Clara Cell Specific Protein Research Articles

Role of von Willebrand factor and Clara cell specific protein as a bio and prognostic marker in patients of traumatic lung injury

Role of von Willebrand factor and Clara cell specific protein as a bio and prognostic marker in patients of traumatic lung injury

Abstract 2873: Epithelial NF-κB signaling is required for different functions in Kras- and EGFR-mediated lung tumorigenesis.

Abstract The transcription factor nuclear factor-κB (NF-κB) is a key mediator of the inflammatory response, and its activation correlates with poor prognosis in non-small cell lung cancer patients. Oncogenic KRAS and epidermal growth factor receptor (EGFR) mutations occur frequently in lung adenocarcinoma and are associated with increased NF-κB activation in lung tumors. Recent papers have shown that inhibition of epithelial NF-κB signaling in a mouse model of Kras-mediated lung tumorigenesis decreases tumor formation. However, the mechanisms linking NF-κB signaling to tumor formation remain unclear. We hypothesize that epithelial NF-κB signaling is a common requirement for lung tumorigenesis. For our studies, we used mice that inducibly express oncogenic KrasG12D or EGFRL858R+T790M as well as a dominant negative inhibitor of κB signaling (DN-IκB) in Clara Cell Specific Protein (CCSP)-expressing lung epithelium (designated KrasDN or EGFRDN mice). EGFRDN mice developed smaller and fewer tumors after two months of doxycycline administration compared to mice expressing EGFRL858R+T790M alone, while KrasDN mice did not develop any tumors. In KrasDN mice, loss of Kras transgene expression and increased cell death were observed after one week of doxycycline, suggesting that NF-κB signaling may be required for survival of Kras mutant cells in vivo. In EGFRDN mice, NF-κB inhibition led to a significant reduction in the percentage of BAL neutrophils when compared to mice expressing EGFRL858R+T790M alone. In addition, NF-κB inhibition led to reduced expression of CXCR2 ligands CXCL1 and CXCL2, which are known NF-κB-regulated neutrophil chemoattractants. In vitro inhibition of CXCR2 did not affect proliferation of EGFR-mutant cells, suggesting that the critical function of CXCL1 and CXCL2 may be inflammatory cell recruitment/activation. Future studies will investigate the effects of CXCR2 inhibition and neutrophil depletion on EGFR-mediated tumor formation in vivo. Together, these results suggest that NF-κB signaling is required for both Kras- and EGFR-mediated lung tumorigenesis, but NF-κB's function in a tumor may depend on the driver mutation. From these studies, we hope to identify NF-κB-regulated genes critical for tumor formation as potential targets for the development of better therapeutic strategies for lung cancer patients. Citation Format: Jamie A. Ausborn, Taylor Sherrill, Rinat Zaynagetdinov, Vasiliy Polosukhin, Frank McMahon, Fiona Yull, Timothy Blackwell. Epithelial NF-κB signaling is required for different functions in Kras- and EGFR-mediated lung tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2873. doi:10.1158/1538-7445.AM2013-2873

Stable cell fate changes in marrow cells induced by lung-derived microvesicles

Background: Interest has been generated in the capacity of cellular-derived microvesicles to alter the fate of different target cells. Lung, liver, heart and brain-derived vesicles can alter the genetic phenotype of murine marrow cells; however, the stability of such changes and the mechanism of these changes remain unclear. In the present work, we show that lung-derived microvesicles (LDMV) alter the transcriptome and proteome of target marrow cells initially by mRNA and regulator(s) of transcription transfer, but that long term phenotype change is due solely to transfer of a transcriptional regulator with target cell.Methods/results: In vivo studies: Whole bone marrow cells (WBM) were co-cultured with LDMV (both isolated from male C57BL/6 mice) or cultured alone (control). One week later, cultured WBM was transplanted into lethally-irradiated female C57BL/6 mice. Recipient mice were sacrificed 6 weeks later and WBM, spleens and livers were examined for the presence of lung-specific gene expression, including surfactants A, B, C and D, aquaporin-5, and clara cell specific protein, via real-time RT-PCR. Immunohistochemistry was also performed on lungs to determine the number of transplanted marrow-derived (Y chromosome+) type II pneumocytes (prosurfactant C+). Mice transplanted with LDMV co-cultured WBM expressed pulmonary epithelial cell genes in the cells of their bone marrow, livers and spleens and over fivefold more transplanted marrow-derived Y+/prosurfactant C+cells could be found in their lungs (vs. control mice). In vitro studies: WBM (from mice or rats) was cultured with or without LDMV (from mice or rats) for 1 week then washed and cultured alone. WBM was harvested at 2-week intervals for real-time RT-PCR analysis, using species-specific surfactant primers, and for Western Blot analysis. Proteomic and microRNA microarray analyses were also performed on cells. LDMV co-cultured WBM maintained expression of pulmonary epithelial cell genes and proteins for up to 12 weeks in culture. Surfactant produced at later time points was specific only to the species of the marrow cell in culture indicating de novo mRNA transcription. These findings, in addition to the altered protein and microRNA profiles of LDMV co-cultured WBM, support a stable transcriptional mechanism for these changes.Conclusions: These data indicate that microvesicle alteration of cell fate is robust and long-term and represents an important new aspect of cellular biology.

Marrow cell genetic phenotype change induced by human lung cancer cells

Microvesicles have been shown to mediate varieties of intercellular communication. Work in murine species has shown that lung-derived microvesicles can deliver mRNA, transcription factors, and microRNA to marrow cells and alter their phenotype. The present studies evaluated the capacity of excised human lung cancer cells to change the genetic phenotype of human marrow cells. We present the first studies on microvesicle production by excised cancers from human lung and the capacity of these microvesicles to alter the genetic phenotype of normal human marrow cells. We studied 12 cancers involving the lung and assessed nine lung-specific mRNA species (aquaporin, surfactant families, and clara cell-specific protein) in marrow cells exposed to tissue in co-culture, cultured in conditioned media, or exposed to isolated lung cancer-derived microvesicles. We assessed two or seven days of co-culture and marrow which was unseparated, separated by ficoll density gradient centrifugation or ammonium chloride lysis. Under these varying conditions, each cancer derived from lung mediated marrow expression of between one and seven lung-specific genes. Microvesicles were identified in the pellet of ultracentrifuged conditioned media and shown to enter marrow cells and induce lung-specific mRNA expression in marrow. A lung melanoma and a sarcoma also induced lung-specific mRNA in marrow cells. These data indicate that lung cancer cells may alter the genetic phenotype of normal cells and suggest that such perturbations might play a role in tumor progression, tumor recurrence, or metastases. They also suggest that the tissue environment may alter cancer cell gene expression.

Persistence of microvesicle-induced gene expression changes in murine marrow cells using an in vitro and in vivo model.

e21086 Background: Microvesicles are subcellular entities that are derived from a variety of cells and contain DNA, RNA and protein. We have previously shown that microvesicles isolated from murine lung tissue (lung-derived microvesicles, LDMV) enter murine whole bone marrow cells (WBM) in culture and WBM that has consumed LDMV produce lung-specific mRNA and protein up to three weeks later. We wished to determine the persistence of these phenotypic changes in a long-term in vitro culture and an in vivo transplant model. Methods: In vivo study: cell-free conditioned media (CM) was made using murine lungs. WBM cells were then co-cultured with either LDMV, minced lung fragments (separated from WBM by a 0.4 micron, cell-impermeable membrane) or neither. One week later, cultured WBM was transplanted into cohorts of lethally-irradiated C57BL/6 mice. Recipient mice were sacrificed six weeks post transplant and WBM isolated from each recipient was lysed and examined for the presence of lung-specific genes, including surfactants A, B, C and D (Sp-A, B, C, D), aquaporin-5 (Aq-5), and clara cell specific protein (CCSP), via real-time PCR. In vitro study: CM was made in same fashion as with the in vivo study. One week later cells were washed and placed into a secondary culture, without lung. WBM was harvested at two week intervals and RNA and protein was extracted for real-time PCR and Western Blot analysis. Results: In vivo study: Mice transplanted with WBM exposed to LDMV express pulmonary epithelial cell genes in the cells of their bone marrow, liver, spleen and thymus 6 weeks after transplantation. In vitro study: WBM maintains expression of pulmonary epithelial cell genes and proteins in vitro up to 12 weeks after exposure to soluble factors released by lung cells. Conclusions: These findings suggest that LDMV-induced phenotypic alteration of marrow cells is not a transient phenomenon and that a lung cell-derived transcriptional agent is transferred to marrow cells in culture and responsible for these stable phenotypic changes.

Stem cell plasticity and microvesicles.

e13504 Background: Marrow cells engrafted into irradiated mice form pulmonary epithelial type II pneumocytes. The capacity to do this is influenced by marrow cell cycle. Marrow derived pulmonary epithelial cells after transplantation are increased three fold at the G1/S interface. Methods: We have also shown that microvesicles derived from irradiated lung tissue enter marrow cells resulting in expression of mRNAs for surfactants A-D, clara cell-specific protein and aquaporin 5 and that cell entry is a prerequisite. Marrow cells interacted with lung-derived microvesicles also show an increased capacity for forming pulmonary epithelial cells after marrow transplantation. Results: We have also shown that marrow stem cells do not have a stable phenotype. They alter their phenotype continuously with cell cycle passage and that the most primitive marrow stem cells are an actively cycling population. Purified marrow stem cells, designated aslong-term hematopoietic stem cells (LT-HSC) are in fact mostly in G0, but when whole unseparated marrow is studied, using either Hoechst 33342/Pyronin Y separations or tritiated thymidine suicide followed by competitive engraftment into lethally irradiated mice, over 50% of these long-term engrafting cells are in S/G2/M. Given that these studies are at either an instantaneous point in time or over a 30 minute time interval, this indicates that the marrow stem cell population is a cycling population This explains the observations that marrow stem cell phenotype is continually changing with regard to marrow renewal, differentiation, marrow homing, and gene expression. Lung-derived microvesicles differentially enter marrow stem cells at different points in cell cycle adding another layer to the complexity of cell fate determination for marrow stem cells. Conclusions: Altogether, these observations indicate that microvesicle cell modulation probably explains the phenomena of stem cell plasticity. Furthermore, the marrow stem cell population is differentially modulated at different points in cell cycle by microvesicle exposure. These observations suggest a complex regulatory system for marrow stem cells with a stable population but continually changing individual components. This represents a new biology.

Jaagsiekte sheep retrovirus infects multiple cell types in the ovine lung.

Ovine pulmonary adenocarcinoma (OPA) is a transmissible lung cancer of sheep caused by Jaagsiekte sheep retrovirus (JSRV). The details of early events in the pathogenesis of OPA are not fully understood. For example, the identity of the JSRV target cell in the lung has not yet been determined. Mature OPA tumors express surfactant protein-C (SP-C) or Clara cell-specific protein (CCSP), which are specific markers of type II pneumocytes or Clara cells, respectively. However, it is unclear whether these are the cell types initially infected and transformed by JSRV or whether the virus targets stem cells in the lung that subsequently acquire a differentiated phenotype during tumor growth. To examine this question, JSRV-infected lung tissue from experimentally infected lambs was studied at early time points after infection. Single JSRV-infected cells were detectable 10 days postinfection in bronchiolar and alveolar regions. These infected cells were labeled with anti-SP-C or anti-CCSP antibodies, indicating that differentiated epithelial cells are early targets for JSRV infection in the ovine lung. In addition, undifferentiated cells that expressed neither SP-C nor CCSP were also found to express the JSRV Env protein. These results enhance the understanding of OPA pathogenesis and may have comparative relevance to human lung cancer, for which samples representing early stages of tumor growth are difficult to obtain.

FOXA1 plays a role in regulating secretoglobin 1a1 expression in the absence of CCAAT/enhancer binding protein activities in lung in vivo

Secretoglobin (SCGB) 1A1, also called Clara cell secretor protein (CCSP) or Clara cell-specific 10-kDa protein (CC10), is a small molecular weight secreted protein mainly expressed in lung, with anti-inflammatory/immunomodulatory properties. Previous in vitro studies demonstrated that CCAAT/enhancer-binding proteins (C/EBPs) are the major transcription factors for the regulation of Scbg1a1 gene expression, whereas FOXA1 had a minimum effect on the transcription. To determine the in vivo role of C/EBPs in the regulation of SCGB1A1 expression, experiments were performed in which A-C/EBP, a dominant-negative form of C/EBP that interferes with DNA binding activities of all C/EBPs, was specifically expressed in lung. Surprisingly, despite the in vitro findings, expression of SCGB1A1 mRNA was not decreased in vivo in the absence of C/EBPs. This may be due to a compensatory role assumed by FOXA1 in the regulation of Scgb1a1 gene expression in lung in the absence of active C/EBPs. This disconnect between in vitro and in vivo results underscores the importance of studies using animal models to determine the role of specific transcription factors in the regulation of gene expression in intact multicellular complex organs such as lung.

Lung-Derived Microvesicles Induce Stable Long-Term Epigenetic Changes In Marrow Cells

AbstractAbstract 4799We have previously shown that microvesicles derived from irradiated murine lung cells enter murine bone marrow cells and induce expression of the pulmonary epithelial cell-specific mRNAs Surfactants A-D (Sp-A-D), Aquaporin-5 (Aq-5) and Clara Cell Specific Protein (CCSP). The early mRNA increases are due to direct delivery of mRNA and due to the transfer of a transcription factor to marrow cells (Aliotta JM, et al, Exp Hematol, 2010). The modulated marrow cells produce pulmonary epithelial cell-specific protein and have an enhanced ability to convert into pulmonary epithelial cells after transplantation into lethally-irradiated mice. In the present studies, we evaluated the stability of pulmonary epithelial cell-specific mRNA expression in cytokine-stimulated liquid culture and after in vivo transplantation into lethally-irradiated mice. Whole bone marrow cells isolated from C57BL/6 mice were cultured for seven days opposite C57BL/6 lung fragments, but separated from them by a 0.4 micron cell-impermeable membrane. Marrow cells were then either infused into lethally-irradiated C57BL/6 mice or established in culture with DMEM supplemented with IL-3, IL-6, IL-11 and steel factor. Engrafted mice were sacrificed six weeks after transplantation and real-time RT-PCR was carried out on marrow, liver, spleen and thymus tissue. Expression of Sp-A, B and D, CCSP and Aq-5 were variably seen in all tissues and increases over control ranged from 12-fold to 58-fold. Liquid culture was analyzed every two weeks out to 12 weeks and high levels of expression of different pulmonary epithelial cell-specific mRNAs and proteins (by western blot and Immunohistochemical staining) were observed. In order to determine the mechanism for the increased expression in culture, we established co-cultures of Fischer-344 rat lung opposite C57BL/6 mouse bone marrow cells and, using species-specific primers, determined whether the increased pulmonary epithelial cell-specific mRNA expression at each time point of secondary cytokine-supported culture was rat or mouse-specific. In every instance, the pulmonary epithelial cell-specific mRNA which was detected was mouse-specific, not rat-specific, and thus the long-term expression of lung-specific mRNA in mouse marrow was due to induced transcription by the mouse marrow cells. These data indicate that lung-derived microvesicles induce long-term stable epigenetic changes in marrow cells and suggests that the genetic changes induced by tissue microvesicles in neighboring cells may be biologically important. Disclosures:No relevant conflicts of interest to declare.

Adhesion Protein Profile of Lung-Derived Microvesicles

AbstractAbstract 4803We have previously shown that murine bone marrow cells co-cultured opposite murine lung cells, but separated from them by a cell-impermeable membrane, express pulmonary epithelial cell-specific mRNAs, including Surfactants A-D, Aquaporin-5 and Clara Cell Specific Protein. This effect appears to be enhanced when the lungs used in co-culture are harvested from previously-irradiated mice. We have also shown that lung cells release membrane-bound particles called microvesicles which contain lung cell-specific proteins and mRNA. Microvesicles are capable of entering marrow cells in culture and inducing lung-specific mRNA and protein expression in marrow cells that have internalized them (Aliotta JM, et al, Stem Cells, 2007). Currently, it is not known how microvesicles gain entry into marrow cells and if this process involves the binding of specific adhesion proteins present on the marrow cells and/or the surface of microvesicles. Microvesicles derived from sources other than the lung have been described as expressing various adhesion proteins which facilitate their entry into target cells. In order to determine the presence of adhesion proteins on lung-derived microvesicles, we ultracentrifuged (100,000 g) cell-free conditioned media made from the lungs of C57BL/6 mice. Pelleted material was labeled with one of several directly-conjugated antibodies to an adhesion protein. Microvesicles which were positive for the adhesion protein of interest were identified by flow cytometry and quantified relative to its isotype control. Adhesion proteins that were found to be present on lung-derived microvesicles included CD29 (β1 integrin), CD41 (α2b integrin), CD49e (α5 integrin), CD49f (α6 integrin), CD51 (αv integrin), CD54 (ICAM-1), CD61 (β3 integrin), CD62L (L-selectin), CD81 (TAPA-1), CD107a (LAMP-1), CD154 (CD40 ligand), CD184 (CXCR4) and CD309 (VEGFR2). Those not present included CD9 (p24), CD11b (Mac-1), CD18 (β2 integrin), CD31 (PECAM-1), CD49d (α4 integrin), CD62P (P-selectin) and CD162 (PSGL-1). Lung-derived microvesicles isolated from mice that had been exposed to 500 centigrey total body irradiation five days prior to sacrifice were enriched with certain populations of adhesion protein positive microvesicles compared to those isolated from non-irradiated mice. These populations included ICAM-1 (78% increase), TAPA-1 (45% increase), CD40 ligand (108% increase) and VEGFR2 (136% increase) positive microvesicles. We are attempting to determine if lung-derived microvesicle entry into marrow cells is mediated by interactions with these adhesion proteins. These data indicate that adhesion proteins are present on lung-derived microvesicles. It is possible that differences in mRNA expression seen in marrow cells exposed to microvesicles derived from irradiated and non-irradiated lung in be related to qualitative and quantitative differences in those microvesicles. Disclosures:No relevant conflicts of interest to declare.

Suppression of embryonic lung branching morphogenesis by antisense oligonucleotides against HOM/C homeobox factors

The role of HOM/C homeobox genes on rat embryonic lung branching morphogenesis was investigated using the lung bud explant culture system in an air/liquid interface. Knock down of homeobox b3 and b4 expression by antisense oligonucleotide treatment repressed airway branch formation, while antisense oligonucleotide against homeobox a3 showed no effect. Addition of antisense Hoxb3 oligonucleotide resulted in upregulation of collagen type III mRNA and fibroblast growth factor 10 mRNA, while that of the T-box regulatory factor-4 was decreased. Consequently, expression of Clara cell-specific secretory protein was decreased. These results suggest a critical role for homeobox b3 and b4 genes in lung airway branching morphogenesis.

Cancers involving the lung mediate human marrow cell phenotype change.

10618 Background: Microvesicles from different tissues can mediate genetic phenotype changes of adjacent normal marrow cells. Methods: We have evaluated excised tissue from cancers involving the lung for their capacity to change the genetic phenotype of co cultured human marrow cells. thirteen cancers were evaluated: 11 were primary non-small cell lung cancers and there was one sarcoma and one melanoma of the lung. Excised tissue was co cultured opposite normal volunteer human bone marrow for 2 or 7 days with the cancer tissue separated from the human marrow by a cell impermeably 0.4 um membrane. Real-time RT-PCR for the following lung specific genes, aquaporin 1, aquaporin 2, aquaporin 3, aquaporin 4, aquaporin 5, surfactant A, B, C, and D, and clara cell-specific protein was carried out on 9 tumors. Results: In each instance, including the sarcoma and the melanoma, lung specific genes were highly expressed in the marrow cells. There was expression of from 1-8 lung-specific genes from different cancers ranging from over 2 fold to over 1000 fold of control. Expression was seen in co cultured whole marrow, ficol separated marrow and marrow subjected to ammonium chloride lysis. Human marrow exposed to conditioned media from lung cancer cells expressed high levels of pulmonary specific genes. Ultracentrifugation (28,000 g) of lung cancer conditioned media, pelleted large numbers of microvesicles (diameter 100-180 nm as shown by electron microscopy). Tumor-derived microvesicles also induced expression of lung-specific mRNA in target marrow cells. Pelleted microvesicles stained by the supravital dyes CFSE (green fluorescence) and PKH26 (red fluorescence) were shown to be taken up by human bone marrow cells. Conclusions: These studies indicate that cancer cells are capable of transferring a lung genetic phenotype to marrow cells. Whether this represents genetic information transferred primarily from lung to cancer cells and then secondarily to target co cultured cells or priamry genetic information from the cancer cells ia at present unknown. The capacity of cancer cells to transfer genetic phenotype suggests important possible modes of cancer progression, resurrence and metastases. It also suggests therapeutic strategies to block these occurrences. No significant financial relationships to disclose.

Homing and Engraftment of Bone Marrow Derived Cells in Irradiated Mouse Lungs.

Abstract 4600To determine the degree to which murine bone marrow migrates to and can repopulate damaged lung, we studied lung stem cell expression of the Clara Cell Specific Protein (CCSP). Several different models have been used to deplete the lung stem cells. We used CCtk mice which expresses the HSV-tk under control of the CCSP promoter. Subcutaneous administration of gancicylovir (50 mg/ml) over a 7 day period using Alzet osmotic pumps results in the reduction of CCSP RNA and CCSP positive cells. To determine if a reduced dose of gancicylovir reduced CCSP cells to a nonfatal degree and opened a stem cell niche for homing of hematopoietic cells, CCtk mice had osmotic pumps placed subcutaneously containing a lower dose of 25 mg/ml of gancicylovir. The lungs were explanted at serial times up to 7 days after pump placement. RNA was extracted and real time polymerase chain reaction (RT-PCR) using primers specific for CCSP was used to quantitate CCSP expression. CCtk mice treated with gancicylovir showed a 70% reduction of lung CCSP expression. CCtk mice implanted with osmotic pumps containing 25 mg/ml gangcicylovir and injected intravenously with bone marrow from sex-mismatched GFP+ transgenic FVB male mouse 2 days later were followed for thirty days. The mice were sacrificed, lungs removed, single cell suspensions prepared, stained with a PE labeled anti-CD45 monoclonal antibody and analyzed by flow cytometry for GFP positive, CD45 negative cells. No GFP+, CCSP+ cells cells were detected. Injection of FVB mice with naphthalene (which kills differentiated but not stem CCSP+ cells) at 200 mg/kg intraperitoneally resulted in an 87% decrease in CCSP expression. When injected with bone marrow cells from GFP+ transgenic mice at serial times to 7 days and sacrificed at day 30, again no GFP+ CCSP+ cells were identified. In a third attempt to remove additional CCSP cells and prepare sites for homing of marrow cells, FVB mice were treated with naphthalene (200 mg/kg) followed by 20 Gy irradiation to the lungs 48 hr later, then injected with GFP+ bone marrow, 24 hr later. The mice were sacrificed 30 days later, and examined for GFP+, CD45- cells. Low numbers of GFP+ CCSP+ cells were detected. The data indicates a low baseline level of hematopoietic stem cell potential to home and differentiate to CCSP positive lung cells.Supported by NIH-2R01CA119927-08A1. Disclosures:No relevant conflicts of interest to declare.

Eosinophilic Inclusions in Rat Clara Cells and the Effect of an Inhaled Corticosteroid

Large eosinophilic cytoplasmic inclusions (ECIs) are occasionally seen in untreated rat Clara cells. Following inhalation exposure to a corticosteroid, the number of ECIs was increased. This is the first histopathological description of rat ECIs and attempted characterization by immunohistochemistry, in situ hybridization, and electron microscopy. ECIs were strongly positive for surfactant protein D (SP-D) and weakly positive for Clara cell specific protein (CCSP). Clara cell cytoplasm was positive for CCSP mRNA regardless of ECIs, but not within ECIs. Corticosteroid treatment and ECI presence did not affect the immunohistochemistry and in situ hybridization staining intensities. Electron microscopy revealed large intracytoplasmic granules with an irregular limiting membrane. The ECI number was microscopically quantified in rats from three-, six-, and twenty-four-month studies. The mean ECI counts in treated rats increased from three- to fifty-four-fold with a positive dose-related trend, when compared with vehicle controls. Although the mechanism is unclear, SP-D and to a lesser extent CCSP accumulate in the ECIs. As human bronchial epithelium does not appear to contain structures analogous to the ECI, it is suggested that the observation of an increased number of ECIs in the treated rats is not likely to be relevant for human clinical risk assessment.

Endogenous Fibroblastic Progenitor Cells in the Adult Mouse Lung Are Highly Enriched in the Sca-1 Positive Cell Fraction

Originally identified as a marker specifying murine hematopoietic stem cells, the Sca-1 antigen has since been shown to be differentially expressed by candidate stem cells in tissues including vascular endothelium, skeletal muscle, mammary gland, and prostate of adult mice. In the adult murine lung, Sca-1 has previously been identified as a selectable marker for the isolation of candidate nonhematopoietic (CD45(-)), nonendothelial (CD31(-)) bronchioalveolar stem cells (BASC) located at the bronchioalveolar duct junction that coexpress surfactant protein C and the Clara cell specific protein. Our systematic analysis of CD45(-)CD31(-)Sca-1(+) cells in fetal, neonatal, and adult lung shows that very few of these cells are detectable prior to birth but expand exponentially postnatally coinciding with the transition from the saccular to the alveolar stage of lung development. Unlike candidate BASCs, the CD45(-)CD31(-)Sca-1(+)CD34(+) cell fraction we describe coexpresses immunophenotypic markers (Thy-1 and platelet-derived growth factor receptor alpha) that define lung fibroblastic rather than epithelial cells. The mesenchymal "signature" of the CD45(-)CD31(-)Sca-1(+)CD34(+) cell fraction is further confirmed by transcriptional profiling, by cell culture studies demonstrating enrichment for clonogenic lipofibroblastic and nonlipofibroblastic progenitors, and by immunohistochemical localization of Sca-1 in perivascular cells of the lung parenchyma. Although the CD45(-)CD31(-)Sca-1(+)CD34(+) cell phenotype does define endogenous clonogenic progenitor cells in the adult murine lung, our data indicate that these progenitors are predominantly representative of mesenchymal cell lineages, and highlights the pressing need for the identification of alternative markers and robust functional assays for the identification and characterization of epithelial and fibroblastic stem and progenitor cell populations in the adult lung.

Actinomycin D Blocks Transcription of Surfactant B mRNA in Marrow Cells Co-Cultured with Lung

We have previously described that RNA-filled microvesicles are shed from lung cells and are capable of entering whole bone marrow (WBM) cells in culture (Aliotta et al, Stem Cells , 2007; 25(9):2245–56). WBM cells cultured with these lung-derived microvesicles appear to express lung cell-specific genes after 7 days in culture. However, it is not known if the lung cell-specific mRNA detected in these cells represents de novo transcription or previously transcribed mRNA carried into the cell via microvesicles. To address this issue, we co-cultured WBM cells extracted from C57BL/6 mice and co-cultured them opposite lung from C57BL/6 mice exposed to 500 centigrey (cGy) of total body irradiation (TBI) or no radiation. The two cell types were separated by a cell-impermeable membrane (pore size, 0.4μm). After 7 days of co-culture, WBM cells were removed, extensively washed and cultured alone for an additional 36 hours, with or without the transcriptional blocking agent Actinomycin-D (Act-D, 10 ug/ml). Control WBM cells were co-cultured without lung and not exposed to Act-D (control). WBM cells were then analyzed for the presence of a variety of hematopoietic and lung-specific markers by Real Time RT-PCR and fold difference in target gene expression was determined (relative to control cells). WBM cells co-cultured with or without lung expressed high levels of hematopoietic markers including Sca-1, c-kit and CD45. This expression was nearly or completely eliminated after exposure to Act-D in culture. Expression of the surfactant protein B (Sp-B) gene was also significantly decreased in WBM co-cultured with lung (irradiated or non-irradiated) after exposure to Act-D (mean ± SE, from 16±4.9 to 1.1±0.4-fold increase vs. control, p=0.012). Interestingly, exposure to Act-D resulted in an increase in the expression of other lung-specific genes, including surfactant protein C (Sp-C) and clara cell specific protein (CCSP), in WBM co-cultured with lung (from 165±103 to 3,700±1,625 and 2±0.9 to 93±38-fold increases vs. control, p<0.015). In separate experiments, WBM cells co-cultured with cell-free lung conditioned media (CM) that was pre-treated with RNase had nearly 70% lower Sp-B expression compared with WBM cells co-cultured with untreated cell-free CM. These data suggest that lung cells release a transcriptional factor that induces marrow cell expression of the Sp-B gene in culture. This transcriptional factor is RNase-sensitive and could potentially represent microRNA transferred to marrow cells via lung-derived microvesicles. The increase in expression of other lung-specific genes after Act-D exposure suggests alternative transcriptional control mechanisms for lung-specific genes other than Sp-B.

Keratinocyte growth factor protects against Clara cell injury induced by naphthalene

Airway epithelial cells are exposed to environmental toxicants that result in airway injury. Naphthalene (NA) causes site-selective damage to Clara cells in mouse distal airways. N-terminally truncated recombinant human keratinocyte growth factor (DeltaN23-KGF) protects against acute lung injury. The present study investigated whether or not DeltaN23-KGF protects against NA-induced acute Clara cell damage by measuring airway responses specifically and in order to identify underlying molecular mechanisms. Mice were treated with DeltaN23-KGF or PBS 33 h prior to injection of 200 mg.kg body weight(-1) NA. Lung function was analysed by head-out body plethysmography. Distal airways isolated by microdissection were assessed for cell permeability using ethidium homodimer-1. Immunohistochemistry of Clara cell-specific protein in conjunction with a physical dissector was used to quantify Clara cell numbers. RNA was isolated from frozen airways in order to analyse gene expression using quantitative RT-PCR. DeltaN23-KGF prevented NA-induced airflow limitation and Clara cell permeability, and resulted in twice as many Clara cells compared with PBS pre-treatment. DeltaN23-KGF-pre-treated mice exhibited increased expression of proliferating cell nuclear antigen mRNA. Cytochrome P(450) isoform 2F2, which converts NA into its toxic metabolite, was reduced by approximately 50%. The present results demonstrate that pre-treatment with N-terminally truncated recombinant human keratinocyte growth factor protects against naphthalene-induced injury. This suggests that N-terminally truncated recombinant human keratinocyte growth factor exerts its beneficial effect through a decrease in the expression of cytochrome P(450) isoform 2F2.

Prenatal Lung Epithelial Cell-Specific Abrogation of Alk3-Bone Morphogenetic Protein Signaling Causes Neonatal Respiratory Distress by Disrupting Distal Airway Formation

Bone morphogenetic proteins (BMPs) play important roles in regulating lung development and function although the endogenous regulatory effects of BMP signaling are still controversial. We found that BMP type I receptor Alk3 is expressed predominantly in airway epithelial cells during development. The function of Alk3 in lung development was determined using an inducible knockout mouse model by crossing epithelial cell-specific Cre transgenic mice SPC-rtTA/TetO-Cre and floxed-Alk3 mice. Abrogation of Alk3 in mouse lung epithelia from either early lung organogenesis or late gestation resulted in similar neonatal respiratory distress phenotypes accompanied by collapsed lungs. Early-induction of Alk3 knockout in lung epithelial cells caused retardation of early lung branching morphogenesis, reduced cell proliferation, and differentiation. However, late gestation induction of the knockout caused changes in cell proliferation and survival, as shown by altered cell biology, reduced expression of peripheral epithelial markers (Clara cell-specific protein, surfactant protein C, and aquaporin-5), and lack of surfactant secretion. Furthermore, canonical Wnt signaling was perturbed, possibly through reduced Wnt inhibitory factor-1 expression in Alk3-knockout lungs. Therefore, our data suggest that deficiency of appropriate BMP signaling in lung epithelial cells results in prenatal lung malformation, neonatal atelectasis, and respiratory failure.

Immunohistochemical Localization of Cystic Fibrosis Transmembrane Regulator and Clara Cell Secretory Protein in Taste Receptor Cells of Rat Circumvallate Papillae

Taste receptor cells (TRCs) are the sensory cells of taste transduction and are organized into taste buds embedded in the epithelium of the tongue, palate, pharynx, and larynx. Several studies have demonstrated that TRCs involved in sweet as well as bitter and umami responses express alpha-gustducin, an alpha-subunit of the G-protein complex. It has been further demonstrated that this typical taste protein is a potent marker of chemosensory cells located in several tissues, including gastric and pancreatic mucosa and the respiratory apparatus. We recently observed that alpha-gustducin and phospholipase C beta 2-immunoreactive cells were colocalized in the airways with cystic fibrosis transmembrane regulator (CFTR) and Clara cell-specific secretory protein of 10 (CC10) and 26 kDa (CC26). This finding suggests that TRCs might themselves express secretory markers. To test this hypothesis, we investigated the expression of CFTR, CC10, and CC26 in rat circumvallate papillae using reverse transcriptase-polymerase chain reaction analysis, immunohistochemistry, and confocal laser microscopy. The results showed that secretory markers such as CFTR, CC10, and CC26 are present in taste cells of rat circumvallate papillae, and their immunoreactivity is expressed, to a different extent, in subsets of taste cells that express alpha-gustducin. The presence of CFTR, CC10, and CC26 in taste bud cells and their coexpression pattern with alpha-gustducin confirms and extends our previous findings in airway epithelium, lending further credence to the notion that chemoreception and secretion may be related processes.

Derivation, Characterization, and In Vitro Differentiation of Canine Embryonic Stem Cells

Canine embryonic stem (cES) cell lines were generated to establish a large-animal preclinical model for testing the safety and efficacy of embryonic stem (ES) cell-derived tissue replacement therapy. Putative cES cell lines were initiated from canine blastocysts harvested from natural matings. Times of harvest were estimated as 12-16 days after the presumed surge in circulating levels of luteinizing hormone. Four lines established from blastocysts harvested at days 13-14 postsurge satisfied most of the criteria for embryonic stem cells, whereas lines established after day 14 did not. One line, Fred Hutchinson dog (FHDO)-7, has been maintained through 34 passages and is presented here. FHDO-7 cells are alkaline phosphatase-positive and express both message and protein for the Oct4 transcription factor. They also express message for Nanog and telomerase but do not express message for Cdx2, which is associated with trophectoderm. Furthermore, they express a cluster of pluripotency-associated microRNAs (miRs) (miR-302b, miR-302c, and miR-367) characteristic of human and mouse ES cells. The FHDO-7 cells grow on feeder layers of modified mouse embryonic fibroblasts as flat colonies that resemble ES cells from mink, a close phylogenetic relative of dog. When cultured in nonadherent plates without feeders, the cells form embryoid bodies (EBs). Under various culture conditions, the EBs give rise to ectoderm-derived neuronal cells expressing gamma-enolase and beta 3-tubulin; mesoderm-derived cells producing collagen IIA1, cartilage, and bone; and endoderm-derived cells expressing alpha-fetoprotein or Clara cell-specific protein.