A novel approach for the construction of multiple shRNA expression vectors

Gou, Deming; Weng, Tingting; Wang, Yang; Wang, Zhixin; Zhang, Honghao; Gao, Li; Chen, Zhongming; Wang, Pengcheng; Liu, Lin

doi:10.1002/jgm.1080

Cited by 40 publications

(51 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Construction of siRNA Adenoviral Vector-A highly efficient siRNA vector expressing four short hairpin RNAs under the control of mU6, hU6, H1, and 7SK promoters was previously developed in our laboratory (22). Four siRNA sequences specific to the coding region of PTN (549 -569, 570 -590, 607-627, and 909 -929) were selected based on siRNA Design Software (SDS).…”

Section: Methodsmentioning

confidence: 99%

“…We silenced PTN in a fetal lung organ culture by using the pK4-shRNA vector (22) and examined the effects of the treatment on lung branching morphogenesis. An adenoviral vector carrying four siRNAs targeted to the different regions of rat PTN was constructed to knock down PTN, and another vector with four non-relevant siRNAs was used as a control.…”

Section: Ptn Promotes Fetal Lung Branchingmentioning

confidence: 99%

See 1 more Smart Citation

Pleiotrophin Regulates Lung Epithelial Cell Proliferation and Differentiation during Fetal Lung Development via β-Catenin and Dlk1

Weng¹,

Gao

Bhaskaran

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The role of pleiotrophin in fetal lung development was investigated. We found that pleiotrophin and its receptor, proteintyrosine phosphatase receptor ␤/, 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 ␤-catenin, promoted ␤-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/␤-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, ␤-catenin, and Notch pathways.Fetal lung development is a complex biological process. Rat lung originates from the foregut endoderm as a bifurcation at embryonic day 10 (E10) and undergoes several generations of dichotomous branching morphogenesis to form a respiratory tree thereafter (1). The columnar epithelial cells differentiate into Clara cells and epithelial cells. Although surfactant protein C is detected as early as E13, fetal alveolar epithelial type II cells (fAEC II) 3 are not fully differentiated until E18 when glycogen pool-enriched cells become cuboidal and differentiate into cells containing lamellar bodies. fAEC II can further differentiate and give rise to the alveolar epithelial type I cells (AEC I).The regulation of fetal lung development includes coordinated regulation of molecular pathways as well as reciprocal interactions among mesenchymal cells, epithelial cells, and the extracellular matrix (2). Precise signals from mesenchymal cells regulate lung branching morphogenesis and lead to cell fate determination and the subsequent generation of cell type diversity in the lung epithelium. For example, mesenchymal cells secrete fibroblast growth factor 10 (3, 4). Fibroblast growth factor 10 in turn binds to its receptor, which is located on the surface of epithelial cells. The binding transduces a message to downstream signaling to regulate cell proliferation, differentiation, migration, and branching morphogenesis. Similarly, the epithelial cells also secrete signal molecules to interact with the mesenchyme. Sonic hedgehog (Shh) is growth factor expressed in the developing epithelium. Its receptor, Patched-1 (Ptc), is located on mesenchymal cells. The interaction between Shh and Ptc has been shown to be required for lung bud formation (5). Other growth factors such as pl...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Ptn Promotes Fetal Lung Branchingmentioning

confidence: 99%

Pleiotrophin Regulates Lung Epithelial Cell Proliferation and Differentiation during Fetal Lung Development via β-Catenin and Dlk1

Weng¹,

Gao

Bhaskaran

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…1 ers (human U6, H1, 7SK, and mouse U6) in a single vector (Fig. 1A) as described previously (17). Two sets of four siRNA sequences against rat annexin A2 were selected for constructing the K4 vectors for silencing annexin A2 (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Each RNA sample was treated with 1 g of DNase and was reverse-transcribed into cDNAs with 0.2 g/l (dT) 17 , 0.3 g/l random hexamer primer, and 200 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen). The real time PCR thermal conditions for all genes were 95°C for 15 min, followed by 40 cycles each at 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, and 77°C for 35 s (signal collection temperature).…”

Section: Methodsmentioning

confidence: 99%

Annexin A2 Interactions with Rab14 in Alveolar Type II Cells

Gou

Mishra

Weng

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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.The alveolar epithelium is composed of morphologically and functionally distinct type I and II cells. Type I cells provide the bulk of the surface area for gas exchange, whereas type II cells secrete lung surfactant to maintain mechanical stability of the alveoli (1-3). Lung surfactant is stored and secreted by exocytosis of lamellar bodies, which are specialized organelles found in type II cells. Exocytosis of the lamellar bodies is triggered by several intracellular signaling pathways. A key component of signaling involves the elevation of cytosolic Ca 2ϩ concentration in type II cells (4, 5). Because lung surfactant secretion is regulated by Ca 2ϩ signals, it is important to identify Ca 2ϩ -regulated proteins for exocytosis.Annexins are a family of Ca 2ϩ -dependent phospholipidbinding proteins. More than 50 different annexin isoforms have been identified (6, 7). Each annexin consists of a short variable N-terminal segment and a conserved C-terminal core domain. They have unique Ca 2ϩ -binding sites, which enable them to bind with negatively charged lipids. Annexins participate in many membrane-related events, such as the organization of membrane domains, the linkages of membrane-cytoskeleton, exocytosis and endocytosis, and the regulation of ion fluxes (7). Annexin A2 is abundant in alveolar type II cells. Several studies have indicated that annexin A2 is involved in Ca 2ϩ -regulated exocytosis (8 -10). In permeabilized chromaffin cells, the timedependent loss of secretory capacity can be blocked by the addition of annexin A2 to the culture medium (9). Knockdown of annexin A2 reduces the Ca 2ϩ -evoked exocytosis of WeibelPalade bodies in endothelial cells (10). In alveolar type II cells, accumulating evidence supports a role for annexin A2 in controlling the fusion of lamellar bodies with the plasma membrane and promoting surfactant secretion (11-14)...

show abstract

“…Adenoviral shRNA vectors were constructed as previously described (Gou et al, 2007). Two shRNA sequences targeting 574-592 and 669-689 of mouse P2X 7 R were chosen (Jun et al, 2007;Lu et al, 2007) and were named as si-X 7 (1) and si-X 7 (2).…”

Section: Knockdown Of P2x 7 Rmentioning

confidence: 99%

Purinergic P2X7 receptor regulates lung surfactant secretion in a paracrine manner

Mishra

Chintagari

Guo

et al. 2011

Journal of Cell Science

Self Cite

View full text Add to dashboard Cite

SummaryAlveolar 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 cocultured 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.

show abstract

A novel approach for the construction of multiple shRNA expression vectors

Cited by 40 publications

References 39 publications

Pleiotrophin Regulates Lung Epithelial Cell Proliferation and Differentiation during Fetal Lung Development via β-Catenin and Dlk1

Pleiotrophin Regulates Lung Epithelial Cell Proliferation and Differentiation during Fetal Lung Development via β-Catenin and Dlk1

Annexin A2 Interactions with Rab14 in Alveolar Type II Cells

Purinergic P2X7 receptor regulates lung surfactant secretion in a paracrine manner

Contact Info

Product

Resources

About