Transcriptional Regulation of Airway Epithelial Cell Differentiation: Insights into the Notch Pathway and Beyond.

The airway epithelium is a critical component of the respiratory system, serving as a barrier against inhaled pathogens and toxins. It is composed of various cell types, each with specific functions essential to proper airway function. Chronic respiratory diseases can disrupt the cellular composition of the airway epithelium, leading to a decrease in multiciliated cells (MCCs) and an increase in secretory cells (SCs). Basal cells (BCs) have been identified as the primary stem cells in the airway epithelium, capable of self-renewal and differentiation into MCCs and SCs. This review emphasizes the role of transcription factors in the differentiation process from BCs to MCCs and SCs. Recent advancements in single-cell RNA sequencing (scRNAseq) techniques have provided insights into the cellular composition of the airway epithelium, revealing specialized and rare cell types, including neuroendocrine cells, tuft cells, and ionocytes. Understanding the cellular composition and differentiation processes within the airway epithelium is crucial for developing targeted therapies for respiratory diseases. Additionally, the maintenance of BC populations and the involvement of Notch signaling in BC self-renewal and differentiation are discussed. Further research in these areas could provide valuable insights into the mechanisms underlying airway epithelial homeostasis and disease pathogenesis.

Early Atf4 activity drives airway club and goblet cell differentiation.

Activating transcription factor 4 (Atf4), which is modulated by the protein kinase RNA-like ER kinase (PERK), is a stress-induced transcription factor responsible for controlling the expression of a wide range of adaptive genes, enabling cells to withstand stressful conditions. However, the impact of the Atf4 signaling pathway on airway regeneration remains poorly understood. In this study, we used mouse airway epithelial cell culture models to investigate the role of PERK/Atf4 in respiratory tract differentiation. Through pharmacological inhibition and silencing of ATF4, we uncovered the crucial involvement of PERK/Atf4 in the differentiation of basal stem cells, leading to a reduction in the number of secretory cells. ChIP-seq analysis revealed direct binding of ATF4 to regulatory elements of genes associated with osteoblast differentiation and secretory cell function. Our findings provide valuable insights into the role of ATF4 in airway epithelial differentiation and its potential involvement in innate immune responses and cellular adaptation to stress.

Next generation sequencing technologies to address aberrant mRNA translation in cancer.

In this review, we explore the transformative impact of next generation sequencing technologies in the realm of translatomics (the study of how translational machinery acts on a genome-wide scale). Despite the expectation of a direct correlation between mRNA and protein content, the complex regulatory mechanisms that affect this relationship remark the limitations of standard RNA-seq approaches. Then, the review characterizes crucial techniques such as polysome profiling, ribo-seq, trap-seq, proximity-specific ribosome profiling, rnc-seq, tcp-seq, qti-seq and scRibo-seq. All these methods are summarized within the context of cancer research, shedding light on their applications in deciphering aberrant translation in cancer cells. In addition, we encompass databases and bioinformatic tools essential for researchers that want to address translatome analysis in the context of cancer biology.

Genome-wide B1 retrotransposon binds the transcription factors dioxin receptor and Slug and regulates gene expression in vivo.

Alterations in tissue-specific gene expression greatly affect cell function. Transcription factors (TFs) interact with cis-acting binding sites in noncoding enhancer promoter regions. Transposable elements (TEs) are abundant and similarly represented among mammalian genomes. TEs are important in gene regulation, but their function is not well understood. We have characterized a TE containing functional TF-binding sites for the carcinogen-activated dioxin receptor xenobiotic responsive element (XRE) and the epithelial-mesenchymal transition regulator Slug (Slug site). A Mus promoter database was scanned for XREs to predict coregulation with other TFs. We identified an overrepresented (1,398 genes) B1 retrotransposon containing XRE and Slug sites within 35 bp of each other (designated as B1-X35S). This B1-X35S retrotransposon differed from classic B1s by the presence of the Slug site and by its differential nucleotide conservation outside the X35S region. Phylogenetically, B1-X35S appeared recently in evolution, close to the B1-B subfamily. Comparative gene expression in 61 mouse tissues revealed that B1-X35S-containing genes had lower median expression levels than those with canonical B1 TEs, suggesting a repressive role for X35S. Indeed, X35S was functional and able to bind aryl hydrocarbon (dioxin) receptor (AhR) and Slug and, importantly, to repress cis-reporter genes. Moreover, AhR and Slug were recruited to X35S in vivo and repressed the endogenous expression of X35S-containing genes. Our results demonstrate the existence of a widely present B1 subfamily in the mouse. Because AhR and Slug are relevant in tumor development and differentiation, X35S may represent a genome-wide regulatory mechanism and a tool to modulate gene expression.

Gonadotropin releasing hormone analogs induce apoptosis by extrinsic pathway involving p53 phosphorylation in primary cell cultures of human prostatic adenocarcinomas.

BACKGROUND: Gonadotropin-releasing-hormone (GnRH) analogs are widely used to block hypothalamic-pituitary-gonadal axis and inhibit blood androgen levels in patients with prostate cancer (PCa). In addition, GnRH analogs induce proliferation arrest and apoptosis through GnRH receptors expressed on the membrane of PCa cells. Possible molecular mechanisms involved in GnRH-mediated apoptosis on prostate cancer cells were studied. METHODS: Primary cultures from PCa and benign prostatic hyperplasia (BPH) (non-malignant control) were derived from samples provided by our Institutional Hospital. Cell cultures were incubated for 24 hr with 20 ng/ml of GnRH agonist Leuprolide (Lp) or antagonist Cetrorelix (Cx). Apoptosis was evaluated by studying the expression of Bax and Bcl-2 and the activation of caspase-9 (intrinsic pathway), caspase-8 (extrinsic pathway), and caspase-3. Also, mRNA level, protein expression and phosphorylation of p53 were studied. RESULTS: Cleaved caspase-8 and -3, but not -9, increased in presence of Lp and Cx in PCa cell cultures. Bax and Bcl-2 mRNA levels showed no changes after GnRH-analog treatments. Only Bax protein showed an increase after Cx treatment in PCa cell cultures. p53 mRNA level was higher in PCa than in BPH cell cultures. Lp and Cx increased p53 expression and phosphorylation in PCa cell cultures. CONCLUSIONS: Apoptosis induced by GnRH analogs seems to be mediated by extrinsic pathway involving p53 phosphorylation. Phosphorylated-p53 might be associated with the increase in apoptotic NGF receptor, p75, previously reported by our laboratory. These findings reinforce the concept of clinical use of GnRH analogs for PCa suggesting that intraprostatic treatment may be more effective.

Regulation of cell survival by resveratrol involves inhibition of NF kappa B-regulated gene expression in prostate cancer cells.

BACKGROUND: Polyphenols have been proposed as antitumoral agents. We have shown that resveratrol (RES) induced cell cycle arrest and promoted apoptosis in prostate cancer cells by inhibition of the PI3K pathway. The RES effects on NF kappaB activity in LNCaP cells (inducible NF kappaB), and PC-3 cells (constitutive NF kappaB) are reported. METHODS: Cells were treated with 1-150 microM of RES during 36 hr. NF kappaB subcellular localization was analyzed by western blot and immunofluorescence. I kappaB alpha was evaluated by immunoprecipitation followed by Western blot. Specific DNA binding of NF kappaB was determined by EMSA assays and NF kappaB-mediated transcriptional activity by transient transfection with a luciferase gene reporter system. RESULTS: RES induced a dose-dependent cytoplasmic retention of NF kappaB mediated by I kappaB alpha in PC-3 cells but not in LNCaP. RES-induced inhibition of NF kappaB specific binding to DNA was more significant in PC-3 cells. NF kappaB-mediated transcriptional activity induced by EGF and TNFalpha were inhibited by RES in both cell lines. LY294002 mimicked RES effects on NF kappaB activity. CONCLUSION: Antiproliferative and apoptotic effects of RES on human prostate cancer cells may be mediated by the inhibition of NF kappaB activity. This mechanism seems to be associated to RES-induced PI3K inhibition. RES could have therapeutic potential for prostate cancer treatment.

Mechanisms involved in resveratrol-induced apoptosis and cell cycle arrest in prostate cancer-derived cell lines.

Resveratrol is a polyphenol found at high concentrations in grapes and red wine with reported anticarcinogenic effects. We studied the molecular mechanism of resveratrol-induced apoptosis and proliferation arrest in prostate derived cells PZ-HPV-7 (nontumorigenic line), LNCaP (androgen-sensitive cancer line), and PC-3 (androgen-insensitive cancer line). Apoptosis and cell cycle distribution were evaluated by flow cytometry and proliferation by MTT assay and direct cell counting. Caspases, bax, bcl-2, cyclins, Cdks, p53, p21, and p27 were measured by Western blot and kinase activities of cyclin/Cdk complexes by immunoprecipitation followed by kinase assays with appropriate substrates. Resveratrol induced a decrease in proliferation rates and an increase in apoptosis in cancer cell lines in a dose- and time-dependent manner. These effects were coincident with cell accumulation at the G0/G1 phase. In LNCaP and PC-3, the apoptosis induced by resveratrol was mediated by activation of caspases 9 and 3 and a change in the ratio of bax/bcl-2. Expressions of cyclin D1, E, and Cdk4 as well as cyclin D1/Cdk4 kinase activity were reduced by resveratrol only in LNCaP cells. In contrast, cyclin B and Cdk1 expression and cyclin B/Cdk1 kinase activity were decreased in both cell lines in the presence of resveratrol. However, modulator proteins p53, p21, and p27 were increased by resveratrol only in LNCaP cells. These effects probably result in the observed proliferation arrest and disruption of cell cycle control. In addition, the specific differences found between LNCaP and PC-3 suggest that resveratrol acts through different mechanisms upon the androgen or estrogen receptor cell status.