Dataset Information

Differential effects of phosphatase inhibitors on the calcium homeostasis and migration of HaCaT keratinocytes.

ABSTRACT: Changes in intracellular calcium concentration ([Ca²?]i) as well as in the phosphorylation state of proteins have been implicated in keratinocyte wound healing revealed in scratch assays. Scratching confluent HaCaT monolayers decreased the number of cells displaying repetitive Ca²? oscillations as well as the frequency of their Ca²?-transients in cells close to the wounded area and initiated migration of the cells into the wound bed. In contrast, calyculin-A (CLA) and okadaic acid (OA), known cell permeable inhibitors of protein phosphatase-1 and 2A, increased the level of resting [Ca²?]i and suppressed cell migration and wound healing of HaCaT cells. Furthermore, neither CLA nor OA influenced how scratching affected Ca²? oscillations. It is assumed that changes in and alterations of the phosphorylation level of Ca²?-transport and contractile proteins upon phosphatase inhibition mediates cell migration and wound healing.

SUBMITTER: Ruzsnavszky O

PROVIDER: S-EPMC3640006 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Differential effects of phosphatase inhibitors on the calcium homeostasis and migration of HaCaT keratinocytes.

Ruzsnavszky Olga O Dienes Beatrix B Oláh Tamás T Vincze János J Gáll Tamás T Balogh Enikő E Nagy Gábor G Bátori Róbert R Lontay Beáta B Erdődi Ferenc F Csernoch Laszlo L

PloS one 20130430 4

Changes in intracellular calcium concentration ([Ca²⁺]i) as well as in the phosphorylation state of proteins have been implicated in keratinocyte wound healing revealed in scratch assays. Scratching confluent HaCaT monolayers decreased the number of cells displaying repetitive Ca²⁺ oscillations as well as the frequency of their Ca²⁺-transients in cells close to the wounded area and initiated migration of the cells into the wound bed. In contrast, calyculin-A (CLA) and okadaic acid (OA), known ce ...[more]

PMID: 23646108

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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

Differential effects of phosphatase inhibitors on the calcium homeostasis and migration of HaCaT keratinocytes.

Publications

Differential effects of phosphatase inhibitors on the calcium homeostasis and migration of HaCaT keratinocytes.

Similar Datasets

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