lesional and non-lesional skin from atopic dermatitis (AD) patients. Our data strikingly suggest that the balance of HA produced by distinct HASs is differentially involved in the regulation of keratinocyte physiology and pathophysiology: HAS1 is responsible for the bulk of HA production in normal keratinocytes, whereas HAS3 is prominent in pathological conditions.…”

Section: Introductionmentioning

confidence: 99%

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Malaisse

Bourguignon

Vuyst

et al. 2014

Journal of Investigative Dermatology

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Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

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Epidermal morphogenesis during progressive in vitro 3D reconstruction at the air–liquid interface

Cited by 74 publications

References 20 publications

Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation

Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation

Defective channels lead to an impaired skin barrier

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

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