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Impact of shRNA-mediated MXD4/MAD4 down-modulation on the transcriptome profile of human keratinocyte precursor cells

ABSTRACT: Characterization at the global transcriptome level of the gene networks ensuring the regulation of the immature status of cutaneous stem- and precursor-cells is a necessary step to further understand the concept of ‘stemness’ in this tissue. Moreover, considering possible clinical applications, the search for molecular targets to control stem cell characteristics and regenerative capacity is a necessary step to improve therapeutic approaches. Cutaneous cell therapy is concerned by this objective, as skin graft bioengineering requires a phase of ex vivo expansion of keratinocytes from donors, during which the preservation of a sufficient stem cell pool is critical for graft take and long-term skin regeneration. We have investigated the role of transcription factor MXD4/MAD4 in native keratinocyte precursors from adult human skin. A stable lentiviral-based shRNA-mediated MXD4 knock‑down (KD) approach was developed and used to study the properties of MXD4-deficient [MXD4KD] versus control [MXD4WT] native keratinocyte precursors. Using long-term cultures and clonal assays, we found that decreased MXD4 expression increased keratinocyte precursor immaturity and clonogenic potential, thus promoting self-renewal. Importantly, [MXD4KD] keratinocyte precursors exhibited an improved capacity for three-dimensional epidermis reconstruction, which was preserved at a late ex vivo expansion stage where this potential was lost in [MXD4WT] keratinocytes. To analyze the biological impact of MXD4 knock‑down at the molecular level, comparative transcriptome profiling of [MXD4WT] and [MXD4KD] cells was performed using next-generation RNA sequencing (RNA-seq). This set of data provides a material that permits the dissection of genetic networks modulated by MXD4 in human keratinocyte precursor cells, and controlling their immature status and epidermis regeneration capacity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE202700 | GEO | 2022/08/02

REPOSITORIES: GEO

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Project description:Cursons2015 - Regulation of ERK-MAPK signaling in human epidermis Model comparing the abundance of phosphorylated MAPK signalling proteins and calcium signalling in the epidermis. This model is described in the article: Regulation of ERK-MAPK signaling in human epidermis. Cursons J, Gao J, Hurley DG, Print CG, Dunbar PR, Jacobs MD, Crampin EJ. BMC Syst Biol 2015; 9: 41 Abstract: The skin is largely comprised of keratinocytes within the interfollicular epidermis. Over approximately two weeks these cells differentiate and traverse the thickness of the skin. The stage of differentiation is therefore reflected in the positions of cells within the tissue, providing a convenient axis along which to study the signaling events that occur in situ during keratinocyte terminal differentiation, over this extended two-week timescale. The canonical ERK-MAPK signaling cascade (Raf-1, MEK-1/2 and ERK-1/2) has been implicated in controlling diverse cellular behaviors, including proliferation and differentiation. While the molecular interactions involved in signal transduction through this cascade have been well characterized in cell culture experiments, our understanding of how this sequence of events unfolds to determine cell fate within a homeostatic tissue environment has not been fully characterized.We measured the abundance of total and phosphorylated ERK-MAPK signaling proteins within interfollicular keratinocytes in transverse cross-sections of human epidermis using immunofluorescence microscopy. To investigate these data we developed a mathematical model of the signaling cascade using a normalized-Hill differential equation formalism.These data show coordinated variation in the abundance of phosphorylated ERK-MAPK components across the epidermis. Statistical analysis of these data shows that associations between phosphorylated ERK-MAPK components which correspond to canonical molecular interactions are dependent upon spatial position within the epidermis. The model demonstrates that the spatial profile of activation for ERK-MAPK signaling components across the epidermis may be maintained in a cell-autonomous fashion by an underlying spatial gradient in calcium signaling.Our data demonstrate an extended phospho-protein profile of ERK-MAPK signaling cascade components across the epidermis in situ, and statistical associations in these data indicate canonical ERK-MAPK interactions underlie this spatial profile of ERK-MAPK activation. Using mathematical modelling we have demonstrated that spatially varying calcium signaling components across the epidermis may be sufficient to maintain the spatial profile of ERK-MAPK signaling cascade components in a cell-autonomous manner. These findings may have significant implications for the wide range of cancer drugs which therapeutically target ERK-MAPK signaling components. This model is hosted on BioModels Database and identified by: BIOMD0000000659. To cite BioModels Database, please use: Chelliah V et al. BioModels: ten-year anniversary. Nucl. Acids Res. 2015, 43(Database issue):D542-8. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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Impact of shRNA-mediated MXD4/MAD4 down-modulation on the transcriptome profile of human keratinocyte precursor cells

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