Claudin 1 Mediates TNFα-Induced Gene Expression and Cell Migration in Human Lung Carcinoma Cells

Epithelial-mesenchymal transition (EMT) is an important mechanism in carcinogenesis. To determine the mechanisms that are involved in the regulation of EMT, it is crucial to develop new biomarkers and therapeutic targets towards cancers. In this study, when TGFβ1 and TNFα were used to induce EMT in human lung carcinoma A549 cells, we found an increase in an epithelial cell tight junction marker, Claudin 1. We further identified that it was the TNFα and not the TGFβ1 that induced the fibroblast-like morphology changes. TNFα also caused the increase in Claudin-1 gene expression and protein levels in Triton X-100 soluble cytoplasm fraction. Down-regulation of Claudin-1, using small interfering RNA (siRNA), inhibited 75% of TNFα-induced gene expression changes. Claudin-1 siRNA effectively blocked TNFα-induced molecular functional networks related to inflammation and cell movement. Claudin-1 siRNA was able to significantly reduce TNF-enhanced cell migration and fibroblast-like morphology. Furthermore, over expression of Claudin 1 with a Claudin 1-pcDNA3.1/V5-His vector enhanced cell migration. In conclusion, these observations indicate that Claudin 1 acts as a critical signal mediator in TNFα-induced gene expression and cell migration in human lung cancer cells. Further analyses of these cellular processes may be helpful in developing novel therapeutic strategies

Functional Redundancy of Two Pax-Like Proteins in Transcriptional Activation of Cyst Wall Protein Genes in Giardia lamblia

The protozoan Giardia lamblia differentiates from a pathogenic trophozoite into an infectious cyst to survive outside of the host. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately induced. Pax family transcription factors are involved in a variety of developmental processes in animals. Nine Pax proteins have been found to play an important role in tissue and organ development in humans. To understand the progression from primitive to more complex eukaryotic cells, we tried to identify putative pax genes in the G. lamblia genome and found two genes, pax1 and pax2, with limited similarity. We found that Pax1 may transactivate the encystation-induced cwp genes and interact with AT-rich initiatior elements that are essential for promoter activity and transcription start site selection. In this study, we further characterized Pax2 and found that, like Pax1, Pax2 was present in Giardia nuclei and it may specifically bind to the AT-rich initiator elements of the encystation-induced cwp1-3 and myb2 genes. Interestingly, overexpression of Pax2 increased the cwp1-3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of nuclear localization, DNA-binding activity, and transactivation activity of Pax2. These results are similar to those found in the previous Pax1 study. In addition, the profiles of gene expression in the Pax2 and Pax1 overexpressing cells significantly overlap in the same direction and ERK1 associated complexes may phosphorylate Pax2 and Pax1, suggesting that Pax2 and Pax1 may be downstream components of a MAPK/ERK1 signaling pathway. Our results reveal functional redundancy between Pax2 and Pax1 in up-regulation of the key encystation-induced genes. These results illustrate functional redundancy of a gene family can occur in order to increase maintenance of important gene function in the protozoan organism G. lamblia

Health relevance of the modification of low grade inflammation in ageing (inflammageing) and the role of nutrition

Ageing of the global population has become a public health concern with an important socio-economic dimension. Ageing is characterized by an increase in the concentration of inflammatory markers in the bloodstream, a phenomenon that has been termed "inflammageing". The inflammatory response is beneficial as an acute, transient reaction to harmful conditions, facilitating the defense, repair, turnover and adaptation of many tissues. However, chronic and low grade inflammation is likely to be detrimental for many tissues and for normal functions. We provide an overview of low grade inflammation (LGI) and determine the potential drivers and the effects of the "inflamed" phenotype observed in the elderly. We discuss the role of gut microbiota and immune system crosstalk and the gut-brain axis. Then, we focus on major health complications associated with LGI in the elderly, including mental health and wellbeing, metabolic abnormalities and infections. Finally, we discuss the possibility of manipulating LGI in the elderly by nutritional interventions. We provide an overview of the evidence that exists in the elderly for omega-3 fatty acid, probiotic, prebiotic, antioxidant and polyphenol interventions as a means to influence LGI. We conclude that slowing, controlling or reversing LGI is likely to be an important way to prevent, or reduce the severity of, age-related functional decline and the onset of conditions affecting health and well-being; that there is evidence to support specific dietary interventions as a strategy to control LGI; and that a continued research focus on this field is warranted

gPax2, a Pax homologue in Giardia lamblia, involved in transcriptional regulation of cyst wall protein 1 and 2 genes

Pax蛋白質家族是一個很大的轉錄因子群，普遍存在於脊椎動物中，主要參與胚胎發育和器官生成。目前的研究中，在原蟲類中並未發現有類似pax基因存在；而我們在梨形鞭毛蟲資料庫中搜尋到兩個類似Pax的蛋白質，gPax1和gPax2。不同於脊椎動物Pax蛋白質家族之paired DNA-binding domain位於N端，gPax1和gPax2的paired domain位於C端。 們發現內生性gpax2 RNA表現量在梨形鞭毛蟲囊體化24小時時期表現會較滋養體時期低。我們製作可大量表現gpax2的質體，並在其C端加有HA-tag，轉染至wild type梨形鞭毛蟲中。我們利用HA抗體作西方墨點法得知HA tagged gPax2在滋養體時期和囊體化24小時時期之蛋白質表現量差不多。其次，我們經由免疫螢光染色觀察到HA tagged gPax2在梨形鞭毛蟲滋養體時期和囊體化24小時時期皆表現於細胞核中。我們利用電泳遷移率分析發現gPax2會專一性結合於梨形鞭毛蟲兩種生活型態轉換的指標性基因-cyst wall protein 1 (cwp1)和cwp2基因之啟動子，這兩個基因會在囊體化時大量表現，皆為組成囊壁的成份之一。我們製作一個突變體，將其C端的paired domain去除，gPax2del，發現gPax2del即失去和cwp1和cwp2基因啟動子結合的能力，而且其在細胞核表現量有變少，散佈在細胞質的vesicles中。大量表現gPax2的細胞株，相較於vector control的細胞株，cwp1和cwp2基因表現的mRNA和Cwp1蛋白質的量增加。相較於大量表現gPax2細胞株，大量表現gPax2del的細胞株則會使cwp1和cwp2基因的mRNA表現量減少，Cwp1蛋白質也觀察到有減少。我們認為gPax2可能參與cwp1和cwp2基因的轉錄調控，且其paired domain也會影響cwp1和cwp2基因表現。另外我們在paired domain中找到兩段疑似nuclear localization signal (NLS)片段，分別將其序列上的正價性胺基酸作突變體，gPax2m1和gPax2m2，大量表gPax2m1和gPax2m2的細胞株中，皆發現gPax2m1和gPax2m2在細胞核的表現量有變少，而且都明顯表現在梨形鞭毛蟲吸盤周圍，我們認為此二段區域可能是gPax2的NLS。和大量表現gPax2細胞株相比，大量表現gPax2m1的細胞株則會使cwp1和cwp2基因的RNA表現量減少，Cwp1蛋白質也觀察到有減少；而大量表現gPax2m2的細胞株則會使cwp1和cwp2基因的RNA表現量增加，Cwp1蛋白質也觀察到有增加。我們認為gPax2的paired domain中存有NLS，並且也會影響cwp1和cwp2基因的表現。們推論，gPax2會促進cwp1和cwp2基因的表現，而且其C端的paired domain可能參與該調控。Pax protein family is a large group of transcription factors in vertebrates. They are mainly involved in embryogenesis and organogenesis. To date, the pax gene has still not been found in protozoa. We have found two Pax-like open reading frames, gPax1 and gPax2, in Giardia lamblia data base. Unlike Pax proteins from vertebrates which have a paired DNA-binding domain near the N terminus, the paired domains of gPax1 and gPax2 were located near the C terminus.e found that the mRNA expression levels of endogenous gpax2 gene during 24 hours encystation were lower than that during vegetative growth in G. lamblia. We transfected a construct that expressed HA-tagged gpax2 into wild type G. lamblia. Western blot showed that HA-tagged gPax2 was expressed at similar levels during vegetative growth and during 24 hr encystation stage in G. lamblia. Immunofluorescence assay showed that the HA-tagged gPax2 was located in nuclei during vegetative growth and 24 hr encystation in G. lamblia. By electrophoretic mobility shift assays, we found that gPax2 specifically bound to the promoters of cyst wall protein 1 (cwp1) and cwp2 which are important genes during stages-changing in G. lamblia. Cwp1 and Cwp2 are two components of cyst wall. The expression of the cwp1and cwp2 mRNA was induced during encystation in G. lamblia. We made a mutant, gPax2del, which lacks the C-terminal paired domain. We found that gPax2del could not bind to the cwp1 and cwp2 promoters. gPax2del was expressed in some vesicles in cytosol, and gPax2del proteins were hardly detected in nuclei. We found that the levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax2 overexpressing cell line increased significantly relative to the levels in the vector control cell line. In addition, we found that the levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax2del overexpressing cell line decreased significantly relative to the levels in the gPax2 overexpressing cell line. We suggest that gPax2 might be involved in the transcriptional regulation of the cwp1 and cwp2 genes, and the paired domain would contribute partially to this regulation.e found two stretches of basic amino acids that may be nuclear localization signals (NLS)-like sequences in the paired domain of gPax2. We made two mutants, gPax2m1 and gPax2m2, by mutating the positive charge amino acids to neutral amino acids. We found that gPax2m1 and gPax2m2 were located in cytosol, and around the sucking disk, but gPax2m1 and gPax2m2 were hardly detected in nuclei. This suggests that these two stretches of basic amino acids in the paired domain could be NLSs. We found that the levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax2m1 overexpressing cell line decreased significantly relative to the levels in the gPax2 overexpressing cell line. The levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax2m2 overexpressing cell line increased significantly relative to the levels in the gPax2 overexpressing cell line. We suggest that two NLSs may be present in the paired domain of gPax2 and they could influence the cwp1 and cwp2 gene expression.e suggest that gPax2 can up-regulate the cwp1 and cwp2 gene expression and the paired domain of gPax2 might be involved in this regulation.摘要 Ibstract III一章 序論 1.1 梨形鞭毛蟲簡介 1.2 Pax蛋白質家族簡介 2.3 本篇論文重點 4二章 方法與步驟 5.1 梨形鞭毛蟲的培養 5.2 質體建構 5.3 質體的轉型與萃取 7.4 梨形鞭毛蟲的轉染(transfection)與選殖(selection) 8.5重組gPax2蛋白質及其突變體蛋白質的質體建構、表現和純化 8.6電泳遷移分析 Electrophoretic Mobility Shift Assays (EMSA) 10.7 RT-PCR和real-time PCR 11.8 西方墨點法和Coomassie blue染色 12.9 免疫螢光染色(Immunofluorescence assay, IFA) 13.10 囊體計數(cyst count) 13三章 實驗結果 15.1 鑑別梨形鞭毛蟲之gpax2基因 15.2 在不同時期的梨形鞭毛蟲中的gpax2基因表現及表現位置 16.3 gPax2可利用paired domain專一性結合cyst wall protein 1基因啟動子 17.4 鑑定gPax2與cwp1基因啟動子之結合位置 19.5 鑑定gPax2之nuclear localization signal (NLS) 20.6 gPax2和其突變株對cwp1表現的影響 21四章討論 23.1 梨形鞭毛蟲基因體中的Pax同源蛋白質 23.2 gPax2在梨形鞭毛蟲中的表現情形 24.3 gPax2會結合AT-rich序列 24.4 鑑定gPax2的nuclear localization signal (NLS) 25.5 gPax2 paired domain的構形會影響DNA結合能力 25.6 gPax2和gPax1之比較 26.7 gPax2可能在梨形鞭毛蟲中所扮演的角色 27.8 預測gPax2的transactivation domain位置 28五章附錄 29六章 附表 46七章參考文獻 4

Recruitment of Pax2 to the <i>cwp1-3</i> and <i>myb2</i> promoters.

<p>(A) Microarray analysis. Microarray data were obtained from the 5′▵5N-Pac and pPPax1 (or pPPax2) cell lines during vegetative growth. Fold changes are shown as the ratio of transcript levels in the pPPax1 (or pPPax2) cell line relative to the 5′▵5N-Pac cell line. Results are expressed as the means ± standard error of at least three separate experiments. (B) Pax2 and Pax1 overexpression generated similar gene expression patterns. The Venn diagrams illustrate the overlap of altered gene expression between the Pax2 and Pax1 overexpressing cells. Thirty eight and 185 genes were up-regulated (i.e. increased levels of gene expression relative to the control) in the Pax1 and Pax2 overexpressing cells, respectively. Among them, nineteen genes overlap. Fifty four and 172 genes were down-regulated in the Pax1 and Pax2 overexpressing cells, respectively. Among them, thirty genes overlap. (C) ChIP assays. The non-transfected WB cells were cultured in growth medium for 24 h and then subjected to ChIP assays. Anti-Pax2 antibody was used to assess binding of Pax2 to endogenous gene promoters. Preimmune serum was used as a negative control. Immunoprecipitated chromatin was analyzed by PCR using primers that amplify the 5′-flanking region of specific genes. At least three independent experiments were performed. Representative results are shown. Immunoprecipitated products of Pax2 yielded more PCR products of <i>pax2</i>, <i>cwp1</i>, <i>cwp2</i>, <i>cwp3</i>, <i>myb2</i>, and <i>ran</i> promoters, indicating that Pax2 was bound to these promoters. The <i>18S ribosomal RNA</i> gene promoter was used as a negative control for our ChIP analysis.</p

Phosphorylation of Pax2 and Pax1 proteins by ERK1 associated complexes.

<p>(A) Diagrams of the pPERK1 and pPERK1m plasmids. The <i>pac</i> gene (open box) expression cassette is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone-0030614-g002" target="_blank">Figure 2C</a>. The <i>erk1</i> gene is under the control of its own 5′-flanking region (open boxes) and the 3′-flanking region of the <i>ran</i> gene (dotted box). ERK1m does not contain the predicted kinase domain (residues 26–202)(gray box). The filled box indicates the coding sequence of the HA epitope tag. (B) Similar levels of immunoprecipitated ERK1 protein from the vegetative and encysting pPERK1 cultures used in kinase assays. The pPERK1 stable transfectants were cultured in growth (Veg, vegetative growth) or encystation medium for 24 h (Enc, encystation) and then subjected to IP-kinase assays using anti-HA antibody. The addition of similar levels of the HA-tagged ERK1 protein from the vegetative and encysting pPERK1 cultures in each kinase reaction was confirmed by Western blot using an anti-HA antibody (left panel). The addition of similar levels of the HA-tagged ERK1 protein from the encysting pPERK1, and pPERK1m cultures in each kinase reaction was confirmed by Western blot using an anti-HA antibody (right panel). Equal amounts of protein loading were confirmed by SDS-PAGE and Coomassie blue staining. (C) Encystation-induced kinase activity of ERK1 for Pax2 substrate. The pPERK1 stable transfectants were cultured in growth (Veg, vegetative growth) or encystation medium for 24 h (Enc, encystation) and then subjected to IP-kinase assays using anti-HA antibody. Kinase activity was measured using purified recombinant Pax2 as a substrate. As a negative control, an IP-kinase assay was performed with the encysting 5′▵5N-Pac cultures which did not express the HA-tagged ERK1 protein (lane 5). Another IP-kinase assay was performed with the encysting pPERK1m cultures which expressed the ERK1m-HA protein without the predicted kinase domain (residues 26–202) (lane 7). To account for ERK1 autophosphorylation, an additional control without substrate but with immunoprecipitated ERK1-HA was also included (lane 1). (D) Encystation-induced kinase activity of ERK1 for Pax1 substrate. IP-kinase assays were performed as described above, except that Pax1 substrate was used.</p

Detection of Pax2 binding sites in multiple promoters.

<p>Electrophoretic mobility shift assays were performed using purified Pax2 and various ³²P-end-lableled oligonucleotide probes as described. Components in the binding reaction mixtures are indicated above the lanes. The arrowhead indicates the shifted complex. The transcription start sites of the <i>cwp2</i>, <i>cwp3</i>, and <i>myb2</i> genes determined from 24-h encysting cells are indicated by asterisks <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Lujan1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Sun1" target="_blank">[14]</a>. The transcription start sites of the <i>ran</i> gene determined from vegetative cells are indicated by asterisks <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Sun2" target="_blank">[26]</a>. The AT-rich Inr elements spanning the transcription start sites are underlined. The translation start sites of the <i>cwp2</i> and <i>cwp3</i> genes are framed. “18S” represents 18S ribosomal RNA. “+”, “+/−”, and “−” represent moderate binding, weak binding, and no binding, respectively. “+++” and “++” represent strong binding.</p

Domain architecture of Pax2 protein and alignment of the paired domains.

<p>(A) Schematic representation of the giardial Pax2 protein. The gray boxes indicate the paired domains. (B) Alignment of the paired domains. The paired domains from members of the human and <i>Drosophila</i> Pax family are analyzed by ClustalW 1.83 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Chenna1" target="_blank">[80]</a>. GenBank accession numbers for human Pax1 to 9 and <i>Drosophila</i> Prd are NM_006192, NM_000278, NM_181458, NM_006193, NM_016734, NM_000280, NM_001135254, NM_003466, NM_006194, and NM_164990, respectively. The open reading frame numbers (GenBank accession numbers) for the giardial Pax1 and Pax2 are 32686 (XM_001704983.1) and 16640 (XM_001709076.1) in the <i>G. lamblia</i> genome data base, respectively. Letters in black boxes, letters in gray boxes, and hyphens indicate identical amino acids, similar amino acids and gaps in the respective proteins, respectively. Gray boxes indicate the α helices in the paired domain of human Pax6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Xu1" target="_blank">[58]</a>. The arrows indicate the key residues contacting the major groove in human Pax6 or <i>Drosophila</i> Prd <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Xu1" target="_blank">[58]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Xu2" target="_blank">[62]</a>. The arrowheads indicate the residues that make contact with the minor groove/phosphodiester backbone in human Pax6 or <i>Drosophila</i> Prd <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Xu1" target="_blank">[58]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Xu2" target="_blank">[62]</a>. Two regions (residues 185–205 and 226–248) rich in basic amino acid residues are underlined by dotted lines. (C) Alignment of N-terminal regions of giardial Pax2 and Pax1 ClustalW 1.83 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030614#pone.0030614-Chenna1" target="_blank">[80]</a>. Letters in black boxes, letters in gray boxes, and hyphens indicate identical amino acids, similar amino acids and gaps in the respective proteins, respectively.</p