Rescue of the Transcription Factors Sp1 and NFI in Human Skin Keratinocytes through a Feeder-Layer-Dependent Suppression of the Proteasome Activity

Duval, Céline; Gaudreault, Manon; Vigneault, François; Touzel-Deschênes, Lydia; Rochette, Patrick J.; Masson-Gadais, Bénédicte; Germain, Lucie; Guérin, Sylvain L.

doi:10.1016/j.jmb.2012.01.021

Cited by 11 publications

(10 citation statements)

References 89 publications

(104 reference statements)

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“…A total of 675- (for Kf7), 1791- (for Km29) and 862 genes (for Km39), corresponding to 1.4%, 3.7% and 1.8% of all the targets contained on the array, respectively, fitted into that category of differentially regulated genes (Figure 6B). Clustering of the in vivo microarray data for all the genes from the Sp1 sub-family expressed in keratinocytes grown with or without i3T3 into a heatmap indicated clearly that there is no significant variation in the expression of these genes, including Sp1 (Figure 6C), a result consistent with those recently reported by our laboratory [16]. The filters from the Arraystar software were then set to select genes with at least a 4.7-fold change in expression (activated or repressed) between keratinocytes grown with or without i3T3.…”

Section: Resultssupporting

confidence: 90%

“…Indeed, gene profiling on microarrays revealed no significant change in the transcription of the Sp1 gene when keratinocytes are grown with a feeder layer (see Figure 6C). However, this result is consistent with our recent finding that human skin keratinocytes maintain a higher amount of Sp1 at the protein level (and also Sp3 and NFI) when grown in the presence of a feeder layer, not through a corresponding change in the transcription of that gene but rather by preventing Sp1 from being degraded by endogenous proteases [16]. Sp1 has been reported as being specifically degraded by a yet unidentified, trypsin-like serine protease in the rat lung and HL60 cells [35,36].…”

Section: Discussionsupporting

confidence: 92%

“…We recently demonstrated that monitoring the expression of general transcription factors such as Sp1 and NFI is of a considerable interest in order to evaluate the quality of keratinocyte cultures prior to their use for the production of reconstructed skin tissues [16,17]. The ubiquitously expressed Sp1 protein is implicated in the regulation of a multitude of cellular processes, notably in the cell cycle [18,19].…”

Section: Introductionmentioning

confidence: 99%

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Irradiated Human Dermal Fibroblasts Are as Efficient as Mouse Fibroblasts as a Feeder Layer to Improve Human Epidermal Cell Culture Lifespan

Bisson

Rochefort

Lavoie

et al. 2013

IJMS

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A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3) can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes.

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Section: Resultssupporting

confidence: 90%

Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

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Irradiated Human Dermal Fibroblasts Are as Efficient as Mouse Fibroblasts as a Feeder Layer to Improve Human Epidermal Cell Culture Lifespan

Bisson

Rochefort

Lavoie

et al. 2013

IJMS

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“…The reason for this is the prevention of proteasome degradation of glycosylated proteins. The resulting increase of Sp1 and NF1 levels plays a positive role in keratinocytes proliferation inhibiting differentiation [44].…”

Section: Post-translational Modifications Of Nf1mentioning

confidence: 99%

New insights into the role of NF1 in cancer

Sabová

Kretova²,

Luciaková³

2013

neo

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NF1 proteins are a family of transcription factors that act either as repressors or as activators. Functional studies indicate that NF1 participate in signaling pathways that regulate cell viability, proliferation and differentiation. Participation in regulation of genes important for tumor progression and metastasizing suggests a potential value of NF1 as a prognostic factor for certain types of cancer.

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“…We recently used irradiated human dermal fibroblasts (iHFL) as a feeder layer in the monolayer culture of human skin keratinocytes and demonstrated that it was as efficient as i3T3 at maintaining the proliferative characteristics of these cells and the number of cell passages for which they could be grown in culture [11,12]. Meanwhile, we also previously reported an association between stem cell differentiation and their expression level of the transcription factors (TFs) Sp1 and NFI for both skin and corneal epithelial cells as well as for corneal endothelial cells [13]. Our goal herein is thus to characterize the impact of different feeder layers on the proliferative properties of hCECs in monolayer cultures.…”

Section: Introductionmentioning

confidence: 99%

Irradiated Human Fibroblasts as a Substitute Feeder Layer to Irradiated Mouse 3T3 for the Culture of Human Corneal Epithelial Cells: Impact on the Stability of the Transcription Factors Sp1 and NFI

Le‐Bel

Ghio

Guérin

et al. 2019

IJMS

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Because of the worldwide shortage of graftable corneas, alternatives to restore visual impairments, such as the production of a functional human cornea by tissue engineering, have emerged. Self-renewal of the corneal epithelium through the maintenance of a sub-population of corneal stem cells is required to maintain the functionality of such a reconstructed cornea. We previously reported an association between stem cell differentiation and the level to which they express the transcription factors Sp1 and NFI. In this study, we investigated the impact of replacing irradiated 3T3 (i3T3) murine fibroblast feeder cells by irradiated human corneal fibroblasts (iHFL) on the expression of Sp1 and NFI and evaluated their contribution to the proliferative properties of human corneal epithelial cells (hCECs) in both monolayer cultures and human tissue engineered corneas (hTECs). hCECs co-cultured with iHFL could be maintained for up to two more passages than when they were grown with i3T3. Western Blot and electrophoretic mobility shift assays (EMSAs) revealed no significant difference in the feeder-layer dependent increase in Sp1 at both the protein and DNA binding level, respectively, between HCECs grown with either i3T3 or iHFL. On the other hand, a significant increase in the expression and DNA binding of NFI was observed at each subsequent passage when hCECs were co-cultured along with i3T3. These changes were found to result from an increased expression of the NFIA and NFIB isoforms in hCECs grown with i3T3. Exposure of hCECs to cycloheximide revealed an increased stability of NFIB that likely resulted from post-translational glycosylation of this protein when these cells were co-cultured with i3T3. In addition, iHFL were as efficient as i3T3 at preserving corneal, slow-cycling, epithelial stem cells in the basal epithelium of the reconstructed hTECs. Furthermore, we observed an increased expression of genes whose encoded products promote hCECs differentiation along several passages in hCECs co-cultured with either type of feeder layer. Therefore, the iHFL feeder layer appears to be the most effective at maintaining the proliferative properties of hCECs in culture most likely by preserving high levels of Sp1 and low levels of NFIB, which is known for its gene repressor and cell differentiation properties.

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Rescue of the Transcription Factors Sp1 and NFI in Human Skin Keratinocytes through a Feeder-Layer-Dependent Suppression of the Proteasome Activity

Cited by 11 publications

References 89 publications

Irradiated Human Dermal Fibroblasts Are as Efficient as Mouse Fibroblasts as a Feeder Layer to Improve Human Epidermal Cell Culture Lifespan

Irradiated Human Dermal Fibroblasts Are as Efficient as Mouse Fibroblasts as a Feeder Layer to Improve Human Epidermal Cell Culture Lifespan

New insights into the role of NF1 in cancer

Irradiated Human Fibroblasts as a Substitute Feeder Layer to Irradiated Mouse 3T3 for the Culture of Human Corneal Epithelial Cells: Impact on the Stability of the Transcription Factors Sp1 and NFI

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