Influence of Sp1/Sp3 expression on corneal epithelial cells proliferation and differentiation properties in reconstructed tissues

PURPOSE : Primary cultured epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. However, as such cells are passaged in culture, they often lose their ability to proliferate by progressing toward terminal cell differentiation, a process likely to be determined by altered expression of transcription factors that have functions critical for cell adhesion and differentiation. This study was designed to determine whether the variable life span of primary cultured human corneal epithelial cells (HCECs) might be the consequence of varying expression levels of the well-known transcription factors Sp1 and Sp3 (Sp1/Sp3). METHODS : HCECs were obtained from donor eyes and cultured on irradiated Swiss-3T3. Sp1/Sp3 expression was monitored by Western blot and electrophoretic mobility shift assay (EMSA). The Sp1/Sp3 regulatory influence was evaluated by transfection of HCECs with a recombinant plasmid bearing the Sp1/Sp3-dependent poly(ADP-ribose) polymerase (rPARP) promoter fused to the CAT reporter gene. HCECs that expressed various levels of Sp1/Sp3 were also used for the production of corneal substitutes. RESULTS : Expression of Sp1/Sp3 was dramatically inconsistent between HCECs isolated from the eyes of different donors. Both factors were highly expressed during one passage and then totally disappeared as cells terminally differentiated. Proper stratification of HCECs on reconstructed tissue substitutes could be obtained only with cells that also had a delayed peak of Sp1/Sp3 expression when cultured in vitro. CONCLUSIONS : Expression of Sp1/Sp3 may represent a good predictor for selecting HCECs that are most likely to proliferate, stratify, and differentiate properly when used for the production of reconstructed corneal substitutes

Transcriptional regulation of the human α6 integrin gene by the transcription factor NFI during corneal wound healing

Purpose. Wound healing of the corneal epithelium is highly influenced by regulation of integrin gene expression. A recent study demonstrated that laminin (LM), a major constituent of the extracellular matrix (ECM), reduces expression of the human α6 integrin subunit gene by altering the properties of the transcription factor (TF) Sp1. In this work, a target site was identified for the TF nuclear factor I (NFI) on the human α6 gene, and its regulatory influence was characterized in corneal epithelial cells. Methods. Plasmids bearing the α6 promoter fused to the CAT gene were transfected into human (HCECs) and rabbit (RCECs) corneal epithelial cells grown on LM. The DNA-binding site for NFI in the α6 promoter was identified by DNase I footprinting. Expression and DNA binding of NFI was monitored by Western blot, RT-PCR, and electrophoretic mobility shift assays (EMSAs), and its function was investigated through RNAi and NFI overexpression assays. Results. All NFI isoforms were found to be expressed in HCECs and RCECs. Transfection analyses revealed that NFI is a repressor of α6 expression in both types of cells. LM increases expression of NFI, whereas inhibition of each NFI isoform increases promoter activity suggesting that NFI is a key repressor of α6 transcription. In addition, the negative influence of NFI appears to be potentiated by the degradation of Sp1 when cells are grown on LM. Conclusions. Repression of α6 expression therefore contributes to the final steps of corneal wound healing by both reducing proliferation and allowing attachment of the epithelium to the basal membrane

Differential binding of the transcription factors Sp1, AP-1, and NFI to the promoter of the human α5 integrin gene dictates its transcriptional activity

Purpose. Damage to the corneal epithelium results in the massive secretion of fibronectin (FN) shortly after injury and induces the expression of its integrin receptor α5β1. The authors reported previously that FN induces α5 expression in human corneal epithelial cells and rabbit corneal epithelial cells by altering the binding of the transcription factor (TF) Sp1 to a regulatory element from the α5 promoter that it is also flanked by binding sites for the TFs NFI and AP-1. Here, they assessed the function of NFI and AP-1 on α5 gene expression and evaluated the contribution of FN to their overall regulatory influence. Methods. TF binding to the α5 promoter was evaluated in vitro by electrophoretic mobility shift assays and in vivo by ligation-mediated PCR or chromatin immunoprecipitation. TFs expression was monitored by Western blot, whereas their influence was assessed by transfection and RNAi analyses. Results. Coexpression of Sp1, NFI, and AP-1 was demonstrated in all cell types, and each TF was shown to bind efficiently to the α5 promoter. Whereas both AP-1 and Sp1 activated expression directed by the α5 promoter, NFI functioned as a potent repressor of that gene. Interestingly, FN could either promote or repress α5 promoter activity in a cell density–dependent manner by differentially altering the ratio of these TFs. Conclusions. These results suggest that α5 gene expression is likely dictated by subtle alterations in the nuclear ratio of TFs that either repress (NFI) or activate (Sp1 and AP-1) α5 transcription in corneal epithelial cells

Medical Pluralism Predicts Non-ART Use among Parents in Need of ART: A Community Survey in KwaZulu-Natal, South Africa

Despite documented common use of traditional healers and efforts to scale up antiretroviral treatment (ART) in sub-Saharan Africa, evidence on whether medical pluralism predicts ART use is inconclusive and restricted to clinic settings. This study quantitatively assesses whether medical pluralism predicts ART use among parents in need of ART in South Africa. 2,477 parents or primary caregivers of children were interviewed in HIV-endemic communities of KwaZulu-Natal. Analysis used multiple logistic regression on a subsample of 435 respondents in need of ART, who reported either medical pluralism (24.6 %) or exclusive public healthcare use (75.4 %). Of 435 parents needing ART, 60.7 % reported ART use. Medical pluralism emerged as a persistent negative predictor of ART utilization among those needing it (AOR [95 % CI] = .556 [.344 - .899], p = .017). Use of traditional healthcare services by those who need ART may act as a barrier to treatment access. Effective intersectoral collaboration at community level is urgently needed

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Messenger RNA Life-Cycle in Cancer Cells: Emerging Role of Conventional and Non-Conventional RNA-Binding Proteins?

Functional specialization of cells and tissues in metazoans require specific gene expression patterns. Biological processes, thus, need precise temporal and spatial coordination of gene activity. Regulation of the fate of messenger RNA plays a crucial role in this context. In the present review, the current knowledge related to the role of RNA-binding proteins in the whole mRNA life-cycle is summarized. This field opens up a new angle for understanding the importance of the post-transcriptional control of gene expression in cancer cells. The emerging role of non-classic RNA-binding proteins is highlighted. The goal of this review is to encourage readers to view, through the mRNA life-cycle, novel aspects of the molecular basis of cancer and the potential to develop RNA-based therapies

The feeder layer-mediated extended lifetime of cultured human skin keratinocytes is associated with altered levels of the transcription factors Sp1 and Sp3

Primary cultured epithelial cells that are used for basic research are often cultivated on plastic whereas those used for clinical purposes are usually cultured in the presence of a feeder layer. Here, we examined the influence of a feeder layer on the expression, affinity and DNA binding ability of the transcription factors, Sp1 and Sp3 in primary cultures of human skin keratinocytes. Co-culturing both newborn and adult skin keratinocytes with lethally irradiated 3T3 cells as a feeder layer contributed to maintain the cell's morphological and growth characteristics and delayed terminal differentiation in vitro. 3T3 also stabilized the DNA binding properties of Sp1 without altering its transcription. Stimulation of Sp1/Sp3 expression appears to be mediated through cell–cell interactions and by factors secreted by 3T3. Thus, we propose that the feeder layer delay terminal differentiation of primary cultured skin keratinocytes by preventing extinction of transcription factors, like Sp1 and Sp3, which play pivotal functions in the cell cycle

Essais de compatibilité seringue/équipements biomédicaux : une affaire de débit

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