Human Skin Keratinocytes on Sustained TGF-β Stimulation Reveal Partial EMT Features and Weaken Growth Arrest Responses

Abstract

Defects in wound closure can be related to the failure of keratinocytes to re-epithelize. Potential mechanisms driving this impairment comprise unbalanced cytokine signaling, including Transforming Growth Factor-β (TFG-β). Although the etiologies of chronic wound development are known, the relevant molecular events are poorly understood. This lack of insight is a consequence of ethical issues, which limit the available evidence to humans. In this work, we have used an in vitro model validated for the study of epidermal physiology and function, the HaCaT cells to provide a description of the impact of sustained exposure to TGF-β. Long term TGF-β1 treatment led to evident changes, HaCaT cells became spindle-shaped and increased in size. This phenotype change involved conformational re-arrangements for actin filaments and E-Cadherin cell-adhesion structures. Surprisingly, the signs of consolidated epithelial-to-mesenchymal transition were absent. At the molecular level, modified gene expression and altered protein contents were found. Non-canonical TGF-β pathway elements did not show relevant changes. However, R-Smads experienced alterations best characterized by decreased Smad3 levels. Functionally, HaCaT cells exposed to TGF-β1 for long periods showed cell-cycle arrest. Yet, the strength of this restraint weakens the longer the treatment, as revealed when challenged by pro-mitogenic factors. The proposed setting might offer a useful framework for future research on the mechanisms driving wound chronification.

Identification of Gene Signatures and Expression Patterns During Epithelial-to-Mesenchymal Transition From Single-Cell Expression Atlas

“…Apart from that, GO:0042060 (wound healing) and GO:0043588 (skin development) have also been shown to enrich optimal genes associated with EMT. According to recent publications, both wound healing which involves cell–cell and cell-extracellular matrix interactions ( Kalluri, 2009 ; Yin et al, 2013 ) and skin development which associates with TGF-β signaling pathways ( Liarte et al, 2020 ) have been reported to be functionally related to the transformation between epithelial and mesenchymal cells ( Kalluri, 2009 ; Yin et al, 2013 ; Liarte et al, 2020 ), validating our prediction.…”

Dissecting the microenvironment around biosynthetic scaffolds in murine skin wound healing

“…S8I-M ). IFEB received Tgfb1 and Tgfb2 (signals playing key roles in regulating epithelial-to-mesenchymal transition (EMT) for keratinocytes) from T cells, macrophages and germinative layer (GL) keratinocytes (55). Interactive plots for other types of cells were summarized in Fig.…”

Carbon Dots Induce Epithelial‐Mesenchymal Transition for Promoting Cutaneous Wound Healing via Activation of TGF‐β/p38/Snail Pathway

“…[ 5,11,18 ] However, because HaCaT cells could express a large number of cell adhesion molecules (i.e., E‐cadherin) during the proliferation, which could form the adherens junctions to bind cells with each other, HaCaT cells tend to aggregate and appear clumped in Figure 2c, which is well consistent with the result of the previous report. [ 53 ] But just because of the confinement of the adhesion molecules, it makes the morphological variation of the HaCaT cells treated by CDots less obvious. Thus, HaCaT cells should be first digested by trypsin and then seeded into culture flasks for the detailed investigations of the morphological variation and the confirmation of EMT process.…”