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1.  Regulation of cell-matrix adhesion by OLA1, the Obg-like ATPase 1 
Attachment of cells to the extracellular matrix induces clustering of membrane receptor integrins which in turn triggers the formation of focal adhesions (FAs). The adaptor/scaffold proteins in FAs provide linkage to actin cytoskeleton, whereas focal adhesion kinase (FAK) and other FA-associated kinases and phosphatases transduce integrin-mediated signaling cascades, promoting actin polymerization and progression of cell spreading. In this study, we explored the role of OLA1, a newly identified member of Obg-like ATPases, in regulating cell adhesion processes. We showed that in multiple human cell lines RNAi-mediated downregulation of OLA1 significantly accelerated cell adhesion and spreading, and conversely overexpression of OLA1 by gene transfection resulted in delayed cell adhesion and spreading. We further found that OLA1-deficient cells had elevated levels of FAK protein and decreased Ser3 phosphorylation of cofilin, an actin-binding protein and key regulator of actin filament dynamics, while OLA1-overexpressing cells exhibited the opposite molecular alterations in FAK and cofilin. These findings suggest that OLA1 plays an important negative role in cell adhesion and spreading, in part through the regulation of FAK expression and cofilin phosphorylation, and manipulation of OLA1 may lead to significant changes in cell adhesion and the associated phenotypes.
PMCID: PMC3959106  PMID: 24486488
OLA1; cell-matrix adhesion; cell spreading; FAK; cofilin
2.  PITX2 associates with PTIP-containing histone H3 lysine 4 methyltransferase complex 
Pituitary homeobox 2 (PITX2), a Paired-like homeodomain transcription factor and a downstream effector of Wnt/β-catenin signaling, plays substantial roles in embryonic development and human disorders. The mechanism of its functions, however, is not fully understood. In this study, we demonstrated that PITX2 associated with histone H3 lysine 4 (H3K4) methyltransferase (HKMT) mixed-lineage leukemia 4 (MLL4/KMT2D), Pax transactivation domain-interacting protein (PTIP), and other H3K4 HKMT core subunits. This association of PITX2 with H3K4 HKMT complex was dependent on PITX2’s homeodomain. Consistently, the PITX2 protein complex was shown to possess H3K4 HKMT activity. Furthermore, the chromatin immunoprecipitation result revealed co-occupancy of PITX2 and PTIP on the promoter of the PITX2’s transcriptional target. Taken together, our data provide new mechanistic perspectives on PITX2’s functions and its related biological processes.
PMCID: PMC3963365  PMID: 24486544
PITX2; MLL4/KMT2D; PTIP; histone H3 lysine 4 (H3K4); histone lysine methyltransferase (HKMT)
3.  The β-domain of cluster 2b streptokinase is a major determinant for the regulation of its plasminogen activation activity by cellular plasminogen receptors 
Cluster 2b streptokinase (SK2b), secreted by invasive skin-trophic strains of Streptococcus pyogenes (GAS), is a human plasminogen (hPg) activator that optimally functions when human plasma hPg is bound, via its kringle-2 domain, to cognizant bacterial cells through the a1a2 domain of the major cellular hPg receptor, Plasminogen-binding group A streptococcal M-like protein (PAM). Another class of streptokinases (SK1), secreted primarily by GAS strains that possess affinity for pharyngeal infections, does not require PAM-bound hPg for optimal activity. We find herein that replacement of the central β-domain of SK2b with the same module from SK1 reduces the dependency of SK2b on PAM, and the converse is true when the β-domain of SK1 is replaced with this same region of SK2b. These data suggest that simple evolutionary shuttling of protein domains in GAS can be employed by GAS to rapidly generate strains that differ in tissue tropism and invasive capability and allow the bacteria to survive different challenges by the host.
PMCID: PMC3984925  PMID: 24486550
Streptokinase; Streptococcus pyogenes; bacterial virulence; human plasminogen; protein domains
4.  Augmenting podocyte injury promotes advanced diabetic kidney disease in Akita mice 
To determine if augmenting podocyte injury promotes the development of advanced diabetic nephropathy (DN), we created mice that expressed the enzyme cytosine deaminase (CD) specifically in podocytes of diabetic Akita mice (Akita-CD mice). In these mice, treatment with the prodrug 5-flucytosine (5-FC) causes podocyte injury as a result of conversion to the toxic metabolite 5-fluorouracil (5-FU). We found that treatment of 4-5 week old Akita mice with 5-FC for 5 days caused robust albuminuria at 16 and 20 weeks of age compared to 5-FC treated Akita controls, which do not express CD (Akita CTLs). By 20 weeks of age, there was a significant increase in mesangial expansion in Akita-CD mice compared to Akita CTLs, which was associated with a variable increase in glomerular basement membrane (GBM) width and interstitial fibrosis. At 20 weeks of age, podocyte number was similarly reduced in both groups of Akita mice, and was inversely correlated with the albuminuria and mesangial expansion. Thus, enhancing podocyte injury early in the disease process promotes the development of prominent mesangial expansion, interstitial fibrosis, increased GBM thickness and robust albuminuria. These data suggest that podocytes play a key role in the development of advanced features of diabetic kidney disease.
PMCID: PMC3985087  PMID: 24491571
diabetes mellitus; diabetic nephropathy; podocyte
5.  Silencing of Cav1.2 gene in neonatal cardiomyocytes by lentiviral delivered shRNA 
Cav1.2 (α1C) and Cav1.3 (α1D) L-type Ca channels are co-expressed in the heart. To date, there are no pharmacological or biophysical tools to separate α1D from α1C Ca currents (ICa-L) in cardiomyocytes. Here, we established a physiological model to study α1D ICa-L in native myocytes using RNA interference. Transfection of rat neonatal cardiomyocytes (RNC) with α1C specific siRNA resulted in low silencing efficiency (50–60%) at the mRNA and protein levels. The use of lentivirus shRNA resulted in 100% transfection efficiency and 92% silencing of the α1C gene by real-time PCR and Western blot. Electrophysiological experiments showed that the total ICa-L was similarly reduced by 80% in lentivirus transfected cells. Both biochemical and functional data demonstrated high transfection and silencing efficiency in the cardiomyocytes using lentiviral shRNA. This novel approach allows for the assessments of the roles of α1C and α1D Ca channels in native myocytes and could be used to examine their roles in physiological and pathological settings.
PMCID: PMC4334931  PMID: 19422800
L-type Ca channel; Silencing; Rat neonatal cardiomyocytes; Cardiac; Lentivirus
6.  Recombinant truncated AniA of pathogenic Neisseria elicits a non-native immune response and functional blocking antibodies 
AniA of the pathogenic Neisseria is glycosylated in its C-terminal repeat region by the pilin glycosylation (pgl) pathway. AniA appears to be unique among bacterial nitrite reductases as it contains an N-terminal extension that includes a lipid modification site as well as a C-terminal extension that is glycosylated. Immunising with various glycoforms of the AniA protein demonstrated a strong humoral immune response to the basal monosaccharide. In addition, when animals were immunised with a truncated form of AniA, completely lacking the glycosylated C-terminal region, the antibody response was directed against AniA regardless of the glycosylation state of the protein. Immuno-SEM confirmed that AniA is expressed on the cell surface in N. gonorrhoeae. Antisera generated against a truncated, non-glycosylated, recombinant form of the AniA protein are capable of blocking nitrite reductase function in a whole cell assay. We propose that recombinant modified AniA has potential as a vaccine antigen for N. gonorrhoeae.
PMCID: PMC4326246  PMID: 23313483
glycosylation; AniA; nitrite reductase; Neisseria meningitidis; Neisseria gonorrhoeae
7.  Insulin-dependent apolipoprotein B degradation is mediated by autophagy and involves class I and class III phosphatidylinositide 3-kinases 
Insulin acutely stimulates the degradation of apolipoprotein B (apo B) which decreases very low density lipoprotein (VLDL) secretion by liver. Insulin-dependent apo B degradation (IDAD) occurs following phosphatidylinositide 3-kinase (PI3K) activation and involves lysosomal degradation. Insulin suppression of apo B secretion is blocked by over-expression of phosphatase and tensin homologue (PTEN) in McArdle RH7777 (McA) cells suggesting the importance of Class I PI3K generated PI (3,4,5) triphosphate (PIP3) in IDAD. Classical autophagy inhibitors including 3-methyladenine, L-asparagine and bafilomycin A1 also blocked the ability of insulin to suppress apo B secretion by rat hepatocytes (RH) suggesting that IDAD occurs through an autophagy-related mechanism. IDAD is also blocked following over-expression in McA cells of a dominant negative kinase-defective Vps34, a class III PI3K that generates PI 3-monophosphate required for autophagy. Vps34 inhibition of IDAD occurs without altering insulin-dependent S473 phosphorylation Akt indicating PI3K/PIP3/Akt signaling is intact. Cellular p62/SQSTM1, an inverse indicator of autophagy, is increased with insulin treatment consistent with the known ability of insulin to inhibit autophagy, and therefore the role of insulin in utilizing components of autophagy for apo B degradation is unexpected. Thapsigargan, an inducer of endoplasmic reticulum (ER) stress, and a recently demonstrated autophagy inhibitor, blocked apo B secretion which contrasted with other autophagy inhibitors and mutant Vps34 results which were permissive with respect to apo B secretion. Pulse chase studies indicated that intact B100 and B48 proteins were retained in cells treated with thapsigargan consistent with their accumulation in autophagosomal vacuoles. Differences between IDAD and ER stress-coupled autophagy mediated by thapsgargin suggest that IDAD involves an unique form of autophagy. Insulin action resulting in hepatic apo B degradation is novel and important in understanding regulation of hepatic VLDL metabolism.
PMCID: PMC4321104  PMID: 23685141
apo B; autophagy; PI3K; Vps34; thapsigargin; PTEN; VLDL; liver
Abdominal aortic aneurysms (AAA) are progressive dilatations of infra-renal aorta causing structural weakening rendering the aorta prone to rupture. AAA can be potentially stabilized by inhibiting inflammatory enzymes such as matrix metalloproteinases (MMP); however, active regression of AAA is not possible without new elastic fiber regeneration. Here we report the elastogenic benefit of direct delivery of polyphenols such as pentagalloyl glucose (PGG), Epigallocatechin gallate (EGCG), and catechin, to smooth muscle cells obtained either from healthy or from aneurysmal rat aorta. Addition of 10 μg/ml PGG and ECGC induce elastin synthesis, organization, and crosslinking while catechin does not. Our results indicate that polyphenols bind to monomeric tropoelastin and enhance coacervation, aid in crosslinking of elastin by increasing lysyl oxidase (LOX) synthesis, and by blocking MMP-2 activity. Thus, polyphenol treatments leads to increased mature elastin fibers synthesis without increasing the production of intracellular tropoelastin.
PMCID: PMC3947410  PMID: 24440697
Abdominal aortic aneurysm; polyphenols; elastin; elastogenesis; vascular smooth muscle cells
9.  Development of a stable cell line with an intact PGC-1alpha/ERRalpha axis for screening environmental chemicals 
The estrogen-related receptor α (ERRα) and the peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) play critical roles in the control of several physiological functions, including the regulation of genes involved in energy homeostasis. However, little is known about the ability of environmental chemicals to disrupt or modulate this important bioenergetics pathway in humans. The goal of this study was to develop a cell-based assay system with an intact PGC-1α/ERRα axis that could be used as a screening assay for detecting such chemicals. To this end, we successfully generated several stable cell lines expressing PGC-1α and showed that the reporter driven by the native ERRα hormone response unit (AAB-Luc) is active in these cell lines and that the activation is PGC-1α-dependent. Furthermore, we show that this activation can be blocked by the ERRα selective inverse agonist, XCT790. In addition, we find that genistein and bisphenol A further stimulate the reporter activity, while kaempferol has minimal effect. These cell lines will be useful for identifying environmental chemicals that modulate this important pathway.
PMCID: PMC3967403  PMID: 24457025
10.  Hydrogen Peroxide Activation of ERK5 Confers Resistance to Jurkat Cells Against Apoptosis Induced by the Extrinsic Pathway 
Reactive oxygen species (ROS) including hydrogen peroxide (H2O2) exhibit both pro-survival and pro-death signaling in leukemic cells. We examined the effect of exogenous H2O2 on Fas ligand (FasL) -induced apoptosis in Jurkat cells. H2O2 applied prior to (pre-conditioning) and during (post-conditioning) FasL stimulation attenuated early apoptosis through activation of EKR5. H2O2 increased the activated caspase-8 sequestered in the mitochondria thereby decreasing cell death through the extrinsic apoptotic pathway. In addition, inhibition of a protein tyrosine phosphatase likely explains the post-conditioning requirement for H2O2. Given that chemotherapeutic agents used for the treatment of acute lymphoblastic leukemia are thought to work partly through production of ROS, a simultaneous inhibition of the ERK5 pathway may abrogate the ROS-initiated pro-survival signaling for an enhanced cell kill.
PMCID: PMC3974591  PMID: 24462874
apoptosis; extrinsic pathway; hydrogen peroxide; Jurkat cell; FAS ligand
11.  [No title available] 
PMCID: PMC3934640  PMID: 24406162
12.  [No title available] 
PMCID: PMC3951502  PMID: 24434143
13.  [No title available] 
PMCID: PMC3962750  PMID: 24434148
14.  Adenosine A3 Receptor Agonists Protect HL-60 and U-937 Cells from Apoptosis Induced by A3 Antagonists 
The effects of novel, selective adenosine (ADO) A3 receptor antagonists of diverse structure on cells of the human HL-60 leukemia and U-937 lymphoma cell lines were examined. Both 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine3,5-dicarboxylate (MRS 1191, 0.5µM) and 6-carboxymethyl-5,9-dihydro-9-methyl-2-phenyl-[1,2,4]-triazolo[5,1-a][2,7]naphthyridine (L-249313, 0.5 µM) induced apoptotic cell death and expression of bak protein. Low concentrations of the A3 receptor agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyluxonamide (Cl-IB-MECA, 10 nM or 1 µM) protected against antagonist-induced cell death. At concentrations ≥ 10 µM, the agonist alone produced apoptosis and bak expression in various cell lines. It is suggested that there exists a tonic low level of A3 receptor activation, possibly induced by release of endogenous adenosine, that results in cell protection.
PMCID: PMC4309999  PMID: 9125172
15.  Matrix rigidity regulates spatiotemporal dynamics of Cdc42 activity and vacuole formation kinetics of endothelial colony forming cells 
Recent evidence has shown that endothelial colony forming cells (ECFCs) may serve as a cell therapy for improving blood vessel formation in subjects with vascular injury, largely due to their robust vasculogenic potential. The Rho family GTPase Cdc42 is known to play a primary role in this vasculogenesis process, but little is known about how extracellular matrix (ECM) rigidity affects Cdc42 activity during the process. In this study, we addressed two questions: Does matrix rigidity affect Cdc42 activity in ECFC undergoing early vacuole formation? How is the spatiotemporal activation of Cdc42 related to ECFC vacuole formation? A fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor was used to examine the effects of the rigidity of three-dimensional (3D) collagen matrices on spatiotemporal activity of Cdc42 in ECFCs. Collagen matrix stiffness was modulated by varying the collagen concentration and therefore fibril density. The results showed that soft (150 Pa) matrices induced an increased level of Cdc42 activity compared to stiff (1 kPa) matrices. Time-course imaging and colocalization analysis of Cdc42 activity and vacuole formation revealed that Cdc42 activity was colocalized to the periphery of cytoplasmic vacuoles. Moreover, soft matrices generated faster and larger vacuoles than stiff matrices. The matrix-driven vacuole formation was enhanced by a constitutively active Cdc42 mutant, but significantly inhibited by a dominant-negative Cdc42 mutant. Collectively, the results suggest that matrix rigidity is a strong regulator of Cdc42 activity and vacuole formation kinetics, and that enhanced activity of Cdc42 is an important step in early vacuole formation in ECFCs.
PMCID: PMC3971637  PMID: 24393843
endothelial colony forming cells (ECFCs); Rho family GTPases; fluorescence resonance energy transfer (FRET); live cell imaging; matrix stiffness; mechanotransduction
16.  Genetic variants affecting alternative splicing of human cholesteryl ester transfer protein 
Cholesteryl ester transfer protein (CETP) plays an important role in reverse cholesterol transport, with decreased CETP activity increasing HDL levels. Formation of an alternative splice form lacking exon 9 (Δ9-CETP) has been associated with two single nucleotide polymorphisms (SNPs) in high linkage disequilibrium with each other, namely rs9930761 T>C located in intron 8 in a putative splicing branch site and rs5883 C>T in a possible exonic splicing enhancer (ESE) site in exon 9. To assess the relative effect of rs9930761 and rs5883 on splicing, mini-gene constructs spanning CETP exons 8 to 10, carrying all four possible allele combinations, were transfected into HEK293 and HepG2 cells. The minor T allele of rs5883 enhanced splicing significantly in both cell lines whereas the minor C allele of rs9930761 did not. In combination, the two alleles did not yield greater splicing than the rs5883 T allele alone in HepG2 cells. These results indicate that the genetic effect on CETP splicing is largely attributable to rs5883. We also confirm that Δ9-CETP protein is expressed in the liver but fails to circulate in the blood.
PMCID: PMC3929938  PMID: 24393849
Cholesteryl ester transfer protein; coronary artery disease; statin; alternative splicing; CETP levels in liver and plasma
17.  S-nitrosothiols increases cystic fibrosis transmembrane regulator expression and maturation in the cell surface 
S-nitrosothiols (SNOs) are endogenous signaling molecules with a broad spectrum of beneficial airway effects. SNOs are normally present in the airway, but levels tend to be low in cystic fibrosis (CF) patients. We and others have demonstrated that S-nitrosoglutathione (GSNO) increases the expression, maturation, and function of wild-type and mutant F508del cystic fibrosis transmembrane conductance regulator (CFTR) in human bronchial airway epithelial (HBAE) cells. We hypothesized that membrane permeable SNOs, such as S-nitrosoglutathione diethyl ester (GNODE) and S-nitroso-N-acetyl cysteine (SNOAC) may be more efficient in increasing the maturation of CFTR. HBAE cells expressing F508del CFTR were exposed to GNODE and SNOAC. The effects of these SNOs on the expression and maturation of F508del CFTR were determined by cell surface biotinylation and Western blot analysis. We also found for the first time that GNODE and SNOAC were effective at increasing CFTR maturation at the cell surface. Furthermore, we found that cells maintained at low temperature increased cell surface stability of F508del CFTR whereas the combination of low temperature and SNO treatment significantly extended the half-life of CFTR. Finally, we showed that SNO decreased the internalization rate of F508del CFTR in HBAE cells. We anticipate identifying the novel mechanisms, optimal SNOs, and lowest effective doses which could benefit cystic fibrosis patients.
PMCID: PMC3974270  PMID: 24393850
Cystic fibrosis; CFTR; S-nitrosothiol; S-nitrosylation; Chaperones; Molecular therapy
18.  Ezetimibe markedly attenuates hepatic cholesterol accumulation and improves liver function in the lysosomal acid lipase-deficient mouse, a model for cholesteryl ester storage disease 
Lysosomal acid lipase (LAL) plays a critical role in the intracellular handling of lipids by hydrolyzing cholesteryl esters (CE) and triacylglycerols (TAG) contained in newly internalized lipoproteins. In humans, mutations in the LAL gene result in cholesteryl ester storage disease (CESD), or in Wolman disease (WD) when the mutations cause complete loss of LAL activity. A rat model for WD and a mouse model for CESD have been described. In these studies we used LAL-deficient mice to investigate how modulating the amount of intestinally-derived cholesterol reaching the liver might impact its mass, cholesterol content, and function in this model. The main experiment tested if ezetimibe, a potent cholesterol absorption inhibitor, had any effect on CE accumulation in mice lacking LAL. In male Lal−/− mice given ezetimibe in their diet (20 mg/day/kg bw) for 4 weeks starting at 21 days of age, both liver mass and hepatic cholesterol concentration (mg/g) were reduced to the extent that whole-liver cholesterol content (mg/organ) in the treated mice (74.3±3.4) was only 56% of that in those not given ezetimibe (133.5±6.7). There was also a marked improvement in plasma alanine aminotransferase (ALT) activity. Thus, minimizing cholesterol absorption has a favorable impact on the liver in CESD.
PMCID: PMC3935496  PMID: 24370824
fatty liver; hepatomegaly; intrahepatic; cholesterol absorption; small intestine; triacylglycerol
19.  Distal NF-kB binding motif functions as an enhancer for nontypeable H. influenzae-induced DEFB4 regulation in epithelial cells 
Among the antimicrobial molecules produced by epithelial cells, DEFB4 is inducible in response to proinflammatory signals such as cytokines and bacterial molecules. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that exacerbates chronic obstructive pulmonary disease in adult and causes otitis media and sinusitis in children. Previously, we have demonstrated that DEFB4 effectively kills NTHi and is induced by NTHi via TLR2 signaling. The 5′-flanking region of DEFB4 contains several NF-κB binding motifs, but their NTHi-specific activity remains unclear. In this study, we aimed to elucidate molecular mechanism involved in DEFB4 regulation, focusing on the role of the distal NF-κB binding motif of DEFB4 responding to NTHi. Here, we show that the human middle ear epithelial cells up-regulate DEFB4 expression in response to NTHi via NF-κB activation mediated by IκKα/β–IκBα signaling. Deletion of the distal NF-κB binding motif led to a significant reduction in NTHi-induced DEFB4 up-regulation. A heterologous construct containing the distal NF-κB binding motif was found to increase the promoter activity in response to NTHi, indicating a NTHi-responding enhancer activity of the distal NF-κB binding motif. Furthermore, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that the p65 domain of NF-κB binds to the distal NF-κB binding motif in response to NTHi. Taken together, our results suggest that NTHi-induced binding of p65 NF-κB to the distal NF-κB binding motif of DEFB4 enhances NTHi-induced DEFB4 regulation in epithelial cells.
PMCID: PMC3940165  PMID: 24368180
20.  Insulin like growth factor 2 regulation of Aryl Hydrocarbon Receptor in MCF-7 breast cancer cells 
Insulin like growth factor (IGF)-1 and IGF-2 stimulate normal growth, development and breast cancer cell proliferation. Cyclin D1 (CCND1) promotes cell cycle by inhibiting retinoblastoma protein (RB1). The aryl hydrocarbon receptor (AHR) is a major xenobiotic receptor that also regulates cell cycle. The purpose of this study was to investigate whether IGF-2 promotes MCF-7 breast cancer proliferation by inducing AHR. Western blot and quantitative real time PCR (Q-PCR) analysis revealed that IGF-2 induced an approximately 2-fold increase (P <.001) in the expression of AHR and CCND1. Chromatin immunoprecipitation (ChIP), followed by Q-PCR indicated that IGF-2 promoted (P < .001) a 7-fold increase in AHR binding on the CCND1 promoter. AHR knockdown significantly (P < .001) inhibited IGF-2 stimulated increases in CCND1 mRNA and protein. AHR knockdown cells were less (P < .001) responsive to the proliferative effects of IGF-2 than control cells. Collectively, our findings have revealed a new regulatory mechanism by which IGF-2 induction of AHR promotes the expression of CCND1 and the proliferation of MCF-7 cells. This previously uncharacterized pathway could be important for the proliferation of IGF responsive cancer cells that also express AHR.
PMCID: PMC3932621  PMID: 24380854
Aryl hydrocarbon Receptor; IGF-2; CCND1; breast cancer cells
21.  Human RON receptor tyrosine kinase induces complete epithelial-to-mesenchymal transition but causes cellular senescence 
The RON receptor tyrosine kinase is a member of the MET proto-oncogene family and is important for cell proliferation, differentiation, and cancer development. Here we created a series of Madin-Darby canine kidney (MDCK) epithelial cell clones that express different levels of RON, and have investigated their biological properties. While low levels of RON correlated with little morphological change in MDCK cells, high levels of RON expression constitutively led to morphological scattering or complete and stabilized epithelial-to-mesenchymal transition (EMT). Unexpectedly, MDCK clones expressing higher levels of RON exhibited retarded proliferation and senescence, despite increased motility and invasiveness. RON was constitutively tyrosine-phosphorylated in MDCK cells expressing high levels of RON and undergoing EMT, and the MAPK signaling pathway was activated. This study reveals for the first time that RON alone is sufficient to induce complete and stabilized EMT in MDCK cells, and overexpression of RON does not cause cell transformation but rather induce cell cycle arrest and senescence, leading to impaired cell proliferation.
PMCID: PMC4296733  PMID: 17588532
recepteur d’origine Nantais (RON); epithelial-to-mesenchymal transition (EMT); cell proliferation; cell migration and invasion; senescence
22.  HER4 Selectively Coregulates Estrogen Stimulated Genes Associated with Breast Tumor Cell Proliferation 
The EGFR-family member HER4 undergoes regulated intramembrane proteolysis (RIP) to generate an intracellular domain (4ICD) that functions as a transcriptional coactivator. Accordingly, 4ICD coactivates the estrogen receptor (ER) and associates with ER at target gene promoters in breast tumor cells. However, the extent of 4ICD coactivation of ER and the functional significance of the 4ICD/ER transcriptional complex is unclear. To identify 4ICD coactivated genes we performed a microarray gene expression analysis of β-estradiol treated cells comparing control MCF-7 breast cancer cells to MCF-7 cells where HER4 expression was stably suppressed using a shRNA. In the MCF-7 cell line, β-estradiol significantly stimulated or repressed by 2-fold or more 726 or 53 genes, respectively. Significantly, HER4/4ICD was an obligate coactivator for 277 or 38% of the β-estradiol stimulated genes. Ingenuity Pathway Analysis of β-estradiol regulated genes identified significant associations with multiple cellular functions regulating cellular growth and proliferation, cell cycle progression, cancer metastasis, decreased hypoplasia, tumor cell migration, apoptotic resistance of tumor cells, and increased transcription. Genes coactivated by 4ICD displayed functional specificity by only significantly contributing to cellular growth and proliferation, cell cycle progression, and decreased hypoplasia. In direct concordance with these in situ results we show that HER4 knockdown in MCF-7 cells results in a loss of estrogen stimulated tumor cell proliferation and cell cycle progression, whereas, estrogen stimulated tumor cell migration was unaffected by loss of HER4 expression. In summary, we demonstrate for the first time that a cell surface receptor functions as an obligate ER coactivator with functional specificity associated with breast tumor cell proliferation and cell cycle progression. Nearly 90% of ER positive tumors coexpress HER4, therefore we predict that the majority of breast cancer patients would benefit from a strategy to therapeutic disengage ER/4ICD coregulated tumor cell proliferation.
PMCID: PMC3908545  PMID: 24333426
Breast cancer; EGFR-family; steroid receptors; cell cycle; p160 steroid receptor coactivator; gene regulation
23.  Akt-independent GSK3 inactivation downstream of PI3K signaling regulates mammalian axon regeneration 
Inactivation of glycogen synthase kinase 3 (GSK3) has been shown to mediate axon growth during development and regeneration. Phosphorylation of GSK3 by the kinase Akt is well known to be the major mechanism by which GSK3 is inactivated. However, whether such regulatory mechanism of GSK3 inactivation is used in neurons to control axon growth has not been directly studied. Here by using GSK3 mutant mice, in which GSK3 is insensitive to Akt-mediated inactivation, we show that sensory axons regenerate normally in vitro and in vivo after peripheral axotomy. We also find that GSK3 in sensory neurons of the mutant mice is still inactivated in response to peripheral axotomy and such inactivation is required for sensory axon regeneration. Lastly, we provide evidence that GSK3 activity is negatively regulated by PI3K signaling in the mutant mice upon peripheral axotomy, and the PI3K-GSK3 pathway is functionally required for sensory axon regeneration. Together, these results indicate that in response to peripheral nerve injury GSK3 inactivation, regulated by an alternative mechanism independent of Akt-mediated phosphorylation, controls sensory axon regeneration.
PMCID: PMC3916952  PMID: 24333443
Axon regeneration; GSK3 signaling; PI3K signaling; In vivo electroporation
24.  Accumulation of Nano-sized Particles in a Murine Model of Angiogenesis 
To evaluate the ability of nm-scaled iron oxide particles conjugated with Azure A, a classic histological dye, to accumulate in areas of angiogenesis in a recently developed murine angiogenesis model.
Materials and Methods
We characterized the Azure A particles with regard to their hydrodynamic size, zeta potential, and blood circulation half-life. The particles were then investigated by Magnetic Resonance Imaging (MRI) in a recently developed murine angiogenesis model along with reference particles (Ferumoxtran-10) and saline injections.
The Azure A particles had a mean hydrodynamic diameter of 51.8 ± 43.2 nm, a zeta potential of -17.2 ± 2.8 mV, and a blood circulation half-life of 127.8 ± 74.7 minutes. Comparison of MR images taken pre- and 24-hours post- injection revealed a significant increase in R2* relaxation rates for both Azure A and Ferumoxtran-10 particles. No significant difference was found for the saline injections. The relative increase was calculated for the three groups, and showed a significant difference between the saline group and the Azure A group, and between the saline group and the Ferumoxtran-10 group. However, no significant difference was found between the two particle groups.
Ultrahigh-field MRI revealed localization of both types of iron oxide particles to areas of neovasculature. However, the Azure A particles did not show any enhanced accumulation relative to Ferumoxtran-10, suggesting the accumulation in both cases to be passive.
PMCID: PMC3909773  PMID: 24321551
Angiogenesis; iron oxide particles; ultrahigh-field MRI; sponge model
25.  TLR4-dependent metabolic changes are associated with cognitive impairment in an animal model of type 1 diabetes 
We investigated the role of Toll-like receptor 4 (TLR4), a major mediator of innate immune responses, on cognitive performance in a type 1 diabetes model (T1D). After administration of streptozotocin, both TLR4 knockout (TLR4 KO) and wild type (WT) diabetic mice displayed metabolic alterations similar to those observed in T1D patients, including increased levels of glucose, cholesterol, triglycerides and ketones. T1D mice exhibited cognitive impairment which was less severe in TLR4 KO mice compared to WT mice. WT mice with higher glucose and those with higher triglyceride levels exhibited significantly more anxiety and impaired memory compared to those with lower levels of glucose and triglycerides; these correlations were absent in TLR4 KO mice. Additional findings suggest roles for TLR4 signaling in modifying the expression of enzymes involved in energy metabolism in brain cells in the setting of T1D. Our data show that TLR4 contributes to the negative impact of T1D on anxiety and cognition.
PMCID: PMC3916215  PMID: 24342620
TLR4; diabetes; ketones; memory; anxiety

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