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Kage H.,Will Rogers Institute Pulmonary Research Center | Flodby P.,Will Rogers Institute Pulmonary Research Center | Gao D.,Will Rogers Institute Pulmonary Research Center | Kim Y.H.,Will Rogers Institute Pulmonary Research Center | And 8 more authors.
American Journal of Physiology - Lung Cellular and Molecular Physiology | Year: 2014

Claudins are tight junction proteins that regulate paracellular ion permeability of epithelium and endothelium. Claudin 4 has been reported to function as a paracellular sodium barrier and is one of three major claudins expressed in lung alveolar epithelial cells (AEC). To directly assess the role of claudin 4 in regulation of alveolar epithelial barrier function and fluid homeostasis in vivo, we generated claudin 4 knockout (Cldn4 KO) mice. Unexpectedly, Cldn4 KO mice exhibited normal physiological phenotype although increased permeability to 5-carboxyfluorescein and decreased alveolar fluid clearance were noted. Cldn4 KO AEC monolayers exhibited unchanged ion permeability, higher solute permeability, and lower short-circuit current compared with monolayers from wild-type mice. Claudin 3 and 18 expression was similar between wild-type and Cldn4 KO alveolar epithelial type II cells. In response to either ventilator-induced lung injury or hyperoxia, claudin 4 expression was markedly upregulated in wild-type mice, whereas Cldn4 KO mice showed greater degrees of lung injury. RNA sequencing, in conjunction with differential expression and upstream analysis after ventilator-induced lung injury, suggested Egr1, Tnf, and Il1b as potential mediators of increased lung injury in Cldn4 KO mice. These results demonstrate that claudin 4 has little effect on normal lung physiology but may function to protect against acute lung injury. © 2014 the American Physiological Society.


Pfaffenbach K.T.,University of Southern California | Pong M.,University of Southern California | Morgan T.E.,University of Southern California | Wang H.,Will Rogers Institute Pulmonary Research Center | And 4 more authors.
Journal of Cellular Physiology | Year: 2012

Glucose regulated protein 78/immunoglobulin binding protein (GRP78/BiP) is an endoplasmic reticulum (ER) chaperone protein and master regulator of the unfolded protein response (UPR). The response of GRP78 to overt pharmacologically induced ER stress is well established, whereas the modulation of GRP78 to physiologic changes is less characterized. In this study, we examined the regulation of GRP78 in response to reduced IGF-1 growth factor signaling, a common consequence of calorie restriction (CR). ER chaperone protein expression was quantified in cell lysates prepared from the livers of calorie restricted (CR) and ad libitum fed mice, as well as MEFs grown in normal medium or serum starved. The requirement of IGF-1 signaling on GRP78 expression was studied using MEFs with IGF-1 receptor overexpression (R+) or deletion (R-), and the regulatory mechanism was examined using mTORC1 and PI3K inhibitors, as well as R- cells with knockdown of transcription factor FOXO1 compared to shRNA control. We observed a 40% reduction in GRP78 protein expression in CR mice and in serum-starved MEF cells. R- cells had drastically reduced AKT phosphorylation and exhibited lower levels of ER chaperones, in particular 80% less GRP78. Despite an 80% reduction in GRP78 expression, R- cells were not under chronic ER stress, but were fully capable of activating the UPR. Neither forced expression of FOXO1-AAA nor knockdown of FOXO1 in R- cells affected GRP78 expression. In conclusion, we report that IGF-1 receptor signaling regulates GRP78 expression via the PI3K/AKT/mTORC1 axis independent of the canonical UPR and FOXO1. © 2012 Wiley Periodicals, Inc.


PubMed | Biochemistry and Molecular Biology and., Washington University in St. Louis, University of Southern California and Will Rogers Institute Pulmonary Research Center
Type: Journal Article | Journal: The Journal of biological chemistry | Year: 2016

Maintenance of stem/progenitor cell-progeny relationships is required for tissue homeostasis during normal turnover and repair. Wnt signaling is implicated in both maintenance and differentiation of adult stem/progenitor cells, yet how this pathway serves these dichotomous roles remains enigmatic. We previously proposed a model suggesting that specific interaction of -catenin with either of the homologous Kat3 co-activators, p300 or CREB-binding protein, differentially regulates maintenance versus differentiation of embryonic stem cells. Limited knowledge of endogenous mechanisms driving differential -catenin/co-activator interactions and their role in adult somatic stem/progenitor cell maintenance versus differentiation led us to explore this process in defined models of adult progenitor cell differentiation. We focused primarily on alveolar epithelial type II (AT2) cells, progenitors of distal lung epithelium, and identified a novel axis whereby WNT5a/protein kinase C (PKC) signaling regulates specific -catenin/co-activator interactions to promote adult progenitor cell differentiation. p300/-catenin but not CBP/-catenin interaction increases as AT2 cells differentiate to a type I (AT1) cell-like phenotype. Additionally, p300 transcriptionally activates AT1 cell-specific gene Aqp-5. IQ-1, a specific inhibitor of p300/-catenin interaction, prevents differentiation of not only primary AT2 cells, but also tracheal epithelial cells, and C2C12 myoblasts. p300 phosphorylation at Ser-89 enhances p300/-catenin interaction, concurrent with alveolar epithelial cell differentiation. WNT5a, a traditionally non-canonical WNT ligand regulates Ser-89 phosphorylation and p300/-catenin interactions in a PKC-dependent manner, likely involving PKC. These studies identify a novel intersection of canonical and non-canonical Wnt signaling in adult progenitor cell differentiation that has important implications for targeting -catenin to modulate adult progenitor cell behavior in disease.


PubMed | Los Angeles CountyUniversity of Southern California Medical Center and Childrens Hospital Los Angeles, Will Rogers Institute Pulmonary Research Center and Saban Research Institute
Type: Journal Article | Journal: Journal of biochemistry | Year: 2015

The role of WNT signalling in metazoan organogenesis has been a topic of widespread interest. In the lung, while the role of canonical WNT signalling has been examined in some detail by multiple studies, the non-canonical WNT signalling has received limited attention. Reliable evidence shows that this important signalling mechanism constitutes a major regulatory pathway in lung development. In addition, accumulating evidence has also shown that the non-canonical WNT pathway is critical for maintaining lung homeostasis and that aberrant activation of this pathway may underlie several debilitating lung diseases. Functional analyses have further revealed that the non-canonical WNT pathway regulates multiple cellular activities in the lung that are dependent on the specific cellular context. In most cell types, non-canonical WNT signalling regulates canonical WNT activity, which is also critical for many aspects of lung biology. This review will summarize what is currently known about the role of non-canonical WNT signalling in lung development, homeostasis and pathogenesis of disease.


Flodby P.,Will Rogers Institute Pulmonary Research Center | Borok Z.,Will Rogers Institute Pulmonary Research Center | Banfalvi A.,Will Rogers Institute Pulmonary Research Center | Zhou B.,Will Rogers Institute Pulmonary Research Center | And 5 more authors.
American Journal of Respiratory Cell and Molecular Biology | Year: 2010

Pulmonary alveolar epithelium is comprised of two morphologically and functionally distinct cell types, alveolar epithelial type (AT) I and AT2 cells. Genetically modified mice with cell-specific Cre/loxP-mediated knockouts of relevant genes in each respective cell type would be useful to help elucidate the relative contributions of AT1 versus AT2 cells to alveolar homeostasis. Cre has previously been efficiently expressed in AT2 cells in mouse lung with the surfactant protein (SP)-C promoter; however, no transgenic mouse expressing Cre in AT1 cells has so far been available. To develop an AT1 cell-specific transgenic Cre mouse, we generated a knockin of a Cre-IRES-DsRed cassette into exon 1 of the endogenous aquaporin 5 (Aqp5) gene, a gene expressed specifically in AT1 cells in the distal lung epithelium, resulting in the mouse line, Aqp5-Cre-IRES-DsRed (ACID). Endogenous Aqp5 and transgenic Cre in ACID mice showed a very similar pattern of tissue distribution by RT-PCR. To analyze Cre activity, ACID was crossed to two Cre reporter strains, R26LacZ and mT/mG. Double-transgenic offspring demonstrated reporter gene expression in a very high fraction of AT1 cells in the distal lung, whereas AT2 cells were negative. As expected, variable reporter expression was detected in several other tissues where endogenous Aqp5 is expressed (e.g., submandibular salivary gland and stomach). ACID mice should be of major utility in analyzing the functional contribution of AT1 cells to alveolar epithelial properties in vivo with Cre/loxP-mediated gene deletion technology.


Shi J.,U.S. National Cancer Institute | Marconett C.N.,University of Southern California | Duan J.,University of Illinois at Chicago | Hyland P.L.,U.S. National Cancer Institute | And 28 more authors.
Nature communications | Year: 2014

The genetic regulation of the human epigenome is not fully appreciated. Here we describe the effects of genetic variants on the DNA methylome in human lung based on methylation-quantitative trait loci (meQTL) analyses. We report 34,304 cis- and 585 trans-meQTLs, a genetic-epigenetic interaction of surprising magnitude, including a regulatory hotspot. These findings are replicated in both breast and kidney tissues and show distinct patterns: cis-meQTLs mostly localize to CpG sites outside of genes, promoters and CpG islands (CGIs), while trans-meQTLs are over-represented in promoter CGIs. meQTL SNPs are enriched in CTCF-binding sites, DNaseI hypersensitivity regions and histone marks. Importantly, four of the five established lung cancer risk loci in European ancestry are cis-meQTLs and, in aggregate, cis-meQTLs are enriched for lung cancer risk in a genome-wide analysis of 11,587 subjects. Thus, inherited genetic variation may affect lung carcinogenesis by regulating the human methylome.


PubMed | Management Information Services Inc., Dana-Farber Cancer Institute, University of Southern California, University of Milan and 6 more.
Type: | Journal: Nature communications | Year: 2014

The genetic regulation of the human epigenome is not fully appreciated. Here we describe the effects of genetic variants on the DNA methylome in human lung based on methylation-quantitative trait loci (meQTL) analyses. We report 34,304 cis- and 585 trans-meQTLs, a genetic-epigenetic interaction of surprising magnitude, including a regulatory hotspot. These findings are replicated in both breast and kidney tissues and show distinct patterns: cis-meQTLs mostly localize to CpG sites outside of genes, promoters and CpG islands (CGIs), while trans-meQTLs are over-represented in promoter CGIs. meQTL SNPs are enriched in CTCF-binding sites, DNaseI hypersensitivity regions and histone marks. Importantly, four of the five established lung cancer risk loci in European ancestry are cis-meQTLs and, in aggregate, cis-meQTLs are enriched for lung cancer risk in a genome-wide analysis of 11,587 subjects. Thus, inherited genetic variation may affect lung carcinogenesis by regulating the human methylome.


PubMed | Norris Comprehensive Cancer Center, University of Southern California, Southern Research Institute, Comprehensive Care and Will Rogers Institute Pulmonary Research Center
Type: Journal Article | Journal: American journal of physiology. Lung cellular and molecular physiology | Year: 2014

Claudins are tight junction proteins that regulate paracellular ion permeability of epithelium and endothelium. Claudin 4 has been reported to function as a paracellular sodium barrier and is one of three major claudins expressed in lung alveolar epithelial cells (AEC). To directly assess the role of claudin 4 in regulation of alveolar epithelial barrier function and fluid homeostasis in vivo, we generated claudin 4 knockout (Cldn4 KO) mice. Unexpectedly, Cldn4 KO mice exhibited normal physiological phenotype although increased permeability to 5-carboxyfluorescein and decreased alveolar fluid clearance were noted. Cldn4 KO AEC monolayers exhibited unchanged ion permeability, higher solute permeability, and lower short-circuit current compared with monolayers from wild-type mice. Claudin 3 and 18 expression was similar between wild-type and Cldn4 KO alveolar epithelial type II cells. In response to either ventilator-induced lung injury or hyperoxia, claudin 4 expression was markedly upregulated in wild-type mice, whereas Cldn4 KO mice showed greater degrees of lung injury. RNA sequencing, in conjunction with differential expression and upstream analysis after ventilator-induced lung injury, suggested Egr1, Tnf, and Il1b as potential mediators of increased lung injury in Cldn4 KO mice. These results demonstrate that claudin 4 has little effect on normal lung physiology but may function to protect against acute lung injury.


PubMed | 5 Biochemistry and Molecular Biology and., 2 Pediatrics., 3 Surgery and. and Will Rogers Institute Pulmonary Research Center
Type: Journal Article | Journal: American journal of respiratory cell and molecular biology | Year: 2016

Bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity, has been linked to endoplasmic reticulum (ER) stress. To investigate a causal role for ER stress in BPD pathogenesis, we generated conditional knockout (KO) mice (cGrp78(f/f)) with lung epithelial cell-specific KO of Grp78, a gene encoding the ER chaperone 78-kD glucose-regulated protein (GRP78), a master regulator of ER homeostasis and the unfolded protein response (UPR). Lung epithelial-specific Grp78 KO disrupted lung morphogenesis, causing developmental arrest, increased alveolar epithelial type II cell apoptosis, and decreased surfactant protein and type I cell marker expression in perinatal lungs. cGrp78(f/f) pups died immediately after birth, likely owing to respiratory distress. Importantly, Grp78 KO triggered UPR activation with marked induction of the proapoptotic transcription factor CCAAT/enhancer-binding proteins (C/EBP) homologous protein (CHOP). Increased expression of genes involved in oxidative stress and cell death and decreased expression of genes encoding antioxidant enzymes suggest a role for oxidative stress in alveolar epithelial cell (AEC) apoptosis. Increased Smad3 phosphorylation and expression of transforming growth factor-/Smad3 targets Cdkn1a (encoding p21) and Gadd45a suggest that interactions among the apoptotic arm of the UPR, oxidative stress, and transforming growth factor-/Smad signaling pathways contribute to Grp78 KO-induced AEC apoptosis and developmental arrest. Chemical chaperone Tauroursodeoxycholic acid reduced UPR activation and apoptosis in cGrp78(f/f) lungs cultured ex vivo, confirming a role for ER stress in observed AEC abnormalities. These results demonstrate a key role for GRP78 in AEC survival and gene expression during lung development through modulation of ER stress, and suggest the UPR as a potential therapeutic target in BPD.


PubMed | Will Rogers Institute Pulmonary Research Center and Medical Center and Childrens Hospital Los Angeles
Type: | Journal: BMC biology | Year: 2016

Epithelial-mesenchymal cross talk is centerpiece in the development of many branched organs, including the lungs. The embryonic lung mesoderm provides instructional information not only for lung architectural development, but also for patterning, commitment and differentiation of its many highly specialized cell types. The mesoderm also serves as a reservoir of progenitors for generation of differentiated mesenchymal cell types that include SMA-expressing fibroblasts, lipofibroblasts, endothelial cells and others. Transforming Growth Factor (TGF) is a key signaling pathway in epithelial-mesenchymal cross talk. Using a cre-loxP approach we have elucidated the role of the TGF type I receptor tyrosine kinase, ALK5, in epithelial-mesenchymal cross talk during lung morphogenesis.Targeted early inactivation of Alk5 in mesodermal progenitors caused abnormal development and maturation of the lung that included reduced physical size of the sub-mesothelial mesoderm, an established source of specific mesodermal progenitors. Abrogation of mesodermal ALK5-mediated signaling also inhibited differentiation of cell populations in the epithelial and endothelial lineages. Importantly, Alk5 mutant lungs contained a reduced number of SMA(pos) cells and correspondingly increased lipofibroblasts. Elucidation of the underlying mechanisms revealed that through direct and indirect modulation of target signaling pathways and transcription factors, including PDGFR, PPAR, PRRX1, and ZFP423, ALK5-mediated TGF controls a process that regulates the commitment and differentiation of SMA(pos) versus lipofibroblast cell populations during lung development.ALK5-mediated TGF signaling controls an early pathway that regulates the commitment and differentiation of SMA(pos) versus LIF cell lineages during lung development.

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