Dataset Information

HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes.

ABSTRACT: Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNF? or IL-1?. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.

SUBMITTER: Colombo I

PROVIDER: S-EPMC5748104 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes.

Colombo Irma I Sangiovanni Enrico E Maggio Roberta R Mattozzi Carlo C Zava Stefania S Corbett Yolanda Y Fumagalli Marco M Carlino Claudia C Corsetto Paola Antonia PA Scaccabarozzi Diletta D Calvieri Stefano S Gismondi Angela A Taramelli Donatella D Dell'Agli Mario M

Mediators of inflammation 20171217

Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the <i>in vitro</i> studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate <i>in vitro</i>, a ...[more]

PMID: 29391667

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Project description:BackgroundKeratinocytes constitute a major part of the melanoma microenvironment, considering their protective role towards melanocytes in physiological conditions. However, their interactions with tumor cells following melanomagenesis are still unclear.MethodsWe used two in vitro models (melanoma-conditioned media and indirect co-culture of keratinocytes with melanoma cells on Transwell inserts) to activate immortalized keratinocytes towards cancer-associated ones. Western Blotting and qPCR were used to evaluate keratinocyte markers and mediators of cell invasiveness on protein and mRNA expression level respectively. The levels and activity of proteases and cytokines were analysed using gelatin-FITC staining, gelatin zymography, chemiluminescent enzymatic test, as well as protein arrays. Finally, to further study the functional changes influenced by melanoma we assessed the rate of proliferation of keratinocytes and their invasive abilities by employing wound healing assay and the Transwell filter invasion method.ResultsHaCaT keratinocytes activated through incubation with melanoma-conditioned medium or indirect co-culture exhibit properties of less differentiated cells (downregulation of cytokeratin 10), which also prefer to form connections with cancer cells rather than adjacent keratinocytes (decreased level of E-cadherin). While they express only a small number of cytokines, the variety of secreted proteases is quite prominent especially considering that several of them were never reported as a part of secretome of activated keratinocytes' (e.g., matrix metalloproteinase 3 (MMP3), ADAM metallopeptidase with thrombospondin type 1 motif 1). Activated keratinocytes also seem to exhibit a high level of proteolytic activity mediated by MMP9 and MMP14, reduced expression of TIMPs (tissue inhibitor of metalloproteinases), upregulation of ERK activity and increased levels of MMP expression regulators-RUNX2 and galectin 3. Moreover, cancer-associated keratinocytes show slightly elevated migratory and invasive abilities, however only following co-culture with melanoma cells on Transwell inserts.ConclusionsOur study offers a more in-depth view of keratinocytes residing in the melanoma niche, drawing attention to their unique secretome and mediators of invasive abilities, factors which could be used by cancer cells to support their invasion of surrounding tissues. Video abstract.

Dataset Information

HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes.

Publications

HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes.

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