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ABSTRACT: Alveolar epithelial cell (AEC) trans-differentiation is a process where type II alveolar epithelial cells (AEC II) trans-differentiate into type I alveolar epithelial cells (AEC I) during lung recovery after various injuries, in which AEC I are damaged. This process is critical for lung tissue repair. MicroRNAs are a group of small RNAs that regulate gene expression at the post-transcriptional level. They have the potential to regulate almost every aspect of cell physiology. However, whether AEC trans-differentiation is regulated by microRNAs is completely unknown. In this study, we found that miR-375 was downregulated during AEC trans-differentiation. The overexpression of miR-375 with an adenoviral vector inhibited alveolar epithelial trans-differentiation as indicated by an increase in the AEC II marker, surfactant protein C, and decreases in the AEC I markers, T1α and advanced glycosylation end product-specific receptor. miR-375 also inhibited the Wnt/β-catenin pathway. The constitutively activation of Wnt/β-catenin signaling with a stabilized form of β-catenin blocked the miR-375 effects. Frizzled 8 was identified as a target of miR-375. In summary, our results demonstrate that miR-375 regulates AEC trans-differentiation through the Wnt/β-catenin pathway. This discovery may provide new targets for therapeutic intervention to benefit lung recovery from injuries.
Nucleic Acids Research 02/2013; · 8.03 Impact Factor
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ABSTRACT: P2X7 receptor (P2X7R) is a purinergic ion-channel receptor. We have previously shown that the activation of P2X7R in alveolar type I cells stimulates surfactant secretion in alveolar type II cells. In this study, we determined whether miR-150 regulates P2X7R-mediated surfactant secretion. The miR-150 expression level in alveolar type II cells was much higher than alveolar type I cells, which was inversely correlated with the P2X7R protein level. An adenovirus expressing miR-150 significantly reduced the P2X7R protein expression in E10 cells, an alveolar type I cell line. Furthermore, pre-treatment of E10 cells with the adenovirus reduced the surfactant secretion induced by E10 cell conditioned medium. Our study demonstrates that miR-150 regulates surfactant secretion through P2X7R.
Biochemical and Biophysical Research Communications 05/2012; 422(4):586-9. · 2.48 Impact Factor
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ABSTRACT: Alveolar epithelium is composed of alveolar epithelial cells of type I (AEC I) and type II (AEC II). AEC II secrete lung surfactant by means of exocytosis. P2X(7) receptor (P2X(7)R), a P2 purinergic receptor, has been implicated in the regulation of synaptic transmission and inflammation. Here, we report that P2X(7)R, which is expressed in AEC I but not AEC II, is a novel mediator for the paracrine regulation of surfactant secretion in AEC II. In primary co-cultures of AEC I and AEC II benzoyl ATP (BzATP; an agonist of P2X(7)R) increased surfactant secretion, which was blocked by the P2X(7)R antagonist Brilliant Blue G. This effect was observed in AEC II co-cultured with human embryonic kidney HEK-293 cells stably expressing rat P2X(7)R, but not when co-cultured with AEC I in which P2X(7)R was knocked down or in co-cultures of AEC I and AEC II isolated from P2X(7)R(-/-) mice. BzATP-mediated secretion involved P2Y(2) receptor signaling because it was reduced by the addition of the ATP scavengers apyrase and adenosine deaminase and the P2Y(2) receptor antagonist suramin. However, the stimulation with BzATP might also release other substances that potentially increase surfactant secretion as a greater stimulation of secretion was observed in AEC II incubated with BzATP when co-cultured with E10 or HEK-293-P2X(7)R cells than with ATP alone. P2X(7)R(-/-) mice failed to increase surfactant secretion in response to hyperventilation, pointing to the physiological relevance of P2X(7)R in maintaining surfactant homeostasis in the lung. These results suggest that the activation of P2X(7)R increases surfactant secretion by releasing ATP from AEC I and subsequently stimulating P2Y(2) receptors in AEC II.
Journal of Cell Science 02/2011; 124(Pt 4):657-68. · 6.11 Impact Factor
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ABSTRACT: Mammalian lung development is a complex biological process, which is temporally and spatially regulated by growth factors, hormones, and extracellular matrix proteins. Abnormal changes of these molecules often lead to impaired lung development, and thus pulmonary diseases. Epithelial-mesenchymal interactions are crucial for fetal lung development. This paper reviews two interconnected pathways, pleiotrophin and Wnt/beta-catenin, which are involved in fibroblast and epithelial cell communication during fetal lung development.
Respiratory research 01/2010; 11:80. · 3.36 Impact Factor
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ABSTRACT: The role of pleiotrophin in fetal lung development was investigated. We found that pleiotrophin and its receptor, protein-tyrosine
phosphatase receptor β/ζ, were highly expressed in mesenchymal and epithelial cells of the fetal lungs, respectively. Using
isolated fetal alveolar epithelial type II cells, we demonstrated that pleiotrophin promoted fetal type II cell proliferation
and arrested type II cell trans-differentiation into alveolar epithelial type I cells. Pleiotrophin also increased wound healing
of injured type II cell monolayer. Knockdown of pleiotrophin influenced lung branching morphogenesis in a fetal lung organ
culture model. Pleiotrophin increased the tyrosine phosphorylation of β-catenin, promoted β-catenin translocation into the
nucleus, and activated T cell factor/lymphoid enhancer factor transcription factors. Dlk1, a membrane ligand that initiates
the Notch signaling pathway, was identified as a downstream target of the pleiotrophin/β-catenin pathway by endogenous dlk1
expression, promoter assay, and chromatin immunoprecipitation. These results provide evidence that pleiotrophin regulates
fetal type II cell proliferation and differentiation via integration of multiple signaling pathways including pleiotrophin,
β-catenin, and Notch pathways.
Journal of Biological Chemistry 10/2009; 284(41):28021-28032. · 4.77 Impact Factor
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ABSTRACT: The role of pleiotrophin in fetal lung development was investigated. We found that pleiotrophin and its receptor, protein-tyrosine phosphatase receptor beta/zeta, were highly expressed in mesenchymal and epithelial cells of the fetal lungs, respectively. Using isolated fetal alveolar epithelial type II cells, we demonstrated that pleiotrophin promoted fetal type II cell proliferation and arrested type II cell trans-differentiation into alveolar epithelial type I cells. Pleiotrophin also increased wound healing of injured type II cell monolayer. Knockdown of pleiotrophin influenced lung branching morphogenesis in a fetal lung organ culture model. Pleiotrophin increased the tyrosine phosphorylation of beta-catenin, promoted beta-catenin translocation into the nucleus, and activated T cell factor/lymphoid enhancer factor transcription factors. Dlk1, a membrane ligand that initiates the Notch signaling pathway, was identified as a downstream target of the pleiotrophin/beta-catenin pathway by endogenous dlk1 expression, promoter assay, and chromatin immunoprecipitation. These results provide evidence that pleiotrophin regulates fetal type II cell proliferation and differentiation via integration of multiple signaling pathways including pleiotrophin, beta-catenin, and Notch pathways.
Journal of Biological Chemistry 09/2009; 284(41):28021-32. · 4.77 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are small endogenous RNAs and are widely regarded as one of the most important regulators of gene expression in both plants and animals. To define the roles of miRNAs in fetal lung development, we profiled the miRNA expression pattern during lung development with a miRNA microarray. We identified 21 miRNAs that showed significant changes in expression during lung development. These miRNAs were grouped into four distinct clusters based on their expression pattern. Cluster 1 contained miRNAs whose expression increased as development progressed, while clusters 2 and 3 showed the opposite trend of expression. miRNAs in cluster 4 including miRNA-127 (miR-127) had the highest expression at the late stage of fetal lung development. Quantitative real-time PCR validated the microarray results of six selected miRNAs. In situ hybridization demonstrated that miR-127 expression gradually shifted from mesenchymal cells to epithelial cells as development progressed. Overexpression of miR-127 in fetal lung organ culture significantly decreased the terminal bud count, increased terminal and internal bud sizes, and caused unevenness in bud sizes, indicating improper development. These findings suggest that miR-127 may have an important role in fetal lung development.
Physiological Genomics 06/2009; 37(3):268-78. · 2.73 Impact Factor
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Nili Jin,
Yujie Guo,
Peng Sun,
Anna Bell,
Narendranath Reddy Chintagari,
Manoj Bhaskaran,
Kimberly Rains,
Pradyumna Baviskar,
Zhongming Chen, Tingting Weng,
Lin Liu
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ABSTRACT: Cl(-) transport is essential for lung development. Because gamma-aminobutyric acid (GABA) receptors allow the flow of negatively-charged Cl(-) ions across the cell membrane, we hypothesized that the expression of ionotropic GABA receptors are regulated in the lungs during development. We identified 17 GABA receptor subunits in the lungs by real-time PCR. These subunits were categorized into four groups: Group 1 had high mRNA expression during fetal stages and low in adults; Group 2 had steady expression to adult stages with a slight up-regulation at birth; Group 3 showed an increasing expression from fetal to adult lungs; and Group 4 displayed irregular mRNA fluctuations. The protein levels of selected subunits were also determined by Western blots and some subunits had protein levels that corresponded to mRNA levels. Further studied subunits were primarily localized in epithelial cells in the developing lung with differential mRNA expression between isolated cells and whole lung tissues. Our results add to the knowledge of GABA receptor expression in the lung during development.
Gene Expression Patterns 08/2008; 8(6):397-403. · 2.02 Impact Factor
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Deming Gou,
Amarjit Mishra, Tingting Weng,
Lijing Su,
Narendranath Reddy Chintagari,
Zhixin Wang,
Honghao Zhang,
Li Gao,
Pengcheng Wang,
Heidi M Stricker,
Lin Liu
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ABSTRACT: Annexin A2, a calcium-dependent phospholipid-binding protein, is abundantly expressed in alveolar type II cells where it plays a role in lung surfactant secretion. Nevertheless, little is known about the details of its cellular pathways. To identify annexin A2-regulated or associated proteins, we silenced endogenous annexin A2 expression in rat alveolar type II cells by RNA interference and assessed the change of the cellular transcriptome by DNA microarray analysis. The loss of annexin A2 resulted in the change of 61 genes. Thirteen of the selected genes (11 down-regulated and 2 up-regulated genes) were validated by real time quantitative PCR. When the loss of rat annexin A2 was rescued by overexpressing EGFP-tagged human annexin A2, six of seven selected targets returned to their normal expression level, indicating that these genes are indeed annexin A2-associated targets. One of the targets, Rab14, co-immunoprecipitated with annexin A2. Rab14 also co-localized in part with annexin A2 and lamellar bodies in alveolar type II cells. The silencing of Rab14 resulted in a decrease in surfactant secretion, suggesting that Rab14 may play a role in surfactant secretion.
Journal of Biological Chemistry 06/2008; 283(19):13156-64. · 4.77 Impact Factor
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ABSTRACT: Arsenic is a carcinogen that is known to induce cell transformation and tumor formation. Although studies have been performed to examine the modulation of signaling molecules caused by arsenic exposure, the molecular mechanisms by which arsenic causes cancer are still unclear. We hypothesized that arsenic alters gene expression leading to carcinogenesis in the lung.
In this study, we examined global gene expression in response to 0.75 microM arsenic treatment for 1-7 days in a rat lung epithelial cell line (L2) using an in-house 10 k rat DNA microarray. One hundred thirty one genes were identified using the one-class statistical analysis of microarray (SAM) test. Of them, 33 genes had a fold change of > or = 2 between at least two time points. These genes were then clustered into 5 groups using K-means cluster analysis based on their expression patterns. Seven selected genes, all associated with cancer, were confirmed by real-time PCR. These genes have functions directly or indirectly related to metabolism, glycolysis, cell proliferation and differentiation, and regulation of transcription.
Our findings provide important insight for the future studies of arsenic-mediated lung cancer.
BMC Genomics 02/2008; 9:115. · 4.07 Impact Factor
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ABSTRACT: The application of RNA interference (RNAi) as a research and therapeutic tool depends on its ability to silence genes in a sequence-specific manner. Recent studies have reported that the effective knockdown of genes can be achieved by multiple short hairpin RNAs (shRNAs) in a single vector. Moreover, this approach can depress several genes simultaneously. However, current methods for the construction of multiple shRNA vectors often suffer from vector instability and are time-consuming. Here, we describe a simple, quick and low-cost approach to construct a single vector expressing four shRNA sequences driven by four different promoters. Using this vector, we were able to improve the gene silencing efficiency and make it possible to silence four different genes simultaneously, further expanding the application spectrum of RNAi, both in functional studies and therapeutic strategies.
The Journal of Gene Medicine 10/2007; 9(9):751-63. · 2.48 Impact Factor
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ABSTRACT: Alveolar epithelial cell (AEC) injury and repair during hyperoxia exposure and recovery have been investigated for decades, but the molecular mechanisms of these processes are not clear. To identify potentially important genes involved in lung injury and repair, we studied the gene expression profiles of isolated AEC II from control, 48-h hyperoxia-exposed (>95% O(2)), and 1-7 day recovering rats using a DNA microarray containing 10,000 genes. Fifty genes showed significant differential expression between two or more time points (P<0.05, fold change >2). These genes can be classified into 8 unique gene expression patterns. Real-time PCR verified 14 selected genes in three patterns related to hyperoxia exposure and early recovery. The change in the protein level for two of the selected genes, bmp-4 and retnla, paralleled that of the mRNA level. Many of these genes were found to be involved in cell proliferation and differentiation. In an in vitro AEC trans-differentiation culture model using AEC II isolated from control and 48-h hyperoxia-exposed rats, the expressions of the cell proliferation and differentiation genes identified above were consistent with their predicted roles in the trans-differentiation of AEC. These data indicate that a coordinated mechanism may control AEC differentiation during in vivo hyperoxia exposure and recovery as well as during in vitro AEC culture.
Free Radical Biology and Medicine 09/2007; 43(4):628-42. · 5.42 Impact Factor
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ABSTRACT: An important mechanism for gene regulation utilizes small non-coding RNAs called microRNAs (miRNAs). These small RNAs play important roles in tissue development, cell differentiation and proliferation, lipid and fat metabolism, stem cells, exocytosis, diseases and cancers. To date, relatively little is known about functions of miRNAs in the lung except lung cancer.
In this study, we utilized a rat miRNA microarray containing 216 miRNA probes, printed in-house, to detect the expression of miRNAs in the rat lung compared to the rat heart, brain, liver, kidney and spleen. Statistical analysis using Significant Analysis of Microarray (SAM) and Tukey Honestly Significant Difference (HSD) revealed 2 miRNAs (miR-195 and miR-200c) expressed specifically in the lung and 9 miRNAs co-expressed in the lung and another organ. 12 selected miRNAs were verified by Northern blot analysis.
The identified lung-specific miRNAs from this work will facilitate functional studies of miRNAs during normal physiological and pathophysiological processes of the lung.
BMC Genomics 02/2007; 8:29. · 4.07 Impact Factor
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ABSTRACT: Polarized distribution of chloride channels on the plasma membrane of epithelial cells is required for fluid transport across the epithelium of fluid-transporting organs. Ionotropic gamma-aminobutyric acid receptors are primary ligand-gated chloride channels that mediate inhibitory neurotransmission. Traditionally, these receptors are not considered to be contributors to fluid transport. Here, we report a novel function of gamma-aminobutyric acid receptors involving alveolar fluid homeostasis in adult lungs. We demonstrated the expression of functional ionotropic gamma-aminobutyric acid receptors on the apical plasma membrane of alveolar epithelial type II cells. gamma-Aminobutyric acid significantly increased chloride efflux in the isolated type II cells and inhibited apical to basolateral chloride transport on type II cell monolayers. Reduction of the gamma-aminobutyric acid receptor pi subunit using RNA interference abolished the gamma-aminobutyric acid-mediated chloride transport. In intact rat lungs, gamma-aminobutyric acid inhibited both basal and beta agonist-stimulated alveolar fluid clearance. Thus, we provide molecular and pharmacological evidence that ionotropic gamma-aminobutyric acid receptors contribute to fluid transport in the lung via luminal secretion of chloride. This finding may have the potential to develop clinical approaches for pulmonary diseases involving abnormal fluid dynamics.
Journal of Biological Chemistry 12/2006; 281(47):36012-20. · 4.77 Impact Factor
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ABSTRACT: Fetal lung development is a complex biological process that involves temporal and spatial regulations of many genes. To understand the molecular mechanisms of this process, we investigated gene expression profiles of fetal lungs on gestational days 18, 19, 20, and 21, as well as newborn and adult rat lungs. For this analysis, we used an in-house rat DNA microarray containing 6,000 known genes and 4,000 expressed sequence tags (ESTs). Of these, 1,512 genes passed the statistical significance analysis of microarray (SAM) test; an at least twofold change was shown for 583 genes (402 known genes and 181 ESTs) between at least two time points. K-means cluster analysis revealed seven major expression patterns. In one of the clusters, gene expression increased from day 18 to day 20 and then decreased. In this cluster, which contained 10 known genes and 5 ESTs, 8 genes are associated with development. These genes can be integrated into regulatory pathways, including growth factors, plasma membrane receptors, adhesion molecules, intracellular signaling molecules, and transcription factors. Real-time PCR analysis of these 10 genes showed an 88% consistency with the microarray data. The mRNA of LIM homeodomain protein 3a (Lhx3), a transcription factor, was enriched in fetal type II cells. In contrast, pleiotrophin, a growth factor, had a much higher expression in fetal lung tissues than in fetal type II cells. Immunohistochemistry revealed that Lhx3 was localized in fetal lung epithelial cells and pleiotrophin in the mesenchymal cells adjacent to the developing epithelium and blood vessel. Using GenMAPP, we identified four regulatory pathways: transforming growth factor-beta signaling, inflammatory response, cell cycle, and G protein signaling. We also identified two metabolic pathways: glycolysis-gluconeogenesis and proteasome degradation. Our results may provide new insights into the complex regulatory pathways that control fetal lung development.
AJP Lung Cellular and Molecular Physiology 12/2006; 291(5):L1027-37. · 3.66 Impact Factor
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ABSTRACT: Although several studies have shown that an induction of insulin-like growth factor (IGF) components occurs during hyperoxia-mediated lung injury, the role of these components in tissue repair is not well known. The present study aimed to elucidate the role of IGF system components in normal tissue remodeling. We used a rat model of lung injury and remodeling by exposing rats to > 95% oxygen for 48 h and allowing them to recover in room air for up to 7 days. The mRNA expression of IGF-I, IGF-II, and IGF-1 receptor (IGF-1R) increased during injury. However, the protein levels of these components remained elevated until day 3 of the recovery and were highly abundant in alveolar type II cells. Among IGF binding proteins (IGFBPs), IGFBP-5 mRNA expression increased during injury and at all the recovery time points. IGFBP-2 and -3 mRNA were also elevated during injury phase. In an in vitro model of cell differentiation, the expression of IGF-I and IGF-II increased during trans-differentiation of alveolar epithelial type II cells into type-I like cells. The addition of anti-IGF-1R and anti-IGF-I antibodies inhibited the cell proliferation and trans-differentiation to some extent, as evident by cell morphology and the expression of type I and type II cell markers. These findings demonstrate that the IGF signaling pathway plays a critical role in proliferation and differentiation of alveolar epithelium during tissue remodeling.
Journal of Cellular Biochemistry 04/2006; 97(5):984-98. · 2.87 Impact Factor
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ABSTRACT: The comparison of organ transcriptomes is an important strategy for understanding gene functions. In the present study, we attempted to identify lung-prominent genes by comparing the normal transcriptomes of rat lung, heart, kidney, liver, spleen, and brain. To increase the efficiency and reproducibility, we first developed a novel parallel hybridization system, in which 6 samples could be hybridized onto a single slide at the same time.
We identified the genes prominently expressed in the lung (147) or co-expressed in lung-heart (23), lung-liver (37), lung-spleen (203), and lung-kidney (98). The known functions of the lung-prominent genes mainly fell into 5 categories: ligand binding, signal transducer, cell communication, development, and metabolism. Real-time PCR confirmed 13 lung-prominent genes, including 5 genes that have not been investigated in the lung, vitamin D-dependent calcium binding protein (Calb3), mitogen activated protein kinase 13 (Mapk13), solute carrier family 29 transporters, member 1 (Slc29a1), corticotropin releasing hormone receptor (Crhr1), and lipocalin 2 (Lcn2).
The lung-prominent genes identified in this study may provide an important clue for further investigation of pulmonary functions.
BMC Genomics 02/2006; 7:47. · 4.07 Impact Factor
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Analytical Biochemistry 08/2005; 342(1):167-9. · 3.00 Impact Factor
