Differentiation Of Basal Cells Research Articles

Role of KRAS in regulating normal human airway basal cell differentiation

BackgroundKRAS is a GTPase that activates pathways involved in cell growth, differentiation and survival. In normal cells, KRAS-activity is tightly controlled, but with specific mutations, the KRAS protein is persistently activated, giving cells a growth advantage resulting in cancer. While a great deal of attention has been focused on the role of mutated KRAS as a common driver mutation for lung adenocarcinoma, little is known about the role of KRAS in regulating normal human airway differentiation.MethodsTo assess the role of KRAS signaling in regulating differentiation of the human airway epithelium, primary human airway basal stem/progenitor cells (BC) from nonsmokers were cultured on air-liquid interface (ALI) cultures to mimic the airway epithelium in vitro. Modulation of KRAS signaling was achieved using siRNA-mediated knockdown of KRAS or lentivirus-mediated over-expression of wild-type KRAS or the constitutively active G12 V mutant. The impact on differentiation was quantified using TaqMan quantitative PCR, immunofluorescent and immunohistochemical staining analysis for cell type specific markers. Finally, the impact of cigarette smoke exposure on KRAS and RAS protein family activity in the airway epithelium was assessed in vitro and in vivo.ResultssiRNA-mediated knockdown of KRAS decreased differentiation of BC into secretory and ciliated cells with a corresponding shift toward squamous cell differentiation. Conversely, activation of KRAS signaling via lentivirus mediated over-expression of the constitutively active G12 V KRAS mutant had the opposite effect, resulting in increased secretory and ciliated cell differentiation and decreased squamous cell differentiation. Exposure of BC to cigarette smoke extract increased KRAS and RAS protein family activation in vitro. Consistent with these observations, airway epithelium brushed from healthy smokers had elevated RAS activation compared to nonsmokers.ConclusionsTogether, these data suggest that KRAS-dependent signaling plays an important role in regulating the balance of secretory, ciliated and squamous cell differentiation of the human airway epithelium and that cigarette smoking-induced airway epithelial remodeling is mediated in part by abnormal activation of KRAS-dependent signaling mechanisms.

NOTCH signaling promotes the survival of irradiated basal airway stem cells.

Radiation-induced lung injury to normal airway epithelium is a frequent side-effect and dose-limiting factor in radiotherapy of tumors in the thoracic cavity. NOTCH signaling plays key roles in self-renewal and differentiation of upper airway basal lung stem cells during development, and the NOTCH pathway is frequently deregulated in lung cancer. In preclinical lung cancer models, NOTCH inhibition was shown to improve the radiotherapy response by targeting tumor stem cells, but the effects in combination with irradiation on normal lung stem cells are unknown. NOTCH/γ-secretase inhibitors are potent clinical candidates to block NOTCH function in tumors, but their clinical implementation has been hampered by normal tissue side-effects. Here we show that NOTCH signaling is active in primary human- and murine-derived airway epithelial stem cell models and when combined with radiation NOTCH inhibition provokes a decrease in S-phase and increase in G1-phase arrest. We show that NOTCH inhibition in irradiated lung basal stem cells leads to a more potent activation of the DNA damage checkpoint kinases pATM and pCHK2 and results in an increased level of residual 53BP1 foci in irradiated lung basal stem cells reducing their capacity for self-renewal. The effects are recapitulated in ex vivo cultured lung basal stem cells after in vivo whole thorax irradiation and NOTCH inhibition. These results highlight the importance of studying normal tissue effects that may counteract the therapeutic benefit in the use of NOTCH/γ-secretase inhibitors in combination with radiation for antitumor treatment.

Abstract 1733: Role of the SIX1 oncofetal protein in endometrial basal cell metaplasia and carcinogenesis following neonatal exposure to diethylstilbestrol

Abstract Early-life exposure to estrogenic chemicals can increase cancer risk, likely by disrupting normal patterns of cellular differentiation. In a model of this process, female mice exposed to the potent synthetic estrogen diethylstilbestrol (DES) on postnatal days 1-5 develop endometrial adenobasal carcinoma as adults. Neoplastic glands are comprised of abnormal populations of basal cells (cytokeratin (CK)14+), luminal cells (CK18+), and low numbers of "mixed” basal/luminal cells (CK14+/18+), all of which express the oncofetal protein sine oculis homeobox 1 (SIX1). We hypothesized that DES-induced SIX1 expression is necessary for aberrant endometrial differentiation patterns and carcinogenesis. To test this hypothesis, a conditional knockout model was generated in which floxed Six1 was excised in the uterus using progesterone receptor (Pgr) cre. The most prominent change in DES-exposed SIX1 knockout (DES-Six1d/d) mice was the absence of basal cells in the uterine horns. There was a greater than 10-fold decrease in CK14 labeling in the uterine horns of DES-Six1d/d mice compared to DES-exposed SIX1 wildtype (DES-Six1+/+) mice as determined by quantitative image analysis. However, DES-Six1d/d mice exhibited a 42% increase in cancer incidence compared to DES-Six1+/+ mice at 6 months of age (16/18 DES-Six1d/d vs. 7/15 DES-Six1+/+). Interestingly, mixed cells were still present in DES-Six1d/d mice. These findings demonstrate that SIX1 is a cellular differentiation factor necessary for DES-induced basal cells but not mixed cell development or cancer. Furthermore, these data suggest that DES-induced SIX1 expression decreases endometrial carcinogenesis by facilitating basal cell differentiation. Studies investigating the mixed cell population as a putative cancer progenitor cell population are ongoing. Interestingly, mixed cells were present in 35% (63/181) of malignant human endometrial tissue biopsies and in 0% (0/29) of normal endometrial tissue biopsies, suggesting that the DES mouse model has significant similarities to human endometrial cancer. Citation Format: Alisa A. Suen, Wendy N. Jefferson, Charles E. Wood, Anna C. Kenan, Carmen J. Williams. Role of the SIX1 oncofetal protein in endometrial basal cell metaplasia and carcinogenesis following neonatal exposure to diethylstilbestrol [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1733.

Loss of Polycomb Repressive Complex 2 impairs lung bronchiolar cell growth and underlies chronic obstructive pulmonary disease

EZH2, the functional enzymatic component of the Polycomb Repressive Complex 2 (PRC2), is a histone methyl transferase that tri-methylates histone H3 at lysine 27 (H3K27me3). This protein plays a significant role in maintaining tissue specificity by silencing gene programs that control lineage fate. We hypothesized that PRC2 controls cell fate of the lung stem cells. In order to test this hypothesis, we generated mice in which we can conditionally delete Ezh2 upon ingestion of doxycycline. To examine growth, differentiation and self-renewal potentials of different lung epithelial cells, we FACS-isolated distal lung bronchioalveolar stem cells (BASCs) and cultured them in air-liquid-interface Matrigel cultures. We observed a 2fold decrease in organoid number of EZH2 null vs wild type bronchiolar cells and a significant difference in organoid diameter between Ezh2 wild type, heterozygous and null bronchiolar cells. To test in vivo potential of EZH2 depleted epithelium to repair, we performed naphthalene injury, which specifically injures the bronchiolar club cells. We observed that 7 days post naphthalene injury, Ezh2 knock-out lungs had significantly less repair, as measured by repopulation of bronchiolar lung cells. Given that we observed that lung stem cells were impaired when PRC2-mediated gene repression was perturbed, we sought to learn if PRC2 is also dysregulated in Chronic Obstructive Pulmonary Disease (COPD). We first performed Gene Set Enrichment Analysis and found that genes up-regulated in COPD patients relative to healthy smokers were also up-regulated in Ezh2 knock-out adenocarcinoma cells. We next obtained lung tissue from healthy patients and patients with COPD, and performed immunohistochemistry for the PRC2 mark H3K27me3 and immunofluorescence for basal and club cells. There was a significant decrease in H3K27me3, coupled with an increase in basal cells and goblet cells that contained high expressions of club cell markers in COPD lung compared to healthy controls. Finally, to determine if loss of PRC2 function drives basal cell fate and goblet cell differentiation, EZH2 transcripts were knocked down with short hairpins in human bronchiolar epithelial cells, and basal cell differentiation capacity was analyzed. We observed an increased expression of a goblet cell marker and decreased expression of a club cell marker in EZH2 knockdown cultures. Together our results indicate that PRC2 plays an important role in lung stem cell function as well as club cell differentiation fate. These mechanisms may also be perturbed in COPD patients. ChIP-seq and RNA-seq studies to understand the transcriptional and epigenetic changes leading to these phenotypes are underway Support or Funding Information NIH grant K22 CA 20103, Molecular Mechanisms of Toxicity Training Grant T32ES07266 There was a 2-fold decrease in organoid number of EZH2 null vs wild type bronchiolar cells. There was a significant difference in organoid diameter between Ezh2 wild type, heterozygous and null bronchiolar cells. Immunohistochemistry staining of EZH2 shows distinct differences in EZH2 expression between wild type, heterozygous and null organoids. Naphthalene exposure to mouse lung has been shown to kill club cells in the trachea and bronchioles. This injury typically results in club cell regeneration and subsequent lung cell repopulation. Club cells without EZH2 were unable to regenerate and repopulate the airway after naphthalene injury. Club cells that did regenerate in the Ezh2-null lungs lacked the GFP tag, indicating that these cells were likely not responding to doxycycline to delete Ezh2. Gene Set Enrichment Analysis was used to compare gene expression between murine Ezh2-deleted adenocarcinoma cells and COPD patients. It revealed that a significant number of the same genes are highly expressed in both states when compared to healthy lungs or the Ezh2 WT setting. Immunohistochemistry shows significantly lower abundance of H3K27me3 in the nuclei of COPD bronchiolar cells compared to normal. Immunofluorescence staining of club (CCSP and SOX2) and basal cells (KRT5 and SOX2) in normal vs COPD lung airway shows a mixture of metaplasia and hyperplasia along with collapsed airways in COPD airway compared to normal. There is an increase in basal cells and goblet cells that also contain high expressions of CCSP (a club cell marker). EZH2 transcripts were knocked down in immortalized, Human Bronchiolar Epithelial Cells (HBEC) which were, then, prompted to differentiate in air-liquid-interface cultures. Acetylated-Mucin5 was highly expressed while club cell secretory protein (CCSP) was lowly expressed in EZH2 knockdown cells compared to wild type. This suggest that EZH2 plays a role in club and goblet cell differentiation. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sox2 regulates palate development by promoting basal cell differentiation

With a frequency of 1:500–2,500, cleft lip with or without cleft palate (CL/P) is the most common congenital craniofacial defect. In mice and humans, a complex gene regulatory network (GRN) controls the different processes required for palatogenesis. Understanding how transcription factors act as the modulators of gene expression and function in palatogenesis will lead to better genetic counseling and genetic therapies for CL/P. Our group has shown that conditionally inactivating Sox2, one of the earliest transcription factors required during embryogenesis, results in clefting of the palate and fusion of the tongue to the mandible (ankylglossia).ObjectiveTo examine the role of Sox2, a stem cell factor, in palatogenesis.MethodsUsing Pitx2cre, Sox2 was conditionally inactivated in the oral epithelial tissue of developing murine embryos. Palate development was studied during the stages of embryogenesis.ResultsThe conditional ablation of Sox2 was sufficient to generate clefting in mice. Further examination revealed that Sox2 is expressed in the P63+ basal cell population of the oral epithelium as well as the periderm, which acts to prevent oral adhesions during development. Inactivating Sox2 resulted in defective periderm formation. The loss of an intact periderm resulted in intraoral adhesions that prevented complete palate shelf extension and ankylglossia.ConclusionsSox2 is required for periderm differentiation. Inactivating Sox2 results in cleft palate and ankylglossia.Support or Funding InformationUniversity of Iowa College of DentistryNIH, NIDCRThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exaggerated BMP4 signalling alters human airway basal progenitor celldifferentiation to cigarette smoking-related phenotypes.

Airway remodelling in chronic obstructive pulmonary disease (COPD) originates, in part, from smoking-induced changes in airway basal stem/progenitor cells (BCs). Based on the knowledge that bone morphogenetic protein 4 (BMP4) influences epithelial progenitor function in the developing and adult mouse lung, we hypothesised that BMP4 signalling may regulate the biology of adult human airway BCs relevant to COPD.BMP4 signalling components in human airway epithelium were analysed at the mRNA and protein levels, and the differentiation of BCs was assessed using the BC expansion and air-liquid interface models in the absence/presence of BMP4, BMP receptor inhibitor and/or small interfering RNAs against BMP receptors and downstream signalling.The data demonstrate that in cigarette smokers, BMP4 is upregulated in ciliated and intermediate undifferentiated cells, and expression of the BMP4 receptor BMPR1A is enriched in BCs. BMP4 induced BCs to acquire a smoking-related abnormal phenotype in vitro mediated by BMPR1A/Smad signalling, characterised by decreased capacity to differentiate into normal mucociliary epithelium, while generating squamous metaplasia.Exaggerated BMP4 signalling promotes cigarette smoking-relevant airway epithelial remodelling by inducing abnormal phenotypes in human airway BCs. Targeting of BMP4 signalling in airway BCs may represent a novel target to prevent/treat COPD-associated airway disease.

LEADeR role of miR-205 host gene as long noncoding RNA in prostate basal cell differentiation

Though miR-205 function has been largely characterized, the nature of its host gene, MIR205HG, is still completely unknown. Here, we show that only lowly expressed alternatively spliced MIR205HG transcripts act as de facto pri-miRNAs, through a process that involves Drosha to prevent unfavorable splicing and directly mediate miR-205 excision. Notably, MIR205HG-specific processed transcripts revealed to be functional per se as nuclear long noncoding RNA capable of regulating differentiation of human prostate basal cells through control of the interferon pathway. At molecular level, MIR205HG directly binds the promoters of its target genes, which have an Alu element in proximity of the Interferon-Regulatory Factor (IRF) binding site, and represses their transcription likely buffering IRF1 activity, with the ultimate effect of preventing luminal differentiation. As MIR205HG functions autonomously from (albeit complementing) miR-205 in preserving the basal identity of prostate epithelial cells, it warrants reannotation as LEADeR (Long Epithelial Alu-interacting Differentiation-related RNA).

DIAGNOSTIC INFORMATIVITY OF IMMUNOHISTOCHEMICAL BIOMARKERS IN CONDUCTING PRIVATIC PATHOLOGICAL AND ANATOMICAL RESEARCH OF PROSTATE CANCER

The results of an in vivo pathoanatomical differential diagnosis of primary acinar adenocarcinoma of the prostate and benign lesions of the prostate gland were analyzed using a transrectal biopsy of the prostate gland using an optimized panel of antibodies to high-molecular cytokeratins (clone 34E12), protein P63, AMACR, PSA and/or PSRA. The greatest diagnostic informativity of the basal cell differentiation marker of the columnar prostate epithelial cells was shown. The use of this set of biomarkers contributes to the establishment of a correct diagnosis and, in addition to the routine morphological examination of the biopsy material, can be recommended for carrying out an intravital pathologic anatomic differential diagnosis of prostate cancer.

Hippo signaling promotes lung epithelial lineage commitment by curbing Fgf10 and β-catenin signaling.

Organ growth and tissue homeostasis rely on the proliferation and differentiation of progenitor cell populations. In the developing lung, localized Fgf10 expression maintains distal Sox9-expressing epithelial progenitors and promotes basal cell differentiation in the cartilaginous airways. Mesenchymal Fgf10 expression is induced by Wnt signaling but inhibited by Shh signaling, and epithelial Fgf10 signaling activates β-catenin signaling. The Hippo pathway is a well-conserved signaling cascade that regulates organ size and stem/progenitor cell behavior. Here, we show that Hippo signaling promotes lineage commitment of lung epithelial progenitors by curbing Fgf10 and β-catenin signaling. Our findings show that both inactivation of the Hippo pathway (nuclear Yap) or ablation of Yap result in increased β-catenin and Fgf10 signaling, suggesting a cytoplasmic role for Yap in epithelial lineage commitment. We further demonstrate redundant and non-redundant functions for the two nuclear effectors of the Hippo pathway, Yap and Taz, during lung development.

SIS-ECM Laden with GMSC-Derived Exosomes Promote Taste Bud Regeneration

Oral cancer has a high annual incidence rate all over the world, and the tongue is the most frequently affected anatomic structure. The current standard care is ablative surgery of malignant neoplasm, followed by tongue reconstruction with free flap. However, such reconstructive modalities with postsurgery radiotherapy or chemotherapy can hardly support the functional recovery of the tongue—particularly, functional taste bud regeneration—in reconstructed areas, thus seriously affecting patients’ prognosis and life quality. Using a critical-sized tongue defect model in rats, we show that combinatory transplantation of small intestinal submucosa–extracellular matrix (SIS-ECM) with gingival mesenchymal stem cells (GMSCs) or their derivative exosomes promoted tongue lingual papillae recovery and taste bud regeneration as evidenced by increased expression of CK14, CK8, and markers for type I, II, and III taste bud cells (NTPdase 2, PLC-β2, and AADC, respectively). In addition, our results indicate that GMSCs or their derivative exosomes could increase BDNF expression, a growth factor that plays an important role in the proliferation and differentiation of epithelial basal progenitor cells into taste bud cells. Meanwhile, we showed an elevated expression level of Shh—which is essential for development, homeostasis, and maintenance of the taste bud organ—in wounded areas of the tongue among animals treated with GMSC/SIS-ECM or exosome/SIS-ECM as compared with SIS-ECM control. Moreover, our data show that GMSCs or their derivative exosomes promoted innervation of regenerated taste buds, as evidenced by elevated expressions of neurofilament and P2X3 at the injury areas. Together, our findings indicate that GMSC/SIS-ECM and exosome/SIS-ECM constructs can facilitate taste bud regeneration and reinnervation with promising potential application in postsurgery tongue reconstruction of patients with tongue cancer.

MEG3 is increased in idiopathic pulmonary fibrosis and regulates epithelial cell differentiation

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease causing fibrotic remodeling of the peripheral lung, leading to respiratory failure. Peripheral pulmonary epithelial cells lose normal alveolar epithelial gene expression patterns and variably express genes associated with diverse conducting airway epithelial cells, including basal cells. Single-cell RNA sequencing of pulmonary epithelial cells isolated from IPF lung tissue demonstrated altered expression of LncRNAs, including increased MEG3. MEG3 RNA was highly expressed in subsets of the atypical IPF epithelial cells and correlated with conducting airway epithelial gene expression patterns. Expression of MEG3 in human pulmonary epithelial cell lines increased basal cell-associated RNAs, including TP63, KRT14, STAT3, and YAP1, and enhanced cell migration, consistent with a role for MEG3 in regulating basal cell identity. MEG3 reduced expression of TP73, SOX2, and Notch-associated RNAs HES1 and HEY1, in primary human bronchial epithelial cells, demonstrating a role for MEG3 in the inhibition of genes influencing basal cell differentiation into club, ciliated, or goblet cells. MEG3 induced basal cell genes and suppressed genes associated with terminal differentiation of airway cells, supporting a role for MEG3 in regulation of basal progenitor cell functions, which may contribute to tissue remodeling in IPF.

AB011. The regulatory effects of androgen in wound healing of the prostatic urethra after thulium laser resection of the prostate

BackgroundThis research aims to determine the role of androgen in wound healing of the prostatic urethra after thulium laser resection of the prostate (TmLRP). Further, the change of basal cell proliferation and differentiation as well as macrophage polarization and the inflammatory response influenced by androgen were explored. Moreover, the macrophage cytokines effected by finasteride were detected in urine specimens from thulium laser prostatectomy patients, thus to reveal the mechanism of androgen in wound repair process after prostatectomy.Methods(I) Twenty-four beagles that received TmLRPs were randomly distributed into a castration group, a testosterone undecanoate (TU) group and a control group. The prostate wound was assessed once a week using a cystoscope. Histological analysis was then carried out to study the re-epithelialization of the prostatic urethra in each group; (II) immunohistochemical and immunofluorescence techniques were used to determine the basal cell proliferation and differentiation influenced by androgen; (III) the macrophage polarization and inflammatory response in the wound tissue and urine was also investigated; (IV) the macrophage cytokines effected by finasteride were detected in urine specimens from thulium laser prostatectomy patients by enzyme-linked immunosorbent assay (ELISA).Results(I) The healing of the prostatic urethra after a TmLRP was more rapid in the castration group and slower in the TU group than that in the control group; (II) castration accelerated re-epithelialization by promoting basal cell proliferation in the wound surface and beneath the wound and by accelerating the differentiation of basal cells into urothelial cells; (III) castration reduced the duration of the inflammatory phase and induced the conversion of M1 macrophages to M2 macrophages, thus accelerating the maturation of the wound. By contrast, androgen supplementation enhanced the inflammatory response and prolonged the inflammatory phase. Moreover, the anti-inflammatory phase was delayed and weakened. TNF-α, IL-6 and IL-12 that secreted by M1 were higher than the other two groups, and IL-10 and TGF-1 that secreted by M2 were obviously postponed compared with the other two groups; (IV) in the urine specimens from thulium laser prostatectomy patients, the expression of TNF-α, IL-6 and IL-12 were lower while the expression of IL-10 and TGF-1 were higher under the influence of finasteride.Conclusions(I) Androgen deprivation promotes re-epithelialization of the wound, regulates the inflammatory response, and accelerates wound healing of the prostatic urethra after a TmLRP; (II) castration accelerates re-epithelialization by promoting basal cell proliferation in the wound surface and beneath the wound and by accelerating the differentiation of basal cells into urothelial cells; (III) androgen inhibits the prostatic urethra wound healing by regulating the macrophage polarization; (IV) this research established foundation to reveal the mechanism and application of anti-androgen in the treatment of wound healing after prostatectomy.

Effects of prostaglandin E2 on gap junction protein alpha 1 in the rat epididymis.

Gap junctions are responsible for intercellular communication. In the adult mammalian epididymis, gap junction protein alpha 1 (GJA1) is localized between basal and either principal or clear cells. GJA1 levels and localization change during the differentiation of basal cells. The present objective was to determine the role of basal cells and prostaglandin E2 (PGE2) on GJA1 in the rat epididymis. Prior to basal cell differentiation, GJA1 is colocalized with TJP1 at the apical lateral margins between adjacent epithelial cells. When basal cells are present, GJA1 becomes associated between basal and principal cells, where it is primarily immunolocalized until adulthood. Basal cells express TP63, differentiate from epithelial cells, and produce prostaglandin-endoperoxide synthase 1 by 21 days of age. Prior to day 21, GJA1and TP63 are not strongly associated at the apical region. However, by day 28, TP63-positive basal cells migrate to the base of the epithelium, and also express GJA1. To assess effects of PGE2 on GJA1, rat caput epididymal (RCE) cells were exposed to PGE2 (50μM) for 3h. PGE2 increased levels of Gja1 mRNA in RCE cells, while levels of Gjb1, Gjb2, Gjb4, and GjB5 were unaltered. Furthermore, PGE2 increased protein levels of GJA1, phospho-GJA1, phospho-AKT, CTNNB1, and phospho-CTNNB1. Total AKT and the tight junction protein claudin1 were also not altered by PGE2. Data suggest that development of the epididymal epithelium and differentiation of epididymal basal cells regulate the targeting of GJA1, and that this appears to be mediated by PGE2.

Stromal Gli signaling regulates the activity and differentiation of prostate stem and progenitor cells

Interactions between cells in the stroma and epithelium facilitate prostate stem cell activity and tissue regeneration capacity. Numerous molecular signal transduction pathways, including the induction of sonic hedgehog (Shh) to activate the Gli transcription factors, are known to mediate the cross-talk of these two cellular compartments. However, the details of how these signaling pathways regulate prostate stem and progenitor cell activity remain elusive. Here we demonstrate that, although cell-autonomous epithelial Shh-Gli signaling is essential to determine the expression levels of basal cell markers and the renewal potential of epithelial stem and progenitor cells, stromal Gli signaling regulates prostate stem and progenitor cell activity by increasing the number and size of prostate spheroids in vitro Blockade of stromal Gli signaling also inhibited prostate tissue regeneration in vivo The inhibition of stromal Gli signaling suppressed the differentiation of basal and progenitor cells to luminal cells and limited prostate tubule secretory capability. Additionally, stromal cells were able to compensate for the deficiency of epithelial Shh signaling in prostate tissue regeneration. Mechanistically, suppression of Gli signaling increased the signaling factor transforming growth factor β (TGFβ) in stromal cells. Elevation of exogenous TGFβ1 levels inhibited prostate spheroid formation, suggesting that a stromal Gli-TGFβ signaling axis regulates the activity of epithelial progenitor cells. Our study illustrates that Gli signaling regulates epithelial stem cell activity and renewal potential in both epithelial and stromal compartments.

CLINICAL AND PATHOMORPHOLOGICAL MANIFESTATIONS OF RESPIRATORY SYNCYTIAL VIRUS INFECTION. CASE REPORT

On the example of clinical observation of severe respiratory syncytial virus (RSV) infection, which occurred with the development of a specific pneumonia complicated by acute respiratory distress-syndrome and disseminated intravascular coagulation syndrome, which caused a lethal outcome in the non-term (33-34 weeks gestation) child of two months of life, the clinical and pathomorphological features of the disease are shown. Immunomorphologic study after death allowed to demonstrate the most important stages of respiratory syncytial viral infection pathogenesis and to specify reasons of long-term airway obstruction, caused by complicated respiratory syncytial viral infection. It was indicated, that bronchial obstruction may be caused by qualitative changes of epithelial lumen of different bronchi, such as direct cytophatic effect of respiratory virus or reactive changes of lumen in the form of basal squamous cell differentiation of epithelial lumen and poor regeneration of respiratory epithelium in the later stages of the disease.

Expansion of Airway Basal Cells and Generation of Polarized Epithelium.

Airway basal stem cells are the progenitor cells within the airway that exhibit the capacity to self-renew and give rise to multiple types of differentiated airway epithelial cells. This stem cell-derived epithelium displays organized architecture with functional attributes of the airway mucosa. A protocol has been developed to culture and expand human airway basal stem cells while preserving their stem cell properties and capacity for subsequent mucociliary differentiation. This achievement presents a previously unrealized opportunity to maintain a durable supply of progenitor cells derived from healthy donors to differentiate into human primary airway epithelium for cellular and molecular-based studies. Further, basal stem cells can be harvested from patients with a specific airway disease, such as cystic fibrosis, enabling investigation of potentially altered behavior of disease-specific cells in the appropriate context of the airway mucosa. Here we describe, in detail, a protocol for the serial expansion of airway basal stem cells to enable the generation of nearly unlimited airway basal cells that can be stored and readily available for subsequent culturing and differentiation. In addition, we describe culturing and differentiation of airway basal stem cells on permeable transwell filters at air-liquid interface to create functional mucociliary pseudostratified polarized airway epithelial mucosa.

AB029. The regulatory effects of androgen in wound healing of the prostatic urethra after thulium laser resection of the prostate

BackgroundThis research aims to determine the role of androgen in wound healing of the prostatic urethra after thulium laser resection of the prostate (TmLRP). Further, the change of basal cell proliferation and differentiation as well as macrophage polarization and the inflammatory response influenced by androgen were explored. Moreover, the macrophage cytokines effected by finasteride were detected in urine specimens from thulium laser prostatectomy patients, thus to reveal the mechanism of androgen in wound repair process after prostatectomy.Methods(I) Twenty-four beagles that received TmLRPs were randomly distributed into a castration group, a testosterone undecanoate (TU) group and a control group. The prostate wound was assessed once a week using a cystoscope. Histological analysis was then carried out to study the re-epithelialization of the prostatic urethra in each group; (II) immunohistochemical and immunofluorescence techniques were used to determine the basal cell proliferation and differentiation influenced by androgen; (III) the macrophage polarization and inflammatory response in the wound tissue and urine was also investigated; (IV) the macrophage cytokines effected by finasteride were detected in urine specimens from thulium laser prostatectomy patients by ELISA.Results(I) The healing of the prostatic urethra after a TmLRP was more rapid in the castration group and slower in the TU group than that in the control group; (II) castration accelerated re-epithelialization by promoting basal cell proliferation in the wound surface and beneath the wound and by accelerating the differentiation of basal cells into urothelial cells; (III) castration reduced the duration of the inflammatory phase and induced the conversion of M1 macrophages to M2 macrophages, thus accelerating the maturation of the wound. By contrast, androgen supplementation enhanced the inflammatory response and prolonged the inflammatory phase. Moreover, the anti-inflammatory phase was delayed and weakened. TNF-α, IL-6 and IL-12 that secreted by M1 were higher than the other two groups, and IL-10 and TGF-β1 that secreted by M2 were obviously postponed compared with the other two groups. 4. In the urine specimens from thulium laser prostatectomy patients, the expression of TNF-α, IL-6 and IL-12 were lower while the expression of IL-10 and TGF-β1 were higher under the influence of finasteride.Conclusions(I) Androgen deprivation promotes re-epithelialization of the wound, regulates the inflammatory response, and accelerates wound healing of the prostatic urethra after a TmLRP; (II) castration accelerates re-epithelialization by promoting basal cell proliferation in the wound surface and beneath the wound and by accelerating the differentiation of basal cells into urothelial cells; (III) androgen inhibits the prostatic urethra wound healing by regulating the macrophage polarization; (IV) this research established foundation to reveal the mechanism and application of anti-androgen in the treatment of wound healing after prostatectomy.

Ectopic expression of the transcription factor MafB in basal keratinocytes induces hyperproliferation and perturbs epidermal homeostasis.

Mammalian epidermis is composed of four morphologically and functionally distinct layers of keratinocytes. The innermost basal layer consists of proliferating self-renewing keratinocytes, which also undergo asymmetric cell division to differentiate into postmitotic suprabasal cells throughout life. Control of the balance between growth and differentiation of basal cells is important for epidermal homeostasis to prevent skin disorders including malignancies; however, the underlying mechanism remains to be elucidated. Recently, MafB was identified as one of the transcription factors that regulate epidermal keratinocyte differentiation. MafB is expressed in postmitotic differentiating keratinocytes, and epidermal differentiation is partially impaired in MafB-deficient mice. To further establish the roles of MafB in the epidermis in vivo, we generated mice transgenic for MafB under the control of the basal cell-specific keratin (Krt) 14 promoter. In the epidermis of transgenic mice at embryonic day 18.5, the number of proliferating Krt14-positive basal-like cells was increased, and the granular and cornified layers were thickened. Furthermore, these MafB transgenic mice developed papillomas spontaneously with age. Therefore, MafB promotes differentiation in postmitotic keratinocytes and simultaneously has potential to promote growth when ectopically expressed in undifferentiated basal keratinocytes.

Essential roles of Tbx3 in embryonic skin development during epidermal stratification.

Stepwise differentiation of epidermal cells is essential for development of stratified epithelium, but the underlying mechanisms remain unclear. Here, we show that Tbx3, a member of the T-box family of transcription factors, plays a pivotal role in this mechanism. Tbx3 is expressed in both basal and suprabasal cells in the interfollicular epidermis of mouse embryos. Epidermis-specific Tbx3 conditional knockout (cKO) embryos are small in size and display a thinner epidermis with an impaired barrier function. In the Tbx3 cKO epidermis, keratin 5-positive undifferentiated cells, which reside in both basal and suprabasal layers of wild-type embryos, are localized exclusively in the basal layer. In addition, mRNA expression levels of granular cell markers are increased in the Tbx3 cKO epidermis, suggesting that Tbx3 prevents premature differentiation of spinous cells. We further show that Tbx3 maintains the proliferative potential of basal cells and ensures their planar-oriented cell division. Moreover, Tbx3 is shown to be required for the expression of Hes1, a well-known Notch signaling target protein that is essential for epidermal development. We therefore propose that Tbx3 functions upstream of Hes1 to regulate proliferation and differentiation of basal and suprabasal cells during epidermal development.

Abstract P6-08-08: GATA3 inhibits breast basal-like tumorigenesis

Abstract BACKGROUND: Breast cancer can be broadly categorized into two groups depending on the cell type affected. Luminal-type tumors are typically estrogen receptor (ER) positive that are associated with better survival and respond to hormone therapies whereas basal-like tumors are ER negative, more aggressive, and associated with a poor prognosis. GATA3 is a transcription factor well studied for its role as a master regulator of cellular differentiation and stem cell self renewal. Loss of Gata3 in mouse mammary glands blocks luminal cell differentiation and induces growth defects, and low levels of GATA3 are associated with basal-like and metastatic human breast cancers with epithelial-to-mesenchymal transition (EMT). Importantly, luminal cells have been shown to be the origin of some basal-like breast cancers. Due to the proliferation defects caused by GATA3 deficiency, it remains elusive how loss of function of GATA3 contributes to breast cancers development and progression. METHODS: We previously demonstrated that p18Ink4c (p18), a cell cycle inhibitor, is a downstream target of GATA3 in regulating mammary luminal cell proliferation and loss of p18 leads to luminal type tumorigenesis. To test the role of Gata3 deficiency in tumorigenesis, we generated p18-/-;Gata3+/- mice. Mammary gland development and tumorigenesis were characterized in vivo using a panel of cellular and molecular assays. Results were further confirmed in vitro with well established cell lines. RESULTS: Loss of p18 rescued mammary growth defects caused by Gata3 heterozygosity. Gata3 heterozygosity impaired luminal, but promoted basal gene expression in mammary epithelial cells. Gata3 heterozygosity in p18 null mice accelerated spontaneous mammary tumorigenesis, reducing the average latency of tumor onset. More importantly, Gata3 heterozygosity transformed the luminal type tumors of p18 null mice into heterogeneous basal-like breast cancers with activated EMT. Conversely, reintroduction of GATA3 inhibited tumor growth and reduced expression of EMT markers in basal-like tumor xenografts. We discovered that expression of GATA3 and Vimentin, an EMT marker, is inversely related in human breast cancers. CONCLUSION: Our data indicates that GATA3 promotes luminal but suppresses basal cell differentiation in the mammary gland and in tumor development. Mechanisms underlying the role of GATA3 in suppressing basal-like tumor development are under investigation.BACKGROUND: Breast cancer can be broadly categorized into two groups depending on the cell type affected. Luminal-type tumors are typically estrogen receptor (ER) positive that are associated with better survival and respond to hormone therapies whereas basal-like tumors are ER negative, more aggressive, and associated with a poor prognosis. GATA3 is a transcription factor well studied for its role as a master regulator of cellular differentiation and stem cell self renewal. Loss of Gata3 in mouse mammary glands blocks luminal cell differentiation and induces growth defects, and low levels of GATA3 are associated with basal-like and metastatic human breast cancers with epithelial-to-mesenchymal transition (EMT). Importantly, luminal cells have been shown to be the origin of some basal-like breast cancers. Due to the proliferation defects caused by GATA3 deficiency, it remains elusive how loss of function of GATA3 contributes to breast cancers development and progression. METHODS: We previously demonstrated that p18Ink4c (p18), a cell cycle inhibitor, is a downstream target of GATA3 in regulating mammary luminal cell proliferation and loss of p18 leads to luminal type tumorigenesis. To test the role of Gata3 deficiency in tumorigenesis, we generated p18-/-;Gata3+/- mice. Mammary gland development and tumorigenesis were characterized in vivo using a panel of cellular and molecular assays. Results were further confirmed in vitro with well established cell lines. RESULTS: Loss of p18 rescued mammary growth defects caused by Gata3 heterozygosity. Gata3 heterozygosity impaired luminal, but promoted basal gene expression in mammary epithelial cells. Gata3 heterozygosity in p18 null mice accelerated spontaneous mammary tumorigenesis, reducing the average latency of tumor onset. More importantly, Gata3 heterozygosity transformed the luminal type tumors of p18 null mice into heterogeneous basal-like breast cancers with activated EMT. Conversely, reintroduction of GATA3 inhibited tumor growth and reduced expression of EMT markers in basal-like tumor xenografts. We discovered that expression of GATA3 and Vimentin, an EMT marker, is inversely related in human breast cancers. CONCLUSION: Our data indicates that GATA3 promotes luminal but suppresses basal cell differentiation in the mammary gland and in tumor development. Mechanisms underlying the role of GATA3 in suppressing basal-like tumor development are under investigation. Citation Format: Pei X-H, Chan HL, Liu S, Scott A, Pimentel E, Slingerland J, Robbins D, Capobianco A, Bai F. GATA3 inhibits breast basal-like tumorigenesis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-08.