Process Of Epithelial Differentiation Research Articles

Metabolic dysfunction mediated by HIF-1α contributes to epithelial differentiation defects in eosinophilic esophagitis

Investigating the contributory role that epithelial cell metabolism plays in allergic inflammation is a key factor to understanding what influences dysfunction and the pathogenesis of the allergic disease eosinophilic esophagitis (EoE). We previously highlighted that the absence of hypoxia signaling through hypoxia-inducible factor (HIF)-1α in EoE contributes to esophageal epithelial dysfunction. However, metabolic regulation by HIF-1α has not been explored in esophageal allergy. We sought to define the role of HIF-1α-mediated metabolic dysfunction in esophageal epithelial differentiation processes and barrier function in EoE. In RNA sequencing of EoE patient biopsy samples, we observed the expression pattern of key genes involved in mitochondrial metabolism/oxidative phosphorylation (OXPHOS) and glycolysis. Seahorse bioenergetics analysis was performed on EPC2-hTERT cells to decipher the metabolic processes involved in epithelial differentiation processes. In addition, air-liquid interface cultures were used to delineate metabolic dependency mechanisms required for epithelial differentiation. Transcriptomic analysis identified an increase in genes associated with OXPHOS in patients with EoE. Epithelial origin of this signature was confirmed by complex V immunofluorescence of patient biopsy samples. Bioenergetic analysis invitro revealed that differentiated epithelium was less reliant on OXPHOS compared with undifferentiated epithelium. Increased OXPHOS potential and reduced glycolytic capacity was mirrored in HIF1A-knockdown EPC2-hTERT cells that exhibited a significant absence of terminal markers of epithelial differentiation, including involucrin. Pharmacologic glucose transport inhibition phenocopied this, while rescue of the HIF-1α-deficient phenotype using the pan-prolyl hydroxylase inhibitor dimethyloxalylglycine resulted in restored expression of epithelial differentiation markers. An OXPHOS-dominated metabolic pattern in EoE patients, brought about largely by the absence of HIF-1α-mediated glycolysis, is linked with the deficit in esophageal epithelial differentiation.

Abstract A049: Fallopian tubes of BRCA carriers are characterized by an alteration of epithelial differentiation

Abstract Patients with a germline mutation in BRCA1/2 are at high risk of developing ovarian cancer, the deadliest gynecologic cancer leading to over 207,000 deaths per year in the world. Although the ovarian surface epithelium was long thought to be the origin of this cancer, it is now widely appreciated that the fallopian tube epithelium harbors the cell-of-origin for high-grade serous “ovarian” cancers (HGSC). Nonetheless the earliest stages of carcinogenesis remain poorly characterized. Our study aims to better understand why patients with highly penetrant inherited mutations in BRCA1/2 are at greater risk of HGSC using state-of-the art single cell genomics and spatial profiling of fallopian tubes removed during prophylactically risk-reducing salpingo-oophorectomies (RRSOs). Fallopian tubes from patients with BRCA1/2 mutations are fully embedded in paraffin for careful pathologic examination, limiting the technologies that can be applied to study the molecular profiles of the cancer-prone epithelium. The first aim of our study was to determine whether the samples of cells from fimbria of fallopian tubes obtained through an exfoliative cytology following RRSO are comparable to histologic preparation at the transcriptomic level. We performed single cell RNA sequencing of brushings from 14 fimbriae removed during RRSOs from high-risk mutation carriers, brushings of 3 non-carriers and 12 tissues from non-carriers. As expected, fimbrial brushing mainly consisted of epithelial cells and immune cells and were depleted of fibroblasts and endothelial cells relative to enzymatically dissociated tissue specimens. Fallopian tubes from high-risk mutation carriers were associated with signatures of altered epithelial differentiation suggesting that altered epithelial differentiation processes proceed and may even be required for cancer development. The immune compartment was also markedly different in mutation carriers compared to matched controls. We have recently described how in mutation carriers the myeloid cell compartment shifts from monocyte-enriched in normal Fallopian tubes to macrophage-enriched in HGSCs (Brand et al). In Fallopian tubes of high-risk mutation carriers, we discovered a novel subset of cancer-free patients with signatures similar to advanced tumors, suggesting myeloid compartment remodeling is a prerequisite for, although not sufficient for, tumor formation. BRCA1/2 carriers with higher macrophages also have features of dysfunctional natural killer and T cells. Ongoing spatial transcriptomic profiling of high and average risk fallopian tubes are enabling dissection of the altered cell-cell communications occurring concomitantly with disordered epithelial differentiation. Together these data are expected to advance our understanding of the cellular and molecular signatures of early stage HGSC. Leveraging this insight could lead to the development of new diagnostic tools for early detection of this cancer, an essential prerequisite for improving the chances of survival for patients. Citation Format: Quentin Chartreux, Marcela Haro, Joshua Brand, Andrew Li, Bobbie J. Rimel, Simon Gayther, Huy Dinh, Fabiola Medeiros, Kate Lawrenson. Fallopian tubes of BRCA carriers are characterized by an alteration of epithelial differentiation [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A049.

Modulation of radiation-induced oral mucositis (mouse) by dermatan sulfate: effects on differentiation processes

PurposeDuring head and neck cancer radiotherapy, oral mucositis is the most frequent early side effect. Systemic dermatan sulfate (DS) administration has been shown to significantly decrease oral mucosal radiation reactions during daily fractionated irradiation (IR) in an established mouse model. The aim of this study was to investigate the mechanism of the oral epithelial differentiation process, during IR alone and in combination with DS treatment in the same mouse model.MethodsFractionated IR 5 × 3 Gy/week was given to the snouts of mice over two weeks, either alone (IR) or in combination with daily DS treatment of 4 mg/kg (IR + DS). Groups of mice (n = 3) were sacrificed every second day over the course of 14 days in both experimental arms. Their tongue was excised and subjected to immunohistochemical processing.ResultsIn the p16 analysis as a proliferation marker, the difference between IR alone and IR + DS in the germinal (proliferation) layer was not significant, not stimulating the proliferation process. For the p21 analysis as a differentiation marker on the functional (differentiation) layer, the difference between IR alone and IR + DS arms was significant, indicating that DS inhibited the differentiation process. In the cytokeratin (CK) analysis as the indicator of cellular skeletal integrity, the percentage of antibody-positive cells was above the normal level in both experimental arms and significantly superior in the IR + DS arm.ConclusionThe mucosal protective activity of DS, instead of stimulating proliferation, is based on prevention of cell loss by a combination of effects leading to the inhibition of cellular differentiation and an increase in the expression of epithelial mechanical strength between intercellular mechanical junctions.

QUALITATIVE AND QUANTITATIVE CHANGES IN THE CELLULAR COMPOSITION OF GUMS IN PATIETS WITH PERIODONTAL DISEASES

Background. The results of a comprehensive study of cytological features of epithelial differentiation processes in gums in patients with generalized periodontitis are presented in the article.Objective. The objects of clinical observations were patients with generalized periodontitis. The disease duration was 3-5 years.Methods. Material for the study was gums epithelium taken from a marginal area of gums in young adults, with generalized periodontitis. The epithelium was removed with a spatula, with subsequent transfer to a glass plate and drying in the open air for 3-5 minutes. The colouring of the material was done according to Romanovskyy-Himza, with a next microscopic and morphological analysis, taking into account the percentage of different forms of epithelial cells in normal and due to an age aspect.Results. One of cytological signs of periodontal pathology in young people is epithelial cells with signs of irritation. It should be noted that these cellular elements belong to the intermediate layer of cells that meet histological sections and ribbed surface layers are characterized by homogenization and vacuolization of cytoplasm as a manifestation of prolonged irritation by bacterial cell aggression.Conclusions. Analysis of cytograms proved general periodontitis in the examined patients, in absence of adequate therapy as automotive continuous process. Its components may regress under the influence of effective treatment and re-activate during exacerbation of the inflammatory process under the influence of pathogenic factors such as microbial, hygienic and immune.

IL-24 regulates the process of epithelial differentiation

IL-24 regulates the process of epithelial differentiation

The Role of MicroRNA-210 in Esophageal Squamous Cell Carcinoma

In this review, we summarize our findings on microRNA-210 (miR-210) and the target gene, and discuss their significance in human esophageal squamous cell carcinoma (ESCC). MicroRNAs are evolutionarily conserved small noncoding RNAs (20-23 nucleotides) that bind to complementary sequences in the 3' UTR of target mRNAs and regulate gene expression by the cleavage of target mRNAs and/or translational inhibition. MicroRNAs play important roles in the initiation and progression of cancer, and it has been shown that the expression of some microRNAs is altered in malignancies. Carcinomas are derived from epithelial cells, and poor prognosis in patients with carcinoma is associated with the disruption of characteristics of differentiated epithelial cells, such as cell junctions and polarity. Here, we identified miR-210 as one of the microRNAs that is markedly differentially expressed during the process of epithelial differentiation, though the clinical roles of miR-210 in carcinomas remained unknown. We show that the expression of miR-210 is downregulated in ESCC and derived cell lines. Marked decreases in the level of miR-210 were observed especially in poorly differentiated carcinomas. Moreover, we found that miR-210 inhibits cancer cell survival and proliferation. Finally, we identified fibroblast growth factor receptor-like 1 (FGFRL1) as a target gene of miR-210 in ESCC, and demonstrated that FGFRL1 accelerates cancer cell proliferation. Taken together, our findings show an important role for miR-210 as a tumor suppressive microRNA with effects on cancer cell proliferation.

Dielectric spectroscopy for non-invasive monitoring of epithelial cell differentiation within three-dimensional scaffolds

In this study, we introduce a cellular differentiation cellular model based on dielectric spectroscopy that characterizes epithelial differentiation processes. Non-invasive cellular monitoring was achieved within a three-dimensional microenvironment consisting of a cell-containing collagen I gel seeded onto microfabricated scaffolds. In this proof-of-concept investigation, Madin–Darby canine kidney cells were cultured within microfabricated, geometrically controlled scaffolds and allowed us to differentiate to hollow cyst-like structures. This transformation within the three-dimensional environment is monitored and characterized through dielectric spectroscopy while maintaining cell culture in vitro.

HIF1α Is Essential for Normal Intrauterine Differentiation of Alveolar Epithelium and Surfactant Production in the Newborn Lung of Mice

Neonatal respiratory distress syndrome (RDS) is mainly the result of perturbation in surfactant production and is a common complication seen in premature infants. Normal fetal lung development and alveolar cell differentiation is regulated by a network of transcription factors. Functional loss of any of these factors will alter the developmental program and impact surfactant production and normal gas exchange. During development, the fetus is exposed to varying oxygen concentrations and must be able to quickly adapt to these changes in order to survive. Hypoxia-inducible factor 1alpha (HIF1alpha) is the primary transcription factor that is responsible for regulating the cellular response to changes in oxygen tension and is essential for normal development. Its role in lung maturation is not well defined and to address this knowledge gap, a lung-specific HIF1alpha knock-out model has been developed. Loss of HIF1alpha early in lung development leads to pups that die within hours of parturition, exhibiting symptoms similar to RDS. Lungs from these pups display impaired alveolar epithelial differentiation and an almost complete loss of surfactant protein expression. Ultrastructural analysis of lungs from HIF1alpha deletion pups had high levels of glycogen, aberrant septal development, and decreased expression of several factors necessary for proper lung development, including HIF2alpha, beta-catenin, and vascular endothelial growth factor. These results suggest that HIF1alpha is essential for proper lung maturation and alteration in its normal signaling during premature delivery might explain the pathophysiology of neonatal RDS.

Activation of Kaposi's Sarcoma-Associated Herpesvirus Lytic Gene Expression during Epithelial Differentiation

The oral cavity has been identified as the major site for the shedding of infectious Kaposi's sarcoma-associated herpesvirus (KSHV). While KSHV DNA is frequently detected in the saliva of KSHV seropositive persons, it does not appear to replicate in salivary glands. Some viruses employ the process of epithelial differentiation for productive viral replication. To test if KSHV utilizes the differentiation of oral epithelium as a mechanism for the activation of lytic replication and virus production, we developed an organotypic raft culture model of epithelium using keratinocytes from human tonsils. This system produced a nonkeratinized stratified squamous oral epithelium in vitro, as demonstrated by the presence of nucleated cells at the apical surface; the expression of involucrin and keratins 6, 13, 14, and 19; and the absence of keratin 1. The activation of KSHV lytic-gene expression was examined in this system using rKSHV.219, a recombinant virus that expresses the green fluorescent protein during latency from the cellular EF-1alpha promoter and the red fluorescent protein (RFP) during lytic replication from the viral early PAN promoter. Infection of keratinocytes with rKSHV.219 resulted in latent infection; however, when these keratinocytes differentiated into a multilayered epithelium, lytic cycle activation of rKSHV.219 occurred, as evidenced by RFP expression, the expression of the late virion protein open reading frame K8.1, and the production of infectious rKSHV.219 at the epithelial surface. These findings demonstrate that KSHV lytic activation occurs as keratinocytes differentiate into a mature epithelium, and it may be responsible for the presence of infectious KSHV in saliva.

Effects of snuff extract on epithelial growth and differentiation in vitro

Snuff is a locally irritative agent causing hyperkeratinization and hyperplasia of the oral epithelium. The aim of the study was to investigate the effects of snuff on epithelial cell growth and differentiation in vitro. Three-dimensional HaCaT cell cultures were grown for 6, 12, 14, and 18 days in the presence of 1% snuff extract. Ki-67, p53 and cytokeratins (Cks) 5, 13, 10, 19, 18, involucrin and filaggrin were studied by means of immunohistochemistry. Ki-67 indices were assessed, and the results analyzed statistically. Marked morphologic changes were seen with advanced culture time in the snuff group, probably as a result of increased toxic effects. Snuff exposure decreased the percentage of Ki-67 positive cells on days 6, 12, and 14, suggesting that snuff does not stimulate proliferation activity in this in vitro model. Cornification-related Ck 10 decreased after snuff exposure, indicating disturbances in the epithelial differentiation process.

High Levels of Zinc Ions Induce Loss of Mitochondrial Potential and Degradation of Antiapoptotic Bcl-2 Protein in in Vitro Cultivated Human Prostate Epithelial Cells

Prostate epithelial cells contain the highest levels of zinc among all organs and tissues in the human body. Zinc is accumulated primarily in the mitochondria, where it is responsible for inhibition of mitochondrial aconitase activity, thereby increasing citrate production. The present study was designed to clarify the role of zinc for human prostate epithelial cell growth and apoptosis. Apoptosis of in vitro cultivated human prostate epithelial cells exposed to ZnCl2 was analyzed by determination of phospholipid membrane asymmetry, nuclear fragmentation, DNA strand breaks, changes of mitochondrial potential and cellular pro/antiapoptotic proteins. Zinc induced apoptosis without involvement of p53 by decreasing mitochondrial transmembrane potential (ΔΨm) and Bcl-2 protein levels in proliferating epithelial cells. Thus, the high local concentrations of zinc ions in the prostatic lumen seem to be necessary to regulate proliferative activities and to enforce epithelial differentiation processes.

Murine clusterin: molecular cloning and mRNA localization of a gene associated with epithelial differentiation processes during embryogenesis.

Clusterin is a broadly distributed glycoprotein constitutively expressed by various tissues and cell types, that has been shown to be involved in cell-cell adhesion and expressed during cellular differentiation in vitro. To assess the suggested participation of clusterin in these processes in vivo, we have cloned the cDNA encoding murine clusterin and studied the cellular distribution of clusterin mRNA during murine embryogenesis. Sequence analysis of the cDNA encoding murine clusterin revealed 92 and 75% sequence identity with the rat and human cDNAs, respectively, and conservation of the predicted structural features which include alpha-helical regions and heparin-binding domains. From 12.5 d of development onwards, the clusterin gene is widely expressed in developing epithelia, and selectively localized within the differentiating cell layers of tissues such as the developing skin, tooth, and duodenum where proliferating and differentiating compartments are readily distinguished. In addition, transient and localized clusterin gene expression was detected in certain morphogenetically active epithelia. In the lung, abundant gene transcripts were detected in cuboidal epithelial cells of the terminal lung buds during branching morphogenesis, and in the kidney, clusterin gene expression in the epithelial cells of comma and S-shaped bodies coincided with the process of polarization. Our results demonstrate the in vivo expression of the clusterin gene by differentiating epithelial cells during murine embryogenesis, and provide novel evidence suggesting that clusterin may be involved in the differentiation and morphogenesis of certain epithelia.

Keratins in malignant mesotheliomas and pleural adenocarcinomas: Comparative immunohistochemical analysis with polyclonal and monoclonal antibodies

The distribution of intra-cellular keratins was studied in normal pleural mesothelium, malignant mesotheliomas and adenocarcinomas. This study was performed on deparaffinized sections of tissue fixed in Bouin's solution by indirect immunofluorescence with a monoclonal antibody (KL1) and a conventional keratin antiserum (AKS). Discrepancies were detected using one antibody or the other. Cells from normal mesothelium and 18 cases of malignant mesotheliomas (papillary, tubulary, solid epithelial type) were strongly labelled only by KL1. The 2 cases of sarcomatoid type were negative with both antibodies. In contrast 5 metastatic adenocarcinomas and 5 lung adenocarcinomas were weakly positive or negative with both antibodies. These data confirm the presence of cytokeratins in epithelial differentiation process. Although a clear-cut distinction between mesotheliomas and adenocarcinomas was not possible using these keratin antibodies. Our data point out the importance of reactivity pattern of the antibody used in such investigations.