Search

Overexpression of nectin cell adhesion protein 4 correlates with cancer progression and poor prognosis in many human malignancies. Enfortumab vedotin (EV) is the first nectin-4-targeting antibody drug conjugate approved by the US Food and Drug Administration for treatment of urothelial cancer. However, inadequate efficacy has limited progress in the treatment of other solid tumors with EV. Furthermore, ocular, pulmonary, and hematological toxic side effects are common in nectin-4-targeted therapy, which frequently results in dose reduction and/or treatment termination. Thus, we designed a second generation nectin-4-specific drug, 9MW2821, based on interchain-disulfide drug conjugate technology. This novel drug contained a site-specifically conjugated humanized antibody and the cytotoxic moiety monomethyl auristatin E. The homogenous drug-antibody ratio and novel linker chemistry of 9MW2821 increased the stability of conjugate in the systemic circulation, enabling highly efficient drug delivery and avoiding off-target toxicity. In preclinical evaluation, 9MW2821 exhibited nectin-4 specific cell binding, efficient internalization, bystander killing, and equivalent or superior antitumor activity compared with EV in both cell-line-derived xenograft and patient-derived xenograft models. In addition, 9MW2821 demonstrated a favorable safety profile; the highest non-severely toxic dose in monkey toxicological studies was 6 mg/kg, with milder adverse events compare to EV. Overall, 9MW2821 is a nectin-4-directed, investigational antibody-drug conjugate based on innovative technology that endowed the drug with compelling preclinical antitumor activity and a favorable therapeutic index. The 9MW2821 antibody-drug conjugate is being investigated in a Phase I/II clinical trial (NCT05216965) in patients with advanced solid tumors.

Objective: Inbreeding depression of reproduction is a major concern in the conservation of native chicken genetic resources. Here, based on the successful development of strongly inbred (Sinb) and weakly inbred (Winb) Langshan chickens, we aimed to evaluate inbreeding effects on reproductive traits and identify candidate genes involved in inbreeding depression of reproduction in Langshan chickens.Methods: A two-sample t-test was performed to estimate the differences in phenotypic values of reproductive traits between Sinb and Winb chicken groups. Three healthy chickens with reproductive trait values around the group mean values were selected from each of the groups. Differences in ovarian and hypothalamus transcriptomes between the two groups of chickens were analyzed by RNA sequencing (RNA-Seq).Results: The Sinb chicken group showed an obvious inbreeding depression in reproduction, especially for traits of age at the first egg and egg number at 300 days (p

Simple Summary Osteosarcoma is the predominant form of primary bone cancer and outcomes for patients with metastatic disease have not improved for several decades. Recent genomic and pharmacological studies have implicated dysregulated histone deacetylases as druggable targets to treat several types of cancers, including osteosarcoma. This study aimed to assess the inhibitory effects of 4SC-202, a next-generation inhibitor for class I histone deacetylases, on human osteosarcoma cell growth in vitro and in vivo. We demonstrated that the anti-tumor effect of 4SC-202 involves combined cell-cycle arrest and apoptosis, as well as a reduction in cell invasion and migration. Moreover, 4SC-202 revised the global transcriptome and induced distinct signatures of gene expression in vitro. Additionally, 4SC-202 decreased tumor growth of established human tumor xenografts in immunodeficient mice in vivo. Our study suggests that 4SC-202 may be exploited as a valuable drug to promote more effective treatment of patients. Abstract Dysregulation of histone deacetylases (HDACs) is associated with the pathogenesis of human osteosarcoma, which may present an epigenetic vulnerability as well as a therapeutic target. Domatinostat (4SC-202) is a next-generation class I HDAC inhibitor that is currently being used in clinical research for certain cancers, but its impact on human osteosarcoma has yet to be explored. In this study, we report that 4SC-202 inhibits osteosarcoma cell growth in vitro and in vivo. By analyzing cell function in vitro, we show that the anti-tumor effect of 4SC-202 involves the combined induction of cell-cycle arrest at the G2/M phase and apoptotic program, as well as a reduction in cell invasion and migration capabilities. We also found that 4SC-202 has little capacity to promote osteogenic differentiation. Remarkably, 4SC-202 revised the global transcriptome and induced distinct signatures of gene expression in vitro. Moreover, 4SC-202 decreased tumor growth of established human tumor xenografts in immunodeficient mice in vivo. We further reveal key targets regulated by 4SC-202 that contribute to tumor cell growth and survival, and canonical signaling pathways associated with progression and metastasis of osteosarcoma. Our study suggests that 4SC-202 may be exploited as a valuable drug to promote more effective treatment of patients with osteosarcoma and provide molecular insights into the mechanism of action of class I HDAC inhibitors.

Background. Primary dysmenorrhea (PD) is the commonest gynecological disorder in young women of reproductive age, and there is not always satisfactory relief of pain treated by common medications. Therefore, acupuncture has been used as an alternative therapy to relieve the symptoms of PD. In clinical practice, a penetrating method of acupuncture with long needle has been shown to be particularly effective for improving primary dysmenorrhea. This study was conducted to evaluate the effectiveness of this technique for pain relief in patients with primary dysmenorrhea as compared with a conventional pain medication. Methods. The present study is a perspective, randomized, ibuprofen-controlled trial. Sixty-two eligible participants were randomly assigned in a 1 : 1 ratio to receive either acupuncture treatment or ibuprofen administration. The treatment lasted for three menstrual cycles for both groups. The primary outcome was the intensity of menstrual pain measured by using the visual analogue scale at the completion of treatment. Secondary outcomes included the severity of symptoms associated with menstrual pain, responder rate, and safety of acupuncture treatment. The clinical outcomes were measured on each menstrual cycle at baseline, treatment course (3 cycles), and follow-up period. Results. Sixty-four patients of primary dysmenorrhea were recruited, and 62 subjects were included in the final analysis. At trial completion, acupuncture was shown to be associated with a significantly lower pain intensity and decreased symptom severity of primary dysmenorrhea as compared with ibuprofen (p<0.05). A significantly higher responder rate was found in the acupuncture group as compared with the control group (p<0.05). No serious adverse events were reported by patients in either group. Conclusions. The penetrating method of acupuncture with long needle may be an effective and safe therapy for pain relief in patients with primary dysmenorrhea. This trial was registered with the Chinese Clinical Trial Registry (ChiCTR-IOR-17012621).

Distinct members of the Ets family of transcription factors act as positive or negative regulators of genes involved in cellular proliferation, development, and tumorigenesis. In human lung cancer, increased ETS1 expression is associated with poor prognosis and metastasis. We tested whether ETS1 contributes to lung tumorigenesis by binding to Twist1, a gene involved in tumor cell motility and dissemination. We used a mouse lung cancer model with metastasis driven by conditionally activated Kras and concurrent tumor suppressor Lkb1 loss (KrasG12D/ Lkb1−/− model) and a similar model of lung cancer that does not metastasize, driven by conditionally activated Kras alone (KrasG12D model). We show that Ets1 and Twist1 gene expression differs between KrasG12D tumors (low Ets1 and Twist1 expression) and KrasG12D/Lkb1−/− tumors (high Ets1 and Twist1 expression). In human lung tumors, ETS1 and TWIST1 expression positively correlates and low combined ETS1 and TWIST1 levels are associated with improved survival compared to high levels. Using mouse cell lines derived from KrasG12D and KrasG12D/Lkb1−/− mouse models and the human lung cancer (A549) cell line, we show that ETS1 regulates Twist1 expression. Chromatin immunoprecipitation assays confirm binding of ETS1 to the Twist1 promoter. Overexpression studies show that ETS1 transactivates Twist1 promoter activity in mouse and human cells. Silencing endogenous Ets1 by siRNA in mouse cell lines decreases Twist1 mRNA levels, decreases invasion, and increases cell growth. Ets1 and Twist1 are at the crossroad of several signaling pathways in cancer. Understanding their regulation may inform the development of therapies to impair lung tumor metastasis.

The study investigates the relationship between air pollution and the status of human mental health using the data of the China Migrant Dynamic Survey (CMDS), 2014. Various econometric methods were used. Particularly, the ordinary least square (OLS) was used to estimate the parametric impact of mental health. Moreover, a robustness test was used to estimate negative impact of air pollution on mental health. Results found that air pollution significantly increased psychological disorder. The concentration of PM2.5 in the air leads to higher prominence of the four negative emotions such as nervous, depression, powerless, and restless or fidgety. After introducing the instrumental variable to solve the endogenous problem, the impact of air pollution on the level of the mental illnesses was still significant and negative. Results are also evident that air pollution has heterogeneity to the negative status of residents’ mental health. Specifically, it has a significant and negative impact on women, rural and urban population, and middle- and low-income groups. The study results suggest that the government must have to revise the environmental regulation policy reduce the negative impact on human health.

The long-lasting phosphorescent glass with the composition of ZnO–P2O5–SiO2: Tb3+ was prepared by melt-quenched method. After irradiation by 254 nm light, a visible greenish light can be observed with the naked eyes, in the dark for up to 9 h after removal of the activating light. The luminescent properties of ZnO–P2O5–SiO2: Tb3+ were studied by fluorescence spectra, afterglow spectra and thermoluminescence spectra. The results show that the luminescent properties of the glasses become better with the increase of ZnO contents. The luminescent properties of glasses increase with the increase of Tb3+ contents. When 2% of ZnO is replaced by 2% of RO (RO=CaO, SrO, BaO), the luminescent property of glasses become worse in the order of CaO, SrO, BaO. The higher the contents of RO (RO=CaO, SrO, BaO) are, the worse the luminescent properties of the glasses become.

In recent years, air pollution in China has posed serious threats to human health. In this study, we investigate the relationship between air pollution and residents’ health by nesting the household registration data of the China Migrant Dynamic Survey in 2014 with city characteristic data and pollution data. The results indicated that an increase in the concentration of air pollution significantly reduced residents’ health levels. After using the instrumental variable (IV)-Oprobit model to solve endogenous problem, we have found that the negative impact of air pollution on residents’ health remained significant. Moreover, the lag term of environmental indexes was introduced and found that air pollution significantly increased health risk. The results also demonstrated that the impact of air pollution on human health was heterogeneous. Men and urban residents were more sensitive to air pollution and more adversely affected. In addition to air pollution, the study found that, among the influencing factors at the individual level, males’ health was better than females’ health, and education positively impacted on residents’ health. The health effects were consistent with the theoretical predictions of the Grossman model. The financial expenditure and medical input were negatively correlated with residents’ health levels. Based on the results, we suggest the Chinese government should implement rigorous air pollution laws and regulations.

The function of most long noncoding RNAs (lncRNAs) is unknown. However, recent studies reveal important roles of lncRNAs in regulating cancer-related pathways. Human antisense lncRNA-NKX2-1-AS1 partially overlaps the NKX2-1/TTF1 gene within chromosomal region 14q13.3. Amplification of this region and/or differential expression of genes therein are associated with cancer progression. Herein we show higher levels of NKX2-AS1 and NKX2-1 in lung adenocarcinomas relative to non-tumor controls but no correlation between NKX2-1-AS1 and NKX2-1 levels across specimens, or with amplification of the 14q13.3 region, suggesting that NKX2-1-AS1 and NKX2-1 are independently regulated. Loss-and-gain of function experiments showed that NKX2-1-AS1 does not regulate NKX2-1 expression, or nearby genes, but controls genes in trans. Genes up-regulated by NKX2-1-AS1-knockdown belong to cell adhesion and PD-L1/PD-1 checkpoint pathways. NKX2-1-AS1 negatively regulates endogenous CD274/PD-L1, a known target of NKX2-1, and the transcriptional activity of -1kb-CD274 promoter-reporter construct. Furthermore, NKX2-1-AS1 interferes with NKX2-1 protein binding to the CD274-promoter, likely by NKX2-1 protein-NKX2-1-AS1 interactions. Finally, NKX2-1-AS1 negatively regulates cell migration and wound healing, but not proliferation or apoptosis. These findings support potential roles of NKX2-1-AS1 in limiting motility and immune system evasion of lung carcinoma cells, highlighting a novel mechanism that may influence tumorigenic capabilities of lung epithelial cells.

Distinct members of the Ets family of transcription factors act as positive or negative regulators of genes involved in cellular proliferation, development, and tumorigenesis. In human lung cancer, increased ETS1 expression is associated with poor prognosis and metastasis. We tested whether ETS1 contributes to lung tumorigenesis by binding to Twist1, a gene involved in tumor cell motility and dissemination. We used a mouse lung cancer model with metastasis driven by conditionally activated Kras and concurrent tumor suppressor Lkb1 loss (Kras

Concomitant ingestion of alcohol and cephalosporin may cause a disulfiram-like reaction; however its fatal outcomes are not commonly known. We retrospectively reviewed 78 patients who had cephalosporin induced disulfiram-like reaction (CIDLR). The patients who had a negative skin test to cephalosporin prior to intravenous antibiotics were included, and those who were allergic to either alcohol or antibiotics were excluded. The average age of 78 patients was 37.8±12.2 (21–60) years. Of the 78 patients, 93.58% of the patients were males, 70.51% of the patients consumed alcohol after use of antibiotics, and 29.49% patients consumed alcohol initially, followed by intravenous antibiotics; however, no significant difference of morbidity was observed in these two groups. All patients were administered antibiotics intravenously. Five of 78 patients (6.41%) developed severe CIDLR too urgently to be rescued successfully. In conclusion, it is important for clinicians to educate patients that no alcohol should be used if one is taking cephalosporin. Also, clinicians should keep in mind that cephalosporin should not be prescribed for any alcoholics.

NiCoCrAlY/Al2O3 and NiCoCrAlY/B4C composite powders were prepared with hydrogen reduction and solid state alloying process. NiCoCrAlY/Al2O3-B4C composite coatings with different contents of B4C were prepared by atmospheric plasma spray technology. The microstructures, mechanical properties, and tribological properties of the composite coatings with different B4C contents were systematically investigated. The results show that the microhardness of the composite coatings increases, while the tensile strength of the composite coatings decreases, with the increase of B4C contents. The wear volume of the composite coatings decreases from room temperature to 800 °C with the increase of B4C contents. Abrasion wear is the main wear mechanism of the NiCoCrAlY/Al2O3-B4C composite coating from room temperature to 800 °C.

SummaryTwo populations of Nkx2-1+ progenitors in the developing foregut endoderm give rise to the entire postnatal lung and thyroid epithelium, but little is known about these cells because they are difficult to isolate in a pure form. We demonstrate here the purification and directed differentiation of primordial lung and thyroid progenitors derived from mouse embryonic stem cells (ESCs). Inhibition of TGFβ and BMP signaling, followed by combinatorial stimulation of BMP and FGF signaling, can specify these cells efficiently from definitive endodermal precursors. When derived using Nkx2-1GFP knockin reporter ESCs, these progenitors can be purified for expansion in culture and have a transcriptome that overlaps with developing lung epithelium. Upon induction, they can express a broad repertoire of markers indicative of lung and thyroid lineages and can recellularize a 3D lung tissue scaffold. Thus, we have derived a pure population of progenitors able to recapitulate the developmental milestones of lung/thyroid development.

Hexagonal boron nitride was pressed and sintered at 2000 degrees C with CaB(2)O(4) as an additive to promote its crystallization, which was used as an abradable sealing coating for aircraft turbo engines. Microstructures, phase compositions and tribological properties of the sintered hBN were tested, and the results show that CaB(2)O(4) can effectively promote crystal growth of hBN at 2000 degrees C for 5 h in N(2) ambience. The friction coefficients of the sintered hBN under atmosphere ambience increase as the temperature increasing from room temperature to 400 degrees C, and then decrease with further increasing of temperature up to 800 degrees C. Under water vapor ambience, friction coefficients of the sintered hBN are much lower than those under atmosphere ambience, which are attributed to a lamella-slip of hBN and the solid lubrication effect of H(3)BO(3). (C) 2011 Elsevier B.V. All rights reserved.

Epigenetic regulation of transcription plays an important role in cell-specific gene expression by altering chromatin structure and access of transcriptional regulators to DNA binding sites. Surfactant protein B (Sftpb) is a developmentally regulated lung epithelial gene critical for lung function. Thyroid transcription factor 1 (Nkx2-1) regulates Sftpb gene expression in various species. We show that Nkx2-1 binds to the mouse Sftpb (mSftpb) promoter in the lung. In a mouse lung epithelial cell line (MLE-15), Nkx2-1 knockdown reduces Sftpb expression, and mutation of Nkx2-1 cis-elements significantly reduces mSftpb promoter activity. Whether chromatin structure modulates Nkx2-1 regulation of Sftpb transcription is unknown. We found that DNA methylation of the mSftpb promoter inversely correlates with known patterns of Sftpb expression in vivo. The mSftpb promoter activity can be manipulated by altering its cytosine methylation status in vitro. Nkx2-1 activation of the mSftpb promoter is impaired by DNA methylation. The unmethylated Sftpb promoter shows an active chromatin structure enriched in the histone modification H3K4me3 (histone 3-lysine 4 trimethylated). The ATP-dependent chromatin remodeling protein Brg1 is recruited to the Sftpb promoter in Sftpb-expressing, but not in non-expressing tissues and cell lines. Brg1 knockdown in MLE-15 cells greatly decreases H3K4me3 levels at the Sftpb promoter region and expression of the Sftpb gene. Brg1 can be co-immunoprecipitated with Nkx2-1 protein. Last, Nkx2-1 and Brg1 with intact ATPase activity are required for mSftpb promoter activation in vitro. Our findings suggest that DNA methylation and chromatin modifications cooperate with Nkx2-1 to regulate Sftpb gene cell specific expression.

Caveolin-1 protein has been called a ‘conditional tumor suppressor’ because it can either suppress or enhance tumor progression depending on cellular context. Caveolin-1 levels are dynamic in non-small-cell lung cancer, with increased levels in metastatic tumor cells. We have shown previously that transactivation of an erythroblastosis virus-transforming sequence (ETS) cis-element enhances caveolin-1 expression in a murine lung epithelial cell line. Based on high sequence homology between the murine and human caveolin-1 promoters, we proposed that ETS proteins might regulate caveolin-1 expression in human lung tumorigenesis. We confirm that caveolin-1 is not detected in well-differentiated primary lung tumors. Polyoma virus enhancer activator 3 (PEA3), a pro-metastatic ETS protein in breast cancer, is expressed at low levels in well-differentiated tumors and high levels in poorly differentiated tumors. Conversely, Net, a known ETS repressor, is expressed at high levels in the nucleus of well-differentiated primary tumor cells. In tumor cells in metastatic lymph node sites, caveolin-1 and PEA3 are highly expressed, whereas Net is now expressed in the cytoplasm. We studied transcriptional regulation of caveolin-1 in two human lung cancer cell lines, Calu-1 (high caveolin-1 expressing) and NCI-H23 (low caveolin-1 expressing). Chromatin immunoprecipitation-binding assays and small interfering RNA experiments show that PEA3 is a transcriptional activator in Calu-1 cells and that Net is a transcriptional repressor in NCI-H23 cells. These results suggest that Net may suppress caveolin-1 transcription in primary lung tumors and that PEA3 may activate caveolin-1 transcription in metastatic lymph nodes.

We previously identified an Ets cis-element in the mouse caveolin-1 promoter that is selectively activated in lung epithelial (E10), but not lung endothelial murine lung endothelial cell line (MFLM-4), cell lines and therefore appears important for differential, cell-specific caveolin-1 transcription. In the present study, we demonstrate that immunostaining of adult mouse lung detects the ETS protein Ets-related molecule (ERM PEA3) in distal lung epithelium in alveolar type I and II cells, but not in bronchial epithelium or lung endothelial cells. We tested ERM and polyomavirus enhancer activator 3 (PEA3) for their ability to increase endogenous caveolin-1 transcripts and to activate caveolin-1 promoter fragments containing the -865 Ets cis-element. Chromatin immunoprecipitation (ChIP) assays show that both ERM and PEA3 bind to the caveolin-1 promoter in murine E10, but not MFLM-4, cells. Normalized luciferase activities show that only ERM activates the caveolin-1 promoter in E10 cells, but neither protein enhances promoter activity in MFLM-4 cells. Mutation of the Ets site blocks ERM-mediated promoter activation in E10 cells. Furthermore, overexpression of ERM increases the cellular content of caveolin-1 mRNA and protein, in E10, but not MFLM-4, cells. The effects of PEA3 on the cellular content of endogenous caveolin-1 expression are variable. These results demonstrate that ERM is involved in caveolin-1 regulation in a murine lung epithelial, but not lung endothelial cell line. We conclude that transcriptional regulation of caveolin-1 differs markedly between lung epithelial and endothelial cell lines, perhaps explaining why the onset of caveolin-1 expression differs in epithelial and endothelial cells during lung development.

In the lung, caveolin-1 is expressed in both type I alveolar epithelial and endothelial cells where it is hypothesized to modulate molecular signaling activities and progression of tumorigenesis. Developmentally, caveolin-1α is expressed in fetal lung endothelial, but not epithelial, cells; in adult lung, both cell types express caveolin-1α. To test the hypothesis that caveolin-1 transcription is differentially regulated in type I and endothelial cells, we characterized the proximal promoter of the mouse caveolin-1 gene in lung cell lines to identify factors that control its cell-specific expression. We show that caveolin-1 expression is regulated by an Ets cis-element in a lung epithelial cell line, but not a lung endothelial cell line, and that three ETS family members, ETS-1, PEA3, and ERM, recognize and bind the Ets site in the epithelial cell line. Based on these findings, we have identified the Ets cis-element as a region that accounts for differential transcriptional regulation of caveolin-1 in lung epithelial and endothelial cells.

T1alpha, a differentiation gene of lung alveolar epithelial type I cells, is developmentally regulated and encodes an apical membrane protein of unknown function. Morphological differentiation of type I cells to form the air-blood barrier starts in the last few days of gestation and continues postnatally. Although T1alpha is expressed in the foregut endoderm before the lung buds, T1alpha mRNA and protein levels increase substantially in late fetuses when expression is restricted to alveolar type I cells. We generated T1alpha null mutant mice to study the role of T1alpha in lung development and differentiation and to gain insight into its potential function. Homozygous null mice die at birth of respiratory failure, and their lungs cannot be inflated to normal volumes. Distal lung morphology is altered. In the absence of T1alpha protein, type I cell differentiation is blocked, as indicated by smaller airspaces, many fewer attenuated type I cells, and reduced levels of aquaporin-5 mRNA and protein, a type I cell water channel. Abundant secreted surfactant in the narrowed airspaces, normal levels of surfactant protein mRNAs, and normal patterns and numbers of cells expressing surfactant protein-B suggest that differentiation of type II cells, also alveolar epithelial cells, is normal. Anomalous proliferation of the mesenchyme and epithelium at birth with unchanged numbers of apoptotic cells suggests that loss of T1alpha and/or abnormal morphogenesis of type I cells alter the proliferation rate of distal lung cells, probably by disruption of epithelial-mesenchymal signaling.

We identified rat developing arteries and neural crest derivatives with multiple epidermal growth factor-like domains ( DANCE) as a developmentally regulated gene using suppression-subtractive hybridization. Northern analysis confirmed a fivefold induction of this mRNA transcript between fetal day 18 and 20 that persisted through postnatal day 17. The level was declining at postnatal day 21 and was similar in adult lung to that at fetal day 18. In adults DANCE mRNA abundance was highest in lung, kidney, and spleen, lower in heart, skeletal muscle, and brain, but absent from liver and thymus. It was abundant in pulmonary artery endothelium and a lung epithelial type 2 cell line, barely detectable in vascular smooth muscle, and absent in fibroblasts. In situ hybridization revealed a regulated pattern of expression in endothelial cells of fetal, postnatal, and adult lung. Because DANCE mRNA was inducible in systemic arteries during recovery from injury, we searched for induction in lung injured by hyperoxia. Mouse DANCE mRNA abundance was unchanged during an acute 3-day exposure period, induced threefold 5 days into the recovery phase, and returned to baseline at days 8, 11, and 14. In situ hybridization at day 5 suggested a diffuse pattern of induction. DANCE may play a role in lung endothelial cell biology during development repair after injury.

SUMMARY Caveolin-1 is a scaffolding protein component of caveolae, membrane invaginations involved in endocytosis, signal transduction, trans- and intracellular trafficking, and protein sorting. In adult lung, caveolae and caveolin-1 are present in alveolar endothelium and Type I epithelial cells but rarely in Type II cells. We have analyzed patterns of caveolin-1 expression during mouse lung development. Two caveolin-1 mRNAs, full-length and a 5 � variant that will translate mainly into caveolin-1 � and – � isoforms, are detected by RT-PCR at embryonic day 12 (E12) and afterwards in the developing and adult lung. Immunostaining analysis, starting at E10, shows caveolin-1 � localized in primitive blood vessels of the forming lung, in an overlapping pattern to the endothelial marker PECAM-1, and later in all blood vessels. Caveolin-1 � is not detected in fetal or neonatal lung epithelium but is detected in adult epithelial Type I cells. Caveolin-1 was previously shown to be expressed in alveolar Type I cells. These data suggest that expression of caveolin-1 isoforms is differentially regulated in endothelial and epithelial cells during lung development. Caveolin-1 � is an early marker for lung vasculogenesis, primarily expressed in developing blood vessels. When the lung is fully differentiated postnatally, caveolin-1 � is also expressed in alveolar Type I cells. (J Histochem Cytochem 50:33–42, 2002)

Several recent observations have suggested that cytosine methylation has a role in the in vivo transcriptional regulation of cell-specific genes in normal cells. We hypothesized that methylation regulates T1alpha, a gene expressed primarily in lung in adult rodents. In fetuses T1alpha is expressed in several organs, including the entire nervous system, but during development its expression is progressively restricted to lung alveolar type I epithelial cells, some osteoblasts and choroid plexus. Here we report that T1alpha is methylated at a key Sp1 site in the proximal promoter in cells and organs, including brain, where no gene expression is detectable. Conversely, in T1alpha-expressing cells, these sites are not methylated. In embryonic brain T1alpha is unmethylated and expressed; in adult brain the gene is methylated and not expressed. In lung epithelial cell lines, methylation of the T1alpha promoter in vitro decreases expression by approx. 50% (the maximum suppression being 100%). Analysis of mutated promoter constructs indicates that a single Sp1 site in the proximal promoter provides all or most of the methylation-sensitive gene silencing. We conclude that, in addition to regulation by transcription factors, cytosine methylation has a role in the complex expression patterns of this gene in intact animals and primary cells.

The T1α gene is one of few markers for the type I cell phenotype in the adult mammalian lung. Type I cells form a large, thin epithelial layer that facilitates gas exchange and transport of fluids between the air spaces and capillaries. The T1α gene has a complex pattern of developmental expression in lung and brain; in vitro studies indicate that expression is regulated in part by thyroid transcription factor 1, forkhead proteins, and Sp1/Sp3 proteins. To explore the mechanisms that confine T1α expression in intact adult animals to alveolar type I and choroid plexus epithelial cells, we generated mice bearing a 1.3-kb T1α promoter-chloramphenicol acetyltransferase (CAT) gene. In situ hybridization and RNase protection assays show that the 1.3-kb promoter confers a pattern of CAT expression that largely matches the endogenous T1α in embryos and mid-term fetuses in lung and central nervous system. However, the 1.3-kb promoter lacks elements important for perinatal up-regulation of T1α in the lung and maintenance of that expression in the adult lung and brain. The final adult pattern of T1α expression may be directed by elements outside the 1.3-kb fragment, perhaps those 5' to the 1.3-kb fragment as we show herein, or in 3' and intronic regions. Dev Dyn 1999;215:319–331. © 1999 Wiley-Liss, Inc.

The Grainyhead family of transcription factors controls morphogenesis and differentiation of epithelial cell layers in multicellular organisms by regulating cell junction- and proliferation-related genes. Grainyhead-like 2 (Grhl2) is expressed in developing mouse lung epithelium and is required for normal lung organogenesis. The specific epithelial cells expressing Grhl2 and the genes regulated by Grhl2 in normal lungs are mostly unknown. In these studies we identified the NK2-homeobox 1 transcription factor (Nkx2-1) as a direct transcriptional target of Grhl2. By binding and transcriptional assays and by confocal microscopy we showed that these two transcription factors form a positive feedback loop in vivo and in cell lines and are co-expressed in lung bronchiolar and alveolar type II cells. The morphological changes observed in flattening lung alveolar type II cells in culture are associated with down-regulation of Grhl2 and Nkx2-1. Reduction of Grhl2 in lung epithelial cell lines results in lower expression levels of Nkx2-1 and of known Grhl2 target genes. By microarray analysis we identified that in addition to Cadherin1 and Claudin4, Grhl2 regulates other cell interaction genes such as semaphorins and their receptors, which also play a functional role in developing lung epithelium. Impaired collective cell migration observed in Grhl2 knockdown cell monolayers is associated with reduced expression of these genes and may contribute to the altered epithelial phenotype reported in Grhl2 mutant mice. Thus, Grhl2 functions at the nexus of a novel regulatory network, connecting lung epithelial cell identity, migration, and cell-cell interactions.

Pneumonia results from bacteria in the alveoli. The alveolar epithelium consists of type II cells, which secrete surfactant and associated proteins, and type I cells, which constitute 95% of the surface area and meet anatomic and structural needs. Other than constitutively expressed surfactant proteins, it is unknown whether alveolar epithelial cells have distinct roles in innate immunity. Because innate immunity gene induction depends on NF-κB RelA (also known as p65) during pneumonia, we generated a murine model of RelA mutated throughout the alveolar epithelium. In response to LPS, only 2 of 84 cytokine transcripts (CCL20 and CXCL5) were blunted in lungs of mutants, suggesting that a very limited subset of immune mediators is selectively elaborated by the alveolar epithelium. Lung CCL20 induction required epithelial RelA regardless of stimulus, whereas lung CXCL5 expression depended on RelA after instillation of LPS but not pneumococcus. RelA knockdown in vitro suggested that CXCL5 induction required RelA in type II cells but not type I cells. Sorted cell populations from mouse lungs revealed that CXCL5 was induced during pneumonia in type I cells, which did not require RelA. TLR2 and STING were also induced in type I cells, with RelA essential for TLR2 but not STING. To our knowledge, these data are the first direct demonstration that type I cells, which constitute the majority of the alveolar surface, mount innate immune responses during bacterial infection. These are also, to our knowledge, the first evidence for entirely RelA-independent pathways of innate immunity gene induction in any cell during pneumonia.

The developmental abnormalities associated with disruption of signaling by retinoic acid (RA), the biologically active form of vitamin A, have been known for decades from studies in animal models and humans. These include defects in the respiratory system, such as lung hypoplasia and agenesis. However, the molecular events controlled by RA that lead to formation of the lung primordium from the primitive foregut remain unclear. Here, we present evidence that endogenous RA acts as a major regulatory signal integrating Wnt and Tgfbeta pathways in the control of Fgf10 expression during induction of the mouse primordial lung. We demonstrated that activation of Wnt signaling required for lung formation was dependent on local repression of its antagonist, Dickkopf homolog 1 (Dkk1), by endogenous RA. Moreover, we showed that simultaneously activating Wnt and repressing Tgfbeta allowed induction of both lung buds in RA-deficient foreguts. The data in this study suggest that disruption of Wnt/Tgfbeta/Fgf10 interactions represents the molecular basis for the classically reported failure to form lung buds in vitamin A deficiency.

Angiotensin converting enzyme (ACE) 2 is a carboxypeptidase that shares 42% amino acid homology with ACE. Little is known about the regulation or pattern of expression of ACE2 in the mouse lung, including its definitive cellular distribution or developmental changes. Based on Northern blot and RT‐PCR data, we report two distinct transcripts of ACE2 in the mouse lung and kidney and describe a 5′ exon 1a previously unidentified in the mouse. Western blots show multiple isoforms of ACE2, with predominance of a 75–80 kDa protein in the mouse lung versus a 120 kDa form in the mouse kidney. Immunohistochemistry localizes ACE2 protein to Clara cells, type II cells, and endothelium and smooth muscle of small and medium vessels in the mouse lung. ACE2 mRNA levels peak at embryonic day 18.5 in the mouse lung, and immunostaining demonstrates protein primarily in the bronchiolar epithelium at that developmental time point. In murine cell lines ACE2 is strongly expressed in the Clara cell line mtCC, as opposed to the low mRNA expression detected in E10 (type I‐like alveolar epithelial cell line), MLE‐15 (type II alveolar epithelial cell line), MFLM‐4 (fetal pulmonary vasculature cell line), and BUMPT‐7 (renal proximal tubule cell line). In summary, murine pulmonary ACE2 appears to be primarily epithelial, is developmentally regulated, and has two transcripts that include a previously undescribed exon. J. Cell. Biochem. 101:1278–1291, 2007. © 2007 Wiley‐Liss, Inc.

Developmentally important genes have recently been linked to tissue regeneration and epithelial cell repair in neonatal and adult animals in several organs, including liver, skin, prostate, and musculature. We hypothesized that developmentally important genes play roles in lung injury repair in adult mice. Although there is considerable information known about these processes, the specific molecular pathways that mediate injury and regulate tissue repair are not fully elucidated. Using a hyperoxic injury model to study these mechanisms of lung injury and tissue repair, we selected the following genes based upon their known or putative roles in lung development and organogenesis: TTF-1, FGF9, FGF10, BMP4, PDGF-A, VEGF, Ptc, Shh, Sca-1, BCRP, CD45, and Cyclin-D2. Our findings demonstrate that several developmentally important genes (Sca-1, Shh, PDGF-A, VEGF, BCRP, CD45, BMP4, and Cyclin-D2) change during hyperoxic injury and normoxic recovery in mice, suggesting that adult lung may reactivate key developmental regulatory pathways for tissue repair. The mRNA for one gene (TTF-1), unchanged during hyperoxia, was upregulated late in recovery phase. These novel findings provide the basis for testing the efficacy of post-injury lung repair in animals genetically modified to inactivate or express individual molecules.

The transcription factor Sp1 plays an important regulatory role in transactivation of the lung type I cell differentiation gene T1alpha. Like other lung cells, type I cells may encounter changes in oxygen concentration during the lifetime of the organism. We found that exposure of mice to hyperoxia rapidly increases expression of T1alpha and other type I cell genes, and that returning the mice to normoxia quickly decreases expression. Likewise hyperoxia increases both endogenous T1alpha expression in lung epithelial cell lines and transcription of luciferase (Luc) from T1alpha promoter deletion constructs. Using wild-type promoter fragments and gel shift assays, we determined that Sp1/Sp3 and a key Sp cis-element in the proximal promoter mediate the hyperoxic response. Mutations of this element and inhibition of Sp-DNA binding by mithramycin block the hyperoxic response. Western analyses of cell homogenates show that the overall abundance of Sp1 and Sp3 proteins is not altered by hyperoxia. However, the abundance of nuclear Sp1 increases after short hyperoxic exposures, suggesting that signaling pathways activated by hyperoxia lead to Sp protein translocation, perhaps as a result of increased Sp phosphorylation.

Alveolar type I epithelial cells form the major surface for gas exchange in the lung. To explore how type I cells differ in gene expression from their progenitor alveolar type II cells, we analyzed transcriptional regulation of T1alpha, a gene expressed by adult type I but not type II cells. In vivo developmental patterns of T1alpha expression in lung and brain suggest active gene regulation. We cloned and sequenced 1.25 kilobase pairs of the T1alpha promoter that can drive reporter expression in lung epithelial cell lines. Deletion analyses identified regions important for lung cell expression. The base pair (bp) -100 to -170 fragment conferred differential regulation in lung epithelial cells compared with fibroblasts. Sequence alignment of this fragment with type II-specific surfactant protein B and C promoters shows similar consensus elements arranged in a different order. Gel retardation studies with alveolar epithelial cell line nuclear extracts, thyroid transcription factor I (TTF-1) homeodomain, hepatic nuclear factor (HNF)-3beta, or Sp1 proteins, and supershift assays were used to characterize TTF-1, HNF-3 (TGT3), and Sp1/Sp3 binding sites. The TGT3 site binds factors with binding properties similar to HNF-3/Fkh (hepatic nuclear factor-3/forkhead) proteins but different from HNF-3alpha or HNF-3beta. Co-transfection with a TTF-1 expression vector moderately transactivated the -170 bp-reporter construct. Mutational analysis of these three binding sites showed reduced transcriptional activity of the -170 bp promoter. Therefore, several regulatory sequences involved in type II cell gene regulation are also present in the T1alpha promoter, suggesting that genes of the peripheral lung epithelium may be regulated by similar factors.

The homeodomain transcription factor Nkx2-1 is essential for normal lung development and homeostasis. In lung tumors, it is considered a lineage survival oncogene and prognostic factor depending on its expression levels. The target genes directly bound by Nkx2-1, that could be the primary effectors of its functions in the different cellular contexts where it is expressed, are mostly unknown. In embryonic day 11.5 (E11.5) mouse lung, epithelial cells expressing Nkx2-1 are predominantly expanding, and in E19.5 prenatal lungs, Nkx2-1-expressing cells are predominantly differentiating in preparation for birth. To evaluate Nkx2-1 regulated networks in these two cell contexts, we analyzed genome-wide binding of Nkx2-1 to DNA regulatory regions by chromatin immunoprecipitation followed by tiling array analysis, and intersected these data to expression data sets. We further determined expression patterns of Nkx2-1 developmental target genes in human lung tumors and correlated their expression levels to that of endogenous NKX2-1. In these studies we uncovered differential Nkx2-1 regulated networks in early and late lung development, and a direct function of Nkx2-1 in regulation of the cell cycle by controlling the expression of proliferation-related genes. New targets, validated in Nkx2-1 shRNA transduced cell lines, include E2f3, Cyclin B1, Cyclin B2, and c-Met. Expression levels of Nkx2-1 direct target genes identified in mouse development significantly correlate or anti-correlate to the levels of endogenous NKX2-1 in a dosage-dependent manner in multiple human lung tumor expression data sets, supporting alternative roles for Nkx2-1 as a transcriptional activator or repressor, and direct regulator of cell cycle progression in development and tumors.