Stratified Squamous Epithelial Cells Research Articles

Epidermal Barrier Development via Corneoptosis: A Unique Form of Cell Death in Stratum Granulosum Cells.

Epidermal development is responsible for the formation of the outermost layer of the skin, the epidermis. The establishment of the epidermal barrier is a critical aspect of mammalian development. Proper formation of the epidermis, which is composed of stratified squamous epithelial cells, is essential for the survival of terrestrial vertebrates because it acts as a crucial protective barrier against external threats such as pathogens, toxins, and physical trauma. In mammals, epidermal development begins from the embryonic surface ectoderm, which gives rise to the basal layer of the epidermis. This layer undergoes a series of complex processes that lead to the formation of subsequent layers, including the stratum intermedium, stratum spinosum, stratum granulosum, and stratum corneum. The stratum corneum, which is the topmost layer of the epidermis, is formed by corneoptosis, a specialized form of cell death. This process involves the transformation of epidermal keratinocytes in the granular layer into flattened dead cells, which constitute the protective barrier. In this review, we focus on the intricate mechanisms that drive the development and establishment of the mammalian epidermis to gain insight into the complex processes that govern this vital biological system.

Single-cell RNA sequencing in cornea research: Insights into limbal stem cells and their niche regulation

The corneal epithelium is composed of stratified squamous epithelial cells on the outer surface of the eye, which acts as a protective barrier and is critical for clear and stable vision. Its continuous renewal or wound healing depends on the proliferation and differentiation of limbal stem cells (LSCs), a cell population that resides at the limbus in a highly regulated niche. Dysfunction of LSCs or their niche can cause limbal stem cell deficiency, a disease that is manifested by failed epithelial wound healing or even blindness. Nevertheless, compared to stem cells in other tissues, little is known about the LSCs and their niche. With the advent of single-cell RNA sequencing, our understanding of LSC characteristics and their microenvironment has grown considerably. In this review, we summarized the current findings from single-cell studies in the field of cornea research and focused on important advancements driven by this technology, including the heterogeneity of the LSC population, novel LSC markers and regulation of the LSC niche, which will provide a reference for clinical issues such as corneal epithelial wound healing, ocular surface reconstruction and interventions for related diseases.

Expression of K-19 Cytokeratin in Gingival Epithelial Cells in Periodontal Health and Disease - An Immunohistochemical Study

Background and aim: CK19, a secondary keratin of simple epithelial cells, is the smallest cytokeratin. Its expression was observed in basal and suprabasal stratified squamous epithelial cells of oral mucosa, especially in case of inflammatory conditions and epithelial dysplasia. The aim of this analytical cross-sectional study was to investigate the expression of CK 19 in the gingival epithelial cells of healthy and periodontitis subjects and to correlate the findings with the clinical parameters. Materials and methods: The tissue samples were collected from a total of 30 patients from the healthy and periodontitis groups, during orthodontic/ 3rd molar extractions and during periodontal surgeries respectively. The tissues were then processed for immunohistochemistry analysis and the results were obtained. Results: The data obtained were statistically analysed (CHI square and ANOVA tests) and the results suggested that, there was no statistically significant difference in the expression of CK-19 between healthy and periodontitis groups. Conclusion: The results of this study further affirm the varied responses to destruction (breakdown and repair) from individual to individual during the host-microbial interactions. The mixed outcome could also be due to the suppression of overt inflammation following phase I therapy. Keywords: basal layers, cytokeratin, immunohistochemistry, inflammation, oral epithelium.

YAP1 activation by human papillomavirus E7 promotes basal cell identity in squamous epithelia.

Persistent human papillomavirus (HPV) infection of stratified squamous epithelial cells causes nearly 5% of cancer cases worldwide. HPV-positive oropharyngeal cancers harbor few mutations in the Hippo signaling pathway compared to HPV-negative cancers at the same anatomical site, prompting the hypothesis that an HPV-encoded protein inactivates the Hippo pathway and activates the Hippo effector yes-associated protein (YAP1). The HPV E7 oncoprotein is required for HPV infection and for HPV-mediated oncogenic transformation. We investigated the effects of HPV oncoproteins on YAP1 and found that E7 activates YAP1, promoting YAP1 nuclear localization in basal epithelial cells. YAP1 activation by HPV E7 required that E7 binds and degrades the tumor suppressor protein tyrosine phosphatase non-receptor type 14 (PTPN14). E7 required YAP1 transcriptional activity to extend the lifespan of primary keratinocytes, indicating that YAP1 activation contributes to E7 carcinogenic activity. Maintaining infection in basal cells is critical for HPV persistence, and here we demonstrate that YAP1 activation causes HPV E7 expressing cells to be retained in the basal compartment of stratified epithelia. We propose that YAP1 activation resulting from PTPN14 inactivation is an essential, targetable activity of the HPV E7 oncoprotein relevant to HPV infection and carcinogenesis.

Skin Erosions in a Newborn.

Skin Erosions in a Newborn.

Changes in Rabbit Conjunctival Mucins Following Exposure to Sulfur Mustard

Sulfur mustard (SM) is a vesicating chemical warfare agent that is relatively easy to synthesize, stockpile, and deliver. It is known to cause severe injury to the eye damaging both the conjunctiva and cornea; developing approaches to counter SM-induced acute and chronic ocular toxicities is a high priority. In the present studies, we characterized the ocular effects of SM in New Zealand white rabbits. Within one day of exposure to SM, edema and hazing of the cornea were observed, followed by neovascularization which persisted for at least 28 days. This was associated with upper and lower eyelid hyperemia, edema and conjunctival inflammation. The conjunctiva is composed of a proliferating epithelium largely consisting of stratified columnar and squamous epithelial cells overlying a well-defined dermis. Superficial layers of the conjunctival epithelium expressed keratin 1, a marker of differentiating squamous epithelium, while keratin 17, a marker of proliferating stratified squamous epithelium, was expressed in cells overlying the basement membrane. SM treatment increased the number of keratin 17 expressing epithelial cells. Mucin 5AC producing goblet cells were interspersed within the superficial layers of the conjunctival epithelium. Alcian blue/periodic acid-Schiff staining revealed that these cells generated both acidic and neutral mucins. SM was found to increase the number of goblet cells producing neutral mucins. SM also increased expression of membrane-associated mucin 1 and mucin 4 in the superficial layers of the conjunctiva. These data demonstrate that ocular exposure of rabbits to SM causes significant damage not only to the cornea, but to the eyelid and conjunctiva, altering conjunctival mucins which are important in protecting the eye from injury. Multiple targets within the eye should be assessed when evaluating the efficacy of potential SM ocular countermeasures.

GATA4 blocks squamous epithelial cell gene expression in human esophageal squamous cells

GATA4 promotes columnar epithelial cell fate during gastric development. When ectopically expressed in the developing mouse forestomach, the tissue emerges as columnar-like rather than stratified squamous with gene expression changes that parallel those observed in the pre-malignant squamous to columnar metaplasia known as Barrett’s esophagus (BE). GATA4 mRNA up-regulation and gene amplification occur in BE and its associated cancer, esophageal adenocarcinoma (EAC), and GATA4 gene amplification correlates with poor patient outcomes. Here, we explored the effect of ectopic expression of GATA4 in mature human esophageal squamous epithelial cells. We found that GATA4 expression in esophageal squamous epithelial cells compromised squamous cell marker gene expression and up-regulated expression of the canonical columnar cell cytokeratin KRT8. We observed GATA4 occupancy in the p63, KRT5, and KRT15 promoters, suggesting that GATA4 directly represses expression of squamous epithelial cell marker genes. Finally, we verified GATA4 protein expression in BE and EAC and found that exposure of esophageal squamous epithelial cells to acid and bile, known BE risk factors, induced GATA4 mRNA expression. We conclude that GATA4 suppresses expression of genes marking the stratified squamous epithelial cell lineage and that this repressive action by GATA4 may have implications in BE and EAC.

Differential Expressions of Aquaporin Subtypes in Female Reproductive Tract of Mice.

Although many aquaporin (AQP) transcripts have been demonstrated to express in the female reproductive tract, the defined localizations and functions of AQP subtype proteins remain unclear. In this study, we investigated the expression of AQP1, AQP3, AQP5, AQP6, and AQP9 proteins in female reproductive tract of mouse and characterized their precise localizations at the cellular and subcellular levels. Immunofluorescence analyses for AQP1, AQP3, AQP6, and AQP9 showed that these proteins were abundantly expressed in female reproductive tract and that intense immunoreactivities were observed in mucosa epithelial cells with a subtype-specific pattern. The most abundant aquaporin in both vagina and uterine cervix was AQP3. Each of AQP1, AQP3, AQP6, and AQP9 exhibited its distinct distribution in stratified squamous or columnar epithelial cells. AQP9 expression was predominant in oviduct and ovary. AQP1, AQP3, AQP6, and AQP9 proteins were mostly seen in apical membrane of ciliated epithelial cells of the oviduct as well as in both granulosa and theca cells of ovarian follicles. Most of AQP subtypes were also expressed in surface epithelial cells and glandular cells of endometrium in the uterus, but their expression levels were relatively lower than those observed in the vagina, uterine cervix, oviduct and ovary. This is the first study to investigate the expression and localization of 5 AQP subtype proteins simultaneously in female reproductive tract of mouse. Our results suggest that AQP subtypes work together to transport water and glycerol efficiently across the mucosa epithelia for lubrication, proliferation, energy metabolism and pH regulation in female reproductive tract.

Abstract 5839: The unfolded protein response gene Ire1α is required for tissue renewal and normal differentiation in the mouse tongue and esophagus

Abstract IRE1α is a well-characterized stress response signaling protein that adapts the cell to conditions of endoplasmic reticulum (ER) stress. In addition to well-established sources of ER stress such as viral infection, hypoxia and nutrient deprivation, it has recently been proposed that the morphological and epigenetic changes associated with routine cell differentiation are also a source of ER stress for the cell, and that IRE1α has an important role in adapting the cell to its newly differentiated state. Here, we provide evidence that IRE1α is essential for the differentiation of stratified squamous epithelium. We utilized mice expressing K14CreERT2 and floxed IRE1α to enable tamoxifen-inducible deletion of IRE1α targeted to the basal layer and stem cells of stratified squamous epithelium. After tamoxifen injection, IRE1α knockout mice displayed severe weight loss resulting in death within 10 days of treatment. Histological analysis revealed that epithelial homeostasis in the tongue and esophagus was severely disrupted leading to significantly decreased food intake and, as a result, death. The tongue epithelium and esophageal mucosal layer were found to be significantly thinner, and filiform papillae were lost from the tongue. Immunohistochemical staining showed significant downregulation of the proliferative marker Ki67, as well as a marker of differentiation, Sox2, suggesting an almost complete loss of tissue renewal as early as four days after IRE1α deletion. This work highlights the complexity of IRE1α signaling beyond adaptation to external sources of stress and proposes a role of IRE1α in the normal physiology of tissue homeostasis. As inhibition of the IRE1α signaling pathway has been proposed as a therapeutic target in certain cancers, an increased understanding of how broad the scope of IRE1α activity is may provide insight into potential side effects resulting from these treatments. Citation Format: Fiona E. Chalmers, Douglas B. Stairs, Adam B. Glick. The unfolded protein response gene Ire1α is required for tissue renewal and normal differentiation in the mouse tongue and esophagus [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5839.

Bovine Papillomavirus Type 2 Infection Associated with Papillomatosis of the Amniotic Membrane in Water Buffaloes (Bubalus bubalis)

Multiple papillomatous nodules were observed scattered over the amniotic membrane in six water buffaloes that had recently aborted. Grossly, some of the nodules had multiple villous projections while others appeared as single prominent conical or cylindrical horns. Histology revealed folded hyperplastic and hyperkeratotic epithelium supported by a narrow fibro-vascular stalk. Using PCR, sequences of the bovine Deltapapillomavirus type 2 (BPV-2) E5 gene were amplified from the amniotic papillomas. Furthermore, expression of the E5 gene was detected using reverse transcription (RT)-PCR. Western blotting revealed BPV-2 E5 oncoprotein as well as L1 protein, suggesting both abortive and productive infection. Additionally, a functional complex composed of BPV-2 E5 oncoprotein and the phosphorylated PDGFβR was detected, which is consistent with the activation of PDGFβR by the interaction with BPV-2 E5 oncoprotein. These results demonstrate that BPV-2 can infect the amnion of water buffaloes and suggest that this infection may cause proliferation of the epithelial cells of the amnion. While the precise pathogenesis in uncertain, it is possible that BPV-2 infection of stratified squamous epithelial cells within squamous metaplasia foci and/or amniotic plaques could lead to papilloma formation. Papillomavirus-associated amniotic papillomas have not previously been reported in any species, including humans.

Putative Adhesion Factors in Vaginal Lactobacillus gasseri DSM 14869: Functional Characterization.

Lactobacilli play an important role in the maintenance of a healthy vaginal microbiota, and some select species are widely used as probiotics. Vaginal isolates of Lactobacillus gasseri DSM 14869 and Lactobacillus rhamnosus DSM 14870 were previously selected to develop the probiotic EcoVag capsules and showed therapeutic effects in women with bacterial vaginosis (BV). However, the molecular mechanisms involved in their probiotic activity are largely unknown. In this study, we identified three cell surface molecules in L. gasseri DSM 14869 that promote adhesion to vaginal epithelial cells (VEC) by constructing dedicated knockout mutants, including exopolysaccharides (EPSs), a protein containing MucBP-like domains (N506_1778), and a putative novel adhesin (N506_1709) with rib/alpha-like domain repeats. EPS knockout mutants revealed 20-fold and 14-fold increases in adhesion to Caco-2 and HeLa cells, respectively, compared with wild type, while the adhesion to VEC was reduced 30% by the mutation, suggesting that EPSs might mediate tissue tropism for vaginal cells. A significant decrease in adhesion to Caco-2 cells, HeLa cells, and VEC was observed in the N506_1778 knockout mutant. The N506_1709 mutant showed no significant difference for the adhesion to Caco-2 and HeLa cells compared with wild type (WT); in contrast, the adhesion to VEC revealed a significant decrease (42%), suggesting that N506_1709 might mediate specific binding to stratified squamous epithelial cells, and this putative novel adhesin was annotated as Lactobacillus vaginal epithelium adhesin (LVEA). Thus, we have discovered an important role for EPSs and a novel adhesin, LVEA, in the adhesive capacity of a vaginal probiotic Lactobacillus strain.IMPORTANCE Lactobacilli are known to contribute to the maintenance of a healthy vaginal microbiota and some are selected as probiotics for the prevention or treatment of urogenital diseases, such as bacterial vaginosis. However, the molecular mechanisms for these health-promoting effects are not fully understood. Here, we functionally identified three cell surface factors of a Lactobacillus gasseri strain potentially involved in its adhesion to vaginal epithelial cells, including exopolysaccharides (EPSs) and two sortase-dependent proteins (N506_1778 and N506_1709). We could demonstrate the tissue-specific adhesion of EPSs to vaginal cells and that N506_1709 might be a novel adhesin specifically mediating bacterial binding to stratified squamous epithelial cells. The results provide important new information on the molecular mechanisms of vaginal Lactobacillus spp. adhesion.

Inside Cover: Contrast of nuclei in stratified squamous epithelium in optical coherence tomography images at 800 nm (J. Biophotonics 9/2019)

The nuclei of epithelial cells in stratified squamous epithelia have been reported to be either low scattering or high scattering. Using micro‐optical coherence tomography, we demonstrate that the nuclei are ‘low scattering’ in the core; those previously reported ‘high‐scattering’ signals are likely from the nucleocytoplasmic boundary.Further details can be found in the article by Si Chen, Xinyu Liu, Nanshuo Wang, et al. (e201900073). image

Jarid1b promotes epidermal differentiation by mediating the repression of Ship1 and activation of the AKT/Ovol1 pathway.

ObjectivesTerminally differentiated stratified squamous epithelial cells play an important role in barrier protection of the skin. The integrity of epidermal cells is maintained by tight regulation of proliferation and differentiation. The aim of this study was to investigate the role of epigenetic regulator H3K4me3 and its demethylase Jarid1b in the control of epithelial cell differentiation.Materials and methodsRT‐qPCR, Western blotting and IHC were used to detect mRNA and protein levels. We analysed cell proliferation by CCK8 assay and cell migration by wound healing assay. ChIP was used to measure H3K4me3 enrichment. A chamber graft model was established for epidermal development.ResultsOur studies showed that H3K4me3 was decreased during epidermal differentiation. The H3K4me3 demethylase Jarid1b positively controlled epidermal cell differentiation in vitro and in vivo. Mechanistically, we found that Jarid1b substantially increased the expression of mesenchymal‐epithelial transition (MET)‐related genes, among which Ovol1 positively regulated differentiation gene expression. In addition, Ovol1 expression was repressed by PI3K‐AKT pathway inhibitors and overexpression (O/E) of the PI3K‐AKT pathway suppressor Ship1. Knockdown (KD) of Ship1 activated downstream PI3K‐AKT pathway and enhanced Ovol1 expression in HaCaT. Importantly, we found that Jarid1b negatively regulated Ship1 expression, but not that of Pten, by directly binding to its promoter to modulate H3K4me3 enrichment.ConclusionOur results identify an essential role of Jarid1b in the regulation of the Ship1/AKT/Ovol1 pathway to promote epithelial cell differentiation.

Loss of TRIM29 Alters Keratin Distribution to Promote Cell Invasion in Squamous Cell Carcinoma.

: TRIM29 (tripartite motif-containing protein 29) is a TRIM family protein that has been implicated in breast, colorectal, and pancreatic cancers. However, its role in stratified squamous epithelial cells and tumors has not been elucidated. Here, we investigate the expression of TRIM29 in cutaneous head and neck squamous cell carcinomas (SCC) and its functions in the tumorigenesis of such cancers. TRIM29 expression was lower in malignant SCC lesions than in adjacent normal epithelial tissue or benign tumors. Lower expression of TRIM29 was associated with higher SCC invasiveness. Primary tumors of cutaneous SCC showed aberrant hypermethylation of TRIM29. Depletion of TRIM29 increased cancer cell migration and invasion; conversely, overexpression of TRIM29 suppressed these. Comprehensive proteomics and immunoprecipitation analyses identified keratins and keratin-interacting protein FAM83H as TRIM29 interactors. Knockdown of TRIM29 led to ectopic keratin localization of keratinocytes. In primary tumors, lower TRIM29 expression correlated with the altered expression of keratins. Our findings reveal an unexpected role for TRIM29 in regulating the distribution of keratins, as well as in the migration and invasion of SCC. They also suggest that the TRIM29-keratin axis could serve as a diagnostic and prognostic marker in stratified epithelial tumors and may provide a target for SCC therapeutics. SIGNIFICANCE: These findings identify TRIM29 as a novel diagnostic and prognostic marker in stratified epithelial tissues.

Molecular basis of Mitomycin C enhanced corneal sensory nerve repair after debridement wounding

The ocular surface is covered by stratified squamous corneal epithelial cells that are in cell:cell contact with the axonal membranes of a dense collection of sensory nerve fibers that act as sentinels to detect chemical and mechanical injuries which could lead to blindness. The sheerness of the cornea makes it susceptible to superficial abrasions and recurrent erosions which demand continuous regrowth of the axons throughout life. We showed previously that topical application of the antibiotic and anticancer drug Mitomycin C (MMC) enhances reinnervation of the corneal nerves and reduces recurrent erosions in mice via an unknown mechanism. Here we show using RNA-seq and confocal imaging that wounding the corneal epithelium by debridement upregulates proteases and protease inhibitors within the epithelium and leads to stromal nerve disruption. MMC attenuates these effects after debridement injury by increasing serpine1 gene and protein expression preserving L1CAM on axon surfaces of reinnervating sensory nerves. These data demonstrate at the molecular level that gene expression changes in the corneal epithelium and stroma modulate sensory axon integrity. By preserving the ability of axons to adhere to corneal epithelial cells, MMC enhances sensory nerve recovery after mechanical debridement injury.

Carcinoma escamoso oral em gato jovem

Background: : : Oral neoplasms are common in cats. Just the percentage of oral squamous cell carcinoma (SCC) accounts for about 70% of all feline oral tumors also it is considered highly invasive and malignant. There are reports that the flea collar increases five times the risk oral SCC development. In addition, smoke exposure is also considered a risk factor, and the animals were exposed to tobacco when makes it self-cleaning. SCC well differentiated is characterized histologically by numerous keratin pearls formation and clearly evident intercellular bridges with poor mitotic activity and nuclear pleomorphism. The median age of cats with oral SCC is about 11-13 years, but, cats as young as three years and as old as twenty-one years have been previously reported. However, it was not found reports of oral SCC in cats at age younger than one year old. The aim of this article is reports one case of oral SCC feline at age of three months old. Five days after surgical procedure the patient returned to the veterinary and the owner report that it returned to eating well. Case: It was exanimate a three months old male mixed breed cat presenting oral volume increased and the biopsy test was suggested. However, the owner returned 3 months later when the animal showed tumor 6 cm in diameter, dysphagia, tongue protrusion, excessive salivation, halitosis and weight loss. It was indicated a wide surgical excision of tumoral mass. The tissue was submitted to histopathology. Discussion: The historical, macroscopic characteristics and predisponents factors may aid in carcinoma diagnosis, however, the cytologic or histological examination of tissue confirms this diagnosis. Therefore, the microscopic evaluation revealed a malignant neoplastic proliferation of stratified squamous epithelial cells desmosomal junctions evident, few foci keratin pearl formation and presence of mononuclear inflammatory reaction perifocal. A well-differentiated squamous cell carcinoma was diagnosed by histological study. Thirty days after the surgical procedure, the animal showed a complete recovery with weight increase and eating normally. Thus, surgery proves to provide excellent quality of life maintenance for a long period of time and, although it had shown no signs of recurrence at the present moment, the literature describes that SCC long-term recurrences are common. Indeed, an important question is the quality of life of the felines with oral SCC, whereas this neoplasm led to a marked decrease in eating and drinking due to pain. Therefore, the treatment target is making the possible to maintain an excellent quality of life as long as possible. With the surgery was possible the maintenance of an excellent quality of life for a longer period. An early diagnosis could have defined the need for surgical procedure before the animal presented clinical signs characteristic, whereas represent a locally invasive neoplasm with poor response to conventional treatments models and unfavorable prognosis. Actually, increased incidences of SCC have been associated with clinical and preventive veterinary medicine improvements, which led to the prolongation of life to this animal, becoming more susceptible to senile disease, but this paper reports one case of oral SCC feline at age of three months old.

Regulation of keratin 5/14 intermediate filaments by CDK1, Aurora-B, and Rho-kinase

We previously reported that vimentin, GFAP, and desmin (type III intermediate filament [IF] proteins) are mitotically phosphorylated by CDK1, Aurora-B, and Rho-kinase. This phosphorylation is critical for efficient separation of these IFs and completion of cytokinesis. Keratin 5 (K5) and K14 form a heterodimer, which constitutes IF network in basal layer cells of stratified squamous epithelia. Here, we report that the solubility of K5/K14 increased in mitosis. The in vitro assays revealed that three mitotic kinases phosphorylate K5 more than K14. We then identified Thr23/Thr144, Ser30, and Thr159 on murine K5 as major phosphorylation sites for CDK1, Aurora-B, and Rho-kinase, respectively. Using site- and phosphorylation-state-specific antibodies, we demonstrated that K5-Thr23 was phosphorylated in entire cytoplasm from prometaphase to metaphase, whereas K5-Ser30 phosphorylation occurred specifically at the cleavage furrow from anaphase to telophase. Efficient K5/K14-IF separation was impaired by K5 mutations at the sites phosphorylated by these mitotic kinases. K5-Thr23 phosphorylation was widely detected in dividing K5-positive cells of murine individuals. These results suggested that mitotic reorganization of K5/K14-IF network is governed largely through K5 phosphorylation by CDK1, Aurora-B, and Rho-kinase.

Functional expression of BMP7 receptors in oral epithelial cells. Interleukin-17F production in response to BMP7

Background: Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily. Recently, BMP7 has been demonstrated to be produced by salivary glands and contribute to embryonic branching in mice. The BMP7 in saliva is thought to be delivered to the oral cavity and is expected to contact with stratified squamous epithelial cells which line the surface of oral mucosa. In this study, we attempted to investigate the effects of BMP7 on oral epithelial cells.Methods: The expression of BMP receptors was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). OSCCs were stimulated with human recombinant BMP7 (hrBMP7) and the phosphorylation status of Smad1/5/8 was examined by western blotting. For microarray analysis, Ca9-22 cells were stimulated with 100 ng/mL of hrBMP7 and total RNA was extracted and subjected to real-time PCR. The 5’-untranslated region (5’-UTR) of IL-17 F gene was cloned to pGL4-basic vector and used for luciferase assay. Ca9-22 cells were pre-incubated with DM3189, a specific inhibitor of Smad1/5/8, for inhibition assay.Results: All isoforms of type I and type II BMP receptors were expressed in both Ca9-22 and HSC3 cells and BMP7 stimulation resulted in the phosphorylation of Smad1/5/8 in both cell lines. The microarray analysis revealed the induction of interleukin-17 F (IL-17 F), netrin G2 (NTNG2) and hyaluronan synthase 1 (HAS1). Luciferase assay using the 5’-UTR of the IL-17 F gene revealed transcriptional regulation. Induced IL-17 F production was further confirmed at the protein level by ELISA. Smad1/5/8 inhibitor pretreatment decreased IL-17 F expression levels in the cells.

Abstract 4794: Reduced Epstein-Barr virus replication in human papillomavirus-immortalized keratinocyte organotypic raft culture

Abstract An epidemic rise in oropharyngeal squamous cell carcinoma (OSCC) infected with human papillomavirus has been observed over the past decade. The majority of HPV+OSCC arise from the tonsils and base of tongue (BOT), which are lymphoid-rich regions that are known to harbor Epstein-Barr virus (EBV). Indeed, we previously reported HPV/EBV co-infection in 25% of tonsillar and 70% of BOT tumors. HPV is a DNA tumor virus that specifically infects proliferating basal cells in stratified squamous epithelia. HPV replication is facilitated by viral oncoproteins E6 and E7, which create an S-phase-like environment via degradation of cell cycle regulators p53 and Rb, respectively. EBV is also an established DNA tumor virus that replicates in differentiated epithelia in its normal lifecycle. However, EBV exhibits a latent infection characterized by oncogene expression and lack of viral replication in EBV-associated carcinomas. E6/E7-mediated alteration of the cell cycle results in an interruption in epithelial differentiation, which is required for EBV replication in epithelial cells. Thus, we hypothesize that HPV immortalization of keratinocytes shifts the EBV life cycle from replication to latency. To test our hypothesis, we developed an EBV/HPV co-infection model utilizing organotypic raft culture to produce stratified epithelia in vitro. Tonsillar keratinocytes immortalized by HPV were EBV-infected by co-culture with IgG-induced EBV-positive Akata Burkitt’s lymphoma cells four days after differentiation induction. Raft tissues were harvested 6 days post-EBV infection for analysis of viral life cycles. EBV infection did not alter HPV replication or gene expression. In HPV-negative tonsillar keratinocytes, robust EBV replication was observed that was blocked by treatment with acyclovir, a nucleoside analog that inhibits EBV lytic replication. In contrast, a dramatic decrease in EBV DNA levels was observed in HPV-positive keratinocytes, which was not further reduced by acyclovir. Human telomerase-immortalized normal oral keratinocytes supported robust EBV replication in organotypic raft culture suggesting a specific effect of HPV rather than immortalization on EBV replication. Expression of E7 alone was sufficient to decrease EBV DNA levels. Preliminary data suggest no change in EBV infectivity between HPV-negative and HPV-positive rafts, and an increase in EBV latency-associated gene expression in HPV-positive cells. The ability of EBV to establish persistent latent infection in HPV-infected epithelial cells would contribute additional viral oncogene expression to explain the rapid development and progression of HPV+OSCC. Citation Format: Joseph T. Guidry, William K. Songock, Xiaohui Ma, Cherie-Ann O. Nathan, Jason M. Bodily, Rona S. Scott. Reduced Epstein-Barr virus replication in human papillomavirus-immortalized keratinocyte organotypic raft culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4794. doi:10.1158/1538-7445.AM2017-4794

Cellular events and behaviors after grafting of stratified squamous epithelial cell sheet onto a hydrated collagen gel.

Autologous stratified squamous epithelial cell sheets have been successfully used to treat epithelial defects in tissues such as the cornea and the esophagus. However, the regenerative cellular events occurring in the grafted epithelial cells are unclear in the early stages of wound healing. In this study, we created an in vitro grafting model using cultured normal human epidermal keratinocyte (NHEK) sheets and a type I collagen gel to investigate the cellular processes that occur within the grafted cell sheet. Cultured NHEK cells successfully became a stratified squamous cell sheet resembling epithelial tissue, retained expression of cellular integrins and adhesion proteins, and adhered successfully to a type I collagen gel. After culture on the collagen gel, expression of E‐cadherin, and β‐catenin decreased in the cells of the basal layer of the grafted cell sheet, resembling events characteristic of a partial epithelial–mesenchymal transition (EMT). These basal cells also induced migration of the cell sheet. Those phenomena are consistent with the essential events that occur in the wound‐healing process observed previously in cell studies. Therefore, the epithelial cell sheet grafted onto a type I collagen gel is a suitable model in vitro to study cellular events and behaviors. Furthermore, we also addressed the therapeutic mechanisms by which the epithelial cell sheet promotes wound healing.