ETS1 regulates Twist1 transcription in a KrasG12D/Lkb1-/- metastatic lung tumor model of non-small cell lung cancer.

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.

A Multigenerational Strategy to Transform Health Education into Community Action.

BACKGROUND: The National Institute for Minority Health and Health Disparities funded Centers of Excellence to address health disparities through research, education and professional training, and community engagement. This article summarizes a decade of multigenerational educational programing embedded in the Community Engagement Core (CEC) of the National Institute for Minority Health and Health Disparities-funded Center for Healthy Communities-Center of Excellence at the University of South Alabama. OBJECTIVES: Our objective is to demonstrate how community-based participatory research (CBPR) initiated the multigenerational approach, uniting the community health education and the educational pipeline programs, and transformed a traditional professional symposium into a mechanism to increase community participation and action. METHODS: Community engagement and education adhered to CBPR principles and methods. A 3-year planning process before full funding of the Center of Excellence allowed the CHC to develop community partnerships and implement pilot projects that would assure community access and participation in COE programs. Program innovation was rooted in community suggestions and community priorities. The annual Regional Health Disparities Symposium (RHDS) was literally transformed through community engagement. CONCLUSIONS: Education programs for adults and youth achieved their goals independently, the STARS AND STRIPES (Student Training for Academic Reinforcement in the Sciences and Special Training to Raise Interest and Prepare for Entry into the Sciences) pipeline program has a success rate of 88% for participants' admission to colleges and universities. CHA-led events have documented an outreach to more than 6,500 community members and the COE has funded eight CHA-led projects directly addressing community action plans developed through CBPR methods during the history of the RHDS. But the real story has emerged from transformative multigenerational interaction via CBPR.

Serines in the intracellular tail of podoplanin (PDPN) regulate cell motility.

Podoplanin (PDPN) is a transmembrane receptor that affects the activities of Rho, ezrin, and other proteins to promote tumor cell motility, invasion, and metastasis. PDPN is found in many types of cancer and may serve as a tumor biomarker and chemotherapeutic target. The intracellular region of PDPN contains only two serines, and these are conserved in mammals including mice and humans. We generated cells from the embryos of homozygous null Pdpn knock-out mice to investigate the relevance of these serines to cell growth and migration on a clear (PDPN-free) background. We report here that one or both of these serines can be phosphorylated by PKA (protein kinase A). We also report that conversion of these serines to nonphosphorylatable alanine residues enhances cell migration, whereas their conversion to phosphomimetic aspartate residues decreases cell migration. These results indicate that PKA can phosphorylate PDPN to decrease cell migration. In addition, we report that PDPN expression in fibroblasts causes them to facilitate the motility and viability of neighboring melanoma cells in coculture. These findings shed new light on how PDPN promotes cell motility, its role in tumorigenesis, and its utility as a functionally relevant biomarker and chemotherapeutic target.

Epigenetic mechanisms modulate thyroid transcription factor 1-mediated transcription of the surfactant protein B gene.

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.

Increased PEA3/E1AF and decreased Net/Elk-3, both ETS proteins, characterize human NSCLC progression and regulate caveolin-1 transcription in Calu-1 and NCI-H23 NSCLC cell lines.

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.

Resident cellular components of the human lung: current knowledge and goals for research on cell phenotyping and function.

The purpose of the workshop was to identify still obscure or novel cellular components of the lung, to determine cell function in lung development and in health that impacts on disease, and to decide promising avenues for future research to extract and phenotype these cells. Since robust technologies are now available to identify, sort, purify, culture, and phenotype cells, progress is now within sight to unravel the origins and functional capabilities of lung cells in developmental stages and in disease. The Workshop's agenda was to first discuss the lung's embryologic development, including progenitor and stem cells, and then assess the functional and structural cells in three main compartments of the lung: (1) airway cells in bronchial and bronchiolar epithelium and bronchial glands (basal, secretory, ciliated, Clara, and neuroendocrine cells); (2) alveolar unit cells (Type 1 cells, Type 2 cells, and fibroblasts in the interstitium); and (3) pulmonary vascular cells (endothelial cells from different vascular structures, smooth muscle cells, and adventitial fibroblasts). The main recommendations were to: (1) characterize with better cell markers, both surface and nonsurface, the various cells within the lung, including progenitor cells and stem cells; (2) obtain more knowledge about gene expression in specific cell types in health and disease, which will provide insights into biological and pathologic processes; (3) develop more methodologies for cell culture, isolation, sorting, co-culture, and immortalization; and (4) promote tissue banks to facilitate the procurement of tissue from normal and from diseased lung for analysis at all levels.

Knowns and unknowns of the alveolus.

Our current alveolar paradigm includes three highly specialized cell populations. Alveolar type I cells are flat, elongated cells that presumably enable gas exchange. Alveolar type II cells are small, cuboidal cells with metabolic, secretory, progenitor, and immunologic functions. Alveolar fibroblasts secrete extracellular matrix proteins that support alveolar structure. These cells work together to facilitate respiration. Many years of high-quality research have defined our understanding of alveolar biology. However, there is much to be determined about the factors controlling cellular phenotypes and crosstalk. Moreover, specific questions remain regarding origin, repopulation, and previously unrecognized functions of each cell. This article summarizes the current data for each cell type and highlights areas that would benefit from further investigation.

Angiotensin converting enzyme 2 is primarily epithelial and is developmentally regulated in the mouse lung.

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.

ERM is expressed by alveolar epithelial cells in adult mouse lung and regulates caveolin-1 transcription in mouse lung epithelial cell lines.

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.

Key developmental regulators change during hyperoxia-induced injury and recovery in adult mouse lung.

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.

Alterations in gene expression in T1 alpha null lung: a model of deficient alveolar sac development.

BACKGROUND: Development of lung alveolar sacs of normal structure and size at late gestation is necessary for the gas exchange process that sustains respiration at birth. Mice lacking the lung differentiation gene T1alpha [T1alpha(-/-)] fail to form expanded alveolar sacs, resulting in respiratory failure at birth. Since little is known about the molecular pathways driving alveolar sacculation, we used expression microarrays to identify genes altered in the abnormal lungs and, by inference, may play roles in normal lung morphogenesis. RESULTS: Altered expression of genes related to cell-cell interaction, such as ephrinA3, are observed in T1alpha(-/-) at E18.5. At term, FosB, Egr1, MPK-1 and Nur77, which can function as negative regulators of the cell-cycle, are down-regulated. This is consistent with the hyperproliferation of peripheral lung cells in term T1alpha (-/-) lungs reported earlier. Biochemical assays show that neither PCNA nor p21 are altered at E18.5. At term in contrast, PCNA is increased, and p21 is decreased. CONCLUSION: This global analysis has identified a number of candidate genes that are significantly altered in lungs in which sacculation is abnormal. Many genes identified were not previously associated with lung development and may participate in formation of alveolar sacs prenatally.

Transcription of the caveolin-1 gene is differentially regulated in lung type I epithelial and endothelial cell lines. A role for ETS proteins in epithelial cell expression.

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-1alpha is expressed in fetal lung endothelial, but not epithelial, cells; in adult lung, both cell types express caveolin-1alpha. 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.

Enhanced binding of Sp1/Sp3 transcription factors mediates the hyperoxia-induced increased expression of the lung type I cell gene T1alpha.

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.

T1alpha, a lung type I cell differentiation gene, is required for normal lung cell proliferation and alveolus formation at birth.

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.

Alveolar type I cells: molecular phenotype and development.

Understanding of the functions and regulation of the phenotype of the alveolar type I epithelial cell has lagged behind studies of its neighbor the type II cell because of lack of cell-specific molecular markers. The recent identification of several proteins expressed by type I cells indicates that these cells may play important roles in regulation of cell proliferation, ion transport and water flow, metabolism of peptides, modulation of macrophage functions, and signaling events in the peripheral lung. Cell systems and reagents are available to characterize type I cell biology in detail, an important goal given that the cells provide the extensive surface that facilitates gas exchange in the intact animal.

Synthesis of 1,25-dihydroxyvitamin D(3) by human endothelial cells is regulated by inflammatory cytokines: a novel autocrine determinant of vascular cell adhesion.

In addition to its calciotropic function, the secosteroid 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) has potent nonclassical effects. In particular, local production of 1,25D(3) catalyzed by the enzyme 1alpha-hydroxylase (1alpha-OHase) may act as an autocrine/paracrine immunomodulatory mechanism. To investigate the significance of this in vascular tissue the expression and function of 1alpha-OHase in human endothelial cells was characterized. Immunohistochemical and in situ hybridization analyses show, for the first time, the presence of 1alpha-OHase mRNA and protein in endothelial cells from human renal arteries as well as postcapillary venules from lymphoid tissue. Reverse transcription-PCR and Western blot analyses confirmed the presence of 1alpha-OHase in primary cultures of human umbilical vein endothelial cells (HUVEC). Enzyme activity in HUVEC (318 +/- 56 fmoles 1,25(OH)(2)D(3)/hr/mg protein) increased after treatment with tumor necrosis factor-alpha (1054 +/- 166, P < 0.01), lipopolysaccharide (1381 +/- 88, P < 0.01), or forskolin (554 +/- 56, P < 0.05). Functional studies showed that exogenously added 1,25(OH)(2)D(3) or its precursor, 25-hydroxyvitamin D(3) (25(OH)D(3)), significantly decreased HUVEC proliferation after 72 h of treatment (33% and 11%, respectively). In addition, after 24 h treatment, both 1,25(OH)(2)D(3) and 25(OH)D(3) increased the adhesion of monocytic U937 cells to HUVEC (159% and 153%, respectively). These data indicate that human endothelia are able to produce active vitamin D. The rapid induction of endothelial 1alpha-OHase activity by inflammatory cytokines suggests a novel autocrine/paracrine role for the enzyme, possibly as a modulator of endothelial cell adhesion.

The alpha-isoform of caveolin-1 is a marker of vasculogenesis in early lung development.

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-1alpha and -beta 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-1alpha 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-1alpha 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-1alpha is an early marker for lung vasculogenesis, primarily expressed in developing blood vessels. When the lung is fully differentiated postnatally, caveolin-1alpha is also expressed in alveolar Type I cells.