FGF10 maintains distal lung bud epithelium and excessive signaling leads to progenitor state arrest, distalization, and goblet cell metaplasia

<p>Abstract</p> <p>Background</p> <p>Interaction with the surrounding mesenchyme is necessary for development of endodermal organs, and Fibroblast growth factors have recently emerged as mesenchymal-expressed morphogens that direct endodermal morphogenesis. The fibroblast growth factor 10 (<it>Fgf10</it>) null mouse is characterized by the absence of lung bud development. Previous studies have shown that this requirement for <it>Fgf10 </it>is due in part to its role as a chemotactic factor during branching morphogenesis. In other endodermal organs <it>Fgf10 </it>also plays a role in regulating differentiation.</p> <p>Results</p> <p>Through gain-of-function analysis, we here find that FGF10 inhibits differentiation of the lung epithelium and promotes distalization of the embryonic lung. Ectopic expression of FGF10 in the lung epithelium caused impaired lung development and perinatal lethality in a transgenic mouse model. Lung lobes were enlarged due to increased interlobular distance and hyperplasia of the airway epithelium. Differentiation of bronchial and alveolar cell lineages was inhibited. The transgenic epithelium consisted predominantly of proliferating progenitor-like cells expressing Pro-surfactant protein C, TTF1, PEA3 and Clusterin similarly to immature distal tip cells. Strikingly, goblet cells developed within this arrested epithelium leading to goblet cell hyperplasia.</p> <p>Conclusion</p> <p>We conclude that FGF10 inhibits terminal differentiation in the embryonic lung and maintains the distal epithelium, and that excessive levels of FGF10 leads to metaplastic differentiation of goblet cells similar to that seen in chronic inflammatory diseases.</p

Functional Wnt Signaling Is Increased in Idiopathic Pulmonary Fibrosis

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease, characterized by distorted lung architecture and loss of respiratory function. Alveolar epithelial cell injury and hyperplasia, enhanced extracellular matrix deposition, and (myo)fibroblast activation are features of IPF. Wnt/beta-catenin signaling has been shown to determine epithelial cell fate during development. As aberrant reactivation of developmental signaling pathways has been suggested to contribute to IPF pathogenesis, we hypothesized that Wnt/beta-catenin signaling is activated in epithelial cells in IPF. Thus, we quantified and localized the expression and activity of the Wnt/beta-catenin pathway in IPF. METHODOLOGY/PRINCIPAL FINDINGS: The expression of Wnt1, 3a, 7b, and 10b, the Wnt receptors Fzd1-4, Lrp5-6, as well as the intracellular signal transducers Gsk-3beta, beta-catenin, Tcf1, 3, 4, and Lef1 was analyzed in IPF and transplant donor lungs by quantitative real-time (q)RT-PCR. Wnt1, 7b and 10b, Fzd2 and 3, beta-catenin, and Lef1 expression was significantly increased in IPF. Immunohistochemical analysis localized Wnt1, Wnt3a, beta-catenin, and Gsk-3beta expression largely to alveolar and bronchial epithelium. This was confirmed by qRT-PCR of primary alveolar epithelial type II (ATII) cells, demonstrating a significant increase of Wnt signaling in ATII cells derived from IPF patients. In addition, Western blot analysis of phospho-Gsk-3beta, phospho-Lrp6, and beta-catenin, and qRT-PCR of the Wnt target genes cyclin D1, Mmp 7, or Fibronectin 1 demonstrated increased functional Wnt/beta-catenin signaling in IPF compared with controls. Functional in vitro studies further revealed that Wnt ligands induced lung epithelial cell proliferation and (myo)fibroblast activation and collagen synthesis. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that the Wnt/beta-catenin pathway is expressed and operative in adult lung epithelium. Increased Wnt/beta-catenin signaling may be involved in epithelial cell injury and hyperplasia, as well as impaired epithelial-mesenchymal cross-talk in IPF. Thus, modification of Wnt signaling may represent a therapeutic option in IPF

Ror2 modulates the canonical Wnt signaling in lung epithelial cells through cooperation with Fzd2

<p>Abstract</p> <p>Background</p> <p>Wnt signaling is mediated through 1) the beta-catenin dependent canonical pathway and, 2) the beta-catenin independent pathways. Multiple receptors, including Fzds, Lrps, Ror2 and Ryk, are involved in Wnt signaling. Ror2 is a single-span transmembrane receptor-tyrosine kinase (RTK). The functions of Ror2 in mediating the non-canonical Wnt signaling have been well established. The role of Ror2 in canonical Wnt signaling is not fully understood.</p> <p>Results</p> <p>Here we report that Ror2 also positively modulates Wnt3a-activated canonical signaling in a lung carcinoma, H441 cell line. This activity of Ror2 is dependent on cooperative interactions with Fzd2 but not Fzd7. In addition, Ror2-mediated enhancement of canonical signaling requires the extracellular CRD, but not the intracellular PRD domain of Ror2. We further provide evidence that the positive effect of Ror2 on canonical Wnt signaling is inhibited by Dkk1 and Krm1 suggesting that Ror2 enhances an Lrp-dependent STF response.</p> <p>Conclusion</p> <p>The current study demonstrates the function of Ror2 in modulating canonical Wnt signaling. These findings support a functional scheme whereby regulation of Wnt signaling is achieved by cooperative functions of multiple mediators.</p

Distinct regulation of c-myb gene expression by HoxA9, Meis1 and Pbx proteins in normal hematopoietic progenitors and transformed myeloid cells

The proto-oncogenic protein c-Myb is an essential regulator of hematopoiesis and is frequently deregulated in hematological diseases such as lymphoma and leukemia. To gain insight into the mechanisms underlying the aberrant expression of c-Myb in myeloid leukemia, we analyzed and compared c-myb gene transcriptional regulation using two cell lines modeling normal hematopoietic progenitor cells (HPCs) and transformed myelomonocytic blasts. We report that the transcription factors HoxA9, Meis1, Pbx1 and Pbx2 bind in vivo to the c-myb locus and maintain its expression through different mechanisms in HPCs and leukemic cells. Our analysis also points to a critical role for Pbx2 in deregulating c-myb expression in murine myeloid cells cotransformed by the cooperative activity of HoxA9 and Meis1. This effect is associated with an intronic positioning of epigenetic marks and RNA polymerase II binding in the orthologous region of a previously described alternative promoter for c-myb. Taken together, our results could provide a first hint to explain the abnormal expression of c-myb in leukemic cells

Human Vav1 Expression in Hematopoietic and Cancer Cell Lines Is Regulated by c-Myb and by CpG Methylation

Vav1 is a signal transducer protein that functions as a guanine nucleotide exchange factor for the Rho/Rac GTPases in the hematopoietic system where it is exclusively expressed. Recently, Vav1 was shown to be involved in several human malignancies including neuroblastoma, lung cancer, and pancreatic ductal adenocarcinoma (PDA). Although some factors that affect vav1 expression are known, neither the physiological nor pathological regulation of vav1 expression is completely understood. We demonstrate herein that mutations in putative transcription factor binding sites at the vav1 promoter affect its transcription in cells of different histological origin. Among these sites is a consensus site for c-Myb, a hematopoietic-specific transcription factor that is also found in Vav1-expressing lung cancer cell lines. Depletion of c-Myb using siRNA led to a dramatic reduction in vav1 expression in these cells. Consistent with this, co-transfection of c-Myb activated transcription of a vav1 promoter-luciferase reporter gene construct in lung cancer cells devoid of Vav1 expression. Together, these results indicate that c-Myb is involved in vav1 expression in lung cancer cells. We also explored the methylation status of the vav1 promoter. Bisulfite sequencing revealed that the vav1 promoter was completely unmethylated in human lymphocytes, but methylated to various degrees in tissues that do not normally express vav1. The vav1 promoter does not contain CpG islands in proximity to the transcription start site; however, we demonstrated that methylation of a CpG dinucleotide at a consensus Sp1 binding site in the vav1 promoter interferes with protein binding in vitro. Our data identify two regulatory mechanisms for vav1 expression: binding of c-Myb and CpG methylation of 5′ regulatory sequences. Mutation of other putative transcription factor binding sites suggests that additional factors regulate vav1 expression as well

Expression of B-RAF V600E in Type II Pneumocytes Causes Abnormalities in Alveolar Formation, Airspace Enlargement and Tumor Formation in Mice

Growth factor induced signaling cascades are key regulatory elements in tissue development, maintenance and regeneration. Perturbations of these cascades have severe consequences, leading to developmental disorders and neoplastic diseases. As a major function in signal transduction, activating mutations in RAF family kinases are the cause of human tumorigenesis, where B-RAF V600E has been identified as the prevalent mutant. In order to address the oncogenic function of B-RAF V600E, we have generated transgenic mice expressing the activated oncogene specifically in lung alveolar epithelial type II cells. Constitutive expression of B-RAF V600E caused abnormalities in alveolar epithelium formation that led to airspace enlargements. These lung lesions showed signs of tissue remodeling and were often associated with chronic inflammation and low incidence of lung tumors. The inflammatory cell infiltration did not precede the formation of the lung lesions but was rather accompanied with late tumor development. These data support a model where the continuous regenerative process initiated by oncogenic B-RAF-driven alveolar disruption provides a tumor-promoting environment associated with chronic inflammation

26S Proteasome Activity Is Down-Regulated in Lung Cancer Stem-Like Cells Propagated In Vitro

Cancer stem cells (CSCs) are a small subset of cancer cells capable of self-renewal and tumor maintenance. Eradicating cancer stem cells, the root of tumor origin and recurrence, has emerged as one promising approach to improve lung cancer survival. Cancer stem cells are reported to reside in the side population (SP) of cultured lung cancer cells. We report here the coexistence of a distinct population of non-SP (NSP) cells that have equivalent self-renewal capacity compared to SP cells in a lung tumor sphere assay. Compared with the corresponding cells in monolayer cultures, lung tumor spheres, formed from human non-small cell lung carcinoma cell lines A549 or H1299, showed marked morphologic differences and increased expression of the stem cell markers CD133 and OCT3/4. Lung tumor spheres also exhibited increased tumorigenic potential as only 10,000 lung tumor sphere cells were required to produce xenografts tumors in nude mice, whereas the same number of monolayer cells failed to induce tumors. We also demonstrate that lung tumor spheres showed decreased 26S proteasome activity compared to monolayer. By using the ZsGreen–cODC (C-terminal sequence that directs degradation of Ornithine Decarboxylase) reporter assay in NSCLC cell lines, only less than 1% monolayer cultures were ZsGreen positive indicating low 26S proteasome, whereas lung tumor sphere showed increased numbers of ZsGreen-positive cells, suggesting the enrichment of CSCs in sphere cultures

Wnt Signaling Is Required for Early Development of Zebrafish Swimbladder

10.1371/journal.pone.0018431PLoS ONE63

Expression of p89c-Mybex9b, an alternatively spliced form of c-Myb, is required for proliferation and survival of p210BCR/ABL-expressing cells

The c-Myb gene encodes the p75c-Myb isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is pc-Mybex9b, which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein–protein interactions and negative regulation. In hematopoietic cells, expression of pc-Mybex9b accounts for 10–15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of pc-Mybex9b and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75c-Myb, pc-Mybex9b is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of pc-Mybex9b enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of pc-Mybex9b reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34+ cells, without affecting the levels of p75c-Myb. Together, these studies indicate that expression of the low-abundance pc-Mybex9b isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells

Intra-Aortic Clusters Undergo Endothelial to Hematopoietic Phenotypic Transition during Early Embryogenesis

Intra-aortic clusters (IACs) attach to floor of large arteries and are considered to have recently acquired hematopoietic stem cell (HSC)-potential in vertebrate early mid-gestation embryos. The formation and function of IACs is poorly understood. To address this issue, IACs were characterized by immunohistochemistry and flow cytometry in mouse embryos. Immunohistochemical analysis revealed that IACs simultaneously express the surface antigens CD31, CD34 and c-Kit. As embryos developed from 9.5 to 10.5 dpc, IACs up-regulate the hematopoietic markers CD41 and CD45 while down-regulating the endothelial surface antigen VE-cadherin/CD144, suggesting that IACs lose endothelial phenotype after 9.5 dpc. Analysis of the hematopoietic potential of IACs revealed a significant change in macrophage CFC activity from 9.5 to 10.5 dpc. To further characterize IACs, we isolated IACs based on CD45 expression. Correspondingly, the expression of hematopoietic transcription factors in the CD45(neg) fraction of IACs was significantly up-regulated. These results suggest that the transition from endothelial to hematopoietic phenotype of IACs occurs after 9.5 dpc